PAWR-mediated suppression of BCL2 promotes switching of 3-azido withaferin A (3-AWA)-induced autophagy to apoptosis in prostate cancer cells

An active medicinal component of plant origin with an ability to overcome autophagy by inducing apoptosis should be considered a therapeutically active lead pharmacophore to control malignancies. In this report, we studied the effect of concentration-dependent 3-AWA (3-azido withaferin A) sensitization to androgen-independent prostate cancer (CaP) cells which resulted in a distinct switching of 2 interrelated conserved biological processes, i.e. autophagy and apoptosis. We have observed 3 distinct parameters which are hallmarks of autophagy in our studies. First, a subtoxic concentration of 3-AWA resulted in an autophagic phenotype with an elevation of autophagy markers in prostate cancer cells. This led to a massive accumulation of MAP1LC3B and EGFP-LC3B puncta coupled with gradual degradation of SQSTM1. Second, higher toxic concentrations of 3-AWA stimulated ER stress in CaP cells to turn on apoptosis within 12 h by elevating the expression of the proapoptotic protein PAWR, which in turn suppressed the autophagy-related proteins BCL2 and BECN1. This inhibition of BECN1 in CaP cells, leading to the disruption of the BCL2-BECN1 interaction by overexpressed PAWR has not been reported so far. Third, we provide evidence that <i>pawr-</i>KO MEFs exhibited abundant autophagy signs even at toxic concentrations of 3-AWA underscoring the relevance of PAWR in switching of autophagy to apoptosis. Last but not least, overexpression of EGFP-LC3B and DS-Red-BECN1 revealed a delayed apoptosis turnover at a higher concentration of 3-AWA in CaP cells. In summary, this study provides evidence that 3-AWA is a strong anticancer candidate to abrogate protective autophagy. It also enhanced chemosensitivity by sensitizing prostate cancer cells to apoptosis through induction of PAWR endorsing its therapeutic potential.

一类源自植物的活性药用成分，若可通过诱导细胞凋亡（apoptosis）拮抗细胞自噬（autophagy），则应被视作调控恶性肿瘤的治疗活性先导药效团（pharmacophore）。本研究探讨了浓度依赖性3-叠氮基睡茄内酯A（3-AWA, 3-azido withaferin A）对雄激素非依赖性前列腺癌（androgen-independent prostate cancer, CaP）细胞的增敏效应，该效应可引发两个相互关联的保守生物学过程——细胞自噬与细胞凋亡——发生显著转换。
本研究中我们观测到三类明确的细胞自噬特征性指标：其一，亚毒性浓度的3-AWA可在前列腺癌细胞中诱导自噬表型，并上调自噬标志物的表达水平，表现为MAP1LC3B与EGFP-LC3B斑点大量聚集，同时伴随SQSTM1的逐步降解；其二，较高毒性浓度的3-AWA可在CaP细胞中激活内质网应激（endoplasmic reticulum stress, ER stress），通过上调促凋亡蛋白PAWR的表达，在12小时内诱导细胞凋亡，而PAWR可进一步抑制自噬相关蛋白BCL2与BECN1的表达。迄今为止，尚无研究报道过在CaP细胞中，过表达的PAWR可通过抑制BECN1破坏BCL2与BECN1的相互作用；其三，本研究证实，即便在毒性浓度的3-AWA处理下，pawr基因敲除小鼠胚胎成纤维细胞（pawr-KO MEFs）仍表现出大量自噬特征，这进一步凸显了PAWR在细胞自噬向细胞凋亡转换过程中的关键作用。最后，通过过表达EGFP-LC3B与DS-Red-BECN1，我们发现高浓度3-AWA处理的CaP细胞中，细胞凋亡周转过程出现延迟。
综上，本研究证实3-AWA是一种强效的抗癌候选化合物，可阻断保护性细胞自噬；同时，其可通过诱导PAWR的表达，使前列腺癌细胞对细胞凋亡更为敏感，进而增强化疗敏感性，这印证了其治疗应用潜力。