蟾毒灵与顺铂协同抑制乳腺癌MCF-7细胞增殖的机制探讨

The mechanism of bufalin and cisplatin synergistically suppress the proliferation of breast cancer MCF-7 cells

目的探讨蟾毒灵(Bufalin)联合顺铂对乳腺癌MCF-7细胞增殖及凋亡的影响,并探讨可能的协同机制。方法采用MTT法检测空白对照组(仅培养液)、单纯细胞对照组和实验组的细胞增殖率,实验组加入不同浓度的顺铂或Bufalin单药或以固定比例(Bufalin∶顺铂=1 nmol/L∶1μmol/L)联合作用24 h。分别采用20 nmol/L Bufalin、20μmol/L顺铂单药或联合作用24 h,用流式细胞术检测细胞凋亡,并用Western Blotting检测活化的Met(p-Met)、Met、活化的Src(p-Src)、Src、PARP及其裂解cleaved PARP蛋白的表达。结果 MTT法检测显示,顺铂和Bufalin均以剂量依赖方式抑制乳腺癌MCF-7细胞增殖。在顺铂≥0.1μmol/L和Bufalin≥0.1 nmol/L时两药联合作用可协同抑制MCF-7细胞增殖(0<CI<0.433)。流式细胞术检测显示,20μmol/L顺铂作用24 h可诱导(10.7±4.8)%的MCF-7细胞凋亡,而20 nmol/L Bufalin作用24 h未明显诱导细胞凋亡,20 nmol/L Bufalin与20μmol/L顺铂联合作用24 h后可诱导(40.8±8.5)%的MCF-7细胞凋亡。Western Blotting检测显示,顺铂作用后可导致Met和Src活化,Bufalin与顺铂联合作用后可抑制顺铂诱导的Met和Src活化,同时上调PARP裂解。结论 Bufalin可能通过抑制顺铂诱导的Met和Src活化与顺铂协同抑制MCF-7细胞增殖,增强顺铂诱导的细胞凋亡。

Objective To investigate the influence of bufalin in combination with cisplatin on the proliferation of breast cancer cells and explore the possible mechanism. Methods MTT assay was used to detect the cell proliferation in blank control group （only medium）, cell control group （only MCF-7 cells） and experimental groups （cells were exposed to 20 nmol/L bufalin or 20 μmol/L cisplatin alone, or their combination in a fixed ratio for 24 h）. And then cell apoptosis was determined by flow cytometry after stained by propidium iodide. Expression of Met, p-Met, Src, p-Sre, PARP and cleaved PARP was analyzed by Western blotting. Re- suits MT-r assay showed that cisplatin and bufalin could both suppress MCF-7 cells proliferation in a dose-dependent manner. Cispla- tin and bufalin could synergistically suppress the proliferation of MCF-7 cells （0〈CI〈0. 433） at concentrations of≥0. 1 p, mol/L and ≥0. 1 nmol/L, respectively. Flow cytometry analysis showed that 20 p, mol/L cisplatin induced （10.7±4. 8）% apoptosis of MCF-7 cells after 24 h. Bufalin （20 nmol/L） did not induce significant apoptosis after 24 h. In contrast, the treatment of 20 nmol/L bufalin and 20 μmol/L eisplatin increased the apoptotic fraction to （40. 8±8.5 ）%. Western Blotting analysis showed that cisplatin treatment activated Met and Src. Bufalin could significantly down-regulate the cisplatin-induced Met and Src activation and up-regulate the cleaved PARP. Conclusion Bufalin and cisplatin can synergistically suppress the proliferation and induce apoptosis of MCF-7 cells through down-reg- ulating the cisplatin-induced activation of Met and Src.