Investigation of Vitamin D2 and Vitamin D3 Hydroxylation by Kutzneria albida

The active vitamin D metabolites 25-OH−D and 1α,25-(OH)₂−D play an essential role in controlling several cellular processes in the human body and are potentially effective in the treatment of several diseases, such as autoimmune diseases, cardiovascular diseases and cancer. The microbial synthesis of vitamin D₂ (VD₂) and vitamin D₃ (VD₃) metabolites has emerged as a suitable alternative to established complex chemical syntheses. In this study, a novel strain, Kutzneria albida, with the ability to form 25-OH−D₂ and 25-OH−D₃ was identified. To further improve the conversion of the poorly soluble substrates, several solubilizers were tested. 100-fold higher product concentrations of 25-OH−D₃ and tenfold higher concentrations of 25-OH−D₂ after addition of 5 % (w/v) 2-hydroxypropyl β-cyclodextrin (2-HPβCD) were reached. Besides the single-hydroxylation products, the human double-hydroxylation products 1,25-(OH)₂−D₂ and 1,25-(OH)₂−D₃ and various other potential single- and double-hydroxylation products were detected. Thus, K. albida represents a promising strain for the biotechnological production of VD₂ and VD₃ metabolites.     

Recent Developments of 19‐Nor‐1,25‐dihydroxyvitamin D3 Analogues

The vitamin D hormone, 1α,25‐dihydroxyvitamin D₃ [1,25‐(OH)₂D₃], exerts its hormonal effects predominantly on intestine, bone, and kidney, where it plays a crucial role in calcium and phosphorus homeostasis and bone mineralization. In addition to its classical actions, 1,25(OH)₂D₃ exerts pleiotropic effects in a wide variety of target tissues and cell types, often in an autocrine/paracrine fashion. These biological activities of 1,25(OH)₂D₃ have suggested a multitude of potential therapeutic applications for the vitamin D hormone in the treatment of hyperproliferative disorders (e.g. cancer and psoriasis), immune dysfunction (autoimmune diseases), and endocrine disorders (e.g. hyperparathyroidism). However, the calcemic effects induced by 1,25(OH)₂D₃—hypercalcemia, increased bone resorption, and soft tissue calcification—limit the use of the natural ligand in these clinical applications. Therefore, numerous 1,25(OH)₂D₃ analogues have been synthesized with the intent of producing therapeutic agents devoid of hypercalcemic and hyperphosphatemic side effects. To this aim, much attention has been focused on the development of 19‐nor‐vitamin D₃ derivatives that lack the ring‐A exocyclic methylene group (C19). In this review, the 19‐nor‐1,25(OH)₂D₃ analogues are classified according to modifications made at the A‐ring, the side chain, or both the A‐ring and side chain, as well as other positions. The biological activities of these 19‐nor‐1,25(OH)₂D₃ analogues are summarized and their structure–activity relationships and binding features with the vitamin D receptor (VDR) are discussed.     

Optimization of Photoreaction for the Production of Vitamin D2

The photochemical formation of vitamin D₂ (VD₂) is complicated, and optimization is essential for commercial production. An optimization methodology is introduced for investigating the factors influencing highly effective production of VD₂. For this research, the irradiation time and the initial concentration of ergosterol (C_E) were fully considered as the significant factors to simulate in the predictive models developed using SPSS and MATLAB software. The data obtained from the designed experiments were fit as two variable equations and their relationships were plotted as three‐dimensional surfaces. The model has a quite satisfactory coefficient (R² > 0.99). The best conditions observed were: C_E = 1.09 g/dm³ and an irradiation time of 64.5 min, while the yield of pre‐vitamin D₂ (P) reached a maximum of 45.83 %. Three experiments were performed and an average yield 45.93 % was obtained. The yield obtained could be compared with the predicted yield satisfactorily.     

Synthesis, structure, and biological activity of des-side chain analogues of 1α,25-dihydroxyvitamin D3 with substituents at C18

An improved synthetic route to 1α,25‐dihydroxyvitamin D₃ des‐side chain analogues 2 a and 2 b with substituents at C18 is reported, along with their biological activity. These analogues display significant antiproliferative effects toward MCF‐7 breast cancer cells and prodifferentiation activity toward SW480‐ADH colon cancer cells; they are also characterized by a greatly decreased calcemic profile. The crystal structure of the human vitamin D receptor (hVDR) complexed to one of these analogues, 20(17→18)‐abeo‐1α,25‐dihydroxy‐22‐homo‐21‐norvitamin D₃ ( 2 a ) reveals that the side chain introduced at position C18 adopts the same orientation in the ligand binding pocket as the side chain of 1α,25‐dihydroxyvitamin D₃.     

