Phytochemicals Target Epigenetic Signaling To Block Cancer Stem Cell-Driven Colon Carcinogenesis

Background: Phytochemicals modulate key cellular signaling pathways and have proven anticancer effects. Alcea rosea (AR; Hollyhock) is an ornamental plant in the Malvaceae family with known anti-inflammatory properties. Its role as an anti-cancer agent however, has not yet been confirmed. The present investigation is focused on the mechanistic insight of AR seed extract as an anti-cancer agent for colon cancer. Methodology: Colon cancer cell lines HCT116 and SW480 and normal colon epithelial cells were used in the study. Cell proliferation, cell cycle and stemness were determined by hexoseaminidase, flow cytometry and spheroid assays, respectively. TOP-Flash reporter and WIF1 promoter activities were performed in HEK-293T cells. For in vivo effects, we used the HCT116-tumor xenograft model for colon tumorigenesis. Real Time PCR, Western blot and immunohistochemistry studies were performed for determining gene expression. Results: The AR seed extract inhibited proliferation and colony formation in a dose and time dependent manner in colon cancer cell lines. The anti-proliferative activity appeared to be mediated by apoptotic mechanisms, as cleavage of PARP and increased expression of Bax accompanied decreased levels of BCL-xl protein. In addition, AR seed extract arrested cells at Go/G1 phase of cell cycle and exhibited decreases in Cyclin D1. Treatment of cells with AR seed extract also reduced the number and size of colonospheres in a dose-dependent manner which coincided with decreases in the expression of ALDH1A1 and Dclk1, markers of CSCs (Cancer Stem Cells). During analyses of pathways critical for CSC maintenance and colon cancer progression, AR seed extract attenuated levels of β-catenin, Jagged1, Notch-ICD, Hes1 and EZH2. TOP-flash reporter activity, a measure of Wnt signaling, decreased significantly in response to treatment while overexpression of wild type but not mutant EZH2, reversed the inhibitory effects. Moreover, WIF1 (a Wnt antagonist) promoter activity increased dramatically following treatment with AR seed extract which phenocopied increases in WIF1 reporter activity following EZH2 knockdown. To determine the effect of AR seed extract on tumor growth in vivo, athymic nude mice bearing tumor xenografts were administered AR seed extract intraperitoneally at 200 mg/Kg daily for 21 days. The AR seed extract significantly inhibited tumor xenograft growth. In addition, there were reduced levels of EZH2, β-catenin, CyclinD1 and Ki-67 along with attenuated levels of Dclk1 in the xenograft tissues. Finally, preliminary run through HPLC yielded three prominent peaks and further identification via NMR spectroscopy is underway to identify the active ingredient(s). Conclusion: These results clearly suggest that AR seed extracts can be an effective preventative/therapeutic agent for colon cancer. Citation Format: Shahid Umar, Ishfaq Ahmed Ahmed, Dharmalingam Subramaniam. Phytochemicals target epigenetic signaling to block cancer stem cell-driven colon carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4656. doi:10.1158/1538-7445.AM2015-4656