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Withanone Rich Combination of Ashwagandha Withanolides

Restricts Metastasis And Angiogenesis Through hnRNP-K

Ran Gao1, Navjot Shah1, Jung-Sun Lee2, Shashank P. Katiyar3, Ling Li1,

Eonju Oh2, Durai Sundar3, Chae-Ok Yun3, Renu Wadhwa1, and Sunil C. Kaul1

Authors’ Affiliations: 1Cell Proliferation Research Group and DBT-AIST

International Laboratory for Advanced Biomedicine, National Institute of Advanced

Industrial Science & Technology (AIST), Tsukuba - 305 8562, Japan; 2Department of

Bioengineering, College of Engineering, Hanyang University, 222 Wangsimni-Ro,

Seongdong-Gu, Seoul 133-791, Korea; and 3Department of Biochemical Engineering

& Biotechnology, Indian Institute of Technology (IIT) Delhi, Hauz Khas, New Delhi

110016, India

Running title: Ashwagandha inhibits Metastasis and Angiogenesis

Key words: Ashwagandha, Withanone, Withaferin A, combination, metastasis

inhibition

Abbreviations:

hnRNP-K, heterogeneous nuclear ribonucleoprotein-K; i-Extract, alcoholic extract;

WA, Withaferin A; WiNA, Withanone and Withaferin A; VEGF, vascular endothelial

growth factor; HUVEC, human umbilical vein endothelial cells; MMP, matrix

metalloproteinase

on April 5, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 18, 2014; DOI: 10.1158/1535-7163.MCT-14-0324

http://mct.aacrjournals.org/

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Financial Information:

This work was partly supported by grants from the Ministry of Knowledge Economy,

Korea (10030051) and the Korea Science and Engineering Foundation

(2013K1A1A2A02050188, 2013M3A9D3045879, 2010-0029220) to C-O Yun and

AIST, Japan Special Budget to S. C. Kaul and R. Wadhwa.

Corresponding authors:

Renu Wadhwa, National Institute of Advanced Industrial Science & Technology

(AIST), Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki - 305 8562, Japan. Tel/Fax: 81-29-

861-2900. E-mail: [email protected]; Sunil C. Kaul, National Institute of

Advanced Industrial Science & Technology (AIST), Central 4, 1-1-1 Higashi,

Tsukuba, Ibaraki - 305 8562, Japan. Tel: 81-298-61-6713. E-mail: [email protected];

and Chae-Ok Yun, Department of Bioengineering, College of Engineering, Hanyang

University, 17 Haengdang-dong, Seongdong-gu, Seoul, Korea. Tel: 82-2-2220-0491;

Fax: 82-2-2220-4850. E-mail: [email protected]

Disclosure of Potential Conflicts of Interest:

No potential conflicts of interest were disclosed.

R. Gao and N. Shah contributed equally to this work.

Current address: Ran Gao, Institute of Laboratory Animal Science, Chinese Academy

of Medical Science (CAMS) & Comparative Medicine Center, Peking Union Medical

College (PUMC), China.

Current address: Navjot Shah, Department of Pediatrics, Darby Children Research




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Institute, Medical University of South Carolina, Charleston, SC 29425, USA.

Word count: 5491

Total number of figures: 6

Abstract

Ashwagandha is an important herb used in Indian system of traditional home

medicine, Ayurveda. Alcoholic extract (i-Extract) from its leaves and its component,

Withanone, were previously shown to possess anticancer activity. In the present

study, we developed a combination of Withanone and Withaferin A, major

withanolides in the i-Extract, that retained the selective cancer cell killing activity,

and found that it also has significant anti-migration, -invasion and -angiogenic

activities, both in in vitro and in vivo assays. Using bioinformatics and biochemical

approaches, we demonstrate that these phytochemicals caused downregulation of

migration-promoting proteins: hnRNP-K, VEGF and metalloproteases, and hence are

candidate natural drugs for metastatic cancer therapy.

Introduction

Metastasis is a complex and multi-step process of cancer cell movement from

their primary to a secondary site within the body through bloodstream or the lymph

system. Cancer itself is extremely complex to understand and treat; metastasis poses

additional hurdles in its therapy. Multiple factors that determine metastasis include

origin, type and stage of the cancer, and in turn determine the type and outcome of the

treatment. The process of metastasis requires (i) an acquisition of migratory

characteristics by tumor cells to leave the primary site, (ii) capacity to proteolytically




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digest the surrounding connective tissues, (iii) characteristics to enter the lymphatic or

blood vessels through which they travel to distant sites of the body and (iv) ability to

proliferate at the new site to establish into tumor. The choice of cancer treatment

(either surgery, chemotherapy, radiation therapy, hormone therapy, laser-

immunotherapy or a combination) depends on the type of primary cancer, size and

location of the metastasis. However, current options to cure metastatic cancers are

very limited. There is a substantial need to understand the phenomenon of metastasis,

uncover the factors that influence cellular migration and adhesion characteristics, and

formulate new strategies and reagents for safe and effective cancer treatment.

