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BiomedicalResearchJournal

APRIL 2015 | VOLUME 2 | ISSUE 1

SCHOOL OF SCIENCE

pISSN: 2349-3666; eISSN: 2349-3674

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BiomedicalResearchJournal

APRIL 2015 | VOLUME 2 | ISSUE 1

SCHOOL OF SCIENCE

EDITORS-IN-CHIEF

Dhananjaya Saranath (Mumbai, India)

Aparna Khanna (Mumbai, India)

SECTION EDITORS

Cancer Biology:

Girish Maru (Navi Mumbai, India)

Stem Cell Biology:

Vaijayanti P. Kale (Pune, India)

Nanotechnology:

Vilas G. Gaikar (Mumbai, India)

Phytochemistry:

Lokesh Bhatt (Mumbai, India)

EDITORIAL BOARD

Ali Syed Arbab (Detroit, USA)

Amit Agarwal (Bangalore, India)

Anjali A. Karande (Bangalore, India)

Basuthkar J. Rao (Mumbai, India)

Hemant Malhotra (Jaipur, India)

Kirti S. Laddha (Mumbai, India)

Mohan C. Vemuri (Frederick, USA)

Nancy Pandita (Mumbai, India)

Paul J. Verma (Rosedale, Australia)

Pritish Bhattacharya (New Jersey, USA)

Purvish M. Parikh (Mumbai, India)

Sai Yendamuri (New York, USA)

Sumitra Chanda (Rajkot, India)

Surinder K. Mehta (Chandigarh, India)

Victoria M. Villaflor (Chicago, USA)

Alpana Ray (Missouri, USA)

Anandwardhan Hardikar (Sydney, Australia)

Ashok B. Vaidya (Mumbai, India)

Dhirendra Bahadur (Mumbai, India)

Karuna Shanker (Lucknow, India)

Mayur Yergeri (Mumbai, India)

Naganand Rayapuram (Evry, France)

Partha Basu (Kolkata, India)

Prasad S. Adusumilli (New York, USA)

Pulok Mukherjee (Kolkata, India)

Ramesh Goyal (Ahmedabad, India)

Sukhinder Kaur Cheema (St. John's, Canada)

Sunita Saxena (New Delhi, India)

Tania Fernandez (San Francisco, USA)

EDITORIAL OFFICE

School of Science, NMIMS (Deemed-to-be University)

Bhaidas Sabhagriha Building,

Bhaktivedanta Swami Marg,

Vile Parle (W), Mumbai 400056, India.

Email: [email protected]

EDITORIAL ASSISTANT

Brijesh S. (Mumbai, India)

Aims and Scope

“Biomedical Research Journal (BRJ)” is a

premier peer reviewed open access journal,

published by School of Science, NMIMS

(Deemed-to-be) University, for promoting the

advancement of ideas in the interdisciplinary

realms of Medicine, Science and Technology.

The goal is to share new discoveries and

translational knowledge with scientists,

academicians, clinicians and students in the

field of Biomedical and Biology/Chemistry/

Biotechnology/Stem Cell Biology/Cancer

Biology in the realm of basic and applied

aspects in the different areas.

BRJ aims at creating a platform to help

advance the domains and frontiers of inter- and

multi-disciplinary research across the various

areas of sciences and recent advances in cross

pollination across biology, chemistry, and

medicine. Integrative science is the present

and future of science, and the journal proposes

to highlight and emphasize contemporary

technology towards understanding various

aspects of the sciences.

The initial focus areas of BRJ include

review articles and original research papers in

cancer biology, stem cell biology, nano-

technology and phytochemistry.

A rigorous peer review process is

implemented to judge the effectiveness,

legitimacy and reliability of the research

content. The papers will be published online as

well as provide hard copy of the Journal issues

to the authors of the papers on request.

Information for Subscribers

BRJ is planned as a six monthly publication

with two issues published in the first year.

Currently, there are no subscription charges for

the journal and can be accessed online. For

submission instructions, subscription and

additional information please visit:

http://science.nmims.edu

Disclaimer

The views and opinions expressed in the

articles published in the journal are the sole

responsibility of the authors. The Publisher,

NMIMS School of Science and the Editors

cannot be held responsible for errors or any

consequences from the use of information

contained in this journal.

Copyright

The Journal grants all users a free, permanent,

worldwide, continuous right of access to, and a

license to copy, use, distribute, perform and

display the work publicly and to make and

distribute derivative works in any digital

medium for any reasonable non-commercial

purpose, subject to proper citation of

authorship and ownership rights. The journal

also grants the right to make a printed copy for

personal non-commercial use only.

Biomedical Research Journal

General Information

Contents April 2015, Volume 2, Issue 1

Dhananjaya Saranath and Aparna Khanna ..........................................................................................1

Uday B. Maachani, Uma Shankavaram, Kevin Camphausen and Anita Tandle ..................................6

Saumya Nigam, Sudeshna Chandra and Dhirendra Bahadur ...........................................................21

Sweta Dash, Raghava Reddy Sunkara and Sanjeev K. Waghmare ..................................................37

Lokesh Gambhir, Rahul Checker, Deepak Sharma and Santosh K. Sandur .....................................57

Deepak N. Modi and Pradeep Bhartiya …………………..…………….............................………….....83

Cody D. Schlaff, W. Tristram Arscott, Ira Gordon, Kevin A. Camphausen and Anita Tandle ............105

Shweta Singh, Suprita Ghode, Moirangthem Ranjita Devi, Lalita Limaye and Vaijayanti Kale ........120

Editorial: Precision/personalized medicine in cancer

Advances in Omics technologies in GBM

Dendrimers based electrochemical biosensors

Developmental signalling in maintenance and regulation of cancer stem cells

Diverging role of Nrf2 in cancer progression and prevention

Physiology of embryo-endometrial cross talk

Human EGFR-2, EGFR and HDAC triple-inhibitor CUDC-101 enhances

radiosensitivity of glioblastoma multiforme cells

Phenotypic and functional characterization of a marrow-derived stromal cell

line, M210B4 and its comparison with primary marrow stromal cells

Biomed Res J 2015;2(1):1-5

Editorial

Precision/Personalized Medicine in Cancer

Recent technological advances have provided

unprecedented opportunities to develop

platforms for implementing 'Precision/

Personalized Medicine', with confluence of

medicine and technology making significant

advances in treatment. The Cancer Genome

Atlas (TCGA), a large scale initiative started in

2006, to generate a comprehensive landscape

for identification of alterations in tumor types

with a view to develop better therapies, is on a

wind down. The next phase is use of the

information generated for 'Precision/

Personalized Medicine'. Implementation of

'Precision/Personalized Medicine' requires

understanding of the biology of each cancer

type with precise definition of the cancer

genome. The different genome alterations will

identify the 'Founder Mutations' involved in

the early phase, but may not be associated with

a fully transformed phenotype; 'Driver

Mutations' required for fully transformed

phenotype; and 'Passenger Mutations'

considered as collateral damage. Treatment

against key oncogenic driver mutations in

individual cases with targeted drugs will be the

benefit of the 'Precision Medicine' approach.

Rational choices for treatment have to be

preceded by full genomic data and expression

data, facilitating combination therapy

decisions. Thus, advanced technology

including 'Next Generation Sequencing'

(NGS) with both availability and affordability

will enable understanding of cancer and other

diseases, a feasible proposition.

Next Generation Sequencing is massively

parallel sequencing enabling rapid sequencing

of the entire genome or exome sequencing on

whole genome or cDNA (RNA-Seq), builds

on the concept of 'NGS taking us into

expanded genomic testing for risk prediction,

diagnosis, prognosis, treatment response and

disease free survival or overall survival in real

time'. The understanding of the mechanisms

and processes in cancer with single aberrations

or cumulative alterations including mutations,

rearrangements, amplifications, deletions,

insertions and other alterations in cancer will

be discerned. It is important to remember that

several countries have initiated studies in this

direction. Prime Minister David Cameroon,

UK, endorsed the 'Genomes Project' for

collection of data for whole genome sequences

from 100,000 individuals, to be completed by

2017, sanctioning USD 475 million for

sequencing studies, with a view to better

understand complex diseases including

cancer. Barack Obama, President, USA,

launched the 'Precision Medicine' initiative

Dhananjaya Saranath and Aparna Khanna

with a USD 215 million for genomic data on

one million volunteers to accelerate patient

powered research that promises to accelerate

biomedical discoveries and provide clinicians

with new tools, knowledge and therapies for

individual patients. A result of better

understanding of cancer, is the current

repertoire of targeted therapy drugs and

personalized medicine on the global scene.

In the past decade the advent of targeted

therapy and new tailored drugs has led to a

revolution in treatment of lung cancer, with

larger benefit, lower toxicity and better quality

of life for the patient. The treatment is often

based on molecular profiling of individual

patients with identified cancer, as also

indicated in other cancers with a similar

molecular profile. Thus, Tyrosine kinase

inhibitors including Erlotinib, Gefitinib,

Afatinib targeting epidermal growth factor

receptors (EGFR), and ALK Inhibitors

including Crizotinib, Ceritinibare beneficially

used in patients with aberration of the genes in

Non-small cell lung cancer, and indicated for

additional cancers with the appropriate

molecular profile. Involvement of Kras,

EGFR, ALK, HER2, Braf, MET, AKTI,

MAP2KI, PI3KC genes have been identified

in lung cancers, opening possibilities of

targeted therapy with consequential benefits.

Thus, an additional aspect which has emerged

is combination therapy using two or more

targeted drugs, or targeted drugs plus the

conventional chemotherapeutic drugs. A few

examples are targeted drugs Dabrafenib and

Trametinib, mitogen activated protein kinase

1/2 (MAPK1/2) for melanoma with

BRAFV600E/K; angiogenesis inhibitor,

Bevacizumab, against vascular endothelial

factor A is a targeted therapy in cancers of the

colon, lung, breast, kidney and brain; whereas

Ramicirumab, a monoclonal antibody against

vascular endothelial growth factor receptor 2,

is used in gastric cancer and Non-small cell

lung cancer, and in combination with

Docetaxel improves outcomes in bladder

cancer. HER2 gene antibody – Herceptin,

shows substantial survival benefits in all

newly diagnosed and recurrent breast cancer

patients with amplification and over-

expression of the gene. Development of

companion diagnostics indicating the

pathogenic molecular alterations and new

targeted drugs go hand-in-hand, and

guidelines for several molecular diagnostic

tests are available. Thus, 'Precision/

Personalized Medicine' will enable the current

oncologists to 'Win the War against Cancer'.

The current issue includes a review paper

and an original article on glioblastoma

multiforme (GBM), one of the most

aggressive brain tumor, a cancer with bad

prognosis and median survival of 15 months.

The conventional treatment of GBM using the

strategy of surgery, radiation and

chemotherapy, has advanced only

incrementally in the last 30 years. With the

advent of molecular biology and consequent

Biomed Res J 2015;2(1):1-5

2 Editorial: Precision/personalized medicine in cancer

improved understanding of basic tumor

biology, targeted therapies have become

cornerstones for cancer treatment. Several

signaling pathways including RTKs/PI3K/

AKT/mTOR/VEGF/VEGFR are deregulated

in GBM, playing a major role in

tumorigenesis, treatment resistance and

progression of GBM. Dr. Anita Tandle and

colleagues from National Cancer Institute,

Bethesda, Maryland, USA, discuss the Omics

of GBM and applications in novel therapies, in

the article, ‘Advances in Omics technologies

in GBM’. The authors survey the technologies

of genomics, transcriptomics, epigenomics,

proteomics, metabolomics and post

transcriptional modifications of microRNAs

in GBM. A comprehensive information in

GBM will lead to better understanding of the

cancer, highlight the various signal

transduction pathways, and identify key

molecules associated with the pathogenesis,

culminating in development of new drugs and

'Personalized treatment'. The original article

by the group, ‘EGFR 2, EGFR and HDAC

triple inhibitor CUDC-101 enhances

radiosensitivity of GBM cells’, convincingly

shows enhancement of in vitro radiosensitivity

of GBM and breast cancer cell lines

selectively, with no effect on normal human

lung fibroblast cell line. The radiosensitization

of the cancer cell lines was attributed to

inhibition of DNA double stranded break

repair and modulation of cell cycle. A better

understanding of the cancer will open avenues

for better contemporary treatment.

Nanomaterials and nanoparticles

including dendrimers, polymers, nanotubes,

oxides, and enzymes and their hybrids as

catalysts for various sensors such as glucose

sensors, DNA sensors, neurotransmitters

sensors, are another facet of technological

advances with tremendous applications in

health sciences. Dendrimers are synthetic

nanoscale compounds with unique properties,

resulting in biomedical and industrial

applications. Dendrimers have a number of

features that make them ideally suited for

sensor applications, such as, its high surface

area, high reactivity, easy dispersability and

rapid fabrication. Dr. Saumya Nigam and Dr.

Dhirendra Bahadur, Indian Institute of

Technology Bombay, Mumbai, along with Dr.

Sudeshna Chandra, NMIMs (Deemed-to-be)

University, present a review on ‘Dendrimers

based electrochemical biosensors’. The

review highlights the advanced development

of effective, rapid and versatile electro-

chemical biosensors based on dendrimers. A

must read review for all to understand the

technology.

The concept of cancer stem cells (CSC)

proposed earlier in the year 2000, are now well

accepted to play a critical role in cancers. The

CSCs are more of an enigma and relatively

more difficult to decode the biology of CSCs.

The conserved Wnt/β-Catenin, Notch and

Sonic Hedgehog pathways regulate stem cell

pluripotency and cell fate decisions during

Biomed Res J 2015;2(1):1-5

Saranath and Khanna 3

normal embryonic development and adult

tissue homeostasis, and aberrant activity

within these pathwaysis displayed in several

cancers. Human cancers contain a relatively

dormant cell population, CSCs, with

characteristics similar to normal stem cells.

Convincing evidence indicates that CSCs are

responsible for chemotherapy/radiation

therapy resistance, maintenance and

consequent recurrence of the cancer. The roles

of Wnt, Notch and Hedgehog pathways in

cancers and their deregulation are of critical

significance, directly linked to CSCs. In order

to target the CSCs therapeutically, it is

imperative to understand the molecular

mechanisms regulating CSCs responsible for

maintenance and recurrence of cancer, and

develop combination therapies to target CSCs

inhibiting the cumulative action of the

deregulated genes. Dr. Sanjeev Waghmare and

his colleagues from Advanced Centre for

Treatment, Research and Education in Cancer,

Navi Mumbai, succinctly review the

intricately complex signalling cascades of

Wnt, Notch and Hedgehog genes, regulation

and maintenance of normal developmental

processes, and their association in cancer, in

the article, ‘Developmental signalling in

maintenance and regulation of cancer stem

cells’. Whereas, the article, ‘Phenotypic and

functional characterization in the

maintenance and regulation of a marrow-

derived stromal cell line, M210B4 and its

comparison with primary marrow stromal

cells’ by Dr. Vaijayanti Kale and colleagues

from the National Centre for Cell Science,

Pune, emphasizes importance of alternative

systems for investigating regulation of

hematopoietic stem cells (HSCs). The authors

showed that the cell line M210B4

unequivocally differentiated towards

adipogenic lineage, and exhibited a higher

HSC-supportive ability and conclude that the

cell line M210B4 is an appropriate substitute

to study HSC regulation in vitro.

The transcription factor Nrf2 containing

the conserved basic leucine zipper structure

belongs to the Cap 'N' Collar family, and plays

a critical role in cell defense and survival

pathways. Nrf2 often protects cells and tissues

from toxicants and carcinogens via

transcription of cytoprotective genes, and

hence considered chemopreventive,

protecting against redox-mediated injury and

carcinogenesis. Paradoxically, the flip side of

Nrf2 is protection of cancer cells from

chemotherapeutic agents and/or radiotherapy

resulting in resistance to the therapy and

cancer progression. Nrf2 is aberrantly

upregulated in several cancer types, and

associated with poor prognosis in cancer

patients. The dilemma of the dual action of

Nrf2 has been well reviewed in the article,

‘Divergent role of Nrf2 in cancer

progression and prevention’ by Dr. Santosh

Sandur and colleagues from Bhabha Atomic

Research Centre, Mumbai. The review

indicates a wider approach with better

Biomed Res J 2015;2(1):1-5

4 Editorial: Precision/personalized medicine in cancer

comprehension of the mechanisms of action of

Nrf2 and consequent design and development

of drugs to handle the upregulation or

downregulation of Nrf2 in the preventive,

protective or destructive niche of normalcy

and diseases. Thus, 'One fit for all' is not a

feasible solution in all conditions indicating

importance of 'Precision/Personalized

Medicine'.

The mechanisms of embryo implantation

and development resulting in pregnancy are

comparable to cancer with respect to the

growth processes and mechanisms of

development. A receptive endometrium,

normal blastocyst, cross talk between fetal and

maternal compartments remodeling uterine

vasculature, and selector activity comprise

innate requirements for successful pregnancy.

Adverse events such as preeclampsia,

infertility and intra-uterine growth retardation

are minimized or avoided in normal fetal

growth. The highly orchestrated embryo-

endometrial cross talk involves a plethora of

molecules including hormones, cytokines,

growth factors, specific immune modulating

factors, to create the appropriate micromileu

for establishing pregnancy. Dr. Deepak Modi

and Mr. Pradeep Bhartiya from the National

Institute for Research in Reproductive Health,

Mumbai, take us through the ‘Physiology of

embryo-endometrial cross talk’ lucidly

highlighting the various processes and

interactions. The networking interactions and

intricate physiology in a pregnancy is very

well explained. The applications in a clinical

scenario for successful implantation,

infertility treatment and development of

contraceptive drugs are discussed.

Biomed Res J 2015;2(1):1-5

Saranath and Khanna 5

INTRODUCTION

Glioblastoma

Brain tumors account for about 85–90% of all

primary central nervous system (CNS) tumors.

Worldwide, approximately 343,175 new cases

of brain and other CNS tumors were diagnosed

in the year 2012 (http://www.cbtrus.org/).

Glioblastoma or glioblastoma multiforme

(GBM) is the most lethal and clinically

challenging of brain tumors. Most patients die

of their ailment in less than a year (Stupp et al.,

2005). Some of the reasons for high fatality are

the complex nature and diffuse character of the

tumor itself and the high rate of disease

recurrence. As the name infers, it is

multiforme microscopically showing regions

of pseudopalisading and hemorrhage,

multiforme genetically with various genetic

alterations leading to its aggressive nature.

The standard of care for treatment of GBM

includes surgical resection followed by

radiation and chemotherapy. The addition of a

chemotherapeutic agent, Temozolamide in

recent years changed the median survival of

for GBM patients to 14.6 months from 12.1

months with surgery and radiotherapy (Stupp

et al., 2005). Also, currently there is no

Key words: Glioblastoma, Omics, Genomics, Transcriptomics, Epigenomics, Proteomics, Metabolomics. *Corresponding Author: Anita Tandle, Radiation Oncology Branch, National Cancer Institute, 10 Center Drive Magnuson Clinical Center Room B3-B100, Bethesda MD 20892, USA.Email: [email protected]

Advances in Omics Technologies in GBM

Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda MD, USA

Glioblastoma multiforme (GBM) is one of the most lethal human cancers and poses a great challenge in the

therapeutic interventions of GBM patients worldwide. Despite prominent recent advances in oncology, on

an average GBM patients survive 12–15 months with conventional standard of care treatment. To

understand the pathophysiology of this disease, recently the research focus has been on omics-based

approaches. Advances in high-throughput assay development and bioinformatic techniques have provided

new opportunities in the molecular analysis of cancer omics technologies including genomics,

transcriptomics, epigenomics, proteomics, and metabolomics. Further, the enormous addition and

accessibility of public databases with associated clinical demographic information including tumor

histology, patient response and outcome, have profoundly improved our knowledge of the molecular

mechanisms driving cancer. In GBM, omics have significantly aided in defining the molecular architecture of

tumorigenesis, uncovering relevant subsets of patients whose disease may require different treatments. In

this review, we focus on the unique advantages of multifaceted omics technologies and discuss the

implications on translational GBM research.

Uday B. Maachani, Uma Shankavaram, Kevin Camphausen, Anita Tandle*

Biomed Res J 2015;2(1):6-20

Review

standard of care available for recurrent disease

and most of the patients die. Hence there is an

urgent need to develop molecular targeted

therapy for this devastating disease.

Some of the molecular alterations

responsible for GBM progression and

therapeutic resistance includes genetic and

epigenetic alterations, activation of stem cell

pathways, and changes in the tumor

microenvironment and cellular metabolism.

However, the functional consequences of

many of these alterations are largely unknown

in GBM tumorigenesis (Frattini et al., 2013;

Schonberg et al., 2013).

Omics

With the sequencing of the human genome, the

study of biological systems underwent a major

genomic revolution. The major technological

breakthroughs in high-throughput assay

development, technological advancements in

instrumentation and bioinformatics data

analysis have reshaped how we view the

cancer genome (Vucic et al., 2012). “Omics”

refers to the study of cancer as a whole entity

focusing on the various micro- and macro-

molecules. It includes (but not limited to)

DNA mutations, copy number changes,

epigenetic changes like DNA methylation,

transcriptome analysis and whole-genome

DNA/RNA sequencing. The omics-based

recent approaches including genomics,

transcriptomics, epigenomics, proteomics and

metabolomics have unveiled the molecular

mechanisms behind various cancers and

assisted in identification of next-generation

molecular markers for early diagnosis,

prognosis, predictive of response to treatments

and predisposition to gliomas (Chin, 2013;

Cho, 2010) (Fig. 1). The publically available

multi-omics databases collected by

International Cancer Genome Consortium

Figure 1: Omics in glioblastoma.

Biomed Res J 2015;2(1):6-20

Maachani et al. 7

(ICGC) and The Cancer Genome Atlas

(http://cancergenome.nih.gov/) a network

group using a sample cohort of several

hundred clinical specimens of GBM further

elaborated the molecular processes funda-

mental to GBM pathogenesis (Hudson et al.,

2010; Verhaak et al., 2010).

Genomics/Transcriptomics

Early work on gene expression analysis of

gliomas employed DNA microarrays and

attempted to correlate mRNA signatures with

the grades of gliomas and their clinical

behavior to aid in overall prognosis and

treatment response of patients (Kim et al.,

2002; Mischel et al., 2004). Transcriptomics is

the study of RNA transcripts that are produced

by the genome, under specific circumstances

or in a specific cell using high-throughput

methods, such as microarray analysis,

allowing the identification of genes that are

differentially expressed in distinct cell

populations. Recent multi-omics (genomics,

transcriptomics and proteomics) data

integration studies have utilized patient

derived samples and cell lines to reveal

heterogeneity among the primary GBM,

suggesting additional molecular subclasses:

neural, proneural, classical and mesenchymal

(Verhaak et al., 2010). These subtypes were

defined on the basis of distinct gene signatures

and also characterized by different molecular

alterations and activated pathways (Brennan et

al., 2013; Verhaak et al., 2010). The proneural

subtype was mostly characterized by

abnormalities in platelet-derived growth

factor receptor-α (PDGFRA) or in isocitrate

dehydrogenase 1 (IDH1); whereas mutation of

the epidermal growth factor receptor (EGFR)

was found in the classical subgroup, and

mutations in neurofibromin 1 (NF1) were

common in mesenchymal tumors. The neural

subtype seemed to be similar to the classical

subtype but with a higher frequency of TP53

mutations (Brennan et al., 2013). Cytogenetic

and molecular studies have also identified a

number of recurrent chromosomal

abnormalities and genetic alterations in

malignant gliomas, as well as novel

candidates, particularly in GBMs. The

identification of molecular subtypes has

revealed a set of core signaling pathways

commonly activated in GBM (Table 1)

(Furnari et al., 2007) and could be used in

molecular targeted therapies (Table 2).

Epigenomics

Epigenetic changes involve enzymatic

modifications of DNA and associated histone

proteins to regulate gene expression. In recent

years these changes have been recognized as

important causes of phenotypic changes in

human cancers (Esteller, 2007). The

epigenetic changes are dynamic in nature and

play an important role in gene expression and

DNA structure. Epigenetic alterations,

especially those related to changes in histone

acetylation, are a recent focus for therapeutic

Biomed Res J 2015;2(1):6-20

8 Advances in omics technologies in GBM

drug targeting in clinical trials. Genomic-array

(microarray) techniques studying DNA

methylation have identified frequent

promoter-associated hypermethylation of

specific loci accompanying tumor suppression

in GBM (Sturm et al., 2014), such as

(CDKN2A), RB1, PTEN, TP53 (Amatya et

al., 2005; Baeza et al., 2003; Costello et al.,

1996; Nakamura et al., 2001) and other

previously unrecognized regulatory genes

EMP3, PDGFB (Alaminos et al., 2005; Bruna

et al., 2007). Most significantly, O-6-methyl-

guanine-DNA methyltransferase (MGMT)

promoter hypermethylation was identified

occurring in ~45% of adult patients with GBM

(Brennan et al., 2013; Esteller et al., 1999).

MGMT hypermethylation leads to gene

silencing, and reduced gene expression levels

which compromises its ability to repair

damaged DNA by alkylating agents like

Temozolomide (Felsberg et al., 2011). Thus,

gene methylation could be used as a biomarker

Table 1: Role of Omics in biomarkers identification and disease prognosis

GBM Biomarkers Role in GBM prognosis

EGFR amplification

EGFRvIII mutation

EGFR amplification is the most common event in primary GBM, with EGFRvIII being the most

prominent mutated receptor tyrosine kinase receptor occurring in ~50% of GBM cases that

overexpress EGFR (Verhaak et al., 2010). A potential predictive biomarker for molecular

therapies.

PDGFRA PDGFRA is mainly mutated and expressed in abnormally high amounts in proneural tumors

(Verhaak et al., 2010) and associated with poor prognosis in IDH1 mutant GBM (Brennan et al.,

2013).

TP53 mutation TP53 gene although mutated, has no predictive or prognostic role. Can distinguish tumor grade

(Brennan et al., 2013).

1p/19q Co-deletion 1p/19q co-deletion is the most common genetic alteration in oligodendroglioma tumors and is

associated with favorable response to chemotherapy, radiation and survival (Alaminos et al.,

2005).

MGMT promoter

methylation

Promoter methylation of MGMT gene, inactivates DNA repair function (Esteller et al., 1999). It is

the first predictive epigenetic biomarker with a putative diagnostic role in detecting

pseudoprogression. MGMT methylation helps in molecular stratification of patients for

Temozolomide therapy (Malstrom et al., 2012).

VEGF VEGF is considered to be the driving factor of tumor angiogenesis and has been identified in

64.1% GBMs. It is a strong predictor of survival, in patients with gliomas (Reynes et al., 2011)

PTEN A gene level biomarker with poor survival outcomes for GBM (Baeza et al., 2003). PTEN is deleted

in 50–70% of primary and 54–63% of secondary GBM. Also mutated in 14–47% primary GBM.

Mutation is linked to resistance to targeted EGFR inhibitors in GBM (Deberardinis et al., 2008).

IDH1/2 mutation IDH1 mutation is now recognized as an important driver in the etiology of low-grade and

secondary brain tumors (48). Has prognostic value in WHO grade III and IV GBM. Accumulation of

oncometabolite 2-hydroxygluatrate (2HG) considered as metabolomic imaging biomarker for

mutant IDH1 gliomas (Chen et al., 2014).

Biomed Res J 2015;2(1):6-20

Maachani et al. 9

to predict sensitivity to chemo- radiotherapy

(Malmström et al., 2012; Wick et al., 2012).

Further, based on DNA methylation patterns,

proneural subtype is classified into CpG island

methylator phenotype (CIMP) positive and

CpG–CIMP-negative GBM subsets which

strongly correlates with IDH1 gene mutation

status (Noushmehr et al., 2010; Turcan et al.,

2012). Glioma CIMP (G-CIMP) is a powerful

determinant of tumor aggressiveness

(Brennan et al., 2013; Riemenschneider et al.,

2010). These epigenomic and other multi-

omic analyses have revealed several

mutations, altered proteins, miRNA

expressions and pathways associated with

GBM pathogenesis and prognosis.

Table 2: Molecular targeted therapies for glioblastoma

Other current strategies tested in GBMs

Pathways targeted Agents Molecular targets

Epidermal Growth Factor Pathway

EGFR is amplified and frequently mutated in ~50% of GBMs and is

overexpressed in many malignant gliomas. Therefore could be used

as a therapeutic targeted agent in GBM patients.

Erlotinib (Roche)

Gefitinib (AstraZeneca)

Kinase inhibitors of

EGFR

VEGF Pathway

Targeting vascular endothelial growth factor (VEGF) pathways to

induce anti-angiogenic effects in the treatment of malignant gliomas

has been in focus for past few years.

Bevacizumab (Avastin;

Genentech)

Recombinant human

neutralizing monoclonal

antibody to VEGF

Vatalanib (Novartis) Kinase inhibitor of

VEGFR/PDGFR

Cediranib (AstraZeneca) pan-VEGFR inhibitor

Transforming Growth Factor β (TGF-β) Pathway

TGF-β is a multifunctional cytokine, which regulates glioma cell

motility, invasion, and immune surveillance. Several small molecule

inhibitors of TGF-β receptors have shown antitumor efficacy in

preclinical models of gliomas.

Trabedersen (Antisense

Pharma)

Anti-sense TGF-β2

mRNA

PI3K–AKT–mTOR Pathways

PI3K pathways regulate several malignant phenotypes including

antiapoptosis, cell growth, proliferation, and invasion. Activated PI3K

phosphorylates several downstream effectors, including AKT. mTOR

is a major player connecting multiple pathways downstream from

AKT.

Rapamycin (Sirolimus)

Temsirolimus (Sirolimus)

Everolimus (Novartis)

inhibitors of m-TOR

PKC Pathways

Protein kinase C (PKC) is a serine/threonine kinase that regulates

cell proliferation, invasion, and angiogenesis.

enzastaurin (Eli-Lilly) PKC-β inhibitor with

activity against glycogen

synthase kinase 3β

Note: Several of the above agents are being evaluated in clinical trials as monotherapies or in combination with other

treatment modalities such as chemotherapy or radiation in patients with malignant gliomas.

Biomed Res J 2015;2(1):6-20


Proteomics

Proteomic profiling represents the large-scale

examination of protein expression, post-

translational modification, and understanding

how different proteins interact with each other.

