Study On The Effect Of Inhibition Autophagy And Enhancement Of Endoplasmic Reticulumstress Increase Sensitivity Of Osteosarcoma Saos-2 Cells To Cannabinoid Receptor Agonist WIN55,212-2

WIN 55,212-2 is a cannabinoid receptor agonist that activates cannabinoid receptors and has been shown to have antitumor effects.WIN can inhibit tumor cell proliferation and induce apoptosis of many tumor cells.However,the role and mechanism of WIN in osteosarcoma remains unclear.Autophagy is a cell lysosomal degradation pathway that is critical in maintaining intracellular homeostasis and regulating cell survival and death.Inhibition of cell autophagy can reduce chemotherapy-induced tumor cell apoptosis.EndopIasmic reticulum stress(ERS)and autophagy is closely related.The combined effect of these two processes play a role in protecting cell survival or triggering cell death.Therefore,the treatment strategy of endoplasmic reticulum stress and autophagy provides a new idea for tumor therapy.In this study,we examined the effects of WIN55,212-2 on cell viability and apoptosis in osteosarcoma Saos-2 cell lines.At the same time,we further explored the role of WIN55,212-2-induced endoplasmic reticulum stress and autophagy in apoptosis.This study includes the following two parts:Part Ⅰ:The biological effects of WIN55,212-2 on osteosarcoma cells.Part Ⅱ:The role of autophagy and endoplasmic reticulum stress in the apoptosis of osteosarcoma cells induced by WIN55,212-2Part 1:The biological effects of WIN55,212-2 on osteosarcoma cellsObjective:to investigate the biological effects of WIN55,212-2 on osteosarcoma cellsMethods:SAOS-2 osteosarcoma cells were treated with WIN.MTT assay was used to detect the cell viability.Cells were double stained with Annexin V-FITC and PI which was examined by flow cytometry after.Mitochondrial membrane potential was detected through JC-1.Expression of apoptosis-related proteins(Cytochrome C,caspase-3 and PARP)were detected by western blot analysis.Results:MTT results showed that WIN significantly reduced SAOS-2 cell survival in a dose-dependent and time-dependent manner.Annexin V-FITC/PI double staining showed that WIN significantly increased the apoptosis rate of SAOS-2 cells.JC-1 staining showed that WIN significantly decreased the rate of mitochondrial membrane integrity in a dose-dependent manner.The results of western blot showed that the expression of cleaved Caspase-3,cleaved PARP and cytochrome C were up-regulated by WIN.Conclusion:WIN55,212-2 can induce Saos-2 apoptosis through mitochondrial apoptosis pathwayPart Ⅱ:The role of autophagy and endoplasmic reticulum stress in the apoptosis of osteosarcoma cells induced by WIN55,212-2Objective:To investigate the role of autophagy and endoplasmic reticulum stress in the apoptosis of osteosarcoma cells induced by WIN55,212-2METHODS:SAOS-2 osteosarcoma cells were treated with WIN alone,and/or endoplasmic reticulum stress agonists and inhibitors,and autophagic inhibitors.MTT assay was used to detect cell viability.The apoptotic rate was measured by cytometry after Annexin V-FITC/PI double staining.Apoptosis-related proteins(caspase-3 and PARP),endoplasmic reticulum stress-related proteins(GRP78,IRE,TRB3)and autophagy-related proteins(LC3,Beclin 1 and p62)were detected by Western blot.Results:After treatment with WIN,the expression of GRP78,IRE and TRB3 increased significantly with the increase of drug concentration.LC3-Ⅱ and Beclin 1 increased significantly with the increase of drug concentration,while the expression of p62 was decreased.Endoplasmic reticulum stress suppressor 4-PBA and TUDC could alleviate the cytotoxicity induced by WIN.In contrast,endoplasmic reticulum stress activators TG and TUNI increased the WIN-induced cytotoxicity.The inhibition of endoplasmic reticulum stress by 4-PBA reduced WIN-induced apoptosis,and TG-induced endoplasmic reticulum stress increased WIN-induced apoptosis.Combined with autophagy inhibitor 3-MA/CQ can further increase the apoptosis rate and the expression of cleaved PARP and cleaved caspase 3Conclusion:WIN55,212-2 can induce osteosarcoma cell apoptosis and induce endoplasmic reticulum stress and autophagy in osteosarcoma cells.Enhancing endoplasmic reticulum stress and inhibit autophagy can significantly enhance WIN55,212-2 induced Osteosarcoma cell apoptosis.Therefore,WIN55,212-2,combined with autophagy inhibitorsor or endoplasmic reticulum stress activator can provide a new strategy for osteosarcoma treatment.