Metformin Inhibits Proliferation And Cytotoxicity,Induces Apoptosis Via The AMPK Pathway In CD19-chimericantigen Receptor (CAR) Modified T Cells

BackgroundMalignant tumor has become one of the most important diseases that endanger human life and health.The incidence and mortality of malignant tumors are increasing year by year in China.However,traditional methods such as surgery,radiotherapy and chemotherapy have their disadvantages.The treatment of them will have a negative impact on the patient's immunity.It is lack of effective treatment for recurrent and refractory cancers.Therefore,it is necessary to find a new and effective method to cure malignant tumor.In recent years,more and more scientists and medical workers pay more attention to tumor cell immunotherapy.Tumor cell immunotherapy is adoptive cell therapy that can reintroduce autologous cell back into patients body.The immune cells in peripheral blood of patients with cancer are obtained by biotechnology and then made by modification.At last,cells will reintroduce into patients.It will enable the body to regain the ability to kill tumor cells.The greatest advantage of immunotherapy for cancer cells,which avoids rejection of autologous cells,is its safety and effectiveness.In view of this,tumor cell immunotherapy has made up for the disadvantages of surgery,chemotherapy and radiotherapy.At the same time,it lays the foundation for the radical cure of malignant tumor.Chimeric antigen receptor(CAR)T cells,which can be transferred into the body so as to realize the specific killing function of autologous cells to tumor cells,are based on gene modification of T cells in the peripheral blood of patients.Therefore,CAR-T cell immunotherapy has become the focus of attention in the field of biotherapy.It brings hope to treat patients with relapsed and refractory tumors.Based on the specificity of CD 19 expression on the surface of B lymphocytes,CD 19 CAR-T cell therapy has enabled many patients to achieve complete remission,and even cure.However,some B lymphocytes are not sensitive to the treatment of CD 19 CAR-T cells,how to improve the therapeutic effect of CD 19 CAR-T cells has become the focus of many experts and scholars.Whether in the endocrine system or in the cardiovascular system,glucose metabolism has become an important link in the development of the disease.At present,some studies have shown that glucose metabolism plays an important role in the proliferation and apoptosis of tumor cells.But glucose metabolism not only affects the proliferation and apoptosis of tumor cells,but also plays an important role in the function of immune cells.Metformin,as a first-line drug for type ? diabetes,has a prominent advantage in regulating glucose metabolism and improving insulin resistance.Studies have shown that metformin can inhibit the glucose metabolism of tumor cells.Thereby,metformin can inhibite the growth of tumor cells and then play a preventive and therapeutic purposes.ObjectiveThe aim of our study was to probe whether metformin could influence the proliferation,apoptosis and cytotoxicity of CD 19 CAR-T cells,and then research occurrence mechanism.Methods1.PGK-EGFP-CD19 CAR lentivirus plasmid construction was used molecular biological method.Liposome transfection method was applyed for virus packaging.Human peripheral blood T lymphocytes were transfected with lentivirus.Then,lentiviral transfection ratio was detected by flow cytometry.2.The effect of metformin on CD 19 CAR-T cell proliferation was evaluated by CCK-8 cell proliferation assay and cell counting plate counting method.3.The effect of metformin on apoptosis of CD19 CAR-T cells was assessed by flow cytometry.4.CD19 CAR-T cells were co-cultured with tumor cells according to the specific target ratio(E:T),and the effect of metformin on the cytotoxicity of CD 19 CAR-T cells was evaluated by flow cytometry.ELISA assay was used to detect the release of cytokines.5.The effect of metformin on the proliferation,apoptosis and cytotoxicity of CD19 CAR-T cells,which was mediated by AMPK-mTOR-HIF1 pathway,was confirmed by Western blot analysis.Results1.Metformin inhibits CD19 CAR-T cell proliferation and induces apoptosis.2.Cytotoxicityof CD 19 CAR-T cells was inhibited by metformin.3.Metformin promotes AMPK phosphorylation and suppresses mTOR and HIF1a expression in CD 19 CAR-T cells.4.AMPK pathway is involved in metformin-regulated CD 19 CAR-T cella poptosis and cytotoxicity.ConclusionMetformin effects the proliferation,apoptosis and cytotoxicity of CD 19 CAR-T cells via AMPK-mTOR-HIF1 pathway.