Abstract 5484: Targeted inhibition of acid ceramidase and C6-ceramide exposure induces synergistic killing in cancer cells
Authors: Margaret Flowers, Gemma Fabriás, Antonio Delgado, Josefina Casas, Myles C. Cabot
Journal: Cancer Research (2011)

Abstract Introduction. In this study, we evaluated the anticancer activity of DM102 [(2R,3Z)-N-(1-hydroxyoctadec-3-en-2-yl)pivalamide], a novel inhibitor of the enzyme acid ceramidase (AC), a key regulator of ceramide metabolism, in combination with C6-ceramide (C6-cer) in human breast cancer cells. Ceramide, a bioactive sphingolipid and potent enhancer of apoptosis, is known to play a role in chemo- and radiation-induced cell death. AC tempers ceramide-induced apoptosis by hydrolysis, producing sphingosine and free fatty acid, thus reducing intracellular ceramide levels and providing substrate for sphingosine kinase, which converts sphingosine to sphingosine 1-phosphate. AC has emerged as a putative anticancer target following reports of upregulation in prostate cancer and in some breast tumors. In the present work, we determined whether the introduction of an AC inhibitor would enhance the apoptosis-inducing effects of C6-cer in breast cancer cells. Methods. Cultured breast cancer cells were pretreated with 10 μM DM102 before exposure to 2.5-10 μM C6-cer. Cell viability was assessed at 72 hr by 96-well colorimetric assays. Cytotoxic synergy was assessed by the Chou-Talalay method of combination index (CI) determined using CalcuSyn® software. Apoptosis was measured by Annexin V/propidium iodide staining using flow cytometry, and by caspase 3/7 activity. Generation of reactive oxygen species (ROS) was measured by flow cytometry. Autophagy was assessed by Western blot for LC3II activation. Lipid metabolic studies were performed using [3H]palmitic acid. Results. Treatment with DM102 (10 μM) for 24 hr increased levels of 3H-palmitic acid-labeled ceramide by 2-fold, compared to controls, in MDA-MB 231 breast cancer cells. As single agents in viability studies, both C6-cer (IC50 5-10 μM) and DM102 (IC50 20 μM) were only moderately cytotoxic in MDA-MB 231, MCF-7, and SKBr3 cells. Co-administration however, produced synergistic decreases in viability (CI &amp;lt;0.5) in all cell lines, at doses that were half the IC50 of either agent alone. Similar cytotoxic profiles were observed with C6-cer and the AC inhibitor B13, (1R,2R)-2-N-(tetradecanoylamino)-1-(4′-nitrophenyl)-1,3-propiandiol, suggesting that blocking AC activity was key to the synergistic effect. At 24 hr, combination C6-cer/DM102 increased ROS levels &amp;gt; 2-fold in MDA-MB 231 cells. This was accompanied by activation of the autophagy marker, LC3II and a decrease in phospho-Akt, a promoter of cell survival and negative regulator of autophagy. At 48 hr, caspase 3/7 activity increased &amp;gt; 2-fold, and by 72 hr there was a &amp;gt;70% increase in Annexin-V positive cells, compared to controls. Conclusions. These data support the efficacy of targeting AC in combination with exogenous short-chain ceramide as an anticancer strategy, and warrant further investigation into the mechanism of action of the C6 ceramide/DM102 drug duo. (Support: NIGMS77391) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5484. doi:10.1158/1538-7445.AM2011-5484