Using various bioinformatics techniques, the

information can be unified into protein

networks. Currently, histopathology

represents the gold standard for the typing and

grading of gliomas and depends largely on

certain architectural similarities of tumor cells

with normal glial cells (Riemenschneider et

al., 2010; Tohma et al., 1998). We feel that the

underlying disease pathology would result

into differential proteomic profiling of

diseased tissue and the surrounding disease-

free normal tissue. Recent technological

advances in proteomics has allowed analysis

of glioma patient biopsies, proximal fluids,

cerebrospinal fluid (CSF) and cyst fluid,

plasma, glioma cell lines. This has allowed a

comprehensive proteomic profiling of glioma

biology to aid the traditional histopathology in

improving our understanding of glioma

processes and to better evaluation of drug

responses to treatment (Somasundaram et al.,

2009). The techniques involve evaluation of

protein arrays, including antibody and aptamer

arrays. This allows simultaneous detection of

multiple proteins/phosphoproteins. These

high throughput techniques can be used for

efficient biomarker validation, treatment

monitoring and can be translated into clinical

applications in an affordable manner. Various

plasma/serum biomarkers have been

identified earlier for GBM including YKL-40,

GFAP and matrix metalloproteinase-9

(Jayaram et al., 2014). Reynes et al. (2011)

reported inflammatory markers (C-reactive

protein, IL-6 and TNF-) and angiogenesis

markers such as VEGF and soluble VEGF

receptor 1 to be significantly elevated in the

plasma of GBM patients. Jung et al. (2007)

identified GFAP as a discriminatory serum

biomarker for GBM. Similarly, serum

osteopontin (OPN), validated using IHC and

ELISA in GBM patients, was shown to

correlate with poor prognosis

(Sreekanthreddy et al., 2010). In an extended

effort, the TCGA group also generated protein

expression data from 214 GBM patient

samples using a high throughput antibody-

based reverse phase protein arrays (RPPAs)

(Brennan et al., 2013) revealing several

mutations, altered genes, proteins and their

pathways underlying GBM pathophysiology

(Dong et al., 2010).

Some of the challenges in using protein

profiling more commonly in characterizing

and quantifying accepted protein biomarkers

includes high costs, lengthy production times

and most importantly lack of high specificity

antibodies. Moreover, the proteomic approach

has the potential to identify novel diagnostic,

prognostic, and therapeutic biomarkers for

human gliomas. The application of proteomics

in neuro-oncology is still in its developing

stage. Please refer recent reviews by Whittle et

Biomed Res J 2015;2(1):6-20

Maachani et al. 11

al. (2007) and Niclou et al. (2010) for more on

the current status of glioma proteomics and its

clinical applications.

Metabolomics

Nearly a century ago, Otto Warburg made a

seminal observation that even in the presence

of adequate oxygen cancer cells metabolize

glucose by aerobic glycolysis, termed as

Warburg effect (Warburg et al., 1924; 1927).

Moreover, very recently disease-related

altered cellular metabolism has come into

forefront of cancer research. Now, there is

increasing evidence that the underlying

genetic alterations contributing to glioma

pathogenesis is also responsible for altered

cellular metabolism (Parsons et al., 2008).

Metabolomics refers to the global quantitative

assessment of endogenous enzyme kinetics,

cellular biochemical reactions, and synthesis

of cellular metabolites within a biologic

system, (Boros et al., 2005; Griffin and

Shockcor, 2004). Although considerable

progress has been made in understanding

GBM biology through genetic analysis, little is

known about the underlying metabolic

alterations in glioma. In recent years, several

biochemical and biophysical techniques such

as Mass Spectrometry (MS), liquid- and gel-

chromatography, Magnetic Resonance

Spectroscopy (MRS), Nuclear Magnetic

Resonance (NMR), have helped in profiling

global metabolomic signatures in cancers

including glioma (Dunn et al., 2005; Serkova

and Niemann, 2006). Several key differences

in metabolite profiles have been identified in

GBM cancer cells when compared to normal

controls, providing a novel insight into GBM

tumorigenesis (Spratlin et al., 2009). As

metabolomics reflect underlying altered

genotype-phenotype, it can be used as a

predictive biomarker for measure of efficacy

and as a pharmacodynamic marker, for both

traditional chemotherapy and hormonal

agents. Using the 1H-NMR spectra and neural

networks, human glioma cell cultures can be

separated into drug-resistant and drug-

sensitive groups before treatment with

nitrosourea treatment (El-Deredy et al., 1997).

Frequent genetic alterations in glioma such as

MYC amplification, PTEN deletion or protein

loss and EFGR amplification are associated

with multiple downstream metabolic targets

(Deberardinis et al., 2008). IDH1 and IDH2

metabolic genes are mutated in ~12% of

primary gliomas, 86% of grade II and III

gliomas and secondary glioblastoma through a

gain-of-function mutation that alters the

enzymatic activity of the protein product,

which results in the production of 2-

hydroxyglutarate (Dang et al., 2009). The

detection of 2-HG metabolic product has been

proposed to be a potential tool for in vivo

distinction of secondary from primary

glioblastomas (Esmaeili et al., 2013). More

recently Chen et al. (2014) showed that

IDH1-mutant glioma growth is facilitated by

overexpression of glutamate dehydrogenase 2

Biomed Res J 2015;2(1):6-20


gene (GLUD2) and it could be targeted for

growth inhibitory effects. Hence,

metabolomics applications in a clinical

perspective may have a favorable impact on

glioma grade, metabolic state and treatment

stratification of glioma patients.

Other Omics: microRNAs

In recent years, microRNAs (miRNAs) have

emerged in the forefront of cancer molecular

biology. MicroRNAs are key post-

transcriptional regulators that inhibit gene

expression by promoting mRNA decay or

suppressing translation (Iorio and Croce,

2012). Experimental and clinical evidence

supports that miRNAs play pivotal role in

cancer gene regulation, proliferation,

apoptosis and metastasis (Cho, 2011; Iorio and

Croce, 2012). The functional role of miRNAs

was first discovered in human gliomas (Li et

al., 2013). Several miRNA expressions are

found to be dysregulated in GBM. TCGA

group identified alterations in 149 miRNAs

(Dong et al., 2010) and an expression

signature comprising 10 miRNAs with

prognostic prediction (Srinivasan et al., 2011).

miR-128, miR-342 and miR-21 are known to

play both oncogenic and tumor suppressive

roles and are being explored as possible

markers for GBM (Dong et al., 2010;

Srinivasan et al., 2011). More recently several

lines of evidence have implicated over-

expression of miR21 with chemo and

radioresistance of GBM cells. Its expression

levels have been associated with glioma grade

and as a candidate independent marker for

overall survival (Chao et al., 2013; Wu et al.,

2013). Thus, integrative omics analysis has

revealed the importance and scope of

translational repression in microRNA-

mediated GBM pathogenesis. Please refer to

additional reviews (Karsy et al., 2012; Nikaki

et al., 2012; Sana et al., 2011) for more

detailed coverage on miRNA expression and

function in GBM.

Omics data integration methods

The post-human genome project era has

generated enormous heterogeneous and large

data sets. As vast gene profiling datasets and

technologies are being developed, they have

created an unprecedented need to develop

technologies to process the data in a

meaningful way. The efforts have yielded

meaningful results in cancer biomarker

discovery, protein interactions and genotype

to phenotype correlations (Park et al., 2005).

However, current omics technologies cannot

model interactions between multiple

molecules by analyzing individual genes,

proteins or metabolites. This is often not very

effective due to the complex and

heterogeneous nature of human cancers.

Cancer is a complex biological system and

requires a better understanding of the disease's

complexity at systems-level (Faratian et al.,

2009; Hu et al., 2013). Pathway and network

based methods have taken more important role

Biomed Res J 2015;2(1):6-20

Maachani et al. 13

in analysis of high-throughput data, that can

provide a global and systematical way to

explore the relationships between biomarkers

and their interacting partners (Wang et al.,

2015). Integration of data from multiple omic

studies can not only help unravel the

underlying molecular mechanism of

carcinogenesis but also identify the signature

of signaling pathway/networks characteristic

for specific cancer types that can be used for

diagnosis, prognosis and designing tumor

targeted therapy.

Most recently, attempts at integration of

multiple high-throughput omics data have

concentrated on comparing data acquired

using various experimental conditions/

platforms to explore functional and regulatory

associations between genes and proteins

(Faith et al., 2007; McDermott et al., 2009).

This has culminated into combining functional

characterization and quantitative interactions

extracted from various biomolecules such as

DNA, mRNA, proteins and metabolites (Chen

et al., 2011; Coban and Barton, 2012; Mitchell

et al., 2013) (Fig. 1). Some analysis utilizes

pathways in the form of connected routes

through a graph-based representation of the

metabolic network (Blum and Kohlbacher,

2008). Other approaches focus on the

functional module of protein interaction

network and analyze experimental data in the

context of pathways using multiple source

omics data (Wang et al., 2012; Blazier and

Papin, 2012; Federici et al., 2013). Although

currently there are tools available to process

large datasets generated by one platform, it is

expected that soon tools combining data

across multiple platforms will be available to

researchers. This will help in integrating

research results into a framework of whole

biological systems to support translation of

research into clinical applications.

Omics advantages in GBM therapy

So far clinical translation of an effective GBM

therapy has been hindered by multiple factors,

including diffuse infiltration at the time of

diagnosis, significant cellular heterogeneity

(both intratumoral and intertumoral),

difficulty in crossing the blood-brain barrier

by effective drugs, and the role of tumor

progenitor cells in reestablishment of resistant

disease following chemo and radiotherapy.

Current standard treatment of GBM consists

of attempted gross total surgical resection

followed by concurrent temozolomide and

radiation therapy (RT) (Clarke et al., 2010).

Although, RT provides good local control, it is

not very beneficial in controlling the disease

recurrence. In case of GBM, majority of

patients die from recurrent disease, as

currently there is no effective therapy for

recurrent GBM. Therefore, the addition of

systemic chemotherapy to RT can help in

controlling recurrence and offering an

additional radiosensitization benefits in GBM,

benefiting both definitive and palliative

strategies for disease management (Clarke et

Biomed Res J 2015;2(1):6-20


al., 2010; Stupp et al., 2006). So far non-omics

studies have identified few GBM targets at the

protein level, but fail to see an overall role of

molecules in signaling pathways, protein-

protein interactions, and role in metabolic

processes. Unfortunately so far only one drug

has been identified (Temozolomide) which

can radiosensitize GBM patients. Thus, non-

omics techniques will compliment whole

genomic/epigenomics/metabolomics approach

of omics technologies. Without publically

available databases, the surge of preclinical

and clinical information seen in the GBM field

over last few years, would have not been

possible. As omics studies expand our

understanding of the molecular pathways

driving GBM tumorigenesis, more druggable

targets will be identified to treat GBM patients.

Also, understanding of ionizing radiation at

the level of molecular biology will lead to

development and production of targeted

radiosensitizers. Temozolomide is currently

the only radiosensitizing agent used for GBM

with class I evidence of benefit (Mrugala and

Chamberlain, 2008). It is a novel oral

bioavailable second-generation alkylating

agent. At physiologic pH it undergoes

hydrolysis to its active form methyl traizeno-

imidazole carboxamid (MTIC). The

mechanism of action of MTIC, is to transfer a

methyl group to the middle guanine in a GGG

sequence to convert it to O6-methylguanine.

Temozolomide exerts its antineoplastic

activity by interfering with repair of damaged

DNA after radiation treatment (Mrugala and

Chamberlain, 2008). In a recent randomized

trial, concomitant and adjuvant

Temozolomide chemotherapy with radiation,

significantly improved progression free

survival from 12.1 months to 14.6 months, for

GBM patients (Clarke et al., 2010; Stupp et

al., 2005). The consequent analysis of these

patients by Hegi et al. (2005) reported that

patients with methylated MGMT gene

promoter were benefited from this treatment

compared to patients with unmethylated

MGMT promoter. The MGMT promoter

methylation silences the gene function

required to reverse the O6-guanine

methylation and therefore cannot counteract

the action of Temozolomide. Thus, omics has

been helpful in predicting tumor response to

Temozolomide and to guide clinical decision

making. The other most common types of

chemotherapies for GBM under investigation

include targeted molecular therapies,

antiangiogenic therapies, immunotherapies,

gene therapies, radiation-enhancement

therapies and drugs to overcome resistance

(Table 2).

Challenges and Prospective

Oncogenic transformation is a complex,

multistep process that differs widely between

and even within cancer types. Advances in the

large scale omics technologies have led to

identification of promising GBM disease

biomarkers. The major challenge is how to

Biomed Res J 2015;2(1):6-20

Maachani et al. 15

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CONFLICT OF INTEREST

The authors claim no conflict of interest.

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Biomed Res J 2015;2(1):6-20


INTRODUCTION

Dendrimers are hyperbranched globular

macromolecules with well-defined, mono-

disperse, three dimensional spatial

conformations, and a wide spectrum of

chemical and physical properties (Tomalia et

al., 1985). These characteristics indicate

significant differences from the classical

polymeric molecules. Structurally, these

macromolecules are divided into three

architectural regions: the central core,

repetitive and radial branching units and the

terminal functional groups. To achieve a high

degree of precision and structural order,

dendrimers are synthesized in a stepwise

fashion. The number of repeat branching

molecules used during the synthesis refers to

the generation of dendrimers, which also

governs the shape and size of the dendrimers.

Generally, two different methods namely,

divergent and convergent, are adopted for the

Key words: Dendrimers, Biosensors, Polyamidoamine, Polypropylene imine, Bioreceptors, DNA sensor. *Corresponding Author: Dhirendra Bahadur, Department of Metallurgical Engineering and Material Science, Indian Institute of Technology Bombay, Powai, Mumbai, India. Email: [email protected]

Dendrimers based Electrochemical Biosensors

Electrochemical biosensors are portable devices that permit rapid detection and monitoring of biological,

chemical and toxic substances. In the electrochemical biosensors, the bioreceptor is incorporated into the

transducer surface; and when in contact with the analyte, generates measurable signals proportional to the

analyte concentration. Materials with high surface area, high reactivity, and easy dispersability, are most

suited for use in biosensors. Dendrimers are nanomaterial gaining importance for fabrication of

electrochemical biosensors. These are synthetic macromolecules with regularly branched tree-like and

globular structure. The potential applications of dendrimers as biosensors are explored due to their

geometric symmetrical structure, chemical stability, controlled shape and size, and varied surface

functionalities, with adequate functional groups for chemical fixation. The current review provides multi-

faceted use of dendrimers for developing effective, rapid, and versatile electrochemical sensors for

biomolecules. The redox centers in the dendrimers play an important role in the electron transfer process

during immobilization of biomolecules on the electrodes. This has led to an intensive use of dendrimer

based materials for fabrication of electrochemical sensors with improved analytical parameters. The review

emphasizes development of new methods and applications of electrochemical biosensors based on novel

nanomaterials.

1 2 1Saumya Nigam , Sudeshna Chandra , Dhirendra Bahadur *

1Department of Metallurgical Engineering and Material Science, Indian Institute of Technology Bombay, Powai,

Mumbai, India2Department of Chemical Sciences, School of Science, NMIMS (Deemed-to-be) University, Vile Parle (W), Mumbai,

India

Biomed Res J 2015;2(1):21-36

Review

synthesis of dendrimers, and classified into

different “generations”. It is the hyper-

branching of the molecule from the centre of

the dendrimer towards the periphery that

results in homostructural layers between the

focal points (branching points). The number of

focal points from the core towards the outer

surface is the generation number. Thus,

generation refers to the number of repeated

branching cycles performed during the

synthesis. The core part of the dendrimer is

denoted generation “zero” (G0). For example

if a dendrimer is made by convergent

synthesis, and the branching reactions are

performed onto the core molecule three times,

the resulting dendrimer is considered a third

generation dendrimer. Each successive

generation results in a dendrimer roughly

twice the molecular weight of the previous

generation.

The two synthetic methods have inherent

advantages and disadvantages. Using the

divergent synthesis method, the dendritic

molecule is formed from a central core which

then extends radially outwards through

addition of branching molecules. The main

advantage of the divergent method is that high

molecular nanoscaffold architecture is

attained with desired repetitive branching

monomers. Thus, the dendrimer can be tailor

made to achieve maximum functionalities and

properties. However, two major challenges are

encountered in divergent synthesis. First, the

number of reaction points increase in

geometric progression with every generation

followed by increase in molecular weight.

This compromises the reaction kinetics,

making it slower and synthesis of high

generation dendrimers becomes difficult,

further lowering the yield of desired product.

Addition of each branching unit requires care

and precision to prevent structural defects and

asymmetry in the dendrimer structure.

Secondly, the separation of desired dendrimer

from the by-products is hindered due to

molecular similarity exhibited by the by-

product as well as the desired dendrimer. On

the other hand, convergent method employs

synthesis of small dendrites from the exterior

and the reaction proceeds inwards to the

central core. The convergent procedure results

in lesser structural defects and easy

purification of dendrimers resulting in high

degree of monodispersity. Despite the

possibility of purer and flawless dendrimers,

the convergent method falls short in synthesis

of higher generation dendrimers. This choice

is limited due to the steric forces crowding the

dendrites around the central core molecule.

Despite the difficulties, these macro-

molecules have gained interest over classical

polymers due to the varied options presented

by dendritic macromolecules. The freedom of

choice of central core, branching monomeric

units and surface functional groups from the

vast pool of molecules gives rise to a

multivalent system. Ethylenediamine, 1,4-

diaminobutane, 1,12-diaminododecane,

Biomed Res J 2015;2(1):21-36

22 Dendrimers based electrochemical biosensors

cystamine, 1,6-diaminohexane and ammonia

are the most common core molecules. The

varied core and branching monomers affect

the internal chemical environment, three

dimensional structures and size of internal

cavities in the dendrimer. Due to the different

structural and chemical properties, these user-

customized dendrimers find applications in

the fields of drug delivery, gene delivery,

antimicrobials, magnetic resonance imaging,

immunosensing and biosensing.

Methyl acrylate alternating with ethylene

diamine forms the most widely synthesized,

studied and used class of polyamidoamine

(PAMAM) dendrimers (Esfand et al., 2002),

with the internal amide groups providing an

abundance of lone pairs of electrons. Another

popular class of amine terminated dendrimers

is the poly (propylene imine) (PPI)

synthesized by Michael's addition of primary

amines to acrylonitrile followed by

subsequent hydrogenation by Raney cobalt or

Raney nickel catalyst (de Brabander-van den

Berg et al., 2003). The interiors of PPI

dendrimers are the tertiary nitrogen atoms with

lone pairs of electron contributing to their

reactive cavities. Both the classes of

dendrimers have primary amine groups on the

surfaces governing the surface properties,

reactivity and surface charge. Thus, any kind

of detection response observed in these

dendrimers is attributed to the amine groups.

The surface of dendrimers is further modified

to enhance the reactivity/interaction and

Biomed Res J 2015;2(1):21-36

sensor response to be used in biosensing

applications. Various molecules like

ferrocene, polystyrene, polyaniline,

carbohydrates, etc. have been explored for

surface modification (Ashton et al., 1997;

Chen et al., 2014; Hung et al., 2013; Yoon et

al., 2000). The conductivity of the moieties

plays an important role in enhancing response

of the dendritic scaffold in sensing various

biomolecules. The most common modifying

molecule is ferrocene which exhibits multi-

electron transfer in various redox interactions.

Ferrocene has been exploited as central core,

branching monomer as well as for surface

groups in various dendritic systems (Mehmet

et al., 2013; Villalonga-Barber et al., 2013).

They behave as non-interacting redox

moieties undergoing redox processes without

decomposition while maintaining the desired

electrochemical reversibility (Sun et al.,

2014).

The molecular recognition of

biomolecules by dendrimers is primarily

governed by the three dimensional

conformation in higher generations. The

branches of lower generation dendrimers tend

to radiate out towards the periphery and exist

in open conformation. On the other hand, as

the number of generation is increased, the

branches tend to retract and adopt globular

conformations in a three dimensional space

with intramolecular hydrogen bonding

governing the structures. The generation

dependent conformational changes confirmed

Nigam et al. 23

by X-ray analysis, demonstrated that the

higher generations are more spherical as

compared to lower linear generations (Percec

et al., 1998). The globular conformations

closely resemble morphology of globular

proteins and are useful in several biosensing

applications associated with the biomimetic

macromolecular architecture. A vast variety of

biomolecular species have been detected using

dendrimer scaffolds. In the following sections,

details of the various sensors using different

types of dendrimers are discussed.

Dendrimers in electrochemical biosensing

By definition, a biosensor is an analytical

device that makes use of bioreceptor molecule

immobilized onto a transducer (recognition)

surface and produces measurable signals in the

presence of an analyte, due to the bio-

recognition event proportional to the

concentration of the analyte. Biosensors are

classified based on either the bioreceptor or

transduction method or both. Common

bioreceptors include enzymes, antibodies and

DNA, while transducers include electro-

chemical, piezoelectrical, optical techniques.

The transducer techniques using electro-

chemical biosensors have an edge over other

methods due to excellent selectivity and

sensitivity, and precise detection of the desired

species. These are relatively cheaper, faster

and more user friendly as compared to other

techniques. The exceptional features render

the electrochemical biosensors increasingly

applicable in several biomedical and

environmental analyses.

a) Peroxide sensor: Copolymers of pyrrole-

PAMAM dendrimer are used for

electrochemical sensing of hydrogen

peroxide. Different generations of

pyrrole-PAMAM with branched amine

periphery and focal pyrrole functionality

are synthesized by divergent method. The

conjugate is covalently attached to the

electrode surface and horseradish

peroxidase (HRP) immobilized on it to

form conducting films for H O sensing. 2 2

The steady state amperometric response is

measured as a function of H O 2 2

concentration at +0.35V vs. Ag/AgCl, and

demonstrated that the dendritic wedge

played an important role for

immobilization of the HRP enzyme

(Mehmet et al., 2012). Yang et al. (2014)

described a magnetic electrochemical

sensor comprising Fe O nanoparticles 3 4

with graphene oxide (GO) and subsequent

modification by PAMAM dendrimers.

The platform was employed for

modification of the gold electrode acting

as the working electrode and used for the

detection of H O in phosphate buffer 2 2

solution by the method of amperometric

i-t curve. The cyclic voltammograms of

Fe O /GO and Fe O /GO–PAMAM 3 4 3 4

showed an increase in current while

displaying steady redox peaks which

confirmed occurrence of a catalytic

Biomed Res J 2015;2(1):21-36


reaction on the electrode interface. H O 2 2

was detected in a linear calibration range –5 –3of 2.0 × 10 –1.0 × 10 M with a

correlation coefficient of 0.9950 and -6detection limit of 2.0 × 10 M. The sensor

platform also displayed excellent recovery

ratios of 96.9–108.1% H O added to milk 2 2

and juice samples. Another amperometric

electro-chemical biosensor for H O was 2 2

developed by modifying gold bead

electrodes with starburst PAMAM

dendrimers of different generations of 2, 3

and 4, followed by absorption of Prussian

blue (PB). The covalently bonded

dendrimer/PB modified electrodes offered

enhanced sensitivity and lower detection

limits (Bustos et al., 2006). Metallic

(Rhodium) nanoparticles stabilized with

N, N-bis-succinamide-based dendrimer

were immobilized on glassy carbon

electrode (GCE) and electrocatalytic

activity towards hydrogen peroxide

reduction investigated using cyclic

voltammetry and chronoamperometry.

The dendrimer stabilized nanoparticles

showed excellent electrocatalytic activity

for H O reduction reactions and a steady-2 2

state cathodic current response was

observed at −0.3 V (vs. SCE) in phosphate

buffer (pH 7.0). The electrochemical

sensor displayed a linear response to H O 2 2

concentrations ranging from 8 to 30 μM

with a detection limit and sensitivity of 5 −6 −1

μM and 0.031 × 10 A μM , respectively

Biomed Res J 2015;2(1):21-36

(Chandra et al., 2009).

b) Glucose Sensor: A dendritic wedge based

on pyrrole-PAMAM dendrimer was used

to immobilize glucose oxidase (GOx) for

the construction of an amperometric

glucose sensor (Mehmet and Cevdet,

2012). Nanobiocomposite based glucose

biosensor was prepared by electro-

polymerization of pyrrole containing

PAMAM encapsulated Pt nanoparticles

(Pt-PAMAM), and GOx. The developed –1sensor had a sensitivity of 164 µA mM

–1cm and a detection limit of 10 nM within

a wide working range from 0.2−600 µM.

Pyrrole provided electrical conductivity,

stability and homogeneity to the thin film,

while PAMAM provided a favorable

microenvironment to maintain bioactivity

of GOx (Tang et al., 2007). Yoon and

colleagues used varying degrees of redox-

active ferrocenyl in combination with

PAMAM dendrimers (Fc-D) as

recognition unit for fabrication of a

glucose sensor (Yoon et al., 2000). GOx

was deposited layer-by-layer on Au-

surface to form an enzymatically active

GOx/Fc-D multilayered assembly. The

bio-electrocatalytic signals from the

multilayer were directly correlated to the

number of layers deposited, confirming

the tunable sensitivity of the electrode and

hence a potential microbiosensor. Cyclic

voltammetry and surface plasmon

resonance (SPR) was used to investigate

Nigam et al. 25

the redox-orientation changes of

ferrocene-tethered dendrimers and GOx.

SPR monitors change in the refractive

index of the medium next to the Au

sensing surface and are used to monitor

immobilization of GOx onto the Au

surface (Frasconi et al., 2009). Redox-

active dendrimer fabricated using

different generations of poly (propylene

imine) core with peripheral octamethyl

ferrocenyl units (Fig. 1) and deposited on

Pt electrodes for immobilizing GOx has

been used for detection of glucose

(Armada et al., 2006). The amperometric

response of all the dendritic mediators

towards glucose was determined at several

applied potentials. Glucose biosensor has

been developed based on bioactive

polyglycerol (PGLD) and chitosan

dendrimer (CHD). Both the dendrimers

were conjugated with GOx to form

PGLD-GOx and CHD-GOx and

entrapped in polyaniline nanotubes

(PANINT's) during template electro-

chemical polymerization of aniline. The

prepared PGLD-GOx/PANINT's and

CHD-GOx/PANINT's biosensors

exhibited strong amperometric response

to glucose concentrations in ranges

observed in human blood. PGLD-

GOx/PANINT's was more sensitive –1(10.41 nA.mM ) as compared to CHD-

–1GOx/PANINT's (7.04 nA.mM ), due to

specific organization of the GOx layer at

the surface of PGLD and distribution of

PANINT's (Santos et al., 2010).

Ferrocenyl dendrimer (PAMAM-Fc)

has also used for fabricating an

amperometric glucose biosensor. Series of

asymmetric PAMAM dendrimers

containing a single ferrocene unit located

in the focal point have been synthesized.

The transducer consisted of a gold

electrode covalently modified with 3-

mercaptopropionic acid, PAMAM-Fc

dendrimers and GOx enzyme. The

PAMAM-Fc/GOx biosensor showed

excellent performance for recognizing

glucose at +0.25 V with a high sensitivity

(6.54 μA/mM) and low response time

(~3s) in the concentration range of 1–22

mM (Mehmet et al., 2013).

Figure 1: Structures of varying generations of octamethyl

ferrocenyl dendrimers for use as electrode material for

determination of glucose (Armada et al., 2006).

Biomed Res J 2015;2(1):21-36


c) DNA Sensor: Dendrimers were also

exploited for their possible use in

fabricating DNA sensors. An electro-

chemical DNA nanobiosensor was

developed by immobilization of 20-mer

thiolated probe ssDNA on electro-

deposited poly (propyleneimine)

dendrimer (PPI) of generation 4 (G4),

doped with gold nanoparticles (AuNP)

(Arotiba et al., 2008). Cyclic voltammetry

showed that the designed platform

(GCE/PPI-AuNP) exhibited reversible

electrochemical behavior in pH 7.2

phosphate buffer saline (PBS) solution

due to PPI. The redox chemistry of PPI

involves a two electron and one proton

process and is pH-dependent. PPI-AuNP

was able to amperometrically detect target

DNA concentrations at 0.05 nM in PBS.

Using electrochemical impedance

spectroscopy (EIS), the biosensor –12 –9exhibited a dynamic linearity of 10 –10

M for target DNA. The probe immobiliza-

tion effectiveness is apparently attributed

to the AuNP's ability to connect to the

thiolatedssDNA on the GCE surface via

Au-S linkages. Further, the electrostatic

interaction between the cationic platform

and the anionic DNA probe improved the

immobilization process. Proposed charge

transfer scheme between the electrolyte,

DNA and PPI-AuNP is shown in Fig. 2.

A DNA biosensor with probe DNA

sequence, immobilized on a multinuclear

Biomed Res J 2015;2(1):21-36

nickel (II) salicylaldimine metallo-

dendrimer on GCE has been reported

(Arotiba et al., 2007). The authors studied

electrochemical characterization on

immobilization layer of the PPI derivative

by impedimetric and amperometric

methods. The metallo-dendrimer was

electroactive with two reversible redox

centers and was a strong DNA adsorbant.

The sensor responded to 10 µL of 5 nM

target DNA with detection limit as low as –12 3.4 × 10 M. Gold electrode has been

modified with 3-mercaptopropionic acid

and reacted with amino-terminated

PAMAM G-4 dendrimer to obtain a thin

film (Li et al., 2009). Recognition layer of

single-stranded 3´-biotin-avidin combina-

tion was immobilized onto the thin film to

detect the complimentary target. Cyclic

voltammetry (CV), differential pulse

voltammetry (DPV) and electrochemical

impedance spectroscopy (EIS) has been

used to study immobilization and

hybridization of DNA. The dynamic

detection range of the sequence-specific –11 –14DNA was 1.4 × 10 –2.7×10 M with a

–14detection limit of 1.4 × 10 M. Sahoo et

al. (2013), demonstrated a label free

Figure 2: Proposed charge transfer scheme between

PBS, DNA and PPI-AuNP (Arotiba et al., 2008).

Nigam et al. 27

impedimetric DNA biosensor based on

third generation G3 PAMAM dendrimer

functionalized GaN nanowires (NWs).

The developed nanosystem provided large

docking sites to immobilize probe (p-)

DNA covalently. The biosensor was

ultrasensitive and showed detection limit

as low as attomolar (aM) concentration of

complementary target (t-) DNA.

Impedance spectroscopy revealed an

increase in the resistance polarization (R ) p

indicating efficient charge transfer due to

strong covalent binding on NWs surface.

Zhu et al. (2006) modified gold electrodes

with sub-monolayers of mercaptoacetic

acid (RSH) and reacted with G-4 PAMAM

dendrimers to obtain thin films of

PAMAM/RSH. DNA probe was then

immobilized onto the thin films to afford

stable recognition layers. DPV was used to

monitor DNA hybridization with

daunomycin (DNR) as indicator. The

PAMAM-modified Au electrodes without

ssDNA showed good electrochemical

response in DNR solution, while on

attachment with ssDNA the modified

electrode showed a decrease in the DPV

response of DNR. This is attributed to less

accessibility of DNR molecules to ssDNA

probe on the electrode surface. Besides

high generation dendrimers, low

generation dendrimers are also used to

develop DNA biosensor. A second

generation PAMAM (G2-PAMAM)

dendrimer was covalently functionalized

onto multi-walled carbon nanotube

(MWNT) and used as electronic

transducer and tether for surface

confinement of probe DNA. Impedance

spectroscopy revealed occurrence of

hybridization between surface confined

ssDNA probe with target DNA in solution

to form double stranded DNA (dsDNA).