Recently, there has been renewed interest in herbal medicines because of the

safety and economic issues on one hand and the traditional history of wide use on the

other. According to the World Health Organization, 80 percent of the world

population do not have access to advanced medical care and use herbs for primary

health and therapeutic purposes. Ashwagandha (Withania somnifera) is often

categorized amongst the world’s most renowned herbs. It is classified in GRAS

(generally regarded as safe) family as a non-toxic edible herb, and is also called as

Indian ginseng and Queen of Ayurveda (Indian traditional home medicine). The main

active constituents of Ashwagandha are alkaloids and steroidal lactones, commonly

known as withanolides. Withaferin A (WA) is a major chemical constituent of

Withania somnifera. It has been shown to possess anti-tumor potentials, induces

apoptosis by activation of caspase-3 and inhibits JNK, Akt, pERK and IL-6 signal

pathways (1-3). Compared to treatment with either Withaferin A or radiation alone,

the combination of both resulted in a significant enhancement of apoptosis in human

renal cancer cells, and hence was suggested as an effective radiosensitizer in cancer

therapy (4). Withaferin A was shown to induce depolymerization of vimentin (5) and




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cause reregulation of Notch1 signaling (6). We have earlier shown that the high dose

of Withaferin A, but not Withanone, was cytotoxic to normal cells (7). Furthermore,

bioinformatics and experimental data suggested the differential binding efficacies of

Withaferin A and Withanone to cellular targets including mortalin, p53, p21 and

NRF2 (8). We had earlier reported that the low dose of alcoholic extract of

Ashwagandha leaves (i-Extract) and its components, Withanone and Withaferin A,

were non-toxic instead induced differentiation in glioma cells (9). Hence, in low

dose, they were proposed as useful in differentiation based milder and effective

glioma therapy. We also found that Withanone, when added along with Withaferin

A, was able to decrease the toxicity of the latter in normal cells (7). Based on these

findings, we undertook the present study to formulate potent combination of

Withanone and Withaferin A that could have stronger anti-metastatic and anti-

angiogenic activities than the i-Extract. The combination, rich in Withanone, was

non-toxic to normal cells and showed potent anti-metastatic and anti-angiogenesis

activities in in vitro and in vivo assays. We demonstrate that such activities are

mediated through inactivation of multifunctional RNA binding protein, heterogeneous

nuclear ribonucleoprotein K (hnRNP-K) and its downstream effectors, matrix

metalloproteinases (MMPs), pERK and vascular endothelial growth factor (VEGF).

Materials and Methods

Cell culture and colony forming assays

Human glioblastoma (A172 and YKG1), osteosarcoma (U20S), fibrosarcoma

(HT1080), neuroblastoma (IMR32); rat glioblastoma (C6) and mouse immortal

fibroblasts (NIH 3T3) were used for this study. Cells were purchased from JCRB

(Japanese Collection of Research Bioresources) Cell Bank, National Institute of




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Biomedical Innovation, Japan, and were maintained in Dulbecco’s Modified Eagle’s

Medium (DMEM, Invitrogen)-supplemented with 10% fetal bovine serum in a

humidified incubator (37�C and 5% CO2). Cells were used within 10-15 passages of

the original stocks and hence no additional authentications were performed. Human

umbilical vein endothelial cells (HUVEC) were cultured in M199 medium (Invitrogen,

Carlsbad, CA) containing 20% FBS, penicillin-streptomycin (100 IU/ml), 3 ng/ml

basic fibroblast growth factor (Upstate Biotechnology, Lake Placid, NY), and 5 U/ml

heparin. HUVECs were used for experiments in passages 2 through 7. Cells (40-

60% confluency) were treated with Withanone (i-Factor) (5 µg/ml; 10.6 µM),

Withaferin A (0.25 µg/ml; 0.531 µM), and their combination WiNA 20-1 for about 48

h, and were harvested for molecular assays as described below. For colony forming

assay, 1000 cells were plated in 10-cm dish, in triplicates. Cells were allowed to

expand and make colonies during the next two weeks with regular replacement of

culture medium (either control or phytochemical-supplemented, as mentioned) with

the fresh medium every alternate day. Colonies were fixed in methanol : acetone

(1:1), stained with Crystal violet, destained with running tap water, and were then

counted. The plates were scanned using Epson GT-9800F scanner. Statistical

significance of the data was calculated from three independent experiments.

