目的：探讨2-脱氧葡萄糖（2-deoxy-glucose，2-DG）和顺铂(cisplatin)药物组合对人黑色素瘤细胞凋亡的影响及其作用机制。方法：用MTT法检测细胞活力，Annexin-V/PI染色和流式细胞仪检测细胞凋亡。Western blotting检测相关蛋白表达。细胞内ATP含量用生物发光试剂盒检测。结果：不同浓度顺铂（5~25 μmol/L）单药处理虽然能以剂量和时间依赖方式抑制A375细胞活力，但与2-DG（10 mmol/L）联合使用，顺铂处理浓度除5 μmol/L外都显示增强对细胞活力的抑制作用。2-DG（10 mmol/L）单独处理并不诱导A375细胞出现明显死亡（<10%），顺铂（20 μmol/L）单独作用导致50%左右的细胞死亡，而两者联合则导致大于80%的细胞死亡，与单独用药组比较差异显著（P<0.01）。顺铂单药或与2-DG联合均诱导Caspase-3和PARP-1裂解，并抑制抗凋亡蛋白Mcl-1的表达。2-DG联合顺铂（20 μmol/L）能明显抑制Ⅱ 型己糖激酶表达，并且使细胞内ATP含量降低于对照组（6.3 vs 33.0 μmol/mg），与单药组比较差异显著（P<0.01）。2-DG联合顺铂不诱导正常人黑色素细胞凋亡。此外该药物联合对另外三种人黑色素瘤细胞（SK-100，C8161和Mum-2C）也有增强杀伤的作用。结论：2-DG能特异地增强顺铂诱导人黑色素瘤细胞凋亡的敏感性，作用机制可能与下调抗凋亡蛋白Mcl-1表达，抑制肿瘤己糖激酶水平和ATP合成有关。

Objective:To explore effect of drug combination of 2-deoxy glucose (2-DG) and cisplatin on apoptosis of human melanoma cells and its mechanism. Methods: MTT assay was used to detect viability of the cells; Annexin-V/PI staining and flow cytometry assay detected apoptosis of the cells and Wetern blotting assay detected expressions of related-apoptosis protein. Intracellular ATP content was detected by a bioluminescence kit. Results: Although cisplatin with variou concentrations (5-25 μmol/L) alone inhibited viability of the A375 cells as concentration and time dependenct patterns, cisplatin (except 5 μmol/L) combined with 2-DG (10 μmol/L) all enhanced inhibition effect on viability of the cells. Single 2-DG (10 μmol/L) did not induce apparent death of the A375 cells (<10%), single cisplatin (20 μmol/L) resulted in death of the cells (about 50%) and both of them resulted in death of the cells (>80%), compairing with single drug groups there was a significant difference (P<0.01). Single cisplatin or combination with 2-DG all induced cleavages of Caspase-3 and PARP-1, and inhibited expression of anti-apoptosis protein Mcl-1. Two-DG combined with cisplatin (20 μmol/L) could significantly inhibit expression of hexokinase Ⅱ and content of intracellular ATP was lower than that in the control group (6.3 μmol/mg protein vs 33 μmol/mg protein), that was siginificantly different from those of single drug groups (P<0.01). Two-DG combined with cisplatin did not introduce apoptosis of nornal human melanocytes. In addition, the combination of both drugs also enhanced activity of killing another three human melanoma cells (SK-100, C8161 and Mum-2C). Conclution: Two-DG could specifically enhance susceptibility of cisplatin to induce apoptosis of human melanoma cells, Its mechanism could relate with down-regulating expression of anti-apoptosis protein Mcl-1 as well as inhibiting level of tumor hexokinase and synthesis of ATP.

国家自然科学基金项目 (No.81241148); 大连市科技计划项目 (No.2014E14SF147)