Cyclophilin D suppresses colorectal cancer progression through the activation of an autophagy-mediated apoptotic pathway.

Colorectal cancer (CRC) remains a leading gastrointestinal malignancy and lacks effective adjuvant therapies. Cyclophilin D (CypD), a mitochondrial matrix protein, has a controversial role in tumorigenesis. While prior studies suggest a CypD-CRC link, its precise function and mechanisms remain undefined. Here we clarify whether CypD acts as a driver or suppressor of CRC and explore its therapeutic implications. Using RT-qPCR, Western blotting, and immunofluorescence, we confirmed significant CypD downregulation in CRC tissues/cells versus non-malignant controls. Through in vitro (CCK-8, colony formation, transwell migration, spheroid formation, apoptosis, and autophagy assays) and in vivo tumor xenograft experiments with CypD-knockdown/overexpressing CRC cells (DLD1 and RKO), knocking down CypD promoted, whereas its overexpression inhibited, malignant phenotypes (proliferation, migration, self-renewal, tumorigenicity). Mechanistically, CypD activated protective autophagy and cell apoptosis, with autophagy inhibitor chloroquine reversing this effect. Critically, knocking down CypD attenuated the anti-CRC efficacy of 5-fluorouracil/epigallocatechin-3-gallate combination therapy, whereas its overexpression enhanced therapeutic response. This study provides the first definitive evidence that CypD functions as a tumour suppressor via autophagy-apoptosis crosstalk. Low CypD expression may serve as a biomarker for poor treatment response. Enhancing CypD activity represents a promising strategy to augment existing CRC treatments. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-025-33774-1.