The Role Of HIF-1? In BCG-stimulated Macrophage Polarization And Antitumor Effect

Tumors are common clinical diseases and can be divided into benign tumors and malignant tumors.Malignant tumors have become one of the main diseases causing human death.The current treatment methods for malignant tumors include surgery,radiation therapy,chemotherapy,traditional Chinese medicine and biological therapy.BCG has obvious efficacy in the treatment of clinical bladder cancer and melanoma,but its related mechanism is not clear.The results showed that patients with bladder cancer had significantly increased white blood cells in their urine after 48 hours of BCG perfusion.The proportion of macrophages is not large,but the increase trend is the most obvious.It suggests that macrophages play an important role in the antitumor effect of BCG.Macrophage polarization and function are closely related to its metabolism,M1macrophages exhibit higher levels of aerobic glycolysis,and HIF-1?is an upstream transcription factor that regulates glycolysis.However,we do not know whether HIF-1?mediated glycolysis is related to BCG stimulation of macrophage polarization and anti-tumor effect.To study the role and related mechanisms of HIF-1?in BCG-stimulated macrophages to exert antitumor effects,we stimulated mouse bone marrow-derived macrophages?BMDM?with BCG in vitro,tested its phenotype and antitumor function.BMDM of LysMCre^-/+HIF-1?^Flox/Flox conditional knockout mice was used to investigate the role and mechanism of HIF-1?in BCG-stimulated macrophages to M1type and exert anti-tumor effect.Based on the above background,we conducted the following research:1.BCG promotes macrophage polarization to M1 and exerts antitumor effect.To clarify the role of BCG on macrophage polarization,after stimulated BMDM with BCG for 24 h,we detected macrophage phenotype by RT-qPCR,ELISA,FACS methods,and the effect of BMDM on the viability of MCA207 and B16-F10 tumor cells was detected by CCK-8 detection kit.The results show that BCG stimulated BMDM expresses high levels of TNF-?,IL-1?and CD86,and had killing effect on MCA207 and B16-F10 tumor cells.2.BCG promotes macrophage polarization and exerts antitumor effects by inducing aerobic glycolysis.In order to explore the related mechanism of BCG-stimulated BMDM to exert antitumor function,we used 2-DG to inhibit the glycolysis of BMDM for 4 hours,and then stimulated BMDM with BCG for 24 hours,then tested the phenotype and antitumor effect of BMDM and used the lactic acid detection kit to detect the lactic acid content in the cell culture supernatant.The results showed that 2-DG inhibited lactic acid secretion from BCG-stimulated BMDM and inhibited BMDM polarization to M1 and its antitumor effect.These results indicated that BCG induced BMDM to polarize to M1 and exert antitumor effects by inducing aerobic glycolysis.3.BCG stimulates macrophages to polarize to M1 type and exert antitumor effects through HIF-1?-mediated glycolysis.3.1 To clarify the role of HIF-1?in antitumor effects of macrophages,We inoculated MCA207 and B16-F10 tumor cells subcutaneously into group cKO(LysMCre^-/+HIF-1?^Flox/Floxlox/Flox mice)and group floxed(LysMCre^-/-HIF-1?^Flox/Flox mice),measured tumor size after 30 days.The results showed that the tumor size of the cKO group was significantly larger than that of the floxed group.It showed that HIF-1?was indispensable in the antitumor effect of macrophages.3.2 To clarify the role of HIF-1?in the antitumor effect of BCG-stimulated macrophages,we stimulated BMDM of LysMCre^-/+HIF-1?^Flox/Flox mice and LysMCre^-/-HIF-1?^Flox/Flox mice with BCG for 24 h,and detected BMDM phenotype and antitumor effects.The results showed that the levels of TNF-?,IL-1?and CD86in BMDM of BCG-stimulated cKO mice were significantly lower than those of BCG-stimulated floxed mice,and the killing effect of BMDM on tumor cells was also suppressed.This indicated that HIF-1?promoted BCG-stimulated BMDM to M1 type polarization and exerted antitumor effect.3.3 In order to investigate whether the role of HIF-1?in the antitumor effect of BCG-stimulated macrophages is achieved through the mediation of macrophage glycolysis,we used 2-DG to inhibit glycolysis of HIF-1?-deficient BMDM for 4 h and then stimulated HIF-1?-deficient BMDM with BCG for 24 h,we tested the phenotype and antitumor effect of BMDM and lactic acid content in cell culture supernatants.The results showed that the effect was no difference in the phenotype and antitumor function of BMDM in cKO and floxed mice stimulated by BCG and2-DG.It showed that BCG stimulated BMDM polarization to M1 type and exerted anti-tumor effects through HIF-1?-mediated glycolysis.According to the above experiment,we found that BCG could stimulate BMDM to polarize to M1 type and kill tumor cells,and this effect was regulated through HIF-1?-mediated glycolysis.This study may provide an idea for the role of BCG in the prevention and treatment of related diseases.