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Abstract 1846: In vitro studies of the effects of a novel ceramide analog on chemo- resistant breast cancer cells


 Globally, breast cancer is the most frequently diagnosed cancer in women, with an estimated 281,550 new cases in the U.S.A in 2021. About 43,600 women in the U.S. are expected to die from the disease by the end of the year. Chemo- and radio-therapies, primarily, exert anti-tumor effects through the activation of apoptosis, or programmed cell death pathways. As many current therapies have severe side effects, and/or lose their effectiveness over time due to developed resistance, there is a need for novel agents that can improve patient outcomes including the quality and length of life. Ceramide-mimicking drugs have such potential. Ceramide, a bioactive sphingolipid, is a powerful tumor suppressor molecule that is thought to induce apoptosis and inhibit proliferation. As part of our ongoing efforts toward the development of a potent anti-cancer drug, ceramide analog 315, (S,E)-3-hydroxy-2-(2-hydroxybenzylidene)amino-N-tetradecylpropanamide, was synthesized. Ceramide analog 315 has been shown to induce apoptosis in vitro. In the present study, an attempt is made to determine the cell death pathway induced during the treatment of chemo-resistant breast cancer cells with this analog. The MCF-7TN-R cell line is a derivative of the MCF-7 cell line obtained after prolonged exposure to increasing concentrations of TNF-α. These chemo-resistant cells, 24 hours after plating, were treated with a 40 µM solution of ceramide analog 315. Caspase 9 and cytochrome c Elisa assays were performed according to the vendor protocols. Cytochrome c and caspase 9 are believed to activate several pro-apoptotic proteins like APAF-1, BAX, and BCL-2, supporting the intrinsic or mitochondrial pathway. Caspase 9 and cytochrome c are both released from the mitochondrial intermembrane space, increasing the mitochondrial outer membrane permeability. Cytochrome c is a heme protein that, when released, binds to apoptotic protease activating factor 1 (APAF-1). APAF-1 then creates an apoptosome that acts as a platform for caspase 9 activation. Caspase 9, in turn, activates the cascade pathway and induces apoptosis. In this study, cytochrome c levels were shown to be decreased. Controls were found to have a concentration of 8.8 +/- 0.268 ng/mL, while treated cells had a concentration of 7.46 +/- 0.181 ng/mL, with a P value of less than .05. Caspase 9 levels were shown to be even more significantly decreased with controls having a concentration of 678,800 +/- 24,462 ng/mL, while treated cells had a concentration of 496,444 +/- 49,542 ng/mL, with a P value of less than 0.10.We further determined the protein level expression in these cells using microarrays, which revealed the upregulation of death receptors TNFR1, and TRAIL. Our findings help us in better understanding the mechanisms of action of ceramide-mimicking agents, and in working toward the development of strategies to improve the efficacy of anti-cancer drugs.
 Citation Format: Teresa C. Beamon, Royce Hooks, Degrick Cheatham, Navneet Goyal, Tulasi Ponnapakkam, Shahensha Shaik, Maryam Foroozesh. In vitro studies of the effects of a novel ceramide analog on chemo- resistant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1846.