Microbial conversion of sterol‐containing soybean oil production waste

Soybean extract residue (scum), a waste of soybean oil production, was examined as a raw material for C₁₇‐ketosteroid production. As a model process, its bioconversion to 9α‐hydroxyandrost‐4‐ene‐3,17‐dione (9‐OH‐AD) by Mycobacterium sp VKM Ac‐1817D was studied. The content of transformable sterols (sitosterol, stigmasterol and campesterol) in scum was estimated at ～14%. The bioconversion of scum to 9‐OH‐AD was characterized by a long lag‐period (300–350 h) followed by 9‐OH‐AD accumulation. The microbial or chemical elimination of fatty non‐identified components resulted in sterol‐enriched scum preparations. Effective conversion of these preparations by Mycobacterium sp was demonstrated: 9‐OH‐AD molar yield ～65% was reached at 60 h from the scum preparation containing 10 g dm^?3 transformable sterols. The process productivity was comparable with that for high quality‐sitosterol of wood origin (tall‐oil sitosterol). Copyright © 2004 Society of Chemical Industry     

Chemical Synthesis of Side-Chain-Hydroxylated Vitamin D3 Derivatives and Their Metabolism by CYP27B1

1α,25-Dihydroxyvitamin D₃ (abbreviated here as 1,25D₃) is a hormonally active form of vitamin D₃ (D₃), and is produced from D₃ by CYP27 A1-mediated hydroxylation at C25, followed by CYP27B1-mediated hydroxylation at C1. Further hydroxylation of 25D₃ and 1,25D₃ occurs at C23, C24 and C26 to generate corresponding metabolites, except for 1,25R,26D₃. Since the capability of CYP27B1 to hydroxylate C1 of side-chain-hydroxylated metabolites other than 23S,25D₃ and 24R,25D₃ has not been examined, we have here explored the role of CYP27B1 in the C1 hydroxylation of a series of side-chain-hydroxylated D₃ derivatives. We found that CYP27B1 hydroxylates the R diastereomers of 24,25D₃ and 25,26D₃ more effectively than the S diastereomers, but shows almost no activity towards either diastereomer of 23,25D₃. This is the first report to show that CYP27B1 metabolizes 25,26D₃ to the corresponding 1α-hydroxylated derivative, 1,25,26D₃. It will be interesting to examine the physiological relevance of this finding.     

Galectin-3 Contributes to the Inhibitory Effect of lα,25-(OH)2D3 on Osteoclastogenesis

The active form of vitamin D, 1α,25-(OH)₂D₃, not only promotes intestinal calcium absorption, but also regulates the formation of osteoclasts (OCs) and their capacity for bone mineral dissolution. Gal-3 is a newly discovered bone metabolic regulator involved in the proliferation, differentiation, and apoptosis of various cells. However, the role of galectin-3 (gal-3) in OC formation and the regulatory effects of 1α,25-(OH)₂D₃ have yet to be explored. To confirm whether gal-3 contributes to the regulatory effects of 1α,25-(OH)₂D₃ on osteoclastogenesis, osteoclast precursors (OCPs) were induced by macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). TRAP staining and bone resorption analyses were used to verify the formation and activation of OCs. qPCR, Western blotting, co-immunoprecipitation, and immunofluorescence assays were used to detect gene and protein expression. The regulatory effects of gal-3 in OC formation after treatment with 1α,25-(OH)₂D₃ were evaluated using gal-3 siRNA. The results showed that 1α,25-(OH)₂D₃ significantly increased gal-3 expression and inhibited OC formation and bone resorption. Expression levels of OC-related genes and proteins, matrix metalloproteinase 9 (MMP-9), nuclear factor of activated T cells 1 (NFATc1), and cathepsin K (Ctsk) were also inhibited by 1α,25-(OH)₂D₃. Gal-3 knockdown attenuated the inhibitory effects of 1α,25-(OH)₂D₃ on OC formation, activation, and gene and protein expression. In addition, gal-3 was co-localized with the vitamin D receptor (VDR). These data suggest that gal-3 contributes to the osteoclastogenesis inhibitory effect of lα,25-(OH)₂D₃, which is involved in bone and calcium homeostasis.     

