Investigation of the Effect of SOD1 Inhibition on the FMD-2Br-DAB Activity in Pancreatic Cancer Cells

Abstract

Femtomedicine (FMD) compounds are a class of non-platinum halogenated molecules that have previously demonstrated selective anticancer activity. Among them, FMD-2Br-DAB has been proposed to act through a halogen reaction cycle mechanism, in which hydrogen peroxide (H₂O₂) initiates reactive halogen species (RHS) generation and nitric oxide (NO) terminates the cycle, together with a dissociative electron transfer (DET) mechanism. Superoxide dismutase 1 (SOD1) converts superoxide anions (O₂•⁻) into H₂O₂ and thereby contributes to intracellular H₂O₂ availability. However, whether SOD1-associated H₂O₂ availability influences the anticancer activity of FMD-2Br-DAB through the proposed reactive halogen cycle has not been experimentally established. To address this, PANC-1 and BXPC-3 pancreatic cancer cells and GM05757 normal fibroblasts were treated with FMD-2Br-DAB alone and in combination with the SOD1 inhibitors DETC and LCS-1. Cell viability was assessed using the MTT assay, intracellular H₂O₂ was measured, and UV–Vis spectroscopy was used to examine spectral changes in mixtures of FMD-2Br-DAB with each inhibitor. FMD-2Br-DAB reduced cell viability in a concentration-dependent manner. BXPC-3 cells showed selective sensitivity relative to GM05757 fibroblasts, whereas PANC-1 cells did not. The SOD1 inhibitors were associated with changes in FMD-2Br-DAB cytotoxicity, with different responses for DETC and LCS-1. FMD–LCS-1 combinations consistently produced viability above the expected additive response, consistent with an H₂O₂-dependent contribution. In contrast, DETC lowered intracellular H₂O₂ relative to FMD alone in all three cell lines, yet FMD–DETC combinations were less viable than FMD alone in every condition tested, and viability was at or only modestly above the expected additive response in five of six conditions (below it in PANC-1 at 100 μM FMD). The reduced H₂O₂-associated fluorescence did not correspond to the predicted reduction in FMD cytotoxicity following DETC treatment. This unexpected response suggests that additional mechanisms may contribute to the effects of the FMD–DETC combination. UV–Vis spectroscopy showed pronounced spectral changes in the FMD–DETC combination, consistent with a possible chemical interaction between the compounds. These findings are consistent with a possible contribution of H₂O₂-dependent mechanisms to FMD 2Br-DAB activity but do not directly establish the proposed halogen reaction cycle. The FMD–DETC response is consistent with the proposed DET mechanism, with DETC acting as an electron donor, whereas the FMD–LCS-1 response is consistent with the proposed mechanism of H₂O₂-regulated halogen reaction cycles. The reduced selectivity in PANC-1 relative to BXPC-3 cells suggests that H₂O₂ availability alone does not fully explain the differences between the two pancreatic cancer cell lines.