莱菔硫烷抑制PMA诱导宫颈癌细胞迁移与侵袭

Inhibitory effect of sulforaphane on PMA-induced migration and invasion of human cervical cancer cells

目的:观察莱菔硫烷(Sulforaphane,SFN)对宫颈癌细胞迁移和侵袭的抑制情况,并探讨其分子机制。方法:体外培养宫颈癌细胞系HeLa细胞,经100 nmol/L佛波脂PMA诱导24 h后,加入不同浓度的SFN继续孵育24 h。MTT法检测HeLa细胞的生长抑制情况;DCF染色法检测细胞内活性氧(Reactive oxygen species,ROS)的产生,细胞伤口愈合试验和小室侵袭实验分析SFN对PMA诱导HeLa细胞的迁移与侵袭情况。提取细胞总RNA,逆转录PCR检测基质金属蛋白酶-9(matrix met-alloproteinase-9,MMP-9)的表达,明胶酶谱实验检检测其酶活性。萤光素酶报告基因法检测SFN对核因子κB(Nuclear Fac-tor kappa B,NF-κB)活性的影响。结果:0～30μmol/L SFN对HeLa细胞的生长增殖无明显毒性作用。DCF染色显示PMA处理能使HeLa细胞ROS的含量增高5倍,而30μmol/L SFN处理能使ROS的产生降低44%;SFN也能使HeLa细胞迁移和侵袭率分别降低60%和75.5%。RT-PCR结果显示SFN能以剂量依赖性方式抑制PMA诱导的MMP-9表达,明胶酶谱实验显示MMP-9酶活性也明显降低。报告基因实验显示SFN能以剂量依赖性方式抑制NF-κB的活性。结论:SFN抑制NF-κB介导的MMP-9表达,从而影响宫颈癌细胞的迁移和侵袭。

Objective： To observe the inhibitory effect of sulforaphane （SFN） on PMA - induced migration and invasion of human cervical cancer cells, and explore the molecular mechanism. Methods： Cervical cancer HeLa cells were cultured in vitro, then they were in- duced by 100 nmol/L of PMA for 24 hours and co - incubated with different concentrations of SFN for 24 hours. The inhibitory effect of growth of HeLa cells was detected by methyl thlazolyl tetrazolium method; the production of reactive oxygen species （ROS） was measured by DCF staining. The inhibitory effect of SFN on PMA - induced migration and invasion of human cervical cancer cells was analyzed by wound healing assay and Matrigel invasion assay. The total RNA was extracted, and the expression of matrix metalloproteinase -9 was detected by reverse transcription - polymerase chain reaction （ RT - PCR）, the activity of metalloproteinase - 9 was analyzed by gelatin enzyme spec- trum. The effect of SFN on NF - KB was detected by luciferase reporter assay. Results： SFN （0 -30 μmol/L） had no significant toxic effect on growth and proliferation of HeLa cells. DCF staining showed that PMA increased the content of ROS in HeLa cells by 5 times, but SFN （30 μmol/L） decreased the content of ROS by 44% ; SFN decreased the migration rate and invasion rate of HeLa cells by 60% and 75.5%, respectively. RT - PCR results showed that SFN inhibited the expression of PMA - induced metalloproteinase - 9 by a dose - de- pendent manner, the results of gelatin enzyme spectrum showed that the activity of metalloproteinase-9 decreased significantly. The results of luciferase reporter assay showed that SFN inhibltedthe activity of NF - KB by a dose - dependent manner. Condusion： SFN can inhibit the expression of NF - KB mediated metalloproteinase - 9, and further affect migration and invasion of human cervical cancer cells.