The Pennsylvania State University

The Graduate School

College of Agricultural Sciences

POLYPHENOL-RICH FOODS AS INHIBITORS OF COLON CANCER STEM CELLS

A Dissertation in

Food Science

by

Venkata Rohit Charepalli

2018 Venkata Rohit Charepalli

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

August 2018

ii

The dissertation of Venkata Rohit Charepalli was reviewed and approved* by the following:

Jairam K.P. Vanamala Associate Professor of Food Science Dissertation Co-Advisor Co-Chair of Committee

Joshua D. Lambert Associate Professor of Food Science Dissertation Co-Advisor Co-Chair of Committee

Gregory R. Ziegler Professor of Food Science

Mary J Kennett Professor of Veterinary and Biomedical Sciences Robert F. Roberts Professor of Food Science Head of the Department of Food Science

*Signatures are on file in the Graduate School

iii

ABSTRACT

The role of cancer stem cells (CSCs) in the initiation, progression and relapse of cancerous

tumors has been studied in the past few years. Epidemiological studies have revealed a causal

association between consumption of a diet rich in fruits and vegetables with reduced risk of colon

cancer. This is believed to be due in part to the presence of polyphenols such as anthocyanins,

procyanidins and phenolic acid derivatives. However, the effect of these compounds on colon

CSCs has not been studied. In the present studies, I investigated the effects of polyphenol-rich

Eugenia jambolana (Java plum), resveratrol-grape seed extract (RSV-GSE) and purple-fleshed

potatoes on colon CSCs. The overall goal of this project was to investigate the anti-cancer effect

of these polyphenolic compounds and polyphenol-rich foods on colon CSCs in vitro and in vivo,

and to explore the underlying mechanisms of action.

Java plum is a tropical fruit rich in anthocyanins and is typically grown in Florida and

Hawaii in the US. I characterized the anthocyanin profile of Java plum using HPLC-MS and

found that Java plum anthocyanin extract (JPE) contains a variety of anthocyanins including

glucosides of delphinidin, cyanidin, petunidin, peonidin and malvidin. To evaluate the anti-cancer

effects JPE, I treated cancer cells and colon CSCs (positive for CD 44, CD 133 and ALDH1b1

markers), with JPE at 30 and 40 μg/mL for 24 hours. Cell viability was assessed using the 3-[4,5-

dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay and enumeration of viable

cells. I evaluated induction of apoptosis by JPE using the TUNEL and caspase 3/7 glo assays. JPE

suppressed proliferation in HCT-116 cells by more than 50 % and elevated apoptosis in both

HCT-116 cells (200 %) and colon CSCs (165 %). JPE also inhibited the colony formation ability

in colon CSCs as evaluated using colony formation assay. These results warrant further

investigation of the anti-colon cancer effects of java plum using animal models of colon cancer.

iv

We have previously shown that anthocyanin-containing baked purple-fleshed potato (PP)

extracts suppressed early and advanced human colon cancer cell proliferation and induced

apoptosis, but their effect on colon CSCs is not known. In my research, both colon CSCs with

functioning p53 and those with shRNA-attenuated p53 were treated with 5.0 μg/mL baked PP

extracts (PA) for 24 hours. Effects of PA were compared to positive control sulindac. Cell

proliferation was assayed using BrdU incorporation and apoptosis was assayed using TUNEL

assay. In vitro, PA suppressed proliferation and elevated apoptosis in a p53 independent manner

in colon CSCs. To evaluate the pathways targeted by PA, after treatment protein fraction of the

cells was extracted and western blotting was used to look at the levels of proteins in Wnt/β-

catenin and mitochondrial apoptotic signaling pathways. PA, but not sulindac, suppressed levels

of Wnt pathway effector β-catenin (a critical regulator of CSC proliferation) and its downstream

proteins (c-Myc and cyclin D1) and elevated Bax and cytochrome c, mitochondria-mediated

apoptotic proteins. These results were extended to the azoxymethane -induced mouse model of

colon cancer. Mice were given diet supplemented with baked PP (20 % w/w). In vivo, PP reduced

the number of crypts containing cells with nuclear β-catenin (an indicator of colon CSCs) via

induction of apoptosis and suppressed tumor incidence similar to that of sulindac after one week

of feeding. Further, four weeks of feeding PP supplemented diet resulted in significant reduction

of tumors. Combined, our data suggests that suppression of Wnt/β-catenin signaling and elevated

apoptosis via mitochondria-mediated apoptotic pathway by PP may contribute to reduced colon

CSCs number and tumor incidence in vivo.

v

We have previously shown that the grape bioactive compound resveratrol (RSV)

potentiates grape seed extract (GSE)-induced apoptosis in HCT-116 colon cancer cells. As part of

my dissertation research, I tested the anti-cancer efficacy of the RSV-GSE against isolated human

colon CSCs in vitro and the AOM-induced mouse model of colon carcinogenesis in vivo. In vitro,

RSV-GSE suppressed - proliferation, sphere formation, nuclear translocation of β-catenin (a

critical regulator of CSC proliferation) similar to sulindac in isolated human colon CSCs. RSV-

GSE, but not sulindac, suppressed downstream proteins levels of Wnt/β-catenin pathway, c-Myc

and cyclin D1. RSV-GSE also induced mitochondrial-mediated apoptosis in colon CSCs

characterized by elevated p53, Bax/Bcl-2 ratio and cleaved PARP. Furthermore, shRNA-

mediated knockdown of p53, a tumor suppressor gene, in colon CSCs did not alter efficacy of

RSV-GSE. In vivo, RSV-GSE supplementation for 4 weeks resulted in suppressed tumor

formation to a similar extent as sulindac, without any gastrointestinal toxicity. Additionally,

RSV-GSE treatment for one week reduced the number of crypts containing cells with nuclear β-

catenin (an indicator of colon CSCs) via induction of apoptosis. Our study has shown that RSV-

GSE combination eliminates colon CSCs in vivo and in vitro similar to that of NSAID sulindac

without any toxicity. Although further investigations are needed to understand more on the

interactions of these agents and on long-term colon cancer chemopreventive or chemotherapeutic

potential of the RSV-GSE, our findings suggest that clinical testing of RSV-GSE against colon

cancer is required.

vi

TABLE OF CONTENTS

LIST OF FIGURES ........................................................................................................ ix

LIST OF TABLES ......................................................................................................... xiv

ACKNOWLEDGEMENTS ............................................................................................ xv

Chapter 1 Literature review ............................................................................................ 1

1.1 Colon cancer ..................................................................................................... 1 1.1.1 Incidence, risk factors and financial impact................................................ 1 1.1.2 Pathogenesis of colon cancer .................................................................... 2 1.1.3 Existing therapeutic approaches and drawbacks ......................................... 5

1.2 Cancer stem cells ............................................................................................... 6 1.2.1 Anatomy of the colon ............................................................................... 6 1.2.2 Cancer stem cell theory ............................................................................ 8 1.2.2 Role of colon cancer stem cells in resistance to chemotherapy and relapse... 9 1.2.4 Wnt/β-catenin signaling pathway in colon cancer stem cells ....................... 10 1.2.5 P53 in colon cancer and cancer stem cells.................................................. 12

1.3 Dietary polyphenols and select polyphenol-rich foods .......................................... 12 1.3.1 Polyphenols ............................................................................................. 12 1.3.2 Java plum (Eugenia jambolana) ............................................................... 15 1.3.3 Potato ..................................................................................................... 16 1.3.4 Resveratrol and grape seed extract ............................................................ 17

1.4 Anti-colon cancer effects of anthocyanins, resveratrol and GSE ............................ 17 1.4.1 Models of cancer ..................................................................................... 17 1.4.2 In vitro studies ......................................................................................... 19 1.4.3 In vivo studies ......................................................................................... 23 1.4.4 Polyphenols against colon cancer stem cells .............................................. 24

1.5 Purpose and significance..................................................................................... 26 1.6 Hypothesis and objectives................................................................................... 28

Chapter 2 Eugenia jambolana (Java plum) fruit extract exhibits anti-cancer activity against early stage human HCT-116 colon cancer cells and colon cancer stem cells* ... 29

2.1 Abstract ............................................................................................................ 30 2.2 Introduction ................................................................................................ 31

2.3 Materials and methods........................................................................................ 33 2.3.1 Extraction and Purification of Anthocyanins from Java Plum ...................... 33 2.3.2 Chemicals................................................................................................ 34 2.3.3 High Performance Liquid Chromatography Mass Spectrometry (HPLC-

MS) Analysis ............................................................................................ 34 2.3.4 Cell Lines ................................................................................................ 35 2.3.5 Cell Viability ............................................................................................ 35 2.3.6 Apoptosis ................................................................................................ 36 2.3.7 Colony Formation Assay.......................................................................... 37

vii

2.3.8 Statistical Analysis ................................................................................... 38 2.4 Results .............................................................................................................. 38

2.4.1 Evaluation of the Bioactive Compound Profile in JPE ................................ 38 2.4.2 JPE Suppressed Proliferation in HCT-116 Cells ......................................... 39 2.4.3 JPE Induced Apoptosis in HCT-116 Cells and Colon CSCs ........................ 40 2.4.5 JPE Suppressed Colony Formation in Colon CSCs..................................... 42

2.5 Discussion ......................................................................................................... 44

Chapter 3 Anthocyanin-containing purple-fleshed potatoes suppress colon tumorigenesis via elimination of colon cancer stem cells*................................................................ 46

3.1 Abstract ............................................................................................................ 47 3.2 Introduction ....................................................................................................... 48 3.3 Materials and methods........................................................................................ 50

3.3.1 Chemicals................................................................................................ 50 3.3.2 Plant material........................................................................................... 50 3.3.3 Potato characterization ............................................................................. 50 3.3.4 Cancer stem cells ..................................................................................... 52 3.3.5 Lentiviral shRNA-mediated attenuation of p53 in colon CSCs .................... 53 3.3.6 Cell proliferation ...................................................................................... 53 3.3.7 TUNEL assay.......................................................................................... 54 3.3.8 Sphere formation assay ............................................................................ 54 3.3.9 Western blot ............................................................................................ 54 3.3.10 Animal study ......................................................................................... 55 3.3.11 AOM carcinogen injection....................................................................... 55 3.3.12 Experimental diets.................................................................................. 55 3.3.13 Colon tissue collection ............................................................................ 56 3.3.14 Immunohistochemistry/Immunofluorescence staining ............................... 56 3.3.15 Statistical design .................................................................................... 57

3.4 Results .............................................................................................................. 58 3.4.1 UPLC-MS profile of phenolic compounds in PP........................................ 58 3.4.2 PA suppressed proliferation and induced apoptosis in colon cancer stem

cells in a p53 independent manner .............................................................. 58 3.4.3 PA suppressed sphere formation ability of colon CSCs............................... 60 3.4.4 PA elevated mitochondria-mediated apoptotis pathway proteins Bax/Bcl-

2 and cytochrome c ................................................................................... 61 3.4.5 PA suppressed Wnt pathway proteins ........................................................ 62 3.4.6 PP induced apoptosis and reduced number of crypts with nuclear β-

catenin accumulated colon CSCs................................................................ 65 3.4.7 PP suppressed AOM induced colon cancer tumors ..................................... 68 3.5 Discussion .................................................................................................. 69

Chapter 4 Grape compounds suppress colon cancer stem cells in vitro and in a rodent model of colon carcinogenesis* ................................................................................ 72

4.1 Abstract ............................................................................................................ 73 4.2 Introduction ....................................................................................................... 73 4.3 Materials and methods........................................................................................ 77

viii

4.3.1 Chemicals................................................................................................ 77 4.3.2 Animal study ........................................................................................... 77 4.3.3 Azoxymethane carcinogen injection........................................................... 78 4.3.4 Experimental diets ................................................................................... 78 4.3.5 Colon tissue collection .............................................................................. 78 4.3.6 Immunofluorescence staining .................................................................... 79 4.3.7 Cancer stem cells ..................................................................................... 80 4.3.8 Lentiviral shRNA-mediated attenuation of p53 in colon CSCs .................... 80 4.3.9 Cell proliferation ...................................................................................... 81 4.3.10 TUNEL assay ........................................................................................ 81 4.3.11 Sphere formation assay .......................................................................... 82 4.3.12 Western blot........................................................................................... 82 4.3.13 Statistical analysis .................................................................................. 82

4.4 Results .............................................................................................................. 83 4.4.1 RSV-GSE suppressed AOM-induced tumor incidence in mice .................... 83 4.4.2 RSV-GSE induced apoptosis and reduced number of crypts with colon

cancer stem cells ....................................................................................... 85 4.4.3 RSV-GSE suppressed proliferation and induced apoptosis in colon cancer

stem cells .................................................................................................. 87 4.4.4 RSV-GSE suppressed sphere formation ability of colon CSCs .................... 88 4.4.5 RSV-GSE suppressed Wnt pathway proteins.............................................. 89 4.4.6 RSV-GSE elevated mitochondrial apoptotic pathway proteins..................... 91 4.4.7 RSV-GSE efficacy is retained even in the absence of p53 ........................... 93

4.5 Discussion ......................................................................................................... 96

Chapter 5 Conclusions .................................................................................................... 100

5.1 Conclusions ....................................................................................................... 100 5.2 Future work ....................................................................................................... 102

5.2.1 Developing evidence for anti-cancer effect of polyphenols from indigenous sources .................................................................................... 102

5.2.2 Future studies involving PP and RSV-GSE ................................................ 103

Bibliography ................................................................................................................... 106

ix

LIST OF FIGURES

Figure 1-1: Colon cancer development. Adapted from Todaro et al. ................................... 4

Figure 1-2: Morphology of the colon. Source: Kasdagly et al............................................. 7

Figure 1-3: Crypt organization. Source: Kasdagly et al ...................................................... 7

Figure 1-4: Canonical Wnt signaling in stem cells. Adapted from S Al-Sohaily et al. .......... 11

Figure 1-5: Types of polyphenols. Source: Agustin G. Asuero et al. ................................... 13

Figure 1-6: Structure of anthocyanins. Source: Miguel et al. .............................................. 15

Figure 2-1: HPLC chromatogram of Java plum fruit extracts (JPE) anthocyanins; the peak number correspond to anthocyanins in table 2-1. ....................................................... 39

Figure 2-2: Java plum fruit extracts (JPE) suppressed proliferation in HCT-116 cells. HCT-116 cells were treated with JPE (30 or 40 µg/mL) for 24 hours, MTT assay (A) and viable cell count (B) were performed as described in methods. Values are in means ± SE. Means that differ by a common letter (a, b, c) differ at p < 0.05. .............. 40

Figure 2-3: Java plum fruit extracts (JPE) induced apoptosis in HCT-116 cells; (A) Percent apoptosis in HCT-116 cells (n=400) as measured by TUNEL assay. (B)

Apoptosis was also assayed using caspase 3/7 glo assay. Values are in means ± SE. Means that differ by a common letter (a, b, c) differ at p < 0.05. (C) Cells fluorescing bright green due to fragmented DNA, indicator of apoptosis using TUNEL assay. Pictures were taken on a fluorescence microscope at 20x magnification (12 fields per treatment and at least 500 cells were counted). Representative pictures are shown for Control, JPE at 30 µg/mL and JPE at 40 µg/mL. .................................................................................................................... 41

Figure 2-4: Java plum fruit extracts (JPE) induced apoptosis in colon cancer stem cells (colon CSCs). Cells were treated with JPE (30 or 40 µg/mL) for 24 hours and caspase 3/7 glo assay was performed. Values are in means ± SE. Means that differ by a common letter (a, b, c) differ at p < 0.05. ........................................................... 42

Figure 2-5: Effect of Java plum fruit extracts (JPE) on the stemness of colon CSCs. (A)

Cells were treated with JPE (30 or 40 µg/mL) for 24 hours and colony formation assay was performed as described in methods. (B) Representative images taken from the colony forming assay for Control and JPE 30 are presented. Results were expressed as mean ± SE for three experiments at each time point. Means that differ by a common letter (a, b) differ at p < 0.05. ............................................................... 43

x

Figure 3-1: PA suppressed proliferation and induced apoptosis in colon cancer stem cells (colon CSCs) independent of p53. A Anti-proliferative effect of PP anthocyanin extract (PA) in colon CSCs with functioning p53 and with attenuated p53. Cells were treated with PA (5 µg/mL) or sulindac (12.5 µg/mL) for 24 hours and BrdU assay was performed as described in the methods. B – D PA induced apoptosis in colon cancer stem cells with functioning p53 and attenuated p53. TUNEL assay was performed and the results are expressed as percentage apoptosis. Cells fluorescing bright green due to fragmented DNA indicate apoptotic cells. Pictures were taken on a fluorescence microscope at 20x magnification (12 fields per treatment and at least 500 cells were counted). Representative pictures are shown for Control and PA at 5.0 µg/mL. PA = Baked purple-fleshed potato extract. Values are in means ± SE. Means that differ by a common letter (a, b, c for CSCs and x, y, z for CSCs with shRNA-attenuated p53) differ (p < 0.05). .................................................................. 59

Figure 3-2: PA suppressed sphere formation of colon cancer stem cells (colon CSCs) similar to that of sulindac (A). Representative pictures taken at 100x magnification are shown for Control, Solvent, Sulindac at 12.5 µg/mL and PA at 5.0 µg/mL (B). PA = Baked purple-fleshed potato extract. Values are in means ± SE. Means that differ by a common letter (a, b, c) differ (p < 0.05). ................................................... 60

Figure 3-3: PA elevated levels of mitochondria-mediated apoptosis pathway proteins. PA elevated Bax/Bcl-2 ratio (A, B); and cytochrome c levels in colon cancer stem cells (colon CSCs) independent of p53 (C, D). Colon CSCs were treated with PA (5 µg/mL) or sulindac (12.5 µg/mL) for 24 hours, and whole-cell lysates were analyzed for Bax (pro-apoptotic), Bcl-2 (anti-apoptotic) and cytochrome c (pro-apoptotic) levels by western blotting. Actin was used as loading control. C = Control; S = Solvent; SU = Sulindac; PA = Baked purple-fleshed potato extract. Values are in means ± SE. Means that differ by a common letter (a, b) differ p < 0.05. ..................... 62

Figure 3-4: PA suppressed cytosolic and nuclear β-catenin levels in colon cancer stem cells (CSCs) with functioning p53 (A, B) and attenuated p53 (C, D). Colon CSCs were treated with PA (5 µg/mL) or sulindac (12.5 µg/mL) for 24 hours, and cytosolic and nuclear lysates were analyzed for β -catenin by western blotting. Actin and Topoisomerase-2 Beta (TOP2B) was used as loading control for cytosolic and nuclear lysates respectively. C = Control; S = Solvent; SU = Sulindac; PA = Baked purple-fleshed potato extract. Values are in means ± SE. Means that differ by a common letter (a, b, c) differ p < 0.05. ...................................................................... 64

Figure 3-5: β-catenin targets c-Myc and cyclin D1 levels were suppressed by PA in colon cancer stem cells (colon CSCs) with functioning p53 (A, B) and attenuated p53 (C,

D). Colon CSCs were treated with PA (5 µg/mL) or sulindac (12.5 µg/mL) for 24 hours, and nuclear lysates were analyzed for c-Myc and cyclin D1 by western blotting. Topoisomerase-2 Beta (TOP2B) was used as loading control. C = Control; S = Solvent; SU = Sulindac; PA = Baked purple-fleshed potato extract. Values are in means ± SE. Means that differ by a common letter (a, b, c) differ p < 0.05. ................. 65

Figure 3-6: Purple-fleshed potato treatment induced apoptosis (A) and reduced number of crypts with nuclear β-catenin accumulated intestinal stem cells similar to that of

xi

sulindac. Mice injected with azoxymethane [119] were fed with control, baked PP (20 % w/w) or sulindac (0.06 % w/w) supplemented diet for 1 week. Distal colon sections from the mice were analyzed for TUNEL positive crypts and β-catenin localization by immunofluorescence. (A) The fractions of crypts containing at least one TUNEL-positive cell were determined. (B) Nuclear β-catenin index was calculated as a percentage of total number of crypts with nuclear β-catenin accumulation. (C) Staining of β-catenin and DAPI (blue; nuclear counterstain) in mice treated with AOM. Circles mark representative colon CSCs with nuclear β-catenin. Values are in means ± SD (n = 5 in each group). At least 300 crypts from each animal were analyzed. Means that differ by a common letter (a, b, c) differ p < 0.05. (Scale bars: 15 μm). ........................................................................................ 67

Figure 3-7: Purple-fleshed potato suppressed tumor incidence in the colon similar to that of sulindac. Mice injected with azoxymethane were fed with control, baked PP (20 % w/w) or sulindac (0.06 % w/w) supplemented diet for 4 weeks and euthanized. Whole colon tissue was resected and observed in a dissection microscope for visible tumors greater than 2 mm in size. Values are in means ± SD (n = 8 in each group). Means that differ by a common letter (a, b) differ p < 0.05. ........................................ 68

Figure 4-1: RSV – GSE suppressed tumor incidence in the colon similar to that of sulindac. (A) Mice injected with AOM consumed control, RSV-GSE or sulindac (positive control) supplemented diet for four weeks and were euthanized. Whole colon tissue was resected and observed under a dissection microscope for visible tumors. SU = Sulindac; RG = RSV-GSE. Values are in means ± S.E. (n = 8 in each group). Means that differ by a common letter (a, b) differ at p < 0.05. (B) Short-term feeding of sulindac resulted in stomach ulcers (hyperplasia of the stomach, black arrows) and subsequent loss of adipose tissue deposits (blue arrows) compared to control. RSV-GSE supplemented diet consuming animals showed neither hyperplasia nor loss of adipose tissue deposits. .......................................................... 84

Figure 4-2: RSV – GSE treatment induced apoptosis and reduced the number of crypts containing cells with nuclear β-catenin (an indicator of colon CSCs). Mice injected with AOM were fed with control, RSV-GSE or sulindac-containing diet for one week. Distal colon sections from the mice were analyzed for TUNEL positive crypts and β-catenin localization by immunofluorescence. (A) The fractions of crypts containing at least one TUNEL-positive cell (indicator of apoptotic cells) were determined. (B) Quantification of crypts with nuclear β-catenin in mice treated with control, RSV-GSE or sulindac supplemented diet for one week. Accumulation of nuclear β-catenin is hallmark of cancer stem cells and hence was used as an indirect measure for evaluating elimination of cancer stem cells. (C) Staining of β-catenin and DAPI (blue) in mice treated with AOM. Circles mark representative colon stem cells with nuclear β-catenin (CSCs). SU = Sulindac; RG = RSV-GSE. Values are in means ± S.E. (n = 5 in each group). At least 300 crypts from each animal were analyzed. Means that differ by a common letter (a, b, c) differ at p < 0.05. (Scale bars: 15 μm)............................................................................................................ 86

Figure 4-3: RSV – GSE suppressed proliferation, induced apoptosis and suppressed sphere formation in colon CSCs similar to that of sulindac. (A) Anti-proliferative

xii

effect of RSV-GSE in colon CSCs. RSV-GSE induced apoptosis in CSCs (B, C) similar to that of sulindac. CSCs were treated with sulindac (6.25, 12.5 and 25 µg/mL) or RSV-GSE (RSV - 9 µM and GSE 6.25, 12.5 and 25 µg/mL) for 24 hours and BrdU assay was performed to assess proliferation. TUNEL assay was performed based on manufacturer protocol (Roche) and the results are expressed as per cent apoptosis. Cells fluorescing bright green due to fragmented DNA indicate apoptotic cells. Pictures taken on fluorescence microscope at 20X magnification. Representative pictures are shown for Control, RSV-GSE at 9 µM and 12.5 µg/mL respectively and sulindac at 12.5 µg/mL. ................................................................... 87

Figure 4-4: Sphere formation was assessed as described in methods. Representative images taken from the sphere formation assay are presented. Results were expressed as mean ± S.E. for three experiments at each time point. Means that differ by a common letter (a, b, c, d, e, f) differ at p < 0.05. ........................................................ 88

Figure 4-5: RSV – GSE suppressed levels of proteins involved in Wnt/β-catenin pathway in colon CSCs with functioning p53. Nuclear β-catenin (A) and its regulator phosphorylated GSK3β (B) levels were suppressed by RSV-GSE similar to that of sulindac. Downstream targets of Wnt/β-catenin pathway – c-Myc (C) and Cyclin D1 (D), in the nucleus were suppressed by RSV-GSE compared to sulindac. Colon CSCs were treated with RSV-GSE at 9 µM and 12.5 µg/mL, or sulindac at 12.5 µg/mL for 24 h, and cytosolic and nuclear cell lysates were analyzed for respective proteins by western blotting. Actin and topoisomerase-2β (Topo II b) were used as loading controls for cytosolic and nuclear proteins respectively. Values are in means ± S.E. Means that differ by a common letter (a, b, c,) differ at p < 0.05. ...................... 90

Figure 4-6: RSV-GSE induced apoptosis via p53 dependent pathway in colon cancer stem cells (CSCs) with functioning p53. Nuclear p53 levels were elevated (A) by RSV-GSE but not sulindac. Cleaved PARP (B) and Bax/Bcl-2 ratio (C) were also elevated by RSV-GSE but not sulindac. Colon CSCs were treated with RSV-GSE at 9 µM and 12.5 µg/mL, or sulindac at 12.5 µg/mL for 24 h, and cytosolic and nuclear cell lysates were analyzed for respective proteins by western blotting. Actin and topoisomerase-2β (Topo II b) were used as loading controls for cytosolic and nuclear proteins respectively. Values are in means ± S.E. Means that differ by a common letter (a, b, c, or x, y, z) differ at p < 0.05..................................................... 92

Figure 4-7: Modulation of Wnt/β-catenin and apoptotic signaling proteins by RSV – GSE in colon CSCs with attenuated p53. β-catenin (A) and its downstream targets c-Myc (B) and cyclin D1 (C) were suppressed by RSV-GSE compared to sulindac. Pro-apoptotic proteins cleaved PARP (D) and cytochrome C (E) levels were elevated by RSV-GSE greater than that of control and sulindac. Colon CSCs were treated with RSV-GSE at 9 µM and 12.5 µg/mL, or sulindac at 12.5 µg/mL for 24 h, and cytosolic and nuclear cell lysates were analyzed. Actin and topoisomerase-2β (Topo II b) were used as loading controls for cytosolic and nuclear proteins respectively. C = Control; SU = Sulindac; RG = RSV-GSE. Values are in means ± S.E. Means that differ by a common letter (a, b, c, or x, y, z) differ p < 0.05. ....................................... 95

xiii

Figure 4-8: RSV – GSE suppressed COX-2 levels in colon CSCs with functioning (A) and attenuated p53 (B). Colon CSCs were treated with RSV-GSE at 9 µM and 12.5 µg/mL respectively or sulindac at 12.5 µg/ml for 24 h, and nuclear cell lysates were analyzed for COX-2 levels by western blotting. Topoisomerase-2β (Topo II b) was used as a loading control. C = Control; SU = Sulindac; RG = RSV-GSE. Values are in means ± S.E. Means that differ by a common letter (a, b, c) differ at p < 0.05. ......... 95

xiv

LIST OF TABLES

Table 2-1: Anthocyanins identified in Java plum fruit extract. ........................................... 39

Table 3-1: Phenolic and anthocyanin composition of white vs purple-fleshed potatoes by UPLC/MS. ............................................................................................................. 52

xv

ACKNOWLEDGEMENTS

I would like to first thank my parents, Malleshwary and Vijaya Charepalli. Their values,

continued support and guidance have helped me become the person I am today. I would also like

to thank my brother Saroj, for being so patient with my long absence and keeping me up to date

on all the good things happening back home.

My heartfelt thanks and gratitude to my advisor Dr. Jairam K.P. Vanamala for seeing the

potential and giving me an opportunity to pursue doctoral studies. His patience and feedback on

my shortcomings made me push harder each day and complete my work with dedication. I would

like to express my gratitude to my co-advisor, Dr. Joshua D. Lambert for his support with

completion of dissertation and thinking about career after PhD.

