甲基莲心碱逆转人结肠癌细胞奥沙利铂耐药的体外研究

Reversal effect of neferine on oxaliplatin-resistance in human colon cancer cells in vitro

目的:探讨甲基莲心碱(Nef)对人结肠癌细胞奥沙利铂(OXA)耐药的逆转作用及机制。方法:采用OXA浓度逐步递增法(2、4、8、12、24、48μmol/L)孵育人结肠癌HCT116细胞诱导构建OXA耐药株HCT116/OXA;检测Nef对HCT116/OXA细胞的细胞毒性,确定Nef的最适作用浓度和时间;分析并比较OXA(IC_(50)浓度)单独处理、Nef(最适作用浓度)单独处理、OXA(IC_(50)浓度)联合Nef(最适作用浓度)处理后,HCT116/OXA细胞的增殖,凋亡情况及凋亡相关蛋白(Bcl-2,Bax,PARP,p-PARP)的表达情况。结果:与亲本HCT116细胞比较,HCT116/OXA细胞较对OXA的耐药性明显增高(IC_(50):21.00μmol/Lvs.112.00μmol/L,P<0.05),耐药指数为5.33。Nef能明显抑制HCT116/OXA的增殖有作用(P<0.05),并呈浓度依赖性,其最适作用浓度、时间分别为5μmol/L、24h(细胞存活率为90%)。与OXA单独处理比较,HCT116/OXA细胞对OXA联合Nef处理的耐受性明显降低(IC_(50):112.00μmol/Lvs.45.47μmol/L,P<0.05),逆转倍数为2.46;Nef单独作用对HCT116/OXA细胞的凋亡影响不明显(P>0.05),但其与OXA联合作用对HCT116/OXA细胞凋亡诱导作用明显强于OXA单独作用(P<0.05);与OXA或Nef单独作用比较,OXA联合Nef作用后,HCT116/OXA细胞抗凋亡蛋白Bcl-2表达明显下降,Bax、p-PARP等凋亡蛋白表达明显上升(均P<0.05)。结论:Nef可逆转HCT116/OXA对OXA的耐药,机制可能与其调节Bcl-2/Bax表达水平,从而与OXA产生协同作用有关。

Objective： To investigate the reversal effect of neferine （Nef） on oxaliplatin （OXA）-resistance in human colon cancer cells and the mechanism. Methods： Human colon cancer HCT116 cells were cultured in a step-wised exposure to increasing concentrations of OXA （2, 4, 8, 12, 24 and 48 μmol/L） to induce and create the OXA-resistant cell line HCT116/ OXA; the cytotoxic effect of Nef on HCT 116/OXA cells was evaluated, and its optimal treatment concentration and time were determined; in HCT 116/OXA cells after exposure to OXA （IC5 concentration） alone, Nef （optimal treatment concentration） alone or OXA （ICs0 concentration） plus Nef （optimal treatment concentration）, the proliferation and apoptosis as well as the expressions of apoptosis-associated proteins （Bcl-2, Bax, PARP and p-PARP） were analyzed and compared. Results： The resistance of HCT116/OXA cells to OXA was significantly increased compared with the parent HCT116 cells （ICs0：21.00 μmol/L vs. 112.00 μmol/L, P〈0.05）, with a resistance index of 5.33. Nef showed significant inhibitory effect on proliferation of HCT116/OXA cells in a concentration-dependent manner （P〈0.05）, and its optimal treatment concentration and time was 5 μmol/L and 24 h, respectively; compared with lone OXA treatment, the tolerance of HCT 116/OXA cells to OXA plus Nef treatment was significantly reduced （IC50：112.00 μmol/L vs. 45.47 μmol/L, P〈0.05）, and the reverse index was 2.46. Nef alone exerted no significant effect on apoptosis of HCTll6/OXA cells （P〉0.05）, but the apoptosis inducing effect on HCTll6/OXA cells by its combination with OXA was significantly greater than that by OXA alone treatment （P〈0.05）; compared with Nef or OXA alone treatment, the expression level of anti-apoptotic protein Bcl-2 was significantly decreased, and the expression levels of apoptotic protein Bax and p-PARP were significantly increased in HCT116/OXA cells after OXA plus Neftreatment （all P〈0.05）. Conclusion： Nef can reverse OXA-resistance in HCT116/OXA cells, and the mechanism may be associated with its regulating Bcl-2/Bax expression, and thereby, producing a synergistic effect with OXA.