Non-calcemic Vitamin Research Articles

Abstract 3588: Androgen synthesis disruption, ferroptosis induction and prostate cancer inhibition by androgen and vitamin D combination

Abstract Background: Metastatic castration-resistant prostate cancer (CRPC) is a fatal condition that progresses to end-of-life within two years, and it is the fifth leading cause of cancer death in men globally. New avenues for preventing or delaying lethal CRPC progression are a critical unmet need. Hormonal vitamin D3 (calcitriol) inhibits prostate cancer - both castration sensitive and castration resistant - in preclinical models, as documented widely. However, vitamin D as an anti-cancer therapeutic is clinically untenable since prostate tumor suppression in patients requires hormone dosing at a supraphysiologic level which induces hypercalcemia and other toxicities. Results: Herein we provide the first evidence that a subnanomolar, non-calcemic calcitriol dose can inhibit CRPC cell proliferation, G1 to S phase transition, clonal growth, migration, and invasion in vitro, and xenograft growth in vivo, only upon concurrent dosing with 5α-DHT at a physiologic or subphysiologic level. Calcitriol as a single treatment is non-inhibitory at a subnanomolar dose. HSD3β1, a hydroxysteroid dehydrogenase, was markedly induced by calcitriol, while DHT cotreatment reduced the induction. HSD3β1 catalyzes conversion of dehydroepiandrosterone, an adrenal androgen, to androstenedione (AED) - a rate-limiting step in the androgen biosynthesis pathway. AKR1C3, an aldo-keto reductase which promotes androgen biosynthesis, was also reduced by calcitriol/DHT cotreatment. In contrast, AKR1D1, an androgen-inactivating 5β-reductase, was robustly induced by a non-calcemic calcitriol dose in the presence of DHT, not by calcitriol singly. AKR1D1 converts AED and testosterone to 5β-diols. HSD3β1 and AKR1C3 are elevated in CRPC. Disruption of androgen biosynthesis and net reduction of the intratumor androgen pool are expected outcomes of reduced HSD3β1 and AKR1C3, and elevated AKR1D1. The hormone pair led to LC3B induction, ferritin degradation, and induction of ALOX-5 and NCoA4 - the genes which regulate ferroptosis, indicating autophagy mediated ferroptosis (ferritinophagy). Apoptosis and necroptosis were absent. We conclude that vulnerabilities created by combined androgen and non-calcemic vitamin D dosing can lead to cell death by ferroptosis, and this hormone pairing may be a new avenue for arresting CRPC clinical progression. Significance: The translational potential of our results is bolstered by the precedent that supraphysiologic testosterone improved serum PSA scores and other clinical parameters for a subset of therapy-inert CRPC patients who received repeated cycles of once-a-month single testosterone dosing- a protocol known as bipolar androgen therapy (BAT)1. Our findings may offer a strategic framework for enhancing the efficacy of BAT by including oral dosing of a non-calcemic level of vitamin D3. W81XWH-21-1-0307 (DoD); IK6 BX004207 (VA). Ref 1: Nat Rev Urol 2023; PMID 36543976 Citation Format: Bobae Park, Jacqueline Vaquiz, Okunola Igbekoyi, Bandana Chatterjee. Androgen synthesis disruption, ferroptosis induction and prostate cancer inhibition by androgen and vitamin D combination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3588.

Design, Synthesis, Biological Activity, and Structural Analysis of Novel Des-C-Ring and Aromatic-D-Ring Analogues of 1α,25-Dihydroxyvitamin D3.

The toxic calcemic effects of the natural hormone 1α,25-dihydroxyvitamin D3 (1,25D3, 1,25-dihydroxycholecalciferol) in the treatment of hyperproliferative diseases demand the development of highly active and noncalcemic vitamin D analogues. We report the development of two highly active and noncalcemic analogues of 1,25D3 that lack the C-ring and possess an m-phenylene ring that replaces the natural D-ring. The new analogues (3a, 3b) are characterized by an additional six-carbon hydroxylated side chain attached either to the aromatic nucleus or to the triene system. Both compounds were synthesized by the Pd-catalyzed tandem cyclization/cross coupling approach starting from alkyne 6 and diphenol 8. Key steps include a stereoselective Cu-assisted addition of a Grignard reagent to an aromatic alkyne and a Takai olefination of an aromatic aldehyde. The new compounds are noncalcemic and show transcriptional and antiproliferative activities similar to 1,25D3. Structural analysis revealed that they induce a large conformational rearrangement of the vitamin D receptor around helix 6.