25(OH)D Is Effective to Repress Human Cholangiocarcinoma Cell Growth through the Conversion of 25(OH)D to 1α,25(OH)2D3

Cholangiocarcinoma (CCA) is a devastating disease without effective treatments. 1α,25(OH)₂D₃, the active form of Vitamin D, has emerged as a new anti-cancer regimen. However, the side effect of hypercalcemia impedes its systemic administration. 25(OH)D is biologically inert and needs hydroxylation by CYP27B1 to form 1α,25(OH)₂D₃, which is originally believed to only take place in kidneys. Recently, the extra-renal expression of CYP27B1 has been identified and in vitro conversion of 25(OH)D to 1α,25(OH)₂D₃ has been found in some cancer cells with CYP27B1 expression. In this study, CYP27B1 expression was demonstrated in CCA cells and human CCA specimens. 25(OH)D effectively represses SNU308 cells growth, which was strengthened or attenuated as CYP27B1 overexpression or knockdown. Lipocalcin-2 (LCN2) was also found to be repressed by 25(OH)D. After treatment with 800 ng/mL 25(OH)D, the intracellular 1α,25(OH)₂D₃ concentration was higher in SNU308 cells with CYP27B1 overexpression than wild type SNU308 cells. In a xenograft animal experiment, 25(OH)D, at a dose of 6 μg/kg or 20 μg/kg, significantly inhibited SNU308 cells’ growth without inducing obvious side effects. Collectively, our results indicated that SNU308 cells were able to convert 25(OH)D to 1α,25(OH)₂D₃ and 25(OH)D CYP27B1 gene therapy could be deemed as a promising therapeutic direction for CCA.     

Polyethylene‐block‐poly(ε‐caprolactone) diblock copolymers: synthesis and compatibility

A strategy is introduced for the synthesis of polyethylene‐block‐poly(ε‐caprolactone) block copolymers by a combination of coordination polymerization and ring‐opening polymerization. First, end‐hydroxylated polyethylene (PE‐OH) was prepared with a one‐step process through ethylene/3‐buten‐1‐ol copolymerization catalyzed by a vanadium(III) complex bearing a bidentate [N,O] ligand ([PhN?C(CH₃)CHC(Ph)O]VCl₂(THF)₂). The PE‐OH was then used as macroinitiator for ring‐opening polymerization of ε‐caprolactone, leading to the desired nonpolar/polar diblock copolymers. The block structure was confirmed by spectral analysis using ¹H NMR, gel permeation chromatography and differential scanning calorimetry. The unusual topologies of the model copolymers will establish a fundamental understanding for structure–property correlations, e.g. compatibilization, of polymer blends and surface and interface modification of other polymers. © 2014 Society of Chemical Industry     

Controlled synthesis of vinylmethylsiloxane-dimethylsiloxane gradient, block and alternate copolymers by anionic ROP of cyclotrisiloxanes

Three methods for the controlled synthesis of vinylmethylsiloxane-dimethylsiloxane copolymers have been explored. These methods were based on the anionic ring-opening polymerisation of 2-vinyl-2,4,4,6,6-pentamethylcyclotrisiloxane (VD₂) and on the copolymerisation of this monomer with hexamethylcyclotrisiloxane (D₃). All of them permit control of the molecular weight of the copolymer, the structure of the chain ends and the distribution of vinyl groups along the chain.Polymerisation of VD₂ was performed using n-butyllithium as an initiator in THF. Studies on the sequencing of the siloxane units in the polymer were performed using ²⁹Si NMR spectroscopy and first order Markov chain statistics. These methods were complemented by determination of the sequencing at the chain end bearing a fragment of initiator. The structure of the copolymer obtained at −30 °C is highly regular. Almost 90% of the monomer is added to the chain as a result of the attack of the silanolate centre on the vinyl-substituted silicon in the monomer.The copolymerisation of VD₂ with D₃ leads to copolymer with gradient distribution of the vinyl groups along the chain. The conversion of both monomers was followed by gas chromatography and the results were analysed by a simulation method and by the classical methods of Mayo-Lewis and of Kelen-Tüdös. These methods gave similar values for the reactivity ratio r_D3=0.22, 0.21 and 0.22, r_VD2=7.8, 7.7 and 8.3 from the M-L, K-T and simulation methods, respectively.The sequential copolymerisation of D₃ with VD₂ gave the diblock AB copolymers. The block formed in the second step may be contaminated with mers coming from the comonomer.     