Cell cycle analysis

For cell cycle analysis, cells (1 X 105) treated with indicated drugs for 48 h

were harvested with trypsin, washed twice with phosphate-buffered saline (PBS) and

fixed with 70% ethanol at 4�C for 12 h. The fixed cells were centrifuged (2000 rpm

for 10 min), washed twice with cold PBS and re-suspended in 0.25 ml PBS. RNA

was removed by RNAse A treatment (5 μl of 10 mg/ml RNAse was added to 250 μl




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of the cell suspension and incubated at 37�C for 1 h). The cell suspension was stained

with propidium iodide (PI) (10 μl of PI, 1 mg/ml) at 4°C in dark for 30 min. The cell

cycle analysis was done using Guava cell cycle flow cytometer (Millipore) following

the manufacturer’s protocol.

Wound-scratch assay

Cell motility was examined using the Wound-scratch assay. Cell monolayers

were wounded by uniformly scratching the surface with a needle (20 gauge).

Movement of the control and treated cells in the scratched area were serially

monitored under a phase contrast microscope with a 10 X phase objective. Migration

capacity was calculated by measuring the percent of open area in 6-10 randomly

captured images.

Cell invasion assays

Invasion assays were carried out in Boyden chambers (pore size of 8 µm:

Corning Inc., Corning, NY) using Matrigel following the manufacturer’s instructions.

For fluorometric determination of Cell Invasion, QCMTM Cell invasion assay kit

(Millipore) was used. It was also performed using xCELLigence System (Roche) that

used dual chamber Matrigel plates equipped with sensor. Automatic scan and the

data were acquired for 48 h following the manufacturer’s instructions.

Immunoblotting

Cells were lysed in RIPA lysis buffer. The protein (20 μg) was

immunoblotted with anti-phospho Rb, -phospho ERK, -phospho p38 (Cell

Signalling), -NCAM (AbCys SA, Paris, France), -MMP-2 (Santa Cruz Biotech) and -




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Actin (Chemicon International, Temecula, CA) antibodies by standard Western

blotting as described earlier (7).

Immunostaining

Cells were cultured and treated on glass coverslips placed in 12-well culture

dish. The cells were stained with anti-VEGF and -hnRNP-K antibodies (Santa Cruz

Biotech), as described previously (7, 9).

Endothelial cell tube formation assay

The formation of HUVEC capillary like structures on a basement membrane

matrix was used to assess the anti-angiogenic activity of i-Extract and its constituent

phytochemicals. The 16-mm diameter tissue culture plates were coated with 250 μl

growth factor-reduced Matrigel (Collaborative Biomedical Products, Bedford, MA) at

37°C for 30 min. HUVECs were seeded on the Matrigel bed (1.5 × 105 cells/well)

and cultured in M199 medium containing either Avastin or Ashwagandha extract or

phytochemical combination in the presence of VEGF165 (10 ng/ml) for 20 h. M199

medium containing VEGF165 alone served as a control. Capillary networks were

photographed, and the area covered by the tube network was quantified by Image-Pro

Plus software (Media Cybernetics, Silver Spring, MD).

Endothelial cell chemotactic migration assay

The effect of Ashwagandha on the chemotactic motility of HUVECs

responding to VEGF165 was assessed using transwell migration chambers (Corning

Costar, Cambridge, MA) with 6.5-mm diameter polycarbonate filters (8 μm pore size).

Cells in M199 medium containing 1% FBS were stimulated with 10 ng/ml VEGF165




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and treated with Avastin or Ashwagandha regents for 30 min at room temperature,

and then seeded into the lower wells. HUVECs, incubated for 4 h in M199 medium

containing 1% FBS were harvested by trypsinization and loaded into the upper wells

(1 × 105 cells/well). The chamber was then incubated at 37 °C for 4 h. Chemotaxis

was quantified by counting the migrated cells under an optical microscope (Olympus

I X 71; Olympus, Melville, NY) in 10 random fields.

VEGF ELISA

Human VEGF-A was quantified in cell supernatants using the human VEGF

Quantikine Immunoassay Kit (R&D Systems, Minneapolis, MN), following the

manufacturer’s protocol.

In vivo study

Nude mice were obtained from Charles River, Japan. HT1080 (1 X 106) cells

were injected subcutaneously into the abdomen. Intraperitoneal (IP) injections of

WiNA (Withanone, 1 mg/kg and Withaferin A, 0.5 mg/kg) were started when small

tumor buds were formed in about 7 days. These concentrations were determined

based on independent experiments that involved testing of different ratios of

Withaferin A and Withanone for intraperitoneal injections. The concentrations higher

than 1 mg/kg Withanone and 0.5 mg/kg Withaferin A in 100 �l volume of 0.5%

DMSO showed precipitation, and hence were considered inappropriate. Injections

were continued every alternate day and the mice were monitored for tumor size until

3-4 weeks. For metastasis assay, cells were injected into the tail vein. After 7 days,

WiNA injections were performed. Mice were sacrificed and the lungs were examined

for the presence of tumors.




