Identification Of Compounds Targeting MMP-2 And MMP-9 From Hydroxyproline Peptidomimetics Derivatives And The Activity Of LY52 To Inhibit Tumor Invasion And Metastasis

Backgrouns;Tumor invasion and metastasis consists of a series of sequential steps resulting in the formation of metastatic foci distant from the primary tumor;1)release from the primary tumor and invasion of the surrounding basement membrane(BM); 2)invasion and penetration of the BM of vascular(or lymphatic)endothelium at the primary site; 3)extravasion,invasion, and penetration of the BM at the metastatic site,and 4)growth at apparently preferred sites. Proteolytic cleavage of BM by MMP-2 and-9 is a key step in the cascading nature of the process,as well as a growing number of key regulatory genes such as cytokines,growth factors,cell surface receptors and adhesion molecules.The enzymes are expressed at various stages of cancer development but are not expressed or,if so,very weakly in normal cells. Therefore,regulation of MMP-2 and-9 is crucial in blocking tumor invasion and metastasis. Three-dimensional structure analysis showed that the S'₁ pocket in human gelatinase is deeper than that of MMP-3.This has provided helpful clues when using structure-based design strategies selectively to block the proteolytic activity of MMP-2 and-9.Previously,Xu Wenfang in the School of Pharcaceutical Sciences of Shandong University reported some series of hydroxyproline peptidomimetics derivatives,which designed based on the analogue reference drug CGS27023A and bestatin,to fit the deeper S'₁ pocket in gelatinase.The compounds included caffeoyl pyrrolidine and galloyl pyrrolidine derivatives; acetamide peptidomimetics derivatives; and pyrrolidine derivatives.Theoretically,the compounds might selectively inhibit the maturity of MMP-2,-9.Herein,we identified the compounds targeting MMP-2 and MMP-9 from hydroxyproline peptidomimetics derivatives and valued the effect of LY52,one of the caffeoyl pyrrolidine derivatives,in inhibition 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.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 supernatants of SKOV3 cell culture.The second part of experiments,we focused on the compound LY52 in inhibition invasion and metastasis via suppressing activity of MMP-2 and MMP-9.Human ovarian cancer cell line SKOV3 and breast cancer cell line MDA-MB-231 were employed and the cell growth inhibition was checked.We examined the adhesion of carcinoma cell to basement membrane using cell adhesion assay and the expression of CD44 on cell surface by flow cytometry analysis.In the experiment of SDS-PAGE gelatin zymography,the activitie of MMP-2 and-9 was measured in the supernatants of the cultured SKOV3.We further examined the activation of pro-MMP-2 to active-MMP-2 in MDA-MB-231 cells which expresses mesenchymal intermediate filament protein vimentin(VIM+).The expression MMP-2 and MMP-9 was valued using Western blotting and immunocytochemical staining.We used 24-well transwell chambers to evaluate the motility and invasive ability of SKOV3 and MDA-MB-231 in vitro.And then the experiments of pulmonary metastatic model of Lewis lung carcinoma and pulmonary metastatic model of B16-F10 melanoma were carried out in C57BL/6 mice.Antiangiogenic assays,the expressions of VEGF,TGF-αand bFGF were measured using Western blotting and immunohistochemical staining.The MVD was valued using CD34 expression.We also observed the decrease of angiogenesis in the chicken chorioallantoic membrane(CAM)after exposure to LY52.Results;Hydroxyproline peptidomimetics derivatives weakly inhibited the growth of SKOV3 cells in the low concentrations(0.1,1,10 and 100μg/ml)for up to 120 h treatment although the significant anti-proliferative effect was observed in the high concentrations of 500 and 1000μg/ml for longer incubation