| Objective To explore the clinical significance and mechanisms by investigating the effects of sphingosine kinase 1 (SphKl) and focal adhesion kinase (FAK) on the epithelial-mesenchymal transition (EMT) of human colon cancer HCT-116 cells in colon cancer.Methods Human colon cancer HCT-116 cells were divided into three groups. FAK suppression group, SphKl suppression group and control group. 5μmol/L N, N-dimethylsphingosine (DMS) was used to suppress the activity of SphKl. 10pmol/L PF573228 was used to suppress the activation of FAK. The cells treated with equal volume of culture medium severed as control group. Forty-eight hours later, the cell viability was measured by MTT assay. The protein expression of SphKl, FAK and the EMT relative protein E-cadherin, N-cadherin, vimentin and MMP2 was analyzed by Western blotting. The mRNA expression of SphKl, SIP, FAK, E-cadherin and vimentin was detected by quantitative real-time PCR. The ability of tumor cell migration and invasiveness were measured by transwell chamber assays and wound-healing assay.Results The cell viability of HCT-116 cells was suppressed by DMS and PF573228 in dose and time dependent manners. Western blotting showed that DMS group significantly./suppressed the expression of SphKl and down-regulated the expression of FAK, N-cadherin, vimentin and MMP2, meanwhile upregulated the expression of E-cadherin. PF573228 suppressed the expression of FAK and reduced the expression of SphKl, N-cadherin, vimentin and MMP2, meanwhile increased the expression of E-cadherin (P<0.01). Follows were the detailed results, the protein expression of SphKl in PF573228 group and DMS group (respectively 0.78±0.03,0.83±0.05) was lower than control group (respectively 1.34±0.06) (P＜0.01). the protein expression of FAK in PF573228 group and DMS group (respectively 0.87±0.04,0.84±0.07) was lower than control group (respectively 1.35±0.03) (P＜0.01), While E-cadherin in PF573228 group and DMS group(respectively 1.28±0.06,1.10±0.03)was higher than control group (0.67±0.06) (P<0.05). And the protein expression of N-cadhern, Vimentin in PF573228 group and DMS group (0.84±0.02,0.73 ± 0.02,0.52+0.01,1.04+0.02) was lower than control group (1.41 ±0.05, 1.36±0.01) (P＜0.01). T the protein expression of MMP2 in PF573228 group and DMS group (respectively 0.98±0.02,0.86±0.06) was lower than control group (respectively 1.14±0.04) (P＜0.01). The cell mRNA results by and real-time fluorescent quantitative PCR showded that the mRNA expressions of SphKl, FAK, SIP and Vimentin descended compared with control group, while the expression of E-cadherin mRNA increased significantly(P<0.05 or 0.01). Follows were the detailed results:compared with control group, the mRNA expression of FAK (1.00± 0.00 vs 0.16±0.01,0.38±0.03), SphKl (1.00±0.00 vs 0.21±0.08,0.55±0.1), S1P(1.00±0.00 vs 0.08±0.01,0.30±0.06), Vimentin (1.00±0.00 vs 0.24±0.01,0.30±0.06) was decreased in PF573228 group and DMS group, meanwhile the expression of E-cadherin (1.00±0.00 vs 1.56±0.21, 2.39±0.30) was increased significantly (P＜0.05 or P＜0.01). In addition, compared with the control (93.87±6.06)μm, PF573228(22.97 ± 9.54)um, DMS(36.76±9.94)μm on 24 hours by wound-healing assay. And the amount of space between cells of 48 hours is the control(139.95 ± 13.4)μm, PF573228(42.95±12.18)μm, DMS(50.09±9.7)μm. The assay showed the migration ability of HCT-116 cells was significantly decreased by treating with DMS and PF573228 (P<0.01).Conclusion SphKl and FAK signaling pathways interacting each other play important role in the occurrence of EMT of colon cancer HCT-116 cells through suppressing the migration and invasion of cancer cell. |
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