Abstract 4907: Atovaquone-induced increase in reactive oxygen species leads to impaired glycolytic functions in ovarian cancer cells

Abstract

Abstract Atovaquone (ATO) is an FDA-approved anti-malarial drug, which has also shown potent anti-cancer effects. We have previously demonstrated ATO as an effective chemotherapy against high-grade serous ovarian cancer in cell lines and mouse model. In the present study, we aim to determine if ATO is asserting its anti-cancer properties by altering cellular metabolism. Glycolysis and oxidative phosphorylation (OXPHOS) are the two major sources of intracellular energy. ATO reduces OXPHOS energy production by inhibiting mitochondrial complex III. We first determined whether there is a shift in energy production when OXPHOS is inhibited by assessing glycolytic function in various human (OVCAR3 and OVCAR5) and mouse (ID8) ovarian cancer cell lines. Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were assessed by the Seahorse XF Analyzer. First, we pretreated cells with ATO at IC70 concentrations for 1 hour prior to metabolic analysis. As expected, pretreatment with ATO reduces OCR in all cell lines. However, ATO treatment increased basal glycolysis but decreased glycolytic capacity and glycolytic reserve. This suggests that ATO-treated cells have an impaired ability to shift energy production towards glycolysis when there is an increase in energy demand. One of the initial events that occur in ATO-treated cells is a surge in reactive oxygen species (ROS) as measured by H2DCFDA ROS assay. In the human cell lines, we found that ATO significantly increased ROS by approximately 100-fold (MFI). Whereas, in the mouse cell line, the ATO-induced increase in ROS were comparatively attenuated. We hypothesized that the increase in ROS leads to inhibition of glycolysis. We tested this by pre-treating cells with 2mM N-acetylcysteine (NAC), an oxygen radical scavenger, for 1 hour before measuring the metabolic responses to ATO. We found that NAC attenuated the inhibition of glycolytic processes in human cell lines but not in the mouse cell line. Our lab has previously shown that ATO induced activation and increased expression of p53. Studies have shown that ROS activate p53, which in turn upregulates the TP53 induced glycolysis and apoptosis regulatory phosphatase (TIGAR), inhibiting the conversion of F6P to F-2,6-BP and ultimately leads to inhibition of glycolysis. Increases in ROS also shuttle G6P towards the phosphate pentose pathways through G6P dehydrogenase for antioxidant production. We found that long-term ATO treatment for 24 hours also resulted in increases in both TIGAR and G6PD mRNA expression. Taken together, these results suggest that ATO decreased plasticity of metabolic capacity in ovarian cancer cells through ROS and ROS associated glycolytic pathways. Further, this study provides mechanistic insights into ATO’s potential as a chemotherapeutic agent. Citation Format: Nicha Boonpattrawong, Sejal Sharma, Rob Schultz, Mayra A. Betancourt Ponce, Lisa M. Barroilhet, Manish Patankar. Atovaquone-induced increase in reactive oxygen species leads to impaired glycolytic functions in ovarian cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4907.