Vitamin D metabolism in human colon adenocarcinoma-derived Caco-2 cells: Expression of 25-hydroxyvitamin D-3-1 alpha-hydroxylase activity and regulation of side-chain metabolism

1 alpha,25-Dihydroxyvitamin D-3 (1 alpha,25(OH)(2)D-3) and its synthetic analogues exhibit structure-related variations in their growth inhibitory actions in human colon adenocarcinoma-derived Caco-2 cells. Because this might be caused by differences in resistance against metabolic degradation, we used high performance liquid chromatography (HPLC) analysis to investigate pathways of vitamin D metabolism in two different Caco-2 cell clones. Importantly, when Caco-2 cells were incubated with tritium-labelled 25-hydroxyvitamin D-3 (25(OH)D-3) for up to 2 h they produced almost exclusively a metabolite, which was identified as 1 alpha,25(OH)(2)D-3 by co-chromatography with the synthetic standard in two different HPLC systems, and by a radioligand assay showing an identical binding affinity to the intestinal nuclear vitamin D receptor. Expression of the 25(OH)D-3-1 alpha-hydroxylase appears to be constitutive because almost identical enzyme activities are observed in any growth phase. 1 alpha,25(OH)(2)D-3 can also activate side chain metabolism in Caco-2 cells: thereby, 1 alpha,25(OH)(2)D-3 or 25(OH)D-3 are metabolized through the C-24 oxidative pathway into 1 alpha,24(R),25(OH)(3)D-3 and 24(R),25(OH)(2)D-3, respectively, which undergo sequential metabolism into 1 alpha,25(OH)(2)-24oxo-D-3 and 24-oxo-25(OH)D-3, Through C-23 oxidation these intermediary metabolites are further converted into 1 alpha,23,25(OH)(3)-24-oxo-D-3 and 23,25(OH)(2)-24-oxo-D-3, Also direct C-23 oxidation of the substrates 1 alpha,25(OH)(2)D-3 and 25(OH)D-3 generates 1 alpha,23(S),25(OH)(3)D-3 and 23(S),25(OH)(2)D-3, respectively. In summary, our results demonstrated the presence of distinct pathways of vitamin D metabolism in Caco-2 cells: apart from metabolizing 1 alpha,25(OH)(2)D-3 along the C-24 and C-23 oxidative pathways, Caco-2 cells are able to !synthesize 1 alpha,25(OH)(2)D-3 from 25(OH)D-3 through constitutive expression of 25(OH)D-3-1 alpha-hydroxylase activity. The relevance of this finding for the intrinsic growth control of neoplastic colonocytes is discussed. (C) 1997 Elsevier Science Ltd.