基于内质网应激途径探讨蛇六谷提取物对大肠癌细胞5-FU耐药的逆转作用

Konjac Glucomannan Mediates Endoplasmic Reticulum Stress to Reverse 5-FU Resistance in Colorectal Cancer Cells

目的:观察蛇六谷主要成分魔芋葡甘露聚糖(konjac glucomannan,KGM)对大肠癌细胞的逆转耐药作用,并探究其可能的作用机制。方法:构建HCT-8/5-FU耐药细胞株,采用MTT法检测并计算HCT-8/5-FU细胞对5-FU的耐药指数(RI),筛选出最佳KGM浓度后检测其对HCT-8/5-FU细胞的逆转耐药作用;采用流式细胞术检测KGM、5-FU单用或联用对HCT-8/5-FU细胞凋亡的影响;Western blot检测各实验组细胞凋亡相关蛋白以及内质网应激信号通路相关蛋白的表达情况。结果:HCT-8/5-FU细胞相对于HCT-8细胞对5-FU的敏感性显著降低,RI为10.12;KGM能够抑制HCT-8/5-FU细胞生长,且呈剂量依赖性,与5-FU联用能够增加HCT-8/5-FU细胞对5-FU的敏感性;与单独用药相比,KGM和5-FU联合用药能够显著增加细胞凋亡率;Western blot结果显示KGM与5-FU联用能够显著上调HCT-8/5-FU细胞内磷酸化半胱氨酸蛋白酶-3(cleaved caspase-3)、磷酸化半胱氨酸蛋白酶-9(cleaved caspase-9)、Bcl-2相关X蛋白(Bax)、蛋白激酶样内质网激酶(PERK)、磷酸化真核翻译起始因子2α(p-elf2α)、转录激活因子4(ATF4)以及C/EBP同源蛋白(CHOP)的蛋白含量,下调B细胞淋巴瘤-2(Bcl-2)蛋白含量。结论:魔芋葡甘露聚糖通过调控PERK-elf2α-ATF4信号通路,介导内质网应激,进而逆转大肠癌细胞5-FU耐药。

Objective:To investigate the reversal effect of drug resistance on the colorectal cancer cells via Konjac Glucomannan(KGM)and the possible mechanism.Methods:HCT-8/5-FU resistant cell lines were constructed,and the resistance index(RI)of HCT-8/5-FU cells to 5-FU was measured by MTT assay.The optimal KGM concentration was selected to detect the reversal of resistance of HCT-8/5-FU cells;Flow cytometry was used to determine the effects of KGM and 5-FU alone or in combination on the apoptosis of HCT-8/5-FU cells;The expressions of apoptosis related proteins and endoplasmic reticulum stress signaling pathway were detected by Western blot assay.Results:The sensitivity of HCT-8/5-FU cells to 5-FU was significantly lower than that of HCT-8 cells,and the RI was 10.12;KGM inhibited the growth of HCT-8/5-FU cells in a dose-dependent manner,and combined with 5-FU increased the sensitivity of HCT-8/5-FU cells to 5-FU;Compared with the single drug,the combination of KGM and 5-FU significantly increased the apoptosis rate;Western blot results showed that KGM combined with 5-FU could significantly up-regulate the expressions of cleaved caspase-3,cleaved caspase-9,Bax,PERK,p-elf2α,ATF4 and CHOP,and down-regulate the expression of Bcl-2.Conclusion:Konjac Glucomannan mediates endoplasmic reticulum stress to reverse 5-FU resistance in colorectal cancer cells by regulating PERK-elf2α-ATF4 signaling pathway.