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Molecular docking and dynamics simulations

Virtual molecular docking of KH3 domain of hnRNP-K protein with

Withaferin A and Withanone was executed using Autodock suite 4.2 (10). KH3

domain of hnRNP-K (KH3_hnRNP-K) protein was downloaded from Protein Data

Bank (PDBID: 1ZZI) and PubChem Compound database was used to retrieve the

structure of Withaferin A [PubChem: 265237] and Withanone [PubChem: 21679027].

Structure files of both the ligands were prepared for molecular docking by defining

the number of torsion angles, addition of hydrogen atoms and conversion into

software specific file format (pdbqt). Similarly, KH3_hnRNP-K protein was also

prepared by removal of ssDNA, removing bad contacts, addition of hydrogen atoms,

removal of needless water molecules, and conversion of file format into pdbqt.

ssDNA binding site on KH3_hnRNP-K was defined as the site of ligand binding over

KH3_hnRNP-K. First, prepared ligands were virtually docked against KH3_hnRNP-

K protein blindly. Further, both the ligands were docked at the defined binding site

using Lamarckian Genetic Algorithm of Autodock 4.2. Top scoring conformations

were inspected against binding at defined binding site.

The GPU accelerated Amber Molecular Dynamics suite with Amber ff99SB

protein force field was used to perform all atoms explicit molecular dynamics

simulations (MD simulations) of protein-ligand complexes

http://ambermd.org/#Amber12 (11-13). Protein-ligand complex molecules were

solvated with TIP3P water model in a cubic periodic boundary box to generate

required systems for MD simulations and systems were neutralized using appropriate

number of counterions. The distance between box wall and protein complex was set

to greater than 10Å to avoid direct interaction with its own periodic image.

Neutralized system was then minimized, heated up to 300 K temperature and




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equilibrated until the pressure and energies of systems were stabilized. Finally,

equilibrated systems were used to run 60 ns long MD simulations.

During the MD simulations, H-bond fluctuations of ligand with protein were

calculated using VMD software (14). Molecular interaction diagrams were generated

using Maestro, version 9.4, Schrödinger LLC, New York, NY, 2013. All simulation

studies were performed on Intel Core 2 Duo CPU @ 3 GHz of HP origin with 1 GB

DDR RAM and DELL T3600 workstation with 8 GB DDR RAM and

NVIDIA GeForce GTX TITAN 6 GB GDDR5 Graphics Card.

Results

Effect of Withanone and Withaferin A combination on cancer cell proliferation

in vitro

Alcoholic extract of Ashwagandha leaves was earlier shown to possess

Withanone and Withaferin A as major, and Withanolide A, Withanolide D, 12-

deoxywitha-stramonolide as minor withanolides. The ratio of these constituents in

leaves varies depending on their source and stages. Whereas Withanone and

Withaferin A were found to be toxic to cancer cell, Withanone rich i-Extract was non-

toxic to normal human cells (7). Based on these findings, we first generated various

combinations of Withanone and Withaferin A, and investigated their effect on cancer

and normal cell viability in culture. We found that the combination of Withanone (5

µg/ml; 10.6 µM) and Withaferin A (0.25 µg/ml; 0.53 µM), at a ratio of Withanone to

Withaferin A as 20:1, called WiNA 20-1 selectively killed cancer cells. Normal cells

remained unaffected (Fig. 1A). In contrary, the combination of Withanone and

Withaferin A either at 5:1 (WiNA 5-1) or 3:1 (WiNA 3-1) were toxic to normal cells

also (Fig. 1A). Furthermore, the combination WiNA 20-1 was cytotoxic to a variety




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of human cancer cells including osteosarcoma (U20S), breast carcinoma (MCF7),

glioblastoma, (A172 and YKG1), fibrosarcoma (HT1080), neuroblastoma (IMR32);

rat glioblastoma (C6) and mouse immortal fibroblasts (NIH 3T3)(Fig. 1A and data not

shown). Therefore, in the present study, 20:1 was determined as the optimum ratio of

Withanone and Withaferin A in the mixture to selectively kill cancer cells in culture.

