绿茶提取物对人乳头瘤病毒16癌蛋白诱导的人宫颈癌细胞缺氧诱导因子1α和血管内皮生长因子表达的影响

目的 研究绿茶提取物(GTE)及其主要成分表没食子儿茶素没食子酸酯(EGCG)干预对人乳头瘤病毒16(HPV-16)癌蛋白(E6和E7)诱导的人宫颈癌C-33A细胞缺氧诱导因子1α(HIF-1α)和血管内皮生长因子(VEGF)表达的影响.方法 将宫颈癌细胞分为瞬时转染空质粒HA-pSG5(空质粒组)、HA-pSG5-HPV-16 E6质粒(16 E6组)、16 E7质粒(16 E7组)和模拟转染组,转染后18、24和48 h以蛋白质印迹法检测各组细胞HIF-1α蛋白的表达,酶联免疫吸附试验检测VEGF蛋白的表达.转染后24 h,分别用不同浓度GTE或EGCG处理16 E6、16 E7组细胞不同时间,并设未用GTE或EGCG处理组(未干预组),同法检测各组细胞HIF-1α和VEGF蛋白的表达,实时PCR检测HIF-1α和VEGF mRNA的表达.结果 转染后24 h,16 E6和16 E7组细胞HIF-1α蛋白表达明显增加;VEGF蛋白分别为(870±66)、(1487±51)pg/ml,均明显高于空质粒组(366±65)pg/ml,均P<0.01].40μg/ml GTE或50μmol/L EGCG处理16 h即可明显抑制16E6、16E7组细胞HIF-1α蛋白表达;16E6组VEGF蛋白分别为(476±34)、(477±65)pg/ml,VEGF mRNA分别为1.208±0.196、1.174±0.208,均明显低于未干预组分别为(870±66)pg/ml、1.805±0.081,均P<0.01);16E7组VEGF蛋白分别为(649±55)、(514±37)pg/ml,VEGF mRNA分别为1.442±0.136、1.399±0.064,均明显低于未干预组分别为(1487±51)pg/ml、2.123±0.120,均P<0.01).80μg/ml GTE或100μmol/L EGCG对VEGF蛋白表达的抑制作用明显强于40μg/ml GTE或50 μmol/L EGCG(均P<0.01).结论 GTE和EGCG对HPV-16癌蛋白诱导的人宫颈癌细胞HIF-1α蛋白、VEGF蛋白及mRNA表达具有明显抑制作用,其中对VEGF蛋白表达的抑制作用呈明显的剂量依赖性.

Effects of green tea extract on expression of human papillomavirus type 16 oncoproteins-induced hypoxia-inducible factor-1α and vascular endothelial growth factor in human cervical carcinoma cells

Objective To investigate the effects of green tea extract (GTE) and its major component (-)-epigallocatechin-3-gallate (EGCG) on the human papillomavirns (HPV) type 16 oncoproteins (E6 and E7)-induced expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in human cervical carcinoma cells. Methods Human cervical carcinoma cells of the line C-33A were divided to 4 groups to be transiently transfected with the plasmid HA-pSG5-HPV-16E6 containing the HPV type 16 oncoprotein E6, HA-pSGS-HPV-16 E7, or empty plasmid HA-pSG5, or just exposed to the tranfection reagent Lipofectamine 2000 as mock transfection control group. Western blotting and ELISA were used to detect the protein expression of HIF-1α and the protein expression of VEGF 18, 24, and 48 h after the transfection. Twenty-four hours after the transfection, the transfected-cells were treated with GTE (40 μg/ml) or EGCG (50 μmol/L) for 8, 16, and 24 h respectively, or treated with GTE of the concentrations of 20, 40, and 80 μg/ml or EC, CG of the concentrations of 25, 50, and 100 μmoL/L respectively for 16 h. The HIF-1α and VEGF protein levels were analyzed by Western blotting and ELISA respectively and the HIF-1α and VEGF mRNA levels were determined with real-time PCR. Results Twenty-four hours after transfeetion, the HIF-1α protein expression of the 16E6 and 16E7 groups were significantly decreased, and the VEGF protein levels of the 16E6 and 16E7 groups were (870± 66) and (1487±51) pg/ml respectively, both significantly higher than that of the empty plasmid group (366±65) pg/ml, both P<0.01]. Treated by 40 μg/ml of GTE or 50 μmol/L of EGCG for 16 h, ①the HIF-1α protein levels of the 16E6 and 16E7 groups were significantly decreased;②the VEGF protein levels of the 16E6 group were (476±34) and (477±65) pg/ml respectively, and the VEGF mRNA levels of the 16E6 group were 1. 208±0.196 and 1. 174±0.208 respectively, all significantly lower than those of the untreated group (870±66) pg/ml and 1. 805±0.081 respectively, all P<0.01]; and ③the VEGF protein levels of the 16E7 group were (649±55) and (514±37) pg/ml respectively, and the VEGF mRNA levels of the 16E7 group were 1. 442±0. 136 and 1. 399±0. 064 respectively, all significantly lower than those of untreated group(1487±51) pg/ml and 2.123±0.120 respectively, all P<0.01]. The 80 μg/ml of GTE or 100 μmoL/L of EGCG showed much stronger effects on the inhibition of VEGF protein expression than 40 μg/ml of GTE or 50 μmol/L of EGCG (both P<0.01). Conclusion GTE and EC, CG can remarkably inhibit the HPV-16 oneoproteins-induced expression of HIF-1α protein, and VEGF protein and mRNA in human cervical carcinoma calls. Moreover, GTE and EGCG decrease the VEGF protein expression dose-dependently.