Suppression of Chloride Channel 3 Expression Facilitates Sensitivity of Human Glioma U251 Cells to Cisplatin Through Concomitant Inhibition of Akt and Autophagy
Suppression of Chloride Channel 3 Expression Facilitates Sensitivity of Human Glioma U251 Cells to Cisplatin Through Concomitant Inhibition of Akt and Autophagy
复制标题
抑制氯通道 3 表达通过同时抑制 Akt 和自噬促进人胶质瘤 U251 细胞对顺铂的敏感性
DOI：
10.1002/ar.22665
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发表时间：
2013-04-01
影响因子：
2
通讯作者：
Sun, Lian-Kun
中科院分区：
文献类型：
--
作者：
Su, Jing;Xu, Ye;Sun, Lian-Kun
顺铂耐药是临床化疗中的难题，顺铂耐药的机制有待进一步研究。在本研究中，我们研究了氯离子通道 3 (ClC-3) 在顺铂耐药中的作用。通过 LC3-II、beclin 1 和 Atg12-Atg5 的积累证明了自噬。电镜下观察超微结构变化。使用吖啶橙或MDC化学染色来检测酸性囊泡细胞器。通过 PI 和膜联蛋白 V 染色检测细胞凋亡的定量。通过蛋白质印迹分析研究了 Akt 通路和自噬所涉及的机制。我们的结果表明，顺铂在人胶质瘤 U251 细胞中诱导 Akt 磷酸化和自噬。 ClC-3 siRNA 对 ClC-3 的特异性抑制使抗凋亡 U251 细胞对顺铂介导的细胞死亡敏感，并下调磷酸化 Akt。有趣的是，尽管 Akt/mTOR 通路失调，但 ClC-3 抑制也抑制了顺铂诱导的自噬。通过 3-甲基腺胺抵消自噬过程增强了顺铂的细胞毒性，揭示自噬在化疗耐药中发挥着关键作用。通过 NADPH 氧化酶抑制剂二苯基碘 (DPI) 抑制 Akt/mTOR 通路表明顺铂诱导的 Akt/mTOR 通路激活需要通过 NADPH 氧化酶产生活性氧 (ROS)。总的来说，我们的结果表明 ClC-3 抑制会导致 Akt 和自噬的抑制，从而增强顺铂在 U251 细胞中的治疗效果。 Anat Rec，296:595603，2013。(c) 2013 Wiley periodicals, Inc.
Cisplatin resistance is a difficult problem in clinical chemotherapy, and the mechanisms involved in cisplatin resistance require further study. In this study, we investigated the role of chloride channel-3 (ClC-3) in cisplatin resistance. Autophagy was demonstrated by accumulation of LC3-II, beclin 1 and Atg12-Atg5. The ultrastructure changes were observed under electron microscope. Chemical staining with acridine orange or MDC was used to detect acidic vesicular organelles. Quantification of apoptosis was detected by PI and Annexin V staining. The mechanisms involved in the Akt pathway and autophagy were studied by western blot analysis. Our results showed that Akt phosphorylation and autophagy were induced by cisplatin in human glioma U251 cells. Specific inhibition of ClC-3 by ClC-3 siRNA sensitized the apoptosis-resistant U251 cells to cisplatin-mediated cell death and downregulated phosphorylated Akt. Interestingly, ClC-3 suppression also inhibited induction of autophagy by cisplatin although the Akt/mTOR pathway was deregulated. Counteracting the autophagic process by 3-methylademine enhanced cytotoxicity of cisplatin, revealing that autophagy plays a key role in chemoresistance. Suppressing the Akt/mTOR pathway by the NADPH oxidase inhibitor diphenyl iodonium (DPI) indicated that cisplatin-induced activation of Akt/mTOR pathway requires generation of reactive oxygen species (ROS) through NADPH oxidase. Collectively, our results suggest that ClC-3 suppression causes the inhibition of Akt and autophagy, which can enhance the therapeutic benefit of cisplatin in U251 cells. Anat Rec, 296:595603, 2013. (c) 2013 Wiley Periodicals, Inc.