Synthesis of diacetoxy acetal derivatives of santonin and their enhancing effects on HL-60 leukemia cell differentiation

Several diacetoxy acetal analogues have been synthesized from santonin and assessed for their ability of inducing or enhancing the differentiation of human HL-60 leukemia cells. The compounds themselves had little effect on HL-60 cell differentiation. However, three analogues, 2a, 3a, and 5b, synergistically enhanced 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-induced HL-60 cell differentiation when combined with 5 nM of dihydroxyvitamin D3 [1,25-(OH)2D3], a well-known differentiation inducer. Especially, the compound 5b profoundly enhanced the 1,25-(OH)2D3]-induced HL-60 cell differentiation.     

Capsaicin potentiates 1,25-dihydoxyvitamin D3- and all-trans retinoic acid-induced differentiation of human promyelocytic leukemia HL-60 cells

Human promyelocytic leukemia HL-60 cells are differentiated into monocytic or granulocytic lineage when treated with 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] or all-trans retinoic acid, respectively. In this study, the effect of capsaicin, an active component of the red pepper of the genus Capsocum, on cell differentiation was investigated in a HL-60 cell culture system. Treatment of HL-60 cells with 5–30 μg/ml capsaicin for 72 h inhibited cell proliferation and induced a small increase in cell differentiation. Interestingly, synergistic induction of HL-60 cell differentiation was observed when capsaicin was combined with either 5 nM 1,25-(OH)₂D₃ or 50 nM all-trans retinoic acid. Flow cytometric analysis indicated that combinations of 1,25-(OH)₂D₃ and capsaicin stimulated differentiation predominantly to monocytes whereas combinations of all-trans retinoic acid and capsaicin stimulated differentiation predominantly to granulocytes. Capsaicin enhanced protein kinase C activity in 1,25-(OH)₂D₃- and all-trans retinoic acid-treated HL-60 cells. In addition, inhibitors for protein kinase C [bisindolylmaleimide (GF-109203X), chelerythrine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7)] and an inhibitor for extracellular signal-regulated kinase [2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one (PD-098059)] significantly inhibited HL-60 cell differentiation induced by capsaicin in combination with either 1,25-(OH)₂D₃ or all-trans retinoic acid. These results indicate that capsaicin potentiates 1,25-(OH)₂D₃- or all-trans retinoic acid-induced HL-60 cell differentiation and that both protein kinase C and extracellular signal-regulated kinase are involved in the cell differentiation synergistically enhanced by capsaicin.     

Synthesis of DAAS derivatives and their enhancement of HL-60 leukemia cell differentiation

DAAS is the diacetoxy acetal derivative of a-santonin and induces HL-60 cell differentiation into granulocytes. In this report, we investigated the structure-activity relationship (SAR) of DAAS derivatives in the differentiation of human HL-60 leukemia cells. Although its derivatives themselves had less effect on HL-60 cell differentiation than DAAS, the monoacetyl derivative, 2, mainly induced HL-60 cell differentiation. Moreover, compound 2 synergistically enhanced all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation when combined with 50 nM ATRA, a well-known differentiation inducer. This enhancing effect is similar to that of DAAS in ATRA-induced differentiation.     

Nargenicin enhances 1,25-dihydroxyvitamin D3- and all-trans retinoic acid-induced leukemia cell differentiation via PKCβI/MAPK pathways

A major goal in the treatment of acute myeloid leukemia (AML) is to achieve terminal differentiation and prevent drug resistance and side effects. Combined treatment with low doses of ATRA or 1,25-(OH)₂D₃ that do not induce toxicity with another drug is one useful strategy for the treatment of AML. Actinomycetes are the well known sources of antibiotics and bioactive molecules. Previously, we isolated nargenicin from the culture broth of an actinomycete isolate, Nocardia sp. CS682. In this study, we evaluated the effects of nargenicin on cellular differentiation in a human myeloid leukemia HL-60 cell system. Nargenicin inhibited cell proliferation and induced HL-60 cell differentiation when administered in combination with 1,25-(OH)₂D₃ or ATRA. In addition, western blot analyses and kinase inhibitor studies demonstrated that nargenicin primarily enhanced leukemia cell differentiation via PKCβ1/MAPK pathways. The results of this study indicate that nargenicin has the ability to induce differentiation and suggest that it may be useful for the treatment of neoplastic diseases.     

