Withaferin-A induces apoptosis and autophagy in colorectal cancer cell lines via down-regulated expression of histone deacetylase 1.

Colorectal cancer (CRC) remains the third most common malig-nancy worldwide, and there is an urgent need for low-toxicity, mechanism-based preventives or adjuvants. Withaferin-A (WA), a plant-derived steroidal lactone, exhibits broad antitumor activity; however, its role in CRC and its interactions with epigenetic regulators, such as histone deacetylase 1 (HDAC1), remain un-clear. Therefore, we investigated whether WA suppresses CRC growth by down-regulating HDAC1 while inducing apoptosis and autophagy. Caco2 and HT-29 cells were treated with 0–5 μM WA; viability, colony formation, and migration de-creased significantly (IC₅₀ 0.70–1.52 μM). Techniques such as Annexin-V/7-AAD flow cytometry, MDC staining, TEM, and LC3B immunofluorescence showed that 1 μM WA notably increased apoptosis and autophagic flux, along with reduced HDAC1 and p62 levels, higher LC3B-II/I ratios, and an increased Bax/Bcl-2 ratio. Overexpression of HDAC1 via a lentiviral vector reversed these effects, confirming dependence on HDAC1. For translational relevance, eight-week-old C57BL/6J mice were first exposed to the food-borne carcinogen IQ (2-amino-3-methyl-3H-imidazo[4,5-f]quinoline, 100 mg/kg) every other day for three weeks to induce aberrant crypt foci (ACF). Starting the day after the first IQ dose, animals re-ceived WA (2 mg/kg) or vehicle (corn oil) by gavage every other day for the same period. WA reduced the number of macroscopic ACF by more than 60%, restored HDAC1-related LC3B and p62 expression to normal levels, and showed no toxic-ity based on body weight or general health assessments. These findings suggest that WA provides potent, low-toxicity chemopreventive effects against CRC lesion formation through HDAC1-dependent induction of apoptosis and autophagy, sup-porting its further consideration as a preventive or adjuvant agent.