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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
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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
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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.
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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.
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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.
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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.
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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.
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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
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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
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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].
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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.
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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
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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.
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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.
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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).
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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).
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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.
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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.
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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).
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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.
.
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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).
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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.
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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).
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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.
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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
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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
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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.
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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.
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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.
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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
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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-
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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
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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.
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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
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