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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(5~25μmol/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5μmol/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(20μmol/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(20μmol/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 33.0μmol/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 investigate the effects of 2-deoxy-glucose (2-DG) and cisplatin on human melanoma cell apoptosis and its mechanism. Methods: Cell viability was detected by MTT assay. Apoptosis was detected by Annexin-V / PI staining and flow cytometry. Western blotting detection of related protein expression. The content of ATP in cells was detected by bioluminescence kit. Results: Monotherapy with different concentrations of cisplatin (5 ~ 25μmol / L) could inhibit the viability of A375 cells in a time-and dose-dependent manner. However, the combination of 2-DG and 10 mmol / L have shown to enhance the inhibitory effect on cell viability. Treatment with 2-DG alone (10 mmol / L) did not induce obvious death (<10%) in A375 cells, and 50% of cells died of cisplatin alone (20 μmol / L) % Of the cell death, compared with the drug group alone was significantly different (P <0.01). Cisplatin alone or combined with 2-DG both induced the cleavage of Caspase-3 and PARP-1, and inhibited the expression of anti-apoptotic protein Mcl-1. The combination of 2-DG and cisplatin (20 μmol / L) significantly inhibited the expression of type 2 hexokinase and decreased the intracellular ATP content in the control group (6.3 vs 33.0 μmol / mg) compared with the single drug group (P < 0.01). 2-DG combined with cisplatin does not induce normal human melanocyte apoptosis. In addition, the combination of the drug and the other three human melanoma cells (SK-100, C8161 and Mum-2C) also enhance the killing effect. Conclusion: 2-DG can enhance the sensitivity of cisplatin-induced apoptosis in human melanoma cells. The mechanism may be related to the downregulation of Mcl-1, the inhibition of tumor hexokinase and ATP synthesis.