Study On The Effect Of Aerobic Glycolysis On Osteosarcoma Cell Death Induced By Proteasome Inhibitor Bortezomib

Ubiquitin proteasome system plays an important role in maintaining intracellular protein stability.Studies have shown that it is closely related to the occurrence and development of cancer.Targeting Ubiquitin proteasome system has become an important target for cancer treatment.Bortezomib is a proteasome inhibitor first used in clinical trials.Studies have shown that bortezomib can inhibit the progression of many tumors,but the study of its regulatory mechanism is not in-depth.Many evidences have shown that aerobic glycolysis of cancer cells is involved in the regulation of biological behavior of cancer cells and in the killing effect of various chemotherapeutic drugs on cancer cells.However,its role in bortezomib-induced cell death remains unclear.The aim of this study was to clarify the role of aerobic glycolysis in bortezomib-induced cell death and to provide a new strategy for clinical combination therapy.Methods(1)CCK8 was used to detect the effects of Bortezomib at different concentrations on the survival and proliferation of human osteosarcoma cells MG63,U2 OS,HOS and OS187.(2)Western blotting was used to detect the effect of Bortezomib at different concentrations on the expression of cleaved caspase-3 protein in MG63 and HOS cells.Kinase detection kit was used to detect the activity of Caspase-3 in cells.(3)The effects of Bortezomib at different concentrations on the aerobic glycolysis of MG63 and HOS cells were detected by glucose concentration and lactate production detection kit.(4)Western blotting was used to detect the expression of HK2 and GLUT1 in MG63 and HOS cells at different concentrations of Bortezomib.(5)The effect of HK2 si RNA inhibition on Bortezomib-induced aerobic glycolysis was observed.(6)HK2 si RNA and aerobic glycolysis inhibitors(2-DG and 3-BP)were used to inhibit cell aerobic glycolysis.CCK8 was used to detect the effect of combined application on cell survival.(7)The effect of inhibitor aerobic glycolysis on the expression of cleaved caspase-3induced by Bortezomib was detected by Western blotting.Results(1)Bortezomib at different concentrations could significantly reduce the survival and proliferation rates of MG63,U2 OS,HOS and OS187 cells.(2)Western blotting results showed that Bortezomib at different concentrations could significantly increase the expression of cleaved caspase-3 in MG63 and HOS cells,and increase the activity of Caspase-3 in cells.(3)The results of detection kit of glucose concentration and lactic acid production showed that Bortezomib at different concentrations could significantly increase glucose uptake and lactic acid survival in MG63 and HOS cells.(4)Western blotting results showed that Bortezomib at different concentrations increased the expression of HK2,an aerobic glycolysis-related protein,in MG63 and HOS cells,but had no significant effect on the expression of GLUT1.(5)Inhibition of HK2 expression could significantly reduce the increase of Bortezomib-induced aerobic glycolysis.(6)CCK8 results showed that inhibition of cell aerobic glycolysis could further increase the cell survival rate induced by Bortezomib.(7)Western blotting results showed that inhibition of cell aerobic glycolysis could further increase the expression of cleaved caspase-3 induced by Bortezomib.Conclusion:Bortezomib can induce the decrease of survival rate and proliferation rate of osteosarcoma cells and the increase of apoptosis.Bortezomib can induce the increase of aerobic glycolysis in osteosarcoma cells by up-regulating HK2 expression.Inhibition of aerobic glycolysis can significantly enhance the cytotoxicity of osteosarcoma cells induced by Bortezomib.Combination of aerobic glycolysis inhibition and Bortezomib can better inhibit the growth of osteosarcoma cells and provide a new strategy for clinical treatment of osteosarcoma.