I am grateful to Dr. Lavanya Reddivari for her help with experiment design, conducting

animal studies and feedback throughout my doctoral program. I am extremely lucky and thankful

to have known Dr. Sridhar Radhakrishnan, he taught me most of the technical and writing skills

required for working in a research laboratory. His critical feedback on experiment design,

discussions about research and life in general have helped me get through the doctoral program.

I thank my committee members, Dr. Gregory R. Ziegler, and Dr. Mary J. Kennett for

their intellectual input, feedback on dissertation and support for this work. I wish to thank the

Department of Food Science here at Penn State for their continued support throughout the

program. A special thanks to administrative staff of food science.

A big thank you to my lab members over the past years – Aaron Massey, Laura

Markham, Abigail Sido, Eranda Karunathilake, Vijaya Indukuri, Chrissy Koestler, Lauriel

xvi

Stewart. Their awesome company made my stay at State College memorable with a lot of great

memories. I thank Dr. Ramakrishna Vadde for his help and support with the animal studies and

carrying out some of the experiments in this dissertation.

I would like to thank all my friends and family from India, Colorado State University, for

making my time during PhD a memorable one. Heartfelt thanks for those whom I have missed to

acknowledge.

Chapter 1

Literature review

1.1 Colon cancer

1.1.1 Incidence, risk factors and financial impact

Colon cancer is the third most commonly diagnosed cancer in both men and women in

the United States [1]. It is estimated that nearly 1.4 million people across the world are affected

by colon cancer [2]. The National Cancer Institute estimates that for the year 2017, 95,520 new

cases of colon cancer will be diagnosed [1]. In addition to premature death due to colon cancer, it

is estimated that nearly US$14.1 billion per year is spent on colon cancer treatment in the US

[13]. On a per person bases it comes out to $150000 per year. This includes visitations, surgeries,

medication, and continued screenings. This number is expected to increase to over US$17.4

billion by the year 2020 due to the aging population [13].

Colon cancer risk is influenced by both genetic and environmental factors. Genetic

conditions like familial adenomatous polyposis (FAP) or hereditary non-polyposis are two main

forms [3]. Individuals with inflammatory bowel diseases are also at a significantly greater risk for

developing colon cancer [4]. Sex, age and race are also factors that affect colon cancer risk. Men

are more prone than woman to develop colon cancer [1]. The risk of being diagnosed with colon

cancer goes up with age with majority of cases diagnosed are after 50 years of age [2]. Incidence

of colon cancer is also high in African American populations and lower in Asian American

populations compared to Caucasian populations [3]. Although the incidence rates among people

2

aged 50 years and over have continued to decline over the past decade due to screening, among

those under 50 years of age, a 22% increase has been reported [1].

Environmental factors play a significant role in development of colon cancer [5].

Smoking, excessive alcohol consumption, obesity, lack of physical activity and diet have all been

identified as risk factors for colon cancer [5, 6]. Together, these factors may be responsible for up

to 80% of colon cancer cases. Diets rich in red and processed meat, refined starches, sugar, and

saturated and trans-fatty acids but poor in fruits, vegetables, fiber, omega-3 fatty acids and whole

grains are closely associated with an increased risk of colon cancer [7-9].

A number of risk factors for colon cancer are highly modifiable, which suggests that

colon cancer is highly preventable. Nutritional recommendations from the American Cancer

Society indicate the importance of adequate intake (2 ½ servings) of fruits and vegetable in a

regular diet [10]. A meta-analysis of case-control studies suggests that fruit and vegetable

consumption in general is associated with a slight decrease in the risk of colon cancer [7, 11, 12].

For example, in a meta-analysis of fruit consumption was associated with a 13% lower colon

cancer risk, and vegetable consumption was associated with a 40% lower risk [7]. The benefits

from consuming a diet rich in fruit and vegetables could be attributed to the plethora of bioactive

compounds present in them.

1.1.2 Pathogenesis of colon cancer

The development of colon cancer like all cancers is comprised of three main phases:

initiation, promotion and progression [14]. Over these three phases, accumulation of genetic

mutations and epigenetic changes transform normal colon epithelial cells into invasive

3

adenocarcinomas [15]. These alterations can either be acquired, as happens in the sporadic forms,

or be inherited, as with genetic syndromes which predispose subjects to cancer development (e.g.

FAP – familial adenomatous polyposis).

During the initiation phase, mutation and epigenetic changes results in activation of

oncogenes and inactivation of tumor suppressor genes. Adenomatous polyposis coli (APC) is one

such tumor suppressor gene that is mutated in majority of colon cancer cases [16]. Accumulation

of these changes result in the formation of clumps of cells called adenomas or polyps (Figure 1-

1). These early stage polyps usually start out benign, failure to repair the damage results in

advancement to promotion phase leading to larger polyps. The formation of a larger polyps

requires further mutations which lead to activation of oncogenes such as Kirsten rat sarcoma viral

oncogene homolog [17] or B-Raf proto-oncogene, serine/threonine kinase (BRAF) (Figure 1-1).

This stage is still reversible as cells can be eliminated by programmed cell death (apoptosis).

Further mutations including modification of genes involved in cell repair and death (apoptosis),

such as TNF-β (tumor necrosis factor) and p53 [18] results in transition from benign adenoma to

malignant adenocarcinoma (Figure 1-1). Initially adenocarcinomas are localized to the colon

epithelium, but, following additional mutations, can adopt an invasive phenotype and eventually

become metastatic [15, 19].

4

Figure 1-1: Colon cancer development. Adapted from Todaro et al.

5

1.1.3 Existing therapeutic approaches and drawbacks

Currently colon cancer is treated with surgical resection when feasible, in combination with

radiation and/or chemotherapy. 5-fluorouracil (5-FU) and irinotecan are the most common

chemotherapeutic drugs used in the treatment of colon cancer [20, 21]. Recently, non-steroidal

anti-inflammatory drugs such as sulindac and celecoxib have also been shown to be effective in

reducing tumor size in genetically predisposed mice model of colon cancer [22]. The common

theme among all these drugs is their ability to target hallmarks of cancer cells. Hallmarks of

cancer are a set of organized principles that were proposed to rationalize the complexities of

cancer disease [23]. The hallmarks of cancer include sustained proliferation (uncontrolled cell

division), evading growth suppressor proteins and immune system, resisting cell death, enabling

replicative immortality, inducing angiogenesis (formation of new blood vessels), and activating

invasion and metastasis. Genetic instability caused by epigenetic changes and mutations fosters

the multiple hallmarks of cancer cells.

When a single drug/treatment is rendered ineffective due to resistance, a combination

therapy is used to treat colon cancer, wherein multiple hallmarks are targeted to induce cell death.

For example folinic acid and oxaliplatin are used in combination with 5-FU called FOLFOX.

Folinic acid enhances 5-FU function in inhibiting DNA replication whereas oxaliplatin crosslinks

DNA. However, there is systemic and local toxicity because the agents are not selective enough

and may also affect healthy tissue [24]. In addition, in later rounds of therapy, the cancer tends to

relapse and metastasize, and often develops resistance to previous therapies. Hence, there is a

need of chemopreventive agents that are able to target multiple hallmarks of cancer while at the

same time exhibit limited or no toxicity.

There are a number of ways that cancer cells gain resistance to chemotherapy and this

can be either intrinsic or acquired through exposure [21]. Further, cancer tumors are a

6

heterogeneous population of cells, which also extends to resistance. Within a tumor, a

subpopulation of cells can survive chemotherapy while others are sensitive and effectively

eradicated. A small subpopulation of cancer cells, referred to as cancer stem cells (CSCs), are

believed to have resistance thus allowing them to survive chemotherapy treatment [24].

1.2 Cancer stem cells

1.2.1 Anatomy of the colon

The colon consists of four layers (moving from the lumen to outside) - mucosa,

submucosa, muscular layer and serosa (Figure 1-2). The outermost colon epithelial layer, at the

luminal surface, is lined by a single layer of columnar epithelial cells folded into finger-like

invaginations that are supported by the lamina propria to form the functional unit of the colon

called crypts (Figure 1-2).

The colon epithelium consists of multiple cell types with varying levels of differentiation.

They are derived from multipotent stem cells which are located at the bottom of the crypt.

(Figure 1-3) [25, 26]. The stem cells of the colon are capable of self-renewal and divide

asymmetrically to give rise to the transit amplifying cells transit-amplifying cell and a new stem

cells. The transit amplifying cell migrate upward from the bottom of the crypt, proliferate and

differentiate into one of the epithelial cell types (columnar, goblet and enteroendocrine cells) of

the colon wall [25]. These cells are eventually sloughed off allowing the colonic epithelium to be

renewed every 3-5 days [26]. A number of important signaling pathways are responsible for

maintaining the cellular hierarchy and normal colon homeostasis. Dysregulation of stem cell

signaling pathways, along with accumulation of genetic mutations and epigenetic changes, leads

to uncontrolled proliferation and ultimately the development of colorectal cancer [27].

7

Figure 1-2: Morphology of the colon. Source: Kasdagly et al.

Figure 1-3: Crypt organization. Source: Kasdagly et al

8

1.2.2 Cancer stem cell theory

It has been hypothesized for over 40 years that cancers contain the same hierarchy of cell

populations that as normal tissues: stem cells, proliferating transit-amplifying cells, and

terminally differentiated mature cells [28]. The idea that cancer arose from stem cells dates back

to the middle of the 1800s as the embryonal rest theory of cancer [28]. It was not, however, until

1997 that cancer stem cells (CSCs) were isolated from patients with acute myeloid leukemia.

Since then CSCs have been shown to be implicated in tumor formation, metastases and disease

recurrence [29].

CSCs are defined by characteristics similar to those of normal colonic stem cells, mainly

their abilities to form heterogenic tumors that consist of both tumorigenic and non-tumorigenic

populations. This is achieved similar to a normal stem cell, via self-renewing, a characteristic that

drives tumorigenesis, and to aberrantly differentiate, a property that generates the bulk of cells

within a tumor. Although CSCs form a small subpopulation (<1%) of the overall cancer cells,

their ability to form and drive tumorigenesis is a crucial component leading to tumor recurrence,

therapy resistance, and metastasis [30]. CSCs may undergo a symmetrical self-renewing cell

division into two identical daughter CSCs or an asymmetrical self-renewing cell division into one

daughter CSC and one differentiated progenitor cell, resulting in number expansion of CSCs as

well as growth of tumor [31].

A number of papers have reported successful isolation of a subpopulation of cells with

the ability to initiate tumors from both established cell lines [32, 33] and primary patient samples

[34, 35]. In addition, tumors arising from these isolated subpopulation have been shown to

recapitulate the heterogeneity of the tumor they were originally isolated from [34, 36-39]. These

results confirm the existence of CSCs in cancer tumors. The origin of CSCs is still actively

debated. CSCs could arise from neoplastic transformation of normal stem cells [40, 41] or de-

9

differentiation in the immature transit amplifying cells [41]. Normal colon stem cells are long-

lived and therefore have a greater chance of acquiring mutations that could be passed on to their

progeny [42, 43]. Therefore, according to the definition and characteristics of CSCs, we can

conclude that the two hallmark features of CSCs are self-renewal and lineage capacity.

1.2.2 Role of colon cancer stem cells in resistance to chemotherapy and relapse

CSCs have been implicated in disease recurrence following chemotherapy treatment due

to their stem cell like properties and heightened protective mechanisms compared to the non-CSC

tumor cells. First-line colon cancer chemotherapeutic agents (5-FU and oxaliplatin) target the

rapid proliferation hallmark of cancer cells [23]. These agents induce DNA damage and disrupt

DNA replication [20, 21]. Although these drugs are effective at reducing tumor size, they do not

effectively target CSCs because CSCs proliferate at a lower rate than non-CSC tumor cells [44-

46].

In addition, CSCs express a number of other drug resistance systems including high

expression of aldehyde dehydrogenase (ALDH) [47] and ATP-dependent drug efflux pumps [48].

Radiation chemotherapy treatment results in the formation of reactive oxygen species (ROS),

which at high levels are toxic to the cell inducing oxidative damage to lipids, proteins and DNA.

Lipid peroxidation produces a number of reactive aldehydes that can further induce cellular

damage. ALDH is an enzyme responsible for the detoxification of aldehydes [49, 50], catalyzing

the conversion to the less reactive carboxylic acids that can subsequently be excreted. ATP-

binding cassette (ABC) transporters are ATP-dependent drug efflux pumps that actively remove

xenobitoics [51, 52] including the chemotherapeutics [53]. Thus, the failure to target CSC

population by traditional chemotherapeutics can lead to resistance and relapse of cancer [44].

10

Thus, it is important to develop chemotherapeutics that are able to target CSCs while exhibiting

lower toxic side effects.

1.2.4 Wnt/β-catenin signaling pathway in colon cancer stem cells

The Wnt/β-catenin, bone morphogenetic protein/transforming growth factor-β,

Hedgehog, nuclear factor (NF)-κB and Akt/mammalian target of rapamycin signaling pathways

play important roles in the normal physiology of colon stem cells [54]. Dysregulation or aberrant

activation of these key pathways, however, can result in the formation of CSCs and lead to

tumorigenesis. The Wnt/β-catenin pathway is one of the most important pathways in colon

carcinogenesis. The canonical Wnt signaling pathway plays an important role in self-renewal and

maintenance of stem cells and CSCs.

The Wnt pathway consists of extracellular signaling proteins (Wnt ligands), the

transmembrane receptor Frizzled (Fzd), co-receptor low-density lipoprotein-related receptor 5/6

(LRP5/6), Dishevelled (Dsh), β-catenin, axis inhibitor (Axin) and the transcription factor T-cell

factor (TCF)/lymphoid enhancer factor [55] (Figure 1-4). In the absence of Wnt, β-catenin

interacts with Axin, APC and glycogen synthase kinase-3β (GSK-3β) to form a destruction

complex and is phosphorylated by GSK-3β. Phosphorylated β-catenin is then ubiquitinated and

degraded by the proteasome. This process maintains a low level of cytoplasmic β-catenin [56-58].

In the presence of Wnt (Figure 1-4), Wnt ligands bind to the Frizzled/Lrp co-receptor complex

and activate the canonical signaling pathway [94]. Axin is recruited to the plasma membrane

resulting in the inactivation of the APC destruction complex and subsequent stabilization of β -

catenin. The free β -catenin translocates into the nucleus, where it binds to TCF-4 [59]. This

results in promotion of the transcription and expression of downstream targets, including c-Myc

and cyclin D1 [60]. c-Myc and cyclin D1 promote cell proliferation and in preventing apoptosis.

11

Coordination of c-Myc with cyclin D1 or its upstream activators not only accelerates tumor

formation, but also may drive tumor progression to a more aggressive phenotype [61].

Oncogenic mutations of β-catenin, or inactivating mutations of APC tumor suppressor results in

the dysregulation of Wnt/β-catenin pathway in CSCs [62]. Dysregulation of Wnt/β-catenin has

been shown to be one of the earliest events in colon CSCs formation [63]. Over 90% of CRC

cases display an over-activation of Wnt signaling [64].APC is inactivated in almost 85% of all

colorectal cases while β-catenin gene mutations account for half of the remaining cases [16].

CSCs have been shown to exhibit high Wnt activity which is associated with high clonogenic

cancer stem cell potential [65]. Research in mice has shown that targeted knockdown of APC in

Figure 1-4: Canonical Wnt signaling in stem cells. Adapted from S Al-Sohaily et al.

12

colon stem cells resulted in the formation of polyps [41]. Thus, chemoprevention strategies that

target Wnt/β-catenin may be an effective way of eliminating colon CSCs.

1.2.5 P53 in colon cancer and cancer stem cells

The p53 protein functions as a “guardian of the genome”, by inhibiting progression of the

cell cycle and promoting DNA repair and/or apoptosis following a genotoxic insult. Over half of

sporadic colon cancers have been reported to have p53 mutation. Loss of p53 function has been

shown to accelerate the growth of precancerous polyps to cancer [66, 67]. In colon cancer, loss of

p53 function allows uncontrolled proliferation and leads to progression from adenoma to

carcinoma [17]. This is ascribed in part to the fact that many tissues undergo p53-dependent

apoptosis to eliminate cells in the organism that exhibit DNA damage during the transformation

process [68]. In addition, p53 has been shown to be a critical mediator of stem cell function

during the cancer initiation stage by suppressing pluripotency and cellular dedifferentiation [69].

A recent study has shown that targeted deletion of p53 in stem cells of mice with colon cancer

results in elevation of proliferation and reduction of apoptosis [69]. Thus, it is important to test

chemoprevention strategies developed against colon CSCs work even in the absence of p53.

1.3 Dietary polyphenols and select polyphenol-rich foods

1.3.1 Polyphenols

Polyphenols are secondary plant metabolites that are found ubiquitously in plants and are

characterized by having at least two aromatic hydroxyl groups [70-72]. These compounds range

from low molecular weight phenolic acids to the large and complex (highly polymerized) tannins

13

and derived polyphenols (Figure 1-5). Polyphenolic compounds play an important role in plant

physiology because they are involved in growth and reproduction and provide plants with

resistance to pathogens and predators and environmental stresses [73]. Plant foods with high

levels of polyphenols include cereals, fruits, vegetables, wines, and teas. Polyphenols are derived

in plants from the phenyl proponoid pathway [74]. Phenylalanine or tyrosine are deaminated to

cinnamic acid derivatives, which then enter the phenylpropanoid pathway.

According to their structure, polyphenols can be divided into three different classes

(Figure 1-5). These three classes include phenolic acids (eg, gallic acid and curcumin), the most

abundant in foods, flavonoids and the less common stilbenes (eg, resveratrol) [75]. Flavonoids

may be further divided into subclasses: anthocyanins (eg, malvidin), flavones (eg, chrysin),

flavonols (eg, quercetin, myricetin, and rutin), and flavan-3-ols (eg, catechin, epicatechin, and

EGCG) (Figure 1-5) [76, 77]. Among these the most commonly studied polyphenols are the

flavonoids.

Figure 1-5: Types of polyphenols. Source: Agustin G. Asuero et al.

14

Proanthocyanidins (PACs) are the second most abundant natural phenolic compound

after lignin and are grouped under a class of flavonoids called flavan-3-ols [78]. PACs are

classified into two types based on their interflavan linkages: A-type and B-type. A-type linkages

have been found in plums, avocados, peanuts, grape seed extract, and cranberries, while B-type

linkages are found more prominently in fruits such as apples, berries and grapes [78, 79]. The

estimated total daily PAC consumption in the US is only 57.7 mg/person [80], which is

significantly lower compared to France and Mediterranean regions. Because of their high

molecular weight, large number of hydroxyl groups, and ability to form large hydrations shells,

PACs have significantly reduced bioavailability [81]. Although it has been reported that PACs

undergo depolymerization in simulated gastric juice in vitro, other studies have shown that PACs

are stable during gastric transit in human subjects [82-84]. This makes them an attractive

polyphenol source for colon cancer chemoprevention as they can reach the intestine in large

concentrations due to their poor bioavailability.

Anthocyanins confer the bright red, blue and purple colors to fruits and vegetables such

as grapes, blueberries, and color-fleshed potatoes [85, 86]. The de-glycosylated or aglycone forms

of anthocyanins are known as anthocyanidins. The six most common anthocyanidin skeletons are

cyanidin, delphinidin, pelarogonidin, malvidin, petunidin, and peonidin (Figure 1-6). In fruits

and vegetables, the anthocyanidins are glycosylated and/or acylated [86, 87]. The sugar

components of anthocyanins are usually conjugated to the anthocyanidin skeleton via the C3

hydroxyl group in ring C (Figure 1-6). The beneficial health effects of anthocyanins from foods

have been studied extensively [88, 89].155,156. The estimated anthocyanin intake in the United

States is between 180 – 215 mg/d. Like PACs, anthocyanins also have poor bioavailability and

reach the colon at higher concentrations [89-91].

15

1.3.2 Java plum (Eugenia jambolana)

Eugenia jambolana also known as Syzygium cumini (Family Myrtaceae) is a tropical fruit

rich in polyphenols particularly flavonoids such as anthocyanins [92]. Other common names for

this fruit are Jambul, Black Plum, Java Plum, Indian Blackberry, Jamblang and Jamun etc. Java

plum trees are found widely throughout the tropical regions of the Asian subcontinent, Eastern

Africa, South America and Madagascar. In the United States, they have been naturalized to

Florida and Hawaii [93]. The tree fruits once in a year and the berries are small purple ovoid in

shape with a sweetish sour astringent taste. The fruits are typically eaten raw or used in health

drinks, making preserves, smoothies, jellies and wine [93]. The fruit is valued for its diverse

chemical constituents as well as medicinal and therapeutic properties [94, 95]. Both the fruit pulp

and seed extracts have a long history of medicinal use and they have been extensively studied

Figure 1-6: Structure of anthocyanins. Source: Miguel et al.

16

[96, 97]. Hence, dietary approaches for cancer prevention, including identification of new or

development of existing dietary bioactive compound-rich foods are required.

1.3.3 Potato

The potato (Solanum tuberosum) is indigenous to the central Andean region of South America

and was introduced into Europe by the Spanish in the 16th century. Currently, the potato is the

world’s 4th largest food crop and the leading vegetable crop in the US, with per capita

consumption of about 126 lbs annually [98-100]. The International Year of the Potato (2008) was

officially launched at United Nations headquarters (New York) in October 2007 to focus world

attention on the role potato can play in providing food security and eradicating poverty [101].

This popularity makes potatoes and potato products an attractive “delivery system” for

polyphenols in humans. Potatoes are often viewed as unhealthy by consumers, nutritionists and

the media due to their high content of carbohydrates and forms in which they are consumed (e.g.

French fries and potato chips) [102]. Potatoes are, however, rich in variety of functional

components such potassium and vitamin C.

Potatoes contain a number of biologically active secondary metabolites including

polyphenols. The polyphenol content of potato tubers ranges from 530-1770 µg/g fresh weight

(gfw) [103]. Major potato phenolic acids include caffeic acid, chlorogenic acid, ferulic acid, and

cryptochlorogenic acid. Depending on skin and flesh color potatoes may also be rich sources of

anthocyanins (15). Purple-fleshed potatoes are rich in phenolic acids and anthocyanins. Previous

studies [104-106] [107, 108] have shown that phenolic acids are present at amounts 5–12 times

higher in purple-fleshed potatoes compared to their white-fleshed counterparts. Purple-fleshed

cultivars have also been shown to exhibit ~ 10–20 times greater anti-oxidant activity compared to

white-fleshed potatoes, which may be attributed mainly to the presence of anthocyanins and

17

greater amounts of phenolic acids [109, 110]. However, the anthocyanin content in these specialty

cultivars varies greatly by variety of the potato (6-300 mg/100 gfw). The major anthocyanins

identified were coumaryl–rutino–glucosides of petunidin, peonidin, malvidin and pelargonidin,

[103].

1.3.4 Resveratrol and grape seed extract

Grapes are one of the most widely consumed fruits in the world and are rich in

polyphenols. Resveratrol (trans-3, 5, 4' trihydroxystilbene, RSV) is a polyphenol found in grapes

and red wine among other plant products [111]. RSV is synthesized in plant species in response

to stress by the enzyme trihydroxystilbene synthase. Fresh grape skins contain 50-100 mg RSV

per gram, and the concentration in wine ranges from 0.2 mg/l to 7.7 mg/l [112].

Approximately 60-70% of the polyphenols in grapes are found in the seeds and take the

form of PAC dimers, trimers and other oligomers. Grape seed PACs possess chemopreventive

and/or chemotherapeutic effects in various cell culture and animal models [113-115] . Grape seed

extract (GSE, lacks RSV) is a bioactive mixture that is commonly consumed as a dietary

supplement and is sold in the form of capsules or tablets (100–500 mg) [116]. The antioxidant

capacity of GSE is greater than known antioxidants such as vitamin C and E [115].

1.4 Anti-colon cancer effects of anthocyanins, resveratrol and GSE

1.4.1 Models of cancer

To study the etiology of cancer and test new chemoprevention strategies pre-clinical

models are widely used. Technological advancements in the 21st century such as genomics,

18

proteomics have allowed new methods for direct evaluation of clinical samples, however

preclinical models are still the most reliable to test new chemoprevention strategies [117]. In

cancer chemoprevention research, cancer cells derived from human cancers and animals are two

of the widely-used pre-clinical models. The development of cervical cancer HeLa cell line

revolutionized the field of cancer research. It was derived from cervical cancer cells taken from

Henrietta Lacks in 1951 [118] and allowed researchers to grow these cells indefinitely (due to

sustained proliferation capability of cancer cells). Since then, hundreds of cancer cell lines from

various types of cancers including colon cancer have been isolated and propagated either in vitro

as monolayer cultures or in vivo as xenografts in mice [117]. Cancer cell lines offer several

advantages, such as they are cost effective, easy to use, provide an unlimited supply of material

and bypass ethical concerns associated with the use of animal and human tissue. Cell lines also

provide a pure population of cells, which is valuable since it provides a consistent sample and

reproducible results. However, they do have certain drawbacks as in – tumor environment is lost

when culturing cells, repeated division of cultured cells results in further mutations making it

difficult to draw direct comparison of different studies. Nevertheless, cell culture models provide

hypotheses for future animal and human studies.

Animal models with features of specific human colorectal cancers offer an advantage

over cell lines and hence are used to test strategies for cancer prevention and treatment. They

offer the advantage of modeling human colon cancer where recapitulation of the molecular

etiology, pathology, and clinical progression of the disease is possible. Animal models used for

intestinal/colon cancer fit into three categories: spontaneous intestinal cancers in various animal

species, chemically or environmentally induced cancers in rodents, and cancers induced by

genetic manipulation of mice. Among these, rodents/mice are most widely used because they are

cost-efficient and high similarity to human genome.

19

Chemically/environmentally induced colon cancer in mice is one of the widely-used

model to test chemoprevention strategies. DNA alkylating agent 1,2 dimethyl-hydrazine and its

derivative azoxymethane [119] are two of the most commonly used chemicals used for inducing

colon cancer in mice [120, 121]. These colon specific carcinogenic agents are typically injected

intraperitoneally or subcutaneously over several weeks to induce development of tumors in the

distal colon [122]. In addition, tumor incidence and multiplicity can be altered by both genetic

background and by diet. This makes the models useful for the study of gene-gene and gene-

environment interactions that influence the pathogenesis of colorectal cancer [120].

There is epidemiological evidence that long-term intakes of polyphenols can reduce the

incidence of cancers and chronic disease [123]. Traditionally, polyphenols were mainly studied

for their organoleptical properties such as color (anthocyanins), astringency (tannins), and

bitterness (flavonols) [124], as well as to their physiological importance to plants [125-129]. Over

the past two decades polyphenols are being increasingly investigated for their ability to reduce the

risk of chronic diseases, because of their free radical scavenging capacity, which, among other

biological effects, increases antioxidant activity and prevents cellular damage to DNA [85]. Since

oxidative damage to DNA is considered as one of the crucial steps to onset of cancer [130], the

anti-oxidant effect of polyphenols lead to many studies investigating their chemopreventive

properties.

1.4.2 In vitro studies

Dietary polyphenols modulate different cellular processes (pleiotropic effects) on cancer

cells in vitro, acting as chemopreventive blocker agents, chemopreventive suppressor agents, or

both [131]. Chemopreventive blocker agents act immediately before or after initiation of

carcinogenesis, and chemopreventive suppressor agents act after initiation, during the prolonged

20

phases of promotion and progression [132]. Polyphenols either as individual compounds,

combination or in whole-food matrix have been shown to affect molecular events involved in the

initiation, promotion and progression of cancer, thereby inhibiting carcinogenesis. In vitro, the

anti-cancer effects of polyphenols are usually tested in colon cancer cells isolated from primary

human colon cancer tumors.

