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 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
1.4 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
2.3 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-
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-
Chapter 4 Grape compounds suppress colon cancer stem cells in vitro and in a rodent model of colon carcinogenesis* ................................................................................ 72
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
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
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
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.
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.
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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
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.
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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).
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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.
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.
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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).
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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).
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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.
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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.
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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.
.
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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.
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.
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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).
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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.
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3.5 Discussion
Our results demonstrate that in vitro PA significantly suppressed proliferation in CSCs
both with functioning p53 and with attenuated p53, suggesting that PA may work even in p53-
independent cancers. PA also upregulated proteins involved in mitochondria-mediated apoptotic
pathway and downregulated proteins involved in the Wnt/β-catenin signaling pathway. PP
eliminated colon CSCs with nuclear β-catenin in vivo via induction of apoptosis and suppressed
tumor incidence in mice with azoxymethane [119]-induced colon cancer lending support to the
anti-cancer properties of PP, warranting further investigation using detailed studies.
Polyphenolic compounds especially anthocyanins derived from fruits and vegetables
demonstrate chemopreventive and chemotherapeutic activity through modulation of multiple
molecular targets making them ideal for the prevention/treatment of cancer [169]. Potatoes are a
rich source of phenolic acids and color-fleshed potatoes also contain other bioactive compounds
such as anthocyanins and carotenoids. UPLC-MS analysis comparing PP and WP showed that
besides higher levels of phenolic acids, only PP contained anthocyanins (compared to WP, Table
1). We also showed previously that PP had more potent anti-cancer activity on early (HCT-116)
and advanced (HT-29) colon cancer cell lines in vitro [226]. However, the effect against colon
cancer stem cells (colon CSCs) is not known and for this purpose we treated colon CSCs with PP
and compared it with sulindac, a positive control.
PA at 5.0 µg/mL suppressed proliferation and induced apoptosis in colon CSCs with and
without functioning p53, however, sulindac demonstrated p53 dependency (Fig. 1A). The p53
dependency of sulindac has been investigated previously in an AOM-induced mouse model with
dysfunctional p53 [237]. Sulindac was not able to restore acute apoptosis response in p53 -/- mice
when compared to that of p53 +/+ mice. This is particularly important because in late/metastatic
stages of colon cancer p53 is mutated [27]. PA induced apoptosis (Fig. 1B-D) was accompanied
70
by elevated Bax/Bcl-2 ratio and cytochrome c (Fig. 3). Bax is a pro-apoptotic protein that binds
Bcl-2 and aids in the release of cytochrome c, a key promoter in mitochondria-mediated apoptosis
[238]. These results indicate that PP induces apoptosis through the mitochondria-mediated
apoptotic pathway. We have also shown that PA suppressed sphere formation, since formation of
colonospheres is a measure of stemness, our results provide the evidence that PA has the potential
to target the self-renewal of colon CSCs.
PA treatment resulted in significant suppression of β-catenin at both nuclear and cytosolic
levels in both colon CSCs with and without functioning p53 (Fig. 4) greater than that of sulindac.
Stabilization of β-catenin and its subsequent accumulation in the nucleus is accompanied by
increased transcriptional activation of proteins such as c-Myc and cyclin D1, which promote
carcinogenesis by increasing cell proliferation [239, 240]. Indeed, PA treated colon CSCs had
suppressed levels of c-Myc (Fig. 5A and 5C) and cyclin D1 (Fig. 5B and 5D) independent of p53.
Several characteristics of colon CSCs may explain the elimination by PP. Stem cells
express high levels of “stemness” factors including the oncoprotein c-Myc [241], which is
overexpressed in colon CSCs [242]. We have also shown in vitro that PA suppressed Wnt
effector β-catenin and its downstream targets c-Myc and cyclin D1 levels in colon CSCs.
Therefore, stem cells with oncogenic alterations, such as accumulation of β-catenin, may be more
sensitive to PA induced apoptosis, relative to differentiated cells with such alterations.
To further test whether PP can eliminate colon CSCs in vivo, we used an AOM-induced
colon cancer mice model. Mice were fed with modified AIN 93G diet containing human relevant
doses of PP (20 % w/w) or sulindac (positive control; 0.06 % w/w) for 1 or 4 weeks. Week 1
euthanized animals were used to study the early molecular mechanism of PP. Week 4 euthanized
animals were used for endpoint analysis of tumor incidence. PP or sulindac fed mice had
significant increase in the number of crypts with TUNEL positive cells (indicator of apoptosis)
compared to AOM control (Fig. 6A). Nuclear β-catenin localization is observed predominantly in
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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
75
demonstrated using a well-established combination index method that a RSV (~ 25 µM) and GSE
(35-50 µg/ml) mixture was potent in suppressing proliferation and elevating apoptosis in the
HCT-116 human colon cancer cell line at lower concentrations compared to RSV or GSE alone
[152, 246]. Combination index methods is based on the classic isobologram equation CI = D1/d1
+ D2/d2. D1 and D2 are the doses of RSV and GSE respectively in the combination system
where as d1 and d2 are the doses of RSV and GSE alone for the same fractional inhibition,
respectively. In addition, RSV potentiated GSE-induced p53-dependent apoptosis via
mitochondrial apoptotic signaling, and demonstrated specificity to cancer cells, as it was non-
toxic in the normal colonic epithelial cell line CRL-1831[152]. Our preliminary results led us to
believe that a combinatorial approach towards colon cancer chemoprevention using bioactive
compounds is a feasible strategy.
Historically, colon tumorigenesis has been viewed as a stochastic model where wide
populations of abnormal colonocytes have an equal propensity to initiate tumor growth [247].
However, the cancer stem cell (CSC) theory suggests that most, if not all, cancerous tumors are
driven by CSCs, probably through dysregulation of self-renewal pathways [40]. CSCs are capable
of self-renewal, cellular differentiation, and maintain their stem cell-like characteristics even after
invasion and metastasis [190]. Furthermore, they are resistant to standard therapies and thus are
thought to be responsible for cancer relapse. The Wnt/β-catenin signaling pathway plays a critical
role in maintenance of stemness, and survival/proliferation of CSCs [217], and as such, targeting
the Wnt/β-catenin signaling is a good strategy for cancer prevention. Aberrant Wnt signaling in
colon cancer is typically followed by mutation in the K-ras gene and loss of the tumor suppressor
p53. It is estimated that p53 is abnormal in 50% to 75% of colorectal cancer cases, and that this
change marks the transition from noninvasive to invasive disease [17, 218]. Thus, treatments,
which act independent of p53 status, are desirable.
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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.
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
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
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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