Loperamide reverses 5‑FU resistance in colorectal cancer by activating autophagy.

5‑Fluorouracil (5‑FU), a cornerstone chemotherapeutic agent used for colorectal cancer therapy, has long been established as a first‑line treatment. However, clinical evidence has suggested that a substantial proportion of patients develop resistance to 5‑FU, notably compromising its therapeutic efficacy. The present study aimed to investigate whether loperamide (LOP) can enhance the sensitivity of colorectal cancer cells to 5‑FU and to elucidate the potential underlying molecular mechanism. First, the IC₅₀ values of LOP were determined in the 5‑FU‑sensitive HCT8 and 5‑FU‑resistant HCT8R colorectal cancer cell lines, using the Cell Counting Kit‑8 assay to evaluate LOP‑induced alterations in 5‑FU sensitivity. The effects of LOP on cell proliferation were subsequently analyzed using 5‑ethynyl‑2'‑deoxyuridine and colony formation assays. Cell migration was assessed through wound healing and Transwell migration assays, and apoptosis was evaluated using flow cytometric analysis with PI/Annexin V staining. Western blot analysis was performed to measure the expression levels of the autophagy‑associated proteins microtubule‑associated protein 1 light chain 3 (LC3) and Beclin, and autophagosome formation following LOP treatment was visualized. The role of autophagy in LOP‑mediated reversal of drug resistance was further examined using autophagy inhibitors. Finally, xenograft experiments in nude mice were performed to investigate the in vivo effects of LOP on the 5‑FU sensitivity of HCT8R cells. Compared with in the parental cell line, HCT8R cells exhibited enhanced migratory capabilities and resistance to 5‑FU. Notably, LOP was revealed to potentiate the sensitivity of HCT8R cells to 5‑FU, as evidenced by reduced rates of cell proliferation, suppressed migratory ability, increased levels of apoptosis, and decreased tumor weight and volume in subcutaneous xenografts in mice. LOP was also shown to induce upregulation of autophagy marker proteins, leading to the accumulation of autophagosomes within the cells. Blocking autophagy with 3‑methyladenine led to a reversal of the inhibitory effect of LOP on HCT8R cell migration. LOP was also shown to enhance the sensitivity of HCT8R cells to 5‑FU by activating cellular autophagy, thereby suppressing resistant cell proliferation and migration, promoting apoptosis and reversing drug resistance. Taken together, these findings provide novel insights into the mechanisms underlying 5‑FU resistance, thereby highlighting potential therapeutic strategies for colorectal cancer.