Lithocholic acid-based design of noncalcemic vitamin D receptor agonists

The hypercalcemic effects of the hormone 1α,25-dihydroxyvitamin D3 (calcitriol) and most of known vitamin D metabolites and analogs call for the development of non secosteroidal vitamin D receptor (VDR) ligands as new selective and noncalcemic agonists for treatment of hyperproliferative diseases. We report on the in silico design and stereoselective synthesis of six lithocholic acid derivatives as well as on the calcemic activity of a potent LCA derivative and its crystallographic structure in complex with zVDR LBD. The low calcemic activity of this compound in comparison with the native hormone makes it of potential therapeutic value. Structure-function relationships provide the basis for the development of even more potent and selective lithocholic acid-based VDR ligands.

A New Modified Experimental Meibomian Gland Injury Model: Partial Loss of Gland Due to Orifice Cauterization and the Alleviating Potential of 22-Oxacalcitriol.

1α,-25-dihydroxy-22-oxacalcitriol (maxacalcitol) is a non-calcemic vitamin D3 analog clinically approved to treat psoriasis, and its role has been increasingly recognized in suppressing keratinocyte proliferation, mediating inflammation, and regulating the immune response. A large number of studies have suggested that vitamin D plays an important role in maintaining ocular surface health. However, its topical effects on the Meibomian gland (MG) has been insufficiently investigated. Here, we introduce an experimental MG orifice injury model, where the partial glandular loss occurred after electrical cauterization on a limited number of MG orifices, and investigate the efficacy and safety of maxacalcitol ointment in treating this MG orifice injury model. We confirm the alleviation of MG atrophy and ductal dilation by maxacalcitol ointment application. The recovery of injured MG visualizing as the residual MG area is significantly better in the maxacalcitol group (p = 0.020) compared with the Vaseline® group, especially during the first two weeks. The cornea and other ocular tissues were not affected by maxacalcitol ointment application during our two-month observation period. Altogether, this work indicates that maxacalcitol has therapeutic potential in the amelioration of initial injury of MG orifices caused by electrocautery.

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

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/β-catenin signaling pathways. 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. 20(OH)D3 and 1,20(OH)2D3 inhibited the growth of CAL-27 and demonstrated inhibition of WNT/β-catenin and the SHH signaling as evidenced by down-regulation of nuclear translocation of glioma-associated oncogene 1(GLI1) and β-catenin. Noncalcemic vitamin D hydroxyderivatives demonstrated antitumor activities against OSCC comparable to those of 1,25(OH)2D3 Their activities against SHH and the WNT/β-catenin pathways provide insight for a possible target for OSCC treatment.

Calcitriol and non-calcemic vitamin D analogue, 22-oxacalcitriol, attenuate developmental and pathological choroidal vasculature angiogenesis ex vivo and in vivo.

Aberrant ocular angiogenesis can underpin vision loss in leading causes of blindness, including neovascular age-related macular degeneration and proliferative diabetic retinopathy. Current pharmacological interventions require repeated invasive administrations, may lack efficacy and are associated with poor patient compliance and tachyphylaxis. Vitamin D has de novo anti-angiogenic properties. Here, our aim was to validate the ocular anti-angiogenic activity of biologically active vitamin D, calcitriol, and selected vitamin D analogue, 22-oxacalcitriol. Calcitriol induced a significant reduction in ex vivo mouse choroidal fragment sprouting. Viability studies in a human RPE cell line suggested non-calcemic vitamin D analogues including 22-oxacalcitriol have less off-target anti-proliferative activity compared to calcitriol and other analogues. Thereafter, the anti-angiogenic activity of 22-oxacalcitriol was demonstrated in an ex vivo mouse choroidal fragment sprouting assay. In zebrafish larvae, 22-oxacalcitriol was found to be anti-angiogenic, inducing a dose-dependent reduction in choriocapillaris development. Subcutaneously administered calcitriol failed to attenuate mouse retinal vasculature development. However, calcitriol and 22-oxacalcitriol administered intraperitoneally, significantly attenuated lesion volume in the laser-induced choroidal neovascularisation mouse model. In summary, calcitriol and 22-oxacalcitriol attenuate ex vivo and in vivo choroidal vasculature angiogenesis. Therefore, vitamin D may have potential as an interventional treatment for ophthalmic neovascular indications.

