Biological Effects of CYP11A1-Derived Vitamin D and Lumisterol Metabolites in the Skin.

Novel pathways of vitamin D3, lumisterol 3 (L3), and tachysterol 3 (T3) activation have been discovered, initiated by CYP11A1 and/or CYP27A1 in the case of L3 and T3. The resulting hydroxymetabolites enhance protection of skin against DNA damage and oxidative stress; stimulate keratinocyte differentiation; exert anti-inflammatory, antifibrogenic, and anticancer activities; and inhibit cell proliferation in a structure-dependent manner. They act on nuclear receptors, including vitamin D receptor, aryl hydrocarbon receptor, LXRα/ß, RAR-related orphan receptor α/γ, and peroxisome proliferator-activated receptor-γ, with selectivity defined by their core structure and distribution of hydroxyl groups. They can activate NRF2 and p53 and inhibit NF-κB, IL-17, Shh, and Wnt/ß-catenin signaling. Thus, they protect skin integrity and physiology.

Malignant Melanoma: An Overview, New Perspectives, and Vitamin D Signaling.

Melanoma, originating through malignant transformation of melanin-producing melanocytes, is a formidable malignancy, characterized by local invasiveness, recurrence, early metastasis, resistance to therapy, and a high mortality rate. This review discusses etiologic and risk factors for melanoma, diagnostic and prognostic tools, including recent advances in molecular biology, omics, and bioinformatics, and provides an overview of its therapy. Since the incidence of melanoma is rising and mortality remains unacceptably high, we discuss its inherent properties, including melanogenesis, that make this disease resilient to treatment and propose to use AI to solve the above complex and multidimensional problems. We provide an overview on vitamin D and its anticancerogenic properties, and report recent advances in this field that can provide solutions for the prevention and/or therapy of melanoma. Experimental papers and clinicopathological studies on the role of vitamin D status and signaling pathways initiated by its active metabolites in melanoma prognosis and therapy are reviewed. We conclude that vitamin D signaling, defined by specific nuclear receptors and selective activation by specific vitamin D hydroxyderivatives, can provide a benefit for new or existing therapeutic approaches. We propose to target vitamin D signaling with the use of computational biology and AI tools to provide a solution to the melanoma problem.

Metabolism of Lumisterol2 by CYP27A1.

Lumisterol2 (L2) is a photoproduct of UVB action on the fungal membrane sterol, ergosterol. Like vitamin D2, it is present in edible mushrooms, especially after UV irradiation. Lumisterol3 is similarly produced in human skin from 7-dehydrocholesterol by UVB and can be converted to hydroxy-metabolites by CYP27A1 and CYP11A1. These products are biologically active on human cells with actions that include photoprotection and inhibition of proliferation. The aim of this study was to test the ability of CYP11A1 and CYP27A1 to metabolise L2. Purified CYP27A1 was found to efficiently metabolise L2 to three major products and several minor products, whilst CYP11A1 did not act appreciably on L2. The three major products of CYP27A1 action on L2 were identified by mass spectrometry and NMR as 24-hydroxyL2, 27-hydroxyL2 and 28-hydroxyL2. Minor products included two dihydroxy L2 species, one which was identified as 24,27(OH)2L2, and another metabolite with one oxo and one hydroxyl group added. A comparison on the kinetics of the metabolism of L2 by CYP27A1 with that of the structurally similar compounds, L3 and ergosterol, was carried out with substrates incorporated into phospholipid vesicles. CYP27A1 displayed a 12-fold lower Km with L2 as substrate compared to L3 and a 5-fold lower turnover number (kcat), resulting in a 2.2 fold higher catalytic efficiency (kcat/Km) for L2 metabolism. L2 was a much better substrate for CYP27A1 than its precursor, ergosterol, with a catalytic efficiency 18-fold higher. The major CYP27A1-derived hydroxy-L2 products, 24-hydroxyL2, 27-hydroxyL2 and 28-hydroxyL2, inhibited the proliferation of melanoma and epidermoid cancer cell lines. In conclusion, this study shows that L2 is not metabolized appreciably by CYP11A1, but it is a good substrate for CYP27A1 which hydroxylates its side chain to produce 3 major products that display anti-proliferative activity on skin-cancer cell lines.

CYP11A1­derived vitamin D hydroxyderivatives as candidates for therapy of basal and squamous cell carcinomas.

