G6PD缺陷诱发人黑色素瘤A375细胞凋亡机制研究  

Mechanism of G6PD deficiency triggering the apoptosis in the human melanoma A375 cell

作　　者：张正[1] 蔡天池 王艳玲[1] 唐琼玲[1] 张春华[1] 陈龙[1] 朱月春[1] 

机构地区：[1]昆明医科大学生化与分子生物学系,云南昆明650500

出　　处：《中华肿瘤防治杂志》2014年第14期1054-1058,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(30860322;81160246);云南省学术带头人基金(2007PY01-13)

摘　　要：目的:探讨葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase,G6PD)缺陷对A375细胞凋亡的影响及其可能的机制。方法:以A375-WT和A375-G6PDΔ细胞为模型,用Real-time PCR检测G6PD mRNA表达,蛋白质印迹法检测G6PD、Bcl-2、Bcl-xL、STAT5和P-STAT5蛋白的表达,紫外分光光度法测定G6PD酶活性,Heochst 33342/PI双染流式细胞仪检测细胞凋亡,免疫组化观察STAT5蛋白核迁移。结果:A375-WT和A375-G6PDΔ细胞中G6PD mRNA分别为0.545±0.13和0.207±0.03,降低了62.02%,t=-5.854,P=0.028;G6PD蛋白分别为0.975±0.16和0.227±0.10,降低了76.72%,t=-21.593,P=0.002;G6PD酶的比活性分别为(0.088±0.023)和(0.024±0.008)U/mg;降低了72.23%,t=-7.390,P=0.018;A375-G6PDΔ细胞的凋亡率为(8.62±1.67)%,比A375-WT细胞的(2.37±0.78)%升高了3.64倍,t=-12.163,P=0.007;A375-G6PDΔ细胞抗凋亡蛋白Bcl-2的表达量为0.245±0.037,比A375-WT细胞的0.578±0.073降低了57.61%,t=-16.021,P=0.004;Bcl-xL的表达量为0.138±0.019,比A375-WT细胞的0.287±0.067降低了51.92%,t=-5.377,P=0.033;A375-G6PDΔ细胞的P-STAT5/STAT5比值为0.72±0.201,比A375-WT细胞的2.28±0.367降低了68.4%(P=0.004),细胞核内STAT5表达为0.051±0.012,比A375-WT细胞核的STAT5蛋白(0.093±0.018)降低了45%(P=0.007),STAT5核迁移减少。结论:转录因子STAT5磷酸化降低、活性下降是G6PD缺陷所诱发的人黑色素瘤A375细胞凋亡的重要因素之一,为黑色素瘤发生及治疗的研究提供了新的思路。OBJECTIVE： To investigate the effect and its possible mechanism of G6PD deficiency on the A375 cell apoptosis. METHODS： With the A375-WT and A375-G6PDA cells, the expression of G6PD mRNA was assayed using Re- al-time PCR. The expressions of G6PD,Bel-2, Bcl-xL, STATS and P-STAT5 protein were detected by Western blotting. G6PD activity was tested through ultraviolet spectrophotometry,accompanied by flow cytometer with Heochst 33342/PI staining to determine cell apoptosis and immunohistochemistry to observe STAT5 protein migration to nucleus. RESULTS： The mRNA level （0. 207±0.03） and protein amount （0. 227±0. 10） of G6PD in the A375-G6PDA cells decreased by 62.02%（P=0. 028） and 76.72%（P=0. 002） ,respectively,as those （0. 545±0.13 and 0. 975±0.16） of the A375-WT ceils. The specific activity of G6PD （0. 024±0. 008） U/rag in the A375-G6PDA ceils reduced by 72. 23% （P=0. 018） as that （0. 088±0. 023） U/rag of the A375-WT cells. Further study showed that the apoptosis rate increased by 3.64 times （P=0. 007） in the A375-G6PDA cells as that of the A375-WT cells. In comparison with the A375-WT cells,the Bel-2（0. 245±0. 037） and Bcl-xL （0. 138±0. 019） in the A375-G6PDA cells decreased by 57.61%（P=0. 004） and 51.92% （P=0. 033）, respectively. Moreover, the A375-G6PDA cells displayed by 68.4% （P = 0. 004） reduction of P-STATS/ STATS ratio （0.72±0. 201） as that （2. 28±0. 367） of A375-WT cells,accompanied by a decrease of STAT5 migrated to nucleus（0. 051±0. 012,0. 093 ± 0. 018, P = 0. 007）. CONCLUSION： The reduction of phosphorylation and activity of STATS is a key factor in G6PD deficiency triggering the apoptosis of the human melanoma A375 cells, which may be a new clue in the study of melanoma tumorigenesis and therapy.

关 键 词：黑色素瘤 G6PD缺陷 STAT5 A375细胞 细胞凋亡 

分 类 号：R739.5[医药卫生—肿瘤]