Wortmannin阻断PI3K／AKT途径对食管癌缺氧诱导因子-1α及糖酵解的影响

Effect of blocking PI3K/AKT pathway by wortmannin on hypoxia-inducible factor la and glycolysis in esophageal carcinoma

目的探讨人食管癌细胞TE1、TEl3中应用wortmannin阻断P13K／AKT通路对缺氧诱导因子（HIF）-1α的抑制效果及对糖酵解相关基因表达的影响，分析P13K／AKT—HIF-1α途径对食管癌细胞糖酵解通路之间的关系。方法wortmannin（2vmol／L）预处理食管癌细胞TE1、TE13后常氧和缺氧培养，分为①常氧组（N）；②缺氧组（H）；③常氧处理组（N＋W）；④缺氧处理组（H＋W）。采用Western印迹检测细胞中HIF-1α蛋白及己糖激酶（HK）-Ⅱ、葡萄糖载体蛋白（GLUT）-1、乳酸脱氢酶（LDH）-A等糖酵解相关基因蛋白的表达；实时定量PCR检测HIF-1α及HK-Ⅱ、GLUT-1、LDH—A等糖酵解相关基因mRNA的表达；分光光度法测定胞液中LDH、HK-Ⅱ活性和培养上清液中乳酸浓度。结果常氧状态下，在TE1细胞中存在HIF-1α蛋白的表达，wortmannin（2μmol／L）能抑制HIF-1α蛋白表达，12h后抑制效应最明显，故选取12h为后续实验的缺氧时间。TE1、TEl3细胞经wortmannin预处理后HIF_1a、HK-Ⅱ、GLUT-1、LDHA蛋白表达较未加药细胞明显减弱（P〈0．05）；HIF-1α、HK-IItuRNA表达较未加药细胞明显减弱（P〈0．05）。常氧和缺氧条件下加用wortmannin的TE1，TE13组食管癌细胞胞液LDH、HK-Ⅱ活性均较未加药细胞组明显减弱（P〈0．05），未加药细胞缺氧后酶活性增强（P〈0．05）。常氧和缺氧条件下加用wortmannin组较未加药组细胞上清液乳酸浓度明显减低（P〈0．05），加wortmannin组细胞缺氧后表达增强（P〈0．05）。结论常氧及缺氧条件下，WOrtmannin能通过抑制食管癌细胞HIF-1α和糖酵解相关基因的表达导致乳酸水平降低，表明P13K／AKT—HIF-1α途径与食管癌细胞糖酵解通路密切相关。

Objective To investigate the inhibitory effect of blocking PI3K/AKT pathway by wortmannin on hypoxia-inducible factor 1α （HIF-1α） and the effect on the expression of glycolysis associated genes in human esophageal carcinoma cell lines TE1 and TEl3, and to analyze the relation between PI3K/AKT-HIF-la pathway and glycolysis in esophageal carcinoma cells. Methods Esophageal carcinoma cell lines TEl and TEl3 pretreated with wortmannin （2 μmol/L） were incubated under normoxic and hypoxic conditions. And each cell line was divided into four groups. The expression of HIF-la and glycolysis associated genes GLUT-l, LDHA and HKⅡ at protein level were measured by Western blot. The expression of HIF-la, GLUT-l, LDHA and HK-Ⅱ at mRNA level was determined by real-time PCR. The activities of LDH and HK-Ⅱ and lactic acid （LA） concentration in the culture supernatant were tested with speetrophotometer method. Results Under normoxic condition, HIF-la was expressed in TEl cells and the expression of HIF-1α was inhibited by wortmannin （2μmol/L）, the most significant inhibitory effect was at 12 hours, therefore 12 hours was selected for the subsequent hypoxia experiment. Compared with untreated cells, the expression of HIF-1α, HK-Ⅱ,GLUT-1, LDH-A at protein level significantly decreased in TEl and TEl3 cells after pretreated with wortmannin （P 〈 0. 05）, and the expression of HIF-1α, HK-Ⅱ at mRNA level significantly decreased （P〈0.05）. Under normoxic and hypoxic conditions, the HK-Ⅱ and LDH activities in TEl and Tel3 esophageal carcinoma cells significantly decreased after treated with wortmannin compared with untreated cells （P〈0.05）. Under hypoxia condition, the enzyme activity increased in untreated cells （P〈0. 05）. Under normoxic and hypoxic conditions, the lactic acid concentration in the culture supernatant obviously decreased in cells treated with wortmannin compared with untreated cells （P〈 0. 05）. Under hypoxia condition, lactic acid concentration increased in wortmannin treated cells （P 〈0. 05）. Conclusions Under normoxic and hypoxie conditions, wortmannin decrease lactic acid concentration through inhibiting the expression of HIF-1α and glycolysis associated genes, which indicate PI3K/AKT-HIF-1α pathway was closely related to glycolysis in esophageal carcinoma cells.