蟾毒灵诱导骨肉瘤U-20S和U-20S／MTX300细胞系的凋亡

Bufalin induces apoptosis in osteosarcoma U-2OS and U-2OS methotrexate 300-resistant cell lines in vitro

目的 研究蟾毒灵对人骨肉瘤细胞株的增殖抑制和诱导凋亡作用.方法 以不同浓度的蟾毒灵分别作用于骨肉瘤细胞U-2OS和甲氨蝶呤（MTX）耐药骨肉瘤细胞U-2OS/MTX300.采用四甲基偶氮唑蓝（MTT）法检测细胞增殖,Hoechst 33258荧光染色检测细胞凋亡,流式细胞仪检测细胞周期,DNA琼脂糖凝胶电泳检测DNA ladder凋亡条带,Western blot法检测凋亡相关蛋白p53、bax和bcl-2的表达.结果 蟾毒灵能明显抑制骨肉瘤细胞的增殖,其对U-2OS和U-2OS/MTX300细胞的IC50值分别为（8.49±2.1）ng/ml和（10.19±1.7）ng/ml（P＞0.05）.蟾毒灵处理U2OS和U-2OS/MTX300细胞48 h后,细胞均出现明显的染色质凝集,有典型的凋亡小体产生.同时,蟾毒灵能诱导骨肉瘤细胞凋亡,其作用机制是通过阻滞细胞周期于G2/M期、上调p53、bax表达和下调bcl-2的表达来实现.结论 蟾毒灵能显著抑制人骨肉瘤细胞U-2OS和U-2OS/MTX300的生长,并诱导细胞凋亡,其抗骨肉瘤作用不受MTX耐药的影响.

Objective To study the growth inhibition and apoptosis induction effects of bufalin on human osteosarcoma cell lines in vitro. Methods U-2OS and U-2OS/methotrexate （MTX） 300-resistant cell lines were treated with bufalin. Cell viability was assessed by MTT assay. Cell-cycle status, apoptosisinducing effects, and the expression of apoptosis-related proteins were evaluated by flow cytometry,fluorescent staining, DNA fragmentation assay, and Western blotting. Results Bufalin inhibited cell growth in both U-2OS and U-2OS/MTX300 cells. The IC50 values of bufalin for U-2OS and U-2OS/MTX300 cells were （8.49 ±2.1 ） ng/ml and （ 10.19 ± 1.7） ng/ml, respectively. The induction of G2/M cell-cycle arrest was also seen in the bufalin-treated cells. The bufalin-indued apoptosis was confirmed by increased expression of rumor suppressor protein p53, bax and decreased expression of bcl-2. Conclusion Bufalin inhibites the growth of and induces apoptosis in both MTX-sensitive and MTX-resistant human osteosarcoma U-2OS cell lines. The apoptosis-inducing effect of bufalin is not influenced by the presence of high levels of DHFR.