从NF-κB信号通路研究至真方对HCT-8/VCR细胞多药耐药的影响及机制

The effect of Zhizhen formula on multidrug resistence in human colorectal cancer cell lines and its mechanism via NF-κB signaling pathway

[目的]观察至真方含药血清对人大肠癌耐长春新碱细胞株HCT-8/VCR多药耐药的影响,并从NF-κB信号通路探讨其作用机制。[方法]用8%、16%、32%体积分数的至真方含药血清干预人大肠癌敏感细胞株HCT-8与耐长春新碱细胞株HCT-8/VCR,CCK8法检测细胞多药耐药性及存活率;ELISA法检测NF-κB活性;AnnexinV/PI流式细胞术检测细胞凋亡率;Western blot检测NF-κB/p65、IκB-α和Caspase-3蛋白的表达。[结果]体积分数>8%时,高、中、低剂量含药血清组对HCT-8/VCR细胞的抑制率与空白血清组比较差异均有统计学意义(P<0.01),且呈浓度递增趋势;8%、16%、32%体积分数的中剂量组至真方含药血清作用于HCT-8/VCR细胞24h后,NF-κB活性较HCT-8/VCR阴性对照组明显降低(P<0.01);至真方含药血清可促进HCT-8/VCR细胞凋亡,且在一定范围内细胞凋亡率呈浓度依赖性;经至真方含药血清处理后,HCT-8/VCR细胞NF-κB/p65蛋白表达较用药前明显下降(P<0.05),IκB-α蛋白表达明显上调(P<0.05),Caspase-3蛋白表达明显上调(P<0.05)。[结论]至真方含药血清可降低HCT-8/VCR细胞NF-κB的活性,一定程度上逆转了大肠癌细胞的多药耐药,其机制可能与抑制该通路分子开关IκB-α的磷酸化,下调NF-κB/p65蛋白的表达,从而上调通路下游的Caspase-3蛋白的表达,诱导HCT-8/VCR细胞凋亡有关。

[Objective]To investigate the effect of serum containing Zhizhen formula in reversing multidrug resistance and regulating the NF-κB signaling pathway.[Methods]Different concentrations of serum containing Zhizhen formula were used in treating HCT-8and HCT-8/VCR cells.Cell counting Kit-8（CCK-8）assay was used to detect the multidrug resistance and survival rate of HCT-8/VCR cells.The cell apoptosis was detected by using flow cytometry with AnnexinV/PI double staining.Expression of NF-κB/p65,IκB-αand Caspase-3 were determined by Western Blot techniques from the protein levels.[Results]The HCT-8/VCR cell inhibition rate of high,medium and low doses groups were significantly higher than that of blank control group（P〈0.01）,and showed a trend of increasing concentration.The activity of NF-κB was decreased 24 hours after 8%,16%,and 32%（volume fraction）of the serum was given compared to that of blank control group（P〈0.01）.The serum containing Zhizhen Formula induced apoptosis in a dosedependent manner under certain conditions.NF-κB/p65 protein expression decreased obviously（P〈0.05）,IκB-αprotein expression was obviously up-regulated（P〈0.05）and Caspase-3protein expression significantly raised（P〈0.05）after treated with the serum containing Zhizhen Formula.[Conclusion]Serum containing Zhizhen Formula can down-regulate the activity of NF-κB,and partly reverse multidrug resistance in HCT-8/VCR,which may be related to inhibiting the pathway molecular switch IκB-αpredominate phosphorylation,decreasing NF-κB/p65 protein expression,thus increasing Caspase-3protein expression,inducing the cell apoptosis of HCT-8/VCR cells.