[ ABSTRACT] AIM:To investigate the effects of sphingosine kinase l ( SphK1) and focal adhesion kinase ( FAK) on the epithelial-mesenchymal transition ( EMT) of human colon cancer HCT 116 cells.METHODS:Human colon cancer HCT116 cells were divided into 3 groups.N, N-dimethylsphingosine (DMS) was used to suppress the activity of SphK1. PF573228 was used to suppress the activation of FAK .The cells treated with equal volume of culture medium severed as control group.The cell viability was measured by MTT assay .The protein expression of SphK1, FAK and the EMT relative protein E-cadherin, N-cadherin, vimentin and matrix metalloproteinase (MMP) 2 was analyzed by Western blot.The mR-NA expression of SphK1, sphingosine-1-phosphate (S1P), FAK, E-cadherin and vimentin was detected by real-time PCR. The ability of tumor cell migration was measured by wound-healing assay.RESULTS:The cell viability of HCT116 cells was suppressed by DMS and PF 573228 in dose and time dependent manners .DMS significantly suppressed the expression of SphK1, FAK, N-cadherin, vimentin and MMP2, meanwhile enhanced the expression of E-cadherin.PF573228 reduced the expression of FAK , SphK1, N-cadherin, vimentin and MMP2, meanwhile increased the expression of E-cadherin (P<0.01).In addition, the migration ability of HCT116 cells was significantly decreased by treating with DMS and PF573228 (P<0.01).Compared with control group , the mRNA expression of FAK, SphK1, S1P and vimentin was de-creased, while the expression of E-cadherin was increased significantly in PF573228 group and DMS group (P<0.05). CONCLUSION:SphK1 and FAK signaling pathways may play an important role in the occurrence of EMT in the colon cancer HCT116 cells.%目的：研究鞘氨醇激酶1（sphingosine kinase l，SphK1）和黏着斑激酶（focal adhesion kinase，FAK）对人结肠癌HCT116细胞上皮间质转化（ epithelial-mesenchymal transition ，EMT）的影响。方法：将人结肠癌HCT116细胞分成3组：采用SphK1抑制剂N， N-二甲基鞘胺醇（ N，N-dimethylsphingosine ，DMS）、FAK抑制剂PF573228和相同体积的培养基分别处理细胞。 MTT法检测细胞活力，Western blot 方法检测SphK1、FAK、E-cadherin、N-cadherin、vimentin和基质金属蛋白酶2（MMP2）蛋白的表达，real-time PCR检测SphK1、鞘氨醇1-磷酸（S1P）、FAK、E-cadherin和vimentin mRNA的表达，并应用细胞划痕实验检测肿瘤细胞的迁移能力。结果：PF573228和DMS均明显抑制人结肠癌HCT116细胞的活力，并呈时间剂量依赖性。 DMS抑制SphK1的表达，同时下调FAK、N-cadherin、vimentin和MMP2蛋白的表达，而上调E-cadherin蛋白表达上调。 PF573228明显抑制FAK的表达，同时抑制SphK1、N-cad-herin、vimentin和MMP2的表达，上调E-cadherin蛋白的表达（ P＜0.01）。划痕实验显示PF573228和DMS显著抑制HCT116细胞的迁移能力（P＜0.01）。与对照组比较，PF573228组和 DMS组FAK、SphK1、S1P以及vimentin mRNA的表达明显下调，而E-cadherin mRNA的表达则明显上调（P＜0.05）。结论：SphK1和FAK信号通路可能在结肠癌HTC116细胞上皮间质转化过程中发挥重要作用。
展开▼
