Berberine mediates tumor cell death by skewing tumor-associated immunosuppressive macrophages to inflammatory macrophages

Berberine is a plant-derived alkaloid with potent anti-cancer activities. Berberine may redirect the tumor-promoting immunosuppressive M2 macrophages, to tumoricidal activated M1 macrophages. But such an anti-tumor function remains to be demonstrated. Polarization of macrophages to an immunosuppressive phenotype within the tumor microenvironment promotes tumor growth and contributes to resistance to chemotherapy. We examined if berberine would target macrophage polarization to reinstate anti-tumor immune response. Using a B16F10 mouse melanoma model, we assessed berberine-induced re-polarization of immunosuppressive M2 macrophages to anti-tumor M1 macrophages and subsequent T-cell activation within the immunosuppressive tumor microenvironment. The B16F10 culture supernatant along with tumor antigen was used as tumor mimicking conditioned medium (CM). The bone marrow-derived macrophages were cultured in CM for 5 days. The CM-induced skewing of macrophages to M2-like phenotype was confirmed by flow cytometry and ELISA. The T-cells were co-cultured with macrophages to decipher the effect of berberine on T-cell differentiation. In vivo efficacy of berberine was analyzed using melanoma model of solid tumor. Berberine inhibited rIL-6-induced STAT-3 phosphorylation and IL-10 release from B16F10 cells. It enhanced tumor antigen-induced IL-1β, IL-12 and TNFα, but suppressed IL-6 and TGF-β release. Berberine significantly prevented the tumor antigen-mediated IL-10-enhanced IL-6 and TGF-β expression. The CM skewed the bone marrow-derived macrophages to CD206-high but MHC-II-low M2-like tumor-associated macrophages. Berberine partially prevented the generation of these macrophages and was associated with reduced C/EBPβ and Egr2 mRNA expression and lowered IL-10 and TGF-β production. Berberine significantly reduced Arginase-1 expression in CM-treated M1 and M2-like macrophages. Berberine increased MHC-II and CD40 expression on the macrophages augmenting the CTL activity and the number of IFNγ-producing CD4⁺ T-cells. Berberine significantly lowered tumor volume, weight and enhanced the frequency of M1-like macrophages in mice. These data indicate that berberine interferes with pro-tumor macrophage polarization and IL-10 and TGF-β release but restores Tcell anti-tumor cytotoxicity in the tumor microenvironment.