Effect Of Multi-kinase C-MET Inhibitor SIM-89 On Tumor Cell Growth, Migration And Metastasis

Background:It has been found that HGF-dependent autocrine c-Met(HGFR)are activated in a wide variety of human primary and second malignancies, including breast carcinoma, glioblastoma, osteosarcoma and melanoma. On the other hand, the metastatic growth potential of tumors can be activated through non autocrine(paracrine) mechanism. The regulation of c-Met expression appears to be different in oncogene transformation from that of normal signal pathway. Overexpression of MET is a potential carcinogenic factor. Recent study has showed that normal osteoblasts can be transformed by elevated c-Met level and activation of c-Met signal pathway. C-Met dysregulation leads to lung cancer development through two critical pathways, which are overexpression and mutation. Overexpression of c-Met can be found both in non small cell lung cancer and small cell lung cancer. Narrow-spectrum kinase inhibitors SU11274, PHA665752, XL880, XL184 have shown potential inhibition effect on lung cancer, which will be further identified in clinical trials. SIM-89 is a small-molecule kinase inhibitor that targets members of the HGF receptor tyrosine kinase families.Object : To indentify the enzymogram which SIM-89 can inhibit effectively. To investigate the effect of SIM-89 on the vitality and migration in human NSCLC cell(A549, H460, H441, H1299) and murine melanoma cell B16F10. To observe the influence of SIM-89 on nude mice xenograft. To determine the mechanism of SIM-89 in tumor and to provide more evidence for further study in vivo and clinical trials in future.Methods:70 kinase enzymogram screening was proceeded for SIM-89 by Z-lyte technique. MOA analysis was completed on the inhibited kinase. Cell vitality was determined on 24 h, 48 h, 72 h after SIM-89 dosage through CCK8 method. Transwell system was used to observe the inhibition of cell migration. Difference of special gene expression between treatment samples and control samples was evaluated by Real-time PCR. Westernblot assay was used to compare the expression difference of c-Met and p-Met. HGF level in culture medium is determined by ELISA. A549, H460 cells were implanted subcutaneous and B16F10 cells were implanted via tail vein injection in nude mice. The growth curve and weight variation was recorded. Vessels in tumor were observed by immunofluorescence assay.Results: SIM-89 can inhibit 3 kinases including c-Met(IC50=297n M), AMPK, TRKA(IC50=150.2n M). SIM-89 has an ATP competive inhibition on c-Met. By Real-time PCR, SIM-89 has been found to inhibit STAT1, JAK1, c-Met gene expression in H460 cell. C-Met protein expression of A549 and B16F10 cell, Met-p Y1349 of B16F10 can be inhibited by SIM-89. Treated with SIM-89, HGF level of supernatant in culture fluid is significantly lower than control group. Vitality of lung cancer cell lines is inhibited dependent on time and concentration by SIM-89(coefficient correlation r=0.656, 0.448, 0.961, 0.82, 0.804, 0.821, P=0.000). Induced by HGF, migration of H460, H1299 cell is inhibited with transwell migration assay;Induced by FBS, B16F10, H1299 cell is inhibited with transwell migration assay.In vivo,H460 tumor growth of nude mice is inhibited by SIM-89,but the difference is not significant. SIM-89 can inhibit B16F10 pulmonary metastasis of nude mice significantly(P=0.000). Immunofluorescence assay has shown blood vessel decreased in subcutaneous tumors and pulmonary metastasis treated with SIM-89.Conclusion: SIM-89 has significant inhibitive effect on c-Met, TRKA kinases. SIM-89 can also inhibit STAT1, JAK1, MET gene expression of H460 cell and c-Met protein expression of A549 and B16F10 cell. In vitro, SIM-89 can inhibit proliferation and migration of lung cancer cell significantly. The autocrine of HGF in supernatant of cancer cell culture fluid is inhibited by SIM-89 significantly. In vivo, SIM-89 can inhibit H460 tumor growth and B16F10 pulmonary metastasis of nude mice. This promising observation about the effect of SIM-89 on NSCLC cells may be considered it as a good approach in search and development of new anti-cancer drug. Further study in vivo should be carried to explore the pharmacokinetics and toxcity of SIM-89 for its clinical application.