Hydroxyderivatives of vitamin D3, including classical 1,25(OH)2D3 and novel CYP11A1­derived hydroxyderivatives, exert their biological activity by acting as agonists on the vitamin D receptor (VDR) and inverse agonists on retinoid­related orphan receptors (ROR)α and γ. The anticancer activities of CYP11A1­derived hydroxyderivatives were tested using cell biology, tumor biology and molecular biology methods in human A431 and SCC13 squamous (SCC)­ and murine ASZ001 basal (BCC)­cell carcinomas, in comparison with classical 1,25(OH)2D3. Vitamin D3­hydroxyderivatives with or without a C1α(OH) inhibited cell proliferation in a dose­dependent manner. While all the compounds tested had similar effects on spheroid formation by A431 and SCC13 cells, those with a C1α(OH) group were more potent in inhibiting colony and spheroid formation in the BCC line. Potent anti­tumorigenic activity against the BCC line was exerted by 1,25(OH)2D3, 1,20(OH)2D3, 1,20,23(OH)3D3, 1,20,24(OH)3D3, 1,20,25(OH)3D3 and 1,20,26(OH)3D3, with smaller effects seen for 25(OH)D3, 20(OH)D3 and 20,23(OH)2D3. 1,25(OH)2D3, 1,20(OH)2D3 and 20(OH)D3 inhibited the expression of GLI1 and ß­catenin in ASZ001 cells. In A431 cells, these compounds also decreased the expression of GLI1 and stimulated involucrin expression. VDR, RORγ, RORα and CYP27B1 were detected in A431, SCC13 and ASZ001 lines, however, with different expression patterns. Immunohistochemistry performed on human skin with SCC and BCC showed nuclear expression of all three of these receptors, as well as megalin (transmembrane receptor for vitamin D­binding protein), the level of which was dependent on the type of cancer and antigen tested in comparison with normal epidermis. Classical and CYP11A1­derived vitamin D3­derivatives exhibited anticancer­activities on skin cancer cell lines and inhibited GLI1 and ß­catenin signaling in a manner that was dependent on the position of hydroxyl groups. The observed expression of VDR, RORγ, RORα and megalin in human SCC and BCC suggested that they might provide targets for endogenously produced or exogenously applied vitamin D hydroxyderivatives and provide excellent candidates for anti­cancer therapy.

Metabolic activation of tachysterol3 to biologically active hydroxyderivatives that act on VDR, AhR, LXRs, and PPARγ receptors.

CYP11A1 and CYP27A1 hydroxylate tachysterol3 , a photoproduct of previtamin D3 , producing 20S-hydroxytachysterol3 [20S(OH)T3 ] and 25(OH)T3 , respectively. Both metabolites were detected in the human epidermis and serum. Tachysterol3 was also detected in human serum at a concentration of 7.3 ± 2.5 ng/ml. 20S(OH)T3 and 25(OH)T3 inhibited the proliferation of epidermal keratinocytes and dermal fibroblasts and stimulated the expression of differentiation and anti-oxidative genes in keratinocytes in a similar manner to 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ]. They acted on the vitamin D receptor (VDR) as demonstrated by image flow cytometry and the translocation of VDR coupled GFP from the cytoplasm to the nucleus of melanoma cells, as well as by the stimulation of CYP24A1 expression. Functional studies using a human aryl hydrocarbon receptor (AhR) reporter assay system revealed marked activation of AhR by 20S(OH)T3 , a smaller effect by 25(OH)T3 , and a minimal effect for their precursor, tachysterol3 . Tachysterol3 hydroxyderivatives showed high-affinity binding to the ligan-binding domain (LBD) of the liver X receptor (LXR) α and ß, and the peroxisome proliferator-activated receptor γ (PPARγ) in LanthaScreen TR-FRET coactivator assays. Molecular docking using crystal structures of the LBDs of VDR, AhR, LXRs, and PPARγ revealed high docking scores for 20S(OH)T3 and 25(OH)T3 , comparable to their natural ligands. The scores for the non-genomic-binding site of the VDR were very low indicating a lack of interaction with tachysterol3 ligands. Our identification of endogenous production of 20S(OH)T3 and 25(OH)T3 that are biologically active and interact with VDR, AhR, LXRs, and PPARγ, provides a new understanding of the biological function of tachysterol3 .

Knocking out the Vitamin D Receptor Enhances Malignancy and Decreases Responsiveness to Vitamin D3 Hydroxyderivatives in Human Melanoma Cells.

