硒-甲基硒代半胱氨酸通过增强缝隙连接蛋白26构成的同型缝隙连接提高依托泊苷的细胞毒性

Methylselenocysteine Promotes Etoposide Cytotoxicity by Enhancing Homotypic Gap Junctions Composed of Connexin 26

目的探究硒-甲基硒代半胱氨酸(methylselenocysteine,MSC)对缝隙连接蛋白(connexin,Cx)26构成的同型缝隙连接(gap junction,GJ)功能的影响及其对化疗药物细胞毒性的调控。方法以转染并稳定表达Cx26的Tet-on HeLa细胞为工具细胞,MTT法观察MSC对细胞生长的影响;细胞接种荧光示踪法测定MSC对GJ功能的影响;Western blot检测MSC对Cx26蛋白表达的影响;标准细胞集落形成分析法观察化疗药物的细胞毒性;在此基础上,分析MSC对化疗药物细胞毒性的影响及其与调控GJ的关系。结果多西环素(doxycycline,Dox)可诱导Tet-on HeLa细胞Cx26表达并形成有功能的GJ。MSC在50μmol/L内对细胞生长无明显影响,无毒浓度的MSC可以浓度依赖性增强GJ,并可在纳摩尔级发挥作用。该效应与其诱导Cx26蛋白的表达有关。在三种不同作用机制的常见化疗药物中,依托泊苷(etoposide,Eto)在有无GJ形成的情况下表现一定的细胞毒性差异。MSC与Eto联合应用对细胞集落形成的抑制作用强于Eto单药,且该效应只在有GJ形成的HeLa细胞中发生。结论MSC可以通过增强Cx26组成的GJ提高Eto的细胞毒性,提示硒化物联合化疗在肿瘤治疗中具有一定的潜在价值。

Objective To investigate the effect of methylselenocysteine(MSC)on the function of homotypic gap junction(GJ)composed of connexin(Cx)26 and its regulation of chemotherapeutic drug cytotoxicity.Methods The Tet-on HeLa cells transfected with and stably expressing Cx26 were used as the tool cells.Effects of MSC on cell growth,GJ function,and Cx26 protein expression were examined by MTT method,parachute assay,and Western blot analysis,respectively.The cytotoxicity of chemotherapeutic drugs was determined by standard colony-forming assay,and the relationship between MSC's effect on cytotoxicity of these chemotherapeutic drugs and its regulation of GJ was further analyzed.Results In Tet-on HeLa cells,doxycycline(Dox)can induce the expression of Cx26,which could then form functional GJs.Within a concentration range of 50μmol/L,MSC had no significant effect on HeLa cell growth.Nontoxic concentrations of MSC can enhance GJs in a concentration-dependent manner and exert its effect at the nanomolar level.This effect was associated with an induction of Cx26 protein expression by MSC.Among the three common chemotherapeutic agents with different mechanisms of action,etoposide(Eto)presented cytotoxicity differences between HeLa cells cultured at low density(nonconfluent,no GJ formed)and high density(confluent,GJ formed).What's more,the inhibitory effect of Eto combined with MSC on HeLa cell colony formation was stronger than that of Eto alone,and this effect occurred only in HeLa cells with GJ formation.Conclusion MSC can potentiate the cytotoxicity of Eto by enhancing the GJs composed of Cx26,indicating that combined strategy of selenide and chemotherapy shows potential value in the treatment of malignant tumors.