Mechanism of hexamethylcyclotrisiloxane polymerization in the presence of siloxanediols

Polymerization of hexamethylcyclotrisiloxane (D₃ ) in CH₂Cl₂ at 30°C, initiated by triflic acid (TfOH) was studied in the presence of siloxanediols (HD₂OH or HD_xOH) which polycondense giving water, or in the presence of water alone for comparison. D represents a siloxane unit OSiMe₂. In the second case, the relative amounts of cyclic oligomers (D₆, D₉) and of linear high polymer (HP) vary strongly with the molar ratio r =[H₂O]/[TfOH]. When r increases from 1 to 100 (in homogeneous phase), D₉ /D₆ increases from 0.2 to 2 and D₆ /HP decreases from 1 to 0.3. The large decrease of D₆ amount and the increase in D₉/D₆ are attributed to the suppression of their formation through oxonium ions and to their exclusive formation, for r>100, by cyclization of silanol-esters, which is more rapid for HD₉OTf than for HD₆OTf. For polymerization of D₃ in the presence of HD₂ OH, there is a fast and limited ring-opening of D₃ (about 10% conversion over 6 min) at the beginning, without formation of cyclics (D₆, D₉ , etc). Then reaction with D₃ stops. Polymerization of HD₂OH takes place simultaneously, at the same rate as in the absence of D₃, with slow formation of high polymer. At the end of the polycondensation (after, eg, 4 h) D₃ polymerization starts again, giving D₆ , D₉, etc, and HP. The inhibition period for D ₃ is attributed to the complexation of triflic acid hydrates by silanol groups. These activated silanol groups do not react with D₃. A comparison of D₃ polymerization with the addition of water, HD₂OH or HD₁₅OH leads to the conclusion that when r is not too large, propagation involves silylester end-groups, but that for large r ratios (100 or higher), it probably occurs mainly on silanol groups reacting with an activated monomer. © 1999 Society of Chemical Industry     

Ketonization of Proline Residues in the Peptide Chains of Actinomycins by a 4‐Oxoproline Synthase

X‐type actinomycins (Acms) contain 4‐hydroxyproline (Acm X₀) or 4‐oxoproline (Acm X₂) in their β‐pentapeptide lactone rings, whereas their α ring contains proline. We demonstrate that these Acms are formed through asymmetric condensation of Acm half molecules (Acm halves) containing proline with 4‐hydroxyproline‐ or 4‐oxoproline‐containing Acm halves. In turn, we show—using an artificial Acm half analogue (PPL 1) with proline in its peptide chain—their conversion into the 4‐hydroxyproline‐ and 4‐oxoproline‐containing Acm halves, PPL 0 and PPL 2, in mycelial suspensions of Streptomyces antibioticus. Two responsible genes of the Acm X biosynthetic gene cluster of S. antibioticus, saacmM and saacmN, encoding a cytochrome P450 monooxygenase (Cyp) and a ferredoxin were identified. After coexpression in Escherichia coli, their gene products converted PPL 1 into PPL 0 and PPL 2 in vivo as well as in situ in permeabilized cell of the transformed E. coli strain in conjunction with the host‐encoded ferredoxin reductase in a NADH (NADPH)‐dependent manner. saAcmM has high sequence similarity to the Cyp107Z (Ema) family of Cyps, which can convert avermectin B1 into its keto derivative, 4′′‐oxoavermectin B1. Determination of the structure of saAcmM reveals high similarity to the Ema structure but with significant differences in residues decorating their active sites, which defines saAcmM and its orthologues as a distinct new family of peptidylprolineketonizing Cyp.     