Vitamin D3 is not only involved in calcium and phosphate metabolism in humans, but it can also affect proliferation and differentiation of normal and cancer cells, including melanoma. The mechanism of the anti-cancer action of vitamin D3 is not fully understood. The nuclear vitamin D receptor (VDR) is crucial for the phenotypic effects of vitamin D hydroxyderivatives. VDR expression shows an inverse correlation with melanoma progression and poor outcome of the disease. In this study we knocked out the VDR in a human melanoma cell line using CRISPR methodology. This enhanced the proliferation of melanoma cells grown in monolayer culture, spheroids or colonies and their migration. Activated forms of vitamin D, including classical 1,25(OH)2D3, 20(OH)D3 and 1,20(OH)2D3, inhibited cell proliferation, migration rate and the ability to form colonies and spheroids in the wild-type melanoma cell line, while VDR KO cells showed a degree of resistance to their action. These results indicate that expression of VDR is important for the inhibition of melanoma growth induced by activated forms of vitamin D. In conclusion, based on our previous clinicopathological analyses and the current study, we suggest that the VDR can function as a melanoma tumor suppressor gene.

Hydroxylumisterols, Photoproducts of Pre-Vitamin D3, Protect Human Keratinocytes against UVB-Induced Damage.

Lumisterol (L3) is a stereoisomer of 7-dehydrocholesterol and is produced through the photochemical transformation of 7-dehydrocholesteol induced by high doses of UVB. L3 is enzymatically hydroxylated by CYP11A1, producing 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3. Hydroxylumisterols function as reverse agonists of the retinoic acid-related orphan receptors α and γ (RORα/γ) and can interact with the non-genomic binding site of the vitamin D receptor (VDR). These intracellular receptors are mediators of photoprotection and anti-inflammatory activity. In this study, we show that L3-hydroxyderivatives significantly increase the expression of VDR at the mRNA and protein levels in keratinocytes, both non-irradiated and after UVB irradiation. L3-hydroxyderivatives also altered mRNA and protein levels for RORα/γ in non-irradiated cells, while the expression was significantly decreased in UVB-irradiated cells. In UVB-irradiated keratinocytes, L3-hydroxyderivatives inhibited nuclear translocation of NFκB p65 by enhancing levels of IκBα in the cytosol. This anti-inflammatory activity mediated by L3-hydroxyderivatives through suppression of NFκB signaling resulted in the inhibition of the expression of UVB-induced inflammatory cytokines, including IL-17, IFN-γ, and TNF-α. The L3-hydroxyderivatives promoted differentiation of UVB-irradiated keratinocytes as determined from upregulation of the expression at the mRNA of involucrin (IVL), filaggrine (FLG), and keratin 14 (KRT14), downregulation of transglutaminase 1 (TGM1), keratins including KRT1, and KRT10, and stimulation of ILV expression at the protein level. We conclude that CYP11A1-derived hydroxylumisterols are promising photoprotective agents capable of suppressing UVB-induced inflammatory responses and restoring epidermal function through targeting the VDR and RORs.

Association among Vitamin D, Retinoic Acid-Related Orphan Receptors, and Vitamin D Hydroxyderivatives in Ovarian Cancer.

Vitamin D and its derivatives, acting via the vitamin D receptor (VDR) and retinoic acid-related orphan receptors γ and α (RORγ and RORα), show anticancer properties. Since pathological conditions are characterized by disturbances in the expression of these receptors, in this study, we investigated their expression in ovarian cancers (OCs), as well as explored the phenotypic effects of vitamin D hydroxyderivatives and RORγ/α agonists on OC cells. The VDR and RORγ showed both a nuclear and a cytoplasmic location, and their expression levels were found to be reduced in the primary and metastatic OCs in comparison to normal ovarian epithelium, as well as correlated to the tumor grade. This reduction in VDR and RORγ expression correlated with a shorter overall disease-free survival. VDR, RORγ, and RORα were also detected in SKOV-3 and OVCAR-3 cell lines with increased expression in the latter line. 20-Hydroxy-lumisterol3 (20(OH)L3) and synthetic RORα/RORγ agonist SR1078 inhibited proliferation only in the OVCAR-3 line, while 20-hydroxyvitamin-D3 (20(OH)D3) only inhibited SKOV-3 cell proliferation. 1,25(OH)2D3, 20(OH)L3, and SR1078, but not 20(OH)D3, inhibited spheroid formation in SKOV-3 cells. In summary, decreases in VDR, RORγ, and RORα expression correlated with an unfavorable outcome for OC, and compounds targeting these receptors had a context-dependent anti-tumor activity in vitro. We conclude that VDR and RORγ expression can be used in the diagnosis and prognosis of OC and suggest their ligands as potential candidates for OC therapy.

