Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India.
Apoptosis. 2011 Feb;16(2):145-61. doi: 10.1007/s10495-010-0554-0.
Okadaic acid (OA) is a specific and potent protein phosphatase inhibitor and tumor promoter. The present study establishes the role of reactive oxygen species (ROS) and mitogen activated protein kinases in cell death induced by okadaic acid. The study showed that okadaic acid is cytotoxic at 10 nM with an IC50 of 100 nM in U-937 cells. The CVDE assay and mitochondrial dehydrogenase assay showed a time dependent cytotoxicity. The phase contrast visualization of the OA treated cells showed the apoptotic morphology and was confirmed with esterase staining for plasma membrane integrity. OA activated caspases-7, 9 and 3, PARP cleavage and induced nuclear damage in a time and dose dependent manner. Compromised mitochondrial membrane potential, release of cytochrome-c and apoptosis inducing factor confirms the involvement of mitochondria. A time dependent decrease in glutathione levels and a dose dependent increase in ROS with maximum at 30 min were observed. ROS scavenger-N-acetyl cysteine, mitochondrial stabilizer-cyclosporin-A, and broad spectrum caspase inhibitor Z-VAD-FMK inhibited the OA induced caspase-3 activation, DNA damage and cell death but caspase-8 inhibitor had no effect. OA activated p38 MAPK and JNK in a time dependent manner, but not ERK½. MAP kinase inhibitors SB203580, SP600125 and PD98059 confirm the role of p38 MAPK and JNK in OA induced caspase-3 activation and cell death. Over all, our results indicate that OA induces cell death by generation of ROS, and activation of p38 MAPK and JNK, and executed through mitochondrial mediated caspase pathway.
岗田酸（OA）是一种特异性和强效的蛋白磷酸酶抑制剂和肿瘤促进剂。本研究确立了活性氧（ROS）和丝裂原活化蛋白激酶在冈田酸诱导的细胞死亡中的作用。研究表明，冈田酸在 10 nM 时具有细胞毒性，在 U-937 细胞中的 IC50 为 100 nM。CVDE 测定和线粒体脱氢酶测定显示出时间依赖性细胞毒性。OA 处理细胞的相差显微镜观察显示出凋亡形态，并通过质膜完整性的酯酶染色得到证实。OA 以时间和剂量依赖性方式激活 caspase-7、9 和 3、PARP 切割，并诱导核损伤。线粒体膜电位受损、细胞色素 c 释放和凋亡诱导因子的释放证实了线粒体的参与。观察到谷胱甘肽水平随时间呈下降趋势，ROS 水平随剂量呈上升趋势，最大时间为 30 分钟。ROS 清除剂-N-乙酰半胱氨酸、线粒体稳定剂环孢菌素 A 和广谱半胱天冬酶抑制剂 Z-VAD-FMK 抑制 OA 诱导的 caspase-3 激活、DNA 损伤和细胞死亡，但 caspase-8 抑制剂没有效果。OA 以时间依赖性方式激活 p38 MAPK 和 JNK，但不激活 ERK½。MAP 激酶抑制剂 SB203580、SP600125 和 PD98059 证实了 p38 MAPK 和 JNK 在 OA 诱导的 caspase-3 激活和细胞死亡中的作用。总的来说，我们的结果表明，OA 通过产生 ROS、激活 p38 MAPK 和 JNK，并通过线粒体介导的半胱天冬酶途径来诱导细胞死亡。
