The Selective Inhibitory Effects And Mechanisms Of Coptisine On Osteosarcoma Cell Growth And Vasculogenic Mimicry

Aim:In this study, we air to investigate the effect of coptisine (COP) on cell proliferation and vasculogenic mimicry of human osteosarcoma cancer cell MG63, and to elucidate its molecular mechanisms.Methods:AlamarBlue assay was used to determine the effect of COP on the proliferation of human osteosarcoma cancer cell MG63. The effect of COP on cell apoptosis of MG63was observed by TUNEL assay. Mouse xenograft model was used to observe the effect of COP on the MG63xenografts in mice, and the side effects of COP in the mice were evaluated. The effect of COP on the cell cycle distribution of MG63was observed by PI assay, and Western blot assay was used to detect the expression of cell cycle related proteins and signal proteins. The effect of COP on the tube-like structures formation in human osteosarcoma cancer cell MG63and normal vascular endothelial cells HLMVEC in vitro were observed by tube formation assay. The effect of COP on the cell migration of MG63was observed by cell wound healing assay. The effect of COP on the cell adhesion and invasion of MG63cell were observed by cell attachment assay and transwell assay, respectively. The effect of COP on the expression of vasculogenic mimicry-related genes and proteins were assessed by both semi-quantitative RT-PCR assay and Western blot. The luminescence Assay was used to check the effect of COP on the promoter activity of VE-cadherin in tumor cells.Results:AlamarBlue results showed that COP could obviously inhibit MG63proliferation with the IC5012.99μM and9.38μM at48h and72h, respectively. Mice tumor xenograft model results indicated that COP could effectively inhibit MG63tumor growth in vivo. The flow cytometry assay and Western blot showed that COP could obviously induced MG63cell cycle arrest at G0/G1phase and markedly downregulated the expression of cell cycle related proteins CDK4and cyclin D1proteins. The signal pathway assay showed that COP significantly reduced STAT3phosphorylation. In addition, COP effectively inhibited human osteosarcoma cancer cell MG63cell adhesion, migration, invasion and vasculogenic mimicry in a dosage-dependent manner; however, it did not significantly affect the tube formation of normal endothelial cells HLMVEC. The semi-quantitative RT-PCR showed that COP markedly inhibited the expression of vasculogenic mimicry-related genes VE-cadherin and Integrin β3. Western blot result showed that COP could reduce VE-cadherin protein level. Luminescence result showed that COP could effectively diminish VE-cadherin promoter activity in a dose-dependent manner. Furthermore, COP has slight inhibition effect on human peripheral blood cell and human lung microvascular endothelial cell proliferation, and has little side effect on body weight and hematological parameters in mice, and it indicated that COP had very low toxicity.Conclusion:1. COP could effectively inhibit human osteosarcoma cell MG63cell proliferation and arrest the cell cycle at G0/G1phase via reduction of the cell cycle key proteins CDK4and cyclin D1and the phosphorylation of STAT3.2. COP effectively inhibited MG63cell attachment, migration, invasion and vasculogenic mimicry, which was closely related to the down-regulation of vasculogenic mimicry master gene VE-cadherin via the inhibition of VE-cadherin promoter activity.3. COP had little effect on normal cell proliferation and normal endothelial cells tube formation, and it had slight effects on mice body weight, organ index, and hematological parameters in an animal model, indicating that COP had minor toxic effects in vitro and in vivo, and COP may be a promising candidate of anti-osteosarcoma drugs.