1.4.2.1 Anthocyanins

Berries were one of the first anthocyanin rich food sources evaluated for their anti-cancer

activity in vitro in cancer cells. Treatment of anthocyanin rich extracts of berries such as

blueberry, bilberries and chokeberries for 48 hours at 10-75 μg/mL have been shown to inhibit

the growth of advanced stage human colon cancer HT-29 cells but did not affect the growth of

non-malignant colon-derived cells [133]. Anthocyanins from tart cherries significantly reduced

proliferation of early and advanced human colon cancer cells HCT-116 and HT-29 respectively

285 μM and 780 μM respectively [134, 135]. To evaluate whether concentration or composition

dictates the anti-cancer activity of anthocyanins, HT-29 cells were treated with several berry

extracts containing different profiles of phenolic compounds (anthocyanins, flavonols and

tannins). All the berry extracts studied decreased the proliferation and induced cell cycle arrest 24

hours post treatment (concentration ranging from 0-60 mg of extract per mL). This correlated

with their anthocyanin concentration supporting the fact that the inhibitory effect of berry extracts

is based on the concentration rather than the composition of anthocyanins [136-138]. Further

investigation into the anti-proliferative effects of anthocyanins revealed the suppression of cell

cycle regulatory proteins (e.g., p53, p21, p27, cyclin D1, cyclin A, etc.) that participate in

proliferation [139]. Apart from berries, anthocyanin extracts from vegetables such as purple corn

[140] , carrot, radish were also shown to inhibit proliferation of HT-29 colon cancer cells [141].

21

Recently, we have shown that anthocyanins from color-fleshed potatoes at 30 μg gallic acid

equivalent (GAE)/mL inhibit proliferation in colon cancer cells HCT-116 and HT-29 even after

storage and processing [142].

Apart from inhibiting cell proliferation of colon cancer cells, anthocyanins have been

shown to exhibit pro-apoptotic activity [143-145]. The induction of apoptosis was via

mitochondrial (intrinsic) or FAS (extrinsic) pathway [145, 146]. In the intrinsic pathway,

anthocyanin treatment of cancer cells resulted in an increase in mitochondrial membrane

potential, cytochrome c release and modulation of caspase-dependent anti- and pro-apoptotic

proteins which aid in induction of apoptosis [139]. It has further been shown that anthocyanin

treatment leads to accumulation of reactive oxygen species, triggering mitochondrial mediated

apoptosis. In the extrinsic pathway, anthocyanins modulate the expression of FAS and FASL

(FAS ligand) in cancer cells resulting in apoptosis [147]. Recently, we have also recently reported

that color-fleshed potato anthocyanins even after processing (such as frying, baking) suppress

proliferation and induce apoptosis in HCT116 and HT29 cancer cells [142].

1.4.2.2 Resveratrol and grape seed extract

The anti-cancer activity of resveratrol was first reported by Jang et al. [148] against skin

cancer in mice. Since, then there have been many studies investigating anticancer and cancer

chemopreventive efficacy in numerous cancer models in cell culture. We previously reported that

RSV suppressed HT-29 human colon cancer cell proliferation and elevated apoptosis even in the

presence of growth factors via suppression of Wnt signaling pathways and activation of p53

[111]. Other studies have also shown that RSV (100 µM) induced apoptosis independently of p53

in HCT-116 human colon cancer cells via mitochondrial intrinsic apoptotic pathway [149]. In

Caco-2 human colon cancer cells, RSV (> 100 µM) inhibited growth and proliferation, induced

22

apoptosis via mitochondrial mediated pathway, and induced cell cycle arrest via modulation of

cyclins and cyclin dependent kinases [150].

Chemopreventive potential of GSE, a widely used dietary supplement has been studied

extensively in a variety of cancer types [115]. GSE (> 50 µg/ml) suppressed proliferation of

human colon cancer cell lines LoVo and HT-29. GSE induced G1 phase arrest and

mitochondrial/caspase mediated apoptosis in cancer cells [151]. Additionally, we have also

shown that GSE induced apoptosis in HCT-116 cells [152]. GSE might thus exert its beneficial

effects by suppressing proliferative and elevating apoptosis pathways.

Phytochemicals administered as individual components didn’t result in a beneficial effect

unlike the epidemiological evidence supporting health benefits of diets rich in fruits, vegetables

and whole grains [153]. Relatively high doses of single bioactive agents may show potent anti-

carcinogenic effects, however, the synergistic interactions between different dietary ingredients

that potentiate the activities of any single constituent better explain the observed benefits of

whole foods and diets in many epidemiological studies [154, 155]. In a recent study that

compared GSE induced anti-cancer effects to the effects of its individual components, the

researchers found that GSE was more potent in growth inhibition compared to its individual

constituents epigallocatechin and PACs [156].

In addition, bioactive compounds may have pleiotropic effects that in combination reduce

the risk of chronic disease. Different compounds might target different pathways and the net

effect might be a greater suppression of cancer cell growth [55, 157]. Over the last few years,

there have been many studies on bioactive components and their synergistic anti-cancer effects

[158-164]. We have previously shown that RSV potentiates the suppression of proliferation and

elevation of apoptosis in HCT-116 p53 +/+ human colon cancer cell lines by GSE. The dose of

RSV was reduced to 25 µM from 100 µM , when combined with GSE at doses of 35-50 µg/ml.

The induction of apoptosis by RSV-GSE combination was via activating p53 (pp53), elevating

23

Bax and suppressing Bcl-2 (increase in the Bax: Bcl-2 ratio) in p53 +/+ cells, which alters

mitochondrial membrane permeability. Such altered mitochondrial membrane permeability

releases cytochrome C into the cytosol [165, 166] that triggers activation of caspase-9, which

accelerates apoptosis by activating other caspases. RSV-GSE combination did not suppress the

proliferation or induce apoptosis of normal colon epithelial cell CRL-1831 line indicating that the

RSV-GSE combination preferentially target cancer cells while sparing their normal counterparts.

Thus, in vivo confirmation of these results to determine the efficacy of the RSV-GSE

combination is necessary.

1.4.3 In vivo studies

The strong anti-colon cancer effect observed in vitro paved way for animal studies using

rodent models. Anthocyanins, resveratrol and GSE have been shown to suppress colon cancer in

mice and rat models of genetically or chemically induced colon cancer.

Early studies in dimethylhydrazine-induced rat colon cancer models showed significantly

decreased total tumors as well as aberrant crypts by anthocyanins from berries and corn [119,

167-170]. ApcMin/+ mice (genetically induced intestinal tumorigenesis) treated with anthocyanin-

rich extracts from berries showed reduction in tumor burden and number [135, 171].

Unfortunately, the dietary dose, when extrapolated by dose/ surface area comparison to humans,

was found to be very high. Further, it was shown that berry extract containing a mixture of

anthocyanins was more efficacious than a single anthocyanin. Furthermore, route of

administration (mixed in diet vs water) was also shown to have a differential effect, with diet

based administration being more effective [135], possibly in part due to unstable nature of

anthocyanins in aqueous solution at neutral pH [172]. Investigation at molecular level revealed

24

that accumulation of β-catenin was inhibited thus resulting in reduction of polyps [173]. In the

azoxymethane (AOM) -induced model of colon cancer in F344 rats[119], diets containing 2.5, 5

and 10% lyophilized black raspberries significantly decreased total tumors (adenomas and

adenocarcinomas) by 42, 45 and 71% respectively [168]. Unfortunately, also in this study, the

dose of berries in the diets was high and could not be easily reached in a human diet

In vivo studies on resveratrol also depicted an anticancer effect on colon cancer.

Resveratrol mixed in drinking water at 200 ug/kg/day suppressed the growth of colorectal

aberrant crypt foci in F344 rats [174]. Treatment with resveratrol for 7 weeks (daily intake

calculated to be between 0.3 and 0.4 mg/mouse per day) in 5-week-old male ApcMin/+ mice

resulted in a 70% reduction in small intestinal tumors compared with vehicle-treated control

animals. Markers for cell cycle progression and proliferation cyclins D1 and D2 were shown to be

decreased [175].

GSE was also shown to significantly inhibit AOM-induced colonic aberrant crypt foci, a

precursor lesion for colon cancer in rat dual-organ tumor model [177]. GSE was able to suppress

the colonic macroscopic and microscopic damage in 2,4,6-trinitrobenzene sulfonic acid-induced

ulcerative colitis in rats [178]. Similar effect was also observed in rats with dextran sodium

sulfate induced colitis, where treatment of GSE resulted in improved colon epithelial health

[179]. However, the synergistic effect of resveratrol and GSE in combination has not been

evaluated in animal models of colon cancer.

1.4.4 Polyphenols against colon cancer stem cells

The emergence of cancer stem cells (CSCs) as the primary drivers of cancer, resistance to

chemotherapy and relapse has led to research for novel chemopreventive strategies that

effectively target CSCs. Polyphenols from a variety of sources have shown antioxidant,

25

antiproliferative, and pro-apoptotic effects on a variety of cancers, including colon cancer [180,

181]. Since almost every cancer has been shown to contain a sub population of CSCs [46], this

paved the way for studies to evaluate the efficacy of polyphenols in targeting CSCs.

In this regard, several phytochemicals, such as curcumin, a common flavoring agent and

an active ingredient of the spice turmeric; quercetin, a major flavonoid in capers and berries;

piperine, an alkaloid found in black pepper; have been recently shown to target colon CSCs

[182].

Curcumin was one of the first plant based compound shown to target colon CSCs.

Curcumin was shown to inhibit the proliferation of CD133+ colon CSCs enriched from HCT-116

and HT-29 cells via suppression of epidermal growth factor receptor (EGFR) and STAT3. EGFR

and STAT3 are involved in cell growth and involved in various hallmarks of cancer. An analogue

of curcumin, GO-Y030 was shown to target sphere formation (tumor formation ability) of

ALDH+/CD133+ colon CSCs when used at 2 to 5 µM concentrations. This ability was also

shown to extend in vivo using immunocompromised mice, where tumor size was reduced by up

to 58.10% upon injection at 50 mg/kg of body weight [183].

Treatment of CD133+ colon CSCs at a concentration of 75 µM with quercetin resulted in

significant (50%) inhibition of proliferation. In addition, it was also shown that when combined

with 50 µM quercetin, doxorubicin doses were more effective at inhibiting CSC proliferation in

vitro than doxorubicin doses three times more concentrated but lacking quercetin [184]. However,

the study did not look at any of the key genes/proteins involved in colon CSCs maintenance and

function.

Piperine, an alkaloid in black pepper induced cell cycle arrest, endoplasmic reticulum

stress, and apoptosis against HT-29 colon cancer cells at concentrations between 75 and 150 µM

[185]. Further, piperine has also been shown to target self-renewal and sphere formation ability of

colon cancer cells, suggesting the inhibiting effect of piperine on CSCs [186].

26

One common theme across all the studies that looked at colon CSCs targeting ability of

plant derived compounds predominantly were based on individual compounds whose

concentration were either too high to achieve in humans. Further, these studies have mainly

focused on the compound(s) ability to target proliferation, apoptosis and sphere formation

abilities of colon CSCs. Their effect on some of the key signaling pathways responsible for colon

CSCs functioning such as the Wnt/β-catenin pathway has not been studied.

1.5 Purpose and significance

Colorectal cancer is the 3rd most common cancer in both men and women constituting

10% of new cancer cases in men and 11% in women. Despite the use of surgical resection and

chemotherapy, nearly 50% of patients develop recurrent disease, highlighting the need for

improved therapies. Further, the cost of treating colon cancer is estimated to cost around

$150,000 per person per year.

One of the major questions we need to ask is “Are we targeting the right cells?” CSCs

possess the capacity for self-renewal, show the potential to develop into any cell in the overall

tumor population, have the ability to drive continued expansion of the population of malignant

cells, and invade and metastasize. Therefore, strategies that target colon CSCs could be effective

in eliminating colon tumors and reducing the risk of relapse and metastasis.

APC in Wnt/β-catenin pathway functions as a gatekeeper tumor suppressor gene.

Mutations in Apc or β-catenin are present in 80 % of colon cancer cases and are sufficient to

promote further progression with additional mutations. Moreover, dysregulation of Wnt/β-catenin

signaling in stem cells, but not in other crypt cells results in polyp formation, identifying the stem

cell as the cell-of-origin of cancer.

27

Current chemotherapeutic approaches are designed to target only a single hallmark of

cancer cells or a combination therapy is used to target multiple hallmarks. Non-steroidal anti-

inflammatory drugs (NSAIDs) such as sulindac have shown to prevent colon cancer in rodent

models. In a mice model of intestinal cancer, sulindac induced apoptosis in intestinal CSCs with

accumulated β-catenin. However, NSAIDs cause side-effects, including gastrointestinal bleeding,

perforation, renal toxicity, and even death.

Anthocyanin rich extracts of Java Plum (Eugenia Jambolana) have been shown to

effectively target breast and prostate cancer cells. Java Plum is a tropical fruit that is widely

consumed in Asia and developing evidence of targeting colon CSCs could pave way for its use

and further studies using indigenous food sources rich in polyphenols across developing countries

where access to high-quality medicare is limited.

Our previous data has shown that anthocyanin rich extracts from PP even after processing

suppressed proliferation and induced apoptosis in colon cancer cell lines HCT-116 and HT-29.

Potatoes are widely consumed in the US with per capita consumption of 56 Kgs and evaluating

the effect of PP against colon CSCs can help increase consumption of color-fleshed varieties over

white-fleshed potatoes. Similarly, we have shown that RSV and GSE suppressed HCT-116 colon

cancer cell growth by multiple hallmarks including induction of apoptosis, suppression of

proliferation and cell cycle arrest; however, limited knowledge is available regarding their effects

on CSCs. Further, there are no studies that have shown the ability of these polyphenols to target

colon CSCs in vivo.

28

1.6 Hypothesis and objectives

Polyphenols from Java plum, purple-fleshed potatoes and resveratrol-grape seed extract

(RSV-GSE) combination have shown chemopreventive effects against colon cancer. However,

their effect against colon CSCs in vitro and in vivo is limited. Therefore, I hypothesize that these

extracts will inhibit colon CSCs in vitro by targeting their self-renewal properties. I further

hypothesize that dietary supplementation with baked purple-fleshed potato or RSV-GSE

combination can reduce tumor formation and colon CSCs numbers via inhibition of the Wnt/β-

catenin signaling pathway in a chemically induced mice model of colon cancer. In order to test

these hypotheses, I propose the following specific aims:

1) To investigate the anti-cancer properties of the anthocyanin extracts of Java Plum on

colon cancer cells HCT-116 and colon CSCs in vitro (Chapter 1).

2) Determine whether purple-fleshed potato even after processing can target colon CSCs in

a AOM-induced mouse model of colon cancer. Further, determine the molecular

pathways of proliferation and apoptosis targeted in colon CSCs (Chapter 2).

3) Evaluate the efficacy of the RSV­GSE combination in targeting colon CSCs in a AOM-

induced mice model of colon cancer in comparision to sulindac. Further, determine the

molecular pathways of proliferation and apoptosis targeted in colon CSCs (Chapter 3).

29

Chapter 2

Eugenia jambolana (Java plum) fruit extract exhibits anti-cancer activity

against early stage human HCT-116 colon cancer cells and colon cancer stem

cells*

* These results have been published as the following manuscripts:

Charepalli V, Reddivari L, Vadde R, Walia S, Radhakrishnan S, Vanamala J. Eugenia jambolana

(Java Plum) Fruit Extract Exhibits Anti-Cancer Activity against Early Stage Human HCT-116

Colon Cancer Cells and Colon Cancer Stem Cells. Cancers. 2016, 8, 3-29.

30

2.1 Abstract

The World Health Organization predicts over 70 % increase in cancer incidents in

developing nations over next decade. Although, these nations have limited access to novel

therapeutics, they do have access to foods that contain chemopreventive bioactive compounds

such as anthocyanins, and as such, consumption of these foods can be encouraged to combat

cancer. We and others have previously characterized the anti-colon cancer properties of dietary

anthocyanins from different sources. Eugenia jambolana (Java plum) is a tropical medicinal fruit

rich in anthocyanins, however, its anti-colon cancer properties are not well characterized.

Furthermore, recent evidence suggests that colon cancer stem cells (colon CSCs) promote

resistance to chemotherapy, relapse of tumors and contribute to poor prognosis. The objectives of

this study were to 1) characterize the anthocyanin profile of Java plum using HPLC-MS; and 2)

determine the anti-proliferative (cell counting and MTT) and pro-apoptotic (TUNEL and caspase

3/7 glo assay) properties of Java plum fruit extract (JPE) using HCT-116 colon cancer cell line

and colon CSCs (positive for CD 44, CD 133 and ALDH1b1 markers). HPLC-MS analysis

showed that JPE contains a variety of anthocyanins including glucosides of delphinidin, cyanidin,

petunidin, peonidin and malvidin. JPE anthocyanins suppressed (P < 0.05) proliferation in HCT-

116 cells and elevated (P < 0.05) apoptosis in both HCT-116 cells and colon CSCs. JPE also

suppressed the stemness in colon CSCs as evaluated using colony formation assay. These results

warrant further assessment of the anti-cancer activity of JPE, and its molecular mechanisms using

pre-clinical models of colon cancer.

31

2.2 Introduction

Colon cancer is the second leading cause of cancer related deaths in the United State. For

the year 2014, the American Cancer Society estimated that there would be about 136,803 new

cases and 50,310 deaths due to colon cancer [187]. Colon cancer is caused by step-wise

accumulation of mutations in tumor suppressor and oncogenic genes, resulting in the formation of

polyps which ultimately leads to adenocarcinoma [188]. There is increasing evidence that most

cancers including colon cancer have a hierarchy of cells with cancer stem cells (CSCs) forming

the core and sustaining the growth of the tumor [189]. CSCs including colon CSCs mimic the

functionality of normal adult stem cells maintaining their un-differentiated state while dividing

non-symmetrically [190]. They are also resistant to conventional therapies, thus leading to relapse

of cancer in most patients [191, 192]. Agents that target CSCs could be more efficacious and aid

in preventing relapse.

Geographic differences in colon cancer rates and temporal changes in risk among

immigrant populations suggest that diet and lifestyle strongly influence the occurrence of colon

cancer. Although research is still accumulating on the role of specific dietary elements on

colorectal cancer risk, current evidence indicates that higher intake of certain diets including high

in fat or red meat and lower intake of diet rich in fruits and vegetables is linked to a higher risk

for colon cancer. However, unlike most cancers, colon cancer has a long latency period before it

is detected (such as aberrant crypts) [193]. There is increasing evidence of preventive/protective

role of dietary bioactive compounds such as anthocyanins from fruits, vegetables, and herbs

against a variety of cancers including colon cancer [11, 194]. Individual anthocyanins, food-

derived anthocyanin extracts and consumption of anthocyanin-rich foods exhibit anti-cancer

properties in both in vitro and in vivo studies [16, 195, 196]. We have previously shown that

anthocyanins from potato extracts suppressed cell proliferation and induced apoptosis in early

32

(HCT-116) and advanced (HT-29) human colon cancer cell lines [142]. However, there is a

dearth of data on the anti-cancer properties of anthocyanins against CSCs including colon CSCs.

The World Health Organization (WHO) has predicted that there will be 70% increase in

cancer incidence in the developing countries [197]. More than 60 percent of the world’s new

cancer cases occur in Africa, Asia, and Central and South America; 70 percent of the world’s

cancer deaths also occur in these regions [198]. Although, these nations have limited access to

latest pharmaceutical drugs, people in these countries have access to foods that contain

chemopreventive bioactive compounds such as anthocyanins, and as such, consumption of these

foods can be encouraged to combat/prevent cancer. In the US and other developed countries,

there is an increased public awareness of complementary and alternative medicinal approaches

for chronic disease prevention, including different cancers. Although, research on targeted

pharmacological approaches is growing, the risk for different cancer does not seem to subside.

National expenditures for cancer care in the United States totaled nearly $125 billion in 2010 and

could reach $156 billion in 2020 [199]. Hence, dietary approaches for cancer prevention,

including identification of new or development of existing dietary bioactive compound-rich foods

are required.

The native Indian tree, Eugenia jambolana (common name: Java Plum) is found widely

in the Asian sub-continent and other tropical regions of the world [200]. In the United States,

Eugenia jambolana is found in Florida and Hawaii (USDA Natural Resource Conservation

Service Plant Database). This underutilized tropical evergreen tree yields small purple ovoid

fleshy fruits with an astringent taste. This fruit is valued for its diverse chemical constituents as

well as medicinal and therapeutic properties [94, 95]. In traditional Indian medicine, both the fruit

pulp and seed extracts have a long history of medicinal use and they have been extensively

studied for their anti-diabetic properties [96, 97]. Previous studies have identified the major

anthocyanins in Java plum fruit pulp/skin as diglucosides of delphinidin, petunidin and malvidin

33

[200-202]. These anthocyanins are responsible for imparting the ripened fruit its bright purple

color. Java plum fruit extract (JPE) has been shown to exhibit anti-proliferative and pro-apoptotic

effects in estrogen dependent/aromatase positive, and estrogen independent breast cancer cells

[202, 203]. However, there is a lack of literature on the anti-colon cancer properties of

anthocyanin-rich Java plum, particularly given the strong evidence of anti-cancer effects of

dietary anthocyanins [139]. Furthermore, the evidence of effect of anthocyanin-rich foods against

colon CSCs remains elusive. Thus, the current study was conducted to 1) characterize the

anthocyanin profile of JPE, and 2) determine the anti-cancer properties of the JPE on HCT-116

and colon CSCs.

2.3 Materials and methods

2.3.1 Extraction and Purification of Anthocyanins from Java Plum

The anthocyanin-rich fruit skin and pulp was carefully removed from the whole fruit and

extracted with acidified methanol (0.1 % HCl). The extract was concentrated under vacuum (40

0C) in a rotary evaporator for complete removal of the solvent. The concentrated extract was then

dissolved in acidified water and partitioned with ethyl acetate to remove phenolics, flavonoids

and/or carotenoid constituents. The aqueous extract was again concentrated under vacuum (40 ± 1

0C) to obtain anthocyanin concentrate. For further purification, anthocyanin-rich concentrate was

adsorbed onto activated XAD-16 Amberlite resin column and eluted with 3 bed volumes of

acidified water (0.1 % HCl) to remove sugars, acids and/or other undesired water-soluble

compounds. Anthocyanins adsorbed on the resin were subsequently eluted with acidified

methanol. The methanolic extract was then concentrated in a rotavapor at 40 °C under vacuum.

The resultant violet concentrate was dissolved in distilled deionized water containing 0.1 %

34

hydrochloric acid and lyophilized to get purified anthocyanin powder. The material was stored at

-40 0C until further investigations.

2.3.2 Chemicals

Fetal bovine serum (FBS) was purchased from HyClone (Pittsburgh, PA). All other

chemicals and reagents were purchased from Sigma (St Louis, MO).

2.3.3 High Performance Liquid Chromatography Mass Spectrometry (HPLC-MS) Analysis

JPE was dissolved in methanol to attain a concentration of 1 mg/mL. Anthocyanin profile

of the powder concentrate was evaluated by a Waters Alliance HPLC (Pittsburg, PA) equipped

with Waters e2695 quaternary pump and 2998 photodiode array detector. Anthocyanin sample

(20 μL injection volume; 1.0 mg/mL concentration) was analyzed on a Phenomenex (Torrance,

CA) RP-18 column (5 μM, 4.6 x 250 mm) using a gradient from solvent A (water, 0.1 %

trifluoroacetic acid (TFA)) to solvent B (water:ceric ammonium nitrate (CAN):TFA – 53:46:1

v/v/v) at a flow rate 0.6 mL/min. Gradient: Initiallly at 20 % B, then increased to 40 % in 26

minutes, and thereafter to 80 % in 4 minutes and held for additional 10 minutes. Anthocyanins

were monitored at 520 nm.

The resulting column eluent was infused into a Micromass Q-Tof Micro MS fitted with

an electrospray source (ESI) and analyzed for its constituents with the help of ESI-MS/MS

spectrometer. Data was collected in positive ion mode, scanning from 50-1200 at a rate of 0.9

scans per second with 0.1 second interscan delay. Calibration was performed prior to sample

analysis via infusion of sodium formate solution, with mass accuracy within 5 ppm. The capillary

voltage was held at 2200 V, the source temp at 130 0C, and the desolvation temperature at 300 0C

35

at a nitrogen desolvation gas flow rate of 400 L/hour. The quadrupole was held at collision

energy of 7 V. Peak identities were obtained by matching their molecular mass [M]+ and MS/MS

fragmentation ions as shown in Table 1 and Fig 1 and by comparison to published data [204].

ESI-MS has been successfully employed earlier for the characterization of bioactive azadirachtins

in neem [22] and anthraquinones in Rheum emodi [23].

2.3.4 Cell Lines

Colon cancer cell lines HCT-116 were a generous gift from Dr. Bert Vogelstein (School

of Medicine, Johns Hopkins University, Baltimore, MD, USA). Cells were maintained at 37 0C in

a humidified atmosphere with 5 % CO2 and grown in McCoy’s F-12 supplemented with 10 %

FBS, 2.2 g/L sodium bicarbonate, 0.2 g/L bovine serum albumin and 10 mL/L streptomycin-

penicillin mix as described earlier [152].

Colon cancer stem cells (colon CSCs), positive for cancer stem cell markers CD 133, CD

44, and ALDH1b1, were obtained from Celprogen (San Pedro, CA). To maintain the cells in their

undifferentiated state, colon CSCs maintenance media and specially coated cell culture flasks

obtained from Celprogen were used. Cells were maintained in incubation at 37 0C and 5 % CO2 as

described earlier [205]. Cell cultures at approximately 80 % confluence were used for all in vitro

experimental procedures.

2.3.5 Cell Viability

MTT Assay

The cellular viability was evaluated using an assay based on the cleavage of the yellow

dye MTT (3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl tetrazolium bromide) to purple formazan

36

crystals by dehydrogenase activity in mitochondria. Briefly, 20,000 HCT-116 cells were seeded

in a 96-well plate and after 24 hours, cells were treated with JPE at 30 and 40 µg/mL. After 24

hours, cells were rinsed with media and then they received MTT diluted in media for 4 hours as

per the manufacturer’s protocol (Roche Diagnostics, Indianapolis, IN). SDS/NaOH was used to

dissolve the purple formazan crystals, and the optical density of the solution was measured at 570

and 690 nm. The experiment was performed in triplicate, and the data were expressed as the mean

± S.E.

Cell Counting

Briefly, 100,000 HCT-116 cells were plated in a 12-well plate for 24 hours. They were

treated with JPE at 30 and 40 µg/mL. After 24 hours, 20 μL of the suspension were put in

specialized slides obtained from Nexcelom Bioscience (Lawrence, MA) and then inserted in

Nexcelom automated cell counter. The experiment was performed in triplicate, and the data were

expressed as the mean ± S.E.

2.3.6 Apoptosis

Caspase Glo 3/7 Assay

Briefly, 100,000 cells (HCT-116 and colon CSCs) were seeded in a 12-well plate and

incubated for 24 hours. They were treated with JPE at 30 and 40 µg/mL, after 24 hours, cells

were trypsinized and approximately 20,000 cells from each treatment were incubated with 100

µL of Caspase Glo 3/7 reagent (Promega, Madison, WI) for 30 minutes in a 96 well plate. The

luminescence of each sample was measured using a BioTek micro plate reader (Winooski, VT).

The experiment was performed in triplicate, and data are expressed as means ± S.E.

TUNEL Assay

37

Apoptosis was also assessed by terminal deoxynucleotidyl transferase-mediated dUTP

nick end labeling (TUNEL) assay using an In Situ cell death detection kit from Roche

Diagnostics. Experiments were carried out in accordance with the manufacturer's recommended

procedures. Briefly, JPE treated HCT-116 cells grown on glass coverslips were fixed with 4 %

paraformaldehyde in PBS and were permeabilized with 0.1 % Triton X-100 in 0.1 % sodium

citrate in PBS. They were then stained with the TUNEL reaction mixture and finally examined

using a fluorescence microscope. At least 400 cells per treatment were counted and the results are

expressed as percentage apoptosis (% ratio of apoptotic cells/total cells). The experiment was

performed in duplicate and data are expressed as means ± S.E.