The interfacial charge-transfer resistance

of the electrode towards the redox

electrolyte changed due to occurrence of

hybridization. The large number of amino

groups of the dendrimer enhanced the

surface binding of the probe DNA which

in turn resulted in increase in the

sensitivity of the impedimetric biosensor

for the target DNA. The interfacial charge-

transfer resistance responded linearly to

the logarithmic concentration of the target

DNA within a concentration range of

0.5–500 pM with a detection limit of 0.1

pM (S/N = 3) (Zhu et al., 2010). Single-

use electrochemical DNA biosensor has

been fabricated based on pencil graphite

electrode modified with succinamic acid

and G2 PAMAM dendrimer (G2-

PS/GCE). Calf thymus double stranded

DNA (ctDNA) and DNA oligonucleotide

(DNA ODN) immobilized on surface of

G2-PS/GCE under optimum conditions,

showed a detection limit of 4.2 µg/mL

(Congur et al., 2014). Besides dendrimers,

dendritic nanostructures have been used

Biomed Res J 2015;2(1):21-36


dendrimer. Thrombin aptamer probe was

immobilized onto activated dendrimer

monolayer film and detection of thrombin

was investigated in the presence of the 3−/4−reversible [Fe(CN) ] redox couple 6

using impedance technique. The results

showed that the charge-transfer resistance

(R ) value had a linear relationship within ct

concentrations range of 1–50 nM

thrombin, and detection limit (S/N = 3) of

0.01 nM (Zhang et al., 2009).

Impedimetric aptasensor based on

succinamic acid-terminated PAMAM

dendrimer was developed for monitoring

interaction between DNA aptamer (DNA-

APT) and its cognate protein, human

as electrode material in biosensing

applications. Li et al. (2011) described

dendrimer-gold (Den-Au) nanostructure

modified electrode by directly placing the

electrode into 2.8 mM HAuCl and 0.1 M 4

H SO solution at –1.5 V. Scanning 2 4

electron microscopic images show growth

evolution of Den-Au at different time

period (Fig. 3). The Den-Au modified

electrode respond to 1 fM complimentary

target DNA within a wide detection range.

Aptamers, as single-stranded DNA or

RNA sequences that bind to specific target

molecules was determined by a label-free

highly sensitive impedimetric aptasensor

based on amino-terminated PAMAM

Figure 3: SEM images of Den-Au electrodes by electrodeposition in 2.8 mM HAuCl and 0.1 M H SO at different time 4 2 4

points (A) 20s, (B) 100s, (C) 300s and (D) 600s (Li et al. 2011).

Biomed Res J 2015;2(1):21-36

Nigam et al. 29

activated protein C (APC), a key enzyme

in the protein C pathway. The dendrimer

modified aptasensor showed detection

limits of 1.81 µg/mL in buffer solution and

0.02 µg/mL in diluted FBS (Erdem et al.,

2014).

d) Coenzyme Sensor: Hyperbranched

carbosilane polymers, polydiallyl methyl

silane (PDAMS) and polymethyl

diundecenyl silane (PMDUS) with

ferrocene moieties were used for

stabilization of Pt nanoparticles and as

electrode material for NADH oxidation.

The modified electrodes worked in wide

linear concentration ranges for NADH

with a detection limit of 4.78 µM for

PDAMS/PtNPs/Pt and 6.18 µM for

PMDUS/PtNPs/Pt. With regard to the

structure of the two carbosilane polymers

and their films, PDAMS with shorter

branches form rougher films and exhibit

higher rate constants (K ) and sensitivity obs

and smaller Michaelis constants (K' ), M

than PMDUS indicating better

electrocatalytic activity towards NADH

oxidation (Jiménez et al., 2014).

e) Other biomolecules: Tang et al. (2007)

reported enzyme based amperometric

biosensor for determination of glutamate.

A self-assembly of glutamate dehydro-

genase (GLDH) and PAMAM dendrimer

encapsulated Pt nanoparticles on carbon

nanotubes (GLDH/Pt-PAMAM) /CNT) n

were used as electroactive material (Fig.

4). The electrochemical activity was

reported to be attractive with large

determination range of glutamate (2–250

µM), short response time (< 3 s), high –1 2sensitivity (433 µA/mM cm ) and

stability.

Figure 4: Schematic showing the procedure of immobilizing Pt-PAMAM onto CNTs (a) layer-by-layer self-assembly of

GLDH and Pt-PAMAM onto CNTs (b) Pt-PAMAM/CNTs heterostructures were covalently attached via EDC (Tang et al.

2007).

Biomed Res J 2015;2(1):21-36


Pt-PAMAM and GLDH were

alternately deposited until suitable layers

were obtained. PAMAM G-4 dendrimers

crosslinked with reduced graphene oxide

were tested for performance as electro-

chemical biosensors by immobilizing

enzyme tyrosinase (Araque et al., 2013).

The bioelectrode showed excellent

electrocatalytic behavior towards

determination of catechol with a response

time of about 6s, linear range of 10 nM to –122 µM, sensitivity of 424 mAM and a

low detection limit of 6 nM (Fig. 5).

PAMAM dendrimer encapsulated

AuNPs were first immobilized to a

conducting polymer with two amine

groups (3',4'-diamine-2,2',5',2''-terthio-

phene (PDATT) through covalent bonding

between –COOH group of PAMAM and

–NH group of PDATT. Laccase was 2

subsequently covalently bonded to the

–COOH of PAMAM dendrimers to form

PDATT/Den (AuNPs)/laccase probe

(Rahman et al., 2008). The modified

electrode displayed direct electron-

transfer (DET) process of laccase and a

catechin biosensor was fabricated based

on the electrocatalytic process of laccase.

The linear range and detection limit for

catechin sensing was 0.1–10 and 0.05 µM,

respectively. An electrochemical

biosensor based on PAMAM dendrimers

was developed for the detection of

fructose in food samples by immobilizing

fructose dehydrogenase (FDH) on

cysteamine and PAMAM dendrimers. The

concentration range of the enzymatic

biosensor was 0.25–5.0 mM fructose

(Damar et al., 2011). PAMAM dendrimers

were also used to enhance signal response

of a nanobiocomposite fabricated to

obtain an immunosensor for alpha-feto

protein (AFP) in human serum (Giannetto

et al., 2011). The binding of the dendrimer

with biologically active molecules like

antibodies can improve the activity and

Figure 5: (A) Amperometric response obtained with Tyr/PAMAM-Sil-rGO/GCE for different catechol concentrations at

E = –150 mV (B) FE-SEM image of Tyr/PAMAM-Sil-rGO (Araqueet al., 2013).app

Biomed Res J 2015;2(1):21-36

Nigam et al. 31

Biomed Res J 2015;2(1):21-36

sensitivity of the system. Response range

and precision were evaluated using cyclic

voltammetry (CV) and double step

chronoamperometry (DSCA) with limit of

detection of 3 ng/mL and limit of

quantification of 15 ng/mL. The enhanced

immunosensor could be useful for

monitoring prognosis of pregnancy and

occurrence of neoplastic diseases.

Recently, a redox-active silver-PAMAM

dendrimer nanostructure was synthesized

in situ by using wet chemistry (Xiaomei et

al., 2013), and functionalized with mono-

clonal mouse anti-human antibody for free

prostate specific antigen (fPSA). Using,

graphite as the working electrode, a layer

of gold nanoparticles modified with

prostate-specific antibody (mAb2). In

presence of the fPSA, specific immuno-

complex was formed on the functionalized

antibody modified electrode. The Ag-

mediated PAMAM dendrimer directly

catalyzed reduction of H O in the 2 2

detection solution. Thus, PSA was

detected primarily due to the antigen-

antibody immunocoupling. Under optimal

conditions, the developed immunoassay

could determine target fPSA in the

dynamic range of 0.005–5.0 ng/mL with a

detection limit (LOD) of 1.0 pg/mL (S/N =

3). In addition, the accuracy of the

electrochemical immunoassay evaluated

for detection of clinical serum specimens,

was in accordance with referenced

enzyme-linked immunosorbent assay

(ELISA) method.

A multi-analyte sensing device based

on PAMAM dendrimer for simultaneous

at-line monitoring of glucose, ethanol,

pO - and cell density was fabricated (Akin 2

et al., 2011). The device consisted of a

dual biosensor, a modified microscope

and a fiber optical pO -sensor integrated 2

into a flow analysis (FA) system. The

electrochemical transducer consisted of

self-assembly of cysteamine on gold

surface. Alcohol oxidase and pyranose

oxidase were immobilized onto the gold

surface by means of PAMAM (poly-

amidoamine) dendrimer via glutar-

aldehyde cross-linking. The responses for

glucose and ethanol were linear up to 0.5

mM. The biosensor was used for

simultaneous determination of ethanol

and glucose in yeast fermentation process.

A highly stable and sensitive ampero-

metric biosensor was developed by

immobilizing alcohol oxidase (AOX)

through PAMAM dendrimers on a

cysteamine-modified gold electrode

surface for determination of ethanol (Akin

et al., 2009). The optimized ethanol

biosensor showed a linearity from

0.025–1.0 mM with 100 s response time

and detection limit (LOD) of 0.016 mM.

The analytical characteristics of the

system were also evaluated for alcohol

determination in flow injection analysis


(FIA) mode for analysis of ethanol in

various alcoholic beverage as well as

offline monitoring of alcohol production

through yeast cultivation (Yuksel et al.,

2012).

PAMAM dendrimer (generation G4)

stabilized with 1-hexadecanethiol was

used for immobilization of acetylcholin

esterase from electric eel, and choline

oxidase from Alcaligenes sp. was used as

electrode material for fabrication of an

amperometric sensor for pesticides

(Snejdarkova et al., 2004). On similar

lines, urea electrochemical biosensor was

developed based on an electro-co-

deposited zirconia-PPI dendrimer

modified screen printed carbon electrode.

Urease enzyme was immobilized onto

electrodes and an amperometric response

in urea concentration from 0.01 mM to

2.99 mM was obtained with sensitivity of –1 –23.89 µA mM cm (Shukla et al., 2014).

PPI dendrimers have also been used to

reduce HAuCl to form core-shell PPI-Au 4

nanoclusters with several PPI molecules

attached on the surface of one gold

nanoparticles (Zhang et al., 2007). PPI-Au

nanoclusters and myoglobin (Mb) were

alternately adsorbed on the surface of

pyrolytic graphite (PG) electrodes

forming {PPI-Au/Mb} layer-by-layer n

films. The multilayer film assembled with

the dendrimer stabilized Au nanoparticles,

provided a new approach to fabricate

biosensors and bioreactors based on direct

electrochemistry of proteins and enzymes.

CONCLUSIONS

Contemporary studies indicate that the most

elementary chemical reaction of electron

transfer is widely prevalent in several

biological systems and more importantly in

nanosystems with redox dendrimers. This is

possible by tailoring the nature and topology

of the dendrimers to precisely control location

of the redox sites within the macromolecule

and study its electron-transfer processes. The

increase in efforts to combine dendrimers with

other molecules like pyrrole, ferrocene,

enzymes, etc. is promising in biosensing

applications.

ACKNOWLEDGEMENTS

The authors acknowledge Department of

Science and Technology, Government of

India, New Delhi, for providing financial

support. The authors also acknowledge the

publishers for providing copyright

permissions for the figures.



Biomed Res J 2015;2(1):21-36

Nigam et al. 33

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Biomed Res J 2015;2(1):21-36


INTRODUCTION

Tumour is composed of a heterogeneous group

of cells with different morphologies and

behaviour. Research in cancer biology

indicates that several cancers are supported by

a small subset of cells with stem cell like

properties and are termed as cancer stem cells

(CSCs) or tumour initiating cells (TIC).

Evidences of CSCs involved in resistance to

conventional therapies, leading to metastasis

and tumour recurrence is abundant (Beck and

Blanpain, 2013; Chandler and Lagasse, 2010;

Prince and Ailles, 2008).

As early as 1937, Furth and colleagues

demonstrated that a single cell was able to

produce a haematopoietic malignancy on

implantation in mice (Furth et al., 1937). This

suggested that certain cells within a tumour

may have the ability to give rise to tumour

growth (Furth et al., 1937). Later, in 1994,

John Dick's group identified human acute

myeloid leukaemia-initiating cells using + -CD34 CD38 markers and showed that these

cells initiated tumour (Lapidot et al., 1994). In

1997, Bonnet and Dick showed for the first + -time that the CD34 CD38 population of cells

had the self-renewal property. The authors

performed limiting dilution assay to show that + -low numbers of CD34 CD38 cells were able

to form tumours in NOD/SCID mice, identical

to donors; whereas considerably higher

Key words: Cancer stem cells (CSCs); EMT, Epithelial to mesenchymal transition; Lineage tracing; β-catenin; NICD, Notch intracellular domain.*Corresponding Author: Sanjeev K. Waghmare, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India.Email: [email protected]

Developmental Signalling in Maintenance and Regulation

of Cancer Stem Cells

Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar,

Navi Mumbai, India

Sweta Dash, Raghava Reddy Sunkara and Sanjeev K. Waghmare*

Tissue stem cells self-renew throughout the life of an organism thereby maintaining tissue homeostasis and

prevent cancer. The major signalling pathways such as Wnt, Notch and Sonic hedgehog control the stem

cell regulation and their deregulation leads to cancer. Recent evidences showed that there exists a subset of

cells within tumour termed as cancer stem cells (CSCs). These CSCs escape the conventional chemo-

radiotherapy and further lead to tumour relapse followed by metastasis. This review focuses on the

developmental signalling pathways that are involved in the regulation and maintenance of normal stem cells

and CSCs. Understanding the molecular mechanism may be useful to specifically target the CSCs while

sparing the normal stem cells to reduce tumorigenecity.

Biomed Res J 2015;2(1):37-56

Review

+ +numbers of non-CSCs (CD34 CD38 ) were

unable to form tumours (Bonnet and Dick,

1997). These cells were coined as cancer stem

cells. In 2003, Michael Clarke's group

reported the first isolation of CSCs from breast

tumour (Al Hajj et al., 2003). Subsequently,

the presence of CSCs in other solid tumours

like melanomas, hepatocellular carcinoma,

glioblastoma, pancreatic cancer, colorectal

cancer and head and neck cancer have been

identified (Keshet et al., 2008; Li et al., 2007;

Ma et al., 2007; Prince et al., 2007; Ricci-

Vitiani et al., 2007; Singh et al., 2004). The

CSC markers from various cancers are listed in

the Table 1. The characterisation of CSCs uses

various assays that include: sphere-forming

assay, serial transplantation assay in NOD/

SCID mice and in vivo lineage tracing. Serial

transplantation assay, is considered as 'gold

standard' assay, and measures self-renewal as

well as the tumorigenic property of CSCs in

vivo (Al Hajj et al., 2003; Beck and Blanpain,

2013; Bonnet, 1997; Prince et al., 2007).

Recently the strongest evidence for existence

of CSCs has come from the lineage tracing

experiments in mice model for various cancers

such as glioblastoma, skin and colon cancers.

The assay showed that the individual

fluorescent tagged cells have the capability to

give rise to a tumour (Chen et al., 2012;

Driessens et al., 2012; Schepers et al., 2012).

Although many different markers for

CSCs have been identified in tumours of

different tissues, cells isolated by using these

markers are not a pure CSC population. Hence,

one of the major challenges is the isolation of a

pure population of CSCs. Recent study on

quantitative proliferation dynamics of hair

follicle stem cells showed the isolation of stem

cells based on their cell division. This suggests

that it may be possible to isolate pure stem cell

population (Waghmare and Tumbar, 2013;

Waghmare et al., 2008). Another challenge is

to understand how these CSC populations are

regulated and maintained. Therefore, it is

important to study the various signalling

pathways that are crucial for survival of CSC

population.

Biomed Res J 2015;2(1):37-56

38 Developmental signalling in cancer cells

Table 1: Cancer stem cell markers in various cancers

Cancer Cancer stem cell markers

Leukaemia CD34+CD38

- (Bonnet, 1997)

Breast Cancer CD44+CD24- (Hajj et al., 2003); ALDH1+ (Ginesteir et al., 2007); CD133+ (Wright et al., 2008)

Head and Neck

Cancer

CD44+ Lin

- (Prince et al., 2007); A1DH1+ (Clay et al., 2010; Krishnamurthy et al., 2010); CD133

+

(Zhang et al., 2010); CD10+ (Fukusumi et al., 2014); CD98+ (Matens de Kemp et al., 2013)

Pancreatic Cancer CD44+CD24-ESA+ (Li et al., 2007); c-Met (Li et al., 2011)

Liver Cancer CD133+ (Ma et al., 2007); CD90+ (Yang et al., 2008); CD13+ (Haraguchi et al., 2010); OV6+ (Yang et al.,

2008)

Glioblastoma CD133+ (Singh et al., 2004); SSEA1+ (Son et al., 2009), MET (De Bacco et al., 2009)

Melanoma ABCB5+ (Keshet et al., 2008)

Colorectal Cancer CD133+ (Ricci-Vitiani et al., 2007); CD166+ (Dalerba et al., 2007; Vermeulen et al., 2008); Lgr5+ (Barker

et al., 2007; Vermeulen et al., 2008), CD44+ (Haraguchi et al., 2008), CD44v6+ (Todaro et al., 2014)

Embryonic developmental process and

cancer stem cells

Development of an organism is regulated at

the molecular level by various signalling

pathways, and deregulation in these molecular

mechanisms leads to cancer formation. Recent

studies have shown various similarities

between cancer and development. During the

normal developmental process, undifferentia-

ted embryonic stem cells further differentiate

and give rise to the differentiated tissues of an

organism. Similarly in cancer, undifferentia-

ted CSCs are involved in tumour progression

that leads to metastasis (Bellacosa, 2013).

The embryonic stem cells have a core

transcriptional network comprising of

transcription factors like Oct4, Sox2 and

Nanog that contribute to self-renewal and

pluripotency (Boyer et al., 2005). Similarly,

lung CSCs showed elevated levels of Oct4 and

Nanog transcription factors (Chiou et al.,

2010). In head and neck cancer, CD44 variant

CD44v3 was shown to interact with Oct4-

Sox2-Nanog leading to CSC like properties

such as self-renewal and cisplatin resistance

(Bourguignon et al., 2012). Recently, it was

shown that the lineage ablation of Sox2-

expressing cells in both benign and malignant

skin squamous cell carcinomas resulted in

tumour regression indicating an important role

of Sox2 in tumour initiation and CSC

functions. Moreover, chromatin immuno-

precipitation analysis identified Sox2 target

genes involved in controlling tumour stemness

(Boumahdi et al., 2014).

Another important phenomena common

to both the CSCs as wells as the embryonic

stem cells is the occurrence of epithelial to

mesenchymal transition (EMT). During EMT,

the cells lose their polarity and acquire

migration capabilities that results in loss of

epithelial marker E-cadherin and simulta-

neous increase in mesenchymal marker N-

cadherin. During embryogenesis, EMT is

associated with gastrulation required for the

formation of the three germ layers. In cancer,

EMT leads to invasion, metastasis and cancer

stem cell-like phenotype (Kalluri, 2009;

Singh, 2010). A recent study showed that

Twist1, an EMT promoter protein, is expressed

during early stages of tumorigenesis and is

required for the initiation of skin tumours

(Beck et al., 2015).

All these indicate that regulation of

embryonic stem cells and CSCs share similar

mechanisms. Therefore, it suggests that

deregulation of various developmental

pathways are involved in cancer formation and

CSC regulation and maintenance. Hence,

studying the developmental signalling

pathways will shed light on the regulation of

CSCs.

Developmental signalling pathways and

CSCs

The various pathways which are deregulated

in cancer include Wnt, Notch, Hedgehog,

EGFR, PI3K, NFκB, etc. Among these, three

Dash et al. 39

Biomed Res J 2015;2(1):37-56

well-known pathways such as Wnt, Notch and

Hedgehog play an important role in the

development and normal homeostasis.

Conversely, deregulation of these pathways is

shown in CSC regulation and maintenance

(Ailles, 2012; Purow, 2012).

Wnt Pathway

Wnt pathway is evolutionarily conserved and

is involved in various organisms. It was first

discovered in Drosophila, when a mutation in

wingless (wg) gene led to a distinct phenotype

including absence of wings and halters. Later,

Nusse's group showed that the insertion of

Mouse Mammary Tumour Virus (MMTV) in

mice led to mammary tumour by proviral

activation of the int oncogene. The int

oncogene was later demonstrated as the mouse

homologue of the Drosophila wg gene. From

these two studies, a new nomenclature Wnt

(combination of wg and int) was obtained

(Nusse et al., 1984; Rijswijk et al., 1987;

Sharma, 2013).

There are 19 highly conserved Wnt

ligands discovered till date. These ligands are

secreted hydrophobic glycoproteins found to

be associated with cell membranes and extra-

cellular matrix. In Wnt producing cells, the

endoplasmic reticulum produces Wnt ligands,

lipid modified by porcupine (Mikels, 2006;

Willert et al., 2003). Wnt ligands can act

through two general categories of pathways:

canonical and non-canonical. The canonical

pathway is β-catenin dependent, while the 2+non-canonical pathways include Wnt/Ca and

Wnt/JNK pathways. In the canonical pathway

shown in Fig. 1, Wnt ligands bind to the

conserved cysteine rich domain (CRD) of the

frizzled receptors (Fz) which in turn forms co-

receptors complexes with low-density

lipoprotein like receptors (Lrp5/6). Further,

this interaction recruits the Dishevelled (Dsh)

Figure 1: Wnt Pathway. A) In the absence of the Wnt ligand, β-catenin is phosphorylated by destruction complex (APC,

CK1α, GSK3 and Axin) and is subjected to proteasomal degradation resulting in no transcription of the Wnt target

genes.B) In the presence of the Wnt ligand, the destruction complex is disrupted and thereby β-catenin enters the

nucleus and brings about the transcription of Wnt target genes. APC: Adenomatous Polyposis Coli; CK1α: Casein kinase

1α; GSK3: Glycogen synthase kinase 3; TCF: T cell factor; LEF: Lymphoid enhancing factor; Dsh: Dishevelled; LRP:

Low-density lipoprotein like receptors.


Biomed Res J 2015;2(1):37-56

protein to the cytoplasmic tail of Fz receptor

and brings about inhibition of destruction

complex surrounding β-catenin. The

components of the destruction complex

comprise of scaffold protein Axin, Glycogen

synthase kinase 3β (GSK3β), Casein kinase 1α

(CK1α) and adenomatous polyposis coli

(APC). In the absence of the Wnt ligands, the

destruction complex hyper-phosphorylates β-

catenin and targets it for proteasomal

degradation by ubiquitination. The binding of

Wnt ligand to Lrp5/6 causes phosphorylation

of the cytoplasmic tail of Lrp6, which in turn

recruits Axin to the receptor complex that

disrupts the destruction complex and stabilises

β-catenin. The stable β-catenin translocates to

the nucleus and binds to the lymphoid

enhancing factor/T-cell factor (LEF/TCF)

thereby transcriptionally activating the

different target genes involved in cell fate

determination during embryonic development

and tissue homeostasis (Mikels, 2006; Willert

et al., 2003).

Wnt signalling in normal development and

cancer

Wnt pathway is involved in different biological

processes such as embryonic development,

self-renewal, proliferation, morphogenesis,

etc. Wnt3a and Wnt1 knock out in mice led to

deficiencies in neural crest derivatives and

neural tube formation during the development

(McMahon et al., 1990; Yoshikawa et al.,

1997). Wnt3 knock out in mice led to early

gastrulation defect and perturbations in the

establishment of apical ectodermal ridge

during development (Liu et al., 1999). Further,

absence of Wnt4 ligand led to defects in female

development, while Wnt7a deletion led to

female infertility in mice (Jeays-Ward et al.,

2004; Parr et al., 1998). Axin1 knockout in

mice led to neuro-ectodermal and cardiac

abnormalities (Zeng et al., 1997). Wnt

signalling was shown to be crucial in hair

follicle development as targeted deletion of β-

catenin in the epidermis led to failure in

placode morphogenesis (Huelsken et al.,

2000). Absence of Lef1 led to defects in the

pro-B-cell proliferation and abnormalities in

several organs like teeth, mammary glands,

whiskers and hair (Reya et al., 2000;

VanGenderen et al., 1994); while the knockout

of Tcf1 led to thymocyte proliferation and

differentiation defects (Schilham et al., 1998).

Using the Wnt reporter, Axin2-LacZ, Wnt

responsive cells were localised to the sub

ventricular zone (SVZ) of the developing brain

and basal layer of the mammary ducts, which

are the stem cells niches. Furthermore, these

Wnt responsive cells showed high sphere

forming ability and were able to differentiate.

Hence, the Wnt pathway plays an important

role in normal development and tissue

homeostasis (Logan, 2004; VanAmerongen et

al., 2009).

There are strong evidences showing

involvement of Wnt pathway in regulation of

various cancers. Frequent somatic mutations

Biomed Res J 2015;2(1):37-56

Dash et al. 41

in β-catenin were observed in both mice and

human hepatocarcinomas (Coste et al., 1998),

prostate cancers and colon cancers (Voeller et

al., 1998). During intestinal adenoma

initiation, the first step was APC inactivation

followed by β-catenin stabilization, while

progression from adenoma to carcinoma

required the synergistic action of k-ras

activation and β-catenin nuclear localization

(Phelps et al., 2009). β-catenin was shown to

be essential for retaining tumorigenecity of

MDA-MB-231 breast cancer cell lines both in

vivo and in vitro. Further, β-catenin

knockdown cells implanted into mice showed

decrease in the tumour size. In addition, an in

vitro study in breast cancer cell lines showed

reduction in aldehyde dehydrogenase 1

(ALDH1) positive cells (Xu et al., 2015).

Wnt3a expression was associated with EMT

and promoted colon cancer progression (Qi et

al., 2014). Moreover, deletion of Axin1 was

reported in sporadic medulloblastomas and

hepatocellular carcinomas (Dahmen et al.,

2001). Increased expression of Dsh protein in

non-small cell lung carcinoma and meso-

thelioma have been reported (Uematsu et al.,

2003).

Wnt signalling in normal and cancer stem

cell regulation and maintenance

Wnt signalling is important in adult stem

regulation and has been shown to be involved

in stem cell proliferation, self-renewal and

maintenance. In hemato-poietic stem cells

(HSC), overexpression of β-catenin increases

the stem cell pool size suggesting that Wnt

pathway is critical to maintain the

hematopoietic stem cell homeostasis (Reya et

al., 2003). In mice hair follicle stem cells, live

cell imaging showed that β-catenin activation

in hair follicle stem cells was involved in hair

follicle tissue growth (Deschene et al., 2014).

Further, Wnt target gene Lgr5, a G-protein

coupled receptor was identified as an intestinal

stem cell marker indicating an important role

of Wnt pathway in the regulation of intestinal

stem cells (Ailles, 2012, Valkenburg, 2011).

The deletion of Tcf4, a Wnt downstream gene

showed loss of stem cell activity and reduced

proliferation of the intestinal epithelium

(Korinek et al., 1998). In addition, Lgr5 was

identified as a marker of hair follicle stem cells

(Jaks et al., 2008) with multipotent properties.

Moreover, Wnt inhibitor SFRP1 was shown to

play an important role in hematopoietic stem

cell maintenance through extrinsic regulation

(Renstrom et al., 2009). Over-expression of

Sfrp1 led to enhanced mesenchymal stem cell

function in angiogenesis (Dufourcq et al.,

2008). Besides, Sfrp1 was over-expressed in

hair follicle stem cells as compared to the non-

stem cells (Tumbar et al., 2004; Zhang et al.,

2009). Recently, it was shown that Sfrp1 gene

is critical for maintaining proper mammary

gland development wherein loss of Sfrp1

promotes mammosphere formation; however

the role in mammary stem cells needs further

investigation (Gauger et al., 2012).


Biomed Res J 2015;2(1):37-56

In cancer, various reports have shown that

deregulation of Wnt pathway is crucial for the

CSC regulation. Human head and neck CSCs

treated with Wnt antagonist, secreted frizzled-

related protein 4 (sFRP4), the CSCs showed

reduction in the sphere-forming capacity and

decrease in the stemness markers like CD44

and ALDH1 (Warrier et al., 2014). In another

report, β-catenin was shown to be required for

maintenance of cutaneous CSCs since deletion

of β-catenin led to reduction in the CSCs and

tumour regression (Malanchi et al., 2008).

CSCs isolated from mammary tumours of

radiation treated p53-null mice showed altered

DNA repair in response to radiation as well as

β-catenin activation (Zhang et al., 2010). In

prostate cancer, Wnt signalling induced

tumour initiation, EMT and metastasis.

Additionally, in prostate cancer cell lines and

primary cultures, Wnt3a treatment increased

the self-renewal capacity of putative prostate

CSCs, emphasizing that Wnt signalling plays

an important role in prostate cancer (Barker,

2006; Valkenburg, 2011; Verras et al., 2004).

Moreover, the inactivation of APC in Lgr5-

positive stem cells at the intestinal crypts led to

transformation within days; while inactivation

of APC in progenitors or differentiated cells

did not lead to tumour formation even after 30

weeks (Barker et al., 2009). In addition, the

deletion of CD44, a CSC marker and a Wnt

target gene in mice having heterogeneous APC Min/+

mutation (APC ), attenuates intestinal

tumorigenesis (Zeilstra et al., 2008).

Notch Pathway

Notch gene was first discovered in Drosophila

by Morgan and Bridges where they showed

that a mutation led to wings notching and

hence the name “Notch” was coined (Morgan

and Bridges, 1916; Mohr, 1919; Poulson,

1940). There are four Notch genes, three

Delta-like and two Jagged genes in mammals,

that are translated into different Notch ligands,

Delta and Jagged. Recently, it was shown that

for cell fate determination during

development, complex of Notch receptor-

Delta-Jagged acts in concert (Fiuza, 2004;

Boaretoa et al., 2015).

Since the Notch ligands such as Delta and

Jagged proteins, as well as Notch receptors are

transmembrane proteins, cell-cell contact is

important for the signalling cascade. The

Notch receptors contain an extracellular

subunit, having multiple EGF-like repeats,

and a transmembrane subunit (Wharton et al.,

1985). When the Notch ligand binds to its

receptor, the extracellular domain of the Notch

receptor is dissociated from the trans-

membrane domain and the S2 cleavage site is

exposed (Fig. 2). This site is cleaved by

ADAM (a disintegrin and metalloprotease)

generating an intermediate that is further

cleaved by γ-secretase to generate Notch

Intracellular Domain (NICD). NICD then

translocates to the nucleus where it binds to

ubiquitous transcription factor CSL (CBF-1,

Suppressor of Hairless, Lag-1). This complex

displaces a co-repressor complex containing

Biomed Res J 2015;2(1):37-56

Dash et al. 43

SKIP, SHARP and histone deacetylases.

Further, it recruits a co-activator complex

containing a MAML (Mastermind-like

protein), p300 and other chromatin modifying

enzymes, thereby bringing about transcription

of different Notch target genes (Ailles, 2012;

Andersson, 2011; Fiuza, 2004).

Notch signalling in normal development

and cancer

Notch pathway is also evolutionarily

conserved and is important in cell to cell

communication that regulates cell fate

determination during development, cell

proliferation, differentiation and apoptosis.

