槲皮素对5-FU诱导的结直肠癌SW480细胞耐药及自噬调控机制研究

目的:探讨槲皮素(Que)调控5-氟尿嘧啶(5-FU)诱导的结直肠癌SW480细胞耐药及自噬的作用及机制。方法:采用浓度递增诱导法建立5-FU耐药细胞株SW480/5-FU,MTT法设置Que高、中、低剂量组(10、20、40 μmol/L)和对照组。MTT法检测各组细胞的5-FU耐药性,TUNEL染色法检测细胞凋亡,免疫荧光检测细胞自噬活性,Western blot检测细胞p糖蛋白(Pgp)、多药耐药相关蛋白1(MRP1)、三磷酸腺苷结合转运蛋白G超家族成员2抗体(ABCG2)、LC3Ⅰ、LC3Ⅱ、p62、丝/苏氨酸激酶(AKT)、p-AKT、雷帕霉素靶蛋白(mTOR)和p-mTOR蛋白表达。结果:5-FU抑制SW480/5-FU细胞的半抑制浓度(IC₅₀)明显高于SW480细胞(P<0.05),耐药指数(RI)=13.74。与对照组相比,Que中、高剂量组5-FU抑制SW480/5-FU细胞的IC₅₀明显下降(P<0.05),耐药逆转倍数分别为2.27和4.03。与对照组相比,Que中、高剂量组细胞抑制率、凋亡率和自噬活性明显升高(均P<0.05),蛋白的表达均明显升高(均P<0.05),P-gp、MRP1、ABCG2、p62、p-AKT/AKT和p-mTOR/mTOR蛋白表达明显下降(均P<0.05)。结论:Que可以以剂量依赖性的方式逆转SW480/5-FU细胞的5-FU耐药性,诱导细胞自噬,其作用机制可能与抑制AKT/mTOR的磷酸化有关。

Regulatory mechanism of quercetin on resistance and autophagy of 5-FU induced colorectal cancer SW480 cells

Objective:To explore the effects and regulatory mechanism of quercetin(Que)on resistance and autophagy of 5-fluorouracil(5-FU)induced colorectal cancer(CRC)SW480 cells.Methods:5-FU drug-resistant cell line SW480/5-FU was established by concentration increasing method.The high-dose,medium-dose and low-dose Que groups(10,20,40 μmol/L),and control group were set up by MTT.The resistance of 5-FU in each group was detected by MTT.The cells apoptosis was detected by TUNEL staining.The autophagy activity of cells was detected by immunofluorescence.The expression of p glycoprotein(Pgp),multidrug resistance associated protein 1(MRP1),ATP-binding transporter G cassette transporer G2(ABCG2),LC3Ⅰ,LC3Ⅱ,p62,serine/threonine kinase(AKT),p-AKT,mammalian target of rapamycin(mTOR)and p-mTOR was detected by Western blot.Results:The half maximal inhibitory concentration(IC₅₀)of 5-FU for SW480/5-FU cells was significantly higher than that for SW480 cells(P<0.05),and the resistance index(RI)was 13.74.Compared with control group,the IC₅₀ of 5-FU in SW480/5-FU cells was significantly decreased in medium-dose and high-dose Que groups(P<0.05),and the resistance reversal folds were 2.27 and 4.03,respectively.Compared with control group,inhibition rate,apoptosis rate and autophagy activity of cells were significantly increased in medium-dose and high-dose Que groups(all P<0.05),expression of LC3Ⅱ/LC3Ⅰ was significantly increased(all P<0.05),and expression of P-gp,MRP1,ABCG2,p62,p-AKT/AKT and p-mTOR/mTOR was significantly decreased(all P<0.05).Conclusion:Que can reverse 5-FU resistance of SW480/5-FU cells with dose-dependence,and induce autophagy.Its mechanism of action may be related to inhibiting AKT/mTOR phosphorylation.