Effect Of COX-2 Inhibitor On Apoptosis Of Human Osteosarcoma Cell Under Hypoxic Condition

Background:Osteosarcoma is the most common bone cancer, derived from mesenchymal tissue, about 20% of primary bone malignancies, incidence rate 1-3 per million per year. Any age may be affected, but occurs in young people. No significant difference between race, genetic factors not closely related. The disease prognosis is poor, mortality and disability due to amputation were higher. Metastasise widley through the blood stream, lymphatic metastasis is one of the main reason of its poor prognosis.Low oxygen as an important characteristic of solid tumors and survival environment. This not only increase the tumor’s own aggressive And tumor resistance to chemotherapy and radiotherapy resistance increases.In recent years found that hypoxia inducible (Hypoxia-inducible factor-1, HIF-1) mediated cellular hypoxia responses is the most crucial nuclear transcription factor. Low oxygen is an important factor in regulating cell apoptosis, Expression of HIF-1a protein in sensitivity to chemotherapy and prognosis of osteosarcoma. When a solid tumor in a low oxygen environment. HIF-la expression and activity increase. Regulating many downstream genes. Currently the target gene of HIF-1 has been found in the blood vessels and generate relevant factors. Glucose uptake and Glycolysis enzymes. Factors associated with tumor invasion and metastasis and tumor proliferation and apoptosis-related proteins. Specifically related to VEGF, EPO, iNOS and glycolytic enzymes. So that the tumor cells adapt to hypoxia micro environment. Promote the regeneration of blood vessels and the strengthening of energy metabolism. Lead to changes in the biological characteristics of tumor cells. Because the ultimate effect of chemotherapy drugs is to induce tumor cell apoptosis, so in the hypoxic tumor cell apoptosis induced by chemotherapeutic drugs and mechanisms of action provides the basis and theoretical basis for clinical treatment.Cyclooxygenase (COX) is an important rate limiting enzyme in the production of endogenous prostaglandin (PG) by prostaglandin decomposition. COX expression was up-regulated in osteosarcoma tissues. COX-2 specific mechanism of carcinogenesis is still not clear, but the current study found that its overexpression increases its metabolite of prostaglandin E2 (PGE2) synthesis of tumor cell growth and proliferation. PGE2 also increases the vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) expression of angiogenic factors and rumor angiogenesis. COX-2 promotes tumor invasion and metastasis. Inhibiting cell apoptosis. Suppress the immune response to tumor development and other aspects were played an extremely important role. The overexpression and tumor with the COX-2 understanding of relationship. Anti-tumor effect of COX-2 inhibitors. May be related to decreased immune suppression of tumor mediated effects, blocking tumor cell cycle, to reduce tumor angiogenesis, inhibition of tumor cell proliferation. In addition, studies have shown that inhibitors of COX-2 inhibition of osteosarcoma cell proliferation by non-COX-2.According to the characteristics of HIF-1 alpha protein expression in osteosarcoma, this study simulated the hypoxia status of osteosarcoma cells in vivo by chemical hypoxia inducible (CoC12), to observe the effect of COX-2 inhibitor NS-398 on the apoptosis of osteosarcoma MG-63 cells under hypoxic condition and to explore its mechanism.Objective:1.To investigate the apoptosis of osteosarcoma MG-63 cells under hypoxic condition.2.To investigate the effects of COX-2 inhibitors on the cell cycle of MG-63 cells under hypoxia.3.To investigate the effect of COX-2 inhibitor on apoptosis of MG-63 cells under hypoxia and its possible mechanism.Method:Selection of chemical hypoxia inducer cobalt chloride (concentration of 200μmol/L) for the treatment of MG-63 cell of osteosarcoma, set up a chemical hypoxia environment, the MG-63 cells were cultured for 48 h to different concentrations of COX-2 inhibitor NS-398 (0μmol/L,25μmol/L,50μmol/L, and 100μmol/L) respectively. After modeling of MG-63 cells. A normal oxygen group, hypoxia model after adding different concentrations of NS-398 were known as the 0μmol/L group.25 μmol/L group,50μmol/L group and 100μmol/L group, the MG-63 cells were cultured to determine to 48 hours.Treatment MG-63 cell proliferation activity of different concentrations of NS-398 with methyl thiazolyl tetrazolium (MTT) assay, MG-63 cells were collected, and then centrifugation to culture medium, through the washing, centrifugation, adding ice pre cooled volume fraction of 70% ethanol fixed, again centrifuged to remove fixative, dark staining, filtering, after flow cytometric detection of NS-398 on MG-63 cell cycle, apoptosis and cell morphology. Western blot to determine the expression of hypoxia inducible factor 1 alpha(HIF-1a) and apoptosis protein Survivin.Result:1. Under the hypoxic environment, the apoptosis of osteosarcoma cells than in normoxic conditions significantly inhibited HIF-la and apoptosis inhibiting protein expression of Survivin was significantly increased.2. Under the hypoxic environment, after different concentrations of NS-398 treatment for 48h, G0/G1 phase cell cycle arrest in MG-63 cells, the proportion of MG-63 cells in G0/G1 phase increased, G2/M phase ratio decreased, the differences were statistically significant decreased(P<0.05).3. Under the hypoxic environment, different concentrations of NS-398 (25μmol/L, 50μmol/L and 100μmol/L) for different time (24h,48h) after in MG-63 cell viability decreased, exerted an inhibitory effect on the growth, and with increasing of concentration of NS-398 increased as a function of time and its inhibitory effect is more obvious. With the increase of NS-398 concentration, the apoptosis rate increased, decreased the expression of hypoxia inducible factor 1 alpha and apoptosis protein Survivin. Hypoxia is one of the important factors inhibit the apoptosis of MG-63 cells under hypoxia, NS-398 can effectively promote the apoptosis of MG-63 cells, and changes with the concentration gradient. Conclusion:Under the hypoxic environment, the apoptosis of MG-63 cells was significantly inhibited, and the expression of HIF-1α and Survivin were increased in the normal oxygen condition.Under the hypoxic environment, after treatment with different concentrations of NS-398 and MG-63 cells occurred in G0/G1 phase cell cycle arrest, with NS-398 concentration increased the ratio of cells in G0/G1 phase increased, reduce the s and G2/M phase cell percentage; with the increase of concentration of NS-398, increased the rate of apoptosis, expression of HIF-1 alpha and Survivin reduced. Hypoxia environment is one of the important factors that inhibit the increase of apoptosis of MG-63 cells, NS-398 can effectively promote the apoptosis of MG-63 cells in hypoxia environment, and change with the concentration gradient.