The Role of Classical and Novel Forms of Vitamin D in the Pathogenesis and Progression of Nonmelanoma Skin Cancers.

Nonmelanoma skin cancers including basal and squamous cell carcinomas (SCC and BCC) represent a significant clinical problem due to their relatively high incidence, imposing an economic burden to healthcare systems around the world. It is accepted that ultraviolet radiation (UVR: λ = 290-400 nm) plays a crucial role in the initiation and promotion of BCC and SCC with UVB (λ = 290-320 nm) having a central role in this process. On the other hand, UVB is required for vitamin D3 (D3) production in the skin, which supplies >90% of the body's requirement for this prohormone. Prolonged exposure to UVB can also generate tachysterol and lumisterol. Vitamin D3 itself and its canonical (1,25(OH)2D3) and noncanonical (CYP11A1-intitated) D3 hydroxyderivatives show photoprotective functions in the skin. These include regulation of keratinocyte proliferation and differentiation, induction of anti-oxidative responses, inhibition of DNA damage and induction of DNA repair mechanisms, and anti-inflammatory activities. Studies in animals have demonstrated that D3 hydroxyderivatives can attenuate UVB or chemically induced epidermal cancerogenesis and inhibit growth of SCC and BCC. Genomic and non-genomic mechanisms of action have been suggested. In addition, vitamin D3 itself inhibits hedgehog signaling pathways which have been implicated in many cancers. Silencing of the vitamin D receptor leads to increased propensity to develop UVB or chemically induced epidermal cancers. Other targets for vitamin D compounds include 1,25D3-MARRS, retinoic orphan receptors α and γ, aryl hydrocarbon receptor, and Wnt signaling. Most recently, photoprotective effects of lumisterol hydroxyderivatives have been identified. Clinical trials demonstrated a beneficial role of vitamin D compounds in the treatment of actinic keratosis. In summary, recent advances in vitamin D biology and pharmacology open new exciting opportunities in chemoprevention and treatment of skin cancers.

Noncalcemic Vitamin D Hydroxyderivatives Inhibit Human Oral Squamous Cell Carcinoma and Down-regulate Hedgehog and WNT/ß-Catenin Pathways.

BACKGROUND/AIM: The hormonally-active form of vitamin D, 1,25(OH)2D3, demonstrated activity against oral squamous cell carcinoma (OSCC). Cytochrome P450scc (CYP11A1)-derived vitamin D hydroxyderivatives, such as 20(OH)D3 and 1,20(OH)2D3, have overlapping beneficial effects with 1,25(OH)2D3 without causing hypercalcemia. This study sought to determine (i) whether 20(OH)D3 and 1,20(OH)2D3 exhibit antitumor effects against OSCC comparable to those of 1,25(OH)2D3 and (ii) whether these effects may stem from down-regulation of sonic hedgehog (SHH) or WNT/ß-catenin signaling pathways. MATERIALS AND METHODS: Effects on CAL-27 cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt and spheroid assays. Signaling pathways were assessed by immunofluorescence and western blotting. RESULTS: 20(OH)D3 and 1,20(OH)2D3 inhibited the growth of CAL-27 and demonstrated inhibition of WNT/ß-catenin and the SHH signaling as evidenced by down-regulation of nuclear translocation of glioma-associated oncogene 1(GLI1) and ß-catenin. CONCLUSION: Noncalcemic vitamin D hydroxyderivatives demonstrated antitumor activities against OSCC comparable to those of 1,25(OH)2D3 Their activities against SHH and the WNT/ß-catenin pathways provide insight for a possible target for OSCC treatment.

Photoprotective Properties of Vitamin D and Lumisterol Hydroxyderivatives.

