Metformin Affects the Proliferation Cell Nuclear Antigen (PCNA) and p21 Protein Interaction Resulting in Direct Anti-Tumor Activity and Enhances the Cytotoxic/Biological Effects of Chemotherapy Agents or Rituximab in Lymphoma in Vitro and in Vivo Pre-Clinical Models

Most cancer cells predominantly produce adenosine triphosphate (ATP) by a high rate of glycolysis followed by lactic acid fermentation in the cytosol, rather than by a comparatively low rate of glycolysis followed by oxidation of pyruvate in mitochondria. This shift in cellular metabolism is know as the Warburg effect and is primarily observed in rapidly growing tumors including aggressive B-cell lymphoma and is thought to be a consequence of the progression to cancer rather than the cause of it. Altering the glucose metabolism in cancer cells appears to be an attractive strategy in cancer medicine. Previously, we demonstrated that the acquirement of rituximab resistance was associated with an increase in the Warburg effect leading to concomitant chemotherapy resistance in lymphoma pre-clinical models. The use of metformin, an oral biguanide widely used to treat insulin resistance conditions, during chemotherapy has been associated with improved clinical outcomes in solid tumor patients receiving chemotherapy. In a retrospective analysis, we demonstrated that the use of metformin during front-line chemo-immunotherapy (i.e. R+CHOP) improved the clinical outcome of diffuse large B-cell lymphoma (DLBCL). In an attempt to characterize the mechanism by which metformin affects the biology of DLBCL, we studied the metabolic and signaling changes in rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL) exposed to metformin. A panel of RSCL and RRCL were exposed to metformin. Changes in Ki67, proliferation cell nuclear antigen (PCNA), and its regulator (p21) were determined by flow cytometry or Western blotting respectively. Ki67/PCNA/p21 changes were correlated to cell cycle distribution, cell viability and chemotherapy sensitivity. For in vivo studies, SCID mice were inoculated via tail vein injection (iv) with Raji cells (day 0) and assigned to observation, rituximab (at 10mg/kg/dose on Day 3,7,10,14), metformin (at 2ug/ml in drinking water from day 3 till the end of experiment) or metformin with rituximab. Differences in survival (measured as the time for limb paralysis development) were evaluated by log-rank test between treatment arms. Metformin in combination with rituximab (mean survival not reached at 69+/- 5.3 days) lead to an improved survival than rituximab (mean survival 57.1+/-4.2 days) (P=0.05). In vitro exposure of RSCL/RRCL to metformin inhibited cell proliferation (measure by, MTT assay, alamar blue reduction, cell titer glow assay, and ki67 staining) in a dose-dependent manner and enhanced the anti-tumor activity of chemotherapy drugs. Perhaps related to this effect, metformin exposure induced G1 phase cell cycle arrest in both RSCL and RRCL. Using the flow cytometry technology (FITC-labeled anti-KI67 antibody/PI-DNA staining), we detected a decrease in Ki67 of RSCL/RRCL distributed in the G1 and S phase following metformin exposure. In vitro exposure of RSCL and RRCL to metformin increased p21 and reduced PCNA levels. Immuno-precipitation of p21 following metformin drug exposure increased in the interaction between p21 and PCNA. Our data suggests that metformin inhibits the proliferation of RSCL or RRCL and enhances the anti-tumor activity of chemotherapy agents or rituximab in lymphoma pre-clinical models (in vitro and in vivo). Perhaps related to the biological effects observed, p21 and PCNA, play a pivotal role on the anti-tumor activity of metformin in lymphoma pre-clinical models. Our finding highlights a potential role for metformin in the treatment of B-cell malignancies. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund)