CAJ | 학술논문

目的探讨人食管癌细胞TE1、TE13中应用wortmannin阻断PI3K/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PI3K/AKT-HIF-1α途径对食管癌细胞糖酵解通路之间的关系.方法 wortmannin(2μmol/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(2 μmol/L)能抑制HIF-1α蛋白表达,12 h后抑制效应最明显,故选取12 h为后续实验的缺氧时间.TE1、TE13细胞经wortmannin预处理后HIF-1α、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-ⅡmRNA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PI3K/AKT- HIF-1α途径与食管癌细胞糖酵解通路密切相关.
목적 탐토인식관암세포TE1、TE13중응용wortmannin조단PI3K/AKT통로대결양유도인자(HIF)-1α적억제효과급대당효해상관기인표체적영향,분석PI3K/AKT-HIF-1α도경대식관암세포당효해통로지간적관계.방법 wortmannin(2μmol/L)예처리식관암세포TE1、TE13후상양화결양배양,분위①상양조(N)；②결양조(H)；③상양처리조(N+W)；④결양처리조(H+W).채용Western인적검측세포중HIF-1α단백급기당격매(HK)-Ⅱ、포도당재체단백(GLUT)-1、유산탈경매(LDH )-A등당효해상관기인단백적표체；실시정량PCR검측HIF-1α급HK-Ⅱ、GLUT-1、LDH-A등당효해상관기인mRNA적표체；분광광도법측정포액중LDH、HK-Ⅱ활성화배양상청액중유산농도.결과 상양상태하,재TE1세포중존재HIF-1α단백적표체,wortmannin(2 μmol/L)능억제HIF-1α단백표체,12 h후억제효응최명현,고선취12 h위후속실험적결양시간.TE1、TE13세포경wortmannin예처리후HIF-1α、HK-Ⅱ、GLUT-1、LDHA단백표체교미가약세포명현감약(P＜0.05)；HIF-1α、HK-ⅡmRNA표체교미가약세포명현감약(P＜0.05).상양화결양조건하가용wortmannin적TE1,TE13조식관암세포포액LDH、HK-Ⅱ활성균교미가약세포조명현감약(P＜0.05),미가약세포결양후매활성증강(P＜0.05).상양화결양조건하가용wortmannin조교미가약조세포상청액유산농도명현감저(P＜0.05),가wortmannin조세포결양후표체증강(P＜0.05).결론 상양급결양조건하,wortmannin능통과억제식관암세포HIF-1α화당효해상관기인적표체도치유산수평강저,표명PI3K/AKT- HIF-1α도경여식관암세포당효해통로밀절상관.
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 TE13,and to analyze the relation between PI3K/AKT-HIF-1α pathway and glycolysis in esophageal carcinoma cells. Methods Esophageal carcinoma cell lines TE1 and TE13 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-1α and glycolysis associated genes GLUT-1,LDHA and HK-Ⅱ at protein level were measured by.Western blot.The expression of HIF-1α,GLUT-1,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 spectrophotometer method.Results Under normoxic condition,HIF-1α was expressed in TE1 cells and the expression of HIF-1α was inhibited by wortmannin (2 μmol/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 TE1 and TE13 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 TE1 and Te13 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 hyp0xic 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 hypoxic 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.