Levofloxacin enhances 5-fluorouracil efficacy in colorectal cancer by activating the DDR-TP53-mitochondrial apoptosis axis

5-Fluorouracil (5-FU)-based regimens are a cornerstone of colorectal cancer (CRC) chemotherapy. Levofloxacin (LVF), a widely used fluoroquinolone antibiotic, has been reported to exhibit anticancer properties. We investigated LVF's ability to enhance CRC chemosensitivity to 5-FU in cell lines, subcutaneous xenografts (Balb/c-nude mice), and spontaneous intestinal tumors (Apcmin/+ mice). LVF potentiated 5-FU-induced apoptosis in vitro and in vivo. Mechanistically, LVF induced mitochondrial dysfunction in CRC cells, marked by reactive oxygen species (ROS) accumulation, increased mitochondrial ROS, and loss of mitochondrial membrane potential. TP53 and downstream effectors NOXA and BBC3 mediate between LVF-induced mitochondrial damage and 5-FU sensitization. LVF-induced DNA damage response (DDR) and subsequent oxidative phosphorylation (OXPHOS) upregulation trigger mitochondrial damage and TP53 activation. Importantly, we confirmed that the canonical LVF target TOP2A and the reported LVF effector ribosomal S6 kinase 4 (RSK4) are dispensable for LVF-mediated sensitization. Thus, LVF acts as a non-canonical 5-FU chemosensitizer in TP53-WT CRC via DDR-TP53-mitochondrial apoptosis activation, supporting clinical repurposing.