2.3.7 Colony Formation Assay

Ability of JPE to alter the stemness of colon CSCs was evaluated through colony

formation assay [205] by counting the number of colonies that can form after treatment. Briefly,

150,000 colon CSCs were seeded per well in a 6-well plate and incubated for 24 hours in

complete growth media. After 24 hours, growth media was removed and cells were treated with

JPE at 30 and 40 µg/mL for 24 hours. Cells were collected by trypsinization. One hundred treated

cells were seeded into each well of a new 6-well plate and incubated for 10 days in complete

growth media. At the end of 10 days, media was removed and cells were fixed using a fixing

solution (3.7 % paraformaldehyde in 70 % ethanol) for 10 minutes. The cells were stained with

0.05 % Coomassie blue for 20 minutes and then rinsed with PBS. Stained colonies were counted

under a dissecting microscope as described earlier [206].

38

2.3.8 Statistical Analysis

Data were analyzed by one-way ANOVA using Tukey least square difference (LSD) with

IBM SPSS software v22.0 (Armonk, NY).

2.4 Results

2.4.1 Evaluation of the Bioactive Compound Profile in JPE

Liya Li et al [204] previously have reported the anthocyanin profile of JPS. They also

reported that concentration and types of anthocyanins in JPE differs from the geographical region

of the berries. Five types of anthocyanins - delphinidin-diglucoside, cyanidin-diglucoside,

petunidin-diglucoside, peonidin-diglucoside, and malvidin-diglucoside were identified in their

paper using HPLC and LC-MS. In our study, we also found the five reported anthocyanins using

HPLC-MS (Figure 2-1 and Table 2-1). On the basis of mass spectral data, the three major

anthocyanin peaks were identified as delphinidin-3, 5-diglucoside (1), cyanidin-3, 5-diglucoside

(2), and petunidin-3, 5-diglucoside [207]. The remaining six minor anthocyanin constituents were

similarly characterized as diglucosides of peonidin (4), and malvidin (5), and monoglucosides of

delphinidin (6), cyanidin (7), petunidin (8) and malvidin (9).

39

2.4.2 JPE Suppressed Proliferation in HCT-116 Cells

We evaluated the anti-proliferative effects of JPE using the MTT assay. There was a dose

dependent suppression of cell proliferation in HCT-116 cells by JPE (data not shown). At 30

Table 2-1: Anthocyanins identified in Java plum fruit extract.

Peak Anthocyanin RT [M]+ ESI-PI(m/z)

1 Delphinidin- 3,5- diglucoside 12.68 627 465 [M-162]+; 303 [M-162-162]+

2 Cyanidin- 3,5- diglucoside 14.44 611 449 [M-162]+; 287 [M-162-162]+

3 Petunidin- 3,5-diglucoside 15.33 641 479 [M-162]+; 317 [M-162-162]+

4 Delphinidin- 3-glucoside 16.22 449 287 [M-162]+

5 Peonidin- 3,5- diglucoside 17.17 625 463 [M-162]+; 301 [M-162-162]+

6 Malvidin- 3,5- diglucoside 17.83 655 493[M-162]+; 331 [M-162-162]+

7 Cyanidin- 3-glucoside 18.74 449 287 [M-162]+

8 Petunidin- 3- glucoside 19.26 479 317 [M-162]+

9 Unknown 20.28 - -

10 Malvidin- 3-glucoside 22.14 493 331 [M-162]+

11 Unknown 24.02 - -

Figure 2-1: HPLC chromatogram of Java plum fruit extracts (JPE) anthocyanins; the peak number correspond to anthocyanins in table 2-1.

40

µg/mL and 40 µg/mL (Figure 2-2A), there was suppression of proliferation (P < 0.05) by over 60

% compared to control. Proliferation was also assessed by cell counting using an automated cell

counter (Nexcelom) by treating the cells with JPE at 30 µg/mL and 40 µg/mL to confirm our

observations with the MTT assay. Both concentrations resulted in more than 50 % reduction in

viable cell number (P < 0.05, Figure 2-2B).

2.4.3 JPE Induced Apoptosis in HCT-116 Cells and Colon CSCs

A hallmark of cancer is the ability of the cancer cells to evade apoptosis. Apoptosis can

be seen as an important barrier to developing cancer; thus avoiding apoptosis is integral to tumor

development and resistance to therapy [23]. In our study, we evaluated whether JPE extract can

induce apoptosis in both HCT-116 colon cancer cells and colon CSCs. Induction of apoptosis was

assayed by TUNEL assay, where fragmented DNA, characteristic of apoptotic cells, is used to

identify apoptotic cells. JPE at 30 µg/mL and 40 µg/mL induced apoptosis (P < 0.05) in HCT-116

cells compared to control (Figure 2-3A). Representative images of fluorescing cells indicating

Figure 2-2: Java plum fruit extracts (JPE) suppressed proliferation in HCT-116 cells. HCT-116 cells were treated with JPE (30 or 40 µg/mL) for 24 hours, MTT assay (A) and viable cell count (B) were performed as described in methods. Values are in means ± SE. Means that differ by a common letter (a, b, c) differ at p < 0.05.

41

apoptosis are presented (Figure 2-3C). Further, apoptosis was also confirmed using Caspase 3/7

Glo assay. The assay measures the activity of caspases 3 and 7, which are responsible for

fragmentation of DNA. JPE at 30 µg/mL and 40 µg/mL elevated (P < 0.05) caspase 3 and 7

dependent apoptosis in HCT-116 cells (Figure 2-3B) compared to control. Data from TUNEL

and Caspase 3/7 Glo assay confirms that JPE induces apoptosis in colon cancer cell line HCT-

116.

Figure 2-3: Java plum fruit extracts (JPE) induced apoptosis in HCT-116 cells; (A) Percent apoptosis in HCT-116 cells (n=400) as measured by TUNEL assay. (B) Apoptosis was also assayed using caspase 3/7 glo assay. Values are in means ± SE. Means that differ by a common letter (a, b, c) differ at p < 0.05. (C) Cells fluorescing bright green due to fragmented DNA, indicator of apoptosis using TUNEL assay. Pictures were taken on a fluorescence microscope at 20x magnification (12 fields per treatment and at least 500 cells were counted). Representative pictures are shown for Control, JPE at 30 µg/mL and JPE at 40 µg/mL.

As colon CSCs are typically resistant to standard care therapies, we evaluated if JPE can induce

apoptosis in colon CSCs using the Caspase 3/7 Glo assay. JPE at 30 µg/mL and 40 µg/mL

42

2.4.5 JPE Suppressed Colony Formation in Colon CSCs

Colon CSCs possess the ability to initiate and drive the growth of tumors due to their

self-renewal capability [208]. To assess the ability of JPE to target this capability, we

used colony formation assay (Figure 2-5). Single cell suspensions of colon CSCs treated

with JPE were grown in culture plates with complete growth media and the number of

colonies was measured as described in the methods. Colon CSCs when treated with JPE

at 30 µg/mL and 40 µg/mL respectively resulted in a dose-dependent suppression in

colony formation (Fig 5A). Figure 5B also shows representative images collected from

induced apoptosis even in colon CSCs (P < 0.05) by more than 75% and 165% respectively

compared to control (Figure 2-4).

Figure 2-4: Java plum fruit extracts (JPE) induced apoptosis in colon cancer stem cells (colon CSCs). Cells were treated with JPE (30 or 40 µg/mL) for 24 hours and caspase 3/7 glo assay was performed. Values are in means ± SE. Means that differ by a common letter (a, b, c) differ at p < 0.05.

43

the colony forming assay and demonstrates the decreased colony number associated with

JPE treatment compared to control. This shows that JPE affects colon CSCs self-renewal

ability and thus demonstrates anti-cancer activities beyond suppressing proliferation and

inducing apoptosis.

Figure 2-5: Effect of Java plum fruit extracts (JPE) on the stemness of colon CSCs. (A) Cells were treated with JPE (30 or 40 µg/mL) for 24 hours and colony formation assay was performed as described in methods. (B) Representative images taken from the colony forming assay for Control and JPE 30 are presented. Results were expressed as mean ± SE for three experiments at each time point. Means that differ by a common letter (a, b) differ at p < 0.05.

44

2.5 Discussion

In this study, we found that anthocyanin composition of JPE may be similar across

locations, however their concentrations might be different. In addition to anthocyanins, other

class of phenolic compounds identified in JPE include flavonols (Quercetin, Myricetin,

Kaempferol, Luteolin, Isorhamnetin), flavanones (Naringenin) and stilbenoid (Resveratrol) (data

not shown). All these compounds have been shown to exhibit anti-cancer properties [209, 210].

Beneath the complexity of every cancer lie critical events including deregulated cell

proliferation and suppressed apoptosis that provides a platform necessary to support further

neoplastic progression. Cell proliferation is essentially an increase in the number of cells as a

result of cell growth and cell division [23]. The suppression of proliferation by JPE in HCT-116

can be attributed to the presence of the identified compounds – anthocyanins, flavonols and

stilbenoids, which have been previously shown to suppress proliferation in colon cancer cells

individually and in combination in in vitro, in vivo and in human studies [111, 196, 211].

Previous studies with anthocyanin rich chokeberry extracts have shown that suppression of

proliferation in HT-29 colon cancer cells occurs via cell cycle arrest [212]. Thus, further

mechanistic studies are required to study molecular mechanism of anti-proliferative action of JPE

against colon cancer cells, including its effect on proliferative pathways and the cell cycle.

The pro-apoptotic effect of JPE can be attributed to mitochondrial-mediated apoptosis, as

the release of mitochondrial protein cytochrome c results in the step-wise activation of caspases

ultimately leading to DNA fragmentation. Indeed, anthocyanin rich extracts of blueberries have

been shown to activate caspase-3 in colon cancer cell line HT-29 [213].

For the first time, we show that anthocyanin rich JPE extract induces apoptosis in human

colon cancer cells HCT-116 and colon CSCs in a dose dependent manner. Our current results

show that anthocyanin rich foods can be used to target CSCs via elevating apoptosis. In addition,

45

recently curcumin - a major polyphenolic compound found in the Indian spice turmeric, was

shown to synergistically act with chemotherapeutic drug – FOLFOX in elimination of colon

CSCs [214]. Thus, further studies are required to evaluate anti-cancer properties of JPE alone or

in combination with chemotherapeutic drugs. The combination approach helps in lowering the

dosage, thus minimizing/eliminating side effects.

46

Chapter 3

Anthocyanin-containing purple-fleshed potatoes suppress colon

tumorigenesis via elimination of colon cancer stem cells*

*These results have been published as the following manuscript: Charepalli V#, Reddivari L#,

Radhakrishnan S, Vadde R, Agarwal R, Vanamala J, Anthocyanin-containing purple-fleshed

potatoes suppress colon tumorigenesis via elimination of colon cancer stem cells. Journal of

Nutritional Biochemistry. 2015, 26, 1641-1649. # equally contributed

47

3.1 Abstract

Cancer stem cells (CSCs) are shown to be responsible for initiation and progression of

tumors in a variety of cancers. We previously showed that anthocyanin-containing baked purple-

fleshed potato (PP) extracts (PA) suppressed early and advanced human colon cancer cell

proliferation and induced apoptosis, but their effect on colon CSCs is not known. Considering the

evidence of bioactive compounds, such as anthocyanins, against cancers, there is a critical need to

study anti-cancer activity of PP, a global food crop, against colon CSCs. Thus, isolated colon

CSCs (positive for CD 44, CD 133 and ALDH1b1 markers) with functioning p53 and shRNA-

attenuated p53 were treated with PA at 5.0 μg/mL. Effects of baked PP (20 % w/w) against colon

CSCs were also tested in vivo in mice with azoxymethane induced colon tumorigenesis. Effects

of PA/PP were compared to positive control sulindac. In vitro, PA suppressed proliferation and

elevated apoptosis in a p53 independent manner in colon CSCs. PA, but not sulindac, suppressed

levels of Wnt pathway effector β-catenin (a critical regulator of CSC proliferation) and its

downstream proteins (c-Myc and cyclin D1) and elevated Bax and cytochrome c, mitochondria-

mediated apoptotic proteins. In vivo, PP reduced the number of crypts containing cells with

nuclear β-catenin (an indicator of colon CSCs) via induction of apoptosis and suppressed tumor

incidence similar to that of sulindac. Combined, our data suggests that suppression of Wnt/β-

catenin signaling and elevated apoptosis via mitochondria-mediated apoptotic pathway by PP

may contribute to reduced colon CSCs number and tumor incidence in vivo.

48

3.2 Introduction

Colon cancer is the third leading cause of cancer related deaths in the United States [187].

There is mounting evidence that most cancers, including colon cancer, have a hierarchy of cells

with cancer stem cells forming the core and sustaining the growth of the tumor [215]. Cancer

stem cells, including colon cancer stem cells (colon CSCs), mimic the functionality of normal

adult stem cells maintaining their un-differentiated state while dividing non-symmetrically [190].

In vivo studies implicate Wnt/β-catenin signaling in the regulation of colon stem cell proliferation

[63]. In the canonical Wnt pathway, mutations in APC, a tumor suppressor gene, leads to

increased nuclear translocation of β-catenin and subsequent activation of Wnt transcriptional

targets ultimately causing adenoma [215, 216]. Nuclear translocation of β-catenin is implicated in

the transformation of stem cells to cancer stem cells in the colon [217]. P53, a critical tumor

suppressor gene called the guardian of the genome, is mutated in over 50% of cancers, including

colon cancer [218]. Mutated p53 allows uncontrolled proliferation and leads to progression from

adenoma to carcinoma [17]. Thus, it is important to test strategies developed against colon CSCs

work even in the absence of p53.

Sulindac, a non-steroidal anti-inflammatory drug (NSAID) eliminated colon stem cells

with nuclear β-catenin, an indicator of colon CSCs, and reduced polyp number in APCMin/+ mice,

a well-established model for colon cancer [219]. However, long-term use of NSAIDs, in

particular sulindac, is associated with adverse gastrointestinal and renal toxicities [220, 221].

Conversely, as colon cancer involves stepwise mutations in multiple genes, there is a long latency

period [222, 223] before it manifests and thus there is an opportunity to target colon cancer by

suitable modification of diet. There is increasing evidence of preventive/protective role of

bioactive components in the food against colon cancer. Purple-fleshed potatoes are a good source

49

of anthocyanins and phenolic acids, compounds that have also demonstrated anti-colon cancer

efficacy in different models [195, 207, 224].

Potato is one of the largest consumed food crops in the United States. Indeed,

consumption of color-fleshed potatoes increased by 17%, due to putative health benefits, while

traditional potatoes decreased during the last 10 years [225]. We have previously shown that PP

contains high levels of polyphenols such as anthocyanins compared to white-fleshed potatoes

(WP) and retain these levels even after baking [226]. Acetylation makes potato anthocyanins

more stable and distinguishable from other food sources such as berries [227, 228]. We also

showed that anthocyanin-containing PP extracts, even after baking suppressed proliferation and

induced apoptosis similar to raw PP extracts, in early and advanced colon cancer cell lines HCT-

116 and HT-29, respectively [226]. Colon CSCs in vitro have been shown to be targeted by

dietary bioactive compounds such as curcumin [214]. However, there are no laboratory studies

investigating the anti-cancer properties of dietary whole foods such as PP on colon CSCs. Given

that the potato is the most consumed vegetable in the US, the establishment of a link between

anthocyanin-containing PP and inhibiting colon CSCs could be very impactful.

Colon CSCs (positive for CD 44, CD 133 and ALDH1b1 markers) isolated from primary

human colon cancer tumors are a useful model for in vitro experiments to screen anti-cancer lead

compounds [229]. Azoxymethane [119] is a DNA alkylating agent and AOM-induced mouse

colon cancer in vivo models have been shown to be the best model to predict chemopreventive

efficacy [121]. AOM-induced tumors also exhibit aberrant APC expression and nuclear

localization of β-catenin [230, 231]. Thus, these in vitro and in vivo models were used to test the

anti-cancer properties of the anthocyanin-containing PP. Furthermore, we examined the possible

molecular mechanisms that underlie its anti-cancer activity.

50

3.3 Materials and methods

3.3.1 Chemicals

Ethanol, methanol and ethyl acetate were purchased from VWR International (Bristol,

CT). Antibodies for Bax, Bcl-2, β-actin (Actin), β-catenin, cyclin D1, c-Myc, and

Topoisomerase-2 Beta (TOP2B) were purchased from Santa Cruz Biotechnology (Santa Cruz,

CA). Cytochrome c was obtained from Cell Signaling Technology (Beverly, MA).

3.3.2 Plant material

Uniform-sized PP tubers (Purple Majesty variety) were baked in a conventional oven

preheated to 204 °C for 1 hour and 15 min. Before baking, each potato was washed, dried,

wrapped in food-grade aluminum foil, and pierced approximately 1.5 cm deep with a knife at 3

cm intervals. Baked potatoes were cooled for 15–20 minutes, diced with skin into pieces

weighing 7 ± 1 g, and stored at −20 °C. For in vitro experiments, ethanolic extracts of

anthocyanin-containing baked PP were prepared as per our published protocols [226]. Equivalent

doses of ethanol were used as solvent control for all in vitro experiments. Another batch of baked

PP was freeze dried, powdered, and stored at −20 °C before incorporation into diets for the mice

study.

3.3.3 Potato characterization

Ultra performance liquid chromatography and mass spectrometry (UPLC-MS) analysis of

white- (Atlantic variety) and purple-fleshed potato extracts (2 µL) was done using a Waters

Acquity UPLC system from Waters (Milford, MA) with a Waters HSS T3 column (1.8 μm, 1.0 ×

51

100 mm) and a gradient from solvent A (100 % water, 0.1 % formic acid) to solvent B (95 %

methanol, 5 % water, 0.1 % formic acid). Column eluent was infused into a Q-Tof Micro mass

spectrometer [116] fitted with an electrospray source. Data were collected similar to our earlier

published protocols [226]. Peak detection was performed using MarkerLynx software [116]. To

identify metabolite differences between potato varieties, we also carried out peak annotation

using METLIN metabolite database (http://metlin.scripps.edu) using simple, fragment, and

neutral loss search elements. Phenolic metabolite differences between white and purple-fleshed

potatoes are presented in Table 3-1.

52

3.3.4 Cancer stem cells

Colon cancer stem cells (colon CSCs), positive for cancer stem cell markers CD 133, CD

44, and ALDH1b1, were obtained from Celprogen (San Pedro, CA). To maintain the cells in their

undifferentiated state, colon CSCs growth media and specially coated cell culture flasks obtained

Table 3-1: Phenolic and anthocyanin composition of white vs purple-fleshed potatoes by UPLC/MS.

Compound

Identity

Molecular

Ion M+ (m/z)

Retention

Time

(minutes)

White-fleshed

Potato

Purple-fleshed

Potato

Phenolic Acids

p-Coumaric acid 165.1 5.55 527.5 ± 41.9 544.5 ± 23.2

Chlorogenic acid 355.1 6.08 6543.5 ± 35.9 15176.6 ± 73.9

Anthocyanins

Pet-3-rut-5-glc 787.3 5.79 0.0 1578.6 ± 105.7

Mal-3-rut-5-glc 801.3 6.19 0.0 237.5 ± 14.8 Cya-3-O(6-O-

malonyl-β-D-glc) 535.1 6.37 0.0 691.1 ± 3.2 Peo-3-(p-coum)-

isophoro-5-glc 933.3 7.26 0.0 2729.2 ± 275.7

Peo-3-rut-5-glc 771.3 7.80 0.0 2871.1 ± 29.9 Pet-3-(p-coum)-rut-

5-glc 933.3 7.92 0.0 28748.5 ± 235.7 Peo-3-caffeyl-rut-

5-glc 933.3 8.03 0.0 27215.4 ± 2295.1 Pel-3-(p-coum)-rut-

5-glc 887.3 8.11 0.0 80.8 ± 5.4 Pel-3-(4'''-ferul-

rut)-5-glc 917.3 8.15 0.0 1569.3 ± 142.7 Peo-3-(p-coum)-

rut-5-glc 917.3 8.21 0.0 1810.2 ± 135.2 Mal-3-(p-coum)-

rut-5-glc 947.3 8.31 0.0 2707.4 ± 204.4

The compounds are reported as the area under the curve per gram dry weight. Values are presented as the means ± S.E. of 6 replicates. Pet – Petunidin; Mal- Malvidin; Cya-

Cyanidin; Peo – Peonidin; Pel –Pelargonidin;

53

from Celprogen were used. Cells were maintained in incubation at 37 °C and 5 % CO2. Cell

cultures at approximately 80 % confluence were used for all in vitro experimental procedures. For

all experiments low passage number (less than 10) cells were used (not more than 3 weeks after

resuscitation).

3.3.5 Lentiviral shRNA-mediated attenuation of p53 in colon CSCs

Colon CSCs were infected with lentiviral particles encoding shRNA targeting p53

obtained from Santa Cruz Biotechnology according to the manufacturer’s protocol. Briefly, colon

CSCs were infected at a multiplicity of infection of 10 in CSC growth medium containing 5

μg/mL of polybrene (for selection of cells with successful lentiviral induction) at 37 °C and 5 %

CO2. After 24 hours, the spent media was replaced with fresh media and the cells were cultured

for 2 days. The transduced cells were selected in the presence of puromycin (7.5 μg/mL) for 5

days.

3.3.6 Cell proliferation

Cell viability was assessed by BrdU (5-bromo-2'-deoxyuridine) assay kit from Cell

Signaling Technology (Danvers, MA). Briefly, cells were plated at a density of 1 X 105 per well

in 12-well plates. Media was replaced after 24 hours with colon CSCs media without serum

(Celprogen) and dosed with PA or Sulindac. After 24 hours, BrdU incorporation was assayed as

per the manufacturer’s protocol. The experiment was carried out in triplicate, and results were

expressed as the means ± S.E.

54

3.3.7 TUNEL assay

Apoptosis was quantified by using fluorescein labeled nucleotide and terminal

deoxynucleotidyl transferase (TdT) to identify DNA fragmentation (characteristic of apoptosis).

Briefly, cells (9 X 104) were seeded in four-chambered glass slides, and after treatment for 12

hours, the in situ cell death detection kit from Roche Diagnostics (Indianapolis, IN) was used for

quantifying apoptosis according to the manufacturer’s protocol. Slides incubated without TdT

served as a negative control. The percentage of apoptotic cells (apoptotic index) was calculated

by counting the stained cells in 12 fields, each containing at least 50 cells. The experiment was

carried out in triplicate, and results were expressed as means ± SE.

3.3.8 Sphere formation assay

Briefly, colon CSCs (10,000 cells per well) were cultured in stem cell specific serum free

media (2mL) in an ultra-low attachment six well plates (Costar) for 10-12 days. PA or sulindac

was added after 6 hours of seeding. At the end of 10 days, the number of spheres was assayed

using a phase contrast microscope.

3.3.9 Western blot

Cells were plated in 6-well plates at a concentration of 3.0 X 105 cells per well in colon

CSCs media. After 24 hours, cells were transferred to serum free medium for 18 hours. Protein

was extracted according to our previously published protocols [232-234]. The blots were

incubated with primary antibodies overnight at 4 °C at a dilution of 1:500. Subsequently,

secondary antibodies incubation was for 2 hours at room temperature at a dilution of 1:10,000.

Blots were imaged and quantified using the Odyssey Infrared Imaging System and software

55

(Lincoln, NE) and normalized to β-actin, a loading control for cytoplasmic proteins and

Topoisomerase-2 Beta (TOP2B) as a loading control for nuclear proteins. Each treatment was

carried out in triplicate, and results were expressed as means ± SE.

3.3.10 Animal study

A/J male mice (6 weeks old; n = 13 per group) purchased from the Jackson Laboratories

(Bar Harbor, ME) were housed in stainless steel wire cages (3 or 4 per cage) with a 12 hour

light/dark cycle. Mice were allowed access to laboratory rodent chow and water ad libitum. After

two weeks of acclimatization all mice were randomly assigned to four groups and fed AIN-93G

diets obtained from Harlan Laboratories (Indianapolis, IN). The Institutional Animal Care and

Use Committee at Colorado State University approved all experimental procedures involving the

use of mice.

3.3.11 AOM carcinogen injection

All mice except saline controls received six weekly subcutaneous injections of AOM

(Sigma Aldrich, St. Louis, MO) in saline for aberrant crypt foci (ACF) induction at 5 mg/kg

starting at eight weeks of age.

3.3.12 Experimental diets

At 16 weeks of age, the AOM-injected animals were fed the following diets – AIN-93G

control, AIN-93G supplemented with baked PP (20 % w/w), AIN-93G supplemented with

Sulindac (0.06 % w/w).

56

3.3.13 Colon tissue collection

After one week of dietary intervention, five animals from each group were euthanized

using isoflurane. The remaining animals (N = 8 / group) were euthanized after four weeks of

dietary intervention. The colon was resected and washed with RNAse free PBS and observed

under a dissection microscope for counting tumors. Tumors greater than 2 mm were recorded.

For immunohistochemistry and immunofluorescence analysis, about 1 cm of the colon

tissue was collected and fixed with 10 % buffered formalin. Specimens were then flattened,

paraffin-embedded and orthogonally sectioned. The tissue was sectioned at four microns

thickness and mounted on positively charged slides.

3.3.14 Immunohistochemistry/Immunofluorescence staining

Pre-treatment of slides

Prior to staining, the paraffin was softened and the tissue specimens fixed additionally by

baking the slides in an oven at 55 °C for 20 minutes. Deparaffinization was performed with

Fisherbrand (Pittsburg, PA) clearing agent citrisolv twice for 5 minutes and hydrated with

decreasing concentrations of ethanol (100-100-95-70 v/v). For target retrieval, the slides were

incubated in citrate buffer at pH 6 (9 mM citrate, 1 mM citric acid) at 95 °C for 20 minutes. To

quench auto fluorescence from formalin residues, slides were pretreated with sodium borohydride

(1 mg/mL) for 5 minutes. Mouse sections were blocked with mouse IgG serum from the M.O.M

kit and avidin/biotin obtained from Vector Labs (Burlingame, CA) as per manufacturer’s

protocol.

β-catenin staining

57

β-catenin staining was performed at 4 °C overnight using a Abcam rabbit anti-β-catenin

antibody (Cambridge, MA). Biotinylated secondary antibody in combination with streptavidin

fluorescein (Vector Labs) was used for visualization. Mounting media with DAPI (Vector Labs)

was used as a counterstain. All images were taken in Olympus BX-63 microscope with the help

of Cell Sens software from Olympus America (Center Valley, PA). Nuclear β-catenin index was

calculated as a percentage of total number of crypts with nuclear β-catenin accumulation as

described previously [219]. At least 300 crypts were counter per animal.

TUNEL staining (apoptosis)

TUNEL staining was performed using a cell death detection kit from Roche Diagnostics

according to the manufacturer’s protocol for formalin fixed paraffin embedded tissues. Apoptotic

index was calculated as a percentage of total number of crypts with at least one TUNEL positive

cell. At least 300 crypts were counter per animal.

3.3.15 Statistical design

Data are expressed as means ± SE for in vitro data and as means ± SD for in vivo data.

Significance was determined by one-way ANOVA with post hoc Tukey analysis using IBM

SPSS software (Armonk, NY) for in vitro data. For animal studies, analysis of data was done

using mixed procedure in SAS v9.4 software (Cary, NC). The p values < 0.05 were considered

significant.

58

3.4 Results

3.4.1 UPLC-MS profile of phenolic compounds in PP

Peak annotations using METLIN metabolite database are presented in Table 1. Phenolic

acids (chlorogenic acid and p-Coumaric acid) were detected in both white- and purple-fleshed

potato varieties; however, the relative abundance was higher in PP. Glycosylated anthocyanins

were only detected in PP. We have previously shown that PP retains anthocyanins even after

processing (baking) [226]. Baked PP extracts (PA) suppressed early (HCT-116) and advanced

(HT-29) human colon cancer cell proliferation and induced apoptosis similar to that of raw PP

extracts and were more potent compared to white-fleshed potato [226]. Hence, for our in vitro

and in vivo experiments, we used baked PP.

