力达霉素增强顺铂诱导人肝癌BEL-7402细胞凋亡作用及机制研究

Potentiation and mechanism of cisplatin-induced apoptosis by lidamycin in human hepatoma BEL-7402 cells

目的 研究抗肿瘤抗生素力达霉素与顺铂的体外协同抗肿瘤作用及其机制。方法 采用克隆形成法、流式细胞术、琼脂糖凝胶电泳、Hoechst3 3 3 42和PI双荧光染色法、Westernblot等方法。结果 力达霉素与顺铂联合对人肝癌BEL 740 2细胞有很强的协同杀伤作用 ;可以使BEL 740 2细胞的DNA出现明显的梯状断裂 ;可以使阻滞于S期的BEL 740 2细胞减少而使凋亡细胞比例明显增多 ;联合用药后BEL 740 2细胞核出现明显的凋亡形态变化 ;使BEL 740 2细胞内Bcl 2的表达显著降低。结论 力达霉素可以增强顺铂的抗肿瘤作用 。

Aim To investigate the synergetic effect and the mechanism of antitumor action of the antibiotic lidamycin in combination with cisplatin in vitro . Methods Cytotoxicity of the drugs was measured by clonogenic assay. Chromatin condensation was observed by co staining with fluorescent dyes, Hoechst 33342 and propidium iodide. Apoptotic sub G1 was detected by flow cytometry and DNA ladder was observed using agarose gel electrophoresis. Bcl 2 protein level was detected by Western blot assay. Results By using clonogenic assay, lidamycin in combination with cisplatin was found to have synergetic effects on the proliferation of human hepatoma BEL 7402 cells. The data showed that BEL 7402 cells treated with cisplatin and lidamycin in combination produced internucleosomal DNA fragmentation analysed by agarose gel electrophoresis. The results of flow cytometry showed that cisplatin and lidamycin administrated in combination showed no obvious change in G1 phase distribution compared with single treatment. However, this combination reduced the S phase arrest and reversed the reduction of G2/M phase induced by single treatment. The results also showed that there was 11 3% or 9 37% of cells undergoing apoptosis in BEL 7402 cells treated with cisplatin or lidamycin, respectively, while it showed 32 4% of apoptotic cells in combination treatment. Cisplatin, lidamycin and combination of cisplatin and lidamycin was shown to induce typical chromatin condensation in BEL 7402 cells. The study showed that 0 5 μmol·L -1 cisplatin or 1×10 -4 μmol·L -1 lidamycin alone decreased Bcl 2 protein level, while lidamycin in combination with cisplatin strongly inhibited expression of Bcl 2 proteins in BEL 7402 cells. Conclusion The results suggest that lidamycin enhancement of cisplatin induced apoptosis associates with decrease of Bcl 2 protein expression, which may be useful for cancer chemotherapy.