SILAC-based quantitative MS approach reveals Withaferin A regulated proteins in prostate cancer

Abstract

Withaferin A (WA) is a steroidal lactone extracted from Withania somnifera, commonly known as Ashwagandha. WA has several therapeutic benefits. The current study aims to identify proteins that are potentially regulated by WA in prostate cancer (PCA) cells. We used a SILAC-based proteomic approach to analyze the expression of proteins in response to WA treatment at 4 h and 24 h time points in three PCA cell lines: 22Rv1, DU-145, and LNCaP. Ontology analysis suggested that prolonged treatment with WA upregulated the expression of proteins involved in stress-response pathways. Treatment with WA increased oxidative stress, reduced global mRNA translation, and elevated the expression of cytoprotective stress granule (SG) protein G3BP1. WA treatment also enhanced the formation of SGs. The elevated expression of G3BP1 and the formation of SGs might constitute a mechanism of cytoprotection in PCA cells. Knockdown of G3BP1 blocked SG formation and enhanced the efficacy of WA to reduce PCA cell survival. SIGNIFICANCE: Withaferin A, a steroidal lactone, extracted from Withania somnifera is a promising anti-cancer drug. Using a SILAC-based quantitative proteomic approach, we identified proteins changed by WA-treatment at 4 h and 24 h in three prostate cancer (PCA) cell lines. WA-treatment induced the expression of proteins involved in apoptosis and reduced the expression of proteins involved in cell growth at 4 h. WA-treatment for 24 h enhanced the expression of proteins involved in stress response pathways. WA-treated cells exhibited increased oxidative stress, reduced mRNA translation and enhanced SG formation. PCA is characterized by higher metabolic rate and increased oxidative stress. PCA with a higher stress tolerance can effectively adapt to anti-cancer treatment stress, leading to drug resistance and cellular protection. Enhancing the level of oxidative stress along with inhibition of corresponding cytoprotective stress response pathways is a feasible option to prevent PCA from getting adapted to treatment stress. WA-treatment induced oxidative stress, in combination with blocking SGs by G3BP1 targeting, offers a therapeutic strategy to reduce PCA cell survival.

Keywords: G3BP1; Natural compounds; Oxidative stress; Prostate cancer; Stress granule; Withaferin A.