The loss of Notch function in vertebrates is

associated with disruption of neurogenesis,

somite formation, angiogenesis, and lymphoid

development. In Drosophila, Notch is shown

to control the fate of various cell types in the

eye. In vertebrates, Notch is involved in the

establishment of the central and peripheral

nervous systems, spermatogenesis, oogenesis,

myogenesis and imaginal disc development

(Artavanis-Tsakonas et al., 1999). In normal

mammary development, Notch pathway

activation is required for regulation of cell

fate, proliferation and stem cell self-renewal.

The Notch pathway is also shown to be

important for tip-cell formation during

mammalian astrocyte differentiation and

angiogenesis. In vertebrates, the Notch

pathway leads to patterning during inner ear

hair cell formation and insulin-secreting

pancreatic β cell production (Ailles, 2012;

Fortini, 2009).

Notch signalling has been shown to be

involved in various cancers. For instance,

Notch1 regulates breast cancer cells by

inducing Slug expression (Shao et al., 2015).

Notch4 promotes growth of gastric cancer

cells through activation of Wnt1, β-catenin

Figure 2: Notch Pathway. A) In the absence of the Notch ligands (Delta and Jagged), the S2 cleavage site remains

hidden and inaccessible to ADAM. Hence, the NICD is not formed, with consequent no transcription of the Notch target

genes. B) In the presence of the Notch ligands, the conformational change in the intracellular subunit of the Notch

receptor takes place exposing the S2 cleavage site, thus leading to the formation of NICD. Further, NICD translocates the

nucleus and brings about transcription of Notch target genes. NICD: Notch intracellular domain; ADAM: A disintegrin and

metalloprotease; MAML: Mastermind-like protein; SKIP: Ski-interacting protein; SHARP: SMRT associated protein.


Biomed Res J 2015;2(1):37-56

(Quian et al., 2015) and the downstream target

such as c-myc and cyclin-D1. In mice, Notch1

activation combined with p53 loss showed

synergistic effect in the formation of

Osteogenic sarcoma (Tao et al., 2014).

Notch signalling in normal and cancer stem

cell regulation and maintenance

Notch has been shown to be involved in self-

renewal, proliferation and differentiation of

adult stem cells in various tissues. In mice

mammary stem cells, the knockdown of Cbf-1,

a canonical Notch effector, showed increased

stem cell activity in vivo suggesting a role in

controlling mammary stem cells. (Bouras et

al., 2008). Notch directly targets the crypt base

columnar cells that maintain stem cell activity

in mice (VanDussen et al., 2012). The Notch

activation maintains the slow cycling property

of neural stem cells; however, blocking Notch

resulted in increased number of stem cell

divisions followed by depletion of the stem

cell pool (Chapouton et al., 2010).

Furthermore, constitutive activation of Notch

signalling promotes self-renewal in muscle

stem cells through upregulation of Pax7 (Wen

et al., 2012). Mice with satellite cell specific

deletion of RBP-Jj (recombining binding

protein-Jj), a nuclear factor required for Notch

signalling, showed depletion of the stem cell

pool and their muscles lacked ability to

regenerate in response to injury (Bjornson et

al., 2012). Notch 3 has been shown to be

expressed in stem cells and disruption led to

defective stem cells proliferation (Kitamoto

and Hanaoka, 2010).

Notch signalling plays an important role in

a number of hematopoietic and solid tumours,

but the strongest evidences for its role in CSC

regulation has been shown in breast cancer,

embryonal brain tumours and gliomas (Fan et

al., 2006; Pannuti et al., 2010). In various

human breast cancer cell lines and primary

patient tissues, a significant decrease in

mammosphere formation after Notch

inhibition has been demonstrated (Abel et al.,

2014). Further, studies on the human

mammary mammospheres have shown a

feedback loop between Her2/Neu and Notch,

as well as promotion of a hypoxia resistant

phenotype (Pannuti et al., 2010). In brain

tumours, blockade of Notch led to a 5-fold +reduction in the CD133 cell fraction and total

depletion of the side population cells.

Additionally, differentiated cell growth was

observed after Notch inhibition, but lacked

formation of tumour xenografts efficiently,

indicating that the CSCs required for tumour

propagation were absent (Fan et al., 2006). A

recent study on primary human pancreatic

xenografts showed upregulation of the notch

pathway components in pancreatic CSCs.

Additionally, inhibition of notch pathway

reduced CSC percentage and tumour-sphere

formation significantly (Abel et al., 2014).

Biomed Res J 2015;2(1):37-56

Dash et al. 45

Sonic-hedgehog Pathway

Hedgehog pathway is delineated in

Drosophila and determines the anterior-

posterior orientation of developing structures

(Nusslein-Volhard and Wieschaus, 1980).

Similar to Wnt and Notch, the key components

of the Hedgehog pathway are evolutionarily

conserved, although differences are observed

in the mammalian and Drosophila Hedgehog

signalling. While Drosophila has only one

hedgehog gene, three homologues have been

identified in vertebrates namely, Sonic (Shh),

Desert (Dhh), and Indian hedgehog (Ihh). The

Sonic Hedgehog pathway is extensively

investigated in the vertebrate system (Chen et

al., 2005; Varjosalo et al., 2006). The other

components of the Hedgehog pathway include

patched (Ptc) and smoothened (Smo),

constituting a 12-pass transmembrane

glycoprotein and a 7-pass transmembrane

protein, respectively (Varjosalo, 2008;

Wicking et al., 1999).

In the Sonic-hedgehog pathway

elaborated in Fig. 3, absence of the hedgehog

ligand, Smoothened (Smo) is inhibited by

being bound to Patched (Ptc). When the

hedgehog ligand binds to Ptc, inhibition of

Smo is released which acts on protein complex

comprising of fused (Fu), suppressor of fused

(Sufu) and cos-2-costa-2 (Wicking et al.,

1999; Merchant, 2010). These proteins are

generally bound with Gli thereby inhibiting its

action. Once the complex is disrupted, Gli

translocates to the nucleus and brings about

transcription of different downstream targets

(Sasaki et al., 1999; Ruiz, 2007; Stecca, 2010).

Sonic-hedgehog signalling in normal

development and cancer

In vertebrates, the Sonic Hedgehog (Shh), is

expressed widely throughout the developing

central nervous system (CNS), limb, gut, teeth

and hair-follicle. Dhh is involved in

development of the germline, while the Ihh is

Figure 3: Hedgehog Pathway. A) In the absence of Hedgehog ligands (Indian, Sonic and Dessert), the Patched

receptor (ptc) exerts inhibitory action on the Smoothened receptor (smo). The Gli complex (Gli1 and Gli2) remains in the

cytoplasm followed by no transcription of Hedgehog target genes. B) In the presence of Hedgehog ligands, the inhibitory

action of Patched (ptc) on Smoothened (smo) is released, and hence Gli complex translocates to the nucleus and brings

about transcription of Hedgehog target genes.Fu: Fused; SuFu: Suppressor of Fused; Cos 2: cos-2costa-2.


Biomed Res J 2015;2(1):37-56

involved in development of the skeletal system

(Bitgood et al., 1996; Wicking et al., 1999).

Shh also plays a role in neural stem cells,

determining the neuronal cell fate (Merkle et

al., 2007). It was demonstrated that during

repair of acute airway injury, the Hedgehog

pathway gets activated in the airway

epithelium (Watkins et al., 2003). Hedgehog

signalling components Ptc, Gli1, and Gli2

were over-expressed when mammary

progenitor cells grow as mammospheres (Liu

et al., 2006). These reports indicate that the

Hedgehog pathway plays a role in normal stem

cell regulation (Ailles, 2012).

Hedgehog signalling is involved in

various cancers. For example, Ptc1 mutation

was observed in patients with medullo-

blastoma and rhabdomyosarcoma (Hahn et al.,

1996; Johnson et al., 1996; Pietsch et al.,

1997). Sufu as well as Smo mutations were

observed in medulloblastoma (Xie et al., 1998;

Taylor et al., 2002); Gli1 and Gli3 mutations

were seen in pancreatic adenocarcinoma; and

Gli1 gene amplification was seen in

glioblastoma (Clement et al., 2007; Jones et

al., 2008). Further, Kern et al showed that Gli

and PI3K/AKT/mTOR signalling act

synergistically to initiate and maintain chronic

lymphocytic leukemia (Kern et al., 2015).

Another report showed that Sonic hedgehog

ligand over-expression led to increased

number and size of intestinal adenomas in

APC (HET) mice, while loss of Indian

Hedgehog almost completely blocks intestinal

adenoma development (Buller et al., 2015).

Sonic-hedgehog signalling in normal and

cancer stem cell regulation and

maintenance

Hedgehog signalling is involved in stem cell

regulation of various tissues. Shh regulates

self-renewal of neural stem cells (Palma et al.,

2005). The components of Hedgehog pathway

such as Ptc, Gli1 and Gli2 are expressed in the

mammary stem cells and down regulated

during differentiation (Liu et al., 2006).

Hedgehog is involved in controlling neural

stem cells through the p53-independent

regulation of Nanog (Po et al., 2005).

In colon carcinoma, Hedgehog signalling

is activated in CSCs with higher expression of

Gli1 and Gli2. In non-small cell lung cancer,

the malignant phenotype of the tumours is

maintained by ligand-dependent Hedgehog

pathway activation (Watkins et al., 2003).

Furthermore, Bmi1, which is a downstream

target of the Hedgehog pathway was activated

in breast CSCs and is also shown to regulate

normal and leukemic stem cells (Liu et al.,

2006; Takebe, 2011).

CONCLUSION

Tumour maintenance and progression is

regulated by a subset of cells that are known as

cancer stem cells (CSCs). Recently, due to

increase in evidences on the existence of

Biomed Res J 2015;2(1):37-56

Dash et al. 47

CSCs, they have gained more attention but

how these CSCs escape the chemo-

radiotherapy is still unknown. Moreover, how

the CSCs are maintained in the tumour micro-

environment remains elusive. Several reports

showed signalling pathways such as Wnt,

Notch and Sonic-hedgehog are deregulated in

cancer, and also involved in the CSC

regulation and maintenance. In addition,

evidence of cross-talk between the signalling

pathways exists. Therefore, understanding

these signalling pathways at the molecular

level will be of utmost importance The study .

will enable counteracting the issue of

signalling cross-talk, and perhaps, multi

targeted drugs approach can be fruitful.

Hence, further detailed research on

deregulation of the developmental pathways

in CSCs needs to be investigated. Eventually,

elucidation of the signalling mechanisms will

enable to specifically target CSCs without

affecting the normal cells.

ACKNOWLEDGEMENTS

The authors acknowledge Mr. Rahul Sarate

and Mr. Gopal Chovatiya for their

suggestions.



Biomed Res J 2015;2(1):37-56

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INTRODUCTION

The transcription factor, nuclear factor

[erythroid-derived 2]-like 2 (Nrf2) was

identified as NF-E2-like basic leucine zipper

transcriptional activator that binds to the

tandem NF-E2/AP1 repeat of the β-globin

locus control regions (Moi et al., 1994). The

Nrf2 gene was cloned and characterized by

using the tandem repeats of nuclear factor like

erythroid factor-2 (NF-E2)/activator protein-1

(AP1) of the β-globulin locus as a recognition

site probe. Nrf2 contains a basic leucine zipper

DNA binding domain at the C-terminus and an

N-terminal acidic domain (rich in glutamic

and aspartic acid residues), which could

potentially function as an acidic

transactivation domain (Moi et al., 1994).

Further characterization demonstrated it as

Cap`n`Collar (CNC) protein involved in the

control of Drosophila head segment

development by basic leucine zipper DNA

binding domain (bZip) homeotic gene. The

CNC family comprises four members, namely

Nrf1, Nrf2, Nrf3 and p45NF-E2. Nrf1 and

Nrf2 are ubiquitously expressed and are

essential for normal development in mice. The

expression of Nrf3 is restricted to placenta and

liver, while p45NF-E2 expression is restricted

to erythrocytes (Ikeda et al., 2004; Motohashi

Key words: Cancer, Nrf-2 transcription factor, Keap-1 protein, β-TrCP protein.*Corresponding Author: Santosh K. Sandur, Free Radical Biology Section, Radiation Biology and Health Sciences Division, Bio-Science Group, Modular Laboratories, Bhabha Atomic Research Centre, Trombay, Mumbai, India.Email: [email protected]

Diverging Role of Nrf2 in Cancer Progression and

Prevention

Radiation Biology and Health Sciences Division, Bio-Science Group, Bhabha Atomic Research Centre, Mumbai,

India

Lokesh Gambhir, Rahul Checker, Deepak Sharma and Santosh K. Sandur*

The role of transcription factor, nuclear factor [erythroid-derived 2]-like 2 (Nrf2), is detoxification of

xenobiotics, overcoming oxidative stress and offering resistance to ionizing radiation induced cell death.

However, the role of Nrf2 in cancer progression remains debatable. Activation of Nrf2 dependent proteins is

crucial in maintaining cellular redox homeostasis and combating toxicity of carcinogens. Thus, employing

natural or synthetic activators of Nrf2 pathway is a promising approach for development of chemopreventive

modalities. Intriguingly, recent reports have highlighted the dark side of Nrf2 suggesting that multiple cancer

cells demonstrate constitutive activation of Nrf2 caused by mutations in Nrf2 or Keap-1 proteins, offering

survival advantage. Additionally, Nrf2 pathway is also up-regulated in chemoresistant cells and may be a

major contributor in acquired chemoresistance. Thus, targeting Nrf2 pathway has emerged as a novel

strategy to improve efficacy of chemotherapeutic drugs. This review discusses the dark and bright sides of

this transcription factor in line with the recent literature.

Biomed Res J 2015;2(1):57-82

Review

et al., 2002). Expression of Nrf1 is essential for

embryonic development and its deficiency

leads to hepatic abnormality. The Nrf2

knockout mice are viable and exhibit no

phenotypic defects, but are sensitive to

oxidative stress (Chan and Kwong, 2000;

Chan et al., 1998; Leung et al., 2003; Ohtsuji

et al., 2008; Ramos-Gomez et al., 2001; Xu et

al., 2005). Human Nrf2 is homologus to

mouse and contains six highly conserved

domains called Nrf2-ECH homology domains

(Neh). Neh1 domain has a nuclear localisation

signal and CNC-type basic leucine zipper

necessary for DNA binding and dimerization.

The Neh2 domain contains a Keap1 (Kelch-

like ECH-associated protein 1a, negative

regulator of Nrf2) binding pocket and has

seven lysine residues that direct ubiquitin

mediated proteasomal degradation of Nrf2

(Fig. 1) (Itoh et al., 1999; Zhang et al., 2004).

Neh3 is essential for interaction of Nrf2 with

CHD6 (a chromo-ATPase/helicase DNA

binding protein) suggesting involvement in

interaction with co-transcription factors (Nioi

et al., 2005). Neh4 and Neh5 are

transactivation domains that interact with the

CREB-binding protein (CBP) (Katoh et al.,

2001). Neh6 domain interacts with β-

transducin repeat-containing protein (β-TrCP)

(Jain and Jaiswal, 2007). Binding of Keap1 to

Nrf2 brings it close to E3 ligase complex

through two major domains: BTB (Bric a

Brac, tramtrack, broad complex) domain

which interacts with Cul3; and kelch domain

58

Biomed Res J 2015;2(1):57-82

Diverging role of Nrf2 in cancer

Figure 1: Structures and functions of Nrf2 and its repressors Keap1 and β-TrCP1. The relative position of the Neh

domains is shown. The DLG and ETGE motifs present in Neh2 domain that bind to Keap1 are represented above with the

numbering of amino acids based on the human cap'n'collar (CNC)-basic-region leucine zipper (bZIP) protein.

which binds to Nrf2. Interaction of Neh2

domain with Keap1 depends on low-affinity

binding via DLG motif and high-affinity

binding of an ETGE motif which results in a

hinge and latch mechanism of binding. The N-

terminal BTB/POZ (Pox virus Zinc finger)

domain forms homodimers enabling

Keap1–Nrf2 interaction (Adams et al., 2000;

Kensler et al., 2007, Li et al., 2004; Lo et al.,

2006; Padmanabhan et al., 2005).

Activation of Nrf2 dependent genes

Exposure of cells to low levels of oxidative

stress, electrophiles or chemopreventive

compounds leads to activation of Nrf2. Upon

activation, Nrf2 dissociates from inhibitory

protein Keap1 and translocates to the nucleus.

In the nucleus it forms a heterodimer with co-

transcription factor Maf and binds to the anti-

oxidant response element (ARE) sequence to

induce transcription of several different genes

(Zhang, 2006). ARE sequence is the 'core'

sequence of 5´-RTGACnnnGCR-3´ identified

using murine GST-Ya ARE. The sequence was

used to identify genes present in the promoter

region (Rushmore et al., 1991). The Nrf2

downstream genes include phase II

detoxifying enzymes like glutathione S-

transferase (GST), NAD(P)H quinone

oxidoreductase-1 (NQO1), and UDP-

glucuronosyltransferase (UGT), intracellular

cytoprotective proteins like glutamate

cysteine ligase (GCL), glutathione peroxidase

(GPx), thioredoxin (Trx), thioredoxin

reductase (TrxR), peroxiredoxin (Prx), heme

oxygenase-1 (HO-1) and transporters like

multidrug resistance-associated protein

(MRP) (Banning et al., 2005; Ishii et al., 2000;

Ishii and Yanagawa, 2007; Kim et al., 2001;

Maher et al., 2005; Moinova and Mulcahy

1999; Sakurai et al., 2005). Phase II enzymes

reduce the toxicity of xenobiotics by making

them water soluble, thereby facilitating their

elimination. Efflux of endogenous molecules

and xenobiotics is also governed by Nrf2

mediated expression of transporters.

Constitutive expression of Nrf2 by tumor cells

may offer an advantage for ambient growth

and detoxification of xenobiotics, the

phenomena coined as “dark side of Nrf2” (Lau

et al., 2008; Wang et al., 2008c). The present

review emphasizes the putative dual role of

Nrf2 pathway during cancer progression and

highlights its potential as a target for

chemoprevention.

Mechanism of Nrf2 Activation

Nrf2 is sequestered in the cytoplasm by Keap1

which regulates Nrf2 stabilization and levels

inside the cell. The interaction between the

two proteins is a dynamic process regulated in

such a manner that enables Nrf2 to control

both the basal and inducible expression of

dependent genes. Under homeostasis

conditions, Nrf2 is maintained at low basal

levels for expression of cytoprotective genes

(Fig. 2) (Itoh et al., 1999). Nrf2 is at low levels

when bound to Keap1 homodimer through its

Gambhir et al. 59

Biomed Res J 2015;2(1):57-82

kelch repeats domains at C terminal, leading to

Cullin3/Rbx1-mediated polyubiquitination

and subsequent proteasomal degradation.

Keap1 protein contains numerous cysteine

(cys) residues with potential to act as a redox

sensor (Hong et al., 2005b).

Role of cys residues in Nrf2 activation

The significance of Keap1 as a central

regulator of Nrf2 activation was revealed

while addressing the negative regulation of

antioxidant machinery by Keap1 dependent

proteasomal degradation of Nrf2 (McMahon

et al., 2003). The half life of Nrf2 increases

from 15 min to 30 min in cells expressing

mutated ETGE motif containing Nrf2 and

Keap1 (Du et al., 2008). Using in vitro

alkylation and in vivo site-directed

mutagenesis, cys151 was identified as the

major site directly alkylated by Nrf2 inducers

along with critical residues cys273 and cys288

(Dinkova-Kostova et al., 2002; Eggler et al.,

2005; Hong et al., 2005a; Levonen et al.,

2004). Mutation at cys151 abolished induction

of Nrf2 by activators like sulforaphane and

tert-butylhydroquinone but had no impact on

Keap1:Nrf2 binding. Keap1-cys151 restores

phenotypes like over-expression of Nrf2 and

post-natal lethality as observed in Keap1 null

mice (Wakabayashi et al., 2004). However,

activation of Nrf2 by arsenite in cys151 Keap1

mutant MDA-MB231 cells, indicated a

possible redox independent mode of Nrf2

induction (Wand et al., 2008b). Further

Biomed Res J 2015;2(1):57-82

Figure 2: Schematic model of Nrf2 activation under normal and oxidative stress conditions.

60 Diverging role of Nrf2 in cancer

cys273ser and cys288ser mutations showed

abrogated repression of Nrf2 by Keap1

(Levonen et al., 2004; Wakabayashi et al.,

2004). These observations demonstrated that

cys151 is required for the activation of Nrf2,

whereas cys273 and cys288 are needed for

Nrf2 inhibition. Besides, a significant

contribution of the critical cysteine residues

during Nrf2 activation and regulation under

oxidative stress was indicated. Several cellular

redox modifiers modulate activation of Nrf2

via modification of the critical cysteine

residues in Keap1. Further, the afore-

mentioned three critical cysteine residues

undergo thiol modifications leading to

conformational change in the Cul3–E3 ligase

complex leading to loss of E3 ligase ubiquitin

activity. The cysteine residues act as redox

sensors to further perturb the efficiency of

nuclear export signal on Keap1, and mutant

form of Keap1 at leu308 and leu310 was

unable to locate in the cytoplasm (Kobayashi

et al., 2009; Nguyen et al., 2005; Velichkova

and Hasson, 2005). These studies suggested

that under normal conditions, the signals from

nuclear export sequence (NES) of Keap1

maintained the Keap1 dimer in association

with Nrf2 in the cytoplasm.

Exposure of cells to oxidative, xenobiotic

or electrophilic stress abrogates Keap1

induced degradation of Nrf2. Perturbation in

the cellular redox status results in

modifications of critical cysteine residues in

Keap1. The conformational change renders

release of Nrf2 from the low affinity binding

motif (Cullinan et al., 2004; Kobayashi et al.,

2006). The change confers stabilisation and

accumulation of Nrf2 in the cytosol followed

by nuclear translocation. According to hinge

and latch model, ETGE motif remains bound

to the Keap1 following activation. This results

in saturation of Keap1 which is no longer able

to compete with free Nrf2 inducing

translocation to the nucleus and binding to

ARE to induce expression of cytoprotective

machinery of the host cell (Jain and Jaiswal,

2006). An alternate model of induction is

attributed to the polyubiquitination of Keap1

at lys63, leading to subverted Cullin3

interaction and dissociation of Nrf2 from

Keap1 (Zhang et al., 2005). The ubiquitin-

specific protease-15 deubiquitinase restored

Keap1 activity (Villeneuve et al., 2013).

Apart from Keap1 and Cul3/Rbx1, other

mediators also contribute in regulating the low

basal levels of Nrf2. Phosphorylation status of

tyr568 on Nrf2 is governed by Src subfamily

kinases Fyn, Src and Fgr, which influence the

nuclear export of Nrf2. Under oxidative stress

conditions, glycogen synthase kinase-3 beta

(GSK-3β), a serine/threonine protein kinase,

plays an important role in the nuclear export of

Nrf2 by phosphorylating Fyn. Another Src

member Bach1 has been shown to govern

export of Nrf2 from the nucleus, thereby

negatively regulating expression of its

dependent genes. Bach1 competes with Nrf2

for binding to ARE sequence, resulting in

Gambhir et al. 61

Biomed Res J 2015;2(1):57-82

suppression of ARE mediated expression of

Nrf2 dependent genes (Jain and Jaiswal, 2006;

Niture et al., 2011).

Keap1 independent activation of Nrf2

Multiple studies have highlighted Keap1

independent activation of Nrf2. Along with

Keap1 dependent degradation of Nrf2, an

alternate mechanism controls activation and

stabilisation of Nrf2 mediated by β-transducin

repeat-containing protein (β-TrCP) (Rada et

al., 2012). Mouse Nrf2 contains two binding

sites for β-TrCP which acts as an adapter for

the Skp1-Cul1-Rbx1 ubiquitin ligase

complex. GSK-3β phosphorylates serine

residue in SCF/β-TrCP destruction motif

“DSGIS” in Neh6 domain leading to Keap1

independent degradation (Jain and Jaiswal,

2007). Post translational modification also

governs Nrf2 activation. Nrf2 contains

multiple serine, threonine and tyrosine

residues which serve as potential sites for

phosphorylation. Different pathways for

activation of Nrf2 are identified including

protein kinase C (PKC), mitogen-activated

protein kinases (MAPK), phosphatidyl

inositol 3-kinase (PI3K), and RNA-dependant

protein kinase-like endoplasmic reticulum

kinase (PERK) (Cullinan and Diehl, 2004; Lee

et al., 2001; Yu et al., 2000). PKC has multiple

isoforms which play essential roles in growth,

differentiation, cytoprotection, apoptosis,

survival and carcinogenesis and PKC can be

activated by oxidative stimuli. PKC phospho-

rylated ser40 residue in the Neh2 domain

leading to disruption of Keap1/Nrf2

interaction in response to oxidative stress

induced by tBHQ and β-naphthoflavone.

Mutation in the serine residue results in

abrogation of PKC induced activation of Nrf2

(Huang et al., 2002). Interestingly,

phosphorylation of ser40 was required for

release of Nrf2 from Keap1, but does not play

a role in nuclear translocation (Bloom and

Jaiswal, 2003). Nuclear localisation sequence

(NLS) and nuclear export sequence in Nrf2

regulates localization in the cell. The NLS

motifs are identified by adapter proteins like

importins that facilitate transfer inside to

nucleus (Theodore et al., 2008). Another

conserved protein kinase that influences Nrf2

activation is casein kinase II (CK2). CK2

possesses an array of potential targets and

plays a role in complex cellular processes

including cytoprotection. Nrf2 contains 13

potential phosphorylation targets for CK2

abundant in Neh4/Neh5 transcriptional

domains. Phosphorylation dependent nuclear

translocation of Nrf2 is sensitive to Ck2

inhibitor (Apopa et al., 2008; Pi et al., 2007).

Role of MAPK in activation of Nrf2

PI3K and extracellular signal-regulated

protein kinase (ERK) are proposed to regulate

Nrf2 pathway (Cullinan et al., 2003; Kang et

al., 2001). tBHQ enhances NQO1 protein

expression and activity in a PI3K dependent

manner in human neuroblastoma cells. tBHQ

Biomed Res J 2015;2(1):57-82


elicited ARE mediated induction of GST in

hepatoma cells in a PI3K dependent manner.

PI3K inhibitor (Ly294002) abrogated tBHQ

mediated NQO1 induction, indicating a role of

PI3K in Nrf2 activation (Lee et al., 2001).

PERK, a transmembrane kinase, phospho-

rylates Nrf2 in vitro leading to dissociation

from Keap1. A pivotal role of PERK mediated

activation of Nrf2 was proposed as a

mechanism for maintenance of glutathione

levels that act as a cytoprotective buffer

against oxidative insult (Cullinan et al., 2003).

An important role of MAPK in the activation

of Nrf2 via phosphorylation has been reported

by several investigators. Yu et al. (2000)

studied MAPK mediated activation of phase II

detoxification enzymes using multiple

inducers (Jeong et al., 2006). In hepatoma

cells, sulforaphane and tBHQ induced

activation of ERK, MAPK kinase and Raf-1,

to mediate induction of phase II detoxification

enzymes via Nrf2/ARE pathway (Yuan et al.,

2006). MAPK/ERK upon activation initiates

phosphorylation cascade that modulates

activity of multiple downstream transcription

factors (Shen et al., 2004; Zipper and

Mulcahy, 2000). Dithiolcarbamate was shown

to activate ERK and p38 resulting in

transcriptional up-regulation of Nrf2

dependent γ-glutamylcysteine synthetase

(Wild et al., 1999). Shen et al. (2004)

investigated the transactivation potential of

different Nrf2 domains and observed

differential effects of multiple MAPKs in

activating Nrf2. The authors further

demonstrated Raf-1 mediated activation of

Nrf2 attributing it to up-regulation of the co-

activator CREB binding protein.

Pro-oncogenic Effects of Nrf2: The Dark

Side

It is well documented that oxidative stress

plays a pivotal role in the initiation and

progression of cancer, with magnitude of

oxidative stress a key determinant of the

response of a cell towards the oncogenic

stimuli. Chronic exposure of cells to oxidative

insult causes cytotoxicity due to irreversible

damage to vital macromolecules; whereas

transient increase leads to the activation of

redox sensitive pro-survival transcription

factors. Therefore, in order to survive and

proliferate, tumor cells maintain a moderate

oxidative intracellular niche achieved by

taking advantage of the antioxidant defense

machinery of the cell like Nrf2 pathway.

Constitutive activation of Nrf2 and expression

of dependent cytoprotective genes, permits

tumor cells to nurture and expand in an

ambient redox niche. High levels of Nrf2

expression is reported in multiple cancers

including cancers of lung, breast, gall bladder,

pancreatic, colorectal and head and neck

(Jaramillo and Zhang, 2013; Lau et al., 2008;

Shelton and Jaiswal, 2013; Sporn and Liby,

2012). Ikeda et al. (2004) demonstrated

constitutive up-regulation of Nrf2 and GSTP1

in hepatocellular carcinoma indicating role of

Gambhir et al. 63

Biomed Res J 2015;2(1):57-82

Nrf2 in cancer promotion. Nrf2 regulates

expression of an exclusive neoplastic lesion

marker GSTP1 in an ARE dependent

mechanism. Higher levels of Nrf2 have been

associated with poor clinical outcome and

poor responsiveness in pancreatic, cervical

and lung cancer (Geismann et al., 2014; Sporn

and Liby, 2012).

Dysregulation of Nrf2 pathway in cancer

Persistent Nrf2 activation is responsible for the

pro-tumorogenic effect due to genetic and

epigenetic alterations in Nrf2/Keap1

(frequencies of up to 30% in lung or ovarian

cancer). Copy number loss in a member of E3

ubiquitin ligase complex or oncogenic

pathways or persistent exposure to oxidative

stress leads to persistent activation (Barbano et

al., 2013; Martinez et al., 2014; Zhang et al.,

2010). A mutation in the Keap1 protein or loss

of heterozygosity has been reported to result in

persistent Nrf2 activation in multiple cancers

(Padmanabhan et al., 2006; Singh et al., 2006).

DNA methylation of CpG sites in the promoter

region of Keap1 was observed in 51% of

breast, 20% of colorectal, and 12% of lung

cancers, accounting for decreased levels of

Keap1 and consequent enhanced Nrf2

activation (Bryan et al., 2013; Wang et al.,

2008a). Approximately 15% patients with

lung cancer posses somatic mutations in

Keap1, resulting in impaired and inefficient

Nrf2 repression (Hayes and McMahon, 2009).

The prevailing Keap1 mutations were

classified based on their functional impact into

passenger mutations, null mutations and

hypomorphic mutations. Passenger mutations

do not have any effect on Keap1/Nrf2

interaction, whereas null mutations

diminished the ability of Keap1 to repress

Nrf2. Most of the mutations do not affect the

Nrf2 levels, but impact the activity as Keap1 is

unable to act as a negative regulator (Hast et

al., 2014; Hayes and McMahon, 2009; Shibata

et al., 2008). Japanese patients with lung

adenocarcinoma demonstrated Keap1

mutations (Ohta et al., 2008). Dysregulated

suppression of Nrf2 by Keap1 in breast cancer

resulted due to mutation in cys23 residue (Nioi

and Nguyen, 2007). Under hypoxic/

reoxygenation conditions Nrf2 was

upregulated and protected cancer cells from

deleterious effects of oxidative stress (Kim et

al., 2007).

