CoCl2诱导黑素瘤A375细胞上皮间质转化、糖酵解及SB-431542干预研究

Study on CoCl2 Induced Epithelial Mesenchymal Transformation and Glycolysis of Melanoma A375 Cells and SB-431542 Intervention

目的探讨CoCl 2刺激的黑素瘤A375细胞上皮间质转化和糖酵解的作用机制及SB-431542对此的干预作用。方法选用本团队前期已证实的安全有效浓度的CoCl 2(50~150μmol/L)刺激黑素瘤A375细胞,建立体外低氧模型,Western Blot检测HIF-1α、Nodal、上皮间质转化相关蛋白E-cadherin、Vimentin、MMP2及糖酵解各组分蛋白LDH-A、LDH-B的表达,以明确CoCl 2对A375细胞上皮间质转化和糖酵解的影响作用。随后研究SB-431542对Nodal-ALK4-ALK7介导A375细胞上皮间质转化和糖酵解反应的抑制作用:本部分拟筛选安全有效浓度的Nodal受体抑制剂(SB-431542)分别作用于CoCl 2介导的低氧模型,通过MTT实验确定SB-431542的安全浓度用于后续实验。Western blot检测SB-431542对CoCl 2诱导细胞上皮间质转化和LDH活化相关蛋白的影响;琼脂糖凝胶电泳检测LDH同工酶谱的变化情况;此外,使用Western blot检测Nodal及其受体(ALK4、ALK7)的表达。结果CoCl 2可显著上调细胞上皮间质转化相关蛋白Vimentin、MMP2和促瘤相关蛋白Nodal及其受体(ALK4、ALK7)的表达,且表达与作用浓度成正相关;CoCl 2能促进糖酵解活化相关蛋白表达,其中LDH-A表达上调,LDH-B的表达受到抑制(P<0.05)。MTT结果显示10μmol/L SB-431542处理48 h对细胞的增殖影响最小(P<0.05);SB-431542可抑制Vimentin、MMP2和Nodal及其受体ALK4、ALK7表达(P<0.05);促进糖酵解途径的相关蛋白LDH-A的表达下调(P<0.05),抑制糖酵解途径相关蛋白LDH-B的表达上调(P<0.05)。结论CoCl 2可诱导黑素瘤细胞上皮间质转化及糖酵解。SB-431542可通过抑制CoCl 2诱导的Nodal-ALK4-ALK7信号通路抑制与上皮间质转化及糖酵解相关分子的表达。

Objective To investigate the mechanism of epithelial mesenchymal transformation and glycolysis of melanoma in A375 cells stimulated by CoCl2 and the effects of SB-431542 on it.Method Firstly,safe and effective concentration of CoCl2(50-150μmol/L)proved by our team was used to stimulate melanoma A375 cells to establish an invitro hypoxia model.Western blot was used to detect HIF-1α,Nodal,E-cadherin,Vimentin,MMP2,LDH-A and LDH-B,which CoCl 2 effect on the epithelial mesenchymal transformation and glycolysis of A375 cells were clarified.Secondly,inhibitory effects of SB-431542 on Nodal-ALK4-ALK7 mediated epithelial mesenchymal transformation and glycolysis of A375 cells were investigated:we selected SB-431542 to A375 cells with different concentrations.According to MTT results,we selected different safe concentrations of SB-431542 for subsequent experiments.SB-431542 with different concentration gradients were added to hypoxia model induced by CoCl 2(150μmol/L).MTT was used to detect the effect of SB-431542 on CoCl 2 induced cell proliferation.Western blot was used to detect SB-431542 effect on proteins expression related to LDH activation and epithelial mesenchymal transformation induced by CoCl 2.Changes of LDH isozymes were detected by agarose gel electrophoresis.In addition,Western blot was used to detect Nodal expression and its receptors(ALK4,ALK7).Results CoCl 2 could promote the expression of glycolytic activation-related proteins,in which LDH-A was up-regulated and LDH-B was inhibited(P<0.05).MTT results showed that treatment with 10μmol/L SB-431542 for 48 hours had the least effect on cell proliferation(P<0.05).SB-431542 could inhibit the expression of Vimentin,MMP2,Nodal and their receptors ALK4 and ALK7(P<0.05).LDH-A,a related protein that could promot glycolysis pathway,was down-regulated(P<0.05).And LDH-B,a related protein that could inhibit glycolysis pathway,was up-regulated(P<0.05).Conclusion CoCl 2 could induce epithelial-mesenchymal transition and glycolysis of melanoma cells.SB-431542 could inhibit molecules expression related to epithelial-mesenchymal transition and glycolysis by inhibiting Nodal-ALK4-ALK7 signaling pathway induced by CoCl 2.