Withaferin A (WA) is a naturally occurring steroidal lactone with proven cancer chemopreventive activity in preclinical models of different cancers including prostate adenocarcinoma. Previously we compared the RNA‐seq data from control and WA‐treated 22Rv1 human prostate cancer cells to identify mechanistic targets of this phytochemical. The Gene Ontology pathway analysis of the RNA‐seq data revealed significant upregulation of genes associated with autophagy upon WA treatment in 22Rv1 cells. In this study, we extended these findings to investigate the mechanism underlying WA‐induced autophagy. Initially, we confirmed autophagy induction by WA treatment by transmission electron microscopy using three prostate cancer cell lines (LNCaP, 22Rv1, and PC‐3). Fourteen common genes altered by 8‐ and 16‐hour exposure to WA were identified from human autophagy PCR array and these results were consistent with the RNA‐seq data. Two key autophagy markers (LC3BII and SQSTM1) were robustly increased in WA‐exposed LNCaP, 22Rv1, and PC‐3 cells as determined by immunoblotting, and this effect was elevated in the presence of autophagy inhibitor bafilomycin A1 (BafA1). BafA1 treatment augmented WA's cytotoxicity and subsequently its proapoptotic potential. WA treatment induced GABARAPL1 (ATG8L) protein expression in all three cell lines and its knockdown by RNA interference attenuated WA‐mediated apoptosis. WA‐induced autophagy was not affected in the presence of an antioxidant (EUK134). Taken together, the present study reveals that WA‐mediated autophagy is cytoprotective and mediated by GABARAPL1.

中文翻译：

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前列腺癌细胞中withaferin A的细胞保护自噬诱导涉及GABARAPL1。

Withaferin A (WA) 是一种天然存在的甾体内酯，在包括前列腺腺癌在内的不同癌症的临床前模型中具有证明的癌症化学预防活性。以前，我们比较了对照和 WA 处理的 22Rv1 人前列腺癌细胞的 RNA-seq 数据，以确定这种植物化学物质的机制目标。RNA-seq 数据的 Gene Ontology 通路分析揭示了 22Rv1 细胞在 WA 处理后与自噬相关的基因显着上调。在这项研究中，我们扩展了这些发现以研究 WA 诱导的自噬的机制。最初，我们使用三种前列腺癌细胞系（LNCaP、22Rv1 和 PC-3）通过透射电子显微镜证实了 WA 治疗的自噬诱导。从人类自噬 PCR 阵列中鉴定了 14 个因暴露于 WA 8 小时和 16 小时而改变的常见基因，这些结果与 RNA-seq 数据一致。通过免疫印迹测定，在暴露于 WA 的 LNCaP、22Rv1 和 PC-3 细胞中，两个关键的自噬标志物（LC3BII 和 SQSTM1）显着增加，并且在自噬抑制剂巴弗洛霉素 A1（BafA1）存在的情况下，这种作用增强。BafA1 处理增强了 WA 的细胞毒性，随后增强了其促凋亡潜力。WA 处理在所有三种细胞系中诱导 GABARAPL1 (ATG8L) 蛋白表达，并且其通过 RNA 干扰的敲低减弱了 WA 介导的细胞凋亡。WA 诱导的自噬在抗氧化剂（EUK134）的存在下不受影响。总之，本研究表明 WA 介导的自噬具有细胞保护作用，并由 GABARAPL1 介导。