Vitamin D metabolism in human colon adenocarcinoma-derived Caco-2 cells: expression of 25-hydroxyvitamin D3-1alpha-hydroxylase activity and regulation of side-chain metabolism.
1Alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) 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 D3 (25(OH)D3) for up to 2 h they produced almost exclusively a metabolite, which was identified as 1alpha,25(OH)2D3 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)D3-1alpha-hydroxylase appears to be constitutive because almost identical enzyme activities are observed in any growth phase. 1Alpha,25(OH)2D3 can also activate side chain metabolism in Caco-2 cells: thereby, 1alpha,25(OH)2D3 or 25(OH)D3 are metabolized through the C-24 oxidative pathway into 1alpha,24(R),25(OH)3D3 and 24(R),25(OH)2D3, respectively, which undergo sequential metabolism into 1alpha,25(OH)2-24oxo-D3 and 24-oxo-25(OH)D3. Through C-23 oxidation these intermediary metabolites are further converted into 1alpha,23,25(OH)3-24-oxo-D3 and 23,25(OH)2-24-oxo-D3. Also direct C-23 oxidation of the substrates 1alpha,25(OH)2D3 and 25(OH)D3 generates 1alpha,23(S),25(OH)3D3 and 23(S),25(OH)2D3, respectively. In summary, our results demonstrated the presence of distinct pathways of vitamin D metabolism in Caco-2 cells: apart from metabolizing 1alpha,25(OH)2D3 along the C-24 and C-23 oxidative pathways, Caco-2 cells are able to synthesize 1alpha,25(OH)2D3 from 25(OH)D3 through constitutive expression of 25(OH)D3-1alpha-hydroxylase activity. The relevance of this finding for the intrinsic growth control of neoplastic colonocytes is discussed.
Cross
H S
HS
Department of General and Experimental Pathology, University of Vienna Medical School, Austria.
Peterlik
M
M
Reddy
G S
GS
Schuster
I
I
D018938:Caco-2 Cells; D002112:Calcifediol / Q000378:metabolism; D002117:Calcitriol / Q000378:metabolism; D053493:Cholestanetriol 26-Monooxygenase; D002851:Chromatography, High Pressure Liquid; D002999:Clone Cells; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D018167:Receptors, Calcitriol / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000096:biosynthesis*; D014807:Vitamin D / Q000031:analogs & derivatives / Q000378:metabolism*
D018167:Receptors, Calcitriol; D014807:Vitamin D; D013250:Steroid Hydroxylases; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; D002117:Calcitriol; D002112:Calcifediol