N1-取代咖啡酰吡咯烷衍生物-LY52抑制MMP-2活性作用研究

 目的以MMP-2为靶点,设计合成了N1-取代咖啡酰吡咯烷衍生物-LY52,研究其对MMP-2活性抑制作用及抗侵袭转移能力。方法MTT法检测LY52对肿瘤细胞生长抑制作用;台盼蓝染色方法鉴别其作用性质。以proMMP-2酶活性实验方法,直接测定LY52对该酶的抑制作用。MTT法研究细胞与不同类型的基底膜蛋白黏附性。以明胶酶谱实验法,分析Hep-2培养上清液MMP-2的表达及活性。免疫细胞化学和免疫组织化学实验分别检测体外培养和接种裸鼠体内Hep-2细胞的MMP-2表达水平。以Transwellchamber小室法,研究LY52对Hep-2细胞穿过重组基底膜的能力。结果与结论LY52对Hep-2细胞仅具有较弱的生长抑制作用。当化合物与proMMP-2接触时,显示直接抑制酶活性,并可能阻滞proMMP-2向活性型转变。与细胞接触1h,LY52明显抑制Hep-2细胞与基底膜蛋白LN,FN及Matrigel的黏附性;接触24h,Hep-2细胞上清液中MMP-2表达水平显著下降;免疫组织化学实验也发现LY52对接种裸鼠体内的Hep-2细胞生长具有一定的抑制作用,同时降低MMP-2表达水平。当与LY52接触24h,Hep-2细胞穿过包被基底膜蛋白的polycarbonate滤膜能力下降,说明LY52具有抗侵袭和癌细胞游走的能力。LY52可能通过特异性结合并降低MMP-2活性,抑制Hep-2细胞的侵袭与转移能力。

Inhibition of a Caffeoyl Pyrrolidine Derivative on the Activity of Matrix Metalloproteinase-2

OBJECTIVE To investigate the effect of LY52,a caffeolpyrrolidine derivative,on expression of matrix metalloproteinase(MMP)-2,as well as in vitro invasion.METHODS MTT assay was used to value Hep-2 cell growth inhibition.The cellular cytotoxic effect of LY52 was measured using trypan blue exclusion.Gelatinase assay was employed to study the activity of MMP-2.MMP-2 expression in Hep-2 cells was analyzed by gelatin zymography.The experiment of immunocytochemistry and immunohistochemistry was used to check the expression of MMP-2 in vitro and in vivo.Anti-invasion and anti-metastasis ability of LY52 were evaluated with penetration of Hep-2 cells through Matrigel-coated membrane in transwell chamber.RESULTS LY52 showed a weak cytostatic effect on Hep-2 cell growth.In contrast,LY52 blocked MMP-2 activity by decreasing the degradation of succinylated gelatin.Attachment of Hep-2 cells to plastic wells pre-coated with fibronectin,laminin,or Matrigel was also significantly inhibited by 1 h LY52 treatment.Gelatin zymography showed that MMP-2 expressions in Hep-2 cells were reduced in the presence of LY52 for 24 h incubation.Inhibition of MMP-2 expression as well as tumor cell growth was also observed in tissues of tumor xenografts in nude mice treated with LY52(25 or 100 mg·kg^-1) orally.Furthermore,LY52 effectively suppressed Hep-2 cell invasion,and the suppression was evaluated by the numbers of cells penetrating polycarbonate filters pre-coated with Matrigel.CONCLUSION LY52 might suppress the invasion of Hep-2 cells via the inhibition of MMP-2 expression and/or proteolytic activity.