Program: Oral and Poster Abstracts
Session: 625. Lymphoma: Pre-Clinical – Chemotherapy and Biologic Agents: Poster III
Methods: A panel of rituximab-sensitive (RSCL), rituximab-resistant (RRCL) cell lines and primary tumor cells isolated from B-cell lymphoma patients were exposed to escalating doses of metformin (0-64mM). Changes in cell viability were determined by Presto Blue (Sigma) assay in cell lines and Titer Glo in patient samples. Changes in MDMX, MDM2 and p53 expression levels were determined by western blotting after exposure cells to metformin. MDMX-MDM2-p53 interactions and p53 ubiquitination following in vitro exposure to metformin were determined by immunoprecipitation of p53 and probing for MDMX, MDM2, ubiquitin and p53 in RSCL and RRCLs. Loss of Dψm or induction of apoptosis following metformin exposure were assessed by DiOC6 or Annexin V/PI staining and flow cytometry. Oxidative stress induced by metformin was measured by flow cytometry using dihydrorhodamine-123 (DHR-123) dye.
Result: Metformin induced a dose-and time- dependent cell death in cell lines and primary patient tumor cells. In vitro exposure of lymphoma cell to metformin resulted in a decrease in MDMX levels. Immunoprecipitation studies demonstrated that following exposure to metformin, MDMX bound less to p53 leading to less p53 ubiquitination. In vitro exposure of RSCL or RRCL to metformin resulted in the expression of p53 regulated BH3 single domain proteins (Noxa and Puma). Moreover, metformin repressed mitochondrial potential, induced reactive oxidative species (ROS) generation and triggered apoptosis.
Conclusion: Our data suggests that metformin had anti-tumor activity against RSCL, RRCL and primary tumor cells isolated from lymphoma patients. The down-regulation of MDMX and re-activation p53 function following metformin exposure may contribute to the disruption in the mitochondria potential, generation of ROS and induction of apoptosis observed in our models RSCL and RRCL. 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 Funds)
Disclosures: Czuczman: MorphoSys: Consultancy ; Boehringer-Ingelheim: Other: Advisory Board ; Celgene: Employment ; Immunogen: Other: Advisory board .
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