Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia.
Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia; Center for Natural Products and Drug Research (CENAR), University of Malaya, Malaysia.
Phytomedicine. 2018 Mar 15;42:144-151. doi: 10.1016/j.phymed.2018.03.027.
Targeting autophagy is emerging as a promising strategy in cancer therapeutics in recent years. Autophagy can be modulated to drive cancer cell deaths that are notoriously resistant to apoptotic-inducing drugs. In addition, autophagy has been implicated as a prosurvival mechanism in mediating cancer chemoresistance. Our previous study has demonstrated that Panduratin A (PA), a plant-derived active compound exploits ER-stress-mediated apoptosis as its cytotoxic mechanism on melanoma.
Our previous proteomics analysis revealed that treatment with PA resulted in the upregulation of an autophagy marker, LC3B in melanoma cells. Therefore, the present study sought to investigate the role of PA-induced autophagy in melanoma cells.
Transmission electron microscopy was performed for examination of autophagic ultra-structures in PA-treated A375 cells. Cytoplasmic LC3B and p62/SQSMT1 punctate structures were detected using immunofluorescene staining. Expression levels of LC3B II, p62/SQSMT1, ATG 12, Beclin 1, phospho S6 (ser235/236), phospho AMPK (Thr172) and cleaved PARP were evaluated by western blotting.
Autophagosomes, autolysosomes and punctuates of LC3 proteins could be observed in PA-treated A375 cells. PA-induced autophagy in A375 melanoma cells was found to be mediated through the inhibition of mTOR signaling and activation of AMPK pathway. Furthermore, we showed that PA-induced apoptosis was increased in the presence of an autophagy inhibitor, signifying the cytoprotective effect of PA-induced autophagy in melanoma cells.
Taken together, results from the present study suggest that the inhibition of autophagy by targeting mTOR and AMPK could potentiate the cytotoxicity effects of PA on melanoma cells.
近年来，靶向自噬已成为癌症治疗中一种很有前途的策略。自噬可以被调节以促使癌细胞死亡，而这些癌细胞对诱导细胞凋亡的药物具有明显的耐药性。此外，自噬已被牵连为介导癌症化疗耐药性的一种促进生存的机制。我们之前的研究表明，植物源性活性化合物 Panduratin A (PA) 通过内质网应激介导的细胞凋亡发挥其细胞毒性作用，对黑色素瘤起作用。
我们之前的蛋白质组学分析表明，PA 处理导致黑色素瘤细胞中自噬标志物 LC3B 的上调。因此，本研究旨在探讨 PA 诱导的自噬在黑色素瘤细胞中的作用。
用透射电子显微镜观察 PA 处理的 A375 细胞中的自噬超微结构。用免疫荧光染色检测细胞质 LC3B 和 p62/SQSMT1 点状结构。用 Western blot 检测 LC3B II、p62/SQSMT1、ATG12、Beclin1、磷酸化 S6（Ser235/236）、磷酸化 AMPK（Thr172）和裂解的 PARP 的表达水平。
在 PA 处理的 A375 细胞中可以观察到自噬体、自溶酶体和 LC3 蛋白的斑点。PA 诱导的 A375 黑色素瘤细胞自噬被发现是通过抑制 mTOR 信号和激活 AMPK 途径介导的。此外，我们表明，在自噬抑制剂存在的情况下，PA 诱导的细胞凋亡增加，表明 PA 诱导的自噬对黑色素瘤细胞具有细胞保护作用。
总之，本研究结果表明，通过靶向 mTOR 和 AMPK 抑制自噬可以增强 PA 对黑色素瘤细胞的细胞毒性作用。