Improved synthesis and properties of hydroxyl‐terminated liquid fluorosilicone

The synthesis of hydroxyl‐terminated poly(trifluoropropylmethyl)siloxane (PTFPMS‐OH) by anionic ring‐opening polymerization of 1,3,5‐tris(trifluoropropylmethyl)cyclotrisiloxane (D₃F) was studied in bulk using potassium hydroxide as an initiator in the presence of several reaction stabilizers. The promoting effect of the reaction stabilizers for the polymerization of D₃F was investigated by GPC and NMR analyses. Results showed that the selected reaction stabilizers exhibited a significant promoting effect. This new process of polymerization produced well‐defined α,ω‐dihydroxylated polysiloxane in very high yields. The addition of reaction stabilizers could almost completely suppress back‐biting reactions during the polymerization. It was found that there lies an exponential decay relationship between the molecular weight of PTFPMS‐OH and amount of end‐capping agent. Thus, by adjusting the reaction conditions strictly, the molecular weight of the fluorosilicone could be controlled accurately, and meanwhile a broad “terminate window” could be implemented. Thermogravimetic (TG) analysis indicated that PTFPMS‐OH could be used in a wide range of operational temperatures. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43220.     

Studies on a novel anion‐exchange membrane based on chitosan and ionized organic compounds with multiwalled carbon nanotubes for alkaline fuel cells

In this work, a novel hydroxyl‐anion‐conducting membrane composed of chitosan (CTS), an ionized organic compound ([QAIM]OH), and hydroxylated multiwalled carbon nanotubes (MWCNTs‐OH) has been fabricated through a blending‐casting method assisted by a glutaraldehyde (GA) crosslinking process that can improve the mechanical properties of the membrane effectively. Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy revealed that [QAIM]OH and MWCNTs‐OH were successfully introduced into the CTS matrix. A chemical crosslinking reaction between CTS and GA could be confirmed by FTIR, X‐ray photoelectron spectroscopy, and contact angle tests. By tuning the mass fraction of [QAIM]OH and MWCNTs‐OH in the membrane, the maximum OH^? conductivity (5.66 × 10^?3 S cm^?1 at room temperature) could be achieved for the composition CTS:[QAIM]OH (1:0.75 in mass) blend doped with 3% MWCNTs‐OH. At a current density of 59.9 mA cm^?2, a membrane electrode assembly fabricated with the CTS/[QAIM]OH/ MWCNTs‐OH membrane (1:0.5/3%) achieved a power density of 31.6 mW cm^?2 in a H₂/O₂ system at room temperature. Under the condition of intermediate temperature (100–140 °C) without water, the conductivities of the membranes increased with increasing temperature and the amount of [QAIM]OH, which acted as an ionic liquid in the membrane, indicating that the ionic transport behaviors could still be occurring. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46323.     

Pramipexole Derivatives as Potent and Selective Dopamine D3 Receptor Agonists with Improved Human Microsomal Stability

Herein we report the synthesis and evaluation of a series of new pramipexole derivatives as highly potent and selective agonists of the dopamine‐3 (D₃) receptor. A number of these new compounds bind to the D₃ receptor with sub‐nanomolar affinity and show excellent selectivity (>10 000) for the D₃ receptor over the D₁ and D₂ receptors. For example, compound 23 (N‐(cis‐3‐(2‐(((S)‐2‐amino‐4,5,6,7‐tetrahydrobenzo[d]thiazol‐6‐yl)(propyl)amino)ethyl)‐3‐hydroxycyclobutyl)‐3‐(5‐methyl‐1,2,4‐oxadiazol‐3‐yl)benzamide) binds to the D₃ receptor with a K_i value of 0.53 nM and shows a selectivity of >20 000 over the D₂ and D₁ receptors in the binding assays using a rat brain preparation. It has excellent stability in human liver microsomes. Moreover, in vitro functional assays showed it to be a full agonist for the human D₃ receptor.     

Gelation and Degelation of PVA in Aqueous BaTiO3 Slurries

Poly(vinyl alcohol) (PVA) gels favorably with boron oxide (B₂O₃) in aqueous slurries of BaTiO₃, resulting in a dilatant rheology, but the gelation and dilatancy can be efficiently reduced by the use of hydroxylated BaTiO₃. The degelation effect caused by the use of hydroxylated BaTiO₃ was studied, and the corresponding degelation mechanisms were clarified. Interestingly, the hydroxylated BaTiO₃ interacts better than the as‐received form with PVA and B₂O₃, and the hydroxylated form is more chemically stable and less soluble in the aqueous slurry. Based on these specific properties of hydroxylated BaTiO₃, especially its good chemical stability, the free concentrations of PVA and ^?B(OH)₄ in the aqueous slurry are efficiently diminished. The decreased free concentrations of PVA and B₂O₃ facilitate the reduction of the gelation opportunity between them, leading to a great improvement in the rheological behavior of the aqueous slurries.     

