Abstract 452: Direct delivery of a synthetic ATP binding protein reduces ATP and induces apoptosis in Hela cells

Abstract

Abstract Introduction: Reprogramming of cellular metabolism is a hallmark characteristic of cancer cells. As opposed to normal cells, which generate most of their adenine triphosphate (ATP) through oxidative phosphorylation, cancer cells generate ATP less efficiently by aerobic glycolysis (Warburg effect). In addition to deciphering metabolic switches, there is growing interest in understanding the multifaceted role ATP plays in cancer development, progression and chemotherapeutic resistance. One approach to untangling these roles is to significantly reduce intracellular ATP and investigate the cellular response. With that, we designed and created a man-made protein that chelates ATP with high specificity and affinity. This synthetic protein, called DX, has been genetically expressed in a living organism, Escherichia coli, where it reduced intracellular ATP to below the levels of detection. Hypothesis: Delivery of the DX protein into HeLa cancer cells will reduce intracellular bioavailable ATP affecting energy metabolism, viability and drug susceptibility. Methods: A cationic lipid mixture was complexed with active, purified DX protein to generate a DX/lipid complex. The integrity of the complexed protein was verified by western blot analysis. HeLa cells were incubated with the DX/lipid complex to allow efficient delivery of DX into the cytoplasm of the HeLa cells. 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. To establish whether DX impacts drug export, the retention of calcein-AM, a substrate of the ATP-dependent p-glycoprotein pump, was measured post DX-delivery. Results: Western blot analysis confirmed the stability of DX prior to- and post- delivery to HeLa cells. 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. Significantly, HeLa cells treated with DX retained calcein-AM, suggesting reduced p-glycoprotein activity. Conclusion: Advances in protein engineering have made it possible to create artificial proteins with specific functions. In addition to direct clinical applications, synthetic proteins are powerful tools that have the potential to reveal something new about biology. Direct delivery of a synthetic ATP-binding protein potentiated apoptosis in HeLa cells contemporaneous with reduced intracellular ATP. Having established DX activity in a living system, future studies will examine the cancer cell metabolome in response to significant ATP stress. Finally, a reduction in p-glycoprotein activity in DX-treated HeLa cells advocates examination of concurrent DX/chemotherapeutic treatment, principally in drug resistant scenarios. Citation Format: Selina M. Martinez, Shanika Kingston, Arlene Ngor, John C. Chaput, Nagaraj J. Vinay, Shaleen B. Korch. Direct delivery of a synthetic ATP binding protein reduces ATP and induces apoptosis in Hela cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 452.