Establishment of osteosarcoma cell models：effects of NS-398 on invasive ability of MG-63 cells under hypoxic condition / 中国组织工程研究

ABSTRACT

BACKGROUND:In anoxic environment, invasive ability of tumor cels significantly increased. The increase in above invasive ability is possibly associated with the changes in urokinase or matrix metaloproteinases. Studies on hypoxia and tumor therapy especialy the effect on chemotherapy have made great progress, and found that hypoxia-inducible factor 1 is a key nuclear factor mediating celular hypoxia response, and also an important factor for regulating cel apoptosis during hypoxia. OBJECTIVE:To observe the effects of cyclooxygenase-2 inhibitor NS-398 on invasive ability of MG-63 cels under hypoxic condition in cel models of hypoxia osteosarcoma. METHODS: Chemical hypoxia inducer cobalt chloride was used to establish chemical hypoxia environment of concentration gradient of 0, 100, 200, 400 μmol/L. Under 200 μmol/L cobalt chloride, concentration gradient of 30, 60, 90 μmol/L of NS-398 were set. Osteosarcoma cels were determined after 48 hours of culture. Quantitative PCR was used to detect the expression of matrix metaloproteinases 9 mRNA and hypoxia-inducible factor 1α mRNA. Western blot assay was applied to measure hypoxia-inducible factor 1α expression. Flow cytometry was utilized to examine cel apoptosis in each group. RESULTS AND CONCLUSION:PCR and western blot assay results demonstrated that with increased concentration of cobalt chloride, matrix metaloproteinases 9 mRNA and hypoxia-inducible factor 1α mRNA and protein expression increased (P < 0.05), showing a concentration-dependent manner. Under cobalt chloride concentration of 200 μmol/L and hypoxia, with increased concentration of NS-398, matrix metaloproteinases 9 mRNA and hypoxia-inducible factor 1α mRNA and protein expression gradualy reduced, showing a concentration-dependent manner (P < 0.05). Flow cytometry results showed that under 400 μmol/L cobalt chloride concentration, cel apoptosis noticeably reduced. Under cobalt chloride concentration of 200 μmol/L and hypoxia, with increased concentration of NS-398, cel apoptosis increased (P < 0.05). Results confirm that anaerobic environment is an important factor for increased invasive ability of osteosarcoma. Under the anaerobic environment, NS-398 could effectively suppress the invasive ability of osteosarcoma and promote cel apoptosis.