| Osteosarcoma (OS) is the most common primary malignant bone tumors, especially in the teens. The incidence is about four over one million, and the prevalence rate of male is about1.4times that of female. At present, except for surgical resection, the main chemotherapeutic drugs are ADM, CDDP and MTX on clinical treatment for OS, with the development of neoadjuvant chemotherapy, the5years survival rate is rising to65-75%. However, the research and development of new drugs for OS are very few, moreover, if pulmonary metastasis or recurrence occurs, the prognosis will be much worse. So, research and development of high-efficiency new drugs or chemotherapy sensitization agents may be a potential field for OS therapy.Vorinostat (SAHA), a histone deacetylase inhibitor, is the first drug which was authorized by FDA to treat cutaneous T-cell lymphoma in2006. SAHA can play a role in arresting cell cycle, induction of cell differentiation and regulation.Cisplatin (CDDP) is a cell cycle non-specific drug, which has cytotoxicity, inhibits DNA replication of tumor cells, destroys the structure of cell membrane, and has a strong anticancer ability which has been widely applied in clinical, meanwhile, it is also the first-line chemotherapy drugs for treatment of OS. As the research and development of new drugs and the enlightmnet of sensitization for chemotherapeutics, and in consideration of action principle of this two drugs, we designed a treatment protocol of SAHA combiniation with CDDP for OS. Our study will focus on three aspects:the proliferation inhibition on OS cells, the effects of tumor suppression in vivo and the study of molecular mechanism after SAHA combine with CDDP. Our study will be greatly significant in revealing the pathogenesis of OS, and producing new ideas for clinical treatment of OS.Our study contains the following3parts:(1) calculating the concentrations of different cells, which is effective for combination index in SAHA combined with CDDP, and observing the proliferation inhibition, cell cycle, apoptosis and autophagy;(2) establishing the xenografts animal models of OS, and studying the effects of anti-cancer in vivo;(3) researching the molecular mechanism. Conclusively, the present study found that SAHA could markedly sensitize CDDP effects on inhibition of OS cells, and this combination not only decreased the dose of CDDP, but also reduced the side effects of CDDP.Part1Study on the SAHA combination with CDDP for inhibition of OS cells proliferationObjective:To observe the combination effects on proliferation inhibition, cell cycle, apoptosis and autophagy in OS cells.Methods:To observe the combination effects on proliferation inhibition by MTS trial, to observe cell cycle arrest and apoptosis by FCM trial, to observe the formation of autophagy by TEM trial. Results:Combining SAHA with CDDP has a remarkable effect on proliferation inhibition in HOS and U2OS cells compared with the control group. The difference has statistical significance (P<0.05). And the cell cycle was arrested in S phase, the ratio of cell apoptosis increased markedly. The proliferation inhibition on human normal hFOB cell was weaker than OS. And the autophagosome were observed at the mean time.Conclusions:SAHA can sensitize CDDP inhibition on HOS and U2OS cells proliferation, but has weaker effects on hFOB cells. The combination can induce autophagy.Part2Study on the SAHA combination with CDDP for the effects of tumor suppression in xenografts animal modelsObjective:To study the effects on tumor suppression in xenografts animal models.Methods:Naked mice were used to establish the xenografts animal model of OS by HOS cells. All animals were divided into four groups:control, SAHA, CDDP and combination group. After treatment though intraperitoneal injection, we observed the variation of tumor volume and body weight of each animal.Results:Combination group had remarkable effects on tumor suppression, the tumor volume was smaller than that of the control group. The difference has statistical significance (P<0.01). The body weight decreased at the beginning and increased at the later phase. Conclusions:The in vivo results suggest that combing SAHA with CDDP has a strong effect on tumor suppression.Part3Study on the molecular mechanism of the SAHA combination with CDDP for inhibition of OS cellsObjective:To study the molecular mechanism of induced apoptosis and autophagy of the combination.Methods:To detect the change of ROS by FCM, to detect the expression of cell cycle related genes by real-time PCR, to detect the expression of proteins which are related to Caspases signal pathway, ER stress and autophagy by Western Blot.Results:The effects on proliferation inhibition of combination could be partly counteracted by the application of NAC. The cell cycle related genes were changed as well, the expressions of p21and CDK2were increased, the expressions of cyclinA2and cyclinE were decreased. Western Blot results revealed that Caspases proteins expression were increased, but the ER stress related proteins, PERK and eIF2a, were changed unconspicuously. In addition, the ratio of LC3Bâ…¡/â… was elevated, meanwhile, after application of the autophagy inhibitor,3-MA and CQ, the expression of apoptosis related proteins were enhanced.Conclusions:ROS participated in the process of cell apoptosis, and the predominant manner was Caspase-dependent apoptosis signal pathway. Autophagy was induced, but has no effect on ER stress. According to the relationship between apoptosis related proteins and autophagy related proteins, we speculated that autophagy was a protective factor in the process of OS cells apoptosis induced by combination of SAHA and CDDP. |
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