Mutations in the DLG motifs of the DC

domain in Keap1 show highest frequency in

lung cancers (Ganan-Gomez et al., 2013).

Interestingly frame shift mutations in Keap1

are frequent in DGR domain (65%) essential

for interaction with Nrf2 (Taguchi et al.,

2011). Other mutations in the intervening

region and BTB domain of Keap1 occur in

prostate, lung and ovarian cancers. Mutation

in these domains influence the critical cysteine

residues that inhibit its interaction with

Cullin3, leading to inhibition of poly-

ubiquitination of Nrf2. Mutation in other

amino acids like ser104, gly186,423 and

Biomed Res J 2015;2(1):57-82


arg320 within the DC, BTB and IVR domains

are cancer derived mutations that results in

impaired homo-dimerization of Keap1 needed

for repression of Nrf2 (Hast et al., 2014). A

single nucleotide deletion in Keap1 gene was

associated with marked drug resistance

against BRAF and cisplatin in melanoma cells

(Miura et al., 2014). Although mutations in

Keap1 play a central role in constitutive Nrf2

activation, deficiency of Keap1 per se does not

result in cancer. Interestingly, Keap1

knockdown mice (floxed Keap1 allele) did not

develop spontaneous cancer and survived for 2

years. Keap1 knockdown mice showed

constitutive activation of Nrf2 in multiple

tissues including lung and liver. These studies

indicated that impaired Nrf2/Keap1 pathway

may result in cancer cell proliferation or

resistance to anti-cancer modalities, but it does

not set off cancer initiation (Taguchi et al.,

2010). In addition, mutations in Nrf2 gene are

focussed in Keap1 binding domain near ETGE

and DLG motifs termed as hot spot regions.

Mutations in Nrf2 were observed in lung, head

and neck, oesophagus and skin cancers, but are

less abundant (Kim et al., 2010; Shibata et al.,

2008). Nrf2 deficient mice were more

susceptible to urethane induced lung cancer

compared to Nrf2 wild type (Bauer et al.,

2013). The Nrf2 mutations are clustered

within ETGE (57%) and DLG (43%) moti,

which were indispensable for Keap1 binding.

Mutations in ETGE motif disrupt the high

affinity binding with Keap1 and thus prevent

Nrf2 from ubiquitination, whereas mutations

in the DLG motif disrupt low affinity binding

but Nrf2 remains bound to Keap1. Both these

mutations result in Nrf2 stabilisation and

accumulation in nucleus (Taguchi et al., 2011).

Along with the somatic mutations in

Keap1/Nrf2, an alternate mechanism for

activation of Nrf2 in tumorigenesis is

mediated by oncogenic signalling. Expression

of oncogenes like Kras, Braf and Myc activate

Nrf2, elevating antioxidant machinery

resulting in depletion of the intracellular ROS

levels, thus providing a conducive reduced

environment for tumor growth (DeNicola et

al., 2011).

Nrf2 in chemoresistance

A distinctive property of constitutive

activation of Nrf2 is chemoresistance,

protecting cancer cells from anti-cancer drugs

used in chemotherapy. Several studies have

highlighted the pivotal role of Nrf2 in

chemoresistance such as cisplatin in ovarian

cancer, cervical cancer or endometrial serous

carcinoma; gemcitabine in pancreatic cancer;

doxorubicin in liver cancer and 5-fluorouracil

in gastric cancer (Chen et al., 2012; Duong et

al., 2014; Jiang et al., 2010; Ma et al., 2012).

Elevated Nrf2 induces autophagy in ovarian

carcinoma imparting resistance against

cisplatin and tamoxefin (Bao et al., 2014). Due

to the cytoprotective and detoxifying

potential, several Nrf2 dependent genes are

implicated in conferring Nrf2 mediated

Gambhir et al. 65

Biomed Res J 2015;2(1):57-82

chemoresistance, e.g., HO-1 is over-expressed

in multiple cancers. Due to the cytoprotective

nature, over-expression is undesirable in

cancer cells. Over-expression of HO-1 was

associated with increased cell proliferation

and endothelial cell division leading to

angiogenesis (Was et al., 2006). Other Nrf2

dependent genes including NQO1, GPX, TrxR

and Prx1 were shown to be up-regulated in

multiple cancer cells. GPx, a selenoprotein

that detoxifies H O , is implicated in the 2 2

control of malignant growth. Elevated GPx

levels were observed in advanced stages of

colorectal cancer, Barrett`s esophageal

mucosa and gastrointestinal cancers

associated with cell proliferation, growth and

inhibition of apoptosis (Banning et al., 2005;

Chu et al., 2004; Was et al., 2006). Peroxi-

redoxins (Prx) are thiol specific antioxidants

that detoxify peroxides and are elevated in non

small lung cancer (NSLC) and thyroid cancer,

a predictive factor for disease and associated

with prognosis (Kim et al., 2007; Yanagawa et

al., 1999). Trx and TrxR collectively form a

redox couple with a pivotal role in maintaining

cellular redox status in cellular functions

(Brigelius-Flohe, 2008). Despite its protective

role as redox couple, TrxR1 was elevated in

gastrointestinal cancer tissues (Arner and

Holmgren, 2006; Iida et al., 2004). TrxR

knockdown lung carcinoma cells showed

reversal of tumorigenicity and invasion.

Enhanced cellular expression of TrxR has been

attributed to cisplatin resistance, and

inhibition in TrxR activity abrogates

resistance against cisplatin (Sasada et al.,

1999). NQO1 is another Nrf2 dependent gene

over-expressed in adrenal gland, bladder,

breast, colon, liver, lung, ovary, and thyroid

cancers (Basu et al., 2004; Siegel and Ross,

2000). Suppression in NQO1 expression

sensitizes A549 cells to etoposide, cisplatin

and doxorubicin (Wang et al., 2008c).

Chemopreventive Effects of Nrf2: The

Bright Side

Several compounds derived from natural or

synthetic origin with chemopreventive

activity act via Nrf2. Administration of

methylcholanthrene reduced cancer incidence

in rats caused by carcinogenic azo dyes, served

as a nucleation point for use of dietary

compounds as chemopreventive agents

(Richardson and Borsos-Nachtnebel, 1951).

Multiple plant derived products possess

chemopreventive effect by inducing Nrf2

activation (Kelloff et al., 2000; Sporn and Suh,

2000; Talalay and Fahey, 2001; Yang et al.,

2001). Nrf2 activation results in increased

expression of cytoprotective proteins

preventing biomolecules from the damaging

effects of oxidative and xenobiotic stress. Nrf2

knockout mice studies strengthened the notion

of Nrf2 serving as a novel chemopreventive

factor controlling sensitivity to carcinogens

(Slocum and Kensler, 2011). Ablation in Nrf2

led to enhanced tissues damage caused by

cigarette smoke, hyperoxia, ischemic

Biomed Res J 2015;2(1):57-82


reperfusion, portal vein embolization, and

chemical toxins (Chan et al., 1996; Cho and

Kleeberger, 2010; Kudoh et al., 2014;

Shirasaki et al., 2014; Zhao et al., 2011). Mice

with Nrf2 over-expression resulting from

Keap1 knockout shows increased resistance to

lung cancer cell metastasis (Satoh et al., 2010).

Nrf2 ablation was associated with enhanced

sensitivity to mutagens and showed increased

carcinogenesis in bladder, skin, hepatocytes

and colon on exposure to nitrosoamine,

ultraviolet, aflatoxin, dextran sulphate sodium

and azoxymethane (Iida et al., 2004; Khor et

al., 2006; Osburn et al., 2007; Saw et al., 2011;

Xu et al., 2006; Yates et al., 2006). Curcumin,

sulforaphane, oltipraz and CDDO-imidazole

activate Nrf2 while exerting chemopreventive

effects and Nrf2 deficiency in mouse models

abrogated their chemopreventive effects

(McMahon et al., 2001; Ramos-Gomez et al.,

2003; Shen et al., 2006; Slocum and Kensler,

2011; Sussan et al., 2009). Nrf2 has also been

implicated in protecting against ROS

dependent genetic lesions that promote

metastasis (Satoh et al., 2010). Under

conditions of increased ROS levels, Nrf2

induced expression of Kruppel-like factor 9

(Klf9), which further enhanced oxidative

stress mediated cell death (Zucker et al.,

2014). The anti tumor potential of Klf9 in

different cancer types has been reported with

inhibition of glioblastoma stemness through

transcriptional repression, and induced

apoptosis in prostate cancer cells by Akt

inhibition (Huang et al., 2015; Ying et al.,

2014). Nrf2 is also an anti-inflammatory

transcription factor and activation of Nrf2 and

dependent genes reduce chronic inflammation

associated cancers like colorectal or

pulmonary cancer. A protective role of Nrf2 is

supported by studies in mice with a single-

nucleotide polymorphism (SNP) in the

promoter region. The polymorphism was

associated with increased susceptibility to

hyperoxia induced lung damage, due to low

expression of Nrf2 (Cho et al., 2002;

Yamamoto et al., 2004).

Though higher levels of Nrf2 are observed

in multiple malignancies, the role in initiation,

promotion or transformation of normal cells

remains contentious. Low levels of Nrf2 were

essential for oncogenic transformation of

mesenchymal stem cells (Funes et al., 2014).

Epigenetic reactivation of Nrf2 attenuated

skin epidermal cell transformation (Su et al.,

2014). Over-expression of Nrf2 in cancer cells

may enable survival under conditions of

oxidative stress, or detoxify xenobiotics

leading to better survival. Nrf2 prevented

initiation of lung cancer, but accelerated

progression through the Kras signalling

pathway. Thus Nrf2 activators may pave the

way for prevention of lung cancer (Satoh et al.,

2013).

Nrf2 as Target for Therapeutic

Interventions

Nrf2 activation and enhanced expression of its

Gambhir et al. 67

Biomed Res J 2015;2(1):57-82

dependent genes associated with redox

regulating proteins, phase II detoxifying

enzymes and transporters are exploited by

cancer cells to survive and proliferate.

Therefore, agents that inhibit Nrf2 expression

in cancer cells may provide a novel strategy for

therapeutic interventions to enhance efficacy

of existing chemotherapeutic drugs. Brusatol,

a plant extract from Bruceajavanica,

selectively inhibits Nrf2 by increasing

ubiquitination and degradation. It reduced

resistance towards cisplatin in cultured

xenografts (Ren et al., 2011). 6-Hydroxy-1-

methylindole-3-acetonitrile (6-HMA)

protected against cisplatin induced oxidative

nephrotoxicity by inhibiting Nrf2 activation

(Moon et al., 2013). Luteolin, a plant derived

flavanoid, inhibited proliferation of tumor

cells and reduced toxicity of cisplatin in a mice

model (Lin et al., 2010; Sun et al., 2012; Tang

et al., 2011). All-trans retinoic acid (ATRA)

inhibited Nrf2 by activating retinoic acid

receptor α, which directly interacts with Nrf2

and restrain binding to ARE (Wand et al.,

2013a). However, the use of Nrf2 inhibitors in

cancer therapy is at a nascent stage and

requires development of specific agents to

minimize non-specific off-target effects.

Assuming Nrf2 as a target for cancer

prevention, several population-based clinical

trials were conducted with diverse

chemopreventive drugs including phenethyl

isothiocyanate, oltipraz, curcumin,

resveratrol, fumaric acid esters and synthetic

oleanane triterpenoids. Administration of

several Nrf2 activators in clinical trials was

well tolerated, resulting in elevated levels of

cytoprotective enzymes (Kensler et al., 2012;

Linker et al., 2011; Palsamy and Subramanian,

2011; Scannevin et al., 2012). In a Chinese

study, aflatoxin intoxication as a risk factor

was reduced by oltipraz (Kensler et al., 2003).

Favourable effects of sulforaphane, a potent

Nrf2 activator, were observed in the

promotion or progression phase of cancer, and

sulforaphane inhibited cancers of multiple

sites including skin, lung, bladder, breast,

colon and stomach (Conaway et al., 2005;

Dinkova-Kostova et al., 2006; Gills et al.,

2006; Hu et al., 2006; Shen et al., 2007).

Chemoprevention with sulforaphane-rich

extracts of broccoli are in clinical trials in

China (Egner et al., 2011). Similarly, synthetic

oleanane triterpenoids reduced progression of

lung, breast and pancreatic cancers, and

delayed onset of tumor driven by Kras, Trp53,

Brca1 and Erbb2 oncogenes (Liby et al.,

2010).

Salutary health effect of phytochemicals

that induce Nrf2 highlights the role of Nrf2-

activating foods and spices in human diet.

Food products like curcumin from turmeric

root, sulforaphane from broccoli, and

seaweed-based extracts from green alga Ulva

lactuca were shown to activate the Nrf2

pathway in vivo. Extract with sulforaphane

concentration that is achieved by dietary

broccoli consumption, offered protection

Biomed Res J 2015;2(1):57-82


against particulate pollution in humans

(Boddupalli et al., 2012; James et al., 2012;

Wang et al., 2013b). Similarly, phytochemical

constituents of garlic, tomatoes, grapes, green

tea, coffee, and berries show Nrf2 activating

properties, indicating beneficial effects by

dietary consumption (Kropat et al., 2013).

Numerous dietary supplement companies

have developed mixtures of known Nrf2

activators to increase the antioxidant system in

body. Protandim (LifeVantage, Inc, Sandy,

UT, USA), reduced oxidative stress in humans

(Nelson et al., 2006).

Apart from the implication of

phytochemicals and dietary intake, life style of

an individual also plays an important role in

Nrf2 activation. A relationship between

physical activity and Nrf2 activation was

established in a mouse model and exercise-

induced oxidative stress was higher in Nrf2

knockout mice (Miller et al., 2012;

Muthusamy et al., 2012; Zhao et al., 2013).

Evidence from several studies provide a strong

incentive for development of novel Nrf2

activators as putative cancer chemopreventive

agents in normal healthy individuals without

affecting pro-survival potential. However,

caution must be exercised as a pro-

tumorogenic role of Nrf2 in various cancers

indicates dual nature of Nrf2 activation. Nrf2

activation may provide a survival advantage to

pre-existing cancer cells and also participate in

resistance to chemotherapy or radiotherapy.

Future perspective and conclusion:

Given the dual role of Nrf2 in cancer, the prime

query is the role of Nrf2 in cancer initiation or

cell transformation. Transient activation of

Nrf2 by pharmacological activators is safe for

the purpose of chemoprevention as the

activators do not seem to increase the tumor

burden. A major concern of use of Nrf2

activators is their cytotoxicity and non-

specific mechanism of action. The activators

show a tendency to modulate cellular redox

and are reactive towards cysteine residues

which may lead to modulation of signalling

pathways. Thus, designing specific Nrf2

activators like ETGE and DLG mimetic, based

on co-crystal structure of Neh2 domain and

Keap1, may reduce the off target effects.

Further, demonstration of miRNA mediated

regulation Nrf2 pathway provides a new

conduit to explore additional targets. Multiple

studies highlight the cross talk of Nrf2 with

other signalling pathways imperative for cell

survival. Results from our laboratory have

demonstrated implication of Nrf2 cross talk

with NF-κB as a prime target for anti-

inflammatory effect (Gambhir et al., 2014).

Thus, novel agents targeting Nrf2 pathway

specifically needs investigation.

SUMMARY

Nrf2 is a redox sensitive transcription factor,

maintained at low basal levels under normal

conditions. Upon activation, it mediates

Gambhir et al. 69

Biomed Res J 2015;2(1):57-82

expression of dependent cytoprotective genes,

phase II detoxifying enzymes and antioxidant

machinery. Evidences illustrating a positive

role of Nrf2 in cancer prevention has been

documented. Thus, efforts are underway to

identify novel agents that can activate Nrf2.

However, constitutive expression of Nrf2 may

prevent death of precancerous lesions and

promote survival of cancer cells under

oxidative stress suggesting a dual role (Fig. 3).

The transient activation of Nrf2 is beneficial,

in countering ill effects of xenobiotics,

oxidative stressors, carcinogens and

mutagens. Whereas, persistent activation of

Nrf2 in tumor cells confers survival advantage

and makes them refractory to chemotherapy

and/or radiotherapy. Evidence to directly

implicate Nrf2 in cancer initiation needs

confirmation. However, Nrf2 facilitates a

reducing environment through up-regulation

of the antioxidant and cytoprotective

machinery. Thus providing armour for cancer

cell to create an ambient growth niche and

resist toxicity of xenobiotics. Hence, Nrf2 may

serve as an additional target for therapeutic

interventions, increasing susceptibility of

cancer in conjuction with chemotherapy or

radiotherapy treatment modalities.

Biomed Res J 2015;2(1):57-82

Figure 3: Dual role of Nrf2. The bright side is indicated by Nrf2 functions in normal cells where it acts as cytoprotective

transcription factor inducing expression of an array of cytoprotective proteins, antioxidants and detoxifying enzymes

leading to increased survival and cancer prevention. In tumor cells, constitutive high levels of Nrf2 provides an ambient

niche for cancer cells to grow by reducing toxicity of endogenous ROS and xenobiotics. High levels of Nrf2 may increase

cancer cell survival and imparts chemoresistance leading to poor clinical outcome.


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ACKNOWLEDGEMENTS

The authors would like to thank Dr. Anu Ghosh

and Dr. S. Jayakumar for proofreading the

review. The authors would also like to

acknowledge Dr. S. Chattopadhyay, Associate

Director, Bioscience Group, Bhabha Atomic

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Biomed Res J 2015;2(1):57-82


INTRODUCTION

Human reproduction is an inefficient process.

Epidemiological evidences suggest that only

30% of all conceptions get clinically

recognized, and a large number of these are

lost spontaneously. Furthermore, the success

of assisted reproduction is low, as reasonably

good quality embryos fail to implant and there

is a high frequency of spontaneous abortions.

These epidemiologic and clinical evidences

indicate that uterine implantation governs

reproductive success and maternal

incompetence at the endometrial level can be a

constraining factor. Thus, understanding the

molecular events of embryo-maternal

interaction is of interest to reproductive

biologists, clinicians and couples affected by

infertility. The understanding is also essential

for designing rational management strategies

Key words: Endometrium, Decidua, Implantation, Biosensor, Invasion, Trophoblast, Abortion.*Corresponding Author: Deepak N. Modi, Molecular and Cellular Biology Laboratory, National Institute for Research in Reproductive Health, Parel, Mumbai, India.Email: [email protected], [email protected]

Physiology of Embryo-Endometrial Cross Talk

Molecular and Cellular Biology Laboratory, National Institute for Research in Reproductive Health, Parel, Mumbai,

India

Deepak N. Modi* and Pradeep Bhartiya

Implantation of the blastocyst stage embryo into the maternal endometrium is a critical determinant and a

rate-limiting process for successful pregnancy. Embryo implantation requires synchronized changes in the

endometrium before and after arrival of blastocyst into the uterine cavity. Extensive cross talks occur

between the fetal and maternal compartments around the time of implantation which are reflected by

morphologic, biochemical and molecular changes in the endometrial cells and the differentiating

trophoblast cells. The embryo induced morphologic changes include occurrence of epithelial plaque

reaction, stromal compaction and decidualization. Embryonic signals also alter the expression of a large

number of transcription factors, growth factors and their receptors and integrins. Thus the embryo

superimposes a unique signature on the receptive endometrium for successful implantation. Functionally,

the embryo-endometrial cross talk is essential for endowing a “selector activity” to the receptive

endometrium to ensure implantation of only a developmentally competent embryo. On selection, the

decidua creates a conducive microenvironment for trophoblast invasion leading to placentation. Clinical

evidences suggest that along with receptivity, a defective “selector” activity of the receptive uterus may be a

cause of infertility and recurrent miscarriages. Defects in trophoblast invasion are associated with

pregnancy complications like preeclampsia and intra-uterine growth retardation. It is envisaged that

understanding of the embryo-endometrial dialogue leading to the “selector” activity, aids in development of

appropriate therapeutic modalities for infertility related disorders and miscarriages. Conversely, it might

also benefit the development of anti-implantation drugs for contraception.

Biomed Res J 2015;2(1):83-104

Review

for implantation failure and treatment of

infertility.

Our current understanding of the process

of embryo implantation and the determinants

of successful pregnancy have mainly stemmed

from animal models and in vitro studies using

human tissues. Based on the data derived it is

clear that endometrial receptivity and embryo

implantation are complex processes involving

a delicately poised balance of maternal

hormones, endometrial factors and embryonic

influences. The current review focuses on

cellular and molecular events associated with

endometrial receptivity and implantation to

accomplish successful conception. The

embryo-endometrial cross talk at the time of

embryo apposition and implantation mainly in

the primates will be discussed. The general

understanding of the processes of endometrial

receptivity and implantation has been a subject

of recent reviews (Gellerson and Brosens,

2014; Ozturk and Demir, 2010; Young, 2013;

Young and Lessey, 2010).

Endometrial receptivity

A mutual communication between the

blastocyst and the uterus is indispensable for

implantation. Akin to many developmental

processes, it involves an elaborate sequence of

genetic and cellular interactions, to be

executed within an optimal temporal frame for

successful pregnancy. In order to receive a

developing embryo, the endometrium endures

a series of morphological and physiological

transformations. At the same time, the

fertilized ovum undergoes several rounds of

cell division and transforms into blastocyst.

The blastocyst has an outermost layer of

specialized cells, trophoblast cells, that

surround the pluripotent inner cell mass. The

trophoblast cells come in direct contact with

the receptive endometrium establishing a firm

attachment with the endometrial epithelial

cells termed as apposition. Subsequently, the

trophoblasts invade the endometrium and

establish contact with the maternal circulation

to form the placenta.

The endometrium is refractory to embryo

implantation throughout the menstrual cycle

except for a few days after ovulation.

Approximately, on days 21–24 of the human

menstrual cycle (8–10 days post ovulation),

the uterus becomes "receptive", enabling

blastocysts to adhere to the luminal

epithelium. Termed as “window of

receptivity”, the achievement of this stage is

highly dependent on the ovarian steroids,

estrogen and progesterone. The estrogen in the

follicular phase leads to proliferation of

endometrial epithelial cells, the progesterone

surge that occurs in response to ovulation leads

to differentiation of the estrogen primed

endometrium to endow receptivity. Any

disturbance in the levels of these hormones

adversely affects endometrial physiology

leading to failure of implantation. The

endocrine regulation of the menstrual cycle

and role of hormones in endowing receptivity

84

Biomed Res J 2015;2(1):83-104

Embryo-endometrial cross talk

to the endometrium has been a subject of

extensive studies (Jabbour et al., 2006; Young,

2013).

Morphologically, the receptive phase

endometrium is characterized by presence of

columnar epithelium with microvilli, an

increase in stromal cell proliferation and

appearance of pinopod-like structures on the

luminal epithelium (Tu et al., 2014). The

morphological features of the “receptive”

endometrium are associated with expression

of a range of biochemical and molecular

markers, crucial for endowment of this phase

of the uterus. Several markers like

transcription factors, integrins and their

ligands, cytokines and growth factors, have

been associated with the receptive phase (Tu et

al., 2014; Wang and Dey 2006; Zhang et al.,

2013,). A molecular signature of the “receptive

window” using global gene profiling

technologies have been identified that can

phenotype different phases of the menstrual

cycle including the receptive stage to

objectively classify the implantation window

(Garrido-Gómez et al., 2013; Haouzi et al.,

2012). These findings open the field for the

diagnosis of the endometrial defects in assisted

reproductive technology programs (Ruiz-

Alonso et al., 2013).

Embryo induced morphologic changes in

the receptive endometrium

In a conception cycle, the egg that has

fertilized in the fallopian tube undergoes a

series of cell divisions and reaches uterine

lumen at the blastocyst stage. At this time the

trophoblast cells are differentiated and the

embryo is ready to hatch. From rodents to

humans, this embryo induces a second round

of differentiation both at the morphologic and

biochemical level (Banerjee and Fazleabas,

2009). Distinct from the “receptive” stage

endometrium, the embryo induced

differentiation of the receptive endometrium is

rather limited and largely derived from

experimental studies in the non-human

primates and endometrial biopsies obtained

from conception cycles in humans. Three

major non-human primate models to identify

and dissect embryo induced morphological

and physiological changes in the receptive

stage endometrium are: 1) Timed hysterect-

omies and/or biopsies of the endometrium of

baboons or rhesus macaques sequentially

mated with males of proven fertility; 2)

Endometria obtained from mated bonnet

monkeys where the presence of the embryo

has been verified using a preimplantation

factor assay; and 3) Endometrial tissue

obtained from baboons where human

chorionic gonadotropin (hCG) has been

infused in the uterine lumen in a manner that

mimics the transit of blastocyst. The models

have inherent advantages and disadvantages

but are highly complementary and provide

valuable information in terms of identification

and deciphering the functional consequence of

embryo induced changes in uterine

receptivity.

Modi and Bhartiya 85

Biomed Res J 2015;2(1):83-104

Epithelial changes: The earliest endometrial

response prior to implantation is characterized

by an increased proliferative activity of the

luminal and glandular epithelium. Distinct

from the epithelial proliferation observed in

the follicular phase, this proliferative activity

in the pregnant uterus is restricted to focal

areas. In the luminal epithelium, there are large

clump of nuclei with distinct entero-

reduplication and poorly packed chromatin

along with loss of basement membrane. These

changes are termed as “epithelial plaque

reaction” (Jones and Fazleabas, 2001; Rosario

et al., 2005a). The formation of epithelial

plaques is restricted to pregnant endometrium

and reported in a variety of primate species

including humans (Rossman, 1940). While

consistently detected in the conception cycle,

the epithelial plaque reaction is hormonally

regulated and does not require presence of an

embryo, as infusion of hCG directly in the

uterus leads to formation of epithelial plaques

similar to those observed in pregnant monkeys

(Fazleabas et al., 1999; Jones and Fazleabas,

2001). The functional significance of

epithelial plaques is not clear. It is speculated

that the plaque may provide nutrition by means

of intracellular glycogen (Enders et al., 1985;

Rossman, 1940). The plaque response may

stimulate precocious development of the

maternal vasculature below the epithelium

(Enders et al., 1985).

Beyond the plaque reaction, thinning of

the basal lamina and thickening and diffusion

of the apical and lateral gap junctions in

luminal epithelial cells has been reported in

pregnant human, bonnet and baboon

endometrium (Demir et al., 2002; Rosario et

al., 2008). Along with these changes, a few

granulocytic stromal cells are also observed in

the luminal epithelium of pregnant bonnet and

rhesus monkeys prior to implantation (Ghosh

et al., 1993; Rosario et al., 2008). It is

conceivable that these changes occur to

promote adhesiveness to the trophoblast cells

at the time of apposition and invasion.

Stromal changes: An almost universal

reaction of the endometrial stromal cells in

response to an embryo is decidualization. In its

broadest sense, decidualization is defined as

the postovulatory endometrial remodeling

which includes secretory transformation of

uterine stroma, influx of specialized uterine

natural killer cells, and vascular remodeling. A

more restricted definition of decidualization is

an epithelioid transformation of the

endometrial stromal cell with highly

specialized and distinctive functions.

Decidualization only occurs in species in

which placentation involves breaching of the

luminal epithelium by the trophoblasts. The

extent of this differentiation process often

correlates with the degree of trophoblast

invasion (Dunn et al., 2003; Gellersen et al.,

2007).

Morphologically, the elongated spindle

like stromal cells of the secretory phase

Biomed Res J 2015;2(1):83-104

86 Embryo-endometrial cross talk

endometrium transform into cobblestone like

enlarged decidual cells with multiple club

shaped projections arising from the cell

surface and contain abundant glycogen stores

and lipid droplets (Welsh et al., 1985; Wynn et

al., 1974). In humans, this transformation

occurs even in absence of an embryo and is

referred to as the pre-decidual response. In a

conception cycle, under the continuous

support of steroid hormones and blastocyst

derived signals, decidualization of the entire

endometrium is observed (Brosens et al.,

2002; Gellersen and Brosens, 2014). The

decidua forms a dense cellular matrix that

allows coordinated trophoblast invasion while

simultaneously protecting the conceptus from

maternal and environmental insults (Kliman,

2000; Redhorse et al., 2004). In the non-

human primates, decidualization is observed

in conception cycle or on treatment with hCG

(Jones and Fazleabas, 2001; Rosario et al.,

2005a). These observations suggest that unlike

in humans, embryo/embryonic factors are

required for the endometrial stromal cells to

undergo decidualization in monkeys.

Vascularization: A characteristic feature of

the endometrium in a conception cycle is the

enhanced microvasculature. In the pregnant

bonnet monkeys, a large number of small

blood vessels are detected in the stroma

underlying luminal epithelium and the

functionalis region of the endometrial bed

(Rosario et al., 2005a). Increased vascularity

and angiogenesis at the implantation site of

rhesus monkeys has been reported (Sengupta

and Ghosh, 2002). A similar increase in the

number of small blood capillaries in the

stroma of the endometrial functionalis have

been demonstrated in baboons infused with

physiological doses of hCG in the uterine

lumen (Banerjee and Fazleabas, 2009; Jones

and Fazleabas, 2001). These observations

suggest that maternal tissues initiate neo-

vascularization which may be required for

immune cell differentiation and infiltration

(See below).

Immune cell infiltration: The leukocyte

population in the endometrium consists of T

cells, macrophages and large granular

lymphocytes. T cells and macrophages

account for a substantial proportion of the

leukocyte population in human endometrium

throughout the menstrual cycle (Jones et al.,

1998; King, 2000). The largest leukocyte

population in the human endometrium are the

large granulated lymphocytes which express

natural killer (NK) cell antigen CD56. The

uterine NK (uNK) cell population is distinct

from peripheral blood NK cells in phenotypic

and molecular characteristics (Cooper et al.,

2001; Fukui et al., 2011; King et al., 1991;

Lysakova-Devine and O'Farrelly, 2014).

Around the time of implantation, uNK cells

comprise 70–80% of the leukocyte population

in the endometrium and numbers increase if

conception occurs (King, 2000; Kodama et al.,


Biomed Res J 2015;2(1):83-104

1998). It remains to be identified whether the

increase in cell number is solely the result of in

situ proliferation or homing from the

peripheral circulation. In a conception cycle,

the uNK cells differentiate into decidual NK

(dNK) cells, functionally distinct from non-

pregnant uterine counterparts (Kodama et al.,

1998). The functional significance of uNK and

dNK cells in the primate endometrium is

largely speculative. Based on mouse studies

and clinical observations, it appears that NK

cells are crucial for pregnancy and failure of

uNK transformation to dNK cells leads to

pregnancy loss (Fukui et al., 2011; Gong et al.,

2014; Quenby and Farquharson, 2006).

Whether this transformation occurs

exclusively in response to embryo derived

signals or due to decidualization of the stromal

cells, needs to be investigated.