We first examined the effect of Withanone and Withaferin A, and their

combination WiNA 20-1 on cancer cell proliferation by colony forming assay using

highly metastatic human fibrosarcoma, HT1080. As shown in Figs. 1B and 1C,

treatment with WiNA 20-1 caused reduction in colony number and size as compared

to either the control or the cells treated with individual purified components. The

effect of WiNA 20-1 was stronger and statistically significant; 35%, 55% and 70%

reduction in colony formation in the presence of Withaferin A, Withanone and WiNA

20-1, respectively (Fig. 1C). The data was supported by cell cycle analysis (Fig. 1D).

Number of cells in G2 increased from 51.33% to 67.38%, to 77.08% and to 85.37% in

control, Withaferin A, Withanone and WiNA 20-1 treated cells, respectively. Cell

number at S phase decreased from control (27.64%) to Withaferin A treated (14.53%)

to Withanone treated (11.06%) and WiNA 20-1 treated (6.06%) cells.

Effect of Withanone and Withaferin A combination on in vitro cancer cell

migration

Wound-scratch assays revealed that the cells treated with either Withanone or

Withaferin A moved slowly to the scratched area as compared to the control.

Furthermore, the migration of cells treated with WiNA 20-1 combination was highly

reduced (Figs. 2A and 2B). Real time measure of cell migration also showed that the

treatment of cells with (i) either Wi-A or Wi-N reduced their migration capacity and




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(ii) WiNA 20-1 combination the effect was ore pronounced (Fig. 2C). Furthermore,

quantitative assays (Figs. 2D and 2E) revealed that WiNA 20-1 treated cells showed

strongest reduction both in their invasion and migration capacity.

Treatment with Withanone and Withaferin A limits migration, invasion and

angiogenic potential of human endothelial cells

Based on the above data, we next investigated the effect of WiNA 20-1 on

migration, invasion and tube formation capacity of human umbilical vein endothelial

cells (HUVEC). Cells were treated with VEGF (10 ng/ml) for induction of migration,

on basement membrane matrix constituted of Matrigel. Avastin (humanized

monoclonal antibody that inhibits vascular endothelial growth factor A, VEGF-A), an

approved inhibitor of angiogenesis was used as a positive control. As shown in Fig.

3A, whereas VEGF induced the tube formation, Avastin showed clear inhibition. Of

note, both i-Extract and WiNA 20-1 strongly inhibited the tube formation even at

lower doses (1/5th of the concentration used for cytotoxicity assays in HT1080 and

YKG-1 cells as described in Fig. 1). We found that the i-Extract and WiNA 20-1 also

limited the migration and invasion capacity of VEGF-stimulated HUVEC cells, as

shown in Figs. 3B and 3C. Most interestingly, the efficacy of inhibition of migration

and invasion was comparable to Avastin (50 μg/ml) suggesting that the i-Extract and

WiNA 20-1 possess highly potent anti-metastatic activities. We next performed

VEGF ELISA in control and treated cells to investigate whether this effect was due to

the direct inhibition of VEGF by i-Extract and WiNA 20-1. As shown in Fig. 3D, we

found that the treatment with i-Extract and WiNA 20-1 both resulted in substantial

downregulation of VEGF in the conditional medium. Western blotting and




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immunostaining of VEGF also showed strong decrease in WiNA 20-1 treated cells

(Figs. 3E and 3F).

Molecular mechanism of anti-migratory activity of Withanone, Withaferin A

and WiNA 20-1: hnRNP-K as a target

As shown in Fig. 3, anti-migration and anti-angiogenesis activities of WiNA

20-1 seemed to be mediated by downregulation of VEGF, an established player of

metastasis and angiogenesis, and is regulated by hnRNP-K (15). We next examined

the expression of hnRNP-K in control and treated cells. Two other proteins, mortalin

(a member of HSP70 stress chaperone family) and ezrin (a member of Ezrin-Radixin-

Moesin (ERM) family), reported to be associated with cancer cell metastasis (16-18),

were also examined. As shown in Fig. 4, the three proteins were downregulated in

WiNA 20-1 treated cells, suggesting their relationship with decreased migration of

cells, as shown in Figs. 2 and 3. Mortalin was earlier shown to be a target of

Withaferin A and Withanone (8). Examination of hnRNP-K by

immunocytochemistry showed that the number of cells with bright nuclear staining

was less in treated cells suggesting an inhibition of its transcriptional activation

function (Figs. 4C and 4D).

