ERK信号通路参与至真方逆转人大肠癌多药耐药细胞HCT-8/VCR耐药作用机制的研究

Research on the role of ERK signaling pathway in the reversing effect of Zhizhen Recipe on multidrug resistance in human colorectal cancer cell lines

[目的]探讨至真方是否通过影响ERK通路活性,下调P-gp蛋白及ERK、MDR1mRNA表达,逆转人大肠癌多药耐药细胞HCT-8/VCR多药耐药。[方法]细胞增殖-毒性检验CCK-8(cell counting kit-8)法鉴定HCT-8/VCR细胞株的多药耐药性及存活率;流式细胞仪测定细胞内罗丹明123(Rh123)的平均荧光强度;Real-time PCR及Western blot检测ERK、p-ERK、P-gp的基因和蛋白的表达。[结果]HCT-8/VCR为多药耐药细胞;至真方含药血清可明显抑制HCT-8/VCR细胞生长;HCT-8/VCR细胞经8%、16%、32%至真方含药血清干预48h后,细胞内Rho123外排减少,波峰右移,细胞内荧光强度明显增强(P<0.01);HCT-8/VCR细胞ERK、MDR1mRNA的表达水平均有所下降,且呈浓度依赖性;p-ERK表达较用药前(0.764±0.001)明显下降(P<0.01),分别为(0.513±0.002)、(0.498±0.001)、(0.471±0.12);ERK表达较用药前(0.771±0.204)分别下降至(0.437±0.004)、(0.413±0.002)、(0.398±0.001),(P<0.01)。P-gp表达较用药前(0.94±0.014)下降至(0.701±0.01)、(0.663±0.01)、(0.508±0.02),(P<0.01)。[结论]至真方可逆转人大肠癌多药耐药细胞株HCT-8/VCR的耐药作用,其机制与降低P-gp外排功能、降低ERK通路活性、下调P-gp蛋白及基因表达有关。

[Objective]To investigate the role of ERK signaling pathway in the reversing effect of Zhizhen Recipe on multidrug resistance in human colorectal cancer cell lines.[Methods]CCK-8assay was used to detect the drug resistance of HCT-8/VCR cells and apotosis rate.FCM was used to detect intracellular concentration of Rho123.Real-time PCR and western blot were adopted to detect the protein levels and gene levels of ERK,P-ERK and P-gp.[Results] Compared with sensitive cells,HCT-8/VCR appeared multidrug resistant.The drug-serum containing Zhizhen Recipe significantly inhibited the growth of HCT-8/VCR cells.After treatment with 8%,16%and 32% of the drug serum for 48 hours,the discharge of intracellular Rho123 by HCT-8/VCR decreased,wave crest was shifted to right,and intracellular fluorescence intensity was strengthened.The expression of ERK and DR1 mRNA decreased,which was correlated to the concentration.Compared with before treatment,expression of p-ERK was markedly decreased respectively from（0.764±0.001）to（0.513±0.002）,（0.498±0.001）,and（0.471±0.12）（P〈0.01）.The expression of ERK was decreased respectively from（0.771±0.204）to（0.437±0.004）,（0.413±0.002）,and（0.398±0.001）（P〈0.01）.The expression of P-gp was markedly decreased respectively from（0.94±0.014）decreased to（0.701±0.01）,（0.663±0.01）,and（0.508±0.02）（P=0.000）.[Conclusion]Drug-serum containing Zhizhen Recipe could reverse the MDR of colon cancer cells.The possible mechanism was by inhibiting the efflux function of P-gp,reducing the activity of ERK pathway,and down regulating the expression of P-gp protein and gene.