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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。
Objective To design and synthesize N1-substituted caffeopyrrolidine derivative-LY52 as a target of MMP-2, and study its inhibitory effect on MMP-2 activity and its anti-invasion and metastasis potential. Methods MTT assay was used to detect the inhibitory effect of LY52 on the growth of tumor cells. Trypan blue staining was used to identify the role of LY52 in tumor cell growth. The proMMP-2 activity test method, direct determination of LY52 inhibition of the enzyme. MTT assay of cells and different types of basement membrane protein adhesion. The gelatin zymography assay was used to analyze the expression and activity of MMP-2 in Hep-2 culture supernatant. Immunocytochemistry and immunohistochemistry were used to detect the expression of MMP-2 in Hep-2 cells in vitro and in nude mice respectively. The ability of LY52 to penetrate Hep-2 cells through a recombinant basement membrane was studied in a Transwell chamber chamber method. RESULTS AND CONCLUSION LY52 had only a weak inhibitory effect on Hep-2 cells. When the compound is contacted with proMMP-2, it shows a direct inhibition of the enzyme activity and may block the proMMP-2 to active conversion. LY52 significantly inhibited the adhesion of Hep-2 cells to the basement membrane proteins LN, FN and Matrigel after being exposed to the cells for 1 h. The expression of MMP-2 in the supernatant of Hep-2 cells was significantly decreased after exposure for 24 h. Immunohistochemistry also found LY52 could inhibit the growth of Hep-2 cells in nude mice and decrease the expression of MMP-2. When exposed to LY52 for 24 h, the ability of Hep-2 cells to pass through the membrane filter coated with basement membrane protein decreased, indicating that LY52 has the ability to resist invasion and migration of cancer cells. LY52 may inhibit the invasion and metastasis of Hep-2 cells through specific binding and decrease of MMP-2 activity.