莱菔硫烷抑制HepG-2细胞MMP-9的活性以及ERK1/2磷酸化

Sulforaphane inhibits matrix metalloproteinase-9 enzymatic activity and ERK1/2 phosphorylation in HepG-2 cells

目的：探讨莱菔硫烷（su lforaphane,SFN）对肝癌细胞系HepG-2细胞基质金属蛋白酶（m atrix m etallo-prote inase,MMP）-9的活性及对细胞外信号调节激酶（extracelluar signal-regu lated k inase,ERK）1/2磷酸化的影响。方法：体外培养HepG-2细胞,用SFN预处理后,四氮唑盐酶还原法检测细胞的增殖情况,以及LDH的漏出率;明胶酶谱实验和实时荧光定量PCR检测MMP-9的酶活性和mRNA表达水平;W estern b lot检测ERK1/2的磷酸化情况;最后采用定量实时PCR法检测SFN对HepG-2细胞表达Raf激酶抑制蛋白（Raf k inase inh ib itorprote in,RK IP）,Spred-1和Spred-2 mRNA的影响。结果：0,20,40,50μM SFN处理24~72h后,均能抑制HepG-2的生长,LHD漏出率实验显示SFN对细胞无明显的毒性;SFN能明显抑制MMP-9的活性和mRNA的表达水平,同时能抑制ERK1/2的磷酸化。另外,SFN能明显提高RK IP、Spred-1和Spred-2 mRNA的表达水平。结论：SFN对HepG-2细胞生长以及MMP-9的活性具有抑制作用,它可能通过抑制ERK1/2磷酸化,诱导RK IP,Spred-1和Spred-2表达而发挥抗肿瘤的作用。

Objective：This study was designated to investigate the effects of sulforaphane on the matrix metalloproteinase（MMP）-9 enzymatic activity and extracelluar signal-regulated kinase（ERK） 1/2 phosphorylation in a hepatocellular carcinoma cell line,HepG-2.Methods：Cultured HepG-2 cells Were incubated with sulforaphane,Microculture tetrazolium test（MTT assay）,Lactate dehydrogenase（LDH） release,Gelatin zymography,Western blot and quantitative real-time RT-PCR were employed to appraise the effect of sulforaphane on cell proliferation,cytotoxicity,metastatic potential,ERK 1/2 phosphorylation,and RKIP,Spred-1,Spred-2 mRNA expression in HepG-2 cells.Results：0,20,40,50μM of SFN inhibited cell proliferation,MMP-9 enzymatic activity,ERK 1/2 phosphorylation in a dose-dependent manner without exerting any cytotoxicity effect,In addition,an expressive increase in mRNA levels of RKIP,Spred-1,Spred-2 were observed.Conclusion：Sulforaphane treatment could inhibit cell proliferation and invasive potential of HepG-2 cells,and it could be through inhibition of ERK 1/2 cascade phosphorylation and through up-regulation of RKIP,Spred-1 and Spred-2.