Sphingosine Kinase1Activating Focal Adhesion Kinase Pathway Plays An Important Role In Progression Of Colon Carcinoma

Objective To study the expression of sphingosine kinase1(SphK1) and Focal Adhesion Kinase (FAK) in colon carcinoma tissues and to explore their correlation with clinicopathological features. Then further investigate their relationship with the cell adhesion, migration and invasion of human colon cancer cell line lovo and its associated mechanism through vitro examination.Methods①Sixty-six paraffin-embedded colon carcinoma samples were tested with immunohistochemistry method, stactistical analysis was then carried out to explore their expression in clinicopathological features.②Cultured lovo cells were divided into three groups:PMA group, DMS group and control group. Cells of PMA group were treated with100nmol/L Phorbol12-myristate13-acetate (PMA), the DMS group was treated with50μmol/L N, N-dimethylsphingosine (DMS), while the control group was treated with equal volume of culture medium. After treatment, cell proliferation was detected by MTT assay and colony formation assay, the migration and invasion capability of the cells were assessed in Transwell chambers. RT-PCR and Western blot were used to evaluate the mRNA and protein expression of FAK.Results①The immunohistochemistry results show:the positive rates of SphKl and FAK in66colon carcinoma samples were72.7%(48/66) and77.3%(51/66), which were higher than those in adjacent tissues[SphK1:51.5%(34/66); FAK37.9%(25/66)] and normal mucosa[SphK1:34.8%(23/66); FAK:22.7%(15/66)], showing a significant statistical difference(P<0.05). There was a close correlation between SphKl and FAK expression levels (r=0.480, P=0.000). Overexpression of SphKl and FAK in colon carcinoma were all related with depth of invasion, differentiation, distant and lymph node metastasis and Dukes’stages (P<0.05).②Vitro tests’results show that PMA significantly enhanced cells proliferation, invasion and migration, whereas DMS suppressed cells proliferation, migration and invasion (the cells viability, cloning rate, migration and invasion cell count of control group, PMA group and DMS group were as follow:cells viability:0.71±0.03vs1.05±0.05vs0.46±0.04; cloning rate:1.32%±0.26%vs2.17%±0.17%vs0.73%±0.13%; migration cell count:72.19±3.36vs98.46±6.25vs40.48±4.27; invasion cell count:75.48±4.12vs143.36±5.73vs38.57±3.24; all P<0.05vs control group). PMA significantly up-regulated the expression and activity of focal adhesion kinase (FAK), while DMS down-regulated the expression and activity of FAK (FAK mRNA:0.151±0.008vs0.212±0.014vs0.114±0.021; FAK protein:0.332±0.022vs0.374±0.029vs0.296±0.018; phosphor-FAK protein:0.186±0.032vs0.234±0.017vs0.112±0.023; P<0.05vs control group).Conclusions1. Overexpression of SphK1and NF-κB may be involved in the occurrence and development of colon carcinoma. Moreover, SphKl and NF-κB may be correlated with the invasion and metastasis of colon carcinoma.2. SphKl enhances cell proliferation, migration and invasion in human colon cancer cell line lovo possibly by activating FAK.