3.4.2 PA suppressed proliferation and induced apoptosis in colon cancer stem cells in a p53

independent manner

Proliferation was assayed by measuring BrdU incorporation and confirmed using cell

counting. For all our experiments on colon CSCs with functioning p53 and shRNA-attenuated

p53, we used a dose of 5.0 µg/mL PA extract and 12.5 µg/mL sulindac. PA at 5.0 µg/mL

suppressed proliferation by 63 % and 32 % compared to control (Figure 3-1A) in colon CSCs

with functioning p53 and shRNA-attenuated p53, respectively. Sulindac treatment at 12.5 µg/mL

resulted in suppression of proliferation by 55 % in colon CSCs with functioning p53 (Figure 3-

1A). However, in colon CSCs with attenuated p53, suppression of proliferation by sulindac was

modest (16 %), indicating p53 dependency. Induction of apoptosis was analyzed using TUNEL

(terminal transferase dUTP nick end labeling) assay. PA induced 28% and 46 % apoptotic cell

59

death in colon CSCs with functioning p53 and shRNA-attenuated p53 (Figure 3-1 B-D). These

results suggest that PA inhibits the growth of colon CSCs independent of p53.

Figure 3-1: PA suppressed proliferation and induced apoptosis in colon cancer stem cells (colon CSCs) independent of p53. A Anti-proliferative effect of PP anthocyanin extract (PA) in colon CSCs with functioning p53 and with attenuated p53. Cells were treated with PA (5 µg/mL) or sulindac (12.5 µg/mL) for 24 hours and BrdU assay was performed as described in the methods. B – D PA induced apoptosis in colon cancer stem cells with functioning p53 and attenuated p53. TUNEL assay was performed and the results are expressed as percentage apoptosis. Cells fluorescing bright green due to fragmented DNA indicate apoptotic cells. Pictures were taken on a fluorescence microscope at 20x magnification (12 fields per treatment and at least 500 cells were counted). Representative pictures are shown for Control and PA at 5.0 µg/mL. PA = Baked purple-fleshed potato extract. Values are in means ± SE. Means that differ by a common letter (a, b, c for CSCs and x, y, z for CSCs with shRNA-attenuated p53) differ (p < 0.05).

60

3.4.3 PA suppressed sphere formation ability of colon CSCs

Self-renewal is a key property of CSCs that is largely measured in functional assays that

require proliferation, making it difficult to distinguish molecules that affect self-renewal vs.

proliferation. Hence, to assess PA ability to target the self-renewal capability of CSCs, sphere

formation assay was used as described previously [235]. We treated colon CSCs with PA or

sulindac at 5.0 µg/mL and 12.5 µg/mL, respectively. PA significantly suppressed sphere

formation similar to that of sulindac (Figure 3-2A). Figure 3-2B shows representative images

from the sphere formation assay demonstrating complete suppression in comparison to the

control. This demonstrates that, in addition to the anti-proliferative and pro-apoptotic activities,

PA inhibits the colon CSCs self-renewal property.

Figure 3-2: PA suppressed sphere formation of colon cancer stem cells (colon CSCs) similar to that of sulindac (A). Representative pictures taken at 100x magnification are shown for Control, Solvent, Sulindac at 12.5 µg/mL and PA at 5.0 µg/mL (B). PA = Baked purple-fleshed potato extract. Values are in means ± SE. Means that differ by a common letter (a, b, c) differ (p < 0.05).

61

3.4.4 PA elevated mitochondria-mediated apoptotis pathway proteins Bax/Bcl-2 and

cytochrome c

Cytosolic cell lysates of colon CSCs with functioning p53 and shRNA-attenuated

p53 treated with PA and sulindac were subjected to western blot analysis. Bax/Bcl-2 ratio was

elevated in PA treated colon CSCs with functioning p53 (Figure 3-3A and B). Cytochrome c

levels were also elevated by PA treatment independent of p53 status (Figure 3-3C and D)

indicating that the induction of apoptosis might be via mitochondria-mediated apoptotic pathway

[236]. Although sulindac induced apoptosis in colon CSCs, it did not result in elevation of

Bax/Bcl-2 or cytochrome c levels.

62

3.4.5 PA suppressed Wnt pathway proteins

Western blot analysis was performed to investigate whether PA induced

inhibition of colon CSCs growth was associated with Wnt/β-catenin pathway. PA suppressed

levels of cytoplasmic and nuclear β-catenin greater than that of sulindac in colon CSCs with

Figure 3-3: PA elevated levels of mitochondria-mediated apoptosis pathway proteins. PA elevated Bax/Bcl-2 ratio (A, B); and cytochrome c levels in colon cancer stem cells (colon CSCs) independent of p53 (C, D). Colon CSCs were treated with PA (5 µg/mL) or sulindac (12.5 µg/mL) for 24 hours, and whole-cell lysates were analyzed for Bax (pro-apoptotic), Bcl-2 (anti-apoptotic) and cytochrome c (pro-apoptotic) levels by western blotting. Actin was used as loading control. C = Control; S = Solvent; SU = Sulindac; PA = Baked purple-fleshed potato extract. Values are in means ± SE. Means that differ by a common letter (a, b) differ p < 0.05.

63

functioning p53 (Figure 3-4A and B) and shRNA-attenuated p53 (Figure 3-4C and D). The

Wnt/β-catenin pathway downstream targets c-Myc (Figure 3-5A and C) and cyclin D1 (Figure

3-5B and D) were suppressed by PA in colon CSCs with functioning p53 and shRNA-attenuated

p53. These results confirm suppression of β-catenin nuclear translocation by PA, thus limiting

colon CSC growth.

64

Figure 3-4: PA suppressed cytosolic and nuclear β-catenin levels in colon cancer stem cells (CSCs) with functioning p53 (A, B) and attenuated p53 (C, D). Colon CSCs were treated with PA (5 µg/mL) or sulindac (12.5 µg/mL) for 24 hours, and cytosolic and nuclear lysates were analyzed for β -catenin by western blotting. Actin and Topoisomerase-2 Beta (TOP2B) was used as loading control for cytosolic and nuclear lysates respectively. C = Control; S = Solvent; SU = Sulindac; PA = Baked purple-fleshed potato extract. Values are in means ± SE. Means that differ by a common letter (a, b, c) differ p < 0.05.

.

65

3.4.6 PP induced apoptosis and reduced number of crypts with nuclear β-catenin

accumulated colon CSCs

Since PA was able to suppress nuclear translocation of β-catenin in vitro we

hypothesized that PP consumption will eliminate stem cells with nuclear β-catenin in mice with

Figure 3-5: β-catenin targets c-Myc and cyclin D1 levels were suppressed by PA in colon cancer stem cells (colon CSCs) with functioning p53 (A, B) and attenuated p53 (C, D). Colon CSCs were treated with PA (5 µg/mL) or sulindac (12.5 µg/mL) for 24 hours, and nuclear lysates were analyzed for c-Myc and cyclin D1 by western blotting. Topoisomerase-2 Beta (TOP2B) was used as loading control. C = Control; S = Solvent; SU = Sulindac; PA = Baked purple-fleshed potato extract. Values are in means ± SE. Means that differ by a common letter (a, b, c) differ p < 0.05.

66

AOM induced colon cancer. PP supplementation for 1 week markedly induced apoptosis detected

by TUNEL staining, with 16 % of crypts containing at least one TUNEL-positive cell,

comparable to 18.5 % in mice receiving sulindac (Figure 3-6A). PA or sulindac treatment

reduced crypts containing cells with nuclear β-catenin by 50 % at week 1 (Figure 3-6B and C).

These results suggest that PP treatment rapidly removes intestinal stem cells or progenitors with

aberrant activation of Wnt signaling.

67

Figure 3-6: Purple-fleshed potato treatment induced apoptosis (A) and reduced number of crypts with nuclear β-catenin accumulated intestinal stem cells similar to that of sulindac. Mice injected with azoxymethane [119] were fed with control, baked PP (20 % w/w) or sulindac (0.06 % w/w) supplemented diet for 1 week. Distal colon sections from the mice were analyzed for TUNEL positive crypts and β-catenin localization by immunofluorescence. (A) The fractions of crypts containing at least one TUNEL-positive cell were determined. (B) Nuclear β-catenin index was calculated as a percentage of total number of crypts with nuclear β-catenin accumulation. (C) Staining of β-catenin and DAPI (blue; nuclear counterstain) in mice treated with AOM. Circles mark representative colon CSCs with nuclear β-catenin. Values are in means ± SD (n = 5 in each group). At least 300 crypts from each animal were analyzed. Means that differ by a common letter (a, b, c) differ p < 0.05. (Scale bars: 15 μm).

68

3.4.7 PP suppressed AOM induced colon cancer tumors

At week four, all the mice that received AOM injections developed tumors. PP treatment

suppressed the incidence of tumors (greater than 2 mm) by 50 % (Figure 3-7) and could be due to

elimination of colon CSCs via apoptosis as seen in animals euthanized at week 1 (Figure 3-6A).

Sulindac also showed potent suppression of tumor incidence (Figure 3-7), however unlike the PP

group, sulindac consuming mice had significant gastrointestinal (GI) toxicity (stomach/intestinal

ulcers) marked with loss of fat deposits (data not shown).

Figure 3-7: Purple-fleshed potato suppressed tumor incidence in the colon similar to that of sulindac. Mice injected with azoxymethane were fed with control, baked PP (20 % w/w) or sulindac (0.06 % w/w) supplemented diet for 4 weeks and euthanized. Whole colon tissue was resected and observed in a dissection microscope for visible tumors greater than 2 mm in size. Values are in means ± SD (n = 8 in each group). Means that differ by a common letter (a, b) differ p < 0.05.

69

3.5 Discussion

Our results demonstrate that in vitro PA significantly suppressed proliferation in CSCs

both with functioning p53 and with attenuated p53, suggesting that PA may work even in p53-

independent cancers. PA also upregulated proteins involved in mitochondria-mediated apoptotic

pathway and downregulated proteins involved in the Wnt/β-catenin signaling pathway. PP

eliminated colon CSCs with nuclear β-catenin in vivo via induction of apoptosis and suppressed

tumor incidence in mice with azoxymethane [119]-induced colon cancer lending support to the

anti-cancer properties of PP, warranting further investigation using detailed studies.

Polyphenolic compounds especially anthocyanins derived from fruits and vegetables

demonstrate chemopreventive and chemotherapeutic activity through modulation of multiple

molecular targets making them ideal for the prevention/treatment of cancer [169]. Potatoes are a

rich source of phenolic acids and color-fleshed potatoes also contain other bioactive compounds

such as anthocyanins and carotenoids. UPLC-MS analysis comparing PP and WP showed that

besides higher levels of phenolic acids, only PP contained anthocyanins (compared to WP, Table

1). We also showed previously that PP had more potent anti-cancer activity on early (HCT-116)

and advanced (HT-29) colon cancer cell lines in vitro [226]. However, the effect against colon

cancer stem cells (colon CSCs) is not known and for this purpose we treated colon CSCs with PP

and compared it with sulindac, a positive control.

PA at 5.0 µg/mL suppressed proliferation and induced apoptosis in colon CSCs with and

without functioning p53, however, sulindac demonstrated p53 dependency (Fig. 1A). The p53

dependency of sulindac has been investigated previously in an AOM-induced mouse model with

dysfunctional p53 [237]. Sulindac was not able to restore acute apoptosis response in p53 -/- mice

when compared to that of p53 +/+ mice. This is particularly important because in late/metastatic

stages of colon cancer p53 is mutated [27]. PA induced apoptosis (Fig. 1B-D) was accompanied

70

by elevated Bax/Bcl-2 ratio and cytochrome c (Fig. 3). Bax is a pro-apoptotic protein that binds

Bcl-2 and aids in the release of cytochrome c, a key promoter in mitochondria-mediated apoptosis

[238]. These results indicate that PP induces apoptosis through the mitochondria-mediated

apoptotic pathway. We have also shown that PA suppressed sphere formation, since formation of

colonospheres is a measure of stemness, our results provide the evidence that PA has the potential

to target the self-renewal of colon CSCs.

PA treatment resulted in significant suppression of β-catenin at both nuclear and cytosolic

levels in both colon CSCs with and without functioning p53 (Fig. 4) greater than that of sulindac.

Stabilization of β-catenin and its subsequent accumulation in the nucleus is accompanied by

increased transcriptional activation of proteins such as c-Myc and cyclin D1, which promote

carcinogenesis by increasing cell proliferation [239, 240]. Indeed, PA treated colon CSCs had

suppressed levels of c-Myc (Fig. 5A and 5C) and cyclin D1 (Fig. 5B and 5D) independent of p53.

Several characteristics of colon CSCs may explain the elimination by PP. Stem cells

express high levels of “stemness” factors including the oncoprotein c-Myc [241], which is

overexpressed in colon CSCs [242]. We have also shown in vitro that PA suppressed Wnt

effector β-catenin and its downstream targets c-Myc and cyclin D1 levels in colon CSCs.

Therefore, stem cells with oncogenic alterations, such as accumulation of β-catenin, may be more

sensitive to PA induced apoptosis, relative to differentiated cells with such alterations.

To further test whether PP can eliminate colon CSCs in vivo, we used an AOM-induced

colon cancer mice model. Mice were fed with modified AIN 93G diet containing human relevant

doses of PP (20 % w/w) or sulindac (positive control; 0.06 % w/w) for 1 or 4 weeks. Week 1

euthanized animals were used to study the early molecular mechanism of PP. Week 4 euthanized

animals were used for endpoint analysis of tumor incidence. PP or sulindac fed mice had

significant increase in the number of crypts with TUNEL positive cells (indicator of apoptosis)

compared to AOM control (Fig. 6A). Nuclear β-catenin localization is observed predominantly in

71

colon CSCs but rarely in other cells of the crypt in APCMin/+ mice [219] (Supplementary Figure

1), hence we looked at the number of crypts containing nuclear β-catenin. More than 50 % of

crypts with nuclear β-catenin accumulated intestinal stem cells were eliminated in mice fed with

PP or sulindac for 1 week when compared to AOM control (Fig. 6B and 6C). In animals fed with

PP or sulindac for 4 weeks, we observed very few stem cells with accumulated nuclear β-catenin.

It has been previously reported that sulindac treatment eliminates colon CSCs with accumulated

nuclear β-catenin via rapid apoptosis, which is not detected after week 1 [219]. At the end of

week 4, PP significantly suppressed tumor incidence (Fig. 7) comparable to that of sulindac.

In summary, this study demonstrated anti-cancer mechanism of PP (vs sulindac) against

colon CSCs in vitro and in vivo involving the induction of mitochondria-mediated apoptosis and

targeting the Wnt/β-catenin signaling.

72

Chapter 4

Grape compounds suppress colon cancer stem cells in vitro and in a rodent

model of colon carcinogenesis*

*These results have been published as the following manuscript: Reddivari L#, Charepalli V,

Radhakrishnan S, Vadde R, Elias R, Lambert J, and Vanamala J. Dietary grape compounds

suppress oncogenic stem cells in a mouse model of chemically-induced colon cancer. BMC

Complementary and Alternative Medicine. 2016, 16:278. # equally contributed

73

4.1 Abstract

We have previously shown that the grape bioactive compound resveratrol (RSV)

potentiates grape seed extract (GSE)-induced colon cancer cell apoptosis at physiologically

relevant concentrations. However, RSV-GSE combination efficacy against colon cancer stem

cells (CSCs), which play a key role in chemotherapy and radiation resistance, is not known. We

tested the anti-cancer efficacy of the RSV-GSE against colon CSCs using isolated human colon

CSCs in vitro and an azoxymethane-induced mouse model of colon carcinogenesis in vivo. RSV-

GSE suppressed tumor incidence similar to sulindac, without any gastrointestinal toxicity.

Additionally, RSV-GSE treatment reduced the number of crypts containing cells with nuclear β-

catenin (an indicator of colon CSCs) via induction of apoptosis In vitro, RSV-GSE suppressed -

proliferation, sphere formation, nuclear translocation of β-catenin (a critical regulator of CSC

proliferation) similar to sulindac in isolated human colon CSCs. RSV-GSE, but not sulindac,

suppressed downstream proteins levels of Wnt/β-catenin pathway, c-Myc and cyclin D1. RSV-

GSE also induced mitochondrial-mediated apoptosis in colon CSCs characterized by elevated

p53, Bax/Bcl-2 ratio and cleaved PARP. Furthermore, shRNA-mediated knockdown of p53, a

tumor suppressor gene, in colon CSCs did not alter efficacy of RSV-GSE. The suppression of

Wnt/β-catenin signaling and elevated mitochondrial-mediated apoptosis in colon CSCs support

potential clinical testing/application of grape bioactives for colon cancer prevention and/or

therapy.

4.2 Introduction

Colorectal cancer is the third most common cancer among both men and women in the

United States. It is also the second most common cause of cancer-related deaths in men and

74

women combined [187]. With regular screening, colon cancer can be detected early, when

treatment is most effective; however, in the majority of cases colon cancers are detected late after

it has been spread. Over 95% of colon cancer cases are considered sporadic thus placing

environmental factors as the major cause [187]. The most important environmental factors among

them are diet and lifestyle. The highest incidence rates of colon cancer are in developed nations

including the U.S. [7]. Diets rich in refined starch, sugar, and saturated and trans-fatty acids but

poor in fruits, vegetables and whole grains (prevalent in developed nations), have been shown to

be closely associated with an increased risk of colon cancer [7-9]. A meta-analysis of case-control

studies suggests that fruit consumption was associated with a 13% decrease in colon cancer risk.

The benefits from consuming a diet rich in fruits and vegetables could be attributed to the

plethora of bioactive compounds present in them [243].

Grapes are consumed around the world and are a rich source of many bioactive

compounds. Red grapes are rich in resveratrol (RSV), a stilbene that has shown anti-cancer

properties in a variety of models, including human studies [244]. We previously reported that

RSV suppressed proliferation and induced apoptosis via p53 activation in HT-29 and SW-480

human colon cancer cell lines, however, it was effective only at higher concentrations (75-100

µM) [233]. Grape seed extract (GSE) is a popular dietary supplement rich in proanthocyanidins

and has been reported to have anti-colon cancer properties in a variety of in vitro and in vivo

models [245]. As bioactive compounds exist in a complex mixture in fruits and vegetables,

laboratory assessment of their biological activity in combination is more relevant to human

exposure. In addition, because these compounds have pleiotropic effects, there is the potential

that they will exert additive or synergistic chemopreventive actions. A recent study that compared

GSE induced anti-cancer effects to the effects of its individual components found that GSE was

more potent in growth inhibition compared to its individual constituents epigallocatechin,

procyanidins and their association [156]. Our previous studies also support this notion, as we

75

demonstrated using a well-established combination index method that a RSV (~ 25 µM) and GSE

(35-50 µg/ml) mixture was potent in suppressing proliferation and elevating apoptosis in the

HCT-116 human colon cancer cell line at lower concentrations compared to RSV or GSE alone

[152, 246]. Combination index methods is based on the classic isobologram equation CI = D1/d1

+ D2/d2. D1 and D2 are the doses of RSV and GSE respectively in the combination system

where as d1 and d2 are the doses of RSV and GSE alone for the same fractional inhibition,

respectively. In addition, RSV potentiated GSE-induced p53-dependent apoptosis via

mitochondrial apoptotic signaling, and demonstrated specificity to cancer cells, as it was non-

toxic in the normal colonic epithelial cell line CRL-1831[152]. Our preliminary results led us to

believe that a combinatorial approach towards colon cancer chemoprevention using bioactive

compounds is a feasible strategy.

Historically, colon tumorigenesis has been viewed as a stochastic model where wide

populations of abnormal colonocytes have an equal propensity to initiate tumor growth [247].

However, the cancer stem cell (CSC) theory suggests that most, if not all, cancerous tumors are

driven by CSCs, probably through dysregulation of self-renewal pathways [40]. CSCs are capable

of self-renewal, cellular differentiation, and maintain their stem cell-like characteristics even after

invasion and metastasis [190]. Furthermore, they are resistant to standard therapies and thus are

thought to be responsible for cancer relapse. The Wnt/β-catenin signaling pathway plays a critical

role in maintenance of stemness, and survival/proliferation of CSCs [217], and as such, targeting

the Wnt/β-catenin signaling is a good strategy for cancer prevention. Aberrant Wnt signaling in

colon cancer is typically followed by mutation in the K-ras gene and loss of the tumor suppressor

p53. It is estimated that p53 is abnormal in 50% to 75% of colorectal cancer cases, and that this

change marks the transition from noninvasive to invasive disease [17, 218]. Thus, treatments,

which act independent of p53 status, are desirable.

76

Sulindac, a nonsteroidal anti-inflammatory drug has shown promising results in

treatment of colon cancer. Recently it was shown to induce SMAC (a mitochondrial apoptogenic

protein)-dependent apoptosis in cells with nuclear β-catenin (an indicator of colon CSCs) and

decrease polyp numbers in the APCMin/+ mouse that is highly susceptible to spontaneous intestinal

adenoma formation [219]. Chemotherapeutic drugs like sulindac are effective against certain

types of cancers but can also have unexpected adverse effects such as gastrointestinal bleeding,

hepatotoxicity [220, 221] and in some cases chronic inflammation promoted colon cancer [248].

This has stimulated active pursuit of new approaches and/or combination strategies for cancer

chemoprevention. Based on our preliminary data, we hypothesized that the combination of RSV

and GSE suppresses proliferation and induces apoptosis in colon CSCs. Azoxymethane [119], a

DNA alkylating agent, induced mouse colon cancer model is a well-established and reproducible

model of sporadic colon carcinogenesis to predict chemopreventive efficacy [121].

Intraperitoneal injection of AOM in A/J mice, a breed that is susceptible to chemically-induced

carcinogenesis, for six weeks resulted in tumor formation within six weeks of the last injection

[122]. Thus, we determined the efficacy of the RSV-GSE combination using A/J mice with six

week AOM injection regimen and compared its effects to those of sulindac. Furthermore, we

examined the possible molecular mechanisms that underlie the anti-cancer activity of RSV-GSE

(and compared to sulindac) using colon CSCs, positive for CD 44, CD 133 and ALDH1b1

markers, isolated from primary human colon cancer tumors.

77

4.3 Materials and methods

4.3.1 Chemicals

Grape seed extract (GSE, ORAC value 9000-13000 µmole Trolox equivalents/g, total

phenolic content > 85% gallic acid equivalents) was a generous gift from San Joaquin Valley

Concentrates (Fresno, CA). We had previously characterized the GSE used in this study using

UPLC-MS and we detected presence of (+)-catechin and (-)-epicatechin monomers and their

oligomers, and their gallate derivatives similar to other published papers [249, 250]. The GSE

used in this study lacks resveratrol (RSV) as described earlier [152]. BrdU Cell Proliferation

Assay Kit was obtained from Cell Signaling Technology (Danvers, MA). Antibodies for PARP

and cleaved PARP, p53, pGSK3β, Bax, Bcl-2, β-actin, β-catenin, cyclin D1, c-Myc, COX-2 and

topoisomerase-2β (Topo ii b) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Cytochrome C was obtained from Cell Signaling Technology (Beverly, MA). All other chemicals

including RSV were obtained from Sigma (St. Louis, MO).

4.3.2 Animal study

A/J male mice (six weeks old; n = 13 per group) purchased from the Jackson

Laboratories (Bar Harbor, ME) were housed in stainless steel wire cages (three or four per cage)

with a 12 hour light/dark cycle. Mice were allowed access to laboratory rodent chow and water

ad libitum. After two weeks of acclimatization, all mice were randomly assigned to four groups

and fed AIN-93G diets obtained from Harlan Laboratories (Indianapolis, IN).

Ethics statement: All experimental procedures on the animals were approved by the

Institutional Animal Care and Use Committee at Colorado State University.

78

4.3.3 Azoxymethane carcinogen injection

All mice except saline controls received six weekly subcutaneous injections of

azoxymethane (AOM, Sigma) in saline for colon carcinogenesis at 5 mg/kg starting at eight

weeks of age.

4.3.4 Experimental diets

At 16 weeks of age i.e. two weeks following the last AOM injection, the animals were

assigned to the following diets – AIN-93G control, AIN-93G supplemented with RSV-GSE (0.03

and 0.12% w/w, respectively) or AIN-93G supplemented with sulindac (0.06% w/w). RSV and

GSE concentrations were chosen based on the earlier human (n = 32) study that showed a

decrease in serum oxidative stress markers in obese subjects orally supplemented with RSV and

GSE separately [251]. Sulindac concentration was chosen based on previous clinical trial study in

humans (n = 12) with familial adenomatous polyposis where administration of sulindac resulted

in significant reduction of polyp number [252]. The saline control animals received AIN-93G

control diets. All animals had free access to food and water.

4.3.5 Colon tissue collection

After one week of dietary intervention, five animals from each group were euthanized

using isoflurane. The remaining animals (n = 8/group) were euthanized after four weeks of

dietary intervention. The colon was resected and washed with RNAse free PBS and observed

under a dissection microscope for counting tumors. Tumor number was recorded for each animal.

At the end of the study the tumor number was averaged for each treatment group and represented

79

as means ± S.D. For immunohistochemistry and immunofluorescence analysis, about 1 cm of the

colon tissue was collected and fixed with 10% buffered formalin. Specimens were then flattened,

paraffin-embedded and orthogonally sectioned. The tissue was sectioned at four microns

thickness and mounted on positively charged slides.

4.3.6 Immunofluorescence staining

Pre-treatment of slides

Prior to staining, the paraffin was softened and the tissue specimens fixed additionally by

baking the slides in an oven at 55°C for 20 minutes. Deparaffinization was performed with

Fisherbrand (Pittsburg, PA) clearing agent citrisolv twice for five minutes and hydrated with

decreasing concentrations of ethanol (100-100-95-70 v/v). For target retrieval, the slides were

incubated in citrate buffer at pH 6 (9 mM citrate, 1 mM citric acid) at 95°C for 20 minutes. To

quench auto fluorescence from formalin residues, slides were pretreated with sodium borohydride

(1 mg/mL) for five minutes. Mouse sections were blocked with mouse IgG serum from the

M.O.M kit and avidin/biotin obtained from Vector Labs (Burlingame, CA) as per the

manufacturer’s protocol.

β-catenin staining

β-catenin staining was performed at 4°C overnight using a Abcam rabbit anti-β-catenin

antibody (Cambridge, MA). Biotinylated secondary antibody in combination with streptavidin

fluorescein (Vector Labs) was used for visualization. Mounting media with DAPI (Vector Labs)

was used as a counterstain. All images were taken in Olympus BX-63 microscope with the help

of Cell Sens software from Olympus America (Center Valley, PA).

TUNEL staining

80

TUNEL staining was performed using a cell death detection kit from Roche Diagnostics

(Indianapolis, IN) according to the manufacturer’s protocol for formalin fixed paraffin embedded

tissues.

4.3.7 Cancer stem cells

Isolated human colon CSCs positive for cancer stem cell markers CD133, CD44, CD34,

aldehyde dehydrogenase, telomerase, Sox2, cKit, and Lin28, were obtained from Celprogen Inc.

(San Pedro, CA). To maintain the cells in their undifferentiated state, colon CSCs maintenance

media and specially coated cell culture flasks obtained from Celprogen were used. Cells were

maintained in incubation at 37°C and 5% CO2. Cell cultures at approximately 80% confluence

were used for all in vitro experimental procedures. For all experiments low passage number (less

than 10) cells were used (not more than three weeks after resuscitation). The authentication

information for the cell line obtained from Celprogen is available under supplemental

information.

4.3.8 Lentiviral shRNA-mediated attenuation of p53 in colon CSCs

Lentiviral particles encoding shRNA targeting p53 obtained from Santa Cruz

Biotechnology were used to attenuate p53 expression in colon CSCs as described earlier [205].

81

4.3.9 Cell proliferation

Cell viability was assessed by BrdU (5-bromo-2'-deoxyuridine) assay kit from Cell

Signaling Technology (Danvers, MA). Briefly, cells were plated at a density of 1 X 105 per well

in 12-well plates. Media was replaced after 24 hours with serum-free colon CSCs media

(Celprogen) and dosed with RSV-GSE and/or sulindac. For all in vitro experiments sulindac

sulfide, the active form of sulindac was used. Preliminary experiments revealed that lower

concentrations of RSV-GSE were potent in suppressing proliferation of colon CSCs compared to

the concentrations used in our earlier study using HCT-116 early colon cancer cells [152].

