Degradation of Mcl-1 through GSK-3β Activation Regulates Apoptosis Induced by Bufalin in Non-Small Cell Lung Cancer H1975 Cells
Degradation of Mcl-1 through GSK-3β Activation Regulates Apoptosis Induced by Bufalin in Non-Small Cell Lung Cancer H1975 Cells
复制标题
Mcl-1 通过 GSK-3 beta 激活降解调节蟾蜍灵诱导的非小细胞肺癌 H1975 细胞凋亡
DOI：
10.1159/000475438
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发表时间：
2017-01-01
影响因子：
--
通讯作者：
Cao, Fei
中科院分区：
文献类型：
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作者：
Kang, Xiao-hong;Zhang, Jing-hang;Cao, Fei
背景/目的：Mcl-1是抗凋亡的Bcl-2家族成员，在非小细胞肺癌（NSCLC）中经常过表达。蟾毒灵有诱导多种肿瘤细胞凋亡的报道。然而，没有报道显示蟾毒灵可以下调NSCLC中Mcl-1的表达。方法：采用细胞计数试剂盒-8 （CCK-8）法检测H1975细胞的增殖情况。流式细胞术检测细胞凋亡。RT-PCR检测Mcl-1 mRNA。western blotting检测H1975细胞凋亡相关蛋白的表达。免疫沉淀法分析Mcl-1泛素化和NOXA水平。结果：蟾毒灵对细胞生长有一定的抑制作用，且具有时间和剂量依赖性。蟾毒灵通过激活caspase级联和下调Mcl-1表达诱导非小细胞肺癌细胞凋亡。然而，过表达Mcl-1可减少蟾毒灵诱导的细胞凋亡。此外，蟾毒灵没有降低H1975细胞中Mcl-1 mRNA的表达，但强烈促进了Mcl-1蛋白的降解。蛋白酶体抑制剂MG132显著阻止Mcl-1降解，阻断蟾毒灵诱导的Mcl-1还原。蟾毒灵对NOXA蛋白水平无显著影响，但下调p-GSK-3 β的表达。GSK-3抑制剂和GSK-3 β siRNA导致Mcl-1水平升高，逆转了蟾毒灵诱导的Mcl-1降解。结论：蟾毒灵可能通过下调Mcl-1介导H1975细胞凋亡。通过GSK-3活化Mcl-1的蛋白酶体降解参与了蟾毒灵诱导的细胞凋亡。(C) 2017作者。巴塞尔，S. kager AG出版。
Background/Aims: Mcl-1, an anti-apoptotic Bcl-2 family member, is often overexpressed in non-small cell lung cancer ( NSCLC). Bufalin has been reported to induce apoptosis in various tumor cells. However, there is no report showing that bufalin could downregulate Mcl-1 expression in NSCLC. Methods: Cell proliferation was analyzed by cell counting kit-8 ( CCK-8) assay in H1975 cells. Cell apoptosis was detected by flow cytometry. Mcl-1 mRNA was detected by RT-PCR. The expression of apoptosis-associated proteins in H1975 cells was detected by western blotting. The levels of Mcl-1 ubiquitination and NOXA were analyzed by Immunoprecipitation assay. Results: Cell growth was inhibited by bufalin in a time and dose-dependent manner. Bufalin induced apoptosis in NSCLC cells by activating caspase cascades and downregulating Mcl-1 expression. However, overexpression of Mcl-1 diminished bufalin-induced apoptosis. Furthermore, bufalin did not reduce Mcl-1 mRNA expression in H1975 cells, but strongly promoted Mcl-1 protein degradation. Proteasome inhibitor MG132 markedly prevented the degradation of Mcl-1 and blocked bufalin-induced Mcl-1 reduction. Bufalin did not significantly affect NOXA protein levels, but downregulated the expression of p-GSK-3 beta. GSK-3 inhibitor and GSK-3 beta siRNA resulted in increased levels of Mcl-1 and reversed the bufalin-induced Mcl-1 degradation. Conclusion: Bufalin induced cell apoptosis in H1975 cells may be through downregulation of Mcl-1. Proteasomal degradation of Mcl-1 via GSK-3 beta activation was involved in bufalin-induced apoptosis. (C) 2017 The Author(s). Published by S. Karger AG, Basel.