Embryo induced molecular trans-

formations in the receptive endometrium

The molecular dialogue between the embryo

and endometrium involves a complex network

of signaling molecules that mediate cell–cell

or cell–extracellular matrix (ECM)

interactions, and include factors such as

cytokines, growth factors, cell-adhesion

molecules and matrix metalloproteinases.

There is some evidence indicating that the

levels of steroid receptors, growth factors and

cytokines are modulated in the endometrium

during early pregnancy. The following section

reviews the in situ molecular changes

occurring in the primate endometrium in

response to embryonic signals.

Estrogen receptors (ER) and Progesterone

receptors (PR)

Sex steroids exert their effects through their

receptors, estrogen receptor (ER) and

progesterone receptor (PR). As compared to

non-conception cycle, both ER and PR

expression is higher in the conception cycle

around implantation in bonnets, baboons and

rhesus (Ghosh and Sengupta, 1988, Rosario et

al., 2008). Post apposition ER expression is

lost in the epithelium and stroma but retained

in the wall of spiral arteries, blood vessels, and

myometrial smooth muscle cells (Hild-Petito

et al., 1992; Perrot-Applanat et al., 1994).

While PR is most abundantly expressed in

the uterine glands and stroma in the receptive

phase, expression of PR is down-regulated in

the glands but present in the stroma

surrounding the glands and spiral arteries, wall

of spiral arteries, blood vessels, and smooth

muscle cells of the myometrium (Ghosh and

Sengupta, 1988; Hild-Petito et al., 1992;

Perrot-Applanat et al., 1994).

Homeobox genes HOXA10 and HOXA11

HOX genes are essential for endometrial

growth, differentiation and receptivity by

mediating some functions of progesterone.

Both HOXA10 and HOXA11 are expressed in

human endometrial epithelial and stromal

cells, and their expression is significantly

Biomed Res J 2015;2(1):83-104


higher in mid- and late-secretory phases,

coinciding with time of embryo implantation

and high levels of estrogen and progesterone

(Daftary and Taylor, 2006; Godbole et al.,

2007; Modi and Godbole, 2009; Taylor et al.,

1998; Xu et al., 2014).

Unlike steroid receptors, the expression of

HOXA10 is induced in the endometria of

bonnet monkeys in the conception cycle.

Abundant expression of HOXA10 protein is

detectable in stromal and glandular cells of the

pregnant bonnet monkeys (Godbole et al.,

2007). Interestingly, treatment of endometrial

cells with spent blastocyst culture medium

and/or hCG resulted in increased transcription

of HOXA10 (Blitek et al., 2011; Fogle et al.,

2010; Sakkas et al., 2003). However, unlike

the glands and the stroma, in luminal

epithelium of the conception cycle, HOXA10

expression is reduced and expression is

virtually absent in the pre-epithelial plaques

(Godbole et al., 2007; Modi and Godbole,

2009). These observations are surprising, as in

the mouse, suppression of HOXA10 in

epithelial cells leads to inhibition of embryo

implantation; overexpression leads to increase

in litter size in mouse (Bagot et al., 2000).

While this might reflect the fundamental

differences in the mechanisms associated with

implantation in rodents and primates,

observations in the monkey indicates that

products of HOXA10-modulated transcript-

ome in luminal epithelium may be inhibitory

for implantation, and hence may be down

regulated by embryonic stimuli. As

transcription factors, HOX genes regulate

other downstream target genes leading to

proper development of endometrium and

receptivity to implantation. A number of

molecular and morphological markers specific

to the implantation window are regulated by

HOX genes, including pinopodes, β3 integrin

and insulin-like growth factor-binding

protein-1 (Daftary and Taylor, 2006; Modi and

Godbole, 2009). All the HOX targets are also

modulated in the endometria in response to the

embryo (Nimbkar-Joshi et al., 2009).

Cytokines and growth factors

Leukemia inhibitory factor (LIF), Interleukin-

6 and -11 (IL-6 and IL-11) are members of a

single family of cytokines that share the signal

transducer receptor unit gp130 in target cells to

elicit biologic effects. All these three cytokines

play key roles in implantation. First identified

in the mouse where targeted disruption of the

LIF gene showed implantation failure

(Stewart et al., 1992), reduced LIF expression/

secretion has been reported in infertile women

with defects in implantation (Mikolajczyk et

al., 2006; Tawfeek et al., 2012). While LIF

seems to be a critical requirement for

implantation, the expression is not modulated

by embryonic signals as the levels do not alter

in the implantation phase endometria of

bonnet monkeys in conception as compared to

non-conception cycle (Rosario et al., 2005b).

However, in rhesus the expression of LIF and


Biomed Res J 2015;2(1):83-104

its receptors are increased in the endometria of

pregnant monkeys as compared to non-

pregnant controls (Sengupta et al., 2003). LIF

is crucial for implantation in primates as

administration of an antagonist for LIF

receptor or antibody against LIF directly in to

the uterine cavity of monkeys and mice, results

in failure of pregnancy (Sengupta et al., 2006;

Terakawa et al., 2011; White et al., 2007). The

results indicate that LIF is essential in the

process of blastocyst implantation. LIF is also

a promotor of trophoblast invasion (Suman et

al., 2013a; 2013b).

IL-6 and IL-11 are pleiotropic cytokines

required for implantation. IL-11 expression is

increased during decidualization (Godbole

and Modi, 2010), recombinant IL-6 and IL-11

promote decidualization of human

endometrial cells in vitro (Dimitriadis et al.,

2005; Menkhorst et al., 2010). IL-6 and IL-11

are also promoters of trophoblast invasion

(Champion et al., 2012; Modi et al., 2011;

Suman et al., 2009; 2013). IL-11 and the

receptor IL-11Rα are detected in the decidua

mainly at implantation sites in cynomolgus

and rhesus monkeys (Champion et al., 2012;

Dimitriadis et al., 2005). It is also detected in

the vascular endothelial cells and epithelial

plaques. Likewise, the expression of IL-6 is

significantly higher in endometria of animals

in the conception cycles as compared to non-

conception of rhesus and bonnet monkeys

(Rosario et al., 2005b; Sengupta et al., 2003).

Several growth factors like Tumor Growth

Factor (TGF) beta, Epidermal Growth Factor

(EGF) and Tumor Necrosis Factor (TNF)

alpha are pro-inflammatory cytokines that

have emerged to be critical mediators for

implantation owing to their direct effects on

immune cells (Dimitriadis et al., 2005;

Omwandho et al., 2010). In the window of

implantation, a significant increase in

endometrial TGF beta and its receptor occurs

in the glandular epithelium of animals in the

conception cycles as compared to non-

conception cycles (Rosario et al., 2005b;

Sachdeva et al., 2001). TNF alpha and its

receptor population increases in the

endometria of animals in the conception

cycles as compared to non-conception cycles

(Nimbkar-Joshi et al., 2009; Rosario et al.,

2005c). EGF and its receptors are detected in

both the glands and stromal compartments of

the receptive phase endometrium; expression

is increased mainly in the stromal cells of the

pregnant animals (Slowey et al., 1994). An

increase in endometrial LIF, EGF, TGF and

TNF by endometrial cells in presence of

embryonic stimuli prior to apposition suggests

induction of an inflammatory like condition in

the implantation phase endometrium, which

may be a requirement for initiation of

pregnancy; the increase in expression in

stromal compartment implies involvement

with decidualization.

Biomed Res J 2015;2(1):83-104


Integrin and their ligands

Integrins are heterodimeric glycoproteins

which undergo dynamic temporal and spatial

changes in their distribution in the

endometrium during the menstrual cycle in

women (Lessey and Arnold, 1998; Reddy and

Mangale, 2003). Likewise the ECM ligands

for these receptors are likely to play a role in

the establishment of a receptive endometrium.

The integrins and their cognate ligands show

dynamic changes in levels of expression and

polarization during early pregnancy. In the

baboon, the collagen receptor alpha1beta1 and

fibronectin receptor alpha4beta1 expressed in

glandular epithelium during window of

receptivity are lost with the establishment of

pregnancy. The vitronectin receptor,

alpha4beta3 is expressed in the glandular

epithelium in pregnant animals. The

osteopontin receptor, alphavbeta3, is

expressed in both glandular epithelium, and

decidualizing stromal cells of pregnant

animals (Fazleabas et al., 1997; Mangale and

Reddy, 2007). In the mouse decidua,

interactions between integrin alphav beta3 and

vitronectin is required to maintain a balance

between cell proliferation and apoptosis, along

with modulation of inflammatory responses

(Mangale et al., 2008).

Recently the dynamics of integrin

expression mainly alphavbeta3 in the uterine

epithelium has been detailed in early

pregnancy using the bonnet model. The results

revealed that expression of alpha v increases in

luminal epithelial cells of pregnant animals,

show a shift in localization at the site of

attachment (Nimbkar-Joshi et al., 2012). At

the non-attachment pole, the alpha(v) integrin

is mainly in the basal zone of the luminal

epithelial cells. However, at the attachment

pole, alphav is redistributed and also detected

in the apical pole. The differential subcellular

distribution of integrin is directed by

embryonic stimuli as treatment of epithelial

cultures with conditioned medium of human

embryos obtained at IVF leads to increased

distribution of alphav on the apical membrane

(Nimbkar-Joshi et al., 2012). These

observations imply that embryonic stimuli not

only directs cellular reprogramming by

changing gene expression, but also controls

intracellular protein trafficking leading to

preferential sorting of proteins.

From the above studies it is clear that

embryo induces distinct changes in the

receptive stage endometrium and affects

almost all the compartments in preparation of

pregnancy. These changes seem to be induced

in response to secretions by the embryonic

cells and are highly localized in nature. A

summary of the morphological and molecular

changes that occur in the receptive

endometrium in presence of an embryo are

shown in Fig 1.


Biomed Res J 2015;2(1):83-104

Functional Consequence of the

Endometrial-Embryo Cross-Talk

From the discussion above it is clear that

remarkable changes occur in the molecular

profile of endometrium at the time of

apposition and implantation, distinct from

those during the window of receptivity. The

changes seem to be induced in response to

secretions by the embryonic cells and are

localized in nature. However, the functional

connotations of such observations remain far

from clear. This is mainly due to our inability

to perform genetic manipulations in the

endometria of primates. Nevertheless, in

recent years, elegant models have been in vitro

designed to decipher functional consequences

of embryo induced changes in endometrial

cells ( . Weimar et al., 2012) While it would be

beyond the scope of this article to review these

studies, the data derived from these studies,

combined with changes seen in vivo, it appears

that the embryo signals the endometrial bed

prior to implantation making it competent for

embryo quality control and trophoblast

invasion.

Decidua as a “selector” for embryo quality

control

The exceptional rate of early pregnancy loss

may be due to the high prevalence of

Biomed Res J 2015;2(1):83-104

Figure 1: Morphological and molecular changes in the endometrium in response to embryonic signals. The

receptive endometrium senses the endometrium and undergoes extensive biochemical and morphological remodeling.

The molecular changes that occur in the stromal and epithelial compartments are highlighted.


chromosomal abnormalities in the embryo.

Genetic analysis of blastomeres taken from

good quality embryos obtained at in vitro

fertilization (IVF) showed that around 70%

harbor complex chromosomal abnormalities

(Chow et al., 2014; Mertzanidou et al., 2013).

Such observations raise the question of how to

safeguard the mother against prolonged

investment in potentially developmentally

abnormal embryos. One school of thought

believes that abnormal embryos by themselves

are incompetent towards implantation

resulting in pregnancy failure. In recent years

however, experimental evidence indicate that

spontaneous decidualization of endometrium

coupled to menstruation is a judicious strategy

to meet the challenge. The decidua may play a

key role in discriminating normal and

abnormal blastocysts to allow pregnancy.

Evidences to support this hypothesis were

obtained from co-culture of decidual cells with

morphologically normal and abnormal human

embryos obtained at IVF. While morpho-

logically normal embryos had no major effects

on production of a selected set of cytokines;

media derived from decidual cells co-cultured

with morphologically arrested or abnormal

blastocysts led to down-regulation of IL-1b, -

6, -10, -17, -18, eotaxin and heparin-binding

EGF-like growth factor (Teklenburg et al.,

2010a). Such down-regulation is associated

with closure of endometrial competence for

implantation and menstruation (Evans and

Salamonsen, 2014). Microarray analysis of

decidual cells challenged with conditioned

medium from good and poor quality embryos,

identified 449 decidual genes deregulated in

response to medium conditioned by poor-

quality embryos (Brosens et al., 2014). One of

the down regulated genes in response to

signals in conditioned media derived from

poor quality embryos was HSPA8. The protein

functions in protein assembly and folding,

clatherin-mediated endocytosis, assembly of

multiprotein complexes, transport of nascent

polypeptides, and regulation of protein folding

(Stritcher et al., 2013). The observation

suggests that soluble signal from

developmentally impaired human embryos

induce endoplasmic reticulum (ER) stress

response in decidualizing cells. An in vivo

proof for the in vitro observations came from

studies in uteri of mice flushed with

conditioned culture medium of development-

ally competent and incompetent embryos.

Analysis of the uterine transcriptome revealed

that medium derived from competent embryos

evoked a supportive intrauterine environment,

whereas medium derived from poor quality

embryos led to ER stress (Brosens et al.,

2014). Thus, it implies that the endometrium

not only senses signals derived from the

embryo and responds to create a pro-

implantation condition, but is also capable of

terminating the window of endometrial

receptivity to enable the mother to dispose of

compromised embryos. The observation adds

another dimension to the potential of


Biomed Res J 2015;2(1):83-104

decidualizing endometrial stromal cells as

sensors of embryo quality during

implantation.

Thus, we propose a dual-phase response of

the endometrium. The steroid primed

receptive phase endometrium responds to the

incoming embryo creating an obligatory

environment for implantation. At the same

time the decidua gains a 'selector' activity to

recognize developmental competence of the

implanting embryo. Based on the blastocyst

competence as judged by the decidua, either

pregnancy is continued or the maternal

response is aborted and culminates in

menstruation.

Regulation of trophoblast invasion

Once the endometrium encounters a

developmentally competent blastocyst and

decides to continue with pregnancy, the

embryo apposes and trophoblast cells begin to

breach the luminal epithelium and invade in to

the maternal decidua to establish placentation.

Trophoblast cells are inherently invasive and

can invade any tissue. However, in the

pregnant endometrium the invasion is highly

controlled. It is believed that the decidualized

stromal cells secrete a complex array of

molecules that permit the controlled

migration/invasion. While several of the

molecules are already expressed by the

receptive endometrium, others are induced

post decidualization and receiving of the

embryonic signals. Co-culture of trophoblast

and decidual cells or spent medium increases

trophoblast invasion (Godbole et al., 2011;

Menkhorst et al., 2012). We have

demonstrated that decidual cell secretome

enhances invasion of trophoblast cells through

altered expression of matrix metalloproteases

(MMPs) and tissue inhibitors of matrix

metalloproteases (TIMPs) (Godbole et al.,

2011). Conversely, in response to

trophoblasts, the decidual cells also gain a

migratory and invasive phenotype (Gellersen

et al., 2010; Weimar et al., 2013). Thus,

decidualization and embryo driven changes in

the uterine cells creates a microenvironment

favorable for implantation and placentation.

Numerous growth factors that regulate the

proliferation and invasion of trophoblast cells

have been identified at the fetal-maternal

interface. The various factors secreted by the

decidual cells and/or the associated cell types

and their influence on trophoblast invasion has

been recently reviewed (Knöfler, 2010; Modi

et al., 2012). Amongst the various factors, IL-

6, LIF and IL-11 are abundantly produced by

the endometrial stromal and decidual cells,

and play a key role in trophoblast invasion

(Fitzgerald et al., 2008; Modi et al., 2012;

Suman et al., 2013a; Suman and Gupta, 2014).

IL-6 and LIF stimulates invasion of primary

trophoblast and JEG-3 choriocarcinoma cells

via the STAT3 signaling pathway (Jovanović

and Vićovac, 2009; Suman and Gupta, 2014).

The role of IL-11 in trophoblast invasion is

less clear as it inhibits the invasion of primary

Biomed Res J 2015;2(1):83-104


trophoblast and HTR-8/SVneo cells, but

increases invasion of the choriocarcinoma

JEG-3 cells (Suman et al., 2009; 2012; 2013b).

The discrepancy may originate from

differences in the transcription factor content

of the two cell lines. However, the data

suggests that locally produced IL-6, LIF and

IL-11 act to finely tune invasion. While the

cumulative effects of various factors and their

roles under in vivo conditions need

investigations, the observations together

suggest that decidualization driven

transformation of endometrial stromal cells

creates a uterine microenvironment that

controls trophoblast invasion.

Clinical Repercussions of the Embryo-

Endometrial Cross-Talk

Endometrial receptivity is a major rate limiting

step and bottleneck for the success of assisted

reproductive technologies. The discovery that

embryonic signals potentiate the already

primed uterus has opened several avenues for

understanding of the process of implantation

and initiation of pregnancy. Given the

experimental evidence demonstrating the

embryo-endometrial cross talk plays a key role

in endowing receptivity as well as selectivity

to the endometrium, a logical consequence of a

reduced ability to recognize embryonic signals

is implantation failure and/or miscarriage.

Suboptimal response to signals of high quality

embryos will result in a suboptimal

environment for subsequent development and

placenta formation, a major cause of

pregnancy related complications like fetal

growth restriction and gestational

hypertension leading to preeclampsia.

In converse, impaired endometrial

selectivity can result in superfertility. The

hypothesis stems from the observations that

women with recurrent miscarriages are highly

fecund and time to pregnancy is reduced in

those women with a history of five or more

miscarriages (Teklenburg et al., 2010b). Since

developmentally incompetent blastocyst

implant (due to failure of selectivity), these

would lead to late first trimester abortions.

Thus lack the “selector” activity in the

endometrium may be a causative factor

towards compromised pregnancy. Indeed, a

loss of selection sensing has been observed in

endometrial stromal cells derived from

women experiencing recurrent miscarriages

(Salker et al., 2010). Furthermore, when

flushed through the mouse uterus, secreted

factors from decidualizing cultures of stromal

cells derived from patients with recurrent

miscarriages prolonged the window of

receptivity and also increased the incidence of

pathological implantation sites, immune

defects and fetal demise (Brosens et al., 2014).

Additionally, endometrial stromal cells from

patients with recurrent miscarriages show

altered responses to hCG, and failure to

discriminate between high and low quality

human embryos (Salker et al., 2010; Weimar

et al., 2012). Thus, the “selector” activity of


Biomed Res J 2015;2(1):83-104

the decidua may be a key to successful

pregnancy and defects in the process may

cause recurrent miscarriages.

Once the embryo has implanted, the

trophoblast cells invade to establish

placentation. Multiple stages of placentation

could be compromised that can lead to

diseases. Pre-eclampsia affecting 3–5% of

pregnancies, which is characterized by

gestational hypertension and severe

proteinuria, and is a major cause of fetal and

maternal deaths. While pre-eclampsia is

detected later in gestation (20 weeks onwards),

its pathogenesis is established early in

gestation where trophoblast invasion is

defective. It has been shown that in women

with preeclampsia shallow placental invasion

and inadequate plugging of the spiral artery

affects blood supply into the intervillous space

and alters the consistency of the blood flow.

This can lead to fluctuations in the supply of

oxygen to the placenta (Ji et al., 2013; Saito

and Nakashima, 2014), triggering a maternal

response by increasing blood pressure and

compromising fetal development (Furuya et

al., 2008). Another disorder caused by defects

in trophoblast invasion is intrauterine growth

restriction (IUGR). IUGR arises as a result of

inadequate blood supply and/or inadequate

transport of nutrients across the placenta to the

fetus, resulting in a range of mechanisms

including reduced uteroplacental blood flow,

compromised feto-placental angiogenesis and

subsequent villous development (Gourvas et

al., 2012). It is believed that 'poor placentation'

along with hypoxic micromilieu of

fetoplacental site, shear stress of

uteroplacental blood flow, and aberrantly

secreted proinflammatory substances into

maternal circulation, synergistically

contribute to progression of preeclampsia and

IUGR (Furuya et al., 2008; Gourvas et al.,

2012; Ji et al., 2013; Saito and Nakashima,

2014). Since trophoblast invasion and

placentation are dependent on proper

decidualization, defective embryo-

endometrial cross talk can lead to improper

decidual response thereby causing poor

trophoblast invasion, shallow placentation and

hence preeclampsia. Preliminary evidence

suggests that decidualization defects might

exist in decidua of women with pregnancies

complicated with preeclampsia (Saito and

Nakashima, 2014).

Thus, there is a need for better

understanding of the basic processes of

placentation and mechanisms that go awry in

women with preeclampsia and IUGR for

effective therapeutic approaches to these

common disorders.

CONCLUSIONS

The embryo-endometrial cross talk has

evolved as a delicately poised mechanism to

respond initially to the hormonal trigger to

achieve receptivity and then to amplify the

decision under embryonic signals to permit

pregnancy. During this critical period, the

Biomed Res J 2015;2(1):83-104


receptive endometrium gains the ability for

selectivity, where response of the luminal

epithelium serves to transduce and amplify

signals coming from competent embryos

renders the underlying decidual layer more

receptive to invasion (Fig. 1). This permits

embryo apposition followed by trophoblast

invasion to establish placentation (Fig. 2). In

the event the endometrium experiences

presence of a poor-quality embryo, the

supportive network is not activated, but the

decidua mount a stress response, leading to

withdrawal of receptivity resulting in

menstruation and failure of pregnancy (Fig. 2).

Such a selector mechanism of the decidua is

highly desirous to avoid investing energy in

pregnancy with abnormal fetuses which may

not survive till term or have compromised

survival ability. Once the receptive and the

selective competence of the endometrium are

ensured and the right blastocyst implants, the

decidua creates a local microenvironment that

is conducive for trophoblast invasion and

placentation (Fig. 2).

The significance of such bimodal and

biphasic endometrial response to the

implanting embryo is potentially far reaching.

To date, treatment of recurrent miscarriages

and implantation failure are inefficacious and

highly empirical. The recent understanding of

the dual processes has revealed that recurrent

implantation failure may be caused by defects

in endometrial embryo cross talk. It will be

necessary to unravel the molecular processes

that control the timely transition of the

receptive uterus to a selective decidua which

Figure 2: Biosensor activity of the implantation stage endometruim. A) In presence of a normal good quality embryo

the endometrium undergoes extensive biochemical and molecular transformation allowing the apposition of the embryo

to the luminal epithelium. Consequently, the secretory factors from the decidua promote invasion of trophoblast cells

allowing plancetation. B) In presence of a poor quality embryo the endometrium responds by activating a strong

inflammatory cascade thereby triggering closure of receptivity leading to menstruation.


Biomed Res J 2015;2(1):83-104

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INTRODUCTION

The development and application of

molecularly targeted radiosensitizers is crucial

for improving the efficacy of radiotherapy as a

cancer treatment modality. Two main

approaches exist regarding the incorporation

of molecular inhibitors: incorporating

multiple drugs known to target multiple

distinct pathways, such as, in the treatment of

hematological malignancies; or using a single

agent to target a specific pathway, such as

erlotinib, gefetinib or lapatinib which

selectively inhibit receptor tyrosine kinases

(RTKs). The latter has historically been the

more popular approach in solid malignancies

for the vast majority of newly investigated

cancer treatment regimens, particularly when

Radiotherapy remains the standard treatment for glioblastoma multiforme (GBM) following surgical

resection. Given the aberrant expression of human epidermal growth factor receptor 2 (HER2) and

epidermal growth factor receptor (EGFR) which may play a role in therapeutic resistance to receptor

tyrosine kinase inhibitors, and the emerging use of histone deacetylase (HDAC) inhibitors as

radiosensitizers, we defined the effects of CUDC-101, a triple inhibitor of HER2, EGFR and HDAC on the

radiosensitivity of GBM cells. Clonogenic survival was used to determine the in vitro radiosensitizing

potential of CUDC-101 on GBM, breast cancer, and normal fibroblast cell lines. Inhibitory activity was

defined using immunoblots and DNA double strand breaks were evaluated using γH2AX foci. Effects of

CUDC-101 on cell cycle and radiation-induced cell kill were determined using flow cytometry and

fluorescent microscopy. CUDC-101 inhibited HER2, EGFR and HDAC and enhanced in vitro

radiosensitivity of both GBM and breast cancer cell lines, with no effect on normal fibroblasts. Retention of

γH2AX foci was increased by CUDC-101 alone and in combination with irradiation for 24 h. Treatment with

CUDC-101 increased the number of cells in G2 and M phase, with only increase in M phase statistically

significant. An increase in mitotic catastrophe was seen in a time-dependent fashion with combination

treatment. The results indicate the tumor specific CUDC-101 enhanced radiosensitization in GBM, and

suggest that the effect involves inhibition of DNA repair.

Key words: Glioblastoma multiforme, Radiosensitization, HDAC, Multi-target therapy, HER2, EGFR.*Corresponding Author: Anita Tandle, Radiation Oncology Branch, National Cancer Institute, 10 Center Drive Magnuson Clinical Center Room B3-B100, Bethesda, MD 20892, USA. Email: [email protected]

Human EGFR-2, EGFR and HDAC Triple-Inhibitor

CUDC-101 Enhances Radiosensitivity of GBM Cells1 1,2,3 1 1 1Cody D. Schlaff , W. Tristram Arscott , Ira Gordon , Kevin A. Camphausen and Anita Tandle *

1Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA2University of Vermont College of Medicine, Burlington, VT, USA3Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program, Bethesda, MD, USA

Biomed Res J 2015;2(1):105-119

Research Article

combined with radiotherapy. Single-target

agents, despite their pharmacokinetic

simplicity, lower cost and reduced risk of

adverse effects, are often clinically limited due

to the genetic heterogeneity and myriad

dysregulated pathways that exist not only

among different malignancies, but from cell to

cell variations within the same histology (Lai

et al., 2010).

Recently, single small molecular inhibitors

have been designed to simultaneously target

multiple critical cellular pathways to induce

cell death. The drug, CUDC-101, was

designed to target two members of the ERBB

family, human epidermal growth factor

receptor 2 (HER2) and epidermal growth

factor receptor (EGFR), as well as function as

a pan-histone deacetylase inhibitor (HDACi)

(Lai et al., 2010). The overexpression of these

two RTKs has been associated with

tumorigenesis and aggressiveness in many

cancers, including glioblastoma multiforme

(GBM) (Cancer Genome Atlas Research,

2008; Kesavabhotla et al., 2012; Lal et al.,

2002; Mittapalli et al., 2013; Pierga et al.,

2013; Wang et al., 2013). Additionally, histone

acetyltransferase inactivation has been

associated with oncogenesis, yet it is the

aberrant HDAC activity that is considered a

potential target for cancer therapy. Depending

on the experimental system, HDACi has been

reported to induce tumor cell differentiation,

apoptosis, and/or growth arrest, putatively via

modulation of gene expression (Shabason et

al., 2011).

CUDC-101 has been shown to be an

effective agent as a monotherapy for treatment

of various tumor cell lines in vitro including

non-small cell lung cancer, pancreatic, breast,

prostate, brain, and liver cancers (Lai et al.,

2010), and is currently undergoing phase I and

Ib clinical trials as a monotherapy or in

combination with radiotherapy for multiple

cancers. As an initial step in evaluating the

potential of CUDC-101 as a clinically

applicable radiosensitizer, we investigated the

effects of CUDC-101 in a panel of cancer and

normal cell lines. The data indicated that

CUDC-101 selectively enhances tumor cell

radiosensitivity in vitro. Moreover, the

mechanism appears to involve inhibition of

DNA double strand break (DSB) repair and

modulation of the cell-cycle.

MATERIALS AND METHODS

Cell lines and treatment

The human GBM cell line U251, breast

carcinoma cell line MDA-MB-231 and

normal human lung fibroblast cell line MRC9

were obtained from American Type Culture

Collection (ATCC). U251 and MDA-MB-231

cells were grown and maintained in

Dulbecco's Modified Eagle Medium (DMEM;

Invitrogen) supplemented with 10% Fetal

Bovine Serum (FBS; Invitrogen) and

maintained at 37 ºC, 5% CO . MRC9 cells 2

were grown and maintained in minimum

essential medium (MEM; Invitrogen)

supplemented with 10% FBS, non-essential

Biomed Res J 2015;2(1):105-119

106 CUDC-101 induced tumor cell radiosensitization

amino acids (NEAA), and sodium pyruvate

(Invitrogen). Lyophilized CUDC-101 (MW:

434.49) was purchased from Selleck

chemicals and 3.43 mg of CUDC-101 was

reconstituted in 1 mL of DMSO (solubility: 20

mg/mL) and stored at -20 ºC at a concentration

of 10 mM. DMSO was not shown to have an

effect and subsequent experiments were done

with a media only control unless otherwise

noted. Cultures were irradiated using the

Pantak source at a dose of 2.27 Gy/min.

Clonogenic Assay

Cultures were dissociated with 0.25%

trypsin EDTA (Invitrogen) to create a single

cell suspension and a f cells specified number o

were seeded into each well of a six well tissue

culture plate. After allowing cells time to

adhere (24 h) cultures were treated with

varying doses of radiation: 0, 2, 4, 6, and 8 Gy,

followed by CUDC-101 (0.5 µM for U251 and

1.0 µM for MDA-MB-231). The drug was not

removed for duration of the assay. Ten to

fourteen days after seeding, colonies were

stained with crystal violet, and the number of

colonies (≥ 50 cells) were determined.

Surviving fractions were calculated and

survival curves generated by normalizing for

the amount of CUDC-101-induced cell death.

The data represents the mean ± standard error

of mean (SEM) of minimum two independent

experiments.

Cell cycle analysis

The evaluation of the cell cycle phase

distribution was performed using the BD

FACSCalibur. Treatment protocols were

identical to the clonogenic treatment regimen

and cells were seeded into 10 cm petri dishes.

Samples were stained with propidium iodide

(PI) and analyzed using flow cytometry. To

determine the activation of the G cell cycle 2

checkpoint, mitotic cells were distinguished

from G cells as previously reported by Xu et 2

al. (2002) utilizing the mouse monoclonal

antibody (Cell Signaling) against phospho-H3

histone (S10) (6G3) followed by staining with ®a Alexa Fluor-488 F(ab`) fragment of goat 2

anti-mouse conjugated secondary antibody

(Invitrogen) (Xu and Kastan, 2004; Xu et al.,

2002). In this assay the increase of M phase

reflects the abrogation of the G checkpoint. 2

The data represents the mean ± SEM of

minimum of three independent experiments.

Apoptotic Cell Death

The BD Annexin V: FITC Apoptosis

Detection Kit (Catalog Number: 556547) was

performed as per the manufacturer's

instructions. In brief, cells were washed twice

with cold PBS and resuspended in 1X binding

buffer. Subsequently, 100 µL of cell

suspension was incubated for 15 min. at room

temperature (25 ºC) in the dark after adding 5

µL of FITC-Annexin V and PI. Samples were

brought to a final volume of 500 µL and run on

the FACSCalibur capturing 10,000 events.

