鞘氨醇激酶1调控p38和c-Jun氨基末端激酶通路影响结肠癌HT-29细胞的凋亡迁移与侵袭

目的 探讨鞘氨醇激酶1(SphK1)对结肠癌HT-29细胞增殖、凋亡、迁移和侵袭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)和N,N-二甲基鞘氨醇(DMS)诱导和抑制人结肠癌HT-29细胞SphK1的活化和表达,采用四甲基偶氮唑蓝(MTT)法测定细胞增殖,流式细胞术分析细胞凋亡,γ-32P-ATP掺入法检测SphK1的活性,Western blot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.结果 PMA和DMS能分别诱导和抑制SphK1活性和表达,在24 h内呈一定的时间依赖性.PMA能抑制细胞的凋亡,100 nmol/L PMA作用0、6、12、24 h后,细胞凋亡率分别为(9.35±0.84)%、(7.61±0.48)%、(5.53±0.76)%和(0.56±0.33)%.DMS能显著抑制细胞的增殖,诱导细胞的凋亡,50 μmol/L DMS作用0、6、12、24 h后,细胞凋亡率分别为(9.18±0.94)%、(12.06±1.41)%、(19.80±2.36)%和(31.85±3.60)%.对照组、PMA组和DMS组的迁移细胞数分别为68.75±6.15、109.33±11.63和10.83±2.48,侵袭细胞数分别为55.42±4.50、90.58±7.06和9.58±2.39,与对照组比较,PMA组迁移和侵袭细胞数明显增多,而DMS组则显著减少.对照组、PMA组和DMS组的p38表达强度分别为0.20±0.03、0.08±0.02和0.31±0.03,磷酸化p38(p-p38)表达强度分别为0.19±0.02、0.09±0.02和0.38±0.05,应激活化蛋白激酶/c-Jun氨基末端激酶(SAPK/JNK)的表达强度分别为0.26±0.03、0.12±0.03和0.43±0.06,PMA显著抑制p38、p-p38和SAPK/JNK蛋白的表达,而DMS则促进p38、p-p38和SAPK/JNK蛋白的表达.结论 SphK1可促进结肠癌HT-29细胞的增殖、迁移和侵袭能力,并抑制细胞的凋亡,其机制可能是通过抑制p38和SAPK/JNK通路而发挥作用.

Abstract：

Objective To investigate the effect of sphingosine kinase 1 (SphK1) on the proliferation, apoptosis, migration and invasion of colon cancer TH-29 cells and to explore its molecular mechanisms. Methods Phorbol 12-myristate 13-acetate (PMA) was used to induce the activity of SphK1 and N, N-dimethylsphingosine (DMS) was used to suppress the activity of SphK1. Cell prolieration and apoptosis were detected by MTT assay and flow cytometry, respectively. The migration and invasion capabilities of the cells were assessed in Transwell chambers. The activity of SphK1 was assayed by autoradiography. Western blot was used to evaluate the protein expression of SphK1, p38, phosphorylated p38 (p-p38) and SAPK/JNK. Results PMA and DMS were able to induce and suppress the activity and protein expression of SphK1 in a time-dependent manner, respectively. PMA enhanced and DMS suppressed the cell viability in a time- and dose-dependent manner. Being treated with 100 nmol/L PMA or 50 μ mol/L DMS for0, 6, 12, 24 h, the cell apoptosis rates of PMA group were (9.35 ±0.84)%, (7.61 ±0.48)%,(5.53 ±0.76) % and (0.56 ±0.33 ) %, contrastlv, that of DMS group were (9.18 ±0.94) %, ( 12.06 ±1. 41 ) %, ( 19.80± 2.36) % and (31.85 ± 3.60) %, respectively. Compared with the control group, the cell migration and invasion capabilities of the PMA group were significantly enhanced, and that of the DMS group were significantly suppressed. The migration cell number of control, PMA and DMS groups were 68.75 ±6.15, 109.33 ± 11.63 and 10. 83 ±2.48, the invasion cell number of control, PMA and DMS groups were 55.42 ± 4.50, 90. 58 t 7.06 and 9.58 ± 2.39, respectively. With the elevating activity and expression of SphK1, the protein expressions of p38, p-p38 and SAPK/JNK were strikingly suppressed. On the contrary, after treating with DMS the protein expressions of p38, p-p38 and SAPK/JNK were enhanced.Conclusions SphK1 potently enhances the prolieration, migration and invasion of colon cancer HT-29 cells, meanwhile suppresses the cell apoptosis. The suppressing of the p38 and SAPK/JNK signalling pathways may be one of its molecular mechanisms.

Effect of sphingosine kinase 1 on the apoptosis, migration and invasion of colon cancer TH-29 cells and its molecular mechanisms

Objective To investigate the effect of sphingosine kinase 1 (SphK1) on the proliferation, apoptosis, migration and invasion of colon cancer TH-29 cells and to explore its molecular mechanisms. Methods Phorbol 12-myristate 13-acetate (PMA) was used to induce the activity of SphK1 and N, N-dimethylsphingosine (DMS) was used to suppress the activity of SphK1. Cell prolieration and apoptosis were detected by MTT assay and flow cytometry, respectively. The migration and invasion capabilities of the cells were assessed in Transwell chambers. The activity of SphK1 was assayed by autoradiography. Western blot was used to evaluate the protein expression of SphK1, p38, phosphorylated p38 (p-p38) and SAPK/JNK. Results PMA and DMS were able to induce and suppress the activity and protein expression of SphK1 in a time-dependent manner, respectively. PMA enhanced and DMS suppressed the cell viability in a time- and dose-dependent manner. Being treated with 100 nmol/L PMA or 50 μ mol/L DMS for0, 6, 12, 24 h, the cell apoptosis rates of PMA group were (9.35 ±0.84)%, (7.61 ±0.48)%,(5.53 ±0.76) % and (0.56 ±0.33 ) %, contrastlv, that of DMS group were (9.18 ±0.94) %, ( 12.06 ±1. 41 ) %, ( 19.80± 2.36) % and (31.85 ± 3.60) %, respectively. Compared with the control group, the cell migration and invasion capabilities of the PMA group were significantly enhanced, and that of the DMS group were significantly suppressed. The migration cell number of control, PMA and DMS groups were 68.75 ±6.15, 109.33 ± 11.63 and 10. 83 ±2.48, the invasion cell number of control, PMA and DMS groups were 55.42 ± 4.50, 90. 58 t 7.06 and 9.58 ± 2.39, respectively. With the elevating activity and expression of SphK1, the protein expressions of p38, p-p38 and SAPK/JNK were strikingly suppressed. On the contrary, after treating with DMS the protein expressions of p38, p-p38 and SAPK/JNK were enhanced.Conclusions SphK1 potently enhances the prolieration, migration and invasion of colon cancer HT-29 cells, meanwhile suppresses the cell apoptosis. The suppressing of the p38 and SAPK/JNK signalling pathways may be one of its molecular mechanisms.