Structure-specific control of differentiation and apoptosis of human promyelocytic leukemia (HL-60) cells by A-ring diastereomers of 2-methyl- 1α,25-dihydroxyvitamin D3 and its 20-epimer

1α,25-Dihydroxyvitamin D₃ (1α,25(OH)₂D₃) has been shown to modulate not only proliferation and differentiation but also apoptosis of malignant cells, indicating that it would be useful for the treatment of hyperproliferative diseases such as cancer and psoriasis. Little information is available concerning structural motifs of the 1α,25(OH)₂D₃ molecule responsible for modulation of differentiation and apoptosis. We synthesized all possible A-ring diastereomers of the 2-methyl-1α,25(OH)₂D₃ and its 20-epimer and evaluated their biological activities in human promyelocytic leukemia (HL-60) cells. Surprisingly, the potent analogues could be clearly divided into two groups: (i) those bearing the 1α- and 3β-hydroxyl groups on the A-ring were potent inducers of differentiation and growth inhibitors of HL-60 cells and (ii) those bearing the 1β-hydroxyl group together with either 3α- or 3β-hydroxyl groups on the A-ring were potent stimulators of apoptosis in these cells. We have clearly identified for the first time the structural motifs on the basis of the stereochemistry of both hydroxyl groups at positions 1 and 3 of the A-ring of the 1α,25(OH)₂D₃ molecule responsible for the induction of differentiation and apoptosis of HL-60 cells. These findings provide useful information not only for structure–function studies of 1α,25(OH)₂D₃ analogues but also for the development of therapeutic agents for the treatment of leukemia and other cancers.     

Differential involvement of protein kinase C in human promyelocytic leukemia cell differentiation enhanced by artemisinin

Artemisinin, a sesquiterpene lactone endoperoxide that exists in several medicinal plants, is a well-known anti-malarial agent. In this report, we investigated the effect of artemisinin on cellular differentiation in the human promyelocytic leukemia HL-60 cell culture system. Artemisinin markedly increased the degree of HL-60 leukemia cell differentiation when simultaneously combined with low doses of 1 alpha,25-dihydoxyvitamin D(3) [1,25-(OH)(2)D(3)] or all-trans retinoic acid (all-trans RA). Artemisinin by itself had very weak effects on the differentiation of HL-60 cells. Cytofluorometric analysis and cell morphologic studies indicated that artemisinin potentiated 1,25-(OH)(2)D(3)-induced cell differentiation predominantly into monocytes and all-trans RA-induced cell differentiation into granulocytes, respectively. Extracellular-regulated kinase (ERK) inhibitors markedly inhibited HL-60 cell differentiation induced by artemisinin in combination with 1,25-(OH)(2)D(3) or all-trans RA, whereas phosphatidylinositol 3-kinase (PI3-K) inhibitors did not. Particularly, protein kinase C (PKC) inhibitors inhibited HL-60 cell differentiation induced by artemisinin in combination with 1,25-(OH)(2)D(3) but not with all-trans RA. Artemisinin enhanced PKC activity and protein level of PKC beta I isoform in only 1,25-(OH)(2)D(3)-treated HL-60 cells. Taken together, these results indicate that artemisinin strongly enhanced 1,25-(OH)(2)D(3)- and all-trans RA-induced cell differentiation in which PKC is differentially involved in arteminisin-mediated enhancement of leukemia cell differentiation.     

