Triple SILAC based quantitative MS approach to determine Withaferin A regulated proteins in prostate cancer

Withaferin A (WA) is a lactone extracted from Withania somnifera commonly known as Ashwagandha. WA has several therapeutic benefits. The current study was aimed to identify biomarkers that could be targeted by WA in prostate cancer (PCA) cells. We have used SILAC approach to identify WA-regulated proteins at 4 h and 24 time points in three PCA cell lines such as LNCaP, 22Rv1 and DU-145. Ontology prediction suggested that WA treatment can upregulate stress-responsive pathways and shutdown translation and cell metabolism to conserve energy. The cytoprotective stress granule (SG) protein G3BP1 showed upregulation in all the three tested cell lines in response to WA treatment, and subsequently, SGs formed at a higher rate in the WA treated cells. Knockdown (KD) of G3BP1 blocked WA-induced SG formation and reduced the cell survival. We speculate that the activation of G3BP1 and the formation of SGs might constitute a mechanism by which PCA cells induce cell protection after WA- treatment. Knock down of SG proteins such as G3BP1 could help to evade the cytoprotective effects of WA and to assist in the sensitization of cells.