We have previously described new pathways of vitamin D3 activation by CYP11A1 to produce a variety of metabolites including 20(OH)D3 and 20,23(OH)2D3. These can be further hydroxylated by CYP27B1 to produce their C1α-hydroxyderivatives. CYP11A1 similarly initiates the metabolism of lumisterol (L3) through sequential hydroxylation of the side chain to produce 20(OH)L3, 22(OH)L3, 20,22(OH)2L3 and 24(OH)L3. CYP11A1 also acts on 7-dehydrocholesterol (7DHC) producing 22(OH)7DHC, 20,22(OH)27DHC and 7-dehydropregnenolone (7DHP) which can be converted to the D3 and L3 configurations following exposure to UVB. These CYP11A1-derived compounds are produced in vivo and are biologically active displaying anti-proliferative, anti-inflammatory, anti-cancer and pro-differentiation properties. Since the protective role of the classical form of vitamin D3 (1,25(OH)2D3) against UVB-induced damage is recognized, we recently tested whether novel CYP11A1-derived D3- and L3-hydroxyderivatives protect against UVB-induced damage in epidermal human keratinocytes and melanocytes. We found that along with 1,25(OH)2D3, CYP11A1-derived D3-hydroxyderivatives and L3 and its hydroxyderivatives exert photoprotective effects. These included induction of intracellular free radical scavenging and attenuation and repair of DNA damage. The protection of human keratinocytes against DNA damage included the activation of the NRF2-regulated antioxidant response, p53-phosphorylation and its translocation to the nucleus, and DNA repair induction. These data indicate that novel derivatives of vitamin D3 and lumisterol are promising photoprotective agents. However, detailed mechanisms of action, and the involvement of specific nuclear receptors, other vitamin D binding proteins or mitochondria, remain to be established.

Protective effects of novel derivatives of vitamin D3 and lumisterol against UVB-induced damage in human keratinocytes involve activation of Nrf2 and p53 defense mechanisms.

We tested whether novel CYP11A1-derived vitamin D3- and lumisterol-hydroxyderivatives, including 1,25(OH)2D3, 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3, 1,20,23(OH)3D3, lumisterol, 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3, can protect against UVB-induced damage in human epidermal keratinocytes. Cells were treated with above compounds for 24 h, then subjected to UVB irradiation at UVB doses of 25, 50, 75, or 200 mJ/cm2, and then examined for oxidant formation, proliferation, DNA damage, and the expression of genes at the mRNA and protein levels. Oxidant formation and proliferation were determined by the DCFA-DA and MTS assays, respectively. DNA damage was assessed using the comet assay. Expression of antioxidative genes was evaluated by real-time RT-PCR analysis. Nuclear expression of CPD, phospho-p53, and Nrf2 as well as its target proteins including HO-1, CAT, and MnSOD, were assayed by immunofluorescence and western blotting. Treatment of cells with the above compounds at concentrations of 1 or 100 nM showed a dose-dependent reduction in oxidant formation. At 100 nM they inhibited the proliferation of cultured keratinocytes. When keratinocytes were irradiated with 50-200 mJ/cm2 of UVB they also protected against DNA damage, and/or induced DNA repair by enhancing the repair of 6-4PP and attenuating CPD levels and the tail moment of comets. Treatment with test compounds increased expression of Nrf2-target genes involved in the antioxidant response including GR, HO-1, CAT, SOD1, and SOD2, with increased protein expression for HO-1, CAT, and MnSOD. The treatment also stimulated the phosphorylation of p53 at Ser-15, increased its concentration in the nucleus and enhanced Nrf2 translocation into the nucleus. In conclusion, pretreatment of keratinocytes with 1,25(OH)2D3 or CYP11A1-derived vitamin D3- or lumisterol hydroxy-derivatives, protected them against UVB-induced damage via activation of the Nrf2-dependent antioxidant response and p53-phosphorylation, as well as by the induction of the DNA repair system. Thus, the new vitamin D3 and lumisterol hydroxy-derivatives represent promising anti-photodamaging agents.

Vitamin D and its low calcemic analogs modulate the anticancer properties of cisplatin and dacarbazine in the human melanoma A375 cell line.

Melanoma represents a significant challenge in cancer treatment due to the high drug resistance of melanomas and the patient mortality rate. This study presents data indicating that nanomolar concentrations of the hormonally active form of vitamin D, 1α,25­dihydroxyvitamin D3 [1α,25(OH)2D3], its non­calcemic analogues 20S­hydroxyvitamin D3 and 21­hydroxypregnacalciferol, as well as the low­calcemic synthetic analog calcipotriol, modulate the efficacy of the anticancer drugs cisplatin and dacarbazine. It was observed that vitamin D analogs sensitized melanoma A375 cells to hydrogen peroxide used as an inducer of oxidative stress. On the other hand, only 1α,25(OH)2D3 resulted in a minor, but significant effect on the proliferation of melanoma cells treated simultaneously with dacarbazine, but not cisplatin. Notably, cisplatin (300 µM) exhibited a higher overall antiproliferative activity than dacarbazine. Cisplatin treatment of melanoma cells resulted in an induction of apoptosis as demonstrated by flow cytometry (accumulation of cells at the subG1 phase of the cell cycle), whereas dacarbazine caused G1/G0 cell cycle arrest, with the effects being improved by pre­treatment with vitamin D analogs. Treatment with cisplatin resulted in an initial increase in the level of reactive oxygen species (ROS). Dacarbazine caused transient stimulation of ROS levels and the mitochondrial membrane potential (Δψm) (after 1 or 3 h of treatment, respectively), but the effect was not detectable following prolonged (24 h) incubation with the drug. Vitamin D exhibited modulatory effects on the cells treated with dacarbazine, decreasing the half maximal inhibitory concentration (IC50) for the drug, stimulating G1/G0 arrest and causing a marked decrease in Δψm. Finally, cisplatin, dacarbazine and 1α,25(OH)2D3 displayed modulatory effects on the expression of ROS and vitamin D­associated genes in the melanoma A375 cells. In conclusion, nanomolar concentrations of 1,25(OH)2D3 only had minor effects on the proliferation of melanoma cells treated with dacarbazine, decreasing the relative IC50 value. However, co­treatment with vitamin D analogs resulted in the modulation of cell cycle and ROS responses, and affected gene expression, suggesting possible crosstalk between the signaling pathways of vitamin D and the anticancer drugs used in this study.