Interestingly, other researchers also showed that dietary bioactive compounds are more potent

against highly proliferating or advanced cancer cells that are distinctly different from normal cells

[142]. Hence, for this study doses of RSV were kept constant at 9 µM, whereas GSE doses in the

combination varied (6.25, 12.5 and 25 µg/mL). Sulindac was dosed at 6.25, 12.5 and 25 µg/mL.

After 24 hours, BrdU incorporation was assayed as described in manufacturer’s protocol. The

experiment was carried out in triplicate, and results were expressed as the means ± S.E.

4.3.10 TUNEL assay

Apoptosis was quantified by using fluorescein labeled nucleotide and terminal

deoxynucleotidyl transferase (TdT) to identify DNA fragmentation (characteristic of apoptosis).

Briefly, cells (9 X 104) were seeded in four-chambered glass slides, and after treatment with

RSV-GSE or sulindac for 12 hours, the in situ cell death detection kit from Roche Diagnostics

was used for quantifying apoptosis based on the manufacturer protocol. The experiment was

carried out in triplicate, and results were expressed as means ± S.E.

82

4.3.11 Sphere formation assay

Briefly, colon CSCs (10,000 cells per well) were cultured in stem cell specific serum free

media in an ultra-low attachment six-well plates. The cells were maintained in similar conditions

as mentioned earlier under the cancer stem cells section. RSV-GSE or sulindac was added six

hours after the cells were added to the six-well plates. At the end of ten days, the number of

spheres was assayed using a phase contrast microscope.

4.3.12 Western blot

Cells were plated in six-well plates at a concentration of 3.0 X 105 cells per well in colon

CSCs media. After 24 hours, cells were transferred to serum free medium for 18 hours. Protein

was extracted according to our previously published protocols [111, 152]. The blots were

incubated with primary antibodies overnight at 4°C at a dilution of 1:500. Subsequently, the blots

were incubated with secondary antibodies for two hours at room temperature at a dilution of

1:10,000. Blots were imaged and quantified using the Odyssey infrared imaging system and

software (Lincoln, NE) and normalized to β-actin, a loading control for cytoplasmic proteins and

topoisomerase-2β as a loading control for nuclear proteins. Each treatment was carried out in

triplicate, and results were expressed as means ± S.E.

4.3.13 Statistical analysis

Data are expressed as means ± S.E. for all the data. Significance was determined by one-

way ANOVA with post hoc Tukey analysis for in vitro data (SPSS v21, IBM, Armonk, NY). For

83

animal studies, analysis of data was done using mixed procedure in SAS v9.4 software (Cary,

NC). The p values < 0.05 were considered statistically significant.

4.4 Results

4.4.1 RSV-GSE suppressed AOM-induced tumor incidence in mice

Mice exposed to AOM developed colon tumors at the end of the study. The incidence of

AOM-induced tumors was suppressed in the RSV-GSE group by over 50% (Figure 4-1A), an

effect similar to that of sulindac. Sulindac treatment resulted in significant gastrointestinal

toxicity (stomach/intestinal ulcers) marked with loss of fat deposits (Figure 4-1B). Such toxicity

was not observed in the animals consuming RSV-GSE. Neither RSV-GSE nor sulindac

significantly affected average weight gain or food intake across the groups.

84

Figure 4-1: RSV – GSE suppressed tumor incidence in the colon similar to that of sulindac. (A) Mice injected with AOM consumed control, RSV-GSE or sulindac (positive control) supplemented diet for four weeks and were euthanized. Whole colon tissue was resected and observed under a dissection microscope for visible tumors. SU = Sulindac; RG = RSV-GSE. Values are in means ± S.E. (n = 8 in each group). Means that differ by a common letter (a, b) differ at p < 0.05. (B) Short-term feeding of sulindac resulted in stomach ulcers (hyperplasia of the stomach, black arrows) and subsequent loss of adipose tissue deposits (blue arrows) compared to control. RSV-GSE supplemented diet consuming animals showed neither hyperplasia nor loss of adipose tissue deposits.

85

4.4.2 RSV-GSE induced apoptosis and reduced number of crypts with colon cancer stem

cells

Previously conducted studies determined a one week time window for analyzing

sulindac-induced apoptosis in intestinal stem cells in APCMin/+ mice because of the rapid and

transient nature of apoptotic events. Here we found that RSV-GSE supplementation for one week

induced apoptosis with 18% of crypts containing at least one TUNEL-positive cell, an effect

comparable to the 18.5% in mice receiving sulindac (Figure 4-2A). In addition, RSV-GSE and

sulindac treatment for one week also reduced the number of crypts containing cells with nuclear

β-catenin (an indicator of colon CSCs) by more than 50% (Figure 4-2B and C). These data

demonstrate that intestinal stem cells with nuclear β-catenin (CSCs) may be targeted for apoptosis

induction following RSV-GSE or sulindac treatment in mice with AOM induced colon

carcinogenesis. This might also explain lower tumor incidence in the RSV-GSE (and sulindac)

groups at the end of the study (Figure 4-1A).

86

Figure 4-2: RSV – GSE treatment induced apoptosis and reduced the number of crypts containing cells with nuclear β-catenin (an indicator of colon CSCs). Mice injected with AOM were fed with control, RSV-GSE or sulindac-containing diet for one week. Distal colon sections from the mice were analyzed for TUNEL positive crypts and β-catenin localization by immunofluorescence. (A) The fractions of crypts containing at least one TUNEL-positive cell (indicator of apoptotic cells) were determined. (B) Quantification of crypts with nuclear β-catenin in mice treated with control, RSV-GSE or sulindac supplemented diet for one week. Accumulation of nuclear β-catenin is hallmark of cancer stem cells and hence was used as an indirect measure for evaluating elimination of cancer stem cells. (C) Staining of β-catenin and DAPI (blue) in mice treated with AOM. Circles mark representative colon stem cells with nuclear β-catenin (CSCs). SU = Sulindac; RG = RSV-GSE. Values are in means ± S.E. (n = 5 in each group). At least 300 crypts from each animal were analyzed. Means that differ by a common letter (a, b, c) differ at p < 0.05. (Scale bars: 15 μm).

87

4.4.3 RSV-GSE suppressed proliferation and induced apoptosis in colon cancer stem cells

Proliferation and apoptotic response were determined in isolated human colon CSCs in

response to RSV-GSE or sulindac treatment using BrdU incorporation and TUNEL, respectively.

Both RSV-GSE and sulindac induced dose-dependent suppression of cell proliferation (Figure 4-

3A) and elevated apoptosis (Figure 4-3B and C) in colon CSCs. The IC(50) values for RSV-

GSE was determined to be 9 µM and 12.5 µg/mL respectively, and for sulindac at 12.5

µg/mLwhich are at physiologically relevant doses. Thus, we used these doses for subsequent

experiments to determine the mechanism of action.

Figure 4-3: RSV – GSE suppressed proliferation, induced apoptosis and suppressed sphere formation in colon CSCs similar to that of sulindac. (A) Anti-proliferative effect of RSV-GSE in colon CSCs. RSV-GSE induced apoptosis in CSCs (B, C) similar to that of sulindac. CSCs were treated with sulindac (6.25, 12.5 and 25 µg/mL) or RSV-GSE (RSV - 9 µM and GSE 6.25, 12.5 and 25 µg/mL) for 24 hours and BrdU assay was performed to assess proliferation. TUNEL assay was performed based on manufacturer protocol (Roche) and the results are expressed as per cent apoptosis. Cells fluorescing bright green due to fragmented DNA indicate apoptotic cells. Pictures taken on fluorescence microscope at 20X magnification. Representative pictures are shown for Control, RSV-GSE at 9 µM and 12.5 µg/mL respectively and sulindac at 12.5 µg/mL.

88

4.4.4 RSV-GSE suppressed sphere formation ability of colon CSCs

To assess RSV-GSE ability to target the self-renewal capability of CSCs, sphere

formation assay was used (Figure 4-3D). Representative images collected from the sphere

formation assay are shown (Figure 4-3D) which demonstrate the decreased number of spheres

associated with the treatments in comparison to the control. RSV-GSE treatment completely

suppressed colon CSCs sphere formation. This demonstrates that, in addition to the anti-

proliferative and pro-apoptotic activities, RSV-GSE alters the stem-like properties by inhibiting

colon cancer stem cell self-renewal as measured using the sphere formation assay.

Figure 4-4: Sphere formation was assessed as described in methods. Representative images taken from the sphere formation assay are presented. Results were expressed as mean ± S.E. for three experiments at each time point. Means that differ by a common letter (a, b, c, d, e, f) differ at p < 0.05.

89

4.4.5 RSV-GSE suppressed Wnt pathway proteins

As the Wnt/β-catenin signaling pathway is critical for stem cell fate, we treated colon

CSCs with RSV-GSE or sulindac and measured proteins in the pathway - pGSK3β (cytoplasmic)

and, β-catenin, c-Myc and cyclin D1 (all nuclear) using western blotting. Both RSV-GSE and

sulindac treatment suppressed protein levels of pGSK3β in the cytoplasm and nuclear levels of β-

catenin. This indicates reduced translocation of β-catenin to the nucleus and thus suppression of

the canonical Wnt/β-catenin signaling that is frequently deregulated in colon cancer (Figure 4-4A

and B). Downstream proteins of β-catenin, c-Myc and cyclin D1, critical in stem cell

proliferation, were also suppressed by RSV-GSE treatment. However, sulindac treatment failed to

induce any changes in c-Myc and cyclin D1 levels (Figure 4C and D).

90

Figure 4-5: RSV – GSE suppressed levels of proteins involved in Wnt/β-catenin pathway in colon CSCs with functioning p53. Nuclear β-catenin (A) and its regulator phosphorylated GSK3β (B) levels were suppressed by RSV-GSE similar to that of sulindac. Downstream targets of Wnt/β-catenin pathway – c-Myc (C) and Cyclin D1 (D), in the nucleus were suppressed by RSV-GSE compared to sulindac. Colon CSCs were treated with RSV-GSE at 9 µM and 12.5 µg/mL, or sulindac at 12.5 µg/mL for 24 h, and cytosolic and nuclear cell lysates were analyzed for respective proteins by western blotting. Actin and topoisomerase-2β (Topo II b) were used as loading controls for cytosolic and nuclear proteins respectively. Values are in means ± S.E. Means that differ by a common letter (a, b, c,) differ at p < 0.05.

.

91

4.4.6 RSV-GSE elevated mitochondrial apoptotic pathway proteins

P53 is a critical transcription factor that controls cell fate in response to various stresses.

In addition, as “the guardian of the genome”, p53 protein plays a critical role in tumor

suppression by inducing growth arrest, apoptosis, and senescence, as well as by blocking

angiogenesis. Nuclear levels of p53 were elevated by RSV-GSE treatment, but not sulindac,

compared to control in colon CSCs (Figure 4-5A). Downstream of p53, Bax, the pro-apoptotic

protein was elevated and Bcl-2, the anti-apoptotic protein, was suppressed by RSV-GSE

treatment indicating mitochondrial-mediated apoptosis. Bax/Bcl-2 ratio was elevated only in the

RSV-GSE group but not sulindac compared to the control (Figure 4-5B) in colon CSCs. Data for

cleaved PARP, indicator of apoptosis, mirrored the Bax/Bcl-2 ratio, with RSV-GSE treatment

showing highest levels of cleaved PARP (Figure 4-5C).

92

Figure 4-6: RSV-GSE induced apoptosis via p53 dependent pathway in colon cancer stem cells (CSCs)

with functioning p53. Nuclear p53 levels were elevated (A) by RSV-GSE but not sulindac. Cleaved PARP

(B) and Bax/Bcl-2 ratio (C) were also elevated by RSV-GSE but not sulindac. Colon CSCs were treated

with RSV-GSE at 9 µM and 12.5 µg/mL, or sulindac at 12.5 µg/mL for 24 h, and cytosolic and nuclear cell

lysates were analyzed for respective proteins by western blotting. Actin and topoisomerase -2β (Topo II b)

were used as loading controls for cytosolic and nuclear proteins respectively. Values are in means ± S.E.

Means that differ by a common letter (a, b, c, or x, y, z) differ at p < 0.05.

93

4.4.7 RSV-GSE efficacy is retained even in the absence of p53

To determine the requirement of p53 in the CSC inhibitory effects of RSV-GSE, we used

a lentiviral p53-shRNA construct to attenuate p53 expression. Reduced p53 expression had no

effect on RSV-GSE-mediated suppression of nuclear levels of β-catenin and its downstream

proteins, c-Myc and cyclin D1 (Figure 4-7A-C). RSV-GSE also induced apoptosis in colon

CSCs as measured using PARP cleavage (Figure 4-7D) and increased cytochrome C expression

greater than that of sulindac (Figure 4-7E). These results indicate that GSE-RSV-induced CSC

apoptosis occurs via a p53-independent mechanism. Similar trend was observed in nuclear levels

of COX-2 in both colon CSCs and colon CSCs with shRNA attenuated p53 – RSV-GSE

treatment was more potent in suppressing COX-2 expression compared to sulindac (Figure 4-8A

and B).

94

95

Figure 4-7: Modulation of Wnt/β-catenin and apoptotic signaling proteins by RSV – GSE in colon CSCs with attenuated p53. β-catenin (A) and its downstream targets c-Myc (B) and cyclin D1 (C) were suppressed by RSV-GSE compared to sulindac. Pro-apoptotic proteins cleaved PARP (D) and cytochrome C (E) levels were elevated by RSV-GSE greater than that of control and sulindac. Colon CSCs were treated with RSV-GSE at 9 µM and 12.5 µg/mL, or sulindac at 12.5 µg/mL for 24 h, and cytosolic and nuclear cell lysates were analyzed. Actin and topoisomerase-2β (Topo II b) were used as loading controls for cytosolic and nuclear proteins respectively. C = Control; SU = Sulindac; RG = RSV-GSE. Values are in means ± S.E. Means that differ by a common letter (a, b, c, or x, y, z) differ p < 0.05.

Figure 4-8: RSV – GSE suppressed COX-2 levels in colon CSCs with functioning (A) and attenuated p53 (B). Colon CSCs were treated with RSV-GSE at 9 µM and 12.5 µg/mL respectively or sulindac at 12.5 µg/ml for 24 h, and nuclear cell lysates were analyzed for COX-2 levels by western blotting. Topoisomerase-2β (Topo II b) was used as a loading control. C = Control; SU = Sulindac; RG = RSV-GSE. Values are in means ± S.E. Means that differ by a common letter (a, b, c) differ at p < 0.05.

96

4.5 Discussion

The objective of present study was to evaluate the anti-cancer efficacy of RSV-GSE in a

mouse model of colon cancer, and to determine the mechanisms of action using human colon

CSCs in vitro. Our results present the first evidence of in vivo anti-colon cancer efficacy of a

combination of the grape bioactive components RSV and GSE in the mouse model with AOM-

induced colon carcinogenesis. Our data in vitro in colon CSCs demonstrate suppression of

nuclear translocation of β-catenin (Wnt/β-catenin signaling pathway) and induction of

mitochondrial-mediated apoptosis.

Our results indicate that RSV-GSE, bioactive components from grapes, suppress

tumor incidence in a mouse model with AOM-induced colon carcinogenesis (Figure 1A).

Furthermore, RSV-GSE consumption had reduced toxicity compared to sulindac, suggesting

specific targeting of cancer cells (Figure 1B). Indeed, clinical trials in humans have shown that

RSV is quite safe [253], similar results have been observed for GSE [254].

Accumulated experimental evidence has suggested that most cancers, including colon

cancer, have a hierarchal organization regulated by a small number of self-renewing cancer cells,

called CSCs [255]. CSCs including colon CSCs have shown to be resistant to conventional

chemotherapeutic regimens that target homogeneous populations of rapidly proliferating

differentiated tumor cells. For e.g., CD133-positive colon CSCs were shown to be resistant to the

conventional cytotoxic drug 5-florouracil and the resistance was shown to be dependent on Wnt

signaling [256]. The proliferation and the acquisition of the stem cell fates is coordinated by a

small number of highly evolutionarily conserved signaling pathways, including the Wnt/β-catenin

signaling pathway, which is commonly deregulated in most colon cancers [257]. Nuclear

accumulation of β-catenin is implicated in the transformation of stem cells to oncogenic stem

cells in the colon [217]. Although, it has been observed that nuclear β-catenin accumulation is

97

also seen in normal colonic stem cells and progenitor cells which are located at the bottom

proliferative compartment of the intestinal crypts [258, 259], a recent study has shown that it is

observed in less than 0.01% of crypts in wild-type mice [219]. Hence, we [260] and others [219]

considered increased number of crypts with colonic stem cells with nuclear β-catenin

accumulation (supplementary figure S1) as a hallmark of colon carcinogenesis and a signature

feature of elevated oncogenic stem cells. Qiu et al reported that one week of sulindac treatment

resulted in a 75% reduction in the number of crypts containing cells with nuclear β-catenin. Most

importantly, a vast majority (98%) of identifiable stem cells with accumulated nuclear β-catenin

in sulindac-treated APCMin/+ mice were TUNEL-positive at this time point [219]. In the current

study, where AOM (a well-known colon specific carcinogen) was used to induce colon

carcinogenesis, RSV-GSE consuming animals had 62% reduction in number of crypts containing

cells that have accumulated nuclear β-catenin (Figure 2A). Additionally, our data suggest that this

could be due to induction of apoptosis (Figure 2B). Efficacy of RSV-GSE was comparable or

better than sulindac. The in vivo data was supported by our in vitro observations where we

noticed that RSV-GSE at physiologically relevant doses suppressed proliferation and induced

apoptosis as well as suppressed sphere formation in colon CSCs (Figure 3A-D).

There is evidence that nuclear accumulation of β-catenin results in accelerated tumor cell

proliferation and tumor progression through the transcriptional activation of target genes

including c-Myc, cyclin D1 and COX-2 [261]. Mechanistic data in vitro confirmed our in vivo

observations as RSV-GSE suppressed nuclear β-catenin accumulation in colon CSCs (Figure

4A). RSV-GSE also suppressed cytoplasmic levels of pGSK3β (Figure 4B) shown to induce

nuclear β-catenin translocation and down-regulated nuclear levels of proteins downstream of

Wnt/β-catenin pathway, c-Myc and cyclin D1 (Figure 4C, D). c-Myc and cyclin D1 are the key

signatory genes of Wnt signaling and both function in the stimulation of cell proliferation and in

preventing apoptosis. Coordination of c-Myc with cyclin D1 or its upstream activators not only

98

accelerates tumor formation, but also may drive tumor progression to a more aggressive

phenotype [61]. Although, previously published research has shown that RSV and GSE

suppressed nuclear β-catenin translocation, to our knowledge, this is the first study to show such

an effect in colon CSCs.

Alterations in Wnt/β-catenin signaling might also explain why RSV-GSE also suppressed

sphere formation ability in vitro (Figure 3D). Because c-Myc and cyclin D1 also play a role in

stemness [262], our data showing suppressed c-Myc and cyclin D1 only by RSV-GSE treatment

might explain its higher potency compared to sulindac. Our data is in line with recent research

showing that dietary compounds including grape seed extract, curcumin, lycopene and resveratrol

are promising chemopreventive agents against various types of cancers owing to their direct and

indirect effects on CSC self-renewal pathways, such as Wnt/β-catenin signaling pathway [263-

266].

P53 plays a critical role in tumor suppression by inducing growth arrest, apoptosis, and

senescence, as well as by blocking angiogenesis. Consistent with the role of p53 as a cell stress-

associated transcription factor [267, 268], we observed increased expression of p53 (Figure 4E)

and p53-responsive Bax (and Bax/Bcl-2 ratio) (Figure 4G) in colon CSCs with RSV-GSE

treatment. This indicates RSV-GSE induced intrinsic apoptotic signaling pathway by Bax-

induced increased permeation of mitochondrial membrane, resulting in release of cytochrome C

and activation of caspases. Whether similar pathway of apoptosis is activated in p53 knockout

cells remains to be seen, although cytochrome C was elevated by RSV-GSE treatment in colon

CSCs with shRNA attenuated p53. Mutational inactivation of p53 is one of the most frequent

events found in over 50−75% of colon cancer cases, and marks transition to metastasis [269-272].

Our results showing that RSV-GSE exerts its biological efficacy, both anti-proliferative and pro-

apoptotic, in colon CSCs independent of their p53 status (Figure 5A-E) confers an advantage to

the use of RSV-GSE for primary and secondary chemoprevention.

99

Preclinical and clinical studies suggest that COX-2 is involved in chronic inflammation

and its activation may be involved in inflammation-mediated stem cell

proliferation/differentiation [273]. Our data suggests that RSV-GSE was more effective compared

to sulindac in suppressing nuclear COX-2 levels (Figure 6A, B) in colon CSCs and colon CSCs

with shRNA attenuated p53 and might further explain higher potency of RSV-GSE combination

compared to sulindac. Further, NSAIDs like sulindac can suppress both COX-1 and COX-2

thereby deplete prostaglandin in tissues, which mediate mucosal bicarbonate production, mucus

secretion, and maintenance of blood flow [274] and thus mucosal healing [275]. This explains the

increased gastrointestinal toxicity (stomach ulcers and loss of adipose tissue deposits) in mice fed

with sulindac. Unlike, sulindac, RSV and GSE (proanthocyanidins) have minimal effects on

COX-1/ PGE2 thus explains the lack of stomach ulcers and adipose tissue loss. Thus, in the future

studies, it is critical to explore whether sulindac eliminates normal colon stem cells along with

colon cancer stem cells whereas RSV-GSE is selective against only colon cancer stem cells. This

aspect could not be assessed in the current study, as we did not include normal control animals

consuming RSV-GSE or sulindac.

100

Chapter 5

Conclusions

5.1 Conclusions

The aim of my dissertation research was to evaluate the anti-cancer effect of a selection of

commonly consumed polyphenols and polyphenol containing foods against colon CSCs using

both in vitro and in vivo models. This was accomplished via the following objectives

Objective 1: To investigate the anti-cancer properties of the anthocyanin-rich extracts of Java

Plum against HCT-116 colon cancer cells and colon CSCs in vitro (Chapter 2).

Anthocyanins have shown potent anti-cancer effects in a variety of models [39, 40], and studies

have shown that anthocyanins selectively inhibit the growth of cancer cells with relatively little or

no effect on the growth of normal cells [41]. This is in contrast to the current standard of care for

colon cancer (i.e. chemotherapy) which is less specific and can induce significant adverse side

effects. In addition, because these treatments do not specifically target CSCs, disease relapse

occurs in the majority of the cases. My results show that anthocyanin-rich JPE exerts cytotoxic

effects not only against the HCT-116 human colon cancer cells but it also induced apoptosis in

and inhibited the self-renewal ability of colon CSCs. The bioavailability of anthocyanins is low

and hence these compounds reach the intestine at high concentrations and it has been suggested

that concentrations in μg/mL dose range in the colon are feasible [42]. Further, these

anthocyanins are metabolized by gut bacteria to various phenolic acids. More studies are required

to understand the mechanism of action and how anthocyanins and their phenolic acids work as

cancer chemopreventive agents.

101

Objective 2: To determine whether baked purple-fleshed potatoes (PP)can selectively target

colon CSCs in the AOM-induced mouse model of colon cancer and investigate potential

mechanisms of anti-CSC activity in vitro using human colon CSCs (Chapter 3).

PP targeted colon CSCs in vitro and in vivo involving the induction of mitochondria-

mediated apoptosis and targeting the Wnt/β-catenin signaling. In vivo, these effects were

associated with decreased tumor incidence. Compared to sulindac, which was used as a positive

control, PP had similar cancer inhibitory activity but had decreased incidence of gastrointestinal

toxicity. Overall, my results indicate a new direction and strategy for future studies of PP

bioactive compounds and the development and application of related natural compounds.

Regardless of the health-benefits, the sensory attributes and consumer acceptance of these new

color-fleshed cultivars should not be discounted. Earlier sensory analysis from our lab, however,

revealed consumers’ readiness to accept purple-fleshed potatoes provided they were educated on

the health benefits [105, 106]. Although my results are promising, it is important to keep in mind

that PP should not be considered a single food approach to cancer prevention, and should be

consumed as part of a varied diet [107, 276].

Objective 3: To evaluate the efficacy of the RSV­GSE combination in targeting colon CSCs in

the AOM-induced mouse model of colon cancer, and determine the underlying molecular

pathways of proliferation and apoptosis targeted in colon CSCs (Chapter 4). My results show that

the RSV-GSE combination decreases CSCs (β-catenin positive crypt cells) and colon

tumorigenesis in vivo to a similar extent as sulindac, the positive control, but without

gastrointestinal toxicity. Specifically, my in vitro mechanistic studies show that RSV-GSE

suppressed proliferation and sphere formation properties of colon CSCs (positive for CD44,

CD133 and ALDH1b1), suppressed pGSK3β and nuclear translocation of β-catenin, a critical

regulator of CSC proliferation, similar to sulindac. RSV-GSE, but not sulindac, suppressed

102

nuclear levels of downstream proteins of Wnt/β-catenin pathway, c-Myc and cyclin D1. RSV-

GSE also induced mitochondrial-mediated apoptosis in colon CSCs characterized by elevated

p53, Bax/Bcl-2 ratio and cleaved PARP. Furthermore, shRNA-mediated knockdown of p53, a

tumor suppressor gene that is mutated in advanced stages of colon cancer, in the colon CSCs did

not alter efficacy of RSV-GSE. The suppression of Wnt/β-catenin signaling and elevated

mitochondrial-mediated apoptosis in colon CSCs supports potential clinical testing/application of

grape bioactives for colon cancer prevention and/or therapy.

5.2 Future work

5.2.1 Developing evidence for anti-cancer effect of polyphenols from indigenous sources

The WHO has predicted that there will be 70% increase in cancer incidence in the developing

countries [277]. More than 60 % of the world’s new cancer cases occur in Africa, Asia, and

Central and South America; 70 % of the world’s cancer deaths also occur in these regions.

Although, these nations have limited access to latest pharmaceutical drugs, people in these

countries have access to foods that contain compounds with potential cancer chemopreventive

bioactivity such as anthocyanins. Thus, development of data showing the efficacy of these

compounds, coupled with dietary recommendation for the consumption of these foods, represents

a potential approach to reducing cancer burden in developing countries. In chapter 2, I evaluated

the in vitro colon cancer and colon CSC inhibitory activity of Java Plum, a fruit commonly

consumed in Asian countries. I found that JPE, an anthocyanin-rich Java Plum extract, induced

apoptosis in both colon cancer cells and colon CSCs. JPE also reduce the colony formation ability

of CSCs (a marker of “stemness”). The next step is to evaluate the anti-colon cancer effect of java

plum in vivo using a mice model of colon cancer. A common approach used for evaluating anti-

103

cancer effects of fruits is typically administration of either extract or supplemented (freeze-dried

form) in diet. Since the fruit is commonly consumed whole vs in the form of an extract,

incorporating in the mice diet would be a feasible approach to test chemopreventive potential of

java plum. The number of in vivo studies demonstrating underlying mechanistic links to the anti-

cancer/chemopreventive properties of phytochemicals derived from dietary foods is relatively low

and thus, there is a need for higher number of studies on how dietary bioactive compounds can

holistically help in preventing colon cancer.

5.2.2 Future studies involving PP and RSV-GSE

In chapter 3 and chapter 4, I tested the colon cancer preventive activity of PP and RSV-

GSE, and evaluated the role of inhibition of CSCs as a potential mechanism of action. My results

show that both PP and RSV-GSE have shown the ability to inhibit colon CSCs in vitro and in

vivo. PP were administered as a whole-food mixed into diet whereas RSV-GSE was in the form

of a supplement mixed in the diet. Although the route of administration to mice in my study has

been similar, both whole-food and supplement based approaches are required for cancer

chemoprevention because depending on the stage of colon cancer (where often in late stages

consumption of solid food is not possible) either a whole-food approach or a pill/powder can be

administered.