LB1061 Novel noncalcemic vitamin D hydroxyderivatives downregulate SHH and Wnt signaling pathways and inhibit spheroid formation in human oral squamous cell carcinoma and murine basal cell carcinoma

LB1061 Novel noncalcemic vitamin D hydroxyderivatives downregulate SHH and Wnt signaling pathways and inhibit spheroid formation in human oral squamous cell carcinoma and murine basal cell carcinoma

24R,25-Dihydroxyvitamin D3 regulates breast cancer cells in vitro and in vivo

BackgroundEpidemiological studies indicate high serum 25(OH)D3 is associated with increased survival in breast cancer patients. Pre-clinical studies attributed this to anti-tumorigenic properties of its metabolite 1α,25(OH)2D3. However, 1α,25(OH)2D3 is highly calcemic and thus has a narrow therapeutic window. Here we propose another metabolite, 24R,25(OH)2D3, as an alternative non-calcemic vitamin D3 supplement. MethodsNOD-SCID-IL2γR null female mice with MCF7 breast cancer xenografts in the mammary fat pad were treated with 24R,25(OH)2D3 and changes in tumor burden and metastases were assessed. ERα66+ MCF7 and T47D cells, and ERα66- HCC38 cells were treated with 24R,25(OH)2D3in vitro to assess effects on proliferation and apoptosis. Effects on migration and metastatic markers were assessed in MCF7. Results24R,25(OH)2D3 reduced MCF7 tumor growth and metastasis in vivo. In vitro results indicate that this was not due to an anti-proliferative effect; 24R,25(OH)2D3 stimulated DNA synthesis in MCF7 and T47D. In contrast, markers of invasion and metastasis were decreased. 24R,25(OH)2D3 caused dose-dependent increases in apoptosis in MCF7 and T47D, but not HCC38 cells. Inhibitors to palmitoylation, caveolae integrity, phospholipase-D, and estrogen receptors (ER) demonstrate that 24R,25(OH)2D3 acts on MCF7 cells through caveolae-associated, phospholipase D-dependent mechanisms via cross-talk with ERs. ConclusionThese results indicate that 24R,25(OH)2D3 shows promise in treatment of breast cancer by stimulating tumor apoptosis and reducing metastasis. General significance24R,25(OH)2D3 regulates breast cancer cell survival through ER-associated mechanisms similar to 24R,25(OH)2D3 effects on chondrocytes. Thus, 24R,25(OH)2D3 may modulate cell survival in other estrogen-responsive cell types, and its therapeutic potential should be investigated in ER-associated pathologies.

Antiproliferative Activity of Non-Calcemic Vitamin D Analogs on Human Melanoma Lines in Relation to VDR and PDIA3 Receptors.

Vitamin D is a precursor for secosteroidal hormones, which demonstrate pleiotropic biological activities, including the regulation of growth and the differentiation of normal and malignant cells. Our previous studies have indicated that the inhibition of melanoma proliferation by a short side-chain, low calcemic analog of vitamin D—21(OH)pD is not fully dependent on the expression of vitamin D receptor (VDR). We have examined the effects of classic vitamin D metabolites, 1,25(OH)2D3 and 25(OH)D3, and two low calcemic vitamin D analogs, (21(OH)pD and calcipotriol), on proliferation, mRNA expression and vitamin D receptor (VDR) translocation in three human melanoma cell lines: WM98, A375 and SK-MEL-188b (subline b of SK-MEL-188, which lost responsiveness to 1,25(OH)2D3 and became VDR−/−CYP27B1−/−). All tested compounds efficiently inhibited the proliferation of WM98 and A375 melanoma cells except SK-MEL-188b, in which only the short side-chain vitamin D analog—21(OH)pD was effective. Overall, 21(OH)pD was the most potent compound in all three melanoma cell lines in the study. The lack of responsiveness of SK-MEL-188b to 1,25(OH)2D3, 25(OH)D3 and calcipotriol is explained by a lack of characteristic transcripts for the VDR, its splicing variants as well as for vitamin D-activating enzyme CYP27B1. On the other hand, the expression of VDR and its splicing variants and other vitamin D related genes (RXR, PDIA3, CYP3A4, CYP2R1, CYP27B1, CYP24A1 and CYP11A1) was detected in WM98 and A375 melanomas with the transcript levels being modulated by vitamin D analogs. The expression of VDR isoforms in WM98 cells was stimulated strongly by calcipotriol. The antiproliferative activities of 21(OH)pD appear not to require VDR translocation to the nucleus, which explains the high efficacy of this noncalcemic pregnacalciferol analog in SK-MEL-188b melanoma, that is, VDR−/−. Therefore, we propose that 21(OH)pD is a good candidate for melanoma therapy, although the mechanism of its action remains to be defined.

Aromatic-Based Design of Highly Active and Noncalcemic Vitamin D Receptor Agonists.

