缝隙连接通讯介导曲马多对顺铂细胞毒性的抑制作用

THE INVOLVEMENT OF GAP JUNCTIONS IN THE INHIBITORY EFFECT OF TRAMADOL ON CISPLATIN CYTOTOXICITY

目的：晚期肿瘤患者常联合使用镇痛药和抗肿瘤药,但至今为止尚不清楚镇痛药对抗肿瘤药物的疗效有何影响,以及这种影响的机理.方法：磺基罗丹明B法观察曲马多本身对人胶质瘤U87细胞的毒性;标准细胞集落形成分析法观察顺铂的毒性并观察曲马多对顺铂毒性的影响;细胞接种荧光示踪法测定曲马多对U87细胞上Cx43组成的缝隙连接功能的影响.结果：磺基罗丹明B法显示曲马多在小于50 μg/ml的浓度范围内无细胞毒性;细胞集落形成分析法显示,0.5 μg/ml顺铂能够抑制U87细胞的集落形成,而且在有缝隙连接形成的细胞顺铂对细胞集落形成的抑制作用[集落形成率（51±3％）]显著高于对无缝隙连接形成细胞集落形成的抑制作用[集落形成率（73±2％）]（P＜0.05）.在有细胞缝隙连接形成的细胞,曲马多（5 μg/ml）与顺铂联合应用对细胞集落形成的抑制作用低于单用顺铂对细胞集落形成的抑制作用;而在无缝隙连接形成细胞,曲马多对顺铂抑制细胞集落形成的作用无影响.细胞接种荧光示踪法显示曲马多能够抑制U87细胞上由Cx43组成的缝隙连接通讯的荧光传递功能.结论：曲马多可以通过抑制Cx43组成的缝隙连接通讯降低顺铂的细胞毒性.

objective：Cancer patients are often concurrently treated with analgesics and antineoplastic drugs,yet the influence of analgesic agent on the therapeutic activity of antineoplastic drugs and its underling mechanisms are largely unknown.Methods：SRB was used to assay the toxicity of tramadol.＂Standard colony-forming assay＂ was used to examine cell survivals of U87 cells treated with cisplatin with or without tramadol.＂Parachute＂ dye-coupling assay was used to examine dye spread through gap junctions in U87 cells.Results：SRB assay showed that tramadol induced toxicity only when its concentration is up to 50 μg/ ml.Cell colony formation analysis showed that 0.5 μg/ml cisplatin inhibited gap junction formation of cell to cell colony formation,the inhibition rate was significantly higher than that of the seamless connection formed cells （P ＜ 0.05）.Standard Colony forming assay showed that cisplatin inhibited the colonogenic survivals of U87 cells and cisplatin-induced inhibition of clonogenic survivals on cells with gap junctioin formation was much greater than on cells without gap junction formation.In cells with gap junctions,5 μg/ml tramadol decreased the inhibitory effect on clonogenic survivals induced by cisplatin,while tramadol had no effect on toxic effect of cisplatin in cells without gap junctions.Parachute assay demonstrated that tramadol inhibited dye-coupling through gap junctions composed of Cx43 in U87 cells.Conclusion：These results demonstrate that tramadol depressed the sensitivity of glioma cells to cisplatin through inhibition of gap junctions composed of Cx43.