Exploring the cytotoxic effects of bioactive compounds from Alcea rosea against stem cell driven colon carcinogenesis

Seven compounds were isolated from ethyl acetate extract of Alcea rosea and were examined for their cytotoxicity against HCT116, HT29 and SW480 colon cancer cells. It was found that two compounds (C4 and C5) exhibited strong anti-colon cancer activities. These two compounds were used to study their properties that include MTT activity (with IC50 of C4 as 74.71, 129.0 and 131.4 µg/ml in HCT116, HT29 and SW480 respectively, whereas IC50 of C5 as 128.1, 168.4 and 225.8 µg/ml in HCT116, HT29 and SW480 cells respectively), colony formation activity, wound healing activity, spheroid formation activity, DAPI-PI staining, acridine-orange and ethidium bromide staining, ROS measurement, and rhodamine-123 staining in both HCT116 and HT29 colon cancer cells. Both the compounds showed significant increase in apoptosis as visualized by 4',6-diamidino-2-phenylindol/propidium iodide (DAPI-PI) and acridine orange/ethidium bromide (AO/EtBr) staining. The induction of apoptosis was further confirmed by the expressions of cleaved PARP and caspase 3. ROS generation and its effect on MMP were measured by staining cells with Dichloro-dihydro-fluorescein diacetate (DCFH-DA) and Rhodamine. Expression levels of EMT associated markers like Cyclin D1, Slug, Vimentin, and E-Cadherin were also studied. Both the compounds down regulate protein levels of Slug, Cyclin D1, and Vimentin in a concentration-dependent manner. Eeffect of C4 and C5 compounds on key signaling protein like Wnt3a, Notch1, and Shh were evaluated. Additionally, mRNA levels of these genes were also analyzed. C4 exhibited the best binding affinity when docked with Shh and Wnt3a and Notch1. Similarly, C5 exhibited - 8.8, -8.2 and - 7.6 kcal⋅mol^{- 1} with Shh, Wnt3a and Notch1. The present findings provide insight and immense scientific support and integrity to a piece of indigenous knowledge. However, validation in living organisms is necessary before progressing to clinical trials and advancing it into a marketable pharmaceutical product.