摘要
目的探讨鞘氨醇激酶1（SphKl）对人结肠癌LoVo细胞增殖、侵袭、迁移、黏着斑激酶（FAK）通路以及细胞间黏附分子1（ICAM-1）和血管细胞黏附分子1（VCAM-1）的影响。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LoVo细胞SphKl的活性，放射自显影法检测SphKl的活性，四甲基偶氮唑蓝（M-TT）法检测细胞的增殖活性，Boyden小室观察细胞迁移和侵袭能力的变化，酶联免疫吸附试验（ELISA）测定可溶性ICAM-1（sICAM-1）和可溶性VCAM-1（sVCAM-1）浓度，逆转录聚合酶链反应（RT-PCR）检测FAK、ICAM-1和VCAM-1mRNA的表达，Westernblot法检测FAK和磷酸化FAK（p-FAK）蛋白的表达。结果PMA和DMS能分别显著地诱导和抑制SphKl活性，PMA呈时间一剂量依赖性促进细胞的增殖，而DMS则呈时间一剂量依赖性抑制细胞的增殖。PMA和DMS处理24h后，对照组、PMA组和DMS组迁移细胞数分别为（75．48±6．12）个、（143．36±8．73）个和（38．574-3．24）个，侵袭细胞数分别为（64．19±5．36）个、（118．46±6．25）个和（32．48±4．27）个，与对照组比较，PMA组迁移和侵袭细胞数明显增多，DMS组则显著减少（均P〈0．01）。对照组、PMA组和DMS组的FAKmRNA相对表达水平分别为0．42±0．04、0．82±0．06和0．23±0．02，ICAM．1mRNA的相对表达水平分别为0．49±0．06、0．74±0．05和0．26±0．03，VCAM-1mRNA的相对表达水平分别为0．69±0．04、0．89±0．09和0．37±0．04，与对照组比较，差异均有统计学意义（均P〈0．01）。对照组、PMA组和DMS组的FAK蛋白相对表达水平分别为0．34±0．04、0．52±0．06和0．20±0．03，p-FAK蛋白相对表达水平分别为0．37±0．05、0．51±0．06和0．09±0．02，与对照组比较，差异均有统计学意义（均P〈0．01）。sICAM-1和sVCAM-1浓度随着SphKl活性的增强而升高，随着SphKl活性的抑制而降低，与对照组比较，差异均有统计学意义（均P〈0．01）。结论SphK1可能通过激活FAK通路上调ICAM-1和VCAM-1的表达，从而促进LoVo细胞的增殖、迁移和侵袭。
Objective To investigate the effects of sphingosine kinase 1 （SphK1） on the proliferation, migration and invasion of human colon cancer LoVo cells, and to explore the related mechanisms. Methods Human colon cancer LoVo ceils were divided into three groups： phorbol 12- myristate 13-acetate （PMA） was used to induce the activation of SphK1 in the PMA group, N, N- dimethylsphingosine （DMS） used to suppress the activity of SphK1 in DMS group, and the ceils treated with equal amount of 0.9 % NaC1 instead of drugs served as the control group. The activity of SphKl was assayed by autoradiography, the cell proliferation was assessed by MTY assay, cell migration and invasion were examined by Boyden chamber assay, concentrations of sICAM-1 and sVCAM-1 were assayed by ELISA, and RT-PCR and Western blot were used to evaluate the mRNA and protein expression in the cells. Results Theactivity of SphK1 was efficiently induced by PMA and significantly suppressed by DMS. PMA induced cell proliferation in a time- and dose-dependent manner. On the contrast, DMS suppressed cell proliferation in a time- and dose-dependent manner. After treating with PMA, the number of migrating and invasing cells were increased to 143.36 ± 8.73 and 118.46 ± 6.25, significantly higher than those of the control group （75.48 ±6.12 and 64.19± 5.36）. After treating with DMS, the number of migrating and invasing cells were decreased to 38.57 ± 3.24 and 32.48 ± 4.27, significantly lower than those of the control group （P 〈 0.01 ）. The relative expression levels of FAK, ICAM-1 and VCAM-1 mRNA in the PMA group were 0.82 ± 0.06, 0. 74 ± 0.05 and 0.89± 0.09, and those in the DMS group were 0.23 ± 0.02, 0.26 ± 0. 03 and 0.37± 0. 04, with significant differences between the PMA, DMS and control groups （P 〈 0. 01）. Compared with the control group, the relative expression levels of FAK and p-FAK proteins in the PMA group （0.52 ±0.06 and 0.51 ±0.06） were significantly elevated, and those of the DMS group （0.20 ± 0.03 and 0. 09 ± 0.02） were significantly decreased. In addition, the concentrations of sICAM-1 and sVCAM-1 were significantly elevated with the activation of SphK1. On the contrary, those of the DMS group were significantly reduced with the suppression of SphK1 （ Both P 〈 0.01 ）. Conclusions SphK1 may enhance the proliferation, migration and invasion of colon cancer LoVo cells through activating FAK pathway and up-regulating the expression of ICAM-1 and VCAM-1.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2013年第5期331-336,共6页
Chinese Journal of Oncology
基金
国家自然科学基金（30760275）
广西卫生厅基金（桂卫重2010016）
关键词
结肠肿瘤
鞘氨醇激酶1
黏着斑激酶
细胞黏附分子
细胞增殖
细胞迁移
细胞侵袭
Colonic neoplasms
Sphingosine kinase 1
Focal adhesion kinase
Cell adhesion molecule
Cell proliferation
Cell migration
Cell invasion
