Withanolide modulates the potential crosstalk between apoptosis and autophagy in different colorectal cancer cell lines

Abstract

Withaferin A (WFA), a withanolide, is isolated from plants of Withania somnifera (L.) Dual (Solanaceae), known as Indian ginseng, Indian winter cherry or Ashwagandha. It has been reported to exert multifaceted anti-neoplastic effects. Here, we analyzed the impact of WFA on apoptosis and autophagy activation in different human colorectal cancer cell lines. We observed that WFA exposure caused an increased aggregation of cells in the subG1 arrest in cell cycle, and increased the number of late apoptotic cells. WFA also induced the apoptosis via PARP and caspase-3 cleavage accompanied with suppression of levels of anti-apoptotic proteins like Bcl-2 and Bcl-xl. The influence of WFA on autophagy was validated by acridine orange, MDC staining, and immunocytochemistry of LC3. It was found that 24 h treatment of WFA increased the acridine and MDC stained autophagosome with induced the LC3 and other autophagy markers Atg7 and beclin-1 activation. We used Z-DEVD-FMK, a caspase-3 blocker, and 3-MA, an autophagy inhibitor, to confirm whether these effects were specific to apoptosis and autophagy, and observed the recovery of both these processes upon exposure to WFA. Moreover, the activation of β-catenin protein was attenuated by WFA. Interestingly, small interfering RNA (siRNA)-promoted β-catenin knockdown augmented the WFA-induced active form of p-GSK-3β, and stimulated autophagy and apoptosis through PARP and LC3 activation. These findings suggested that WFA could stimulate activation of both apoptosis and autophagy process via modulating β-catenin pathway.

Keywords: Apoptosis; Autophagy; Colorectal cancer; GSK3β; Withaferin A; β-catenin.