Based on the above data, we hypothesized that Withaferin A and Withanone

may bind to hnRNP-K and result in an inhibition of its metastatic signaling, as shown

in Fig. 5A. We, therefore, investigated the binding by molecular dynamics

simulations. The crystal structure of KH3_hnRNP-K was available as DNA-protein

complex with ssDNA in PDB. The analysis of ssDNA and KH3_hnRNP-K complex

revealed the binding site of ssDNA/RNA over KH3_hnRNP-K. ssDNA was found to

interact with residues Lys22, Asp23, Ala25, Ile29, Lys31, Arg40, Lys47, Ile48,




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Asp50, Tyr84, and Ser85 of KH3_hnRNP-K protein (Fig. 5B). Before docking to

ssDNA/RNA binding site, Withaferin A and Withanone were docked randomly over

KH3_hnRNP-K protein to identify most preferable binding site of ligands over

protein. We found that the binding sites of both the ligands were coinciding with

ssDNA/RNA binding site of the protein suggesting that both Withaferin A and

Withanone have tendency to hinder the binding of ssDNA/RNA at KH3_hnRNP-K

protein.

Docking of Withaferin A and Withanone specifically at ssDNA/RNA binding

site generated the bound conformation of ligands within the protein with docking

score of -8.92 and -9.19. Withaferin A was in contact with residues Gly26, Ser27,

Gly30, Lys31, Gln34, Gln83, and Ser85 of KH3_hnRNPK via Hydrogen bonds (H-

bonds) and hydrophobic interactions (Figs. 5C and 5D). Molecular docking of

Withanone with KH3_hnRNP-K resulted in two high affinity conformations (docking

score -9.1 and -8.9) that were docking at minutely different positions within same

binding site. A comparison of Withanone of docking score -9.1 (Withanone_c1) and

Withanone of docking score -8.92 (Withanone_c2) with ssDNA bound conformation

revealed that Withanone_c1 was binding completely at ssDNA binding site but

Withanone_c2 was only partially interfering the binding of ssDNA (data not shown).

Because both the conformations were interfering with the binding of hnRNP-K to

ssDNA, we confirmed the binding characteristics of Withanone, using its two

conformations by MD simulations. Significance of binding of Withaferin A and

Withanone was revealed by the fact that the second nucleotide Thymine (DT) of

ssDNA also interacts with residues Tyr84 and Ser85. Binding of any of the ligands at

these amino acid residues of KH3_hnRNP-K is expected to hinder its binding to

ssDNA/RNA (Figs. 5C and 5D). It may either decrease the binding affinity of




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RNA/ssDNA to hnRNP-K or may make the interaction of RNA/ssDNA and hnRNP-

K completely non-functional because of the involvement of first few nucleotides that

play key role in its transcription activation/deactivation function.

Stability of the protein-ligand complex was further verified by long MD

simulations. Withaferin A was found highly stable at its place during 60 ns MD

simulations with little or no fluctuation. All the interactions of Withaferin A with

KH3_hnRNP-K were conserved during and after the 60 ns MD simulations and

involved the ssDNA binding site of hnRNP-K (Figs. 5C and 5D). Withanone_c1

showed a slight shift in its binding position within ssDNA binding site, attaining a

more stable conformation. Stabilized Withanone_c1 was found interacting with

residues Gly26, Ser27, Ile29, Gly30, Lys31, Arg35, Ser84, Tyr85, and Lys87 of

KH3_hnRNP-K protein, strongly hindering the binding of the protein to ssDNA (Figs.

5C and 5D). Withanone_c2 was also found stable at its binding site during MD

simulation. Based on higher binding efficacy of Withanone_c1 and binding site on

the ssDNA, Withanone_c1 conformation appeared as a highly efficacy ligand (Fig.

5D). hnRNP-K was earlier shown to regulate Erkp44/42 and MMP2 signaling (19).

Western blotting revealed significant decrease in the expression of MMPs and

phospho-Erkp44 in WiNA 20-1 treated cells, as compared to the ones treated with

either Withanone or Withaferin A (Fig. 5E). On the other hand, the cell adhesion

protein, NCAM, showed maximum increase in WiNA 20-1 treated cells (Fig. 5E)).

In vivo validation of anti-metastasis activity of WiNA 20-1 and hnRNP-K as a

target

We next determined the effect of Withaferin A, Withanone and their

combination on cancer cell proliferation and migration in in vivo using nude mice




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HT1080 subcutaneous xenograft and lung metastasis models. Toxicity as a result of

intraperitoneal (IP) injections of either Withanone (1 mg/kg), Withaferin A (0.5

mg/kg) or their combination (WiNA) in 100 �l of 0.5% DMSO was first tested by

visual observations and body weight measurements of mice. The combination with

constituents higher than 1 mg/kg Withanone and 0.5 mg/kg Withaferin A in 100 �l of

injection volume showed precipitation, and hence were considered inappropriate for

in vivo study. There was no significant difference in the body weight of Withaferin