We report the design, synthesis, biological evaluation, and structural analysis of a new class of vitamin D analogues that possess an aromatic m-phenylene D-ring and an alkyl chain replacing the C-ring. A key feature of the synthetic strategy is a stereoselective Pd-catalyzed construction of the triene system in aqueous medium that allows the rapid preparation of small amounts of VDR ligands for biological screening. Analogues with the shorter (2a) and longer (2d, 2e) side chains attached to the triene system have no calcemic activity. Compound 2a binds to VDR with the same order of magnitude than calcipotriol and oxacalcitriol. It also reduces proliferation in normal and tumor cells similarly to the natural hormone 1α,25-dihydroxyvitamin D3, calcipotriol, and oxacalcitriol, suggesting preclinical studies related to hyperproliferative disorders such as psoriasis and cancer.

The effects of Femarelle and Vitamin D Analog on Lipoxygenase Products and Reactive Oxygen Species (ROS) formation in the Female and Male Human-Derived Cultured Osteoblastic Cells

1. AbstractWe have previously reported that human primary cultured bone cells (hObs) respond to estradiol-17β (E2) and different phytoestrogens and vitamin D analogs by modulating cell proliferation. In the present study hObs derived from biopsies of pre- or post- menopausal females or males were cultured and treated with different estrogens and with the non-calcemic vitamin D JK-1624F2-2 (JKF) and assayed for the expression of 12LO and 15LO mRNA as well as for the synthesis of 12 and 15HETEs and the formation of ROS. All cells express 12LO and 15LO and produce 12 and 15 HETEs. Treatment with all estrogens: E2, daidzein (D) and femarelle (F) as well as JKF modulated the expression of both 12 and 15LO mRNA in pre- or post-menopausal female hObs and to some extent in male hObs. 12 and 15HETEs production was also modulated by these compounds in these cells. ROS formation was increased by all hormones in pre- and post- but not in male hObs. ROS formation was also increased by 12 and 15HETEs in pre- and post- ...

Vitamin D Stimulates Epithelial Cell Proliferation and Facilitates Wound Closure via A Cathelicidin Independent Pathway In Vitro

Objective: An important property of vitamin D is its ability to induce the antimicrobial peptide, LL-37, a human cathelicidin. LL-37 also promotes innate immunity, reduces levels of pro-inflammatory cytokines, and stimulates proliferation and migration of epithelial cells. Despite its beneficial effects, vitamin D can cause hypercalcemia. In this study, effects of vitamin D and a non-calcemic vitamin D analog on gingival epithelial cell proliferation and LL-37 production, and in an in vitro wound healing model, were determined. Methods: Human gingival epithelial cells were exposed to vitamin D [1,25(OH)2 D3] or an analog [20(OH)D3]. Effects on cell viability (activity of a mitochondrial enzyme), proliferation (incorporation of BrdU into DNA) and LL-37 production (by ELISA) were determined, and effects on wound healing were evaluated in vitro via a “scrape” wound healing model. Data were analyzed using ANOVA and Scheffe’s F procedure for post hoc comparisons. Results: Vitamin D and the analog had no significant effect on viability, and both increased LL-37 production. Vitamin D, but not the analog, increased proliferation (~50% increase; p<0.03). Neutralizing anti-LL-37 did not significantly alter proliferation. Vitamin D and the analog significantly increased the rate of in vitro wound closure, which was not affected by the presence of the neutralizing LL- 37 antibody, indicating other mechanisms besides LL-37 were responsible for the increase rate of wound closure. Conclusion: While vitamin D and the vitamin D analog increased production of LL-37 20-30%, the increased rates of in vitro wound closure caused by vitamin D and the analog, and stimulation of cell proliferation by vitamin D, probably occur through mechanisms other than LL-37 production.

New vitamin D analogs as potential therapeutics in melanoma

Extensive evidence shows that the active form of vitamin D3 – 1α,25-dihydroxyvitamin D3 – plays an important role in cancer prevention, has tumorostatic activity and may potentially be used in therapy for melanoma. Vitamin D3 and its analogs (secosteroids) exert multiple effects on cancer cells, including inhibition of cell growth and induction of differentiation. Activity of secosteroids depends on multiple cellular factors, including expression of the vitamin D receptor. Despite its endogenous origin, the key drawback for the use of pharmacologically effective doses of 1α,25-dihydroxyvitamin D3 is its hypercalcemic effect leading to profound toxicity. The solution may lie in properties of vitamin D3 analogs with modified side chains, which demonstrate low calcemic activity but conserve the anti-tumor properties. Noncalcemic vitamin D compounds were found to be potent in multiple studies that mandate further clinical testing. Finally, recent studies revealed alternative metabolic pathways for secosteroids and new targets in the cells, which opens up new therapeutic possibilities.

