[Metformin inhibits proliferation and functions of regulatory T cells in acidic environment].

OBJECTIVE: To investigate the regulatory effect of metformin on regulatory T cells (Treg) in acidic environment.
METHODS: CD4+ CD25+ Treg cells were obtained by magnetic bead sorting. Treg and conventional T cells (Tcon) cells were cultured for 24-72 h in pH 7.4 or pH 6.7 medium, and the cell proliferation, apoptosis and Foxp3 expression were detected by flow cytometry. Real-time PCR was used to detect the expression levels of the genes related with glucose metabolism. Thirty-two C57BL/6 male mouse models bearing subcutaneous prostate cancer xenograft derived from RM-1 cells were randomized into 4 equal groups for treatment with PBS, metformin, tumor vaccine, or both metformin and the vaccine. The treatment started on the 4th day following tumor cell injection, and metformin (100 mg/kg) or PBS was administered by intraperitoneal injection on a daily basis; the vaccine was intramuscularly injected every 4 days. The tumor size was continuously monitored, and the mice were euthanized on day 25 after tumor implantation to obtain tumor and blood samples. Flow cytometry was used to detect the changes in CD4+, CD8+, CD4+Foxp3+ cell subsets in the tumor tissue and peripheral blood.
RESULTS: Treg cells showed significantly enhanced proliferation (P < 0.05) while the proliferation of Tcon cells was suppressed in acidic medium (P < 0.001). Treg cells cultured in acidic medium showed significantly increased expressions of OXPHOS-related genes pgc1a (P < 0.001) and cox5b (P < 0.01), which did not vary significantly in Tcon cells in acidic medium. Treg cells exhibited significantly decreased apoptosis in acidic medium (P < 0.01) with increased Foxp3+ cells (P < 0.001) and intracellular alkaline levels (P < 0.01). Metformin obviously reversed the acid tolerance of Treg cells without producing significant effect on Tcon cells. In the animal experiment, both metformin (P < 0.05) and vaccine (P < 0.01) alone reduced the tumor volume, but their combined treatment more potently reduced the tumor volume (P < 0.001). Metformin alone did not obviously affect CD4+ cells or CD8+ cells but significantly decreased the percentage of CD4+Foxp3+ (P < 0.05); the vaccine alone significantly increased CD4+ cells and CD8+ cells (P < 0.001) and also the percentage of CD4+Foxp3+ cells (P < 0.05). The combined treatment, while reducing the percentage of CD4+Foxp3+cells to a level lower than that in the vaccine group (P < 0.01), produced the strongest effect to increase CD4+ cells and CD8+ cells (P < 0.01).
CONCLUSIONS: Metformin can inhibit the proliferation and function of regulatory T cells in an acidic environment and enhance the effect of tumor vaccine by reducing the proportion of Treg cells in vivo to achieve the anti-tumor effect.