The serum vitamin D metabolome: What we know and what is still to discover.

Vitamin D, referring to the two forms, D2 from the diet and D3 primarily derived from phototransformation in the skin, is a prohormone important in human health. The most hormonally active form, 1α,25-dihydroxyvitamin D (1α,25(OH)2D), formed from vitamin D via 25-hydroxyvitamin D (25(OH)D), is not only important for regulating calcium metabolism, but has many pleiotropic effects including regulation of the immune system and has anti-cancer properties. The major circulating form of vitamin D is 25(OH)D and both D2 and D3 forms are routinely measured by LC/MS/MS to assess vitamin D status, due to their relatively long half-lives and much higher concentrations compared to 1α,25(OH)2D. Inactivation of both 25(OH)D and 1α,25(OH)2D is catalyzed by CYP24A1 and 25-hydroxyvitamin D3 3-epimerase. Initial products from these enzymes acting on 25(OH)D3 are 24R,25(OH)2D3 and 3-epi-25(OH)D3, respectively, and both of these can also be measured routinely in some clinical laboratories to further document vitamin D status. With advances in LC/MS/MS and its increased availability, and with the help of studies with recombinant vitamin D-metabolizing enzymes, many other vitamin D metabolites have now been detected and in some cases quantitated, in human serum. CYP11A1 which catalyzes the first step in steroidogenesis, has been found to also act on vitamins D3 and D2 hydroxylating both at C20, but with some secondary metabolites produced by subsequent hydroxylations at other positions on the side chain. The major vitamin D3 metabolite, 20S-hydroxyvitamin D3 (20S(OH)D3), shows biological activity, often similar to 1α,25(OH)2D3 but without calcemic effects. Using standards produced enzymatically by purified CYP11A1 and characterized by NMR, many of these new metabolites have been detected in human serum, with semi-quantitative measurement of 20S(OH)D3 indicating it is present at comparable concentrations to 24R,25(OH)2D3 and 3-epi-25(OH)D3. Recently, vitamin D-related hydroxylumisterols derived from lumisterol3, a previtamin D3 photoproduct, have also been measured in human serum and displayed biological activity in initial in vitro studies. With the current extensive knowledge on the reactions and pathways of metabolism of vitamin D, especially those catalyzed by CYP24A1, CYP27A1, CYP27B1, CYP3A4 and CYP11A1, it is likely that many other of the resulting hydroxyvitamin D metabolites will be measured in human serum in the future, some contributing to a more detailed understanding of vitamin D status in health and disease.

CYP27A1 acts on the pre-vitamin D3 photoproduct, lumisterol, producing biologically active hydroxy-metabolites.

