Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China.
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, China.
Cell Prolif. 2018 Aug;51(4):e12450. doi: 10.1111/cpr.12450. Epub 2018 Mar 1.
Our previous in vitro study showed that 5-(3, 4, 5-trimethoxybenzoyl)-4-methyl-2-(p-tolyl) imidazol (BZML) is a novel colchicine binding site inhibitor with potent anti-cancer activity against apoptosis resistance in A549/Taxol cells through mitotic catastrophe (MC). However, the mechanisms underlying apoptosis resistance in A549/Taxol cells remain unknown. To clarify these mechanisms, in the present study, we investigated the molecular mechanisms of apoptosis and autophagy, which are closely associated with MC in BZML-treated A549 and A549/Taxol cells.
Xenograft NSCLC models induced by A549 and A549/Taxol cells were used to evaluate the efficacy of BZML in vivo. The activation of the mitochondrial apoptotic pathway was assessed using JC-1 staining, Annexin V-FITC/PI double-staining, a caspase-9 fluorescence metric assay kit and western blot. The different functional forms of autophagy were distinguished by determining the impact of autophagy inhibition on drug sensitivity.
Our data showed that BZML also exhibited desirable anti-cancer activity against drug-resistant NSCLC in vivo. Moreover, BZML caused ROS generation and MMP loss followed by the release of cytochrome c from mitochondria to cytosol in both A549 and A549/Taxol cells. However, the ROS-mediated apoptotic pathway involving the mitochondria that is induced by BZML was only fully activated in A549 cells but not in A549/Taxol cells. Importantly, we found that autophagy acted as a non-protective type of autophagy during BZML-induced apoptosis in A549 cells, whereas it acted as a type of cytoprotective autophagy against BZML-induced MC in A549/Taxol cells.
Our data suggest that the anti-apoptosis property of A549/Taxol cells originates from a defect in activation of the mitochondrial apoptotic pathway, and autophagy inhibitors can potentiate BZML-induced MC to overcome resistance to mitochondrial apoptosis.
我们之前的体外研究表明，5-(3,4,5-三甲氧基苯甲酰基)-4-甲基-2-(对甲苯基)咪唑（BZML）是一种新型的秋水仙碱结合位点抑制剂，通过有丝分裂灾难（MC）对 A549/Taxol 细胞中的凋亡抵抗具有强大的抗癌活性。然而，A549/Taxol 细胞中凋亡抵抗的机制尚不清楚。为了阐明这些机制，本研究在 BZML 处理的 A549 和 A549/Taxol 细胞中，研究了与 MC 密切相关的凋亡和自噬的分子机制。
使用 A549 和 A549/Taxol 细胞诱导的异种移植 NSCLC 模型，评估 BZML 的体内疗效。使用 JC-1 染色、Annexin V-FITC/PI 双重染色、caspase-9 荧光比色测定试剂盒和 Western blot 评估线粒体凋亡途径的激活情况。通过确定自噬抑制对药物敏感性的影响，区分不同功能形式的自噬。
我们的数据表明，BZML 对体内耐药性 NSCLC 也表现出良好的抗癌活性。此外，BZML 在 A549 和 A549/Taxol 细胞中均引起 ROS 生成和 MMP 丢失，随后导致细胞色素 c 从线粒体释放到细胞质中。然而，BZML 诱导的 ROS 介导的涉及线粒体的凋亡途径仅在 A549 细胞中被完全激活，而在 A549/Taxol 细胞中未被完全激活。重要的是，我们发现自噬在 BZML 诱导的 A549 细胞凋亡中作为一种非保护性自噬起作用，而在 BZML 诱导的 A549/Taxol 细胞 MC 中作为一种细胞保护性自噬起作用。
我们的数据表明，A549/Taxol 细胞的抗凋亡特性源于线粒体凋亡途径激活缺陷，自噬抑制剂可以增强 BZML 诱导的 MC 以克服对线粒体凋亡的抵抗。