A, Withanone and WiNA-injected mice as compared to the un-injected control (data

not shown). Hence the three reagents, at the doses used, were considered non-toxic in

vivo. Mice with small HT1080 tumor buds (7 days post-injection of cells) were given

the intraperitonial injections of either Withaferin A, Withanone or combination on

every alternate day. As shown in Figs. 6A and 6B, WiNA injected mice showed

strong suppression of subcutaneous HT1080 tumor-xenografts. In the lung metastasis

model, big tumors were detected only in control mice (Fig. 6C). Taken together,

these data suggested that WiNA has significant anti-cancer and anti-metastasis

activities in vivo. We also performed Western blotting of tumor excised from the

control and treated mice, and found that hnRNP-K was decreased in WiNA treated

small tumors as compared to the control big tumors. Furthermore, in agreement with

the in vitro data (Figs. 4 and 5), small tumors showed decrease in hnRNP-K

downstream effectors VEGF, Erkp44/42 and MMP2.

Discussion

Tumor metastasis involves dissociation of cancer cells from the primary tumor

site, followed by migration, invasion, adhesion and proliferation at a distant site.

Matrix metalloproteinases (MMPs), critical regulators of extracellular matrix,




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metastasis and angiogenesis (20), are regulated by cytokines, growth factors via

interlinked signaling pathways (19, 21, 22). hnRNP-K is a multifunctional protein

that regulates ERK1/2, MMPs and VEGF, and contribute to cell migration, invasion

and ascites formation (19, 23-25). ERK1/2 and VEGF have been connected by

positive autocrine feedback loop (26-28).

Several studies have shown that the alcoholic extract of Ashwagandha leaves,

and its components, Withaferin A and Withanone, are cytotoxic to cancer cells, and

possess radio-sensitizing, immunomodulatory, anti-inflammatory, anti-metastasis and

anti-angiogenic properties, suggesting their potential as anticancer drug (1-6, 29-31).

Mechanisms of these activities are only beginning to be resolved. It was shown that

Withaferin A inhibits pro-metastatic intermediate filament protein - vimentin, an

EMT signaling protein (5, 32), pAkt signaling pathway and MMP-9 (33), STAT3 and

its downstream effectors Bcl-xL, Bcl-2, cyclin D1 (34), pAkt and pERK signaling (2),

oncogenic transcription factor STAT3 (35) and NF-kappa B (36). It was shown to

induce extracellular pro-apoptotic tumor suppressor protein, Par-4 (2), oxidative stress

to cancer cells (37-39). In spite of these beneficial anticancer effects, cytotoxicity of

Withaferin A, in high dose, to normal human cells has been a concern (7, 8, 40). A

closely related withanolide, Withanone, on the other hand, caused selective

cytotoxicity to human cancer cells (7, 40). In view of this, we investigated the

cytotoxicity of Withanone and Withaferin A in various combinations in normal and

cancer cells. The combination WiNA 20-1 (Withanone -10.6 µM and Withaferin A -

0.53 µM) was selectively toxic to cancer cells and showed potent anti-migratory and

anti-angiogenic activities in vitro. These activities were supported by molecular

analysis of marker proteins including MMPs that play crucial role in the process of

cancer invasion and metastasis. In an earlier study, we had reported that compared to




19

Withanone, Withaferin A possess stronger affinity for target proteins including

mortalin, p53, p21 and Nrf2, and that might account for its toxicity to normal cells (8).

In the present study, we demonstrate that Withaferin A targets hnRNP-K, an upstream

regulator of MMPs, Erk-p44/42 and VEGF (Fig. 5A), and inhibits its metastasis

signaling. Immunofluorescence analysis revealed decrease in the number of cells

with bright nuclear hnRNP-K, suggesting its compromised transcriptional function

resulting in decreased levels of MMPs, ERK, VEGF, as shown in Figs. 3-6. The

latter might also be due to, at least in part, decreased level of ezrin and mortalin

proteins that are enriched in cancer cells (16, 41-46) (Figs. 5A and 5B), associated

with tumor metastasis as discussed above. Whereas the mechanism(s) of effect of

these phytochemicals on ezrin warrant further studies, mortalin-p53 complex has been

shown to be targeted by Withaferin A and Withanone resulting in activation of p53

function (41). Furthermore, Withanone was also shown to target TPX2 oncogene, a

prime regulator of Aurora A kinase that plays a critical role during mitosis and

cytokinesis (47). These mechanisms are also expected to contribute to the anticancer

and anti-metastasis activities of Withaferin A, Withanone and WiNA 20-1. In in vivo

tumor formation assays, injections of Withanone-rich combination of Withanone and

Withaferin A (1 mg and 0.5 mg/kg BW, respectively) showed significant inhibition of

tumor growth and metastasis. Taken together, we report that (i) Withanone-rich

combination of Withanone and Withaferin A limits cancer cell growth, migration and

angiogenesis in vitro and in vivo (ii) the anti-metastasis activity is mediated by

targeting multifunctional RNA binding protein, hnRNP-K.