periods.The results of gelatinase assay showed that most of the compounds tended to withstand the degradation of succinylated gelatin and demonstrated the inhibition effect on the activity of MMP-2 and MMP-9.Caffeoyl pyrrolidine derivatives LY52,No21 and No26,11i,11r,Pro-B,9+HNE62,16A,No4,DBC,5+Ala,11a and 11u,the acetamide peptidomimetics derivatives,and A18,A11,A13 and A2 were identified as the inhibitors of MMP-2 and MMP-9 using SDS-PAGE gelatin zymography assay in SKOV3 cells.However,compounds A3 and A5,the pyrrolidine derivatives,were found to increase the activity of MMP-2 and MMP-9 in degradation of succinylated gelatin.We focused on the compound LY52 in inhibition invasion and metastasis via suppressing activity of MMP-2 and MMP-9.LYS2 weakly inhibited the growth of SKOV3 and MDA-MB-231 cells.LY52 tended to withstand the degradation of succinylated gelatin in the presence of p-aminophenylmercuric acetate in vitro.The inhibition rates of substrate hydrolysis by gelatinase increased with increasing concentrations of LY52.The IC₅₀was around 11.9 ng/ml.The expressions of MMP-2 and-9 activities in the supernatants of the cultured SKOV3 cells were reduced in a dose-dependent manner by treatment with LY52. LY52 inhibited the activity of active-MMP-2 via blocking the stimulation of pro-MMP-2 using CoA in MDA-MB-231 cells.The result of blocking active-MMP-2 activity was confirmed by Western blotting and immunocytochemical staining.However,LY52 weakly inhibitied the expression of MMP-2 and MMP-9 observed in SKOV3 cells.Moreover,the adhesion of SKOV3 and MDA-MB-231 cells to FN,LN and Matrigel was significantly prevented by pre-treatment with LY52 although there was no significantly change in the expression of CD44 in SKOV3 and MDA-MB-231 cells.We then examined the activity of invasion and migration of cancer cells after treatment with LY52.The invasive potential of SKOV3 and MDA-MB-231 cells was significantly diminished in a dose-dependant manner by 12 h pre-treatment of LY52 observed in 24-well transwell chambers.The activity of anti-invasion and metastasis of LY52 were confirmed using the metastatic models of C57BL/6 mice transplanted with Lewis lung carcinoma(LLC)cells and B16-F10 melanoma cells.LY52 significantly reduced the pulmonary metastatic nodules in the surface of lung originated from the right footpad of mice after 21 days treatment.In the assay of pulmonary metastatic model of B16-F10 melanoma,mice were intravenously injected with B16-F10 cells into the tail vein.The quantity of metastases was significantly decreased after 21 days treatment with LY52 at dosages of 50 and 100 mg/kg.In the assay of tumor angiogenesis,the expression of TGF-αand b-FGF was reduced in culture cells after 12 h incubation.In vivo study,VEGF and MVD was checked in the cancer tissue of SKOV3 and MDA-MB-231 xenografted in nude mice.All the assays were performed using the tissues which removed from nude mice after treatment of LY52.The expression of VEGF and MVD was inhibited. The decrease of angiogenesis was also observed in the chicken chorioallantoic membrane (CAM)after 72 h exposure to LY52.Conclusion;Caffeoyl pyrrolidine derivative LY52,No21 and No26,11i,11r,Pro-B, 9+HNE62,16A,No4,DBC,5+Ala,11a and 11u,the acetamide peptidomimeties derivatives, and A18,A11,A13 and A2 were identified as the inhibitors of MMP-2 and MMP-9 using SDS-PAGE gelatin zymography assay in SKOV3 cells.Compounds A3 and AS,the pyrrolidine derivatives,were found to increase the activity of MMP-2 and MMP-9 in degradation of succinylated gelatin.The LY52 might inhibit the activity of MMP-2 via blocking active-MMP-2.As the results,the inhibition of invasion and metastasis of carcinoma cells was observed in vitro and in vivo.Moreover,the adhesion of carcinoma cells to BM proteins was inhibited.The angiogenesis of tumour was prevented.