Expression and activity of vitamin D-metabolizing cytochrome P450s (CYP1alpha and CYP24) in human nonsmall cell lung carcinomas.

Extrarenal 25-hydroxyvitamin D3-1alpha-hydroxylase is believed to play a major role in the pathogenesis of hypercalcemia associated with various types of granulomatous and lymphoproliferative diseases and certain solid tumors. In this paper, we describe the cloning of the cytochrome P450 component of the extrarenal enzyme from a human nonsmall cell lung carcinoma, SW 900. The cytochrome P450 for the extrarenal 1alpha-hydroxylase has an amino acid sequence identical to that of the cytochrome P450 component of the CYP1alpha, the renal form of the enzyme, and appears to be a product of the same gene. CYP1alpha messenger RNA (mRNA) and 1alpha-hydroxylase enzyme activity were detected in two (SW 900, SK-Luci-6) of a series of five nonsmall cell lung carcinoma cell lines. All five lung cell lines were cultured with the same medium under the same conditions, but only two of the five expressed 1alpha-hydroxylase enzyme; two others (WT-E, Calu-1) expressed high levels of the reciprocally regulated enzyme, 25-hydroxyvitamin D3-24-hydroxylase, with its specific cytochrome P450 component, CYP24. Although under basal conditions the lung cell line SW 900 expressed only CYP1alpha and showed 1alpha-hydroxylase enzyme activity, when treated with small concentrations of 1alpha,25-dihydroxyvitamin D3 or high concentrations of 25-hydroxyvitamin D3, it began to express CYP24 and exhibit 24-hydroxylase enzyme activity. Somewhat surprisingly, SW 900 cells still had detectable CYP1alpha mRNA some 24 h after vitamin D treatment despite the fact that 1alpha-hydroxylase enzyme activity was unmeasurable. These data are consistent with the emerging hypothesis that vitamin D through its active form does not directly turn off CYP1alpha mRNA production but, rather, strongly stimulates CYP24, thereby masking CYP1alpha activity. The factor(s) responsible for the basal expression of CYP1alpha in SW 900 and SK-Luci-6 is currently unknown.

Unique 24-hydroxylated metabolites represent a significant pathway of metabolism of vitamin D2 in humans: 24-hydroxyvitamin D2 and 1,24-dihydroxyvitamin D2 detectable in human serum.

We have produced evidence for a new metabolic pathway for vitamin D2 in humans involving the production of 24-hydroxyvitamin D2 (24OHD2) and 1,24-dihydroxyvitamin D2 [1,24-(OH)2D2]. These metabolites were produced after either a single large dose (10(6) IU) of vitamin D2 or repeated daily doses between 10(3) and 5 x 10(4) IU. We developed assay systems for the metabolites in human serum and showed that in some chronically treated patients, the concentration of 1,24-(OH)2D2 equalled that of 1,25-(OH)2D2 at about 100 pmol/L. The metabolites were identified by high performance liquid chromatography with diode array spectrophotometry for 24OHD2 and by high resolution gas chromatography-mass spectrometry for 1,24-(OH)2D2. We show that 1,24-(OH)2D2 synthesis can be stimulated by PTH, indicating a renal origin for this metabolite and postulate that it is formed from 24OHD2, which may be synthesized in liver. We conclude from this study that vitamin D2 gives rise to two biologically active products, 1,24-(OH)2D2 and 1,25-(OH)2D2, and that 1,24-(OH)2D2 could be an attractive naturally occurring analog of 1,25-(OH)2D3 for clinical use.

Metabolism of a 20-methyl substituted series of vitamin D analogs by cultured human cells: apparent reduction of 23-hydroxylation of the side chain by the 20-methyl group.

We describe here for the first time the effect of introducing a 20-methyl group on the side-chain metabolism of the vitamin D molecule. Using a series of 20-methyl-derivatives of 1alpha,25-(OH)2D3 incubated with two different cultured human cell lines, HPK1A-ras and HepG2, previously shown to metabolize vitamin D compounds, we obtained a series of metabolic products that were identified by comparison to chemically synthesized standards on HPLC and GC-MS. 24-Hydroxylated-, 24-oxo-hydroxylated-, and 24-oxo-23-hydroxylated products of 20-methyl-1alpha,25-(OH)2D3 were observed, but the efficiency of 23-hydroxylation was low as compared with that of the natural hormone and, in contrast to 1alpha,25-(OH)2D3, no truncated 23-alcohol was formed from the 20-methyl analog. These data, taken together with results from other analogs with changes in the vicinity of the C17-C20 positions, lead us to speculate that such changes must alter the accessibility of the C-23 position to the cytochrome P450 involved. Using the HepG2 cell line, we found evidence that the 24S-hydroxylated product of 20-methyl-1alpha,25-(OH)2D3 predominates, implying that the liver cytochrome involved in metabolism is a different isoform. Studies with a more metabolically resistant analog of the series, 20-methyl-Delta(23)-1alpha,25-(OH)2D3, gave the expected block in 23- and 24-hydroxylation, and evidence of an alternative pathway, namely 26-hydroxylation. 20-Methyl-Delta(23)-1alpha,25-(OH)2D3 was also more potent in biological assays, and the metabolic studies reported here help us to suggest explanations for this increased potency. We conclude that the 20-methyl series of vitamin D analogs offers new perspectives into vitamin D analog action, as well as insights into the substrate preferences of the cytochrome(s) P450 involved in vitamin D catabolism.