Acknowledgements




20

Authors thank Roche for providing the xCELLigence System and Masumi Maruyama

for technical help.

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27

Legends to the Figures

Figure 1. Withanone and Withaferin A combination (WiNA 20-1) is selectively

toxic to cancer cells. (A) Cell morphology (B and C) colony forming efficacy (D) cell

cycle distribution of control and treated cells showing that the combination of

Withanone (Wi-N) and Withaferin A (Wi-A) in the ratio of 20:1 was selectively

cytotoxic to cancer cells. The combinations in the ratio of 5:1 and 3: 1 were toxic to

normal cells. The differences, between Withaferin A and WiNA 20-1 as well as

Withanone and WiNA 20-1 in (C) and (D), were statistically significant (C, as shown;

and D, p<0.05).

Figure 2. Withanone and Withaferin A combination (WiNA 20-1) has anti-

migration activity. (A) Wound scratch assay showing migration of control and treated

HT1080 cells. Open area of the wound revealed that the treated cells moved slowly

as compared to the control cells. (B) Quantitation of the open area from three

independent experiments. (C) Real time measure of the migration capacity of cells

and (D and E) Quantitative cell invasion assays showing strong inhibition by WiNA

20-1.

Figure 3. Withanone and Withaferin A combination (WiNA 20-1) has anti-

angiogenic activity. (A) Tube formation capacity, (B) migration, and (C) invasion of

control and treated HUVECs. Quantitation from three independent experiments is

shown. *P < 0.05, **P < 0.01, ***P < 0.001 compared with VEGF165. VEGF

expression, as determined by ELISA (D), Western blotting (E) and immunostaining

(F), is shown. Quantitation from three independent experiments was performed using




28

Imaging J. Actin was used as an internal control (E). An unpaired Student t test was

used to determine the statistical significance of the data.

Figure 4. Treatment with Withanone and Withaferin A combination (WiNA 20-

1) downregulates the level of ezrin, hnRNP-K and mortalin. (A) Western blotting

showing decrease in ezrin, hnRNP-K and mortalin in the treated cells. Actin was

used as an internal control. (B) Quantitation of the signals on Western blots was

performed from three independent experiments using Imaging J. (C) Immunostaining

showing decrease in the number of cells with bright nuclear hnRNP-K in treated cells

(D). An unpaired Student t test was used to determine the statistical significance of

the data.

Figure 5. Withanone and Withaferin A target hnRNP-K. (A) A schematic

diagram showing the interaction of hnRNP-K with DNA and its downstream effectors

involved in cancer cell migration. Model also shows the abrogation of hnRNP-K and

DNA complex and inhibition of cell migration by Withaferin (WA) or Withanone

(WN). (B) Interaction diagram of KH3 domain of hnRNP-K protein with ssDNA is

shown. Amino acid residues of KH3 domain of hnRNP-K protein, such as Gly26,

Ser27, Gly30, Ile36, Lys37, Tyr84, and Ser85 were seen to interact with ssDNA. (C)

Interactions of Withaferin A with amino acid residues of KH3_hnRNP-K protein are

shown (left). Interactions of stabilized Withanone after 60 ns MD simulation with

KH3_hnRNP-K protein are shown (right). In both the cases, most of the residues

were the ones involved in interaction of the protein with ssDNA. (D) Binding

conformations of Withaferin A and Withanone with KH3_hnRNP-K are shown.

Hindrance in interaction of hnRNP-K with ssDNA is shown in the superimposed




29

structures of Withanone-KH3_hnRNP-K complex over ssDNA-KH3_hnRNP-K

complex. (E) Western blot showing decrease in the level of phospho-ERKp44/42 and

MMP2, and increase in the cell adhesion protein NCAM in WiNA 20-1 treated

HT1080.

Figure 6. In vivo anti-metastasis activity of Withaferin A, Withanone and WiNA

20-1. (A) Nude mice showing the suppression of tumor growth in response to WiNA

20-1 treatment. (B) Quantitation of tumor volume from six mice each. (C)

Suppression of lung tumors in WiNA 20-1 injected mice as compared to control. (D)

Western blot showing decrease in the level of hnRNP-K, VEGF, ERKp44/42 and

MMP2 expression in small tumors excised from the treated mice as compared to the

large tumors excised from the control mice.




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Published OnlineFirst September 18, 2014.Mol Cancer Ther Ran Gao, Navjot Shah, Jung-Sun Lee, et al. Restricts Metastasis And Angiogenesis Through hnRNP-KWithanone Rich Combination of Ashwagandha Withanolides

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