Abstract 4639: Metformin decreases cellular ceramides in MCF-7 and MDA-MB 231 breast cancer cell lines by inhibition of ceramide synthetic enzymes

Abstract

Abstract Metformin, an anti-diabetic biguanide, has been shown to have cytotoxic and chemopreventive properties in in vitro and epidemiological studies, respectively. Metformin's predominant cytotoxic functions may be through activation of 5′ adenosine monophosphate-activated protein kinase (AMPK) with subsequent cell apoptosis. Ceramides, a group of waxy lipids found in the cellular membrane, are key players in many elements of cell signaling, including the regulation of cell differentiation, proliferation, and apoptosis. While traditionally thought of as being pro-apoptotic, this notion has been questioned with the discovery of pro-survival ceramides, as well as the demonstration that certain species of ceramides are increased in breast cancer cells. Metformin reduces ceramides in insulin-resistant mouse myoblasts and may reduce the vascular complications of diabetes mellitus. MCF-7 and MDA-MB-231 breast cancer cell lines were treated with increasing concentrations of metformin and cytotoxicity was determined by MTT cell culture experiments after 72 hours of drug exposure. Metformin induces cytotoxicity in these breast cancer cells at a lowest concentration of 1.25 mM, and percentage cytotoxicity increases in a dose-dependent manner. We then determined the effect of metformin on cell ceramide synthesis by analyzing key ceramide synthetic enzymes. Using western immunoblot and polymerase chain reaction (PCR) analysis, we determined that metformin induces a reduction in serine palmitoyl transferase, ceramide synthase, dihydroceramide desaturase, and neutral sphingomyelinase in MCF-7 and MDA-MB-231 breast cancer cells. We also analyzed the distribution of ceramides in treated breast cancer cells using polyclonal antibodies against ceramides. By confocal microscopy, ceramides are globally decreased in MCF-7 and MDA-MB-231 cells treated with metformin. This data suggests that the pro-apoptotic effect of metformin may be partly mediated through its disruption of the balance of ceramides and/or through the reduction of pro-survival ceramides within breast cancer cells. It is also possible that the predominant ceramides in MCF-7 and MDA-MB-231 breast cancer cells are pro-survival and metformin suppresses these ceramides. Further work is necessary to characterize the ceramide content of MCF-7 and MDA-MB-231 cancer cells before and after metformin treatment. Citation Format: Daniel Wann, Victoria Palau, Janet Lightner, Marianne Brannon, William Stone, Koyamangalath Krishnan. Metformin decreases cellular ceramides in MCF-7 and MDA-MB 231 breast cancer cell lines by inhibition of ceramide synthetic enzymes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4639. doi:10.1158/1538-7445.AM2015-4639