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葡糖-6-磷酸脱氢酶(G6PD)在许多肿瘤细胞中高表达,但其发生的作用机理目前仍然不明确.以正常人表皮黑色素细胞(HEM)、野生型人黑色素瘤A375细胞(A375-WT)和G6PD缺陷的A375细胞(A375-G6PDΔ)为对象,经real-time PCR、Western印迹和紫外分光光度法分析显示,A375-WT细胞的mRNA、G6PD蛋白和G6PD活性分别是HEM细胞的1.89倍(P<0.05)、6.86倍(P<0.01)和2.30倍(P<0.05).Annexin V/PI流式细胞仪和Western印迹测定表明,A375-G6PDΔ的凋亡率是A375-WT的5.10倍(P<0.01),活化半胱氨酸蛋白酶3(caspase-3)增高1.84倍(P<0.01)以及89 kD多聚二磷酸腺苷核糖聚合酶-1(PARP-1)生成增加2.87倍(P<0.01).分光光度法分析显示,A375-G6PDΔ的NADPH和GSH分别降低了72.30%(P<0.01)和27.39%(P<0.05),并伴有75.43%的H2O2增高(P<0.01).结果提示,G6PD在黑色素瘤细胞中高表达和高活性,而敲减G6PD表达通过caspase-3和PARP-1信号诱发人黑色素瘤细胞凋亡,这为深入揭示黑色素瘤的发生机理提供了新思路。
Glucose-6-phosphate dehydrogenase (G6PD) is overexpressed in many tumor cells, but its mechanism of action is still unclear.With normal human epidermal melanocytes (HEM), wild-type human melanoma A375 cells (A375- WT) and G6PD-deficient A375 cells (A375-G6PDΔ). Real-time PCR, Western blotting and UV spectrophotometry showed that the mRNA, G6PD and G6PD activities of A375-WT cells were 1.89 (P <0.05), 6.86-fold (P <0.01) and 2.30-fold (P <0.05) .Annexin V / PI flow cytometry and Western blotting showed that the apoptosis rate of A375-G6PDΔwas 5.10 (P <0.01), increased 1.84 folds of caspase-3 (P <0.01) and 2.87 fold increase of 89 kD PARP-1 (P <0.01) .Analysis by spectrophotometry showed that NADPH and GSH of A375-G6PDΔ decreased by 72.30% (P <0.01) and 27.39% (P < ). The results suggest that G6PD is highly expressed and highly active in melanoma cells, while knockdown of G6PD expression induces apoptosis in human melanoma cells through caspase-3 and PARP-1 signaling, The mechanism of occurrence provides a new idea.