Prolonged exposure of the skin to UV radiation causes previtamin D3, the initial photoproduct formed by opening of the B ring of 7-dehydrocholesterol, to undergo a second photochemical reaction where the B-ring is reformed giving lumisterol3 (L3), a stereoisomer of 7-dehydrocholesterol. L3 was believed to be an inactive photoproduct of excessive UV radiation whose formation prevents excessive vitamin D production. Recently, we reported that L3 is present in serum and that CYP11A1 can act on L3 producing monohydroxy- and dihydroxy-metabolites which inhibit skin cell proliferation similarly to 1α,25-dihydroxyvitamin D3. In this study we tested the ability of human CYP27A1 to hydroxylate L3. L3 was metabolized by purified CYP27A1 to 3 major products identified as 25-hydroxyL3, (25R)-27-hydroxyL3 and (25S)-27-hydroxyL3, by NMR. These three products were also seen when mouse liver mitochondria containing CYP27A1 were incubated with L3. The requirement for CYP27A1 for their formation by mitochondria was confirmed by the inhibition of their synthesis by 5ß-cholestane-3α,7α,12α-triol, an intermediate in bile acid synthesis which serves as an efficient competitive substrate for CYP27A1. CYP27A1 displayed a high kcat for the metabolism of L3 (76 mol product/min/mol CYP27A1) and a catalytic efficiency (kcat/Km) that was 260-fold higher than that for vitamin D3. The CYP27A1-derived hydroxy-derivatives inhibited the proliferation of cultured human melanoma cells and colony formation with IC50 values in the nM range. Thus, L3 is metabolized efficiently by CYP27A1 with hydroxylation at C25 or C27 producing metabolites potent in their ability to inhibit melanoma cell proliferation, supporting that L3 is a prohormone which can be activated by CYP-dependent hydroxylations.

On the role of classical and novel forms of vitamin D in melanoma progression and management.

Melanoma represents a significant clinical problem affecting a large segment of the population with a relatively high incidence and mortality rate. Ultraviolet radiation (UVR) is an important etiological factor in malignant transformation of melanocytes and melanoma development. UVB, while being a full carcinogen in melanomagenesis, is also necessary for the cutaneous production of vitamin D3 (D3). Calcitriol (1,25(OH)2D3) and novel CYP11A1-derived hydroxyderivatives of D3 show anti-melanoma activities and protective properties against damage induced by UVB. The former activities include inhibitory effects on proliferation, plating efficiency and anchorage-independent growth of cultured human and rodent melanomas in vitro, as well as the in vivo inhibition of tumor growth by 20(OH)D3 after injection of human melanoma cells into immunodeficient mice. The literature indicates that low levels of 25(OH)D3 are associated with more advanced melanomas and reduced patient survivals, while single nucleotide polymorphisms of the vitamin D receptor or the D3 binding protein gene affect development or progression of melanoma, or disease outcome. An inverse correlation of VDR and CYP27B1 expression with melanoma progression has been found, with low or undetectable levels of these proteins being associated with poor disease outcomes. Unexpectedly, increased expression of CYP24A1 was associated with better melanoma prognosis. In addition, decreased expression of retinoic acid orphan receptors α and γ, which can also bind vitamin D3 hydroxyderivatives, showed positive association with melanoma progression and shorter disease-free and overall survival. Thus, inadequate levels of biologically active forms of D3 and disturbances in expression of the target receptors, or D3 activating or inactivating enzymes, can affect melanomagenesis and disease progression. We therefore propose that inclusion of vitamin D into melanoma management should be beneficial for patients, at least as an adjuvant approach. The presence of multiple hydroxyderivatives of D3 in skin that show anti-melanoma activity in experimental models and which may act on alternative receptors, will be a future consideration when planning which forms of vitamin D to use for melanoma therapy.

Characterization of a new pathway that activates lumisterol in vivo to biologically active hydroxylumisterols.

Using LC/qTOF-MS we detected lumisterol, 20-hydroxylumisterol, 22-hydroxylumisterol, 24-hydroxylumisterol, 20,22-dihydroxylumisterol, pregnalumisterol, 17-hydroxypregnalumisterol and 17,20-dihydroxypregnalumisterol in human serum and epidermis, and the porcine adrenal gland. The hydroxylumisterols inhibited proliferation of human skin cells in a cell type-dependent fashion with predominant effects on epidermal keratinocytes. They also inhibited melanoma proliferation in both monolayer and soft agar. 20-Hydroxylumisterol stimulated the expression of several genes, including those associated with keratinocyte differentiation and antioxidative responses, while inhibiting the expression of others including RORA and RORC. Molecular modeling and studies on VDRE-transcriptional activity excludes action through the genomic site of the VDR. However, their favorable interactions with the A-pocket in conjunction with VDR translocation studies suggest they may act on this non-genomic VDR site. Inhibition of RORα and RORγ transactivation activities in a Tet-on CHO cell reporter system, RORα co-activator assays and inhibition of (RORE)-LUC reporter activity in skin cells, in conjunction with molecular modeling, identified RORα and RORγ as excellent receptor candidates for the hydroxylumisterols. Thus, we have discovered a new biologically relevant, lumisterogenic pathway, the metabolites of which display biological activity. This opens a new area of endocrine research on the effects of the hydroxylumisterols on different pathways in different cells and the mechanisms involved.

