鞘氨醇激酶-1调控ERK和NF-κB通路促进HT-29细胞的增殖和侵袭

Sphingosine kinase 1 regulates colon cancer TH-29 cell apoptosis,migration and invasion via ERK and NF-κB signaling pathways

目的探讨鞘氨醇激酶-1(Sphk1)对结肠癌HT-29细胞的增殖、凋亡和侵袭转移的影响及其作用机制。方法采用(12-)十四酸佛波酯(-13-)乙酸盐(PMA)诱导人结肠癌HT-29细胞Sphk1的活化和表达,N,N-二甲基鞘氨醇(DMS)抑制Sphk1的活化和表达;采用MTT法测定细胞的增殖,流式细胞术分析细胞的凋亡,γ-32P-ATP掺入法检测Sphk1的活性,Western杂交检测蛋白的表达,Transwell小室模型观察细胞迁移和侵袭能力的变化,ELISA法检测细胞基质金属蛋白酶(MMP)-2、MMP-9和尿激酶纤维蛋白溶酶原激活剂(uPA)的分泌。结果 PMA和DMS能分别有效地诱导和抑制HT-29细胞Sphk1活性和蛋白的表达;同时,PMA能促进细胞的增殖、迁移和侵袭,并抑制细胞的凋亡;DMS则抑制细胞的增殖、迁移和侵袭,并促进细胞的凋亡。诱导Sphk1的活化以及表达可促进细胞ERK、p-ERK和NF-κBp65的蛋白表达以及MMP-2、MMP-9和uPA的分泌,而抑制Sphk1则抑制ERK、p-ERK和NF-κBp65的蛋白表达以及MMP-2、MMP-9和uPA的分泌。结论 Sphk1可能是通过激活ERK和NF-κB通路,上调MMP-2、MMP-9和uPA的分泌,从而促进结肠癌细胞的增殖、迁移和侵袭,并抑制细胞的凋亡。

[Objective] To investigate the effect of sphingosine kinase 1（Sphk1） on the apoptosis,migration and invasion of colon cancer TH-29 cell and to explore its molecular mechanisms.[Methods] Phorbol 12-myristate 13-acetate（PMA） was exploited to induce the activity and expression of Sphk1 and N,N-dimethylsphingosine（DMS） was utilized to suppress the activity and expression of Sphk1.Cell proliferation was detected by the method of MTT and cell apoptosis was examined by flow cytometry.Migration and invasion capabilities of cells were assessed by the modified transwell chambers model.The activity of Sphk1 was assayed by the method of autoradiography.Western blotting was exploited to evaluate the protein expression of SphK1,ERK,phosphorylated ERK（p-ERK） and NF-κB p65.The method of ELISA was used to detect the secretion of matrix metalloproteinase（MMP）-2,MMP-9 and urokinase plasminogen activator（uPA）.[Results] PMA and DMS were able to induce and suppress the activity and protein expression of Sphk1,respectively.After treated with PMA,cell proliferation,migration and invasion capabilities were significantly enhanced,and cell apoptosis was markedly suppressed.In contrast,DMS suppressed the cell proliferation,migration and invasion,and promoted cell apoptosis significantly.With the elevating of the activity and protein expression of Sphk1,the protein expressions of ERK,p-ERK and NF-κB p65,the secretions of MMP-2,MMP-9 and uPA were strikingly enhanced.With the suppression of the activity and protein expression of Sphk1,the protein expressions of ERK,p-ERK and NF-κB p65,the secretions of MMP-2,MMP-9 and uPA were strikingly decreased.[Conclusions] Sphk1 potently enhances colon cancer HT-29 cell proliferation,migration and invasion,meanwhile suppresses the cell apoptosis.Activation of ERK and NF-κB signaling pathways resulted in up-regulation of MMP-2,MMP-9 and uPA maybe one of its molecular mechanisms.