绿原酸通过影响自噬及耐药蛋白表达逆转5-氟尿嘧啶结肠癌细胞的耐药性

Chlorogenic acid reverses the drug resistance of 5-fluorouracil colon cancer cells by affecting autophagy and drug resistance protein expression

关键词:  绿原酸  细胞自噬  耐药  结肠癌细胞

Key words:  chlorogenic acid  autophagy  drug resistance  colon cancer cell

摘要:

[目的] 观察绿原酸（ChA）对耐5-氟尿嘧啶（5-FU）结肠癌细胞（HCT116-R）中多药耐药及自噬蛋白的影响，探讨绿原酸对HCT116-R细胞系的耐药逆转作用及相关作用机制。[方法] 体外传代培养HCT116、HCT116-R细胞系，采用CCK8法检测了ChA和5-FU对HCT116细胞增殖活力的影响；采用100、400 mg/L ChA干预HCT116-R细胞系48 h后，采用实时荧光定量逆转录聚合酶链反应（RT-PCR）和蛋白免疫印迹法（Western Blot）分析细胞中耐药、自噬相关基因及相关蛋白的表达。[结果] ChA可以诱导5-FU对结肠癌HCT116细胞增殖活力的抑制作用；Western Blot实验结果表明HCT116-R细胞中P-糖蛋白（P-gp）、肺抗药性相关蛋白（LRP）、自噬相关基因Beclin-1及高迁移率族蛋白B1（HMGB1）表达相对于HCT116细胞显著上调（P<0.05）；进一步分析发现ChA呈剂量性的抑制HCT116-R细胞中P-gp、LRP、Beclin-1及HMGB1 mRNA和蛋白表达（P<0.05），并且ChA还降低了p-Akt1、Akt1、哺乳动物雷帕霉素靶蛋白mTOR表达（P<0.05）。[结论] ChA通过影响Akt1/mTOR分子通路活化降低耐药相关蛋白P-gp、LRP及自噬相关蛋白Beclin1、HMGB1表达，进而逆转5-FU结肠癌细胞系的耐药性。

Abstract:

[Objective] To observe the effect of chlorogenic acid (ChA) on multidrug resistance and autophagy protein in 5-fluorouracil (5-FU)-resistant colon cancer cells (HCT116-R),and to explore the reversal effect of chlorogenic acid on HCT116-R cell line and its related mechanism.[Methods] HCT116 and HCT116-R cells were sub-cultured in vitro,and the effects of ChA and 5-FU on the proliferation of HCT116 cells were detected by CCK8 method. HCT116-R cell line was intervened by 100 mg/L and 400 mg/L ChA for 48 h,and the expression of drug resistance,autophagy related genes and related proteins were analyzed by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western Blot.[Results] ChA can induce 5-FU to inhibit the proliferation of colon cancer HCT116 cells. The results of Western Blot showed that the expressions of p-glycoprotein (P-gp),lung drug resistance related protein (LRP),autophagy related gene Beclin-1 and high mobility group protein B1(HMGB1) in HCT116-R cells were significantly up-regulated compared with those in HCT116 cells (P<0.05). Further analysis showed that ChA inhibited the mrna and protein expressions of P-gp,LRP,Beclin-1 and HMGB1 mRNA HCT116-R cells in a dose-dependent manner (P<0.05),and ChA also decreased the expressions of p-Akt1,Akt1 and mTOR,a mammalian target protein of rapamycin (P<0.05).[Conclusion] ChA decreased the expression of drug resistance related proteins P-gp,LRP and autophagy related proteins Beclin1 and HMGB1 by affecting the activation of Akt1/mTOR molecular pathway,thus reversing the drug resistance of 5-FU colon cancer cell line.