Potentiation of 1,25-dihydroxyvitamin D(3)-induced differentiation of human promyelocytic leukemia cells into monocytes by costunolide,a germacranolide sesquiterpene lactone

Costunolide, a germacranolide sesquiterpene lactone that exists in several medicinal plants, is known to be a possible anti-cancer and chemopreventive agent for tumorigenesis. In this report, we investigated the effect of costunolide on cellular differentiation in the human promyelocytic leukemia HL-60 cell culture system. Costunolide markedly increased the degree of HL-60 leukemia cell differentiation when simultaneously combined with 5nM 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)). Costunolide by itself had very weak effects on the differentiation of HL-60 cells. Cytofluorometric analysis and cell morphologic studies indicated that costunolide potentiated 1,25-(OH)(2)D(3)-induced cell differentiation predominantly into monocytes. Inhibitors for PKC, PI3-K, and ERK markedly inhibited HL-60 cell differentiation induced by costunolide in combination with 1,25-(OH)(2)D(3). In addition, pretreatment of HL-60 cells with costunolide before the 1,25-(OH)(2)D(3) addition also potentiated cell differentiation in a concentration- and time-dependent manner, and the enhanced levels of cell differentiation closely correlated with the inhibitory levels of NF-kappaB-binding activity by costunolide. These results indicate that PKC, PI3-K, ERK and NF-kappaB may be involved in 1,25-(OH)(2)D(3)-mediated cell differentiation enhanced by costunolide.     

Induction of human promyelocytic leukemia HL-60 cell differentiation into monocytes by silibinin: involvement of protein kinase C

The effect of silibinin, an active component of Silybum marianum, on cellular differentiation was investigated in the human promyelocytic leukemia HL-60 cell culture system. Treatment of HL-60 cells with silibinin inhibited cellular proliferation and induced cellular differentiation in a dose-dependent manner. Cytofluorometric analysis and morphologic studies indicated that silibinin induced differentiation of HL-60 cells predominantly into monocytes. Importantly, strongly synergistic induction of differentiation into monocytes was observed when silibinin was combined with 5 nM 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], a well-known differentiation inducer of HL-60 cells into the monocytic lineage. Silibinin enhanced protein kinase C (PKC) activity and increased protein levels of both PKCalpha and PKCbeta in 1,25-(OH)(2)D(3)-treated HL-60 cells. PKC and extracellular signal-regulated kinase (ERK) inhibitors significantly inhibited HL-60 cell differentiation induced by silibinin alone or in combination with 1,25-(OH)(2)D(3), indicating that PKC and ERK may be involved in silibinin-induced HL-60 cell differentiation.     

Synergistic induction of 1,25-dihydroxyvitamin D(3)- and all-trans-retinoic acid-induced differentiation of HL-60 leukemia cells by yomogin,a sesquiterpene lactone from Artemisia princeps

Many anti-inflammatory agents are known to significantly enhance the terminal differentiation of some cancer cells such as leukemia cells. In this study, the effect of yomogin, a eudesmane sesquiterpene lactone isolated from Artemisia princeps with anti-inflammatory activity, was investigated in human promyelocytic leukemia HL-60 cells. Yomogin by itself induced small increases in cell differentiation, with less than 19 % of the cells attaining a differentiated phenotype. Importantly, yomogin synergistically enhanced differentiation of HL-60 cells in a dose-dependent manner when combined with either 5 nM 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2) D(3)] or 50 nM all- trans retinoic acid (all- trans RA). Cytofluorometric analysis and morphologic studies indicated that the combinations of yomogin and 1,25-(OH)(2) D(3) stimulated differentiation to monocytes whereas the combinations of yomogin and all- trans RA stimulated differentiation to granulocytes. These results suggest that yomogin may be useful in combination with 1,25-(OH)(2) D(3) or all- trans-RA in the differentiation therapy for myeloid leukemias. Abbreviations. 1,25-(OH)(2) D(3) :1,25-dihydroxyvitamin D(3) FITC:fluorescein isothiocyanate NBT:nitroblue tetrazolium RA:retinoic acid PE:phytoerythrin     

Capsaicin potentiates 1,25-dihydoxyvitamin D3- and all-trans retinoic acid-induced differentiation of human promyelocytic leukemia HL-60 cells.

