去甲斑蝥素诱导人黑色素瘤A375S2细胞凋亡

Norcantharidin induced A375-S2 melanoma cell apoptosis: involvement of caspases and Bcl-2 family

目的:研究去甲斑蝥素(NCTD)诱导人黑色素瘤A375-S2细胞凋亡的机制。方法:采用MTT法、形态学观察、DNA凝胶电泳及Westernblot检测法。结果:去甲斑蝥素可诱导A375-S2细胞发生凋亡。半胱氨酸天冬氨酸酶(caspase)-3,-9抑制剂可以部分的抑制NCTD诱导的细胞死亡。细胞凋亡时caspase-3,8,-9酶活力升高,caspase3底物caspase-3激活的DNA酶抑制物(ICAD)蛋白表达下降,同时Bcl2/Bax、BclxL/Bax蛋白表达比率明显降低。结论:去甲斑蝥素通过激活caspase和Bcl-2家族诱导A375-S2细胞凋亡。

OBJECTIVETo examine the apoptotic pathway of norcantharidin(NCTD)-induced A375-S2 cell death. METHODSMTT, photomicroscopical observation, DNA agarose gel electrophoresis and Western blot analysis were carried out. RESULTSNCTD exhibited markedly antiproliferative effect on A375-S2 cells in a dose-and time-dependent manner. After 24 h treatment with NCTD, blebbing nuclei and granular apoptotic bodies appeared. Typical DNA fragmentation was observed in NCTD-treated A375-S2 cells. The inhibitory effect of NCTD on A375-S2cells was partially reversed by the inhibitors of caspase-3 and-9 and after NCTD treatment, the inhibition decreased from (69.56%) to (30.96%) and (51.50%), respectively. The activities of caspase-3,-8 and-9 significantly increased after treatment with NCTD at different time. The expression of the inhibitor of caspase-3 activated DNase (ICAD) decreased in a time dependent manner. At the same time, the ratio of Bcl-2/Bax or Bcl-x_(L)/Bax decreased. CONCLUSIONNCTD induced A375-S2 cell apoptosis. The activation of caspases and Bcl-2 family pathways were involved in the process of A375-S2 cell apoptosis which was induced by NCTD.