Potent,Metabolically Stable 2‐Alkyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐adenines as Adenosine A2A Receptor Ligands

Inhibition of adenosine A_2A receptors has been shown to elicit a therapeutic response in preclinical animal models of Parkinson’s disease (PD). We previously identified the triazolo‐9H‐purine, ST1535, as a potent A_2AR antagonist. Studies revealed that ST1535 is extensively hydroxylated at the ω‐1 position of the butyl side chain. Here, we describe the synthesis and evaluation of derivatives in which the ω‐1 position has been substituted (F, Me, OH) in order to block metabolism. The stability of the compounds was evaluated in human liver microsomes (HLM), and the affinity for A_2AR was determined. Two compounds, (2‐(3,3‐dimethylbutyl)‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐6‐amine ( 3 b ) and 4‐(6‐amino‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐2‐yl)‐2‐methylbutan‐2‐ol ( 3 c ), exhibited good affinity against A_2AR (K_i=0.4 nM and 2 nM , respectively) and high in vitro metabolic stability (89.5 % and 95.3 % recovery, respectively, after incubation with HLM for two hours).     

Antibodies against Mucin‐Based Glycopeptides Affect Trypanosoma cruzi Cell Invasion and Tumor Cell Viability

This study describes the synthesis of glycopeptides NHAc[βGal]‐(Thr)₂‐[αGalNAc]‐(Thr)₂‐[αGlcNAc]‐(Thr)₂Gly‐OVA ( 1 ‐OVA) and NHAc[βGal‐αGalNAc]‐(Thr)₃‐[αLacNAc]‐(Thr)₃‐Gly‐OVA ( 2 ‐OVA) as mimetics of both T. cruzi and tumor mucin glycoproteins. These glycopeptides were obtained by solid‐phase synthesis, which involved the prior preparation of the protected glycosyl amino acids αGlcNAc‐ThrOH ( 3 ), αGalNAc‐ThrOH ( 4 ), βGal‐ThrOH ( 5 ), αLacNAc‐ThrOH ( 6 ), and βGal‐αGalNAc‐ThrOH ( 7 ) through glycosylation reactions. Immunizations of mice with glycopeptides 1 ‐OVA and 2 ‐OVA induced high antibody titers (1:16 000), as verified by ELISA tests, whereas flow cytometry assays showed the capacity of the obtained anti‐glycopeptides 1 ‐OVA and 2 ‐OVA antibodies to recognize both T. cruzi and MCF‐7 tumor cells. In addition, antisera induced by glycopeptides 1 ‐OVA and 2 ‐OVA were also able to inhibit T. cruzi fibroblast cell invasion (70 %) and to induce antibody‐mediated cellular cytotoxicity (ADCC) against MCF‐7 cells, with 50 % reduction of cell viability.     

Antitumor Effects of Vitamin D Analogs on Hamster and Mouse Melanoma Cell Lines in Relation to Melanin Pigmentation

Deregulated melanogenesis is involved in melanomagenesis and melanoma progression and resistance to therapy. Vitamin D analogs have anti-melanoma activity. While the hypercalcaemic effect of the active form of Vitamin D (1,25(OH)₂D₃) limits its therapeutic use, novel Vitamin D analogs with a modified side chain demonstrate low calcaemic activity. We therefore examined the effect of secosteroidal analogs, both classic (1,25(OH)₂D₃ and 25(OH)D₃), and novel relatively non-calcemic ones (20(OH)D₃, calcipotriol, 21(OH)pD, pD and 20(OH)pL), on proliferation, colony formation in monolayer and soft-agar, and mRNA and protein expression by melanoma cells. Murine B16-F10 and hamster Bomirski Ab cell lines were shown to be effective models to study how melanogenesis affects anti-melanoma treatment. Novel Vitamin D analogs with a short side-chain and lumisterol-like 20(OH)pL efficiently inhibited rodent melanoma growth. Moderate pigmentation sensitized rodent melanoma cells towards Vitamin D analogs, and altered expression of key genes involved in Vitamin D signaling, which was opposite to the effect on heavily pigmented cells. Interestingly, melanogenesis inhibited ligand-induced Vitamin D receptor translocation and ligand-induced expression of VDR and CYP24A1 genes. These findings indicate that melanogenesis can affect the anti-melanoma activity of Vitamin D analogs in a complex manner.