Human promyelocytic leukemia HL-60 cells are differentiated into monocytic or granulocytic lineage when treated with 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] or all-trans retinoic acid, respectively. In this study, the effect of capsaicin, an active component of the red pepper of the genus Capsocum, on cell differentiation was investigated in a HL-60 cell culture system. Treatment of HL-60 cells with 5–30 μg/ml capsaicin for 72 h inhibited cell proliferation and induced a small increase in cell differentiation. Interestingly, synergistic induction of HL-60 cell differentiation was observed when capsaicin was combined with either 5 nM 1,25-(OH)₂D₃ or 50 nM all-trans retinoic acid. Flow cytometric analysis indicated that combinations of 1,25-(OH)₂D₃ and capsaicin stimulated differentiation predominantly to monocytes whereas combinations of all-trans retinoic acid and capsaicin stimulated differentiation predominantly to granulocytes. Capsaicin enhanced protein kinase C activity in 1,25-(OH)₂D₃- and all-trans retinoic acid-treated HL-60 cells. In addition, inhibitors for protein kinase C [bisindolylmaleimide (GF-109203X), chelerythrine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7)] and an inhibitor for extracellular signal-regulated kinase [2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one (PD-098059)] significantly inhibited HL-60 cell differentiation induced by capsaicin in combination with either 1,25-(OH)₂D₃ or all-trans retinoic acid. These results indicate that capsaicin potentiates 1,25-(OH)₂D₃- or all-trans retinoic acid-induced HL-60 cell differentiation and that both protein kinase C and extracellular signal-regulated kinase are involved in the cell differentiation synergistically enhanced by capsaicin.     

Comparative effects of 1α‐hydroxyvitamin D3 and 1,25‐dihydroxyvitamin D3 on transporters and enzymes in fxr(+/+) and fxr(‐/‐) mice

Previous studies have shown that 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] treatment in mice resulted in induction of intestinal and renal Cyp24a1 and Trpv6 expression, increased hepatic Cyp7a1 expression and activity, as well as higher renal Mdr1/P‐gp expression. The present study compared the equimolar efficacies of 1α‐hydroxyvitamin D₃ [1α(OH)D₃] (6 nmol/kg i.p. q2d × 4), a lipophilic precursor with a longer plasma half‐life that is converted to 1,25(OH)₂D₃, and 1,25(OH)₂D₃ on vitamin D receptor (VDR) target genes. To clarify whether changes in VDR genes was due to VDR and not secondary, farnesoid X receptor (FXR)‐directed effects, namely, lower Cyp7a1 expression in rat liver due to increased bile acid absorption, wildtype [fxr(+/+)] and FXR knockout [fxr(‐/‐)] mice were used to distinguish between VDR and FXR effects. With the exception that hepatic Sult2a1 mRNA was increased equally well by 1α(OH)D₃ and 1,25(OH)₂D₃, 1α(OH)D₃ treatment led to higher increases in hepatic Cyp7a1, renal Cyp24a1, VDR, Mdr1 and Mrp4, and intestinal Cyp24a1 and Trpv6 mRNA expression in both fxr(+/+) and fxr(‐/‐) mice compared to 1,25(OH)₂D₃ treatment. A similar induction in protein expression and microsomal activity of hepatic Cyp7a1 and renal P‐gp and Mrp4 protein expression was noted for both compounds. A higher intestinal induction of Trpv6 was observed, resulting in greater hypercalcemic effect following 1α(OH)D₃ treatment. The higher activity of 1α(OH)D₃ was explained by its rapid conversion to 1,25(OH)₂D₃ in tissue sites, furnishing higher plasma and tissue 1,25(OH)₂D₃ levels compared to following 1,25(OH)₂D₃‐treatment. In conclusion, 1α(OH)D₃ exerts a greater effect on VDR gene induction than equimolar doses of 1,25(OH)₂D₃ in mice. Copyright © 2013 John Wiley & Sons, Ltd.     