Anti-proliferative activity and target cell catabolism of the vitamin D analog 1 alpha,24(S)-(OH)2D2 in normal and immortalized human epidermal cells.

Vitamin D analogs represent valuable new agents for the suppression of proliferation of a variety of cell types, including those of the skin. One such analog is the vitamin D2 metabolite, 1 alpha,24(S)-dihydroxyvitamin D2, which binds strongly to the vitamin D receptor and induces vitamin D-dependent gene expression in vitro. In the work described here, we studied the anti-proliferative activity and target cell metabolism of 1 alpha,24(S)-dihydroxyvitamin D2 in cells of human epidermal origin. We found this analog to be equally potent in its anti-proliferative effect to the hormone 1 alpha,25-dihydroxyvitamin D3. Furthermore, 1 alpha,24(S)-dihydroxyvitamin D2 was metabolized by the human keratinocyte cell line HPK1A-ras at a slower rate than either 1 alpha,25-dihydroxyvitamin D3 or calcipotriol, a drug used effectively in the treatment of psoriasis. We characterized the metabolic products of 1 alpha,24(S)-dihydroxyvitamin D2 as a mixture of side-chain truncated and hydroxylated products. The main product was identified by GC-MS and NMR techniques as 1 alpha,24(S),26-trihydroxyvitamin D2. The biological activity of this main product was determined in a vitamin D-dependent, growth-hormone reporter gene expression system to be lower than that of the parent molecule. We conclude from these data that 1 alpha,24(S)-dihydroxyvitamin D2 is a valuable new anti-proliferative agent with a slower rate of catabolism by cells of epidermal origin. Preliminary evidence suggests that the parent molecule, and not its products, is responsible for this biological activity in vitro.

Target cell metabolism of 1,25-dihydroxyvitamin D3 to calcitroic acid. Evidence for a pathway in kidney and bone involving 24-oxidation.

1,25-dihydroxyvitamin D3 is converted to calcitroic acid before being excreted in the bile. Biosynthesis of calcitroic acid has been demonstrated in two target cells of vitamin D, in the kidney and the osteoblastic cell line UMR-106. Calcitroic acid was identified by combinations of h.p.l.c., u.v. spectroscopy and mass spectrometry. Evidence is presented that calcitroate is derived from the 24-oxidation pathway, possibly through the intermediate 24,25,26,27-tetranor-1,23-dihydroxyvitamin D3. The 24-oxidation pathway to calcitroic acid in bone cells is stimulated by 1,25-dihydroxyvitamin D3. The pathway in both bone cells and perfused kidney operates at physiological concentrations of substrate and appears to be capable of rapid clearance of the hormone.

1 alpha,24(S)-dihydroxyvitamin D2: a biologically active product of 1 alpha-hydroxyvitamin D2 made in the human hepatoma, Hep3B.

A major metabolite of the vitamin D analogue 1 alpha-hydroxyvitamin D2 in human liver cells in culture has been identified as 1 alpha,24(S)-dihydroxyvitamin D2 [1 alpha,24(S)-(OH)2D2]. 1 alpha-Hydroxyvitamin D3 incubated with the same cells gives rise to predominantly 25- and 27-hydroxylated products. Our identification of 1 alpha,24(S)-dihydroxyvitamin D2 is based on comparisons of the liver cell metabolite with chemically synthesized 1 alpha,24(S)-(OH)2D2 and 1 alpha,24(R)-(OH)2D2 by using HPLC, GC and GC-MS techniques. The stereochemical orientation of the 24-hydroxyl group was inferred after X-ray-crystallographic analysis of the 24(R)-OH epimer. 1 alpha,24(S)-Dihydroxyvitamin D2 binds strongly to the vitamin D receptor and is biologically active in growth hormone and chloramphenicol acetyltransferase reporter gene expression systems in vitro, but binds poorly to rat vitamin D-binding globulin, DBP. We suggest that this metabolite, 1 alpha,24(S)-(OH)2D2, possesses the spectrum of biological properties to be useful as a drug in the treatment of psoriasis, metabolic bone disease and cancer.

In vitro metabolism of the vitamin D analog, 22-oxacalcitriol, using cultured osteosarcoma, hepatoma, and keratinocyte cell lines.

Using four cultured cell models representing liver, keratinocyte, and osteoblast, we have demonstrated that the vitamin D analog, 22-oxacalcitriol is degraded into a variety of hydroxylated and side chain truncated metabolites. Four of these metabolic products have been rigorously identified by high pressure liquid chromatography, diode array spectrophotometry, and gas chromatography-mass spectrometry analysis as 24-hydroxylated and 26-hydroxylated derivatives as well as the cleaved molecules, hexanor-1alpha,20-dihydroxyvitamin D3 and hexanor-20-oxo-1alpha-hydroxyvitamin D3. Comparison with chemically synthesized standards has revealed the stereochemistry of the biological products. Although differences exist in the amounts of products formed with the different cell types, it is apparent that 22-oxacalcitriol is subject to metabolism by both vitamin D-inducible and noninducible enzymes. Time course studies suggest that the truncated 20-alcohol is derived from a side chain hydroxylated molecule via a hemiacetal intermediate and the 20-oxo derivative is likely formed from the 20-alcohol. Biological activity measurements of the metabolites identified in our studies are consistent with the view that these are catabolites and that the biological activity of 22-oxacalcitriol is due to the parent compound. These results are also consistent with recent findings of others that the biliary excretory form of 22-oxacalcitriol is a glucuronide ester of the truncated 20-alcohol.