Expression Of Autophagy In Osteosarcoma And The Effects Of Its Regulation On The Chemotherapeutic Sensitivity

OBJECTIVE To explore the expression of Beclinl and Microtubule-associated protein light chain 3Ⅱ(LC3Ⅱ) in osteosarcoma and investigate the role of autophagy in carcinogenesis of osteosarcoma.METHODS The expression of Beclinl and LC3Ⅱin group A (28 specimens of osteosarcoma) and group B (19 specimens of normal bone tissues) were detected by immunohistochemical staining.RESULTS The positive rates of Beclinl were 67.85% in group A and 94.73% in group B. Its expression was lower in osteosarcoma than normal bone tissues, with a significant difference between them (P<0.05). The positive rates of LC3Ⅱwere 64.29% in group A and 94.73% in group B. Its expression was lower in osteosarcoma than normal bone tissues, with a significant difference between them (P＜0.05).CONCLUSIONS The positive rates Beclinl and LC3Ⅱwere lower in osteosarcoma than normal bone tissues. This study demonstrates that autophagy may be related to carcinogenesis of osteosarcoma. OBJECTIVE To study the induction of autophagy and apoptosis by cisplatin(DDP) in osteosarcoma cell line MG63.METHODS The cell proliferation inhibition rate of MG63 cells with DDP in different concentrations and time was evaluated by using the MTT assay. The apoptosis ratio of MG63 cells with DDP in different time was detected by flow cytometry. The change of ultrastructure in MG63 cells with DDP was observed by transmission electron microscope. The expression of autophagy in MG63 cells treated with DDP in serious time was viewed by LC3II fluoresecne stain and flow cytometry to quantitate. The expression of Beclinl mRNA in MG63 cells treated with different concentrations of DDP was examined with RT-PCR.RESULTS (1) There was a positive correlation between proliferation inhibition rates of the cell and the concentration and intervention time of DDP. (2) There was a positive correlation between apoptosis ratios of the cell and the intervention time of DDP. (3) MG63 cells were observed under transmission electron microscope 12 hours after DDP administration, and found a large number of autophagic bodies in the cytoplasm and the accumulation of intracellular glycogen. (4) DDP can induce autophagy in MG63 cells and LC3II fluorescence intensity was the strongest in 12 hours. (5) RT-PCR showed that the expression of Beclinl mRNA in the cells treated with high-dose DDP were higher than that in the non-treated cells, and no significant difference in the expression of Beclinl mRNA was found between the cells treated with low-dose DDP and the non-treated cells.CONCLUSIONS DDP can effectively inhibit the proliferation of osteosarcoma cells MG63, and there are dose-and time-dependent. The mechanisms may include apoptosis and autophagy. Autophagy induced by DDP may be correlated with the time. OBJECTIVE To explore the relationship between autophagy and apoptosis in osteosarcoma cells MG63 induced by cisplatin.METHODS After down-regulation of autophagy in MG63 cells by an autophagy inhibitor,3-methyladenine (3-MA), in different times, and up-regulation of autophagy by an autophagy sensitizer, rapamincy, the cell proliferation inhibition rate of MG63 cells with DDP was evaluated by using the MTT assay. The apoptosis ratio of MG63 cells with DDP was detected by flow cytometry. The distribution of autophagy in MG63 cells was observed by LC3II fluorescence staining and confocal laser microscopy. The level of autophagy in MG63 cells was detected by MDC staining and flow cytometry. The expression of LC3II and Caspase-3 was investigated by western blot.RESULTS (1)DDP could effectively inhibit the proliferation of MG63 cells; the proliferation inhibition rate was enhanced by 3MA before DDP administration; the proliferation inhibition rate was decreased by 3 MA after DDP administration; and the proliferation inhibition rate was enhanced by Rapamycin before DDP administration. (2) 3MA own effected on apoptosis of MG63 cells weakly; the ratio of apoptosis was enhanced by 3MA before DDP administration, and declined delay. (3) LC3II punctate green fluorescence was scattered in the cytoplasm, and the green fluorescence intensity in 3MA+DDP group was weaker than the DDP group. Fluorescence intensity of DDP group and the other four groups were statistically different; 3MA+DDP group and the DDP+3MA group and the other three groups were statistically significant difference, but it was no statistical difference between the two groups. (4) DDP could increase the expression of LC3II and Caspase-3 protein in MG63 cells, while the intervention of 3MA reduced LC3II but increased the expression of Caspase-3.CONCLUSIONS The down-regulated autophagy could increase chemotherapeutic sensitivity of DDP to osteosarcoma and the up-regulated autophagy could decrease it. DDP can induce autophagy and apoptosis, and DDP plays a different role in different stages. In the early stage, autophagy could play the protective role. In the late stage, autophagy is as another death form to promote the cells death together with apoptosis.OBJECTIVE To investigate the effects of regulation of autophagy on the chemotherapeutic sensitivity in osteosarcoma in vivo.METHODS The animal models of osteosarcoma in nude mice were established. 3MA and DDP were injected into the animals. The growth of osteosarcoma was observed by its size. The pathologic manifestations were detected by HE staining.RESULTS There were significant differences among the control group, DDP group and 3MA+DDP group. DDP could be able to effectively inhibit tumor growth. And 3MA could enhance the sensitivity of DDP. HE staining showed that there was a clear area of necrosis in the transplanted tumor tissue in 3MA+DDP group.CONCLUSIONS The combination of 3MA and DDP could increase cells death than exposure to DDP alone in osteosarcoma.