Prolactinemia is uncoupled from central D<Subscript>2</Subscript>/D<Subscript>3</Subscript> dopamine receptor occupancy in amisulpride treated patients

Rationale Atypical antipsychotic drugs are classically associated with lower propensity to extrapyramidal symptoms (EPS) and hyperprolactinemia than typical antipsychotic drugs. It has not been clarified why some atypical antipsychotic drugs, such as amisulpride, induce prolactin plasma concentration (PRL) elevation, but little EPS. Previous studies have found an association between striatal D₂/D₃ receptor occupancy and PRL in typical antipsychotic treated patients suggesting that PRL is a marker of central D₂/D₃ receptors blockade.Objective We have evaluated the relationship between PRL and central (striatum, temporal cortex and thalamus) D₂/D₃ receptor occupancy in amisulpride treated schizophrenic patients.Methods Single photon emission tomography (SPET) and [¹²³I]-epidepride were used to determine D₂/D₃ receptor occupancy in eight amisulpride treated patients. PRL was measured concurrently with the scans.Results The mean PRL was 1166 (range 499–1892 mIU/l) for a mean amisulpride dose of 406 mg/day (range 150–600 mg/day). Amisulpride plasma concentration and central D₂/D₃ receptor occupancy were positively correlated (r=0.83–0.89, df=4, P<0.05). No significant correlations were observed between PRL and amisulpride (daily dose or plasma concentration, P>0.05), or between PRL and central D₂/D₃ receptor occupancy (P>0.05).Conclusions Our findings show that amisulpride-induced hyperprolactinemia is uncoupled from central D₂/D₃ receptor occupancy. Amisulpride has poor blood–brain barrier penetration and reaches much higher concentration at the pituitary, which is outside the blood–brain barrier. Higher D₂/D₃ receptor occupancy at the pituitary gland than at central regions is a possible explanation for amisulpride PRL elevation with low EPS. Further studies evaluating pituitary D₂/D₃ receptor occupancy in vivo are necessary to confirm this hypothesis.This study was partially presented (poster) at the International Congress on Schizophrenia Research, Colorado Springs, USA, 2003 and received the Young Investigator Award.     

Non-uniform blockade of intrastriatal D<Subscript>2</Subscript>/D<Subscript>3</Subscript> receptors by risperidone and amisulpride

Rationale Atypical antipsychotic drugs have been shown to preferentially affect extrastriatal (mesolimbic) D₂/D₃ receptors over those within the striatum (nigrostriatal). The striatum does not contain exclusively nigrostriatal dopamine tracts, however. The caudate nucleus and ventral parts of the striatum primarily contain limbic and associative dopamine pathways more relevant to psychosis. Objectives We tested the hypothesis that two pharmacologically distinct atypical antipsychotic drugs, amisulpride and risperidone, would preferentially occupy of D₂/D₃ dopamine receptors in limbic and associative regions of the striatum. Methods Eight amisulpride-treated patients, six risperidone-treated patients and six age- and sex-matched healthy controls were recruited. Dynamic SPET studies were performed after bolus injection of [¹²³I]epidepride. Binding potential (BP) images were generated using a modified Logan method and aligned between subjects. Regions of interest (ROIs) were placed around head of caudate and putamen bilaterally on an average BP map derived from aligned control images. These ROIs were then applied user-independently to the BP maps for each subject to calculate BP for head of caudate and putamen. Mean occupancy of D₂/D₃ receptors in each ROI was determined by reference to the drug-free healthy volunteer group. Occupancy values for head of caudate and putamen were compared using paired Student’s t test. Results D₂/D₃ receptor occupancy was 42% in caudate and 31% in putamen for risperidone (t=5.9, df=11, p=0.0001) and 51% in caudate and 37% in putamen for amisulpride (t=11.1, df=15, p<0.0001). Conclusions Amisulpride and risperidone both show selective occupancy for limbic and associative D₂/D₃ receptors within the striatum.     

