Diterpenoids Combination With COX-2 On Human Of Osteosarcoma MG-63 Cells In Vitro

Osteosarcoma is the most common primary malignant tumor is more common in children and adolescents, depending on the affected elements There are three main types, osteoblasts, chondrocytes and fibroblasts. The main method of treatment of osteosarcoma is surgery and adjuvant chemotherapy, although with advances in adjuvant therapy, a substantial increase in the survival rate of patients, however, appear more metastasis, drug resistance and side effects of chemotherapy can result in poor prognosis, and therefore urgent need to develop a new alternative is very necessary. In this study, first with kaurane diterpenoids Oridonin joint COX-2 Inhibitor Celecoxib intervention osteosarcoma MG-63 to observe the growth of tumor cells, and eriocalyxin B and it was cutting celecoxib this example further understanding of the mechanism of the two compounds osteosarcoma.Chapter I:Experimental study of oridonin in combination with Cox-2 inhibitors inhibit human osteosarcoma MG-63 cells in vitroObjective: To explore a diterpenoid oridonin combined with COX-2 inhibitor celecoxib, on proliferative inhibition and apoptosis of human osteosarcoma cell MG-63 and Probable Mechanism in vitro.Methods: The proliferation inhibition rates of MG-63 cells after treatment with different concentrations of oridonin and celecoxib alone and their combination for different times were detected by MTT assay. The apoptotic rates of MG-63 cells after treatment with oridonin and celecoxib alone and their combination were detected by flow cytometry,and the expression levels of COX-2 and apoptosis regulatory factors (Bcl-2, Survivin and Bax) mRNAs and proteins were analyzed by real-time PCR and Western blotting, respectively.Results: The proliferation inhibition rate of MG-63 cells in alone and combination of oridonin and celecoxib group was higher than control group, in a dose-dependent manner, and the two drugs have a synergistic effect. The apoptotic rate of MG-63 cells after treatment with the combination of oridonin and celecoxib group was higher than the control group (without any treatment) and alone groups. The expression levels of COX-2、Bcl-2 and Survivin mRNAs and proteins in combination of oridonin and celecoxib group were lower than the control group (without any treatment) and alone groups, whereas the Bax was higher.Conclusion:Oridonin in combination with celecoxib synergistically inhibit MG-63 cells, the process that may be associated with the expression of COX-2 inhibition and apoptosis regulatory factors (Bcl-2, Survivin and Bax) expression.Chapter II:Eriocalyxin B induces apoptosis and autophagy through ERK1/2 Activation in MG-63 Human Osteosarcoma CellsObjective:To investigate a diterpenoid eriocalyxin B (Eriocalyxin B, EriB) on proliferative inhibition, apoptosis and autophagy of human osteosarcoma cell MG-63 and Probable Mechanism in vitro.Methods:The inhibitory rates of proliferation of MG-63 cells after treatment with different concentrations of EriB for different times were detected by MTT assay. The apoptotic rates of MG-63 cells after treatment with EriB (1.5μmol/L) were detected by flow cytometry. After treatment with EriB (1.5μmol/L)group or (U0126+EriB) group [MG-63 cells treated with U0126 (50μmol/L) for 30min and then treated with EriB (1.5μmol/L)] the expression levels of apoptosis regulatory factors Bcl-2 and Bax and autophagy regulatory factors Beclin-1 mRNA were detected by real-time PCR.The phosphorylation levels of ERK1/2 after treatment with EriB(1.5μmol/L) for different times and after treatment with EriB or(U0126+EriB)the expression levels of apoptosis regulatory factors survivin and autophagy regulatory factors microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) proteins were detected by Western blotting.Results:The proliferation inhibition rates of MG-63 cells in different concentrations of EriB for different times groups were increased as compared with control group (without any treatment) in a time and dose-dependent manner. The the apoptotic rate of MG-63 cells was higher than the control group after treatment with the EriB, the rate of apoptosis in a dose-dependent manner. The expression levels of Bcl-2 mRNA and survivin protein in the EriB group were significantly down-regulated as compared with the control group (without any treatment), whereas the expressions of Bax andBeclin-1 mRNA and LC3-Ⅱ protein were up-regulated;The ratio of p-ERK1/2 and ERK1/2 was highest at 6h after treatment with EriB; The expression levels of Bcl-2 mRNA and Survivin protein in (U0126+EriB) group were significantly up-regulated as compared with the EriB group, whereas the expressions of Bax andBeclin-1 mRNA and LC3-Ⅱ protein were down-regulated; the expressions of mRNAs and proteins in (U0126+EriB) group compared with U0126 group have Little changes.Conclusion:eriocalyxin B could inhibit proliferation and induce apoptosis and autophagy through ERK1/2 activation in human osteosarcoma MG-63 cells.Chapter Ⅲ:mavacoxib-induced apoptosis via the P38 signaling pathway in human osteosarcoma MG-63 cellsObjective:To investigate the effect of a long-acting COX-2 inhibitor mavacoxib on the growth of human osteosarcoma MG-63 cells and Possible mechanisms in vitro.Methods:The inhibitory rates of MG-63 treated with different concentrations of mavacoxib at different times were detected by MTT assay. MG-63 cells after treatment with mavacoxib:the proteins and mRNAs expression of Bcl-2, Survivin and Bax were evaluated by Western Blot and Real-time PCR; the proteins expression of P38 was evaluated by Western Blot. Results:The proliferation inhibition rate of MG-63 cells in mavacoxib group was higher than control group. The expression levels of Bcl-2 and Survivin proteins and mRNAs in mavacoxib group were lower than the control group (without any treatment),whereas the Bax was higher.The proteins expression of P38 in MG-63 cells after treatment with mavacoxib were increased.Conclusion:Mavacoxib promote apoptosis of human osteosarcoma MG-63 cells by pathway P38 activation.