The mice model currently used was azoxymethane-induced carcinogen-induced colon

cancer which is similar to sporadic colon cancer seen in humans. Mice develop colon tumors

usually after 6-8 weeks of exposure to azoxymethane. However, humans don’t get exposed to

such carcinogens. Further, in humans, 80% of colon cancer cases are sporadic that typically

develop in older age, hence long-term animal studies are required for a comprehensive evaluation

104

of cancer chemoprevention ability of PP and RSV-GSE. Long-term studies (1 – 2 years) in mice

fed a western diet (representative of US population dietary patterns that are rich in refined sugar

and fat while low in fiber, minerals and vitamins) have shown increased incidence of sporadic

colon and small intestinal tumors compared to mice fed a standard balanced diet [278, 279]. The

tumor incidence, multiplicity and ratio of adenomas to carcinomas were shown to be similar to

that of sporadic human colon cancer; i.e. after two-thirds of the mice’s lifespan. Thus,

incorporating PP or RSV-GSE representative of human consumption patterns in western diet and

long-term feeding could help generate additional evidence.

The development of advanced molecular techniques such as gene expression arrays and

high-throughput non-targeted LC-MS/MS proteomics have given the ability to simultaneously

evaluate the expression levels of various signaling pathways that are altered in cancer cells when

compared to normal cells. Application of microarrays on the RNA samples and LC-MS/MS

proteomics on protein samples of the colon from this study could help shed light on the additional

pathways targeted by PP and RSV-GSE. In the current study, I showed that PP and RSV-GSE

combination targets Wnt/β-catenin stem cell signaling pathway. Wnt/β-catenin is also related to

other key pathways responsible for CSCs self-renewal and metastatic phenotype such as Notch,

nuclear factor kappa beta (NF-κB) and EGFR signaling pathway. Notch pathway activation

inhibits apoptosis of colon CSCs by repressing the cell cycle inhibitor p27. In addition, Notch can

maintain self-renewal and inhibit differentiation through repressing secretory cell lineage [280].

NF-κB recruits CREB-binding protein (CBP) to bind to RelA/p65, this promotes β-catenin

translocation to nucleus, thus activating Wnt and inducing the dedifferentiation of non-cancer

stem cells [281]. The interaction between EGF and EGFR promotes the expression of stem cell-

related molecules such as Notch and Wnt [282]. By evaluating the levels of proteins in these

signaling pathways using advanced techniques such as microarray and proteomics, can give a

comprehensive view of the anti-colon CSCs effect of PP and RSV-GSE. This information could

105

be useful in generation of additional hypothesis driven studies using polyphenols/polyphenol-rich

foods in not only colon cancer but other cancers involving CSCs.

Bibliography

1. Siegel, R.L., et al., Colorectal cancer statistics, 2017. CA: a cancer journal for clinicians, 2017. 67(3): p. 177-193.

2. López, P.J.T., J.S. Albero, and J.A. Rodríguez-Montes, Primary and secondary prevention of colorectal cancer. Clinical medicine insights. Gastroenterology, 2014. 7: p. 33.

3. Winawer, S.J., et al., Risk of colorectal cancer in the families of patients with adenomatous polyps. New England Journal of Medicine, 1996. 334(2): p. 82-87.

4. Bernstein, C.N., et al., Cancer risk in patients with inflammatory bowel disease. Cancer, 2001. 91(4): p. 854-862.

5. Center, M.M., A. Jemal, and E. Ward, International trends in colorectal cancer incidence rates. Cancer Epidemiology and Prevention Biomarkers, 2009. 18(6): p. 1688-1694.

6. Ferrari, P., et al., Lifetime and baseline alcohol intake and risk of colon and rectal cancers in the European prospective investigation into cancer and nutrition (EPIC). International journal of cancer, 2007. 121(9): p. 2065-2072.

7. Marshall, J.R., Prevention of colorectal cancer: diet, chemoprevention, and lifestyle. Gastroenterology clinics of North America, 2008. 37(1): p. 73-82, vi.

8. Bruce, W.R., T.M. Wolever, and A. Giacca, Mechanisms linking diet and colorectal cancer: the possible role of insulin resistance. Nutr Cancer., 2000. 37(1): p. 19-26.

9. Cappellani, A., et al., Strong correlation between diet and development of colorectal cancer. Frontiers in bioscience, 2013. 18: p. 190-8.

10. Society, A.C. Diet and activity factors that affect risks for certain cancers. ACS Guidelines on Nutrition and Physical Activity for Cancer Prevention 2012.

11. Aune, D., et al., Nonlinear reduction in risk for colorectal cancer by fruit and vegetable intake based on meta-analysis of prospective studies. Gastroenterology, 2011. 141(1): p. 106-118.

12. Koushik, A., et al., Fruits, vegetables, and colon cancer risk in a pooled analysis of 14 cohort studies. Journal of the National Cancer Institute, 2007. 99(19): p. 1471-1483.

13. Mariotto, A.B., et al., Projections of the cost of cancer care in the United States: 2010–2020. Journal of the National Cancer Institute, 2011. 103(2): p. 117-128.

14. Kinzler, K.W. and B. Vogelstein, Gatekeepers and caretakers. Nature, 1997. 386(6627): p. 761-763.

15. Karin, M. and F.R. Greten, NF-κB: linking inflammation and immunity to cancer development and progression. Nature Reviews Immunology, 2005. 5(10): p. 749-759.

16. Rajagopalan, H., et al., The significance of unstable chromosomes in colorectal cancer. Nature reviews cancer, 2003. 3(9): p. 695-701.

17. Bedeir, A. and A.M. Krasinskas, Molecular diagnostics of colorectal cancer. Arch Pathol Lab Med, 2011. 135(5): p. 578-87.

18. Subramaniam, R., A. Mizoguchi, and E. Mizoguchi, Mechanistic roles of epithelial and immune cell signaling during the development of colitis-associated cancer. Cancer research frontiers, 2016. 2(1): p. 1.

107

19. Valko, M., et al., Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chemico-biological interactions, 2006. 160(1): p. 1-40.

20. Longley, D.B., D.P. Harkin, and P.G. Johnston, 5-fluorouracil: mechanisms of action and clinical strategies. Nature Reviews Cancer, 2003. 3(5): p. 330-338.

21. Siddik, Z.H., Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene, 2003. 22(47): p. 7265-7279.

22. Qiu, W., et al., Chemoprevention by nonsteroidal anti-inflammatory drugs eliminates oncogenic intestinal stem cells via SMAC-dependent apoptosis. Proceedings of the National Academy of Sciences, 2010. 107(46): p. 20027-20032.

23. Hanahan, D. and R.A. Weinberg, Hallmarks of cancer: the next generation. cell, 2011. 144(5): p. 646-674.

24. Dragu, D.L., et al., Therapies targeting cancer stem cells: Current trends and future challenges. World journal of stem cells, 2015. 7(9): p. 1185.

25. Barker, N., et al., Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature, 2007. 449(7165): p. 1003-1007.

26. Blanpain, C., V. Horsley, and E. Fuchs, Epithelial stem cells: turning over new leaves. Cell, 2007. 128(3): p. 445-458.

27. Fearon, E.R. and B. Vogelstein, A genetic model for colorectal tumorigenesis. Cell, 1990. 61(5): p. 759-767.

28. Sell, S., History of cancer stem cells, in Regulatory networks in stem cells. 2009, Springer. p. 495-503.

29. Dick, J.E., Stem cell concepts renew cancer research. Blood, 2008. 112(13): p. 4793-4807.

30. Todaro, M., et al., Colon cancer stem cells: promise of targeted therapy. Gastroenterology, 2010. 138(6): p. 2151-2162.

31. Khalek, F.J.A., G.I. Gallicano, and L. Mishra, Colon cancer stem cells. Gastrointestinal cancer research: GCR, 2010(Suppl 1): p. S16.

32. Patrawala, L., et al., Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells. Oncogene, 2006. 25(12): p. 1696-1708.

33. Yeung, T.M., et al., Cancer stem cells from colorectal cancer-derived cell lines. Proceedings of the National Academy of Sciences, 2010. 107(8): p. 3722-3727.

34. Al-Hajj, M., et al., Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Academy of Sciences, 2003. 100(7): p. 3983-3988.

35. O’Brien, C.A., et al., A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature, 2007. 445(7123): p. 106-110.

36. Collins, A.T., et al., Prospective identification of tumorigenic prostate cancer stem cells. Cancer research, 2005. 65(23): p. 10946-10951.

37. Singh, S.K., et al., Identification of human brain tumour initiating cells. nature, 2004. 432(7015): p. 396-401.

38. Chu, P., et al., Characterization of a subpopulation of colon cancer cells with stem cell‐like properties. International journal of cancer, 2009. 124(6): p. 1312-1321.

39. Liu, Y.-S., et al., Lgr5 promotes cancer stemness and confers chemoresistance through ABCB1 in colorectal cancer. Biomedicine & Pharmacotherapy, 2013. 67(8): p. 791-799.

40. Barker, N., et al., Crypt stem cells as the cells-of-origin of intestinal cancer. Nature, 2009. 457(7229): p. 608-611.

41. Visvader, J.E., Cells of origin in cancer. Nature, 2011. 469(7330): p. 314-322. 42. Roy, S. and A.P. Majumdar, Signaling in colon cancer stem cells. Journal of molecular

signaling, 2012. 7(1): p. 11.

108

43. Ricci-Vitiani, L., et al., Colon cancer stem cells. Journal of Molecular Medicine, 2009. 87(11): p. 1097.

44. Beck, B. and C. Blanpain, Unravelling cancer stem cell potential. Nature Reviews Cancer, 2013. 13(10): p. 727-738.

45. Yan, J. and D. Tang, Prostate cancer stem-like cells proliferate slowly and resist etoposide-induced cytotoxicity via enhancing DNA damage response. Experimental cell research, 2014. 328(1): p. 132-142.

46. Visvader, J.E. and G.J. Lindeman, Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nature reviews cancer, 2008. 8(10): p. 755-768.

47. Clark, D.W. and K. Palle, Aldehyde dehydrogenases in cancer stem cells: potential as therapeutic targets. Annals of translational medicine, 2016. 4(24).

48. Litman, T., et al., The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2). J Cell Sci, 2000. 113(11): p. 2011-2021.

49. Vasiliou, V. and D.W. Nebert, Analysis and update of the human aldehyde dehydrogenase (ALDH) gene family. Human genomics, 2005. 2(2): p. 138.

50. Alnouti, Y. and C.D. Klaassen, Tissue distribution, ontogeny, and regulation of aldehyde dehydrogenase (Aldh) enzymes mRNA by prototypical microsomal enzyme inducers in mice. Toxicological sciences, 2007. 101(1): p. 51-64.

51. Keysar, S.B. and A. Jimeno, More than markers: biological significance of cancer stem cell-defining molecules. Molecular cancer therapeutics, 2010. 9(9): p. 2450-2457.

52. Fletcher, J.I., et al., ABC transporters in cancer: more than just drug efflux pumps. Nature Reviews Cancer, 2010. 10(2): p. 147-156.

53. Wang, Z., et al., Dihydromyricetin reverses MRP2-mediated MDR and enhances anticancer activity induced by oxaliplatin in colorectal cancer cells. Anti-cancer drugs, 2017. 28(3): p. 281-288.

54. Pan, T., J. Xu, and Y. Zhu, Self-renewal molecular mechanisms of colorectal cancer stem cells. International journal of molecular medicine, 2017. 39(1): p. 9-20.

55. Kris-Etherton, P.M., et al., Bioactive compounds in nutrition and health-research methodologies for establishing biological function: the antioxidant and anti-inflammatory effects of flavonoids on atherosclerosis. Annual review of nutrition, 2004. 24: p. 511-38.

56. Gao, C., et al., Induction of Gsk3β-β-TrCP interaction is required for late phase stabilization of β-catenin in canonical Wnt signaling. Journal of Biological Chemistry, 2014. 289(10): p. 7099-7108.

57. Aberle, H., et al., β‐catenin is a target for the ubiquitin–proteasome pathway. The EMBO journal, 1997. 16(13): p. 3797-3804.

58. Liu, C., et al., Control of β-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell, 2002. 108(6): p. 837-847.

59. Sebio, A., M. Kahn, and H.-J. Lenz, The potential of targeting Wnt/β-catenin in colon cancer. 2014, Taylor & Francis.

60. Krausova, M. and V. Korinek, Wnt signaling in adult intestinal stem cells and cancer. Cellular signalling, 2014. 26(3): p. 570-579.

61. Liao, D.J., et al., Perspectives on c-Myc, Cyclin D1, and their interaction in cancer formation, progression, and response to chemotherapy. Critical Reviews™ in Oncogenesis, 2007. 13(2).

62. Reya, T. and H. Clevers, Wnt signalling in stem cells and cancer. Nature, 2005. 434(7035): p. 843-850.

63. Fevr, T., et al., Wnt/β-catenin is essential for intestinal homeostasis and maintenance of intestinal stem cells. Molecular and cellular biology, 2007. 27(21): p. 7551-7559.

109

64. Nusse, R., et al. Wnt signaling and stem cell control. in Cold Spring Harbor symposia on quantitative biology. 2008. Cold Spring Harbor Laboratory Press.

65. Vermeulen, L., et al., Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. Nature cell biology, 2010. 12(5): p. 468-476.

66. Halazonetis, T.D., V.G. Gorgoulis, and J. Bartek, An oncogene-induced DNA damage model for cancer development. science, 2008. 319(5868): p. 1352-1355.

67. Halberg, R.B., et al., Tumorigenesis in the multiple intestinal neoplasia mouse: redundancy of negative regulators and specificity of modifiers. Proceedings of the National Academy of Sciences, 2000. 97(7): p. 3461-3466.

68. Liu, D., et al., Puma is required for p53-induced depletion of adult stem cells. Nature cell biology, 2010. 12(10): p. 993-998.

69. Davidson, L.A., et al., Targeted deletion of p53 in Lgr5-expressing intestinal stem cells promotes colon tumorigenesis in a preclinical model of colitis-associated cancer. Cancer research, 2015. 75(24): p. 5392-5397.

70. Tsao, R., Chemistry and biochemistry of dietary polyphenols. Nutrients, 2010. 2(12): p. 1231-1246.

71. Pereira, D.M., et al., Phenolics: From chemistry to biology. 2009, Molecular Diversity Preservation International.

72. Crozier, A., I.B. Jaganath, and M.N. Clifford, Dietary phenolics: chemistry, bioavailability and effects on health. Natural product reports, 2009. 26(8): p. 1001-1043.

73. Bravo, L., Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutrition reviews, 1998. 56(11): p. 317-333.

74. Signorelli, P. and R. Ghidoni, Resveratrol as an anticancer nutrient: molecular basis, open questions and promises. The Journal of nutritional biochemistry, 2005. 16(8): p. 449-466.

75. Manach, C., et al., Polyphenols: food sources and bioavailability. The American journal of clinical nutrition, 2004. 79(5): p. 727-747.

76. Nijveldt, R.J., et al., Flavonoids: a review of probable mechanisms of action and potential applications. The American journal of clinical nutrition, 2001. 74(4): p. 418-425.

77. Ramos, S., Cancer chemoprevention and chemotherapy: dietary polyphenols and signalling pathways. Molecular nutrition & food research, 2008. 52(5): p. 507-526.

78. Gu, L., et al., Screening of foods containing proanthocyanidins and their structural characterization using LC-MS/MS and thiolytic degradation. Journal of Agricultural and Food Chemistry, 2003. 51(25): p. 7513-7521.

79. Santos‐Buelga, C. and A. Scalbert, Proanthocyanidins and tannin‐like compounds–nature, occurrence, dietary intake and effects on nutrition and health. Journal of the Science of Food and Agriculture, 2000. 80(7): p. 1094-1117.

80. Gu, L., et al., Concentrations of proanthocyanidins in common foods and estimations of normal consumption. The Journal of nutrition, 2004. 134(3): p. 613-617.

81. Manach, C., et al., Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. The American journal of clinical nutrition, 2005. 81(1): p. 230S-242S.

82. Spencer, J.P., et al., Decomposition of cocoa procyanidins in the gastric milieu. Biochemical and biophysical research communications, 2000. 272(1): p. 236-241.

83. Rios, L.Y., et al., Cocoa procyanidins are stable during gastric transit in humans. The American journal of clinical nutrition, 2002. 76(5): p. 1106-1110.

110

84. Monagas, M., et al., Insights into the metabolism and microbial biotransformation of dietary flavan-3-ols and the bioactivity of their metabolites. Food & function, 2010. 1(3): p. 233-253.

85. Bueno, J.M., et al., Analysis and antioxidant capacity of anthocyanin pigments. Part I: general considerations concerning polyphenols and flavonoids. Critical Reviews in Analytical Chemistry, 2012. 42(2): p. 102-125.

86. Bueno, J.M., et al., Analysis and antioxidant capacity of anthocyanin pigments. Part II: chemical structure, color, and intake of anthocyanins. Critical Reviews in Analytical Chemistry, 2012. 42(2): p. 126-151.

87. Miguel, M.G., Anthocyanins: Antioxidant and/or anti-inflammatory activities. 2011. 88. Kong, J.-M., et al., Analysis and biological activities of anthocyanins. Phytochemistry,

2003. 64(5): p. 923-933. 89. Valenti, L., et al., Dietary anthocyanins as nutritional therapy for nonalcoholic fatty liver

disease. Oxidative medicine and cellular longevity, 2013. 2013. 90. Hidalgo, M., et al., Metabolism of anthocyanins by human gut microflora and their

influence on gut bacterial growth. Journal of Agricultural and Food Chemistry, 2012. 60(15): p. 3882-3890.

91. Miyazawa, T., et al., Direct intestinal absorption of red fruit anthocyanins, cyanidin-3-glucoside and cyanidin-3, 5-diglucoside, into rats and humans. Journal of agricultural and food chemistry, 1999. 47(3): p. 1083-1091.

92. Santos, D.T., et al., Extraction of polyphenols and anthocyanins from the jambul (Syzygium cumini) fruit peels. Food and Public Health, 2013. 3(1): p. 12-20.

93. de Moura, S.C.S.R., et al., Degradation kinetics of anthocyanin of traditional and low-sugar blackberry jam. Food and Bioprocess Technology, 2012. 5(6): p. 2488-2496.

94. Benherlal, P.S. and C. Arumughan, Chemical composition and in vitro antioxidant studies on Syzygium cumini fruit. Journal of the Science of Food and Agriculture, 2007. 87(14): p. 2560-2569.

95. Sagrawat, H., A. Mann, and M. Kharya, Pharmacological potential of Eugenia jambolana: A review. Pharmacognosy magazine, 2006. 2(6): p. 96.

96. Helmstädter, A., Syzygium cumini (L.) SKEELS (Myrtaceae) against diabetes–125 years of research. Die Pharmazie-An International Journal of Pharmaceutical Sciences, 2008. 63(2): p. 91-101.

97. Ayyanar, M. and P. Subash-Babu, Syzygium cumini (L.) Skeels: A review of its phytochemical constituents and traditional uses. Asian Pacific journal of tropical biomedicine, 2012. 2(3): p. 240-246.

98. NPC, Potato statistical yearbook. 2005, National Potato Council (NPC). 99. Potato statistical yearbook. 2006, National Potato Council (NPC). 100. NPC, Potato statistical yearbook. 2007, National Potato Council (NPC). 101. UN. International Year of the Potato 191 session 60. 2008; Available from:

http://www.undemocracy.com/A-RES-60-191/page_1/rect_230,647_688,680. 102. Halton, T.L., et al., Potato and french fry consumption and risk of type 2 diabetes in

women. Am J Clin Nutr, 2006. 83(2): p. 284-90. 103. Reddivari, L., Influence of genetic variability on specialty potato functional components

and their effect on prostate cancer cell lines, in Horticulture. 2007, Texas A&M Universtiy.

104. Madiwale, G., Effect of genotype, storage and processing on the polyphenolic content, composition, in vitro anti-cancer activity and sensory attributes of colored-flesh potatoes., in Department of Food Science and Human Nutrition. 2012, Colorado State University: Fort Collins.

111

105. Madiwale, G.P., et al., Storage elevates phenolic content and antioxidant activity but suppresses antiproliferative and pro-apoptotic properties of colored-flesh potatoes against human colon cancer cell lines. J Agric Food Chem, 2011. 59(15): p. 8155-66.

106. Madiwale, G.P., et al., Combined effects of storage and processing on the bioactive compounds and pro-apoptotic properties of color-fleshed potatoes in human colon cancer cells. J Agric Food Chem, 2012. 60(44): p. 11088-96.

107. Brown, C.R., Antioxidants in potato. Am J Potato Res, 2005. 82: p. 163-172. 108. Rodriguez-Saona, L.E., M.M. Giusti, and R.E. Wrolstad, Anthocyanin pigment

composition of red-fleshed potatoes. Journal of Food Science, 1998. 63(3): p. 458-465. 109. Radhakrishnan, S., et al., Purple potato anthocyanins exert anti-cancer properties and

suppress oxidative stress even after processing. Potato Association of America - PAA 2011, 2011.

110. Madiwale, G.P., et al., Combined effects of storage and processing on the composition and anti-cancer properties of color-fleshed potatoes in vitro. J. Agric. Food Chem., 2012. Just Accepted.

111. Vanamala, J., et al., Resveratrol suppresses IGF-1 induced human colon cancer cell proliferation and elevates apoptosis via suppression of IGF-1R/Wnt and activation of p53 signaling pathways. BMC Cancer, 2010. 10: p. 238.

112. Osei-Mensah, M., Synthesis of Resveratrol Ester Derivatives Using Selective Enzymatic Hydrolysis., in Master of Science in Chemistry 2011, East Tennessee State University.

113. Nomoto, H., et al., Chemoprevention of colorectal cancer by grape seed proanthocyanidin is accompanied by a decrease in proliferation and increase in apoptosis. Nutrition and cancer, 2004. 49(1): p. 81-88.

114. Kaur, M., et al., Grape seed extract inhibits in vitro and in vivo growth of human colorectal carcinoma cells. Clinical Cancer Research, 2006. 12(20): p. 6194-6202.

115. Kaur, M., C. Agarwal, and R. Agarwal, Anticancer and cancer chemopreventive potential of grape seed extract and other grape-based products. The Journal of nutrition, 2009. 139(9): p. 1806S-1812S.

116. Kennedy, J.A., et al., Composition of grape skin proanthocyanidins at different stages of berry development. Journal of Agricultural and Food Chemistry, 2001. 49(11): p. 5348-5355.

117. Gillet, J.-P., S. Varma, and M.M. Gottesman, The clinical relevance of cancer cell lines. Journal of the National Cancer Institute, 2013. 105(7): p. 452-458.

118. Gey, G., Tissue culture studies of the proliferative capacity of cervical carcinoma and normal epithelium. Cancer Res., 1952. 12: p. 264-265.

119. Hagiwara, A., et al., Pronounced inhibition by a natural anthocyanin, purple corn color, of 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP)-associated colorectal carcinogenesis in male F344 rats pretreated with 1, 2-dimethylhydrazine. Cancer letters, 2001. 171(1): p. 17-25.

120. Bissahoyo, A., et al., Azoxymethane is a genetic background-dependent colorectal tumor initiator and promoter in mice: effects of dose, route, and diet. Toxicological Sciences, 2005. 88(2): p. 340-345.

121. Corpet, D.E. and F. Pierre, How good are rodent models of carcinogenesis in predicting efficacy in humans? A systematic review and meta-analysis of colon chemoprevention in rats, mice and men. Eur J Cancer, 2005. 41(13): p. 1911-22.

122. Ilsley, J.N., et al., Dietary iron promotes azoxymethane-induced colon tumors in mice. Nutrition and cancer, 2004. 49(2): p. 162-169.

123. Arts, I.C. and P.C. Hollman, Polyphenols and disease risk in epidemiologic studies. The American journal of clinical nutrition, 2005. 81(1): p. 317S-325S.

112

124. Haslam, E., Vegetable tannins–Lessons of a phytochemical lifetime. Phytochemistry, 2007. 68(22): p. 2713-2721.

125. Grotewold, E., The genetics and biochemistry of floral pigments. Annu. Rev. Plant Biol., 2006. 57: p. 761-780.

126. Manetas, Y., Why some leaves are anthocyanic and why most anthocyanic leaves are red? Flora-Morphology, Distribution, Functional Ecology of Plants, 2006. 201(3): p. 163-177.

127. Schaefer, H.M. and G. Rolshausen, Plants on red alert: do insects pay attention? BioEssays, 2006. 28(1): p. 65-71.

128. Treutter, D., Significance of flavonoids in plant resistance: a review. Environmental Chemistry Letters, 2006. 4(3): p. 147.

129. Mazza, C.A., et al., Functional significance and induction by solar radiation of ultraviolet-absorbing sunscreens in field-grown soybean crops. Plant physiology, 2000. 122(1): p. 117-126.

130. Reuter, S., et al., Oxidative stress, inflammation, and cancer: how are they linked? Free Radical Biology and Medicine, 2010. 49(11): p. 1603-1616.

131. Kok, T., S. Breda, and M. Manson, Mechanisms of combined action of different chemopreventive dietary compounds. European Journal of Nutrition, 2008. 47.

132. Johnson, I.T., Phytochemicals and cancer. Proceedings of the Nutrition Society, 2007. 66(2): p. 207-215.

133. Zhao, C., et al., Effects of commercial anthocyanin-rich extracts on colonic cancer and nontumorigenic colonic cell growth. Journal of Agricultural and food Chemistry, 2004. 52(20): p. 6122-6128.

134. Marko, D., et al., The substitution pattern of anthocyanidins affects different cellular signaling cascades regulating cell proliferation. Molecular nutrition & food research, 2004. 48(4): p. 318-325.

135. Kang, S.-Y., et al., Tart cherry anthocyanins inhibit tumor development in Apc Min mice and reduce proliferation of human colon cancer cells. Cancer letters, 2003. 194(1): p. 13-19.

136. Coates, E.M., et al., Colon-available raspberry polyphenols exhibit anti-cancer effects on in vitro models of colon cancer. Journal of Carcinogenesis, 2007. 6(1): p. 4.

137. Johnson, J.L., et al., Effect of black raspberry (Rubus occidentalis L.) extract variation conditioned by cultivar, production site, and fruit maturity stage on colon cancer cell proliferation. Journal of agricultural and food chemistry, 2011. 59(5): p. 1638-1645.

138. Wu, Q.K., et al., Berry phenolic extracts modulate the expression of p21WAF1 and Bax but not Bcl-2 in HT-29 colon cancer cells. Journal of agricultural and food chemistry, 2007. 55(4): p. 1156-1163.

139. Wang, L.-S. and G.D. Stoner, Anthocyanins and their role in cancer prevention. Cancer letters, 2008. 269(2): p. 281-290.

140. Mazewski, C., K. Liang, and E.G. de Mejia, Inhibitory potential of anthocyanin-rich purple and red corn extracts on human colorectal cancer cell proliferation in vitro. Journal of Functional Foods, 2017. 34: p. 254-265.

141. Jing, P., et al., Structure− function relationships of anthocyanins from various anthocyanin-rich extracts on the inhibition of colon cancer cell growth. Journal of agricultural and food chemistry, 2008. 56(20): p. 9391-9398.

142. Madiwale, G.P., et al., Combined effects of storage and processing on the bioactive compounds and pro-apoptotic properties of color-fleshed potatoes in human colon cancer cells. Journal of agricultural and food chemistry, 2012. 60(44): p. 11088-11096.

113

143. Olsson, M.E., et al., Inhibition of cancer cell proliferation in vitro by fruit and berry extracts and correlations with antioxidant levels. Journal of agricultural and food chemistry, 2004. 52(24): p. 7264-7271.

144. Seeram, N.P., et al., Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro. Journal of agricultural and food chemistry, 2006. 54(25): p. 9329-9339.

145. Reddivari, L., et al., Anthocyanin fraction from potato extracts is cytotoxic to prostate cancer cells through activation of caspase-dependent and caspase-independent pathways. Carcinogenesis, 2007. 28(10): p. 2227-2235.

