塞来昔布联合紫杉醇对人乳腺癌MCF-7/Taxol耐药细胞株多药耐药的逆转作用及机制的探讨

Effect of Combination of Taxol and Celecoxib on Reversing Multidrug Resistance Human Breast Cancer Cells (MCF-7/Taxol) and Explore Its Underlying Mechanism

目的观察环氧化酶-2(COX-2)选择性抑制剂塞来昔布(Celecoxib)联合紫杉醇(Taxol)对人乳腺癌MCF-7/Taxol耐药细胞多药耐药(multiple drug resistance,MDR)的逆转作用,并初步探讨其作用机制。方法体外诱导建立人乳腺癌MCF-7/Taxol耐药细胞株,CCK-8法检测Taxol、塞来昔布对MCF-7/Taxol细胞的毒性作用及塞来昔布对MCF-7/Taxol细胞多药耐药的逆转作用。实验分为:同步传代的MCF-7细胞组(A组),MCF-7/Taxol细胞阴性对照组(B组),单用Taxol无毒剂量的MCF-7/Taxol细胞组(C组),Taxol无毒剂量联合低逆转浓度塞来昔布的MCF-7/Taxol细胞组(D组),Taxol无毒剂量联合高逆转浓度塞来昔布的MCF-7/Taxol细胞组(E组),单用低逆转浓度塞来昔布的MCF-7/Taxol细胞组(F组),单用高逆转浓度塞来昔布的MCF-7/Taxol细胞组(G组)。RT-PCR和Western blot检测各组细胞MDR1基因和乳腺癌耐药蛋白(breast cancer resistance protein,BCRP)基因及其表达产物P-糖蛋白(P-glycoprotein,P-gp)和BCRP,以及COX-2蛋白表达的变化。结果成功建立具有较高耐药性的人乳腺癌MCF-7/Taxol细胞。塞来昔布与Taxol联合作用于细胞(D组、E组)与单用Taxol(C组)相比,对细胞的杀伤效应升高(P<0.05);与B组相比,Taxol(C组)能上调细胞P-gp表达及其mRNA水平(P<0.05),塞来昔布(F组、G组)能下调细胞P-gp、BCRP蛋白表达及其mRNA水平(P<0.05),以及下调COX-2蛋白表达(P<0.05),此外,塞来昔布(D组、E组)能抑制Taxol对细胞P-gp和其mRNA水平的上调作用。结论塞来昔布能有效逆转MCF-7/Taxol细胞的肿瘤多药耐药,其机制可能是通过降低细胞COX-2表达,从而降低P-gp及BCRP蛋白表达。

Objective To investigate the reversal effect of Celecoxib and Taxol on multidrug resistance（MDR） human breast cancer cells（MCF-7/Taxol） and its underlying mechanism.Methods After establishing the resistance cell lines of human breast cancer on Taxol（MCF-7/Taxol）,the effects of the drugs on the toxicity of MCF-7/Taxol cells and the reversal effect of Celecoxib on MDR were determined by CCK-8 assay.The cells were divided into seven groups（A： MCF-7;B： MCF-7/Taxol;C： MCF-7/Taxol＋ 0.03 μg/mL Taxol;D： MCF-7/Taxol＋0.03 μg/mL Taxol＋3 μg/mL Celecoxib;E： MCF-7/Taxol＋0.03 μg/mL Taxol＋6 μg/mL Celecoxib;F： MCF-7/Taxol＋3 μg/mL Celecoxib;G： MCF-7/Taxol ＋6 μg/mL Celecoxib）.The mRNA levels of MDR1 and BCRP in these treated cells were also determined by reverse transcription-polymerase chain reaction（RT-PCR）,the protein levels of P-gp and BCRP in these treated cells were also determined by Western blot method.Results Compared with the Taxol control,the cytotoxicity effects was obviously increased by combination of Taxol and Celecoxib（P〈0.05）.Compared with the vehicle control,Taxol up-regulated mRNA and protein levels of P-gp,whereas Celecoxib down-regulated mRNA and protein levels of P-gp and BCRP（P〈0.05）.Conclusion Celecoxib has reversal effect on MDR in MCF-7/Taxol cells,it＇s possible mechanism might be related to reduce the protein expression of COX-2,the inhibition of P-gp,BCRP mRNA and protein overexpression.