Biomed Res J 2015;2(1):105-119

Schlaff et al. 107

Immunofluorescent staining for γH2AX

Immunofluorescent staining and counting of

γH2AX nuclear foci was performed as

previously described (Xu and Kastan, 2004;

Xu et al., 2002). Cells were seeded on four

well chamber slides and treated with 2 Gy

irradiation followed by treatment with CUDC-

101. Slides were examined on a Leica upright

fluorescent microscope. Images were

imported into ImageJ image software for

analysis. For each treatment condition γH2AX

foci were counted in 50 cells. The data

represents the mean ± SEM of minimum three

independent experiments.

Mitotic Catastrophe

The presence of fragmented nuclei was used to

define cells undergoing mitotic catastrophe.

Cells were seeded on cover slips and treated

with 2 Gy irradiation followed by treatment

with CUDC-101. To visualize nuclear

fragmentation cells were fixed with a 10%

neutral buffered formalin solution and

incubated with α-tubulin followed by anti-

mouse Alexa-555 and mounted with Prolong

gold antifade reagent containing DAPI.

Normal cells and cells undergoing mitotic

catastrophe were manually counted as cells

with nuclear fragmentation. For cells to be

considered positive for mitotic catastrophe,

cells with greater than 2 lobes were used as the

limiting criteria. For each condition 100 cells

were scored. The data represents the mean ±

SEM of minimum three independent

experiments.

Immunoblotting and antibodies

Cells were seeded onto 60-mm dishes, lysed in

radioimmunoprecipitation assay (RIPA)

buffer (Sigma) containing phosphatase and

protease inhibitors (Roche, Indianapolis, IN).

Protein concentrations were quantified using a

DC Protein Assay kit (Bio-Rad Laboratories,

Hercules, CA). Thirty micrograms of protein

was resolved on 4–20% Tris-Glycine gels and

transferred to nitrocellulose membranes (Bio-

Rad Laboratories) and probed with the

indicated antibodies. Primary antibodies were:

actin, CD9 (Millipore, Germany), phospho-

/total EGFR, and acetyl-/total histone H3 (Cell

Signaling Technology, Danvers, MA) and

phospho-/total HER2 (Upstate Cell Signaling

Solutions, Lake Placid, NY). Blots with

phosphorylated targets were visualized with

Super-Signal West Femtoluminol substrate

(Thermo Scientific, Rockfold IL) and ECL

Prime luminol reagent (GE Health-care,

Pittsburgh, PA) was used for total protein.

Actin was used as a loading control.

Statistical Analysis

In vitro experiments were repeated minimum

twice and Student's t-test was used for

statistical analyses. Data are presented as

mean ± SEM. A α value of p < 0.05 was

considered significant. Analyses were done in

GraphPad version 6 (Prism).

Biomed Res J 2015;2(1):105-119


RESULTS

CUDC-101 can effectively target intended

ligands

To assess the effects of CUDC-101 on the

radiosensitivity of tumor cells, a cytotoxicity

assay was performed initially to determine

appropriate dosage with minimal toxic effects.

For this study, cells were plated at clonogenic

density, allowed to attach overnight, and

treated with 4 Gy irradiation followed

immediately by treatment with CUDC-101

using a dose range including the previously

reported average IC values of U251 and 50

MDA-MB-231 cells. U251 and MDA-MB-

231 cells were kept in drug containing media

for duration of the assay (10–14 days). Cells

were then stained with crystal violet and

assessed for inhibition of colony formation.

Dosing CUDC-101 at 0.5 μM and 1.0 μM

immediately post-radiation for the duration of

the entire assay was most effective in

inhibiting colony formation while having

minimal single agent toxic effect on U251 and

MDA-MB-231 respectively (Data not shown).

These concentrations were used to further

investigate the radiosensitive effects of

CUDC-101.

To confirm that CUDC-101 inhibited the

molecule's intended RTK targets, CUDC-101

was given alone or immediately following

irradiation (4 Gy) to U251 cells and RTK

inhibition was analyzed via immunoblot.

Results confirmed that CUDC-101 targeted

the intended ligands (Fig. 1). CUDC-101

treatment reduced the activation of

phosphorylated HER2 (pHER2) dose

dependently, irrespective to the addition of

irradiation, while pEGFR levels were only

modulated at 0.5 µM and no further decrease

in ligands level with combination treatment

was observed (Fig. 1A). Interestingly

however, the levels of total EGFR decreased

dose dependently in both single agent and

combination treatment cohorts. Also, as

expected of an HDACi, treatment with

CUDC-101 increased levels of acetylated H3

in a dose dependent manner. The protein levels

are quantitated and normalized against a

loading control, actin (Fig. 1B).

CUDC-101 treatment inhibits clonogenic

survival tumor specifically

The hallmark of radiosensitivity is reduced

clonogenicity. In both the tumor cell lines,

treatment with CUDC-101 following

irradiation resulted in an increase in

radiosensitivity as assessed by colony forming

ability 10–14 days later. U251 cells treated

with 0.5 μM CUDC-101 yielded a surviving

fraction (SF) of 0.67 ± 0.12; while treatment of

MDA-MB-231 cells with 1.0 μM gave a SF of

0.65 ± 0.07. These values indicate an

approximate value for the degree of

cytotoxicity for the evaluation of CUDC-101

in combination with radiation. As shown in

Fig. 2A and B, a dose enhancement factor

(DEF) of 1.42 in U251 and 1.37 in MDA-MB-

231 was observed at a SF of 0.1.

Biomed Res J 2015;2(1):105-119

Schlaff et al. 109

Accordingly, the potential for tumor-

specific actions of CUDC-101 on

radiosensitivity was determined. The normal

fibroblast cell line MRC9 was treated with

both 0.5 μM and 1.0 μM using the same

treatment schedule and colony formation was

assessed as previously mentioned. CUDC-101

treatment reduced the MRC9 SF to 0.79 ± 0.15

and 0.87 ± 0.007 respectively; in contrast to

the tumor cell lines, CUDC-101 had little

effect on the radiosensitivity (1.13 at 0.5 μM;

1.25 at 1.0 μM) of MRC9 (Fig. 1C). Albeit

some sensitivity was induced it was not

statistically significant. Since the effect seen in

GBM was significant compared to drug alone

and the DEF was greater than that seen in the

Figure 1: CUDC-101 effectively targets the intended ligands. The U251 was radiated (0 or 4 Gy) and incubated with

0.1 or 0.5 µM of CUDC-101 for 24 h. (A) Levels of phosphorylated HER2 and EGFR, and acetylated histone H3 were

analyzed via immunoblot analysis. (B) Results show a decrease in phosphorylated HER2 and total EGFR in the presence

or absence of radiation at 0.5 µM; however no effect was seen on pHER2 and pEGFR at 0.1 µM. Furthermore, acetyl-H3

is maintained and increased in the presence of CUDC-101 with/without radiation at both concentrations tested,

supporting the effect seen by clonogenic survival.

Biomed Res J 2015;2(1):105-119


breast cancer cell line, we focused on this

histology. Further experiments to investigate

the radiosensitization effects of CUDC-101

used U251 cells.

CUDC-101 treatment increased retention

of γH2AX foci

The most lethal form of injury to cells is DNA

damage. Thus drugs inhibiting or retarding

DNA repair have the potential of effective

radiosensitizers. Therefore, the rate of DNA

repair via resolution of γH2AX foci was

measured. The γ-variant of the H2A family

was shown to be a biomarker of DNA double

stranded breaks (DSBs) and delayed

resolution of the foci indicate inhibition of

DNA repair mechanisms (Kuo and Yang,

2008). CUDC-101 was added following

irradiation (2 Gy) and at 1, 6 and 24 h and

U251 nuclei were visualized via

immunofluorescence for inhibition of γH2AX

resolution. Representative images at 24 h are

shown in Fig. 3A. At 1 h and 6 h a significant

difference in the average number of foci per

cell was not observed (Fig. 3B). However,

there was a significant increase in foci

between cells treated with drug only compared

to no treatment at 24 h in U251 (6.33 ± 0.73; p

= 0.003) was observed. Furthermore, at 24 h

there was a significant difference in the

Figure 2: CUDC-101 radiosensitivity is tumor cell specific. Cells were radiated with increasing doses of radiation

(closed circles) or post radiation incubated with various concentrations of CUDC-101 for 10 to 14 days (open circles).

Colony-forming ability was assessed and survival curves generated after normalizing for the cytotoxicity of CUDC-101.

(A) U251 cells were given 0.5 µM CUDC-101 and (B) MB231 cells were given 1.0 µM. (C) Normal fibroblast cell line

MRC9 was irradiated with increasing doses of radiation (closed circles) or 0.5 µM CUDC-101 (open circles) or 1.0 µM

CUDC-101 (closed triangles) following irradiation. Survival curves show that the radiosensitive activity of CUDC-101 is

tumor cell specific, whereas MRC9 cells were not affected. Dose enhancement factors (DEFs) were assessed at

surviving fractions (SFs) 0.1 and 0.01 where applicable. Data represents three-independent experiments (A B) and two-–

independent experiments (C) with points representing mean, and error bars SEM.

Biomed Res J 2015;2(1):105-119

Schlaff et al. 111

average number of foci per cell in those cells

treated with combination radiation and 0.5 μM

CUDC-101 (14.40 ± 0.92; p = 0.0003). As

expected at 24 h almost complete resolution of

DNA DSBs was seen in cells that were given

irradiation only, as the average number of foci

per cell returned to near baseline levels. The

significant inhibition of γH2AX resolution at

24 h suggests that CUDC-101 inhibits repair of

DNA DSBs.

CUDC-101 post irradiation redistributes

GBM cells within cell cycle

Progress through the cell cycle depends on

regulated set of checkpoints, which become

activated in the event of DNA damage. It is

known that radiation has an effect on inducing

G /M delay (Hall and Giaccia, 2006). 2

Therefore, the distribution of tumor cells

within the cell cycle was assessed by flow

cytometry (Fig. 4A–B). As shown in Fig. 4C,

at 24 h an increase of cells in G was observed, 2

although the increase was not significant (25.8

± 3.5% to 40.5 ± 7.0%; p = 0.134). Addition-

ally, at 24 h a significant increase in M phase

from 2.30 ± 0.24% to 6.70 ± 1.1% was

observed with CUDC-101 alone (p = 0.02).

The addition of radiation to CUDC-101,

however, did not augment this increase in M

phase. In fact, a slight decrease in M phase in

cells treated with combination treatment (8.37

± 1.1% to 6.86 ± 0.08%) was observed,

however this difference was not statistically

significant (p = 0.247). Based on these results,

it may be possible that treatment with CUDC-

101 may push cells into M phase by having

off-target effects in altering the activity of

critical regulators of this checkpoint (e.g. ATM

Figure 3: Treatment with CUDC-101 plus radiation impairs the DNA damage repair response. U251 cells seeded in

chamber slides were exposed to 2 Gy irradiation followed by 0.5 μM CUDC-101 and fixed at specified times for

immunoflourescent analysis of nuclear γH2AX foci retention. Foci were evaluated in ≥ 50 nuclei per treatment per

experiment. (A) Representative images obtained from media at 24 h (top left panel), 2 Gy irradiation at 24 h (top right

panel), 0.5 μM CUDC-101 treatment after 24 h exposure (bottom left panel), and 0.5 μM CUDC-101 immediately

following 2 Gy irradiation at 24 h exposure (bottom right panel). Significant retention of γH2AX foci occurs with drug alone

and combination therapy after 24 h with the combinatory effect significantly increased over drug only. (B) Data represents

three independent experiments. Columns represent the mean and error bars are the SEM. * p < 0.05 ** p < 0.001.

Biomed Res J 2015;2(1):105-119


Figure 4: CUDC-101 increases the percentage of cells in more sensitive phases of the cell cycle. U251 cells were

seeded in 10-cm petri dishes, stained with PI and pH3 to differentiate mitotic cells and analyzed by flow cytometry. (A)

Representative histogram of cell cycle distribution of U251 cells treated with drug or irradiation alone or combination

treatment from an independent experiment. (B) Representative dot plots of cell cycle distribution of U251 cells from an

independent experiment, gating and analysis was done using FloJo analysis software. (C) Treatment with 0.5 µM CUDC-

101 in the absence of irradiation significantly increased the number of cells in M-phase; however this effect was not

augmented by the addition of irradiation (2 Gy). Additionally an increase in the G2 phase was observed for both CUDC-

101 alone and in combination with irradiation but was not statistically significant. Data represents three independent

experiments. Columns represent the mean and error bars are the SEM. *p < 0.05; **p < 0.001.

Biomed Res J 2015;2(1):105-119

and Chk2) regardless of the amount of DNA

damage, and may partially account for CUDC-

101-mediated enhancement in radiation-

induced cell killing.

CUDC-101 increases mitotic catastrophe as

mode of cell death

Lai et al. (2010) reported that treatment with

CUDC-101 induced expression of

proapoptotic and antiproliferative proteins in

breast and colon cancer cells lines. To

determine whether the increase in

radiosensitivity resulting from CUDC-101

treatment was due to an enhancement of

radiation-induced apoptosis, Annexin V

staining 24 h after treatment was measured. As

expected for a solid tumor cell line, radiation

induced little apoptotic cell death; treatment

with CUDC-101 yielded essentially identical

levels of apoptosis, and the combination of

irradiation (2 Gy) and CUDC-101 had no

effect on the frequency of apoptotic cell death

events, indicating that the CUDC-101-

mediated radiosensitization of U251 glioma

cells does not involve enhanced susceptibility

to apoptosis (Data not shown).

The apparent inhibition of DSB repair,

increase of cells in M phase, and no increase in

radiation-induced apoptosis suggests that

CUDC-101 induced radiosensitization

involves an enhancement of mitotic

catastrophe. Cells with nuclear fragmentation,

Schlaff et al. 113

defined as the presence of 2 or more distinct

nuclear lobes within a single cell, were

classified as going through mitotic

catastrophe. As shown in the representative

fluorescent micrograph in Fig. 5A, cells

undergoing mitotic catastrophe could be

distinguished after treatment of irradiation (2

Gy), CUDC-101, and combination treatment.

There was a time dependent increase in the

number of cells undergoing mitotic

catastrophe after the treatment with either

radiation or CUDC-101 up to 72 h. In cells

receiving the combination treatment, a

significantly greater number of cells

undergoing mitotic catastrophe was detected

at 48 and 72 h, 61.6 ± 11.1% and 70.3 ± 6.2%

respectively. Furthermore, this increase in

mitotic catastrophe was greater than additive

as compared to irradiation and CUDC-101

alone. These data suggest that the CUDC-101

mediated radiosensitization is achieved by an

inhibition in DNA DSB repair resulting in an

increase in cells undergoing mitotic

catastrophe. Additionally, the data supports

the observations seen in cell cycle analysis

indicating that the increase in M phase seen

with drug alone, maintained when combined

with irradiation at 24 h, was indicative of an

increase in mitotic catastrophe at later time-

points.

DISCUSSION

GBM (WHO Grade IV) is the most common

malignant central nervous system tumor with

Biomed Res J 2015;2(1):105-119

Figure 5: CUDC-101 increases mitotic catastrophe. U251 cells were grown on cover slips and were irradiated (2 Gy)

and exposed to CUDC-101. At 24, 48 and 72 h after treatment cells were fixed for immunocytochemical analysis of mitotic

catastrophe. Nuclear fragmentation (defined as the presence of two or more distinct lobes within a single cell) was

evaluated in at least 150 cells per cohort. Representative fluorescent micrographs are of cells fixed at 72 h after treatment

for (A) medium, (B) 0.5 µM CUDC-101, (C) 2 Gy irradiation and (D) 0.5 µM CUDC-101 following 2 Gy irradiation. (E)

Graph shows percent mitotic catastrophe; *p < 0.05; ***p < 0.0001. Analysis was done using a two-way ANOVA with

Bonferroni multiple comparisons post-test.


an incidence of 0.4–2.8 per year per 100,000

persons, and typified by nuclear atypia,

mitosis, endothelial proliferation and necrosis

(Mineo et al., 2006). Despite aggressive

therapies, prognoses for GBM remains poor,

and average overall survival remains 12–16%

(Stupp et al., 2009). A majority of these

patients will at some point undergo

radiotherapy typically combined with a

radiosensitizing agent. Attempts to develop

clinically relevant radiosensitizers have

traditionally been limited to cytotoxic

chemotherapeutic agents (Camphausen et al.,

2005).

Recently, agents targeting HDAC have

gained in popularity, as they are shown to

enhance the radio response by relaxing the

chromatin, leaving it more susceptible to DNA

damage, among other mechanisms. Lai et al.

(2010) showed the potent inhibition of

multiple oncogenic pathways with CUDC-101

as a monotherapy, and are currently

investigating the effects of this compound in a

dose escalation study for various cancers as a

single agent (NCT01171924), and in

combination with radiation and cisplatin for

locally advanced head and neck cancer

(NCT01384799). We extended the

investigation to assess the radiosensitizing

potential of CUDC-101 in GBM.

In GBM, EGFR is genomically amplified

in 40–50% of tumors, often followed by gene

rearrangement resulting in a ligand-

independent, constitutively phosphorylated

and cell surface localized receptor tyrosine

kinases (RTK) that enhances tumorgenicity

(Heimberger et al., 2005; Lal et al., 2002;

Lopez-Gines et al., 2010; Sugawa et al., 1990;

Wikstrand et al., 1997). Furthermore, recent

preclinical and clinical studies showed that

EGFR may play a role in radioresistance

through activation of downstream signaling

cascades such as, PI3K/Akt/mTOR, and its

involvement in regulating autophagy

(Palumbo et al., 2014). Targeting HER2 is a

well established target for breast cancer

therapy and a negative prognostic factor for

cancers of breast, lung and brain (Hiesiger et

al., 1993; Tateishi et al., 1991).

In regards to GBM, however, HER2 is not

expressed in adult glial cells, but its expression

has been shown to increase with the degree of

astrocytoma degeneration (Mineo et al.,

2006). Taken with the fact that the major

action of HER2 is the heterodimerization of

HER2 with other tyrosine kinase family

members like EGFR; and this HER2

heterodimerization, produces a more potent

RTK with higher ligand affinity and tyrosine

kinase activity, and a lower internalization and

degradation rate, inhibiting HER2 may not

only decrease HER2 activity, but could also

affect the activity of its dimerization partners

(EGFR), thus helping to explain the effects

observed here with CUDC-101. Limited

evidence exists showing the role of HER2 in

Biomed Res J 2015;2(1):105-119

Schlaff et al. 115

radio response; however, a study by Duru et al.

(2012) showed that a pro-survival network

initiated by HER2 was responsible for

radioresistance in breast cancer stem cells.

Additionally, irradiation of breast cancer cell

lines has been associated with increased

expression of EGFR and HER2, which augments

the response to HER2-targeted therapy with

trasztuzumab (Wattenberg et al., 2014).

Interestingly, we also saw an increased

expression of HER2 and EGFR in U251 cells

treated with irradiation alone.

We show CUDC-101 inhibited HER2,

EGFR and HDAC in U251 and MDA-MB-

231 cell lines, which was accompanied by

enhancement in radiosensitivity, while no

significant radiation-induced cell kill was seen

in a normal fibroblast cell line. Although

0.1µM CUDC-101 effectively maintained Ac-

H3, it did not affect HER2 and EGFR

deactivation or reduce colony formation. 0.5

µM CUDC-101 maintained or increased Ac-

H3 levels, decreased levels of pHER2 and total

EGFR, and achieved a significant DEF. Thus

the question arises, if some combinatorial

permutation of these targets is essential for

inducing radiosensitization, or is it solely an

HDACi effect? Albeit an important question,

it was beyond the scope of this study, and

needs further investigation.

The apparent mechanism of CUDC-101-

induced radiosensitization appears to involve

an inhibition of DNA DSB repair. γH2AX has

been shown to correlate with amount of DNA

DSBs (Kuo and Yang, 2008). When X-rays

induce DNA damage γH2AX is recruited to

the damage site and repair proteins such as

ATM or DNA-PK phosphorylate γH2AX, and

p53BP, MRN and BRCA1 are recruited to

complete the repair complex (Mah et al.,

2010). We have previously shown that the

HDACi valproic acid induces retention in

γH2AX foci on the sites of DNA DSBs yet, the

sites of damage are being repaired as observed

by comet assay (Camphausen et al., 2005). A

similar effect may occur with CUDC-101.

Additionally, based on the increase of retained

foci at 24 h and cells in the M-phase of the cell

cycle, CUDC-101 may be inducing DNA

damage as a single agent, and therefore

causing an abrogation of the G /M checkpoint. 2

Alternatively, CUDC-101 may show indirect

effects on critical regulators of the checkpoint.

Therefore, this conflict between the DNA

repair complex remaining on the DNA damage

sites after repair, and the cells still being

pushed into M-phase, may explain the

increase in mitotic catastrophe and the CUDC-

101-induced radiosensitization.

In order for a putative radiosensitizing

agent to be effective clinically, the effects seen

in vitro must be replicated under in vivo tumor

xenograft models. Aside from defining tumor

radioresponsiveness, the ability of the agent to

target its intended ligands is critical. The

radiosensitizing ability seen in vitro by

clonogenic survival with a DEF of 1.42 was

not replicated in vivo (DEF 1.2) due to a high

Biomed Res J 2015;2(1):105-119


degree of toxicity and substantial weight loss

(data not shown). Various other agents have

shown promise in vitro, but their effects were

unable to be translated to in vivo models or

clinically, limited by the toxicity (Camphausen

et al., 2005). Much of the biological

consequences and mechanisms of action

explaining this phenomenon are unclear.

Additionally, in the case of CUDC-101, whose

targets are nuclear (HDAC) and surface

receptors (EGFR and HER2), CUDC-101 may

not effectively reach and inhibit the target(s)

responsible for enhancing radiosensitization

in sufficient number of tumor cells in vivo to

achieve a significant response. It may be

feasible to combine multiple drugs together as

single agents (i.e. lapatinib + erlotinib +

vorinostat) with radiotherapy, as reported in

various haematologic cancers.

The evaluation of the radiosensitizing

potential of CUDC-101, provides the basis for

additional preclinical exploration of the

radiosensitizing potential. Further

investigation and understanding of the

specific molecular mechanisms addressing the

necessity to target all three ligands to achieve

clinically applicable sensitization, is

warranted.

ACKNOWLEDGEMENTS

This research was supported by the Intramural

Research Program of the NIH, National

Cancer Institute, National Institutes of Health.

The authors acknowledge NIH CCR Confocal

Microscropy Core Facility, Radiation Biology

Branch and Dr. Deedee Smart for use of the

equipment.



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INTRODUCTION

Hematopoiesis takes place in bone marrow, a

complex micro-environment comprised of

various cells like osteoblast, endothelial cells,

CXCL12-abundant reticular (CAR) cells,

adipocytes, mesenchymal stromal cells

(MSCs), pericytes, etc. (Calvi et al., 2003;

Ding et al., 2012; Kiel et al., 2005; Mendez-

Ferrer et al., 2010; Omatsu et al., 2010; Park et

al., 2012; Sacchetti et al., 2007; Sugiyama et

al., 2006; Zhang et al., 2003). Hematopoietic

stem cells (HSCs) occur in close contact with

the cells of the microenvironment, which

control the fate of the HSCs via secretion of

various cytokines and extra-cellular molecules

(ECMs). Various signaling mechanisms

emanating from these cells also contribute

actively in the HSC fate decision (Blank et al.,

2008; Eckfeldt et al., 2005). Mesenchymal

stromal cells (MSCs) form a very important

part of the HSC microenvironment. Although

these cells have been used as feeder layers for

several years (Jang et al., 2006), their precise

participation in the HSC niche was

documented recently (Jing et al., 2010;

Mehrasa et al., 2014; Walenda et al., 2010).

Subsequent studies have demonstrated that

Key words: Mesenchymal stromal cells, M210B4, HSC. *Corresponding Author: Vaijayanti Kale, National Centre for Cell Science, NCCS complex, University of Pune Campus, Ganeshkhind, Pune, India.Email: [email protected], [email protected]

Phenotypic and Functional Characterization of a

Marrow-derived Stromal Cell Line, M210B4 and its

Comparison with Primary Marrow Stromal Cells

Stem Cell Laboratory, National Centre for Cell Science, NCCS Complex, University of Pune Campus, Ganeshkhind,

Pune, India

Shweta Singh, Suprita Ghode, Moirangthem Ranjita Devi, Lalita Limaye and Vaijayanti Kale*

In vitro co-culture system consisting of bone marrow stromal cells (BMSCs) or mesenchymal stromal cell

lines of marrow origin has provided important clues about the regulation of hematopoietic stem cells (HSCs)

by their microenvironment or niche. In the current studies, we have compared phenotypic and functional

characters of a marrow-derived mesenchymal stem cell line, M210B4, with BMSCs. We demonstrate that

M210B4 resembles BMSCs in terms of phenotypic characters. Unlike the BMSCs, M210B4 differentiated

only towards adipogenic lineage, and was refractory towards osteogenic differentiation. However, M210B4

cells exhibited a higher HSC-supportive ability as assessed by flow cytometry analyses of the output cells

from co-cultures. We observed that M210B4 cells show a constitutively higher activation of p44/42 and p38

MAPK pathways compared to BMSCs, contributing to their higher HSC-support in vitro. Overall, the results

show that M210B4 forms a suitable in vitro system to study HSC regulation in vitro.

Biomed Res J 2015;2(1):120-133

Research Article

MSCs actively participate in regulation of

hematopoiesis and play an important role in

homing and engraftment of transplanted HSCs

(Bensidhoum et al., 2004; Sohni et al., 2013).

In vitro co-culture of HSCs with stromal

feeder layers forms an excellent model to

study molecular mechanisms involved in the

regulation of hematopoiesis in general, and

HSC fate in particular. Murine stromal cells

were isolated and characterized by Tropel et

al. (2004) and constituted an important in vitro

tool to study stromal cell biology. To have a

constant supply of feeder cells, several stromal

cell lines were generated. Some of the clonal

stromal cell lines that have been established

are PA6 (Piacibel et al., 1996), M210B4

(Sutherland et al., 1991), S17 (Winwman et

al., 1993), and MS5 (Tordjman et al., 1999).

These cells have been successfully used as

feeder layers and possess hematopoiesis-

supportive ability in vitro.

The M210B4 cell line is a clone derived

from bone marrow stromal cells from

(C57BL/6J × C3H/HeJ) F1 mouse (Lemoine

et al., 1988), and supports hematopoiesis when

used as feeder layer for long-term culture-

initiating cell (LTC-IC) assay (Burroughs et

al., 1994). Our group has used this cell line to

study various aspects of hematopoiesis (Bajaj

et al., 2011; Hinge et al., 2010).

In spite of its extensive use in LTC-IC

assays, these cells have not been critically

evaluated in comparison with primary

marrow-derived stromal cells. It is not known

whether they express MSC-like phenotype and

support hematopoiesis with efficiency at par

with BMSCs. In the present study, we have

compared M210B4 cell line with the primary

bone marrow-derived stromal cells (BMSCs)

using phenotypic and functional parameters.

MATERIALS AND METHODS

Cells

The protocols used in animal experimentation

were approved by the institutional animal

ethics committee (IAEC). The C57BL/6J

(CD45.2) and B6.SJL-PtprcaPepcb/BoyJ

(Ptprc; CD45.1) mice (The Jackson

Laboratory, Bar Harbor, USA) were housed

and bred in our experimental animal facility

(EAF). BMSCs were isolated from bone

marrow of 6–8 weeks C57BL6/J mice by

flushing the femurs with complete medium,

constituting Iscove's modified Dulbecco's

medium (IMDM) (HiMedia, Mumbai, India)

supplemented with 20% mesenchymal stem

cell FBS (Mesen-FBS; Stem Cell Technology,

Vancouver, British Columbia, Canada) and

plated in a petridish. After 8–10 days of

incubation, with intermittent removal of non-

adherent cells and addition of fresh medium,

the adherent cells were used for the

experiments (Anjos-Afonso et al., 2008).

M210B4 cell line was purchased from ATCC

and maintained in RPMI1640 (HiMedia,

Mumbai, India) supplemented with 10% FBS

(GIBCO, Invitrogen, Carlsbad, California, –USA). Lineage negative (lin ) cells were

Biomed Res J 2015;2(1):120-133

Singh et al. 121

isolated from bone marrow mononuclear cells

(MNCs) of Ptprc mice by using biotin-labelled

anti-mouse lineage antibody cocktail prepared

from Biotin Mouse Lineage panel (BD

Pharmingen, San Diego, California, USA) and ®Dynabeads biotin binder (Invitrogen,

Calrsbad, California, USA).

Differentiation towards adipocytes and

osteoblasts

For adipocytic differentiation, BMSCs or

M210B4 cells were treated with standard

adipogenic differentiation medium

comprising insulin (4 μg/ml), 3-isobutyl, 1-

methyl xanthine (IBMX) (500 µM),

dexamethasone (0.25 μM), indomethacin (200

µM) and 2 µg/ml insulin for 15–18 days. The

adipogenic differentiation was confirmed by

staining lipid droplets with Oil Red O dye

(Bajaj et al., 2011). For osteoblastic

differentiation, BMSCs or M210B4 cells were

treated with β glycerophosphate (10 nmol/L),

dexamethasone (100 nmol/L) and ascorbic

acid (0.05 nmol/L) for 15–18 days. To confirm

osteoblastic differentiation, cells were stained

with Alizarin Red S to detect calcium deposits

(Sila-Asna et al.,2007).

Co-culture assay

BMSCs or M210B4 cells were seeded in

collagen-coated (50 µg/ml) 24-well plate as 5 –feeder layer. After 24 h, 1 × 10 lin cells

isolated from mouse bone marrow were co-

cultured with either non-irradiated or

60irradiated (8000 rads of gamma ray, Co )

feeders for 7 days in either IMDM

supplemented with 10% MSC-FBS (MSC

qualified FBS, Invitrogen, Carlsbad,

California, USA) or Myelocult medium (Stem

Cell Technology, Vancouver, British

Columbia, Canada). The medium was

supplemented with 25 ng/ml murine IL-6, 25

ng/ml murine SCF and 10 ng/ml murine IL-3

(Peprotech, Rocky Hill, USA). We compared

the HSC-supportive ability of M210B4 and

BMSCs under four different culture

conditions: A) Non-irradiated BMSCs/

M210B4 in (IMDM + 10% Mesen-FBS),

B) Irradiated BMSCs/M210B4 in (IMDM +

10% Mesen-FBS), C) Non-irradiated

BMSCs/M210B4 in Myelocult medium and

D) Irradiated BMSCs/M210B4 in Myelocult

medium. After 7 days of co-culture, the cells –were harvested and analyzed for LSK (lin

+ +Sca1 c-Kit ) stem cell population using flow

cytometry. The LSK population was further

analyzed as long-term HSCs (LT-HSCs) or

short-term HSCs (ST-HSCs) based on CD34

expression. The absolute numbers were

calculated based on percentage of cells

obtained on flow cytometer and total yield,

while the fold increase was calculated by

comparing absolute numbers of input

population to that of the output population.

