Induction of protective autophagy against apoptosis in HepG2 cells by isoniazid independent of the p38 signaling pathway.
Induction of protective autophagy against apoptosis in HepG2 cells by isoniazid independent of the p38 signaling pathway.
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DOI：
10.1039/c5tx00470e
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发表时间：
2016-05
影响因子：
2.1
通讯作者：
Tian-Guang Zhang;Yi-Mei Wang;Jun Zhao;Mingyu Xia;S. Peng;T. Ikejima
中科院分区：
文献类型：
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作者：
Tian-Guang Zhang;Yi-Mei Wang;Jun Zhao;Mingyu Xia;S. Peng;T. Ikejima
异烟肼（INH）作为一线抗结核药物，临床上常出现肝毒性。然而，这种副作用的详细机制仍不清楚。细胞色素P450 2 E1参与INH肝毒性的传统理论仍存在争议，因此需要研究INH发挥肝毒性的其他机制。在目前的研究中，我们表明，在体外治疗人肝癌HepG 2细胞与INH诱导caspase依赖性凋亡通过外在和内在的途径。其特征在于通过流式细胞术使用Annexin V/碘化丙啶（PI）双重染色的凋亡细胞的数量增加，以及通过Western印迹法激活半胱天冬酶8、9、3和聚（ADP-核糖）聚合酶（PARP）蛋白。INH处理还诱导自噬，如通过微管相关蛋白1轻链3-II（LC 3-II）的上调水平、增加的GFP-LC 3点状物和升高的单丹酰尸胺（MDC）荧光强度所示。使用氯喹（CQ）的自噬通量的测量证实，INH刺激自噬，但不通过损害溶酶体降解来抑制自噬。CQ阻断自噬可显著加重INH诱导的细胞凋亡。进一步的研究表明，INH处理下调了哺乳动物雷帕霉素靶蛋白（mTOR）的蛋白磷酸化，mTOR是自噬的关键负调节因子。此外，INH诱导p38信号转导激活。p38抑制剂SB 203580通过增加caspase 9、3和PARP的裂解而有效地增强INH诱导的细胞凋亡，但不影响自噬。综上所述，我们首次发现INH诱导的保护性自噬与mTOR通路的抑制有关，并且INH通过下调caspase 9、3和PARP通路而诱导p38信号激活以抑制凋亡，但不抑制自噬。因此，自噬和p38信号传导的激活可能是INH诱导的肝毒性的治疗策略。
Isoniazid (INH), one of the first-line anti-tuberculosis drugs, is adversely associated with hepatotoxicity in the clinic. However, the detailed mechanism of this side effect is still unclear. The traditional theory that cytochrome P450 2E1 is involved in INH-induced hepatotoxicity remains controversial, therefore other mechanisms by which INH exerts hepatotoxicity need to be investigated. In the current study, we showed that in vitro treatment of human hepatocarcinoma HepG2 cells with INH induced caspase-dependent apoptosis through extrinsic and intrinsic pathways. It was characterized by the increased population of apoptotic cells using Annexin V/propidium iodide (PI) double staining by flow cytometry, and by the activation of caspases 8, 9, 3 and poly (ADP-ribose)-polymerase (PARP) proteins by western blotting. INH treatment also induced autophagy as shown by the upregulated levels of microtubule-associated protein 1 light chain 3-II (LC3-II), increased GFP-LC3 punctates, and elevated monodansylcadaverine (MDC) fluorescence intensity. The measurement of the autophagic flux using chloroquine (CQ) confirmed that INH stimulated autophagy but did not inhibit it by impairing lysosomal degradation. The blockage of autophagy with CQ exacerbated INH-induced apoptosis significantly. Further study showed that INH treatment down-regulated the protein phosphorylation of the mammalian target of rapamycin (mTOR), the key negative regulator of autophagy. In addition, INH induced p38 signaling activation. SB203580, a p38 inhibitor, effectively enhanced INH-induced apoptosis by increasing the cleavages of caspases 9, 3 and PARP, but did not affect autophagy. In summary, we firstly found that INH induced a protective autophagy which was associated with the inhibition of the mTOR pathway, and that INH induced p38 signaling activation to inhibit apoptosis by down-regulation of caspases 9, 3 and PARP pathways, but not that of autophagy. Thus, activation of autophagy and p38 signaling is presumably a therapeutic strategy for INH-induced hepatotoxicity.