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Abstract Introduction: Chemotherapeutic resistance and tumor recurrence remain the leading causes of poor prognosis in colorectal cancer (CRC). Herein, we evaluated the anti-cancer effect of a novel repurposed drug HLN* in CRC. Methods: The changes in membrane potential were studied using DiSC (3) fluorescence by confocal microscopy. Cytotoxic activity of compound was studied using Sulphorhodamine-B (SRB) assay. Changes in mitochondrial membrane potential were studied using JC-1 dye. ROS accumulation was analyzed using DCFDA fluorescence using FACS analysis. Ferrorange assay was used to check the ferrous ion influx within the cell. Western blot was used to identify mode of cell death enacted. Results: Our results show that HLN treatment reduced the survival of human and murine CRC cells (CT26, HCT116, Colo 205 and MC38) with IC50 ranging 1-4 µM. HLN further inhibited proton motive force (PMF) by sequentially depolarizing and hyperpolarizing the cell membrane, as examined by cellular membrane potential using DiSC (3) dye by confocal microscopy. Disruption of the PMF leads to changes in various cellular signaling cascades including ROS production and mitochondrial membrane potential (MMP) changes. The MMP was significantly reduced by HLN treatment in CRC cells. Furthermore, we also observed an increase in ROS production by HLN. Pretreatment with N-acetyl cysteine (NAC), an antioxidant, blocked the effects of HLN. Interestingly, ferroptotic cell death markers GPX4, GPX1, SLC7A11/xCT, NRF2 and KEAP1 were significantly inhibited by HLN treatment in CRC cells as observed by western blotting. Ferroptotic induction was then confirmed using ferrorange assay wherein we observed a concentration dependent increase in ferrorange intensity indicating an increase in Fe+2. Overall, HLN dissipates ΔpH by modulating iron influx leading to ferroptotic cell death through ROS. Conclusion: Taken together, our results demonstrate the anti-cancer activity of a novel repurposed drug HLN and its unique mechanism of action. Our results provide a promise for developing this drug as a potential treatment option for CRC, through a novel anti-cancer mechanism. * Due to Intellectual property issued, the name of the drug is not provided Citation Format: Shreyas R. Gaikwad, Mohamed A. Eltokhy, Morgan Moore, Keerthi Kailath, Sanjay K. Srivastava. Disrupting cytoplasmic proton motive force induces ferroptotic cell death in colorectal cancer: A novel anti-cancer mechanism abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (7Suppl): Abstract nr LB267.
Gaikwad et al. (Fri,) studied this question.