Flow Cytometry

For the phenotypic characterization, BMSCs

and M210B4 cells were stained with APC-

Biomed Res J 2015;2(1):120-133

122 Characterization of a marrow-derived stromal cell line, M210B4

conjugated anti-mouse CD44, PE-conjugated

anti-mouse CD73, APC-conjugated anti-

mouse CD90.1, PE-conjugated anti-mouse

CD105, APC-conjugated anti-mouse CD106

(eBioscience, San Diego, California, USA),

PE-conjugated anti-mouse Sca-1, FITC-

conjugated anti-mouse CD45 and FITC-

conjugated anti-mouse CD34 (BD

Pharmingen, San Diego, California, USA).

For LSK profiling, cells were stained with

APC-conjugated mouse lineage antibody

cocktail, PECy7-conjugated anti-mouse

CD117(c-Kit), PE-conjugated anti-mouse

Sca-1 (BD Pharmingen, San Diego,

California, USA) and FITC-conjugated anti-

mouse CD34 (eBioscience, San Diego,

California, USA). The isotype-stained cells

were used as controls. The stained cells were

acquired on FACS Canto II (Becton

Dickinson, New Jersey, USA) and analyzed

using BD FACS-DIVA SOFTWARE version

5.0.

Western blots

Whole cell lysates were prepared using RIPA

lysis buffer supplemented with Phosphatase

Inhibitor-1(PI-1), Phosphatase Inhibitor-2 (PI-

2), Protease Inhibitor Cocktail (PIC), 1 mM

Phenyl Methyl Sulphonyl Fluoride (PMSF), 1

mM Sodium-orthovanadate and 1 mM

Sodium Fluoride (NaF) (Sigma-Aldrich, St.

Louis, Missouri, USA). The samples having

equal protein concentration were separated by

9% SDS-PAGE and transferred to PVDF

membranes (Biorad, California, USA). The

blots were incubated with specific primary

antibodies – anti-p44/42, anti-p-p44/42

(Thr202/Tyr204), anti-p38, and anti-p-p38

(Thr180/Tyr182); and horseradish peroxidase-

conjugated secondary antibodies (Cell

Signaling Technology, Danvers, Massachu-

settes, USA). The signals were detected using

Lumiglo reagent (Cell Signaling Technology,

Danvers, Massachusettes, USA) and the

signals were captured on X-Ray films.

Statistical analysis

Data were analyzed by Sigma Stat software

using one-way repeated measure analysis of

variance (One-Way RM ANOVA; Jandel

Scientific Software, California, USA). The

plots represent the values as mean ± standard

error of mean (SEM), and p value ≤ 0.05 was

considered significant.

RESULTS

M210B4 cell line is comparable to BMSCs

at phenotypic level

BMSCs are phenotypically characterized by + + +surface markers as Sca1 CD44 CD73

+ + + – –CD90.1 CD105 CD106 CD45 CD34

(Chamberlain et al., 2007; Boxall SL et al.,

2012; Mabuchi et al., 2013). When M210B4

cells and BMSCs were subjected to

phenotypic analyses using the standard

markers, we observed that both BMSCs as

well as M210B4 are highly positive for CD44,

CD106 and Sca-1; and moderately positive for

Biomed Res J 2015;2(1):120-133

Singh et al. 123

CD73, CD90.1 and CD105 (Fig. 1A). In terms

of percentages, more number of BMSCs

expressed CD73, CD90.1 and CD105. Both

cells were negative for CD34 and CD45. The

data showed that M210B4 cells compare well

with BMSCs at phenotypic level, albeit with

minor numerical differences (Table 1).

M210B4 cell line differentiates towards

adipocytic lineage

The marrow-derived Mesenchymal stromal

cells are expected to differentiate towards

osteoblastic, and adipocytic lineages under

appropriate stimuli (Prockop DJ., 1997;

Pittenger MF et al., 1999; Dominici et al.,

2006). However, when these cells are cultured

for long periods they lose their differentiation

capacity. So we investigated whether M210B4

cells possess both adipogenic and osteogenic

differentiation potential. We observed that

M210B4 cells differentiated into adipocytes

(Fig. 1B), and not towards the osteogenic

lineage. The data showed that M210B4

efficiently differentiates towards adipogenic

lineage, but not towards the osteoblastic one.

M210B4 cells expand long-term HSCs in

vitro

The results showed that in type 'A' (Non-

irradiated feeders in IMDM supplemented

with 10% MSC-FBS) and type 'B' (irradiated

feeders in IMDM supplemented with 10%

MSC-FBS) co-cultures, the total number of

cells harvested was significantly low in

M210B4 set (Fig. 2A-a, 2B-a). The absolute –numbers of LT-HSCs (LSK CD34 ) were

significantly increased, whereas the absolute + +numbers of LSK HSC (Lin-Sca-1 c-Kit ), and

Table 1: Percent and mean fluorescence intensity (MFI) of different surface markers in M210B4 and BMSCs.

S. No. Surface Markers M210B4 BMSCs

1 CD44 a) % population 99.99 73.89

b) MFI 35445 1680

2 CD73 a) % population 8.92 25.84

b) MFI 216.43 178.41

3 CD90.1 a) % populati1on 16.17 28.73

b) MFI 253.06 173.34

4 CD105 a) % population 31.48 56.75

b) MFI 230.91 595.31

5 CD106 a) % population 99.96 93

b) MFI 4872 2432

6 Sca-1 a) % population 98.99 86.51

b) MFI 24088 5751

7 CD34 a) % population Nil Nil

b) MFI Nil Nil

8 CD45 a) % population Nil Nil

b) MFI Nil Nil


Biomed Res J 2015;2(1):120-133

Figure 1: Characterization of murine bone marrow stromal cells and M210B4 cell-line. A) Flow cytometric analysis

for the MSC surface markers expressed on BMSCs versus M210B4 cell-line; B) The panel illustrates images of

adipocytes formed from M210B4 cells and BMSCs stained with Oil Red O. The panel shows that BMSCs differentiate

towards the osteoblastic lineage as evident by Alizarin Red S staining; M210B4 cells do not show such differentiation.

(Original magnification 100X).

Singh et al. 125

Biomed Res J 2015;2(1):120-133

+ST-HSC (LSK CD34 ) were significantly

decreased in the M210B4 set as compared to

the BMSCs set (Fig. 2A-b and 2B-b; Table 2).

In terms of fold increase over input, the result

was the same as that of the absolute numbers

(Fig. 2A-c and 2B-c). There was no significant

difference in M210B4 set versus BMSCs set in

type 'C' (non-irradiated feeders in Myelocult

medium) co-cultures with respect to LSK-

HSC, LT-HSC and ST-HSC populations in

Figure 2: Co-culture of murine hematopoietic stem cells with BMSCs versus M210B4 cell-line. Total numbers of

hematopoietic cells obtained in various co-cultures are depicted (a). Quantitative data showing absolute number of

HSCs (b) and fold change over input cells (c) with respect to total cells harvested, LSK, LT-HSC and ST-HSC, at 4

different conditions (2A) Non-irradiated BMSCs/M210B4 in (IMDM+10%MSC-FBS), (2B) Irradiated BMSCs/M210B4 in

(IMDM+10%MSC-FBS), (2C) Non-irradiated BMSCs/M210B4 in Myelocult medium and (2D)Irradiated BMSCs/M210B4

in Myelocult medium. The data of one representative experiment are depicted and are represented as mean ± SEM. N=3.

Biomed Res J 2015;2(1):120-133


terms of absolute numbers and fold increase

over input (Fig. 2C-b–c; Table 2), with the

exception of significantly lower total cells

harvested in M210B4 as compared to that of

BMSCs (Fig. 2C-a). Type 'D' co-cultures

(irradiated feeders in Myelocult medium) did

not show any difference in the total cells

harvested in M210B4 and BMSCs (Fig. 2D-a),

but the absolute numbers and fold increase

over input with respect to LSK and LT-HSC

were significantly higher in M210B4 (Fig. 2D-

b–c; Table 2). The absolute number and fold

increase over input of ST-HSC between

M210B4 and BMSCs was non-significant.

Flow cytometry graphs for all the 4 sets of co-

culture conditions (A–D) are depicted in

supplementary Fig. S1. The data showed that

M210B4 cells have a better HSC-supportive

potential in vitro as compared to BMSCs under

A, B and D culture conditions used in the

experiments, with respect to the primitive LT-

HSC population (Table 2). Under the culture

condition C, however, both BMSCs and

M210B4 gave comparable output of various

HSC populations (Fig. 2C).

Signaling pathways operative in M210B4

vs. BMSCs

Earlier data showed that M210B4 exhibits a

better HSC-supportive ability compared to the

BMSCs. To examine whether this was related

to the difference in their signaling gamut, we

subjected the lysates of these cells to western

blot analyses to detect phosphorylation of p38

and p44/42 MAPK. The four different culture

conditions described above were used.

We observed that under most of the culture

conditions the levels of phospho p38

Table 2: The absolute numbers (mean ± SEM, n=3) and fold increase over input from one representative

experiment have been tabulated.

Absolute numbers × 103

Culture condition

LSK LT-HSC ST-HSC

BMSCs M210B4 BMSCs M210B4 BMSCs M210B4

A 30.53±0.85 13.81±0.93 3.22±0.25 10.59±1.26 26.84±1.02 2.11±0.57

B 19.15±1.12 12.67±0.61 1.10±0.07 4.40±0.52 17.94±1.06 7.95±0.10

C 9.69±1.06 13.38±3.86 7.57±1.03 11.63±3.52 1.31±0.07 0.90±0.23

D 3.27±0.15 10.42±0.44 1.88±0.16 7.99±0.34 1.17±0.06 1.75±0.30

Fold increase over input

Culture condition

LSK LT-HSC ST-HSC

BMSCs M210B4 BMSCs M210B4 BMSCs M210B4

A 10.60±0.29 4.80±0.32 1.62±0.12 5.33±0.63 26.62±1.01 2.10±0.56

B 6.65±0.39 4.40±0.21 0.55±0.03 2.22±0.26 17.80±1.05 7.88±0.09

C 3.36±0.37 4.65±1.34 3.81±0.52 5.85±1.77 1.29±0.07 0.89±0.23

D 1.14±0.05 3.62±0.15 0.95±0.08 4.02±0.17 1.16±0.06 1.74±0.29

Biomed Res J 2015;2(1):120-133

Singh et al. 127

(Thr180/Tyr182) were higher in M210B4 as

compared to the BMSCs (Fig.3A). The levels

of phospho p44/42 (Thr202/Tyr204) were

higher in M210B4 under culture conditions A,

B and D as compared to BMSCs (Fig. 3B).

Under the culture condition C, the level of p-

p38 was high in M210B4, whereas the level of

p-p44/42 was comparable to BMSCs.

Collectively, the data suggests that

increased levels of p-p38 and p-p44/42 in

M210B4 may have conferred upon M210B4 a

better HSC-supportive ability under culture

conditions A, B and D.

DISCUSSION

In vitro co-culture system using non-irradiated

or irradiated feeder layers formulated with

primary marrow derived mesenchymal

stromal cells or cell lines, is a useful tool to

understand the stromal cell-mediated

regulation of HSC fate. The cell line models

become especially useful, when one needs to

use a genetic approach to over-express or

silence any particular gene. Primary cells can

also be genetically modified, but need viral

vectors to get sufficient numbers of modified

cells. Secondly, being primary cells, the

modified cells wither off, necessitating their

reestablishment. Cell lines give a distinct

advantage of unlimited supply of cells, and

also allow use of simple plasmid-based system

for gene manipulations. They also facilitate

generation of independent clones showing

stable expression.

Several cell lines like PA6 (Piacibel et al.,

1996), M210B4 (Sutherland et al., 1991), S17

(Winwman et al., 1993), and MS-5 (Tordjman

et al., 1991) have been established and

successfully used in co-culture studies. Our

group has mainly used M210B4 cell line in our

Figure 3: Western blot analyses of phosphorylated forms of p38 and p44/42 in BMSCs versus M210B4 cell-line.

Total cell lysate of 48 h cultured BMSCs and M210B4 at different conditions, as shown in the figure were subjected to

Western blot analyses. The blots were probed with (i) anti p-p38 and total p38 and (ii) anti p-p44/42 and total p44/42

antibodies. Equal input of proteins was ensured by probing the blots with antibody to α-tubulin. The intensity of the bands

was quantitated by densitometry using ImageJ software.

Biomed Res J 2015;2(1):120-133


previous work (Bajaj et al., 2011; Hinge et al.,

2010). Presently, our group is involved in

genetically modifying these cells using

plasmid vectors expressing various molecules

involved in regulation of hematopoiesis.

When we initiated these studies, we noticed

that though this cell line has been frequently

used in experiments (Sutherland et al., 1991),

its phenotype in comparison with BMSCs has

not been established. Secondly, its

hematopoiesis support in comparison with

BMSCs has not been examined.

Therefore we compared phenotype of

M210B4 with BMSCs. We showed that these

cells are phenotypically comparable to

primary BMSCs. On examination of the

differentiation towards osteoblastic and

adipocytic lineages, we observed that the

M210B4 cells differentiated into adipocytes

(Fig. 1B), but were refractive for osteoblastic

differentiation. BMSCs are most commonly

known to differentiate into osteoblastic,

adipocytic and chondrogenic lineages. We

have shown such tri-lineage differentiation of

placental MSCs (Sharma et al 2012).

Adipocytes and osteoblasts form important

components of the HSC niche and thus the

differentiation of BMSCs towards these two

lineages is relevant in the context of

hematopoiesis. Therefore, in the present study

we examined the ability of M210B4 to

differentiate towards adipogenic and

osteoblastic lineages. However, it will be

interesting to see whether these cells

differentiate towards chondrocytic, and also

towards other lineages like neural, muscular,

etc.

We subjected both M210B4 and primary

stromal cells in co-culture studies. Since the

feeder cells can be used in non-irradiated or

irradiated form, we employed both the

conditions and used two different kinds of

media typically used in such experiments. The

data showed that though under most of the

culture conditions the total output of

hematopoietic cells in M210B4 set was low,

except in co-culture condition 'D', the fold

increase of LT-HSCs over the input was

consistently high in M210B4 set, except under

condition 'C'. The data suggest that M210B4

cells possess better ability to support the LT-

HSCs. In culture condition 'C', the absolute

number and the fold increase over input for LT-

HSCs were higher than in the BMSCs set, but

the data did not reach significance. This could

be related to milder activation of p44/42

MAPK activation under this culture condition

(Fig. 3B). Adipocytes are considered as

negative modulators of hematopoiesis

(Naveiras et al., 2009), but M210B4 cells in

spite of being able to differentiate to

adipocytes supported HSC proliferation. This

shows that differentiated adipocytes, but not

pre-adipocytes, may exert negative effect on

the HSCs. The overall low output of

hematopoietic cells thus appears to be

primarily due to low proliferation of

committed progenitors.

Biomed Res J 2015;2(1):120-133

Singh et al. 129

The signaling mechanisms operative in

the stromal cells are known to affect HSC fate.

Our data showed that constitutively activated

p44/42 and p38 pathways in M210B4 cells

under all culture conditions except for the

levels of p-p44/42 under the culture condition

'C,' may be responsible for their better HSC

support. Stromal cells regulate the HSC fate

via secretion of various cytokines and ECM

molecules (Scadden, 2006; Baraniak et al.,

2010). Cell–cell interactions are also known to

play important role in this process. In our

future experiments we propose to examine

whether M210B4 and BMSCs show

differential ECM and adhesion molecule

profile.

Our group has shown that p44/42 and p38

pathways are coupled and inversely regulated

in primitive stem cells (Kale, 2004; Kale et al.,

2004; Kale, 2005). Although the cell

autonomous role of MAPK signaling

pathways in the regulation of hematopoiesis is

known (Geest et al., 2009), our study showed

that alteration of p44/42 and p38 pathways in

the stromal cells can affect HSC fate.

In summary, our study shows that

M210B4 cell line shows phenotypic similarity

with the primary BMSCs and has higher HSC-

supportive properties by the virtue of the

signaling gamut present in them. This cell line

thus forms a suitable model to examine the

stromal cell-mediated regulation of stem cell

fate. Similarly, this cell line is a suitable model

to study adipogenesis (Bajaj et al., 2011).

ACKNOWLEDGEMENTS

The authors acknowledge Department of

Biotechnology (DBT), Government of India,

New Delhi (Grant BT/PR14036/MED/31/

101/2010); Director, NCCS; FACS core

facility; and Council of Scientific and

Industrial Research (CSIR) for fellowship to

SS, SG and MRD).



Biomed Res J 2015;2(1):120-133

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explicitly by the responsible authorities where the work

was carried out, and that, if accepted, it will not be

published elsewhere in the same form, in English or in any

other language, without the written consent of the

Publisher.

Information for AuthorsBiomedical Research Journal

School of Science, Bhaidas Sabhagriha Building,Bhaktivedanta Swami Marg,

Vile Parle (W), Mumbai - 400056, INDIA.

Email: [email protected]

Ethics

Work on human beings that is submitted to Journal should

comply with the principles laid down in the Declaration of

Helsinki: Recommendations guiding physicians in biomedical

research involving human subjects, adopted by the 18th World

Medical Assembly, Helsinki, Finland, June 1964, amended by

the 29th World Medical Assembly, Tokyo, Japan, October, 1975,

the 35th World Medical Assembly, Venice, Italy, October 1983,

and the 41st World Medical Assembly, Hong Kong, September

1989. The manuscript should contain a statement that the work

has been approved by the appropriate ethical committees related

to the institution(s) in which it was performed and that subjects

gave informed consent to the work. Studies involving

experiments with animals must state that their care was in

accordance with institution guidelines. Patients' and volunteers'

names, initials and hospital numbers, should not be used and

patient confidentiality must be maintained.

Conflict of Interest

By means of a "Conflict of interest statement", all authors must

disclose any financial and personal relationships with other

people or organizations that could inappropriately influence

(bias) their work. If there are no conflicts of interest, please state

"No conflict of interest declaration". This document should be

uploaded as a separate file alongside the submitted manuscript.

Role of the Funding Source

All sources of funding should be declared as an

acknowledgment at the end of the text.

Authorship and Acknowledgments

All authors must be accredited on the paper and all must submit a

completed Author Form with their submission. The form must

be signed by the corresponding author on behalf of all authors

and can be scanned and uploaded.

Copyright

Upon acceptance of an article, Authors will be asked to transfer

copyright. This transfer will ensure the widest possible

dissemination of information. A letter will be sent to the

corresponding Author confirming receipt of the manuscript. A

form facilitating transfer of copyright will be provided.

If excerpts from other copyrighted works are included, the

Author(s) must obtain written permission from the copyright

owners and credit the source(s) in the article.

General Points

We accept Word format of submitted manuscript. Always keep a

backup copy of the electronic file for reference and safety. Save

your files using the default extension of the program used. It is

important that the file be saved in the native format of the word

processor used. The text should be in single-column format.

Keep the layout of the text as simple as possible. Most

formatting codes will be removed and replaced on processing

the article. In particular, do not use the word processor's options

to justify text or to hyphenate words. However, do use bold face,

italics, subscripts, superscripts etc. Do not embed "graphically

designed" equations or tables, but prepare these using the word

processor's facility. When preparing tables, if you are using a

table grid, use only one grid for each individual table and not a

grid for each row. If no grid is used, use tabs, not spaces, to align

columns. The electronic text should be prepared in a way very

similar to that of conventional manuscripts. Do not import the

figures into the text file but, instead, indicate their approximate

locations directly in the electronic text and on the manuscript.

See also the section on Preparation of electronic illustrations.

To avoid unnecessary errors you are strongly advised to

use the "spellchecker" function of your word processor.

Presentation of Manuscript

Please write your text in good English (American or British

usage is accepted, but not a mixture of these). Italicize

expressions of Latin origin, for example, , ., . in vivo et al per se

Use decimal points (not commas).

Title Page

Provide the following data on the title page:

Title

Concise and informative. Titles are often used in information-

retrieval systems. Avoid abbreviations and formulae where

possible.

Author names and affiliations

Where the family name may be ambiguous (e.g., a double

name), please indicate this clearly. Present the Authors'

affiliation addresses (where the actual work was done) below

the names. Indicate all affiliations with a lower-case superscript

letter immediately after the Author's name and in front of the

appropriate address. Provide the full postal address of each

affiliation, including the country name, and, if available, the e-

mail address of each Author.

Corresponding Author

Clearly indicate who is willing to handle correspondence at all

stages of refereeing and publication, also post-publication.

Ensure that telephone and fax numbers (with country and area

code) are provided in addition to the e-mail address and the

complete postal address.

Present/permanent address

If an Author has moved since the work described in the article

was done, or was visiting at the time, a "Present address"' (or

"Permanent address") may be indicated as a footnote to that

Author's name. The address at which the Author actually did the

work must be retained as the main, affiliation address.

Superscript Arabic numerals are used for such footnotes.

Suggestions for reviewers

Please supply the names of up to three potential reviewers for

your manuscript. Please do not suggest reviewers from your

own institution, previous or current collaborators. Please

provide full names, addresses and email addresses of suggested

reviewers. Please note: the final choice of reviewers is that of the

Editor and the journal reserves the right for choice of final

reviewers.

Abstract

A concise and factual abstract of no more than 250 words is

required. The abstract must be structured for original research

articles. The abstract should be divided by subheadings as

follows: Objectives, Materials and Methods, Results,

Discussion and Conclusion.

The abstract should not be structured for review articles.

The abstract should state briefly the purpose of the research, the

principal results and major conclusions. An abstract is often

presented separate from the article, so it must be able to stand

alone.

Keywords

After the abstract provide a maximum of six keywords, to be

chosen from the Medical Subject Headings from Index

Medicus. These keywords will be used for indexing purposes

Abbreviations

Define abbreviations or acronyms that are not standard in this

field at their first occurrence in the article; in the abstract and

also in the main text after it. Ensure consistency of abbreviations

throughout the article.

Text

This should start on the third page and should be subdivided into

the following sections: Introduction, Patients or Materials and

Methods, Resul ts , Discuss ion and Conclusions ,

Acknowledgements.

References

Responsibility for the accuracy of bibliographic citations lies

entirely with the authors. Please ensure that every reference

cited in the text is also present in the reference list (and vice

versa). Any references cited in the abstract must be given in full.

"Unpublished data" and "Personal communications" are not

allowed. As an alternative, say in the text, for example, '(data not

shown)' or '(Dr D. Saranath, School of Science, NMIMS

(Deemed-to-be) University, Mumbai)'. Citation of a reference as

"in press" implies that the item has been accepted for publication

and a copy of the title page of the relevant article must be

submitted.

Indicate references by (first author, year) in the text.

Examples:

Kulkarni J, Khanna A. Functional hepatocyte-like cells derived

from mouse embryonic stem cells: A novel in vitro

hepatotoxicity model for drug screening. Toxicol In Vitro

2006;20:1014-1022.

Bhatnagar R, Dabholkar J, Saranath D. Genome-wide disease

association study in chewing tobacco associated oral

cancers. 2012;48(9):831-835.Oral Oncol

Molinolo AA, Hewitt S, Amornphimoltham PI, Keelawat S,

Saranath D, Gutkind JS . Dissecting the Akt/mTOR et al

signaling network: emerging results from the head and

neck cancer tissue array initiative. Clin Cancer Res

2007;13:4964-4973.

Saranath D. Integrated Biology and Molecular Pathology of

Oral Cancer. : Saranath D, editor. Contemporary Issues In

in Oral Cancer. Oxford Press, 2001:30-71.

List all authors if the total number of authors is seven. For more

than seven authors, first six authors should be listed, followed by

" " For further details you are referred to "Uniform et al.

Requirements for Manuscripts submitted to Biomedical

Journals" ( 1997;277:927-934).J Am Med Assoc

Figure Captions, Tables, Figures and Schemes

Present these, in the given order, at the end of the article. They

are described in more detail below. High-resolution graphics

files must always be provided separate from the main text file.

Footnotes

Footnotes should be used sparingly. Number them

consecutively throughout the article, using superscript Arabic

numbers. Many word processors build footnotes into the text,

and this feature may be used. Should this not be the case,

indicate the position of footnotes in the text and present the

footnotes themselves on a separate sheet at the end of the article.

Do not include footnotes in the Reference list.

Table footnotes

Indicate each footnote in a table with a superscript lowercase

letter.

Tables

Number tables consecutively in accordance with their

appearance in the text. Place footnotes to tables below the table

body and indicate them with superscript lowercase letters.

Avoid vertical rules. Be sparing in the use of tables and ensure

that the data presented in tables do not duplicate results

described elsewhere in the article.

Nomenclature and Units

Follow internationally accepted rules and conventions: use

the international system of units (SI). If other quantities are

mentioned, give their equivalent in SI.

Preparation of Electronic Illustrations

· Make sure you use uniform lettering and sizing of your

original artwork.

· Save text in illustrations as "graphics" or enclose the font.

· Only use the following fonts in your illustrations: Arial or

Times Roman.

· Number the illustrations according to their

sequence in the text.

· Use a logical naming convention for your artwork files.

· Provide all illustrations as separate files and as hardcopy

printouts on separate sheets.

· Provide captions to illustrations separately.

· Produce images near to the desired size of the printed

version.

Formats

Regardless of the application used, when your electronic

artwork is finalised, please "save as" or convert the images to

one of the following formats (Note the resolution requirements

for line drawings, halftones, and line/halftone combinations

given below):

EPS: Vector drawings. Embed the font or save the text as

"graphics".

TIFF: Colour or greyscale photographs (halftones): always use

a minimum of 300 dpi.

TIFF: Bitmapped line drawings: use a minimum of 1000 dpi.

TIFF: Combinations bitmapped line/half-tone (colour or

greyscale): a minimum of 500 dpi is required.

DOC, XLS or PPT: If your electronic artwork is created in any of

these Microsoft Office applications please supply "as is".

Please do not

· Supply embedded graphics in your wordprocessor

(spreadsheet, presentation) document;

· Supply files that are optimised for screen use (like GIF,

BMP, PICT, WPG); the resolution is too low;

· Supply files that are too low in resolution;

· Submit graphics that are disproportionately large for the

content.

If, together with your accepted article, you submit usable colour

figures then it will be ensured that at no additional charge these

figures will appear in colour on the Web (e.g., ScienceDirect and

other sites) in addition to colour reproduction in print.

Captions

Ensure that each illustration has a caption. Supply captions

separately, not attached to the figure. A caption should comprise

a brief title (not on the figure itself) and a description of the

illustration. Keep text in the illustrations themselves to a

minimum but explain all symbols and abbreviations used.

Line drawings

The lettering and symbols, as well as other details, should have

proportionate dimensions, so as not to become illegible or

unclear after possible reduction; in general, the figures should

be designed for a reduction factor of two to three. The degree of

reduction will be determined by the Publisher. Illustrations will

not be enlarged.

Do not use any type of shading on computer-generated

illustrations.

Photographs (halftones)

Remove non-essential areas of a photograph. Do not mount

photographs unless they form part of a composite figure. Where

necessary, insert a scale bar in the illustration (not below it), as

opposed to giving a magnification factor in the caption. Note

that photocopies of photographs are not acceptable.

Preparation of supplementary data

Electronic supplementary material to support and enhance your

scientific research is accepted as supplementary file.

Supplementary files offer the Author additional possibilities to

publish supporting applications, movies, animation sequences,

high-resolution images, background datasets, sound clips and

more. Supplementary files supplied will be published online

alongside the electronic version of your article. In order to

ensure that your submitted material is directly usable, please

ensure that data is provided in one of our recommended file

formats. Authors should submit the material in electronic format

together with the article and supply a concise and descriptive

caption for each file.

Proofs

when your manuscript is received by the Publisher it is

considered to be in its final form. Proofs are not to be regarded as

"drafts". One set of page proofs in PDF format will be sent by e-

mail to the corresponding author, to be checked for

typesetting/editing. No changes in, or additions to, the accepted

(and subsequently edited) manuscript will be allowed at this

stage. Proofreading is solely your responsibility.

The corrected article will be published as quickly and accurately

as possible. In order to do this we need your help. When you

receive the (PDF) proof of your article for correction, it is

important to ensure that all of your corrections are sent back to

us in one communication. Subsequent corrections will not be

possible, so please ensure your first sending is complete. Note

that this does not mean you have any less time to make your

corrections just that only one set of corrections will be accepted.

School of Science was started in 2007 with a view

to provide undergraduate and post graduate

students an opportunity to be a part of the unique

learning methodology of the university, which lays

emphasis on academic excellence combined with

industry oriented training. With the boom in

information technology and more and more

sophistication in instrumentation techniques, there

is now a very thin dividing line between the various

disciplines of science. Therefore, there is a greater

need for flexibility in scientific thought as well as

training manpower on an interdisciplinary plane.

With this thought in view, the SVKM's NMIMS

introduced, highly innovative and unique

interdisciplinary courses at the School of Science

from the academic year 2007-2008. The goal of the

School of Science is to be a Center of Excellence in

the domain of Pure and Applied Science by

providing quality education and research.

About School of Science

Courses Offered

Ph.D. in Biological Sciences, Chemistry (Regular and Professional) and Physiotherapy

Integrated M.Sc.-Ph.D. in Biological Sciences and Chemistry

M.Sc. in Biological Sciences, Chemistry (Analytical and Organic) and Statistics

Master of Physiotherapy [In collaboration with Nanavati Super Speciality Hospital, Mumbai, India]

Post-Graduate Diploma in Physician Assistance (2 years), Operation Theatre

Technology (1 year), Non-Invasive Cardiology (1 year) and Central Sterile Services (1

year) [In collaboration with Asian Heart Institute and Research Centre, Mumbai, India]

Diploma in Clinical Research (Part time: 1 year) [In collaboration with C. B. Patel Research Centre,

Mumbai, India]

Certificate Courses in Molecular Medicine and Molecular Oncology (Part time: 6

months) [for medical/science graduates]

Advanced Course in Clinical Data Management (Part time: 3 months) [In collaboration with

C. B. Patel Research Centre, Mumbai, India]

Salient FeaturesResearch constitutes a major thrust in all the courses offered at the School

Courses oriented to fulfill needs/demands of Research Institutions/Industry

Thrust Areas in ResearchCell Biology, Stem Cell Biology, Molecular Oncology, Reproductive Biology, Microbiology,

Immunology, Pharmacology, Phytochemistry, Nanosciences, Applied Chemistry, Colloidal

Chemistry and Applied Statistics

For More Information Please Contact:

School of Science, NMIMS (Deemed-to-be) University

Tel: 91-22-4219 9943/50; Fax: 91-22-2611 4512; E-mail: [email protected];

Visit us at: http://science.nmims.edu

SVKM’s

Narsee Monjee Institute of Management Studies

Deemed to be UNIVERSITY

V. L. Mehta Road, Vile Parle (W), Mumbai-400 056, INDIA.Tel: 91-22-4235555 | Fax: 91-22-26114512Email: [email protected] | Website:www.nmims.edu

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