An Wei-wei, Gong Xian-feng, Wang Min-wei, Tashiro Shin-ichi, Onodera Satoshi, Ikejima Takashi
China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, China.
Acta Pharmacol Sin. 2004 Nov;25(11):1502-8.
To investigate the mechanism of norcantharidin (NCTD)-induced HeLa cell apoptosis.
HeLa cell growth inhibition was measured by MTT method. Apoptosis was detected by Hoechst 33258 staining and agarose gel electrophoresis. Caspase activities were assayed using caspase apoptosis detection kit. Western blot analysis was used to evaluate the level of ICAD, ERK/p-ERK, JNK/p-JNK, and Bcl-X(L)/Bax expression.
Norcantharidin inhibited HeLa cell growth in a time- and dose-dependent manner. HeLa cells treated with norcantharidin showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. Caspase family inhibitor (z-VAD-fmk), caspase-8, -9 inhibitor (z-IETD-fmk, Ac-LEHD-CHO, respectively) and caspase-3 inhibitor (z-DEVD-fmk) partially prevent norcantharidin-induced apoptosis, but initiator caspase-1 inhibitor (Ac-YVAD-fmk) did not. The activities of caspase-3, -8, and -9 were up-regulated after norcantharidin treatment. Furthermore, NCTD-induced activation of caspase-3 resulted in the degradation of the inhibitor of caspase-activated DNase (ICAD). Up-regulation of mitochondrial Bax expression and down-regulation of Bcl-x(L) expression also participated in the apoptosis induced by NCTD. Although p38 MAPK inhibitor (SB203580) failed to block cell death, ERK MAPK inhibitor (PD98059) and JNK MAPK inhibitor (SP600125) had marked inhibitory effects on norcantharidin-induced apoptosis. Moreover, the phosphorylation of JNK were up-regulated followed by delayed ERK phosphorylation after treatment with NCTD, suggesting that ERK and JNK were both responsible for NCTD-induced apoptosis in HeLa cells and worked at different stages.
The cytotoxic effect of NCTD on HeLa cells was mainly due to apoptosis. The anti-tumor mechanism of NCTD might involve caspses, mitochondrial, and MAPKs pathways.
研究去甲斑蝥素（NCTD）诱导HeLa细胞凋亡的机制。
采用MTT法检测HeLa细胞生长抑制情况。通过Hoechst 33258染色和琼脂糖凝胶电泳检测细胞凋亡。使用半胱天冬酶凋亡检测试剂盒检测半胱天冬酶活性。采用蛋白质免疫印迹分析评估ICAD、ERK/p-ERK、JNK/p-JNK和Bcl-X(L)/Bax的表达水平。
去甲斑蝥素以时间和剂量依赖性方式抑制HeLa细胞生长。用去甲斑蝥素处理的HeLa细胞表现出典型的凋亡特征，包括形态学变化和DNA片段化。半胱天冬酶家族抑制剂（z-VAD-fmk）、半胱天冬酶-8、-9抑制剂（分别为z-IETD-fmk、Ac-LEHD-CHO）和半胱天冬酶-3抑制剂（z-DEVD-fmk）部分阻止去甲斑蝥素诱导的凋亡，但起始半胱天冬酶-1抑制剂（Ac-YVAD-fmk）无效。去甲斑蝥素处理后，半胱天冬酶-3、-8和-9的活性上调。此外，NCTD诱导的半胱天冬酶-3激活导致半胱天冬酶激活的脱氧核糖核酸酶（ICAD）降解。线粒体Bax表达上调和Bcl-x(L)表达下调也参与了NCTD诱导的凋亡。虽然p38丝裂原活化蛋白激酶抑制剂（SB203580）未能阻断细胞死亡，但细胞外信号调节激酶丝裂原活化蛋白激酶抑制剂（PD98059）和应激活化蛋白激酶丝裂原活化蛋白激酶抑制剂（SP600125）对去甲斑蝥素诱导的凋亡有显著抑制作用。此外，用NCTD处理后，JNK的磷酸化上调，随后ERK磷酸化延迟，表明ERK和JNK均参与NCTD诱导的HeLa细胞凋亡，且作用于不同阶段。
NCTD对HeLa细胞的细胞毒性作用主要是由于凋亡。NCTD的抗肿瘤机制可能涉及半胱天冬酶、线粒体和丝裂原活化蛋白激酶途径。
