title: Metabolism of 1alpha,25-dihydroxyvitamin D(3) in human promyelocytic leukemia (HL-60) cells: in vitro biological activities of the natural metabolites of 1alpha,25-dihydroxyvitamin D(3) produced in HL-60 cells.
abstract: The secosteroid hormone, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], induces differentiation of the human promyelocytic leukemia (HL-60) cells into monocytes/macrophages. At present, the metabolic pathways of 1alpha,25(OH)(2)D(3) and the biologic activity of its various natural intermediary metabolites in HL-60 cells are not fully understood. 1alpha,25(OH)(2)D(3) is metabolized in its target tissues via modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway initiated by hydroxylation at C-24 leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways initiated by hydroxylations at C-23 and C-26 respectively together lead to the formation of the end product, 1alpha,25(OH)(2)D(3)-lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 to form 1alpha,25-dihydroxy-3-epi-vitamin-D(3). We performed the present study first to examine in detail the metabolism of 1alpha,25(OH)(2)D(3) in HL-60 cells and then to assess the ability of the various natural intermediary metabolites of 1alpha,25(OH)(2)D(3) in inducing differentiation and in inhibiting clonal growth of HL-60 cells. We incubated HL-60 cells with [1beta-(3)H] 1alpha,25(OH)(2)D(3) and demonstrated that these cells metabolize 1alpha,25(OH)(2)D(3) mainly via the C-24 oxidation pathway and to a lesser extent via the C-23 oxidation pathway, but not via the C-3-epimerization pathway. Three of the natural intermediary metabolites of 1alpha,25(OH)(2)D(3) derived via the C-24 oxidation pathway namely, 1alpha,24(R),25-trihydroxyvitamin D(3), 1alpha,25-dihydroxy-24-oxovitamin D(3) and 1alpha,23(S),25-trihydroxy-24-oxovitamin D(3) [1alpha,23(S),25(OH)(3)-24-oxo-D(3)] were almost as potent as 1alpha,25(OH)(2)D(3) in terms of their ability to differentiate HL-60 cells into monocytes/macrophages. We then selected 1alpha,23(S),25(OH)(3)-24-oxo-D(3) which has the least calcemic activity among all the three aforementioned natural intermediary metabolites of 1alpha,25(OH)(2)D(3) to examine further its effects on these cells. Our results indicated that 1alpha,23(S),25(OH)(3)-24-oxo-D(3) was also equipotent to its parent in inhibiting clonal growth of HL-60 cells and in inducing expression of CD11b protein. In summary, we report that 1alpha,25(OH)(2)D(3) is metabolized in HL-60 cells into several intermediary metabolites derived via both the C-24 and C-23 oxidation pathways but not via the C-3 epimerization pathway. Some of the intermediary metabolites derived via the C-24 oxidation pathway are found to be almost equipotent to 1alpha,25(OH)(2)D(3) in modulating growth and differentiation of HL-60 cells. In a previous study, the same metabolites when compared to 1alpha,25(OH)(2)D(3) were found to be less calcemic. Thus, the findings of our study suggest that some of the natural metabolites of 1alpha,25(OH)(2)D(3) may be responsible for the final expression of the noncalcemic actions that are presently being attributed to their parent, 1alpha,25(OH)(2)D(3).
authors.affiliation: Department of Pediatrics, Women and Infants' Hospital of Rhode Island, Brown University School of Medicine, Providence, RI 02905, USA.
authors.forename: D S
authors.initials: DS
authors.lastname: Rao
authors.forename: M J
authors.initials: MJ
authors.lastname: Campbell
authors.forename: H P
authors.initials: HP
authors.lastname: Koeffler
authors.forename: S
authors.initials: S
authors.lastname: Ishizuka
authors.forename: M R
authors.initials: MR
authors.lastname: Uskokovic
authors.forename: P
authors.initials: P
authors.lastname: Spagnuolo
authors.forename: G S
authors.initials: GS
authors.lastname: Reddy
mesh_terms: D002117:Calcitriol / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D018922:HL-60 Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006887:Hydroxycholecalciferols / Q000494:pharmacology; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism / Q000473:pathology*; D016177:Macrophage-1 Antigen / Q000187:drug effects / Q000378:metabolism; D010084:Oxidation-Reduction
chemical_list: D006887:Hydroxycholecalciferols; D016177:Macrophage-1 Antigen; C038633:1,23,25-trihydroxy-24-oxo-vitamin D3; D002117:Calcitriol