The unabsorbed cholesterol, along with that of bile secretions and flaked colon cells, can be metabolized by the colonic microbiota. The generated metabolites have been proposed as promoters of colorectal cancer (CRC). In this study, the cytotoxicity (by means of MTT assay) of the main commercially available cholesterol-derived metabolites (coprostanol, cholestanol, coprostanone and cholestenone) on human colon cancer (Caco-2) and non-tumor (CCD-18Co) cells was evaluated at different concentrations (9.4-300 µM) and incubation times (24-72 h). In general, the metabolites that most reduced cell viability were coprostanone, values ranging 54-85 % in Caco-2 and 20-81 % in CCD-18Co, and cholestenone (7-91 % in Caco-2 and 10-81 % in CCD-18Co). These two metabolites (coprostanone and cholestenone) are, in turn, the most hydrophobic, thus reflecting a possible relationship between hydrophobicity and cytotoxicity. On the other hand, cholestenone (IC₅₀ at 72 h: 5.1 ± 1 µg / mL) should be considered cytotoxic since it showed an IC₅₀ close to the one considered toxic (< 4 µg / mL). Furthermore, CCD-18Co cells (non-tumor cells) were more vulnerable to the cytotoxic effect of cholesterol metabolites. Possible compensatory responses, attenuating the reduction in cell viability caused by cholesterol metabolites, were observed, however these reactions could favour inflammation and cellular proliferation, likely contributing to the development of CRC. In conclusion, cholesterol metabolites, mainly the most hydrophobic, could act as promoters of CRC through their cytotoxic activity.

Acknowledgement: This work was funded by the Spanish Ministry of Science and Innovation and the European Regional Development Fund (FEDER) (PID2019-104167RB-I00 project).