Molecular Mechanism Of Metformin And Metabolomics Of Zoledronic Acid In Inhibiting Osteosarcoma

Section Ⅰ Molecular Mechanism of Metformin in Osteosarcoma Background and PurposeOsteosarcoma is the most common primary malignant bone tumor.Although the treatment methods such as surgery and chemotherapy have improved significantly in the past 30 years,the overall survival rate of the disease has not been significantly improved.Metformin is one of the most commonly used drugs for the treatment of type 2 diabetes.Previous literature reported that it has anticancer effects in a variety of tumors,but its exact mechanism still remains unclear.MethodsIn this study,we firstly explored the effect of metformin on the proliferation of osteosarcoma cells by CCK-8,plate cloning experiment and flow cytometry.Then we explored the effect of metformin on apoptosis of osteosarcoma cells by flow cytometry,JC-1 staining of mitochondrial membrane and WB assay.We performed GFP-LC3 lentivirus infection on osteosarcoma cells and observed whether metformin can induce osteosarcoma cell autophagy by transmission electron microscopy and WB experiment.Subsequently,we analyzed potential downstream signaling pathways by immunofluorescence,flow cytometry,WB assay and other experimental techniques.Finally,the effects of metformin on osteosarcoma were investigated at the level of animal experiments by constructing tumor-bearing models of tibial plateau in nude mice.ResultsWe demonstrated that metformin could inhibit the proliferation of osteosarcoma cells by inducing cell cycle G2/M phase arrest and promote apoptosis and autophagy in osteosarcoma cells.By further analysis,we found that inhibition of metformin-induced autophagy was related to promote osteosarcoma apoptosis.Metformin caused osteosarcoma mitochondrial membrane potential changes,resulting in excessive generation of ROS,which in turn activated the downstream JNK/c-Jun signaling pathway,thereby inducing osteosarcoma cell apoptosis and autophagy.Finally,at animal level,we demonstrated that a safe dose of metformin could significantly inhibit osteosarcoma proliferation.ConclusionThrough this study,we found that metformin could induce osteosarcoma cell cycle arrest,apoptosis and autophagy through ROS-dependent JNK / c-Jun signaling in vitro and in vivo.Our experiments further proved that the AMPK signaling pathway was not the only signal pathway of anti-tumor effect of metformin,furthermore,metformin’s molecular mechanism of anti-tumor in this study laid the foundation for finding potential therapeutic targets of osteosarcoma.Section Ⅱ Metabolomics of Zoledronic Acid in Osteosarcoma Background and PurposeZoledronic acid is the most representative member of bisphosphonate family.In recent years,more and more evidence shows that it has direct anti-proliferative and pro-apoptotic effects on various tumors.In this study,the mechanism of zoledronic acid in inhibiting the growth of bone tumors was investigated through the use of metabolomics techniques.MethodsIn this study,we first explored the effect of zoledronic acid on the proliferation of osteosarcoma at different concentrations and time by CCK-8 assay and flow cytometric assay to determine the most suitable conditions.Afterwards,liquid chromatography-mass spectrometry was used to analyze the differential changes of intracellular metabolites of osteosarcoma cells treated with zoledronic acid,followed by cluster analysis of metabolites with significant changes and KEGG pathway enrichment analysis.ResultsFirstly,the results of CCK-8 showed that zoledronic acid had inhibitory effects on the proliferation and growth of osteosarcoma cells,and this effect was time and concentration dependent.Flow cytometry showed that the drug can induce G1 phase arrest of osteosarcoma.Subsequently,15 significant differential metabolites and 8 differential metabolites were found in the positive ion mode by liquid chromatography mass spectrometry,while 21 significant differential metabolites and 11 differential metabolites were found in the negative ion mode.The above metabolites are mainly involved in the nucleic acid metabolism,amino acid metabolism and TCA cycle and other important biological processes.Finally,the clustering analysis and KEGG pathway enrichment analysis of the significant difference metabolites revealed that the metabolites involve various important metabolic pathways such as central carbon metabolism in cancer,pyrimidine metabolism,and ABC transporters.ConclusionIn this study,we first used liquid-phase mass spectrometry to analyze the metabolomics in osteosarcoma treated with zoledronic acid with the aim to further explore the mechanism of zoledronic acid in cancer and to find potential therapeutic targets for osteosarcoma.