Cellular oxidative stress and sirtuins mediate regulation of senescence and neuronal differentiation by withaferin A

Abstract

Withaferin A (WA) and Withanone (WN), the steroidal lactones are pharmacologically established for anticancer and chemopreventive effects in certain cancers. However, their effects on redox modulations, mechanisms stimulating senescence and neuronal differentiation in neuroblastoma cells are less understood. Here we examined the influence of WA on perturbations in the molecular architecture of growth, differentiation and senescence of human brain cancer cell SH SY5Y in vitro and test its efficacy in mouse tumor models. We found senescence induction amplified by WA as determined by a senescence-associated β-galactosidase assay. This led us to evaluate DNA damage which was enhanced as measured by phospho-γH2AX foci formation, directed by reactive oxygen species (ROS) production as determined by flow cytometry and confocal imaging. Furthermore, we assessed the influence of DNA damage on cell cycle arrest and DNA repair. Neurosphere formation assay was performed to demonstrate the stem cell inhibitory potential of WA. Subcutaneous xenograft of neuroblastoma cells in athymic Balb/c mice was performed followed by treatment with WA and tumor growth inhibition was established. Withania somnifera (WS) extract and WA induced alterations in ROS, triggering DNA damage and concomitantly regulated SIRTs expression leading to activation of senescence in SH SY5Y cells. Upon prolonged incubation, differentiation into neuronal lineages was confirmed by using differentiation markers such as neurofilament medium, nestin, MAP2 and synaptophysin as measured by immunofluorescence and flow cytometry. The results suggest a complex interplay between the induction of senescence and concurrent neuronal differentiation of SH SY5Y cells mediated by early alterations in SIRT1 and SIRT3. Thus, we report the senescence and differentiation potential of WS extracts and WA through ROS that are mediated via modulation of SIRT1, SIRT3 and mitochondria function.