146. Chang, Y.-C., et al., Hibiscus anthocyanins rich extract-induced apoptotic cell death in human promyelocytic leukemia cells. Toxicology and Applied pharmacology, 2005. 205(3): p. 201-212.

147. Feng, R., et al., Cyanidin-3-rutinoside, a natural polyphenol antioxidant, selectively kills leukemic cells by induction of oxidative stress. Journal of Biological Chemistry, 2007. 282(18): p. 13468-13476.

148. Jang, M., et al., Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science, 1997. 275(5297): p. 218-220.

149. Mahyar-Roemer, M., et al., Resveratrol induces colon tumor cell apoptosis independently of p53 and precede by epithelial differentiation, mitochondrial proliferation and membrane potential collapse. International journal of cancer. Journal international du cancer, 2001. 94(5): p. 615-22.

150. Wolter, F., et al., Downregulation of the cyclin D1/Cdk4 complex occurs during resveratrol-induced cell cycle arrest in colon cancer cell lines. J Nutr., 2001. 131(8): p. 2197-203.

151. Kaur, M., et al., Grape seed extract induces cell cycle arrest and apoptosis in human colon carcinoma cells. Nutrition and cancer, 2008. 60(S1): p. 2-11.

152. Radhakrishnan, S., et al., Resveratrol potentiates grape seed extract induced human colon cancer cell apoptosis. Front Biosci (Elite Ed), 2011. 3: p. 1509-1523.

153. !!! INVALID CITATION !!! (de Kok, 2008 #174;Hennekens, 1996 #9). 154. de Kok, T.M., S.G. van Breda, and M.M. Manson, Mechanisms of combined action of

different chemopreventive dietary compounds: a review. European journal of nutrition, 2008. 47 Suppl 2: p. 51-9.

155. Liu, R.H., Potential synergy of phytochemicals in cancer prevention: mechanism of action. J Nutr., 2004. 134(12 Suppl): p. 3479S-3485S.

156. Dinicola, S., et al., Antiproliferative and Apoptotic Effects Triggered by Grape Seed Extract (GSE) versus Epigallocatechin and Procyanidins on Colon Cancer Cell Lines. International journal of molecular sciences, 2012. 13(1): p. 651-64.

157. Majumdar, A.P., et al., Curcumin synergizes with resveratrol to inhibit colon cancer. Nutr Cancer, 2009. 61(4): p. 544-53.

158. Aggarwal, B.B., et al., Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer research, 2004. 24(5A): p. 2783-840.

159. Chuang, C.C. and M.K. McIntosh, Potential mechanisms by which polyphenol-rich grapes prevent obesity-mediated inflammation and metabolic diseases. Annu Rev Nutr, 2011. 31: p. 155-76.

160. Mertens-Talcott, S.U., S.T. Talcott, and S.S. Percival, Low concentrations of quercetin and ellagic acid synergistically influence proliferation, cytotoxicity and apoptosis in MOLT-4 human leukemia cells. J Nutr., 2003. 133(8): p. 2669-74.

114

161. Del Follo-Martinez, A., et al., Resveratrol and quercetin in combination have anticancer activity in colon cancer cells and repress oncogenic microRNA-27a. Nutr Cancer., 2013. 65(3): p. 494-504.

162. Mertens-Talcott, S.U. and S.S. Percival, Ellagic acid and quercetin interact synergistically with resveratrol in the induction of apoptosis and cause transient cell cycle arrest in human leukemia cells. Cancer letters, 2005. 218(2): p. 141-51.

163. Vanamala, J., et al., Dietary fish oil and pectin enhance colonocyte apoptosis in part through suppression of PPARdelta/PGE2 and elevation of PGE3. American Journal of Potato Research, 2008. 29(4): p. 790-6.

164. Yang, J. and R.H. Liu, Synergistic effect of apple extracts and quercetin 3-beta-d-glucoside combination on antiproliferative activity in MCF-7 human breast cancer cells in vitro. J Agric Food Chem, 2009. 57(18): p. 8581-6.

165. Lomonosova, E. and G. Chinnadurai, BH3-only proteins in apoptosis and beyond: an overview. Oncogene, 2008. 27 Suppl 1: p. S2-19.

166. Kang, M.H. and C.P. Reynolds, Bcl-2 inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy. Clin Cancer Res, 2009. 15(4): p. 1126-32.

167. Hagiwara, A., et al., Prevention by natural food anthocyanins, purple sweet potato color and red cabbage color, of 2-amino-1-methyl-6-phenylimidazo [4, 5-B] pyridine (phip)-associated colorectal carcinogenesis in rats. The Journal of toxicological sciences, 2002. 27(1): p. 57-68.

168. Harris, G., et al., Effects of lyophilized black raspberries on azoxymethane-induced colon cancer and 8-hydroxy-2′-deoxyguanosine levels in the Fischer 344 rat. Nutrition and cancer, 2001. 40(2): p. 125-133.

169. Lala, G., et al., Anthocyanin-rich extracts inhibit multiple biomarkers of colon cancer in rats. Nutrition and cancer, 2006. 54(1): p. 84-93.

170. Magnuson, B., et al. Anthocyanin-rich extracts inhibit growth of human colon cancer cells and azoxymethane-induced colon aberrant crypts in rats: implications for colon cancer chemoprevention. in CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION. 2003. AMER ASSOC CANCER RESEARCH 615 CHESTNUT ST, 17TH FLOOR, PHILADELPHIA, PA 19106-4404 USA.

171. Bobe, G., et al., Dietary anthocyanin-rich tart cherry extract inhibits intestinal tumorigenesis in APCMin mice fed suboptimal levels of sulindac. Journal of agricultural and food chemistry, 2006. 54(25): p. 9322-9328.

172. Cooke, D., et al., Effect of cyanidin‐3‐glucoside and an anthocyanin mixture from bilberry on adenoma development in the ApcMin mouse model of intestinal carcinogenesis—Relationship with tissue anthocyanin levels. International journal of cancer, 2006. 119(9): p. 2213-2220.

173. Misikangas, M., et al., Three Nordic berries inhibit intestinal tumorigenesis in multiple intestinal neoplasia/+ mice by modulating β-catenin signaling in the tumor and transcription in the mucosa. The Journal of nutrition, 2007. 137(10): p. 2285-2290.

174. Tessitore, L., et al., Resveratrol depresses the growth of colorectal aberrant crypt foci by affecting bax and p21 CIP expression. Carcinogenesis, 2000. 21(8): p. 1619-1622.

175. Schneider, Y., et al., Resveratrol inhibits intestinal tumorigenesis and modulates host-defense-related gene expression in an animal model of human familial adenomatous polyposis. Nutrition and cancer, 2001. 39(1): p. 102-107.

176. Ziegler, C.C., et al., Dietary resveratrol does not affect intestinal tumorigenesis in ApcMin/+ mice. The Journal of nutrition, 2004. 134(1): p. 5-10.

115

177. Singletary, K.W. and B. Meline, Effect of grape seed proanthocyanidins on colon aberrant crypts and breast tumors in a rat dual-organ tumor model. Nutrition and cancer, 2001. 39(2): p. 252-258.

178. Li, X.-L., et al., Therapeutic effect and mechanism of proanthocyanidins from grape seeds in rats with TNBS-induced ulcerative colitis. Canadian journal of physiology and pharmacology, 2008. 86(12): p. 841-849.

179. Cheah, K.Y., et al., Grape seed extract reduces the severity of selected disease markers in the proximal colon of dextran sulphate sodium-induced colitis in rats. Digestive diseases and sciences, 2013. 58(4): p. 970-977.

180. Stefanska, B., et al., Epigenetic mechanisms in anti‐cancer actions of bioactive food components–the implications in cancer prevention. British journal of pharmacology, 2012. 167(2): p. 279-297.

181. Patel, B.B. and A.P. Majumdar, Synergistic role of curcumin with current therapeutics in colorectal cancer: minireview. Nutrition and cancer, 2009. 61(6): p. 842-846.

182. Taylor, W.F. and E. Jabbarzadeh, The use of natural products to target cancer stem cells. American journal of cancer research, 2017. 7(7): p. 1588.

183. Lin, L., et al., Targeting colon cancer stem cells using a new curcumin analogue, GO-Y030. British journal of cancer, 2011. 105(2): p. 212-220.

184. Atashpour, S., et al., Quercetin induces cell cycle arrest and apoptosis in CD133+ cancer stem cells of human colorectal HT29 cancer cell line and enhances anticancer effects of doxorubicin. Iranian journal of basic medical sciences, 2015. 18(7): p. 635.

185. Yaffe, P.B., et al., Piperine, an alkaloid from black pepper, inhibits growth of human colon cancer cells via G1 arrest and apoptosis triggered by endoplasmic reticulum stress. Molecular carcinogenesis, 2015. 54(10): p. 1070-1085.

186. Samykutty, A., et al., Piperine, a bioactive component of pepper spice exerts therapeutic effects on androgen dependent and androgen independent prostate cancer cells. PLoS One, 2013. 8(6): p. e65889.

187. Siegel, R., et al., Cancer statistics, 2014. CA: a cancer journal for clinicians, 2014. 64(1): p. 9-29.

188. Markowitz, S.D. and M.M. Bertagnolli, Molecular origins of cancer: Molecular basis of colorectal cancer. N Engl J Med, 2009. 361(25): p. 2449-60.

189. Reya, T., et al., Stem cells, cancer, and cancer stem cells. Nature, 2001. 414(6859): p. 105-11.

190. Todaro, M., et al., Colon cancer stem cells: promise of targeted therapy. Gastroenterology, 2010. 138(6): p. 2151-62.

191. Oh, P.S., et al., Schlafen-3 decreases cancer stem cell marker expression and autocrine/juxtacrine signaling in FOLFOX-resistant colon cancer cells. Am J Physiol Gastrointest Liver Physiol, 2011. 301(2): p. G347-55.

192. Zhang, M. and J.M. Rosen, Stem cells in the etiology and treatment of cancer. Curr Opin Genet Dev, 2006. 16(1): p. 60-4.

193. Society, A.C., Colorectal Cancer Facts & Figures 2011-2013. American Cancer Society, 2011.

194. Boffetta, P., et al., Fruit and vegetable intake and overall cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC). Journal of the National Cancer Institute, 2010. 102(8): p. 529-537.

195. Wang, L.S. and G.D. Stoner, Anthocyanins and their role in cancer prevention. Cancer Lett, 2008. 269(2): p. 281-90.

196. Thomasset, S., et al., Pilot study of oral anthocyanins for colorectal cancer chemoprevention. Cancer prevention research, 2009. 2(7): p. 625-633.

116

197. Stewart, B. and C. Wild, World Cancer Report 2014. Lyon, France: International Agency for Research on Cancer. World Health Organization, 2014.

198. Siegel, R.L., K.D. Miller, and A. Jemal, Cancer statistics, 2015. CA: A Cancer Journal for Clinicians, 2015. 65(1): p. 5-29.

199. Torre, L.A., et al., Global cancer statistics, 2012. CA: A Cancer Journal for Clinicians, 2015. 65(2): p. 87-108.

200. Veigas, J.M., et al., Chemical nature, stability and bioefficacies of anthocyanins from fruit peel of Syzygium cumini Skeels. Food Chemistry, 2007. 105(2): p. 619-627.

201. De Brito, E.S., et al., Anthocyanins present in selected tropical fruits: acerola, jambolão, jussara, and guajiru. Journal of Agricultural and Food Chemistry, 2007. 55(23): p. 9389-9394.

202. Li, L., et al., Eugenia jambolana Lam. berry extract inhibits growth and induces apoptosis of human breast cancer but not non-tumorigenic breast cells. Journal of agricultural and food chemistry, 2009. 57(3): p. 826-831.

203. Nazif, N.M., The anthocyanin components and cytotoxic activity of Syzygium cumini (L.) fruits growing in Egypt. Natural Product Sciences, 2007. 13(2): p. 135-139.

204. Li, L., et al., Eugenia jambolana Lam. berry extract inhibits growth and induces apoptosis of human breast cancer but not non-tumorigenic breast cells. J Agric Food Chem, 2009. 57(3): p. 826-31.

205. Massey, A.R., L. Reddivari, and J. Vanamala, The Dermal layer of sweet sorghum (Sorghum bicolor) stalk, a byproduct of biofuel production and source of unique 3-deoxyanthocyanidins, has more antiproliferative and proapoptotic activity than the pith in p53 variants of HCT116 and colon cancer stem cells. Journal of agricultural and food chemistry, 2014. 62(14): p. 3150-3159.

206. Apel, A., et al., Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy. Cancer research, 2008. 68(5): p. 1485-1494.

207. Wang, L.S., et al., Modulation of genetic and epigenetic biomarkers of colorectal cancer in humans by black raspberries: a phase I pilot study. Clinical cancer research : an official journal of the American Association for Cancer Research, 2011. 17(3): p. 598-610.

208. Todaro, M., et al., Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4. Cell stem cell, 2007. 1(4): p. 389-402.

209. Galati, G. and P.J. O'Brien, Potential toxicity of flavonoids and other dietary phenolics: significance for their chemopreventive and anticancer properties. Free Radical Biology and Medicine, 2004. 37(3): p. 287-303.

210. Vanamala, J., et al., Resveratrol suppresses human colon cancer cell proliferation and induces apoptosis via targeting the pentose phosphate and the talin-FAK signaling pathways-A proteomic approach. Proteome Sci, 2011. 9(1): p. 49.

211. Yun, J.M., et al., Delphinidin, an anthocyanidin in pigmented fruits and vegetables, induces apoptosis and cell cycle arrest in human colon cancer HCT116 cells. Mol Carcinog, 2009. 48(3): p. 260-70.

212. Malik, M., et al., Anthocyanin-rich extract from Aronia meloncarpa E. induces a cell cycle block in colon cancer but not normal colonic cells. Nutrition and cancer, 2003. 46(2): p. 186-196.

213. Srivastava, A., et al., Effect of anthocyanin fractions from selected cultivars of Georgia-grown blueberries on apoptosis and phase II enzymes. Journal of agricultural and food chemistry, 2007. 55(8): p. 3180-3185.

214. Yu, Y., et al., Elimination of Colon Cancer Stem-Like Cells by the Combination of Curcumin and FOLFOX. Transl Oncol, 2009. 2(4): p. 321-8.

117

215. Barker, N., et al., Crypt stem cells as the cells-of-origin of intestinal cancer. Nature, 2009. 457(7229): p. 608-11.

216. van de Wetering, M., et al., The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell, 2002. 111(2): p. 241-50.

217. Schepers, A. and H. Clevers, Wnt signaling, stem cells, and cancer of the gastrointestinal tract. Cold Spring Harbor perspectives in biology, 2012. 4(4): p. a007989.

218. Chen, Z., et al., Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature, 2005. 436(7051): p. 725-30.

219. Qiu, W., et al., Chemoprevention by nonsteroidal anti-inflammatory drugs eliminates oncogenic intestinal stem cells via SMAC-dependent apoptosis. Proc Natl Acad Sci U S A, 2010. 107(46): p. 20027-32.

220. Rao, P. and E.E. Knaus, Evolution of nonsteroidal anti-inflammatory drugs (NSAIDs): cyclooxygenase (COX) inhibition and beyond. Journal of Pharmacy & Pharmaceutical Sciences, 2008. 11(2): p. 81s-110s.

221. Narsinghani, T. and R. Sharma, Lead Optimization on Conventional Non‐Steroidal Anti‐Inflammatory Drugs: An Approach to Reduce Gastrointestinal Toxicity. Chemical biology & drug design, 2014.

222. Davies, R.J., R. Miller, and N. Coleman, Colorectal cancer screening: prospects for molecular stool analysis. Nat Rev Cancer, 2005. 5(3): p. 199-209.

223. de Jong, A.E., et al., Prevalence of adenomas among young individuals at average risk for colorectal cancer. Am J Gastroenterol, 2005. 100(1): p. 139-43.

224. Chen, T., et al., Randomized phase II trial of lyophilized strawberries in patients with dysplastic precancerous lesions of the esophagus. Cancer prevention research, 2012. 5(1): p. 41-50.

225. Potato statistical yearbook. 2008, National Potato Council (NPC). 226. Madiwale, G.P., et al., Combined effects of storage and processing on the bioactive

compounds and pro-apoptotic properties of color-fleshed potatoes in human colon cancer cells. Journal of agricultural and food chemistry, 2012. 60(44): p. 11088-96.

227. Xiao, J. and P. Hogger, Stability of Dietary Polyphenols under the Cell Culture Conditions: Avoiding Erroneous Conclusions. J Agric Food Chem, 2015.

228. Eichhorn, S. and P. Winterhalter, Anthocyanins from pigmented potato (Solanum tuberosum L.) varieties. Food Research International, 2005. 38(8–9): p. 943-948.

229. Kasdagly, M., et al., Colon carcinogenesis: Influence of Western diet-induced obesity and targeting stem cells using dietary bioactive compounds. Nutrition, 2014. 30(11): p. 1242-1256.

230. Maltzman, T., et al., AOM-induced mouse colon tumors do not express full-length APC protein. Carcinogenesis, 1997. 18(12): p. 2435-9.

231. Takahashi, M., et al., Frequent mutations of the beta-catenin gene in mouse colon tumors induced by azoxymethane. Carcinogenesis, 2000. 21(6): p. 1117-20.

232. Radhakrishnan, S., et al., Resveratrol potentiates grape seed extract induced human colon cancer cell apoptosis. Front Biosci (Elite Ed), 2011. 3: p. 1509-23.

233. Vanamala, J., et al., Resveratrol suppresses IGF-1 induced human colon cancer cell proliferation and elevates apoptosis via suppression of IGF-1R/Wnt and activation of p53 signaling pathways. BMC cancer, 2010. 10(1): p. 238.

234. Vanamala, J., et al., Resveratrol suppresses human colon cancer cell proliferation and induces apoptosis via targeting the pentose phosphate and the talin-FAK signaling pathways-A proteomic approach. Proteome Science, 2011. 9(1): p. 49.

118

235. Kumar, S., et al., Silibinin strongly inhibits the growth kinetics of colon cancer stem cell-enriched spheroids by modulating interleukin 4/6-mediated survival signals. Oncotarget, 2014. 5(13): p. 4972.

236. De Angelis, P., et al., Apoptosis and expression of Bax, Bcl-x, and Bcl-2 apoptotic regulatory proteins in colorectal carcinomas, and association with p53 genotype/phenotype. Molecular Pathology, 1998. 51(5): p. 254.

237. Hu, Y., R.K. Le Leu, and G.P. Young, Sulindac corrects defective apoptosis and suppresses azoxymethane-induced colonic oncogenesis in p53 knockout mice. International Journal of Cancer, 2005. 116(6): p. 870-875.

238. Jurgensmeier, J.M., et al., Bax directly induces release of cytochrome c from isolated mitochondria. Proceedings of the National Academy of Sciences of the United States of America, 1998. 95(9): p. 4997-5002.

239. Tetsu, O. and F. McCormick, Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature, 1999. 398(6726): p. 422-6.

240. Kanazawa, S., et al., c-Myc recruits P-TEFb for transcription, cellular proliferation and apoptosis. Oncogene, 2003. 22(36): p. 5707-5711.

241. Kanwar, S.S., et al., The Wnt/beta-catenin pathway regulates growth and maintenance of colonospheres. Mol Cancer, 2010. 9: p. 212.

242. Roy, S. and A.P. Majumdar, Signaling in colon cancer stem cells. J Mol Signal, 2012. 7(1): p. 11.

243. Liu, R.H., Dietary bioactive compounds and their health implications. Journal of food science, 2013. 78(s1): p. A18-A25.

244. Bishayee, A., Cancer prevention and treatment with resveratrol: from rodent studies to clinical trials. Cancer prevention research, 2009. 2(5): p. 409-418.

245. Agarwal, C., R.P. Singh, and R. Agarwal, Grape seed extract induces apoptotic death of human prostate carcinoma DU145 cells via caspases activation accompanied by dissipation of mitochondrial membrane potential and cytochrome c release. American Journal of Potato Research, 2002. 23(11): p. 1869-76.

246. Sharma, G., et al., Synergistic anti-cancer effects of grape seed extract and conventional cytotoxic agent doxorubicin against human breast carcinoma cells. Breast cancer research and treatment, 2004. 85(1): p. 1-12.

247. Kemper, K., C. Grandela, and J.P. Medema, Molecular identification and targeting of colorectal cancer stem cells. Oncotarget, 2010. 1(6): p. 387-395.

248. Mladenova, D., et al., The NSAID sulindac is chemopreventive in the mouse distal colon but carcinogenic in the proximal colon. Gut, 2010: p. gut. 2010.208314.

249. Weber, H.A., et al., Comparison of proanthocyanidins in commercial antioxidants: grape seed and pine bark extracts. J Agric Food Chem, 2007. 55(1): p. 148-56.

250. Agarwal, C., et al., Fractionation of high molecular weight tannins in grape seed extract and identification of procyanidin B2-3,3'-di-O-gallate as a major active constituent causing growth inhibition and apoptotic death of DU145 human prostate carcinoma cells. Carcinogenesis, 2007. 28(7): p. 1478-84.

251. De Groote, D., et al., Effect of the intake of resveratrol, resveratrol phosphate, and catechin-rich grape seed extract on markers of oxidative stress and gene expression in adult obese subjects. Annals of Nutrition and Metabolism, 2012(61): p. 15-24.

252. Cruz–Correa, M., et al., Long-term treatment with sulindac in familial adenomatous polyposis: a prospective cohort study. Gastroenterology, 2002. 122(3): p. 641-645.

253. Boocock, D.J., et al., Phase I dose escalation pharmacokinetic study in healthy volunteers of resveratrol, a potential cancer chemopreventive agent. Cancer Epidemiology Biomarkers & Prevention, 2007. 16(6): p. 1246-1252.

119

254. Bentivegna, S. and K. Whitney, Subchronic 3-month oral toxicity study of grape seed and grape skin extracts. Food and Chemical Toxicology, 2002. 40(12): p. 1731-1743.

255. Ajani, J.A., et al., Cancer Stem Cells: The Promise and the Potential. Seminars in Oncology, 2015. 42, Supplement 1(0): p. S3-S17.

256. Deng, Y., et al., 5-Fluorouracil upregulates the activity of Wnt signaling pathway in CD133-positive colon cancer stem-like cells. Chin J Cancer, 2010. 29(9): p. 810-815.

257. Holland, J.D., et al., Wnt signaling in stem and cancer stem cells. Current opinion in cell biology, 2013. 25(2): p. 254-264.

258. van Noort, M., et al., Wnt signaling controls the phosphorylation status of β-catenin. Journal of Biological Chemistry, 2002. 277(20): p. 17901-17905.

259. Van De Wetering, M., et al., The β-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell, 2002. 111(2): p. 241-250.

260. Charepalli, V., et al., Anthocyanin-containing purple-fleshed potatoes suppress colon tumorigenesis via elimination of colon cancer stem cells. The Journal of Nutritional Biochemistry, 2015.

261. Tatsuguchi, A., et al., Differential expression of cyclo‐oxygenase‐2 and nuclear β‐catenin in colorectal cancer tissue. Alimentary Pharmacology & Therapeutics, 2006. 24(s4): p. 153-159.

262. Kanwar, S.S., et al., The Wnt/β-catenin pathway regulates growth and maintenance of colonospheres. Molecular cancer, 2010. 9(1): p. 212.

263. Velmurugan, B., et al., Dietary feeding of grape seed extract prevents intestinal tumorigenesis in APC min/+ mice. Neoplasia, 2010. 12(1): p. 95-102.

264. Yu, Y., et al., Elimination of colon cancer stem-like cells by the combination of curcumin and FOLFOX. Translational oncology, 2009. 2(4): p. 321-328.

265. Chen, B.-J., et al., Small Molecules Targeting c-Myc Oncogene: Promising Anti-Cancer Therapeutics. International journal of biological sciences, 2014. 10(10): p. 1084.

266. Pistollato, F., F. Giampieri, and M. Battino, The use of plant-derived bioactive compounds to target cancer stem cells and modulate tumor microenvironment. Food and Chemical Toxicology, 2015. 75: p. 58-70.

267. Vogelstein, B., D. Lane, and A.J. Levine, Surfing the p53 network. Nature, 2000. 408(6810): p. 307-310.

268. Hoffman, B. and D. Liebermann, Apoptotic signaling by c-MYC. Oncogene, 2008. 27(50): p. 6462-6472.

269. Worthley, D.L., et al., Colorectal carcinogenesis: road maps to cancer. World Journal of Gastroenterology, 2007. 13(28): p. 3784.

270. Markowitz, S.D. and M.M. Bertagnolli, Molecular basis of colorectal cancer. New England Journal of Medicine, 2009. 361(25): p. 2449-2460.

271. Bedeir, A. and A.M. Krasinskas, Molecular diagnostics of colorectal cancer. Archives of pathology & laboratory medicine, 2011. 135(5): p. 578-587.

272. Sarasqueta, A.F., et al., Integral analysis of p53 and its value as prognostic factor in sporadic colon cancer. BMC Cancer, 2013. 13(1): p. 277.

273. Thanan, R., et al., Nuclear Localization of COX-2 in relation to the Expression of Stemness Markers in Urinary Bladder Cancer. Mediators of Inflammation, 2012. 2012: p. 8.

274. Cheung, D.Y., et al., Proanthocyanidin from Grape Seed Extracts Protects Indomethacin-Induced Small Intestinal Mucosal Injury. Gastroenterology Research and Practice, 2014. 2014: p. 8.

120

275. Takeuchi, K., Prostaglandin EP receptors and their roles in mucosal protection and ulcer healing in the gastrointestinal tract. Advances in clinical chemistry, 2010. 51: p. 121.

276. Camire, M.E., S. Kubow, and D.J. Donnelly, Potatoes and Human Health. Critical Reviews in Food Science and Nutrition, 2009. 49(10): p. 823-840.

277. Stewart, B. and C. Wild, World cancer report 2014. 2014. Lyon CEDEX, France, 2014. 278. Peregrina, K., et al., Vitamin D is a determinant of mouse intestinal Lgr5 stem cell

functions. Carcinogenesis, 2014. 36(1): p. 25-31. 279. Newmark, H., et al., A Western-style diet induces benign and malignant neoplasms in the

colon of normal C57Bl/6 mice. Carcinogenesis, 2001. 22(11): p. 1871-1875. 280. Ghaleb, A.M., et al., Notch inhibits expression of the Krüppel-like factor 4 tumor

suppressor in the intestinal epithelium. Molecular Cancer Research, 2008. 6(12): p. 1920-1927.

281. Schwitalla, S., et al., Intestinal tumorigenesis initiated by dedifferentiation and acquisition of stem-cell-like properties. Cell, 2013. 152(1): p. 25-38.

282. Feng, Y., et al., EGF signalling pathway regulates colon cancer stem cell proliferation and apoptosis. Cell proliferation, 2012. 45(5): p. 413-419.

VITA

Venkata Charepalli

Education

• PhD in Food Science, Pennsylvania State University August 2013 – December 2018

• MS in Biochemistry, Colorado State University August 2010 – December 2012

• B.Tech in Biotechnology, JNTU, India August 2006 – June 2010

Publications

• Pigs, unlike mice, have two distinct colonic stem cell populations similar to humans that

respond to high-calorie diet prior to insulin resistance. AACR Cancer Prevention Research.

2017, 10(8), 442-450.

• Grape compounds suppress colon cancer stem cells in vitro and in a rodent model of colon

carcinogenesis. BMC Complementary and Alternative Medicine. 2016, 16:278.

• Eugenia jambolana (Java Plum) fruit extract exhibits anti-cancer activity against early stage

human HCT-116 colon cancer cells and colon cancer stem cells. Cancers 2016, 8(3), 29.

• Anthocyanin-containing purple-fleshed potatoes suppress colon tumorigenesis via elimination

of colon cancer stem cells. Journal Nutritional Biochemistry 2015, 26(12), 1641-1649.

Honors and awards

• First place – IFT national college bowl food science quiz competition 2017

• PSU college of agricultural sciences scholarship recipient 2017

• First place – PAA graduate student research presentation competition 2016

• PSU college of agricultural sciences travel award 2016