Introduction: Altered cellular energy metabolism is a crucial characteristic of most cancers regardless of tissue or cellular origin. In contrast to normal cells, which generate most of their adenosine triphosphate (ATP) through oxidative phosphorylation, cancer cells are dependent on aerobic glycolysis to meet energy demands (Warburg effect). However, recent studies demonstrate an upregulation in mitochondrial activity (O2 consumption and ATP production) when cancer cells are subjected to stress, such as radiation/chemotherapy treatment. To understand the significance of ATP produced by oxidative phosphorylation in cancer cells, a synthetic ATP-binding protein, DX, was targeted to the mitochondria in HeLa cells to reduce bioavailable ATP. Hypothesis: Reduced available ATP, due to DX in the mitochondria of HeLa cells, will negatively affect cellular metabolism, viability and chemoresistance. Methods: For controlled expression, DX was cloned into the pcDNA™5/TO expression vector under the control of a tetracycline (Tet)-regulated promoter. The DX construct was designed to translocate into the matrix of the mitochondria using the mitochondria targeting sequence and the 3’ UTR of a nuclear SOD2 gene. Cultured T-REx™ HeLa cells (which stably express the Tet-repressor protein) were transfected with pcDNA™5/TO::DX-MITO. As a control, T-REx™ HeLa cells were transfected with the parent plasmid, pcDNA™5/TO, and cultured under identical conditions. RT-PCR was performed to confirm DX expression, while western blot analysis was performed to confirm DX localization to the mitochondria. The impact of DX on cell viability and morphology was assessed using a tetrazolium-based colorimetric assay, caspase 3/7 assay, and by light/fluorescent microscopy. To correlate phenotypic change with DX activity, the level of bioavailable ADP /ATP was measured at specific time points following DX induction. Results: RT-PCR and western blot analysis confirmed DX expression and translation in HeLa cells, respectively, following Tet induction. Cells expressing DX experienced a significant reduction in viability and alteration in morphology compared to control HeLa cells. Contemporaneous with DX expression, bioavailable ATP decreased and the levels of caspases 3/7 increased suggesting that a majority of cells undergo apoptosis in response to energy crisis. Conclusion: Reduced intracellular ATP levels, mediated by a synthetic ATP-binding protein, negatively affects the viability of cervical cancer cells. The consequence of this on biochemical pathways, mitochondrial function and cell viability requires further investigation. We suggest DX, could be used to control and regulate energy metabolism and represents a new approach for cancer chemotherapy.
Citation Format: Eddie Khav, Bernardo Chavira, Matt Hamada, Nagaraj Vinay Janthakahalli, Shaleen Korch. Intracellular ATP depletion in cancer cells by a synthetic ATP-binding protein. [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 214.