POLYPHENOL-RICH FOODS AS INHIBITORS OF COLON CANCER STEM CELLS
Open Access
- Author:
- Charepalli, Venkata Rohit
- Graduate Program:
- Food Science
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 08, 2017
- Committee Members:
- Jairam Kp Vanamala, Dissertation Advisor/Co-Advisor
Jairam Kp Vanamala, Committee Chair/Co-Chair
Joshua D Lambert, Committee Member
Gregory Ray Ziegler, Committee Member
Mary J Kennett, Outside Member - Keywords:
- Cancer stem cells
polyphenols
purple-fleshed potatoes
cancer prevention
resveratrol
grape seed extract
β-catenin
colon cancer - 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. 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. 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.