EB1089 inhibits the parathyroid hormone–related protein–enhanced bone metastasis and xenograft growth of human prostate cancer cells

Parathyroid hormone-related protein (PTHrP) plays a major role in prostate carcinoma progression and bone metastasis. Once prostate cancers become androgen-independent, treatment options become limited. Vitamin D analogues represent a potentially valuable class of agents in this clinical context. Using the prostate cancer cell line C4-2 as a model, we studied the effects of PTHrP and the noncalcemic vitamin D analogue EB1089 on markers of prostate cancer cell progression in vitro and in vivo. C4-2 is a second-generation androgen-independent LNCaP subline that metastasizes to the lymph nodes and bone when injected into nude mice and produces mixed lytic/blastic lesions, mimicking the in vivo situation. We report that PTHrP increases cell migration and invasion, and that a pathway via which EB1089 inhibits these processes is through down-regulation of PTHrP expression. PTHrP also increases anchorage-independent cell growth in vitro and xenograft growth in vivo; EB1089 reverses these effects. The in vivo PTHrP effects are accompanied by increased tumor cell proliferation and survival. Treatment with EB1089 reverses the proliferative but not the antiapoptotic effects of PTHrP. PTHrP also increases intratumor vessel density and vascular endothelial growth factor expression; EB1089 reverses these effects. Intracardially injected C4-2 cells produce predominantly osteoblastic lesions; PTHrP overexpression decreases the latency, increases the severity and alters the bone lesion profile to predominantly osteolytic. EB1089 largely reverses these PTHrP effects. A direct correlation between PTHrP immunoreactivity and increasing tumor grade is observed in human prostate cancer specimens. Thus, decreasing PTHrP production by treatment with vitamin D analogues may prove therapeutically beneficial for prostate cancer.

Are suberythemal doses of ultraviolet B good for your skin?

Ultraviolet radiation B (UVB; wavelength 280–320 nm), while representing only ∼5% of the ultraviolet spectrum of the solar radiation reaching the surface of the earth, is very efficient at cutaneous biological effects (Bjorn, 2008; Slominski and Wortsman, 2000). Because of its involvement in the aetiology of epidermal cancers (basal and squamous cell carcinomas) and in melanomagenesis (Bikle, 2008), a negative health impact of UVB has been emphasized recently, especially in the pigment cell biology field. In this context, the paper by Hong et al. (2008) on the biopositive effects of UVB on the epidermis deserves special attention. The authors have clearly demonstrated that exposure of hairless mice to UVB at 40 mJ cm−2 (approximately ½ minimal erythema dose) daily for 1–3 days clearly strengthens the epidermal barrier function and stimulates antimicrobial peptide production. This indicates that suberythemal doses of UVB may have direct beneficial effects on functions of the skin as well as serving as a useful strategy in treatment of atopic dermatitis, which is in contrast to its negative effect at high doses (Hong et al., 2008). The proposed mechanism of action includes stimulation of the vitamin D3 (D3) signalling system, as UVB induces the expression of the vitamin D receptor and CYP27B1 (encoding 1α-hydroxylase) genes, and use of 1α-hydroxylase inhibitor ketoconazole attenuated these affects apparently via inhibition of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3 formation (Hong et al., 2008). The photochemical isomerization of 7-dehydrocholesterol (7DHC) after absorption of UVB photons to a pre-vitamin D3 intermediate, followed by its slow isomerization to three main products including D3, tachysterol and lumisterol, represents the most fundamental reactions in the photobiology of the skin [reviewed in Holick (2007); Bikle (2008); Bjorn (2008)] (Fig. 1). This conversion and the relative ratio of products depend on the dose of absorbed energy, temperature, wavelength, the presence of singlet oxygen, chemical milieu or of biological membranes [reviewed in Holick (2007); Bikle (2008); Bjorn (2008)]. For example, higher doses of UV can trigger further isomerization of tachysterol to isotachysterols, which is followed by autoxidation, or further degradation/isomerization of D3 with production of suprasterols I and II [reviewed in Holick (2007); Bjorn (2008)]. Also, the presence of singlet oxygen and photosensitizers can transform 7DHC to corresponding 5,7,9(11)-triene (9DDHC) (Fig. 1). Photolysis of 7-dehydrocholesterol (cholesta-5,7-diene-3β-ol; pro-vitamin D3). Vitamin D3 after entering circulation is successively hydroxylated in the liver and kidney to 1,25(OH)2D3 to regulate body calcium metabolism as well as to mediate several pleiotropic effects [reviewed in Holick (2007); Bikle (2008)], serves as an example of endocrine function of the skin (Slominski and Wortsman, 2000). 1,25(OH)2D3 is also produced in the epidermis from D3 and has significant local actions on formation of the skin barrier, and functional differentiation of adnexal structures, and modulation of the skin immune system (reviewed in Holick (2007); Bikle (2008)). These local activities are consistent with the results and conclusions of the paper by Hong et al. (2008), which challenges the current dermatological opinion that UVB is only deleterious for the skin, and that orally delivered D3 can replace all of the functions of the cutaneously formed derivatives of D3 [see variety of UVB induced transformation of pro-vitamin D3 (Fig. 1)]. Furthermore, the pharmaceutical industry is investing significant effort to develop synthetic non-calcemic vitamin D analogues that could serve as potent anticancer drugs. In the above context it is also important to mention that the skin expresses CYP11A1 (Slominski et al., 2004b), which can modify the side chain of D3 (Slominski et al., 2005) or cleave the side chain of 7DHC to produce 7-dehydropregnenolone that can further be modified by steroidogenic enzymes and such products can undergo photochemical transformation to vitamin D-like products without the side chain (Fig. 2) (Slominski et al., 2004b). Photolysis of pregna-5,7-diene-steroidal products of P450scc and other steroidogenic enzymes action on 7-dehydrocholesterol and 7-dehydropregnolone (Slominski et al., 2004b). Thus, the correct question to be asked is how much UVB is bad or good for the skin taking into consideration its level of pigmentation. This presents new and exciting experimental opportunities for pigment biologists, as skin pigmentation affects UVB-induced D3 production and D3 (and perhaps other derivatives presented in 1, 2) can affect skin pigmentation and activities of normal and malignant melanocytes [reviewed in Slominski et al. (2004a)].

