Hedgehog信号通路异常对人结肠腺癌多药耐药性的影响

Effec t of abnormal Hedgehog signaling pathway on multidrug resistance of human colon adenocarcinoma cells

目的:探讨Hedgehog(Hh)信号通路与人结肠腺癌细胞多药耐药的关系。方法:以结肠腺癌敏感细胞株HCT-8和多药耐药细胞株HCT-8/VCR为研究对象,采用Hh信号通路激动剂SAG及抑制剂GANT61进行干预。实验分为4组:HCT-8组、HCT-8/VCR组、HCT-8+SAG组(3nmol/L SAG干预48h)以及HCT-8/VCR+GANT61组(5μmol/L GANT61干预48h)。采用CCK-8(cell counting kit-8)法检测细胞耐药性及存活率。实时荧光定量PCR和蛋白质印迹法分别从基因和蛋白水平检测P-糖蛋白(P-glycoprotein,P-gp)及Hedgehog信号通路相关蛋白Smo和Gli1的表达。结果:HCT-8/VCR组Smo、Gli1和P-gp基因及蛋白表达高于HCT-8组(P<0.05);HCT-8+SAG组24和48h时的细胞存活率及Smo、Gli1、P-gp基因和蛋白表达水平均高于HCT-8组(P<0.05);HCT-8/VCR+GANT61组24和48h时的细胞存活率及Smo、Gli1、P-gp基因和蛋白表达均低于HCT-8/VCR(P<0.05)。结论:人结肠腺癌细胞多药耐药性与Hh信号通路异常激活有关。

Objective： To investigate the correlation between Hedgehog （Hh） signaling pathway and multidrug resistance of human colon adenocarcinoma cells. Methods： Human colon adenocarcinoma cell line HCT-8 and its vincristine （VCR）-resistant variant HCT-8/VCR cells were treated by agonist SAG or antagonist GANT61 of Hh signaling pathway, respectively. Four groups were designed for the purpose of this study： HCT-8 group, HCT-8/VCR group, HCT-8＋SAG group （treated with 3 nmol/L SAG for 48 h）, and HCT-8/VCR＋GANT61 group （treated with 5 μmol/L GANT61 for 48 h）. Then CCK-8 （cell counting kit-8） assay was used to detect the multidrug resistance and the survival rate of the cells. The expression levels of Hh signaling members Smo （Smoothened protein） and Gli1 （Gloma-associated oncoprotein-1） and P-gp （P-glycoprotein） mRNAs and proteins were determined by real-time fluorescence quantitative PCR and Western blotting, respectively. Results： The expressions of Smo, Gli1 and P-gp mRNAs and proteins in HCT-8/VCR group were higher than those in HCT-8 group （P 〈 0.05）. Compared with HCT-8 group, the survival rate and the mRNA and protein expression levels of Smo, Gli1 and P-gp in HCT-8＋SAG group were significantly up-regulated （P 〈 0.05）. Compared with HCT-8/VCR group, the survival rate and the mRNA and protein expression levels of Smo, Gli1 and P-gp in HCT-8/VCR＋GANT61 group were significantly down-regulated （P 〈 0.05）. Conclusion： The multidrug resistance of human colon adenocacinoma cells may be correlated with abnormal activation of Hh signaling pathway.