Revisiting hydrogen peroxide as radiosensitizer for solid tumor cells.

Background and purpose: Tumor hypoxia is the principal cause of clinical radioresistance. Despite its established role as radiosensitizer, hydrogen peroxide (H₂O₂) encounters clinical limitations due to stability and toxicity concerns. Recent advancements in drug delivery combine H₂O₂ with sodium hyaluronate (SH), enabling intratumoral administration of H₂O₂. This study investigates the radiomodulatory pathways of Kochi Oxydol-Radiation for Unresectable Carcinomas (KORTUC) (H₂O₂ + SH) under hypoxia.

Materials and methods: CT26 and 4T1 tumor cells were exposed to H₂O_2, SH and KORTUC under hypoxic conditions. Toxicity levels were determined using MTT and live-cell analysis. KORTUC's radiomodulatory properties were evaluated by colony formation assay and in spheroids. Reactive oxygen species (ROS) levels, DNA damage, apoptosis and ferroptosis were analyzed using flow cytometry. Oxygen consumption rate (OCR) and mitochondrial complex activity were assessed by Seahorse Analyzer. Oxygen levels were investigated using fiber-optic sensors. The in vitro findings were validated in CT26-bearing mice.

Results: KORTUC demonstrated less cytotoxicity than H₂O₂-alone. KORTUC radiosensitized hypoxic tumor cells in a dose-dependent manner with enhancement ratios of 3.1 (CT26) and 2.7 (4T1). Dose-dependent OCR reduction following KORTUC exposure correlated with complex I and II inhibition, accompanied by mitochondrial ROS elevation. KORTUC injection into a 2D hypoxic tumor model surged O₂ levels. KORTUC radiosensitized CT26-tumors, delaying growth by 14 days.

Conclusions: SH in KORTUC mitigates H₂O₂ cytotoxicity. We demonstrate that KORTUC overcomes hypoxia-induced radioresistance through inhibition of OCR, via complex I- and II-blockade, leading to tumor reoxygenation. Understanding KORTUC's pathways is essential for developing effective cancer combination therapies.