Vitamin D Modulation of the Activity of Estrogenic Compounds in Bone Cells In Vitro and In Vivo

Vitamin D analogs modulate different organs, including modulation of energy metabolism, through the induction of creatine kinase (CK) activity. Skeletal organs from vitamin D-depleted rats showed lower constituent CK than those from vitamin D-replete rats. Moreover, estradiol-17beta (E2) or dihydrotestosterone (DHT), which increased CK in organs from intact female or male rats, respectively, stimulated much less CK in vitamin D-depleted rats. Treatment of intact female rats with noncalcemic vitamin D analogs significantly upregulated E2- and DHT-induced CKresponse. These analogs upregulated the CK response to selective estrogen receptor modulators (SERMs) in organs from intact or ovariectomized (Ovx) female rats but abolished SERMs' inhibitory effect on E2-induced CK. These analogs significantly increased estradiol receptor alpha (ERalpha) protein in skeletal organs as well as histomorphological and biochemical changes due to this treatment followed by E2 or DHT. The analogs alone markedly altered the growth plate and the trabeculae and increased trabecular bone volume (%TB V) and trabecular width. The addition of E2 or DHT to this treatment restored all parameters as well as increased %TBV and cell proliferation. Treatment of Ovx female rats with JK 1624 F2-2 (JKF) decreased growth-plate width and increased %TB V, whereas QW1624 F2-2 (QW) restored growth-plate width and %TB V. Treatment of E2 with JKF restored %TBV and growth-plate width, whereas E2 with QW restored all parameters, including cortical width. There was also upregulation of the response of CK to E2 in both combined treatments. Our human-derived osteoblast (hObs)-like cell cultures respond to estrogenic compounds, and pretreating them with JKF upregulated the CK response to E2, raloxifene (Ral), and some phytoestrogens. ERalpha and ERbeta proteins, as well as mRNA, were modulated by CB 1093 (CB) and JKF. JKF increased specific nuclear E2 binding in female hObs but inhibited specific membranal E2 binding. hObs express 25 hydroxyvitamin D3-1alpha hydroxylase (1-OHase)-mRNA and its biological activity, which are both modulated by parathyroid hormone (PTH) and estrogenic compounds. Our results demonstrate mutual interaction between vitamin D and estrogenic compounds. We therefore conclude that combined treatment with less-calcemic analogs of vitamin D and estrogenic compounds might be superior for treatment of bone damage caused by ovariectomy in female rats, with possible application for postmenopausal osteoporosis.

