Methyl methanesulfonate induces necroptosis in human lung adenoma A549 cells through the PIG-3-reactive oxygen species pathway
Methyl methanesulfonate induces necroptosis in human lung adenoma A549 cells through the PIG-3-reactive oxygen species pathway
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甲磺酸甲酯通过PIG-3-活性氧途径诱导人肺腺瘤A549细胞坏死性凋亡
DOI：
10.1007/s13277-015-3531-y
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发表时间：
2016-03-01
期刊：
影响因子：
--
通讯作者：
Yang, Jun
中科院分区：
文献类型：
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作者：
Jiang, Ying;Shan, Shigang;Yang, Jun
甲基磺酸甲酯（MMS）是一种烷基化剂，可通过细胞凋亡和坏死坏死诱导细胞死亡。mms诱导细胞凋亡的分子机制已被广泛研究；然而，关于mms诱导的坏死性下垂的机制知之甚少。因此，我们首先利用人肺癌A549细胞建立了mms诱导的坏死下垂模型。研究发现，在24 h内，虽然浓度为50、100、200、400和800 μM的MMS可以诱导DNA损伤，但只有高浓度（400和800 μM）的MMS才会导致A549细胞坏死，这是因为MMS可以被特异性的坏死抑制剂necrostatin-1抑制，而特异性的凋亡抑制剂carbobenzoxy-valyl-alanyl-aspartyl-[o -甲基]- fluorom甲基酮（Z-VAD-fmk）抑制。mms诱导的坏死下垂进一步证实了坏死下垂生物标志物的诱导，包括细胞NADH和ATP的消耗以及LDH的泄漏。这种坏死细胞死亡还伴随着p53、p53诱导基因3 （PIG-3）、高迁移率组盒1蛋白（HMGB1）和受体相互作用蛋白激酶（RIP）的表达增加，但不伴有凋亡相关的caspase-3和caspase-9蛋白的表达增加。活性氧（ROS）水平升高也参与了这一过程，因为特异性ROS抑制剂（4-氨基-2,4-吡咯烷-二羧酸（APDC））可以抑制坏死细胞的死亡。有趣的是，通过小干扰RNA （siRNA）处理降低猪-3的表达可以抑制ROS的产生。综上所述，这些结果表明MMS可能通过PIG-3-ROS途径诱导A549细胞坏死。
Methyl methanesulfonate (MMS) is an alkylating agent that can induce cell death through apoptosis and necroptosis. The molecular mechanisms underlying MMS-induced apoptosis have been studied extensively; however, little is known about the mechanism for MMS-induced necroptosis. Therefore, we first established MMS-induced necroptosis model using human lung carcinoma A549 cells. It was found that, within a 24-h period, although MMS at concentrations of 50, 100, 200, 400, and 800 μM can induce DNA damage, only at higher concentrations (400 and 800 μM) MMS treatment lead to necroptosis in A549 cells, as it could be inhibited by the specific necroptotic inhibitor necrostatin-1, but not the specific apoptotic inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk). MMS-induced necroptosis was further confirmed by the induction of the necroptosis biomarkers including the depletion of cellular NADH and ATP and leakage of LDH. This necroptotic cell death was also concurrent with the increased expression of p53, p53-induced gene 3 (PIG-3), high mobility group box-1 protein (HMGB1), and receptor interaction protein kinase (RIP) but not the apoptosis-associated caspase-3 and caspase-9 proteins. Elevated reactive oxygen species (ROS) level was also involved in this process as the specific ROS inhibitor (4-amino-2,4-pyrrolidine-dicarboxylic acid (APDC)) can inhibit the necroptotic cell death. Interestingly, knockdown of PIG-3 expression by small interfering RNA (siRNA) treatment can inhibit the generation of ROS. Taken together, these results suggest that MMS can induce necroptosis in A549 cells, probably through the PIG-3-ROS pathway.