Multiple signal transduction pathways in okadaic acid induced apoptosis in HeLa cells
Multiple signal transduction pathways in okadaic acid induced apoptosis in HeLa cells
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DOI：
10.1016/j.tox.2008.11.013
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发表时间：
2009-02-04
期刊：
影响因子：
4.5
通讯作者：
Rao, P. V. Lakshmana
中科院分区：
文献类型：
--
作者：
Jayaraj, R.;Gupta, Nimesh;Rao, P. V. Lakshmana
冈田酸（OA）是腹泻性贝类中毒毒素的主要成分，也是蛋白磷酸酶1和2A的有效抑制剂。我们研究了OA诱导HeLa细胞死亡的信号转导通路。在100 nM的IC50下，OA诱导细胞毒性和凋亡。OA治疗导致活性氧的时间依赖性增加和细胞内谷胱甘肽水平的减少。线粒体膜通透性的丧失导致bax、细胞色素c和AIF从线粒体转移到细胞质中。荧光显微镜下细胞呈典型的凋亡形态，染色质浓缩，细胞核碎裂。我们研究了线粒体介导的caspase级联。Western blot观察bax、caspase -8、10、9、3和7蛋白的激活和裂解的时间依赖性。除了caspase依赖性通路外，AT介导的caspase非依赖性通路也参与OA介导的细胞死亡。OA还引起蛋白磷酸酶2A活性和p38和p42/44 MAP激酶磷酸化的时间依赖性抑制。ac - devoo - cho和Z-VAD-FMK抑制剂研究不能阻止p38和p42/44 MAP激酶的磷酸化。我们的实验表明，caspase抑制剂Ac-DEVD-CHO、Z-IETD-FMK和Z-VAD-FMK可以抑制caspase - 3,8的裂解，但不能阻止oa诱导的细胞凋亡和DNA断裂。同样，环孢素-a和n-乙酰半胱氨酸预处理也不能防止DNA断裂。综上所述，我们的研究结果表明，OA诱导多种信号转导途径独立或同时作用导致细胞凋亡。2008爱思唯尔爱尔兰有限公司版权所有。
Okadaic acid (OA) is the major component of diarrhetic shell fish poisoning toxins and a potent inhibitor of protein phosphatase 1 and 2A. We investigated the signal transduction pathways involved in OA induced cell death in HeLa cells. OA induced cytotoxicity and apoptosis at IC50 of 100 nM. OA treatment resulted in time dependent increase in reactive oxygen species and depleted intracellular glutathione levels. Loss of mitochondrial membrane permeability led to translocation of bax, cytochrome-c and AIF from mitochondria to cytosol. The cells under fluorescence microscope showed typical apoptotic morphology with condensed chromatin, and nuclear fragmentation. We investigated the mitochondrial-mediated caspase cascade. The time dependent activation and cleavage of of bax, caspases-8, 10, 9, 3 and 7 was observed in Western blot analysis. In addition to caspase-dependent pathway AT mediated caspase-independent pathway was involved in OA mediated cell death. OA also caused time dependent inhibition of protein phosphatase 2A activity and phosphorylation of p38 and p42/44 MAP kinases. Inhibitor studies with Ac-DEVO-CHO and Z-VAD-FMK could not prevent the phosphorylation of p38 and p42/44 MAP kinases. Our experiments with caspase inhibitors Ac-DEVD-CHO, Z-IETD-FMK and Z-VAD-FMK inhibited capsase-3, 8 cleavages but did not prevent OA-induced apoptosis and DNA fragmentation. Similarly, pretreatment with cyclosporin-A and N-acetylcysteine could not prevent the DNA fragmentation. In summary, the results of our study show that OA induces multiple signal transduction pathways acting either independently or simultaneously leading to apoptosis. (C) 2008 Elsevier Ireland Ltd. All rights reserved.