Prohibitin Is a Novel Target Gene of Vitamin D Involved in Its Antiproliferative Action in Breast Cancer Cells

Previously, we showed that N-methyl-N-nitrosourea-transformed MCF12F breast epithelial cells exhibited differential expression of several genes, including up-regulation of prohibitin and elevated sensitivity to a relatively noncalcemic vitamin D analogue, 1alpha-hydroxyvitamin D5 [1alpha(OH)D5]. In this report, we evaluated the functional significance of prohibitin in relation to the cellular response to vitamin D. The in silico screening for putative transcription factor binding sites identified two vitamin D receptor (VDR)/retinoid X receptor binding sites in the 1-kb promoter region of prohibitin. Prohibitin up-regulation by 1alpha(OH)D5 treatment at both transcriptional and translational levels was confirmed by real-time reverse transcription-PCR and Western blot analysis in breast cancer cells, identifying prohibitin as a vitamin D target gene. Confocal microscopic analysis showed that prohibitin was localized in the nuclei of MCF-7 cells and a portion of prohibitin was colocalized with VDR, but direct physical interaction between VDR and prohibitin in cell lysates was not detectable. In MCF-7 cells expressing tetracycline-inducible prohibitin (Tet-On model), the overexpression of prohibitin inhibited cell proliferation and enhanced vitamin D-induced antiproliferative activity. Knockdown of prohibitin was accompanied by increased number of cells incorporating bromodeoxyuridine in the whole population and increased cell distribution in the S phase of cell cycle. In addition, prohibitin level had no significant effect on the vitamin D-induced transactivation of CYP24, a VDR target gene. This is the first report to suggest that prohibitin serves as a novel vitamin D target gene, which is involved in the antiproliferative action of vitamin D without affecting CYP24 transactivation in breast cancer cells.

Calcium signaling in cancer and vitamin D

Calcium signals induced by the Ca 2+ regulatory hormone 1,25(OH) 2D 3 may determine the fate of the cancer cell. We have shown that, in breast cancer cell lines, 1,25(OH) 2D 3 induces a sustained increase in concentration of intracellular Ca 2+ ([Ca 2+] i ) by depleting the endoplasmic reticulum (ER) Ca 2+ stores via inositol 1,4,5-trisphosphate receptor/Ca 2+ release channel and activating Ca 2+ entry from the extracellular space via voltage-insensitive Ca 2+ channels. In normal cells, 1,25(OH) 2D 3 triggered a transient Ca 2+ response via activation of voltage-dependent Ca 2+ channels, which were absent in breast cancer cells. The normal cells, but not breast cancer cells, expressed the Ca 2+ binding/buffering protein calbindin-D 28k and were capable of buffering [Ca 2+] i increases induced by a mobilizer of the ER Ca 2+ stores, thapsigargin, or a Ca 2+ ionophore, ionomycin. The 1,25(OH) 2D 3-induced sustained increase in [Ca 2+] i in breast cancer cells was associated with induction of apoptotic cell death, whereas the transient [Ca 2+] i increase in normal cells was not. The forced expression of calbindin-D 28k in cytosol or increase in the cytosolic Ca 2+ buffering capacity with the cell-permeant Ca 2+ buffer BAPTA prevented induction of apoptosis with 1,25(OH) 2D 3 in cancer cells. The sustained increase in [Ca 2+] i in breast cancer cells was associated with activation of the Ca 2+-dependent apoptotic proteases, μ-calpain and caspase-12, as evaluated with antibodies to active (cleaved) forms of the enzymes and the fluorogenic peptide substrates. Selective inhibition of the Ca 2+ binding sites of μ-calpain decreased apoptotic indices in the cancer cells treated with 1,25(OH) 2D 3, thapsigargin, or ionomycin. The μ-calpain activation preceded expression/activation of caspase-12, and calpain was required for activation/cleavage of caspase-12. Certain non-calcemic vitamin D analogs (e.g., EB 1089) triggered a sustained [Ca 2+] i increase, activated Ca 2+-dependent apoptotic proteases, and induced apoptosis in breast cancer cells in a fashion similar to that of 1,25(OH) 2D 3. The 1,25(OH) 2D 3-induced transient Ca 2+ response in normal mammary epithelial cells was not accompanied by activation of μ-calpain and caspase-12. In conclusion, we have identified the novel apoptotic pathway in breast carcinoma cells treated with 1,25(OH) 2D 3: increase in [Ca 2+] i → μ-calpain activation → caspase-12 activation → apoptosis. Our results support the hypothesis that 1,25(OH) 2D 3 directly activates this apoptotic pathway by inducing a sustained increase in [Ca 2+] i . Differences of Ca 2+ regulatory mechanisms in cancer versus normal cells seem to allow 1,25(OH) 2D 3 and vitamin D analogs to induce Ca 2+-mediated apoptosis selectively in breast cancer cells. Thus, deltanoids may prove to be useful in the treatment of tumors susceptible to induction of Ca 2+-mediated apoptosis.

