褪黑素通过下调PD-L1表达抑制胶质瘤细胞免疫逃逸

Melatonin attenuated the immune evasion of glioma cells by down-regulating PD-L1 expression

目的研究褪黑素对胶质瘤细胞免疫逃逸的抑制作用及机制。方法以不同浓度的褪黑素处理T98G、U251、U118三种胶质瘤细胞,CCK-8法检测细胞活性,观察褪黑素抑制胶质瘤细胞活性的剂量-效应关系。予褪黑素单独处理胶质瘤细胞,或先予γ-干扰素(IFN-γ)预处理模拟肿瘤免疫微环境,再予褪黑素干预,采用Western blot与流式细胞术检测胶质瘤细胞程序性死亡受体-配体1(PD-L1)总蛋白与膜蛋白表达情况。将IFN-γ预处理或未预处理的胶质瘤细胞与激活的外周单核细胞(PBMC)共培养,用CCK-8法及结晶紫染色反映胶质瘤细胞的存活情况。结果褪黑素可剂量依赖性地抑制T98G、U251、U118三种胶质瘤细胞的活性,对正常环境下的胶质瘤细胞PD-L1总蛋白与膜蛋白表达无显著影响。然而,在IFN-γ诱导的肿瘤免疫微环境下,褪黑素可显著下调肿瘤细胞的PD-L1总蛋白与膜蛋白表达,并显著抑制胶质瘤细胞的免疫逃逸能力,增强PBMC免疫细胞的杀肿瘤细胞作用。结论褪黑素可下调肿瘤免疫微环境中胶质瘤细胞的PD-L1表达,抑制胶质瘤细胞的免疫逃逸能力,进而发挥间接的抗肿瘤作用。

Objective To determine the effect of melatonin on the immune evasion of glioma cells and the underlying mechanism.Methods Three glioma cell lines were used and treated with melatonin of different concentrations.The cells were pretreated with IFN-γto imitate the immune microenvironment of glioma and cocultured with peripheral blood mononuclear cell(PBMC).The cell viability was determined by CCK-8 assay.The total and membrane PD-L1 protein were detected by Western blot and flow cytometry,respectively.Survived glioma cells were stained by crystal violet assay.Results Melatonin inhibited the cell viability of T98G,U251,and U118 glioma cells in a dosedependent manner.No significant changes were observed in the total or membrane PD-L1 expression of melatonin-treated glioma cells in a normal environment.Nevertheless,under the IFN-γinduced immune microenvironment,melatonin remarkably down-regulated both the total and membrane expression of PD-L1 and attenuated the immune evasion of glioma cells shown by notably eliminated glioma cells by PBMCs.Conclusion Melatonin may attenuate the immune evasion of glioma cells by down-regulating the expression of PD-L1 in glioma cells,which provides evidence for the application of melatonin in glioma treatment.