Abstract 2442: Antitumorigenic effects of phenformin in human ovarian cancer cell lines.
Authors: Jackson Amanda L. , Kilgore Joshua E. , Qiu Haifeng , Zhou Chunxiao , Gehrig Paola A. , Bae-Jump Victoria L. 
Published: August 2014

Abstract
Introduction: Anti-diabetic biguanide drugs, such as metformin, have been shown to have anti-tumorigenic effects by behaving as AMPK activators and mTOR inhibitors. Phenformin is another biguanide with anti-diabetic activity that was withdrawn from the market due to a risk of lactic acidosis that was higher than that seen with metformin. In vitro and in vivo studies suggest that phenformin may be more potent for inhibiting tumor growth than metformin. Thus, our objective was to assess the effect of phenformin on proliferation and apoptosis in ovarian cancer cell lines.
Methods: Three ovarian cell lines, SKOV3, IGROV1, and HEY, were used. Cell proliferation was assessed by MTT assay after exposure to phenformin and after exposure to phenformin under varying glucose levels. Apoptosis was analyzed by Annexin V-FITC assay. Cell cycle progression was evaluated by Cellometer. Western immunoblotting was performed to determine expression of the downstream targets of phenformin, including AMPK, ribosomal protein S6, cyclin D and E2, cyclin dependent kinase (CDK) 4 and 6, p21 and p27. Inhibition of adhesion by phenformin was assessed by in vitro adhesion assay.
Results: Phenformin potently inhibited proliferation in a dose-dependent manner in all three ovarian cancer cell lines (IC50 = 2.5mM for SKOV3 and HEY, 1mM for IGROV1 at 48 hours) (p = 0.0035 – 0.00001). Inhibition of proliferation was found at normal, low and high glucose concentrations, but greater potency was observed under low glucose concentrations. Treatment with phenformin resulted in G1 cell cycle arrest and induction of apoptosis. Cellular adhesion was decreased by 14-32% in the ovarian cancer cell lines at a phenformin dose of 2.5mM (p = 0.009 – 0.0001). Western immunoblot analysis demonstrated that phenformin induced phosphorylation of AMPK, its immediate downstream mediator, within 18 hours of exposure. In parallel, treatment with phenformin decreased phosphorylation of the S6 protein, a key target of the mTOR pathway. Phenformin was also found to increase the cell cycle inhibitors, p21 and p27, and decrease cyclin D, cyclin E2, CDK4 and CDK 6, corresponding with G1 arrest in these cell lines.
Conclusion: Phenformin potently inhibited ovarian cancer cell growth via G1 arrest and increased apoptosis. Although the risk/benefit ratio clearly favors metformin over phenformin for the treatment of diabetes, this may not hold true for the treatment of cancer if phenformin were found to have superior anti-tumorigenic activity. Continued work is needed to explore the benefits of both metformin and phenformin for the treatment of ovarian cancer and the optimal therapeutic index.
Citation Format: Amanda L. Jackson, Joshua E. Kilgore, Haifeng Qiu, Chunxiao Zhou, Paola A. Gehrig, Victoria L. Bae-Jump. Antitumorigenic effects of phenformin in human ovarian cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2442. doi:10.1158/1538-7445.AM2013-2442