Inhibition of prostate cancer-meditated osteoblastic bone lesions by the low-calcemic analog 1α-hydroxymethyl-16-ene-26,27-bishomo-25-hydroxy vitamin D 3

Prostate cancer metastasizes almost exclusively into the bone whereby it induces primarily an osteoblastic response. Non-calcemic vitamin D analogs have been shown to inhibit proliferation of prostate cancer cells in culture and inhibit their growth as subcutaneous xenografts in mice. However, their effect on prostate cancer cell growth in the bone has not been examined. In the present study, we inoculated the osteoblastic prostate cancer cell line MDA-PCa 2b into the bone of male SCID mice and examined the effect of the low-calcemic hybrid analog 1α-hydroxymethyl-16-ene-26,27-bishomo-25-hydroxy vitamin D 3 (JK-1626-2) on their ability to induce bone lesions. We found that 7 weeks after inoculation of MDA-PCa 2b cells, 90% of the mice in the vehicle-treated group had significant bone lesions that were detectable by micro-computed tomography and characterized by thickening of the cortical bone and ossification of the epiphysis. Only 30% of the mice in the analog-treated group (daily injections of 4 μg/kg, 5 days/week for up to 7 weeks) had detectable bone lesions. Histological examination of the decalcified tumor-bearing bones has shown that tumor cells completely replaced the bone marrow in the diaphysis, and destroyed the trabecular bone in the metaphysis in 90% of the vehicle-treated mice. In contrast, the metaphysis of 60% of analog-treated mice appeared normal, although tumor cells were still found in the diaphysis of 70% of the bones in the analog-treated group. There was no evidence of hypercalcemia in any of the analog-treated mice. In a co-culture, MDA-PCa 2b cells induced a profound mitogenic response in osteoblasts followed by enhanced differentiation. However, in the presence of the analog the mitogenic response of the osteoblasts to the malignant cells was significantly attenuated. These experiments led to the hypothesis that, in vivo, JK-1626-2 prevented the metastatic bone lesions by inhibiting the mitogenic response of osteoblasts to growth factors produced by MDA-PCa 2b cells.

19-Nor-1,25(OH)2D2 (a Novel, Noncalcemic Vitamin D Analogue), Combined with Arsenic Trioxide, Has Potent Antitumor Activity against Myeloid Leukemia

Recently, we reported that a novel, noncalcemic vitamin D analogue (19-nor-1,25(OH)2D2; paricalcitol) had anticancer activity. In this study, we explored if paricalcitol enhanced anticancer effects of other clinically useful drugs in vitro against a large variety of cancer cells. Paricalcitol, when combined with As2O3, showed a markedly enhanced antiproliferative effect against acute myeloid leukemia (AML) cells. This combination induced monocytic differentiation of NB-4 acute promyelocytic leukemia (APL) cells and HL-60 AML cells and caused both to undergo apoptosis associated with down-regulation of Bcl-2 and Bcl-x(L). Paricalcitol induced monocytic differentiation of U937 AML cells, which was partially blocked by inducing expression of APL-related PML-retinoic acid receptor alpha (RARalpha) chimeric protein in the U937 cells containing a Zn2+-inducible expression vector coding for this fusion protein (PR9 cells). Exposure to As2O3 decreased levels of PML-RARalpha in PR9 cells, and the combination of paricalcitol and As2O3 enhanced their monocytic differentiation in parallel with the As2O3-mediated decrease of PML-RARalpha. Furthermore, As2O3 increased the transcriptional activity of paricalcitol probably by increasing intracellular levels of paricalcitol by decreasing the function of the mitochondrial enzyme 25-hydroxyvitamin D3-24-hydroxylase, which functions to metabolize the active vitamin D in cells. In summary, the combination of paricalcitol and As2O3 potently decreased growth and induced differentiation and apoptosis of AML cells. This probably occurred by As2O3 decreasing levels of both the repressive PML-RARalpha fusion protein and the vitamin D metabolizing protein, 25-hydroxyvitamin D3-24-hydroxylase, resulting in increased activity of paricalcitol. The combination of both of these Food and Drug Administration-approved drugs should be considered for treatment of all-trans retinoic acid-resistant APL patients as well as those with other types of AML.