Vitamin D signaling and melanoma: role of vitamin D and its receptors in melanoma progression and management.

Ultraviolet B (UVB), in addition to having carcinogenic activity, is required for the production of vitamin D3 (D3) in the skin which supplies >90% of the body's requirement. Vitamin D is activated through hydroxylation by 25-hydroxylases (CYP2R1 or CYP27A1) and 1α-hydroxylase (CYP27B1) to produce 1,25(OH)2D3, or through the action of CYP11A1 to produce mono-di- and trihydroxy-D3 products that can be further modified by CYP27B1, CYP27A1, and CYP24A1. The active forms of D3, in addition to regulating calcium metabolism, exert pleiotropic activities, which include anticarcinogenic and anti-melanoma effects in experimental models, with photoprotection against UVB-induced damage. These diverse effects are mediated through an interaction with the vitamin D receptor (VDR) and/or as most recently demonstrated through action on retinoic acid orphan receptors (ROR)α and RORγ. With respect to melanoma, low levels of 25(OH)D are associated with thicker tumors and reduced patient survival. Furthermore, single-nucleotide polymorphisms of VDR and the vitamin D-binding protein (VDP) genes affect melanomagenesis or disease outcome. Clinicopathological analyses have shown positive correlation between low or undetectable expression of VDR and/or CYP27B1 in melanoma with tumor progression and shorter overall (OS) and disease-free survival (DFS) times. Paradoxically, this correlation was reversed for CYP24A1 (inactivating 24-hydroxylase), indicating that this enzyme, while inactivating 1,25(OH)2D3, can activate other forms of D3 that are products of the non-canonical pathway initiated by CYP11A1. An inverse correlation has been found between the levels of RORα and RORγ expression and melanoma progression and disease outcome. Therefore, we propose that defects in vitamin D signaling including D3 activation/inactivation, and the expression and activity of the corresponding receptors, affect melanoma progression and the outcome of the disease. The existence of multiple bioactive forms of D3 and alternative receptors affecting the behavior of melanoma should be taken into consideration when applying vitamin D management for melanoma therapy.

Noncalcemic 20-hydroxyvitamin D3 inhibits human melanoma growth in in vitro and in vivo models.

A novel pathway of vitamin D3 (D3) metabolism, initiated by C20-hydroxylation of D3 by CYP11A1, has been confirmed to operate in vivo. Its major product, 20(OH)D3, exhibits antiproliferative activity in vitro comparable to that of 1,25(OH)2D3, but is noncalcemic in mice and rats. To further characterize the antimelanoma activity of 20(OH)D3, we tested its effect on colony formation of human melanoma cells in monolayer culture and anchorage-independent growth in soft agar. The migratory capabilities of the cells and cell-cell and cell-extracellular matrix interactions were also evaluated using transwell cell migration and spheroid toxicity assays. To assess the antimelanoma activity of 20(OH)D3in vivo, age-matched immunocompromised mice were subcutaneously implanted with luciferase-labelled SKMel-188 cells and were randomly assigned to be treated with either 20(OH)D3 or vehicle (n=10 per group). Tumor size was measured with caliper and live bioimaging methods, and overall health condition expressed as a total body score scale. The following results were observed: (i) 20(OH)D3 inhibited colony formation both in monolayer and soft agar conditions, (ii) 20(OH)D3 inhibited melanoma cells in both transwell migration and spheroid toxicity assays, and (iii) 20(OH)D3 inhibited melanoma tumor growth in immunocompromised mice without visible signs of toxicity. However, although the survival rate was 90% in both groups, the total body score was higher in the treatment group compared to control group (2.8 vs. 2.55). In conclusion, 20(OH)D3, an endogenously produced secosteroid, is an excellent candidate for further preclinical testing as an antimelanoma agent.

Synthesis and Biological Evaluation of Vitamin D3 Metabolite 20S,23S-Dihydroxyvitamin D3 and Its 23R Epimer.

The vitamin D3 metabolite, 20S,23S-dihydroxyvitamin D3, was chemically synthesized for the first time and identified to be the same as the enzymatically produced metabolite. The C23 absolute configurations of both 20S,23S/R-dihydroxyvitamin D3 epimers were unambiguously assigned by NMR and Mosher ester analysis. Their kinetics of CYP27B1 metabolism were investigated during the production of their 1α-hydroxylated derivatives. Bioactivities of these products were compared in terms of vitamin D3 receptor activation, anti-inflammatory, and antiproliferative activities.