Dietary flavonoid fisetin induces apoptosis, down regulates COX2 and suppresses Wnt signaling pathway in HT29 colon cancer cells

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Colorectal cancer is one of the common malignancy and represents the third leading cause of cancer deaths in both men and women in the U.S. The Wnt pathway has long been suggested to play a crucial role in colorectal cancer. It is well known that the genetic predisposition such as mutations of APC (adenomatous polyposis coli) or beta-catenin results in uncontrolled proliferation of intestinal epithelial cells through the constitutively active Wnt signaling pathway. Overexpression of COX-2 has been frequently observed in colon tumors. Many studies have implicated that PGE2 (prostaglandin E2), the metabolite of COX-2 enzyme, is a potent mitogen and contributes to colon cancer development via stimulating the growth through Wnt pathway. Recent studies show that selective COX-2 inhibitors possess great potential as chemopreventive agents for colon cancer. Fisetin, 3,3',4',7-tetrahydroxyflavone, is a naturally occurring flavonoid commonly found in various vegetables and fruits such as onion, cucumber, apple, persimmon and strawberry. It has been reported that fisetin induces cell cycle arrest via inhibiting cyclin dependent kinases in HT29 human colon cancer cell line. We found that the treatment of HT29 cells with fisetin (0 ~ 120 μM) resulted in induction of apoptosis in a dose-dependent manner as assayed using dUTP-FITC labeled FACS analysis, annexin-V-Fluorescein and propidium iodide immunofluorescent staining, and by assessing the cleavage of PARP (polyADP ribosyl polymerase) and caspase 3 using western blot analysis. Fisetin treatment down regulated the mRNA and protein expression of COX-2 without affecting COX-1. Decreased expression of COX-2 protein following fisetin treatment was also confirmed by immunofluorescent imaging of cells stained with Alexa Fluor-594 conjugated COX-2 antibody and DAPI. Treatment of fisetin also decreased the promoter activity of COX-2 and the transcriptional activity of T cell factor (TCF)/beta-catenin as assessed by luciferase assay using corresponding reporter plasmids. In further experiments, we found that fisetin treatment of cells resulted in an increase in the cytoplasmic level of phospho-beta-catenin with a concomitant decrease in nuclear level of nonphospho-beta-catenin as assessed by western blot analysis. As expected, the expression of cyclin D1, the most well known target of beta-catenin pathway, was down regulated in fisetin treated cells. Fisetin treatment of cells also inhibited the formation of colonies when assessed in the semi solid agar gel. On an average, fisetin (120 μM) treated cells formed only 37 colonies compared to 138 colonies formed by control cells over the 4 weeks of incubation. Taken together, fisetin can induce apoptosis and suppress the growth of colon cancer cells via inhibiting COX-2 and Wnt signaling pathway. Fisetin could be a useful agent for prevention and treatment of colon cancer.