川楝素与γδ T细胞协同抗结直肠癌的作用及机制研究

Toosendanin exerts synergistic effect on γδ T cell-induced cytotoxicity to colorectal cancer by suppressing expression of MCL-1

目的:探讨γδT细胞对结直肠癌细胞的杀伤活性,并研究川楝素对γδT细胞的协同效应。方法:体外培养人γδT细胞并用流式细胞术进行鉴定。乳酸脱氢酶(LDH)释放实验检测γδT细胞和川楝素对人结直肠癌SW480细胞的杀伤活性。Western blot实验检测川楝素对SW480细胞中Bcl-2、Bcl-x L和MCL-1表达水平的影响。ELISA实验检测川楝素是否影响γδT细胞肿瘤坏死因子相关凋亡诱导配体(TRAIL)和Fas配体(Fas L)的分泌。流式细胞术检测川楝素和γδT细胞处理后SW480细胞的线粒体膜电位和凋亡情况。Western blot实验检测川楝素和γδT细胞处理后SW480细胞caspase-9和caspase-3的活化。结果:体外培养的γδT细胞高表达CD3和γδT细胞受体(TCR)。川楝素处理能显著增强γδT细胞对SW480的杀伤活性。川楝素不影响γδT细胞TRAIL和Fas L的表达。川楝素不影响SW480细胞中Bcl-2和Bcl-x L的蛋白水平但显著抑制MCL-1的表达。转染MCL-1质粒能显著抑制川楝素对γδT细胞的协同效应。川楝素通过抑制MCL-1的表达促进γδT细胞对SW480细胞线粒体膜电位的损伤和凋亡的诱导。川楝素通过抑制SW480细胞MCL-1的表达促进γδT细胞对SW480细胞caspase-9和caspase-3的活化。结论:川楝素通过抑制MCL-1的表达发挥对γδT细胞的协同抗结直肠癌作用。

AIM：To investigate the synergistic effect of toosendanin on regulating the cytotoxicity of γδ T cells to colorectal cancer cells.METHODS：γδ T cells amplified in vitro were identified by flow cytometry.Lactate dehydrogenase（LDH） release was detected to evaluate the cytotoxicity of γδ T cells and toosendanin to SW480 cells.The role of toosendanin in regulating the protein expression of Bcl-x L,Bcl-2 and MCL-1 was determined by Western blot.The effect of toosendanin on regulating the secretion of TNF-related apoptosis-inducing ligand（TRAIL） and Fas ligand（Fas L） by γδ T cells was evaluated by ELISA.The mitochondrial membrane potential and apoptosis in SW480 cells treated with γδ T cells and toosendanin were analyzed by flow cytometry.The activation of caspase-9 and caspase-3 were determined by Western blot.RESULTS：CD3 and γδ T-cell receptor（TCR） were highly expressed in the γδ T cells amplified in vitro.Combination with toosendanin significantly enhanced the cytotoxicity of γδ T cells to SW480 cells.Toosendanin did not influence the secretion of TRAIL and Fas L secreted by γδ T cells.Toosendanin did not regulate the expression of Bcl-x L and Bcl-2 but suppressed the expression of MCL-1 in SW480 cells.In addition,enforced expression of MCL-1 obviously suppressed the synergistic effect of toosendanin on γδ T cell-induced cell death in SW480 cells.Meanwhile,co-treatment with toosendanin was able to enhance the γδ T cell-induced apoptosis and decrease of mitochondrial membrane potential.γδ T cell-dependent activation of caspase-9 and caspase-3 was significantly enhanced by toosendanin co-treatment in SW480 cells.CONCLUSION：Toosendanin exerts synergistic effect on γδ T cell-induced cytotoxicity to colorectal cancer by suppressing the expression of MCL-1.