Mechanism Of Hypoxic Microenvironment Induced Osteosarcoma Radioresistance

Part one Contribution of Hypoxia to Radioresistance in Human Osteosarcoma CellsObjective: To investigate whether hypoxic pretreatment contributes to the radioresistance in human osteosarcoma cells.Methods: Human osteosarcoma cells(MG-63) were pretreated under hypoxic condition(1% O2) and then irradiated by X-ray at different dosages,using no hypoxic pretreatment as the negative control. Cellular survival and apoptosis rates were examined under the microscope or measured by the MTT assay.Results: The radiation(6Gy) inhibited the proliferation of MG-63 cells and caused significant cellular apoptosis. However, the cellular sensitivity to irradiation was significantly reduced when the cells were pre-incubated in the hypoxic environment(1% O2), in which there were decreased apoptosis and increased cellular survival rate were observed.Conclusions: Hypoxic may play an important role in enhancing the radioresistance of osteosarcoma cells. Radiation(6Gy) inhibits the proliferation of MG-63 cells and caused cellular apoptosis. Hypoxic environment can be reduced apoptosis and increased cell survival.Part two The Mechanism of Hypoxia-induced Radioresistance in Human Osteosarcoma cellsObjective: To investigate the potential mechanism of hypoxia-induced radioresistance in human osteosarcoma cells.Methods: Autophagy was examined in the MG-63 cells under hypoxic pretreatment and after irradiation. Cells were first transfected with GFP tagged protein LC-1 whose expression level, distribution pattern andautophagic modification were observed underthe fluorescent microscope or determined by Western blotting. The mitochondrial ROS production was visualized by Mito SOX staining, and the functional impairment of mitochondria was assessed by the mitochondrial membrane potential assay.3-Methyladenine(3-MA) was used to inhibit autophagy to confirm its role in mediating radioresistance of the MG-63 cells.Results: The consequential events induced by hypoxic pretreatment in MG-63 cells: 1) increased autophagy; 2) reduced DNA damage; 3) decreased cellular ROS production; 4) reserved mitochondrial function during irradiation; 5) abolished cellular resistance to irradiation upon inhibition of autophagy.Conclusions: Hypoxic enhanced the tolerance to radiation damage of osteosarcoma cells by inducing autophagy which leaded radioresistance.Autophagy may mediate protective effects of hypoxic microenvironment of osteosarcoma cells withstand radiation damage.Part three Expression of HIF-1 is Associated with Prognosis in Patients with OsteosarcomaObjective: To determine whether the protein expression levels of HIF-1,LC3-II and p62 in human osteosarcoma tissues were correlated with the survival duration of each corresponding patient.Methods: HIF-1 level was evaluated by immunohistochemical staining in 89 patients with osteosarcoma. And we detected the expression levels of LC3-II and P62 in osteosarcoma specimens from patients who received surgery and radiotherapy only. The Kaplan-Meier method was used to analyze overall survival differences among the high, moderate or low expression levels of HIF-1, LC3-II and P62.Results: Low HIF-1 expression was detected in 20.2%(18/89) patients with osteosarcoma, moderate in 24.7%(22/89) and high expression in 55.1%(49/89). HIF-1 expression was significantly associated with the Enneking stage(P<0.01), presence of distant metastasis(P<0.01). And has nosignificant correlation with other clinical pathological features. Low LC3-Ⅱexpression was detected in 22.2%(8/36) patients with osteosarcoma,moderate in 25%(9/36) and high expression in 52.8%(19/36). Respectively,P62 expression was 52.8%(19/36), 25%(9/36), 22.2%(8/36). The expression of LC3-Ⅱ and P62 were significantly associated with the Enneking stage(p <0.01) and presence of distant metastasis(P<0.01). And has no significant correlation with other clinical pathological features.Conclusions: High HIF-1, LC3-Ⅱ expression and low P62 expression were significantly associated with shorter overall survival. The level of autophagy in the tissues of osteosarcoma patients is related to the prognosis of radiotherapy.