Abstract 15: Metabolomic profiling of cell death in human lung cancer cells by a novel digitoxin analog

Abstract

Abstract Background: Digitoxin, a cardiac glycoside had shown considerable promise as an anti-cancer therapeutic. However, doses of digitoxin required to inhibit cancer cell proliferation lead to cardiotoxic side effects. We have previously shown that a novel analog of digitoxin, MonoD, activates apoptosis in human lung cancer cells at much lower concentrations as compared to digitoxin. We postulate that treatment with MonoD leads to distinct metabolic signatures on lung cancer cells as compared to digitoxin, which will help delineate the pathways that dictate MonoD action. Experimental Approach: NCI-H460 cells from identical passages were used for metabolite analysis using an optimized version of the methanol/water (80:20) protocol for metabolite extraction. The supernatant containing metabolites were suspended in 0.1% formic acid spiked with an internal standard for Q-Exactive analysis. The data was analyzed using SIEVETM 2.0 software with the component extraction algorithm that has been specifically designed for optimal data analysis in untargeted metabolomics experiments. Metabolites that were found to be significantly different between treatment groups were detected via Chemspider/HMDB database search. Results: We observed several metabolites along the prostaglandin synthesis pathway which were downregulated in MonoD treated lung cancer cells. Prostaglandins play a key role in the generation of the inflammatory response. Chronic inflammation has been shown to be a causative factor in a variety of cancers. Cyclooxygenase-2 (COX-2) derived prostaglandin has been previously shown to promote tumor growth by binding its receptors and activating signaling pathways which control cell proliferation, migration, apoptosis, and angiogenesis. Using the metabolite profiling data, we posited that COX-2, a key enzyme involved in prostaglandin synthesis, in addition to NFκB, a key transcription factor of proinflammatory signaling pathway are downregulated with MonoD treatment. Immunoblotting confirmed the downregulation of COX-2 and NFκB in MonoD-treated cells. Conclusion: Using metabolite profiling, we have elucidated an important mode of action for MonoD in lung cancer cells, and shown that MonoD exerts its anti-tumorigenic properties by downregulating key factors involved in inflammatory response. Citation Format: Yogesh Kulkarni, Neelam Azad, Vivek Kaushik, Juan Sebastian Yakisich, Rajkumar Venkatadri, Clayton Wright, Yon Rojanasakul, George O’Doherty, Anand Krishnan V Iyer. Metabolomic profiling of cell death in human lung cancer cells by a novel digitoxin analog. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 15.