摘要
目的研究华蟾酥毒基（cinobufagin）对多种骨肉瘤细胞系的增殖抑制和诱导凋亡作用，并探讨其相关机制。方法不同浓度的华蟾素毒基分别处理骨肉瘤细胞系U20S、MG63和Sa02后，四甲基偶氮唑蓝（MTT）法观察不同骨肉瘤细胞系的增殖活性，流式细胞仪检测细胞周期的变化，细胞核荧光染色及AnnexinV／PI染色检测细胞凋亡，Westernblot检测IAPs和Bcl．2家族凋亡相关蛋白凋亡相关蛋白Bax、cleaved．PARP、xlAP、clAP．1、survivin及p65的表达。结果MTT检测显示，华蟾酥毒基可明显抑制骨肉瘤细胞U20S、MG63和Sa02的增殖，其抑制细胞增殖的作用呈剂量和时间依赖性，其对U20S、MG63和Sa02细胞的48hIC，。值分别为（104．83~16．96）nmo^L、（47．07~7．5）nmo^L和（136．72~10．08）nmol／L。100nmol／L华蟾酥毒基处理骨肉瘤细胞12h后，流式细胞仪检测可见骨肉瘤U20S、MG63和Sa02的G0／G。期细胞比例下降，G2／M期细胞比例增加，表明华蟾素毒基主要通过将细胞阻滞在G2／M期，以抑制骨肉瘤细胞的增殖。进一步Hoechst33258细胞核染色发现，华蟾素毒基可诱导骨肉瘤细胞产生典型凋亡小体，AnnexinV／PI检测100nm01]L华蟾酥毒基作用48h后，U20S、MG63和Sa02细胞凋亡率分别为33．6％±6．4％、36．4％±7．8％及29．3％±5．1％。表明华蟾酥毒基的抗骨肉瘤效果是通过诱导骨肉瘤细胞凋亡来实现。在探讨其诱导骨肉瘤细胞凋亡的相关机制中，通过Westernblot检测发现其诱导凋亡的作用机制是通过上调IAPs和Bcl．2家族凋亡相关蛋白Bax、cleaved-PARP，下调xIAP、cIAP．1、survivin及p65蛋白来实现。结论华蟾酥毒基可明显抑制骨肉瘤细胞株U20S、MG63和Sa02细胞增殖，阻滞细胞在GJM期，并诱导其凋亡，其诱导凋亡的作用机制为调节IAPs和Bcl．2凋亡相关家族蛋白的活性。
Objective To study the growth inhibition, apoptosis induction effects of cinobufagin（CB）on human osteosar-coma（OS） cell line U2OS, MG63 and SaO2 in vitro and the underlying mechanism of action of cinobufagin in OS cells. Methods Cell viability was assessed by MTT assay. Cell-cycle status, apoptosis-inducing effects were evaluated by flow cytometry, fluores-cent staining and DNA fragmentation assays. Inhibitors of apoptosis proteins （IAPs） and Bcl-2 family proteins including Bax, cleaved-PARP,xIAP,eIAP-1, survivin and p65 were tested by Western blot. Results MTI＇ assay showed that CB could inhibited the growth of U2OS, MG63 and SaO2 cells in a dose- and time-dependent manner. The 48 h IC^0 of CB on U2OS, MG63 and SaO2 cells were （104.83＋ 16.96） nmol/L, （47.07~7.5） nmol/L, and （136.72~ 10.08） nmol/L respectively. The induction of GJM cell-cycle arrest was seen in the ceils treated with CB. After cells were cultured for 12 h in the presence of 100 nmol/L CB, the percentages of cells in the G0/GI phase were decreased, while GJM phase were increased in U2OS, MG63 and SaOS2 cells, respectively. The results showed CB inhibited the proliferation of osteosarcoma cells through blocking the cell cycle in GriM phase. Induction of apoptosis was confirmed by Hoeehst 33258 and Annexin V/PI staining. After treating with 100 nmol/L CB for 48 h, the extents of apoptosis were 33.6%~6.4%, 36.4%~7.8% and 29.3%~5.1%, respectively. These results indicate that the anti-tumor activity of cinobufagin in osteosarcoma cells was due to a Grim cell cycle arrest and apoptosis inducing effect. Western blot showed that CB could induce the apoptosis related family proteins Bax, cleaved-PARP up-regulation, xIAP, cIAP-1, survivin and p65 down- regulation in OS cells. Conclusion CB can inhibit the cell viability and induce Grim cell cycle arrest and apoptosis in U2OS, MG63 and SaO2 cells. The apoptosis-inducing effect of CB is confirmed by the regulation of apoptosis related proteins lAPs and Bel-2 in vitro.
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2014年第4期472-477,共6页
Chinese Journal of Orthopaedics
基金
国家自然科学基金资助项目（81102040.81372866）
