Abstract 2332: Modifying bioavailability of ATP and measuring metabolic response in HeLa cells

Abstract Introduction: Since studies by Otto Warburg demonstrated that cancer cells had markedly reduced oxidative phosphorylation (OXPHOS) under conditions of high oxygen tension (Warburg Effect/aerobic glycolysis), metabolic reprogramming has become a fundamental hallmark of cancer. While numerous discoveries have revealed highly specific metabolic differences between cancer cells, utilization of aerobic glycolysis as the primary means of ATP generation remains one of the most common metabolic alterations observed across a range of cancers. There is growing interest in understanding the multifaceted role ATP plays in cancer development, progression and chemotherapeutic resistance. Using the man-made protein DX, which chelates ATP with high specificity and affinity, we have begun to untangle these roles by significantly reducing cytosolic ATP and investigating cellular response. Hypothesis: Delivery of DX into HeLa cancer cells will reduce intracellular bioavailable ATP and induce an adaptive metabolic response. Methods: A cationic lipid mixture was complexed with active, purified DX protein to generate a DX/lipid complex capable of delivering the protein to the cytoplasm of HeLa cells. To confirm the successful delivery of DX, immunofluorescent microscopy was performed (DYKDDDDK tag monoclonal antibody, Alexa Fluor 647) at various time points following DX transfection. The impact of DX on cell viability was determined using a tetrazolium-based colorimetric cell viability assay and a caspase 3/7 assay. To correlate phenotypic/viability change with DX activity, bioavailable ATP levels were measured at specific time points following DX delivery. Additionally, the relative contribution of glycolysis and OXPHOS to the total ATP production rate was measured using the label-free XF Real Time ATP Rate Assay (XFe96 Seahorse, Agilent Technologies) over time post DX transfection. Results: In a time- and dose- dependent manner, DX negatively impacted cell growth and induced cell death via apoptosis, at a time concomitant with a decrease in bioavailable ATP. In response to DX over time, the total ATP production rates in HeLa cells significantly decreased. Importantly, this reduction in the rate of ATP production was associated with a metabolic program that was primarily glycolytic (70%) with a smaller contribution from OXPHOS (30%) 24 h post DX transfection. Conclusion: Advances in protein engineering have made it possible to create artificial proteins with specific functions. In addition to direct clinical applications, synthetic proteins can be developed into powerful experimental tools to resolve biological enigmas. Direct delivery of DX potentiated energy flux in HeLa cells in an unbiased manner, independent of metabolic pathway interference. This work establishes DX as a useful tool for examination of cancer cell metabolism in response to ATP stress. Citation Format: Taha Muhammad, Ashley Brown, Selina Martinez, John C. Chaput, Jeffrey Norris, Shaleen Korch. Modifying bioavailability of ATP and measuring metabolic response in HeLa cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2332.