Identification Of Compounds Targeting MMP-2 And MMP-9 From Galloyl Cyclic-imide Derivatives And The Activity Of 4i To Inhibit Tumor Invasion And Metastasis

Backgrounds:Tumor invasion and metastasis consist of a series of sequential steps resulting in the formation of metastatic foci distant from the primary tumor.Therefore, proteolytic cleavage of BM and ECM by the proteinases released by the tumor cells is a key step in the cascading nature of the process.The typeâ…£collagen,the main component of BM,can't be degraded by most of the proteinases but typeâ…£collagenase because of its unique structure.The typeâ…£collagen,which is also named gelatinase(matrix metalloproteinase-2,-9),belongs to the matrix metalloproteinase family.These enzymes mediate the stability of cells and have been proved to play key roles in the processes of tumor growth,invasion,metastasis and angiogenesis. Therefore,it is believed that the growth and metastasis can be controlled by gelatinase inhibitors which are expected to be useful for the treatment of cancer.Accordingly, research on inhibitors targeting at gelatinase(MMP-2,9)is a very promising strategy in the development of current anti-metastasis therapy of tumor.Based on the 3D structures of known gelatinase and binding modes of compounds in complex with MMPs,Xu Wenfang et al in School of Pharmaceutical Sciences of Shandong University reported the series of galloyl cyclic-imide derivatives with the long and stable structure,which is designed based on the analogue reference drug antineoplaston A10 and thalidomide,to fit the deeper S1' pocket in gelatinase. Theoretically,the compounds might selectively inhibit the maturity of MMP-2,-9. Herein,we identified the compounds targeting MMP-2 and MMP-9 from galloyl cyclic-imide derivatives and valued the effect of 4i,one of the galloyl cyclic-imide derivatives,in inhibition of invasion and metastasis via suppressing matrix metalloproteinase activity.Methods:In the first part of the experiments,we aimed to identify the compounds targeting MMP-2 and MMP-9 from galloyl cyclic-imide derivatives.Hydrolysis of succinylated gelatin by gelatinase was measured in the presence of compounds and inhibitory rate of compounds was valued.The cell growth inhibition of human ovarian cancer cell line SKOV3 was assayed using MTT method.We used SDS-PAGE gelatin zymography to evaluate the effects of compounds on inhibition of MMP-2 and -9 in the supematants of SKOV3 cell culture.The second part of experiments,we focused on the compound 4i in inhibition invasion and metastasis via suppressing activity of MMP-2 and MMP-9.Human ovarian cancer cell line SKOV3 was employed and the cell growth inhibition was checked by MTT assay and trypan blue staining.We examined the adhesion of carcinoma cell to basement membrane using cell adhesion assay.In the experiment of SDS-PAGE gelatin zymography,the activity of MMP-2 and -9 was measured in the supematants of the cultured SKOV3.We further examined the expression MMP-2 and MMP-9 using Western blotting and immunocytochemical staining.The level of MMP-2 mRNA and MMP-9 mRNA were valued by RT-PCR.We used 24-well transwell chambers to evaluate the motility and invasive ability of SKOV3 in vitro.And then the experiments of pulmonary metastatic model of murine hepatocellular carcinoma H₂₂ cells were carried out in Kunming mice and the artifical pulmonary metastatic model of human SKOV3 cells were used in nude mice.In the assay of angiogenesis,the expression of VEGF was measured using Western blotting.We also observed the decrease of angiogenesis in the chicken chorioallantoic membrane(CAM)exposed to 4i.Results:Galloyl cyclic-imide derivatives weakly inhibited the growth of SKOV3 cells although the significant anti-proliferative effect(48.54%)was observed in the high concentration(200μg/ml).Most of the compounds displayed the inhibitory effect on the activity of MMP-2 and MMP-9 using assay of gelatinase.Compounds 4i,4r,pro- 13, 9+HNEt6,4a,4q were identified as the inhibitors of MMP-2 and MMP-9 using SDS-PAGE gelatin zymography assay in SKOV3 cells.4i is the most promising inhibitor among these compounds.We focused on the effect of compound 4i on inhibition of MMP-2 and MMP-9,as well as invasion and metastasis in vitro and in vivo.The inhibition rates of substrate hydrolyzed by gelatinase were increased with increasing concentrations of 4i exposed for 30 min.The IC50 was around 394.71±1.58 ng/ml.The activity of MMP-2,-9 in the supematants of SKOV3 was reduced in a dose-dependent manner by treatment with 4i for 24h.The inhibition rates by 5,25,50,100,150μg/ml of 4i were 12.7,16.9,34.8, 63.9,and 82.53%,respectively,for MMP-9 and 8.3,14.8,25.8,26.9 and 35.8%, respectively,for MMP-2.The highest inhibitory rates of MMP-2 and MMP-9 obtained from the immunocytochemical staining were 78.77%and 75.21%,respectively.The down-regulated expression of MMP-9,pro-MMP-2,MMP-2 was also valued using the assay of Western blot(P＜0.05).Moreover,the low level of mRNA of MMP-9 and MMP-2 in SKOV3 was observed at the concentration of 150μg/ml.The invasive potential of SKOV3 was significantly diminished in a dose-dependant manner by 24 h treatment of 4i observed in 24-well transwell chambers.The activity of anti-invasion and metastasis of 4i was confirmed using the metastatic models of mice intravenously injected with H₂₂in Kunming mice and SKOV3 cells in nude mice into the tail vein.4i significantly reduced the pulmonary metastatic nodules in the surface of lung.It was decreased by 21.1%,35.3%in Kunming mice and by 46.5%in nude mice after many weeks oral administration with 4i at dosages of 25 and 100 mg/kg,respectively. The adhesion of SKOV3 cells to FN,LN and Matrigel was significantly prevented by pre-treatment with 4i.In addition,the low level of VEGF in SKOV3 cells was observed and the decrease of angiogenesis was measured in the chicken chorioallantoic membrane(CAM)exposed to 4i. Conclusion:Compounds 4i,4r,pro-13,9+HNEt6,4a,4q were identified as the inhibitors of MMP-2 and MMP-9 using SDS-PAGE gelatin zymography assay in SKOV3 cells.Compounds 4i might inhibit invasion and metastasis of tumor cells via decreasing the activity and expression of MMP-2,-9.4i might be a candidate compound for preventing tumor invasion and metastasis.