粉防己碱联合顺铂抑制宫颈癌细胞生长的作用和机制研究

Study on the inhibitory effect and mechanism of tetrandrine combined with cisplatin on cell growth in cervial cancer cells

目的:研究电压门控钾离子通道Kv10.1在宫颈癌中的表达,探讨Kv10.1特异性抑制剂粉防己碱(tetrandrine,TET)单独或与顺铂(cisplatin,DDP)联合使用对宫颈癌细胞生长抑制的作用及可能的分子机制。方法:免疫组化检测宫颈癌及正常宫颈组织中Kv10.1蛋白的表达情况。不同浓度的TET、DDP单药或联合处理宫颈癌SiHa细胞24 h后,采用MTT法检测各组细胞的增殖能力,采用流式细胞技术检测细胞周期,采用Western Blot法检测各组细胞中Eag1及PI3K/Akt信号通路相关蛋白表达水平。向TET组与对照组加入PI3K激动剂740Y-P,采用细胞克隆技术检测细胞生长情况,采用Transwell实验检测细胞侵袭能力,采用划痕实验检测细胞迁移能力,采用实时荧光定量PCR检测细胞Kv10.1表达水平,并进行统计学分析。结果:Kv10.1在宫颈癌组织中的表达明显高于正常宫颈组织,两者差异具有统计学意义(P<0.05),TET、DDP单药及双药联合组均能抑制SiHa细胞的增殖,促进细胞凋亡,其效应呈浓度依赖性,以联合用药组效果最为显著(P<0.05)。Western Blot显示TET单药可降低Kv10.1、p-PI3K和p-Akt蛋白表达,与DDP联合作用后效果更显著(P<0.05);与TET组相比,TET+740Y-P组克隆、侵袭、迁移细胞数量及Kv10.1表达水平均明显增高(P<0.05);与740Y-P组相比,TET+740Y-P组克隆、侵袭、迁移细胞数量及Kv10.1表达水平明显下降(P<0.05)。结论:Kv10.1在宫颈癌组织中的表达显著上调,提示其可能与宫颈癌的发生发展相关。Kv10.1特异性抑制剂TET可抑制PI3K/Akt信号通路,下调Kv10.1,从而提高宫颈癌细胞对顺铂治疗的敏感性,为宫颈癌治疗中DDP的减毒增效提供策略。

Objective:To study the expression of the voltage-gated potassium channel Kv10.1 in cervical cancer,and to investigate the effects and possible molecular mechanisms of tetrandrine(TET),a specific inhibitor of Kv10.1,alone and in combination with cisplatin(DDP) on the inhibition of cervical cancer cells growth.Methods:Expression of Kv10.1 protein in cervical cancer and normal cervical tissues detected by immunohistochemistry.The SiHa cells were treated with different concentrations of tetrandrine and cisplatin 24 hours later the cell proliferation was detected by MTT assay.The cell cycle was detected by flow cytometry.The protein expressions of Eag1 and PI3K/Akt signaling pathway were detected by Western Blot.The agonist 740Y-P of PI3K was added to tetrandrine group and control group,cell clonogenesis ablility was detected by clonogenesis assay,cell invasion ability was detected by Transwell assay,cell migration ability was detected by scratch assay,and cell Kv10.1 expression level was detected by quantitative real-time PCR.Results:Kv10.1 expression was significantly higher in cervical cancer tissues than in normal cervical tissues,and the difference between the two was statistically significant(P < 0.05).TET,DDP monotherapy and TET combined with DDP could remarkably inhibit the proliferation of Si Ha cells and promote apoptosis in a concentration-dependent manner.The combination drug group had the most significant effect(P < 0.05).Western Blot showed that the protein expression levels of Kv10.1,p-PI3K and p-Akt in the TET group were significantly decreased,and the combined effect with DDP was more significant(P < 0.05).Compared with tetrandrine group,the number of clonal,invasive,migratory cells and the expression level of Kv10.1 in TET + 740Y-P group were significantly increased(P < 0.05).Compared with 740Y-P group,the number of clonal,invasive,migratory cells and the expression level of Kv10.1 in TET + 740Y-P group were significantly decreased(P < 0.05).Conclusion:The expression of Kv10.1 was significantly up-regulated in cervical cancer tissues,suggesting that it may be associated with the occurrence and development of cervical cancer TET,a Kv10.1-specific inhibitor,can improve the sensitivity of cervical cancer cells to DDP treatment by down-regulating the expression level of Kv10.1 and inhibiting the PI3K/Akt signaling pathway,which can provide a new strategy for the reduction of toxicity and increase of efficacy of DDP in cervical cancer treatment.