Auranofin promotes retinoic acid- or dihydroxyvitamin D3-mediated cell differentiation of promyelocytic leukaemia cells by increasing histone acetylation

BACKGROUND AND PURPOSE: To investigate the molecular mechanism for the effect of auranofin on the induction of cell differentiation, the cellular events associated with differentiation were analysed in acute promyelocytic leukaemia (APL) cells. EXPERIMENTAL APPROACH: The APL blasts from leukaemia patients and NB4 cells were cotreated with auroanofin and all-trans-retinoic acid (ATRA) at suboptimal concentration. The HL-60 cells were treated with auroanofin and a subeffective dose of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2 vit D3) in combination. The effect of auroanofin was investigated on histone acetylation at the promoter of differentiation-associated genes and expression of cell cycle regulators. KEY RESULTS: Treatment with auroanofin and ATRA cooperatively induced granulocytic differentiation of fresh APL blasts isolated from patients and NB4 cells. The combined treatment also increased reorganization of nuclear PML bodies and histone acetylation at the promoter of the RARbeta2 gene. Auroanofin also promoted monocytic differentiation of the HL-60 cells triggered by subeffective concentration of 1,25(OH)2 vit D3. The combined treatment of auroanofin and 1,25(OH)2 vit D3 stimulated histone acetylation at p21 promoters and increased the accumulation of cells in the G0/G1 phase. Consistent with this, the expressions of p21, p27 and PTEN were increased and the levels of cyclin A, Cdk2 and Cdk4 were decreased. Furthermore, the hypophosphorylated form of pRb was markedly increased in cotreated cells.Conclusions and implications:These findings indicate that auroanofin in combination with low doses of either ATRA or 1,25(OH)2 vit D3 promotes APL cell differentiation by enhancing histone acetylation and the expression of differentiation-associated genes.     

Clorgyline-induced switch from locomotion to mouthing in sensitization to the dopamine D<Subscript>2</Subscript>/D<Subscript>3 </Subscript>agonist quinpirole in rats: role of sigma and imidazoline I<Subscript>2</Subscript> receptors

Rationale. The monoamine oxidase inhibitor (MAOI) clorgyline, blocks locomotor sensitization to the D₂/D₃ dopamine agonist quinpirole and sensitizes self-directed mouthing behavior in rats by a mechanism independent of MAO inhibition. Clorgyline has a high affinity for imidazoline I₂ and sigma receptors, which could account for its effects on quinpirole sensitization. Objectives. To examine whether the effect of clorgyline on quinpirole sensitization is attributed to stimulation of either I₂ or sigma receptors. Methods. In one experiment, rats received injections of the I₂ receptor agonist 2-BFI (0.2 mg/kg, IP) or vehicle, 90 min prior to each injection of quinpirole (0.5 mg/kg, SC, × 8, twice weekly) or saline. A similar protocol was used to examine the effects of the MAOI Ro 41-1049 (10 mg/kg, SC) on quinpirole sensitization. Unlike clorgyline, Ro 41-1049 has no affinity for sigma or I₂ sites. An initial experiment demonstrated that intermittent injections of clorgyline (1 mg/kg, SC) are as effective as a continuous clorgyline administration (1 mg/kg per day via osmotic mini-pump) on quinpirole sensitization. Results. Like clorgyline, Ro 41-1049, but not 2-BFI, blocked the development of quinpirole-induced locomotor sensitization and induced instead sensitization of self-directed mouthing. Conclusions. Because Ro 41-1049 produced the same effects as clorgyline, and 2-BFI had no effects on quinpirole sensitization, it is unlikely that clorgyline exerts its effects via an action at sigma or I₂ receptors. Our results are consistent with the suggestion that clorgyline and Ro 41-1049 affect the behavioral response to quinpirole via the MAOI-displaceable quinpirole binding (MQB) site, and the hypothesis that the MQB site selects what motor output becomes sensitized to repeated injections of quinpirole. Electronic Publication     

Potencies of vitamin D analogs, 1α‐hydroxyvitamin D3, 1α‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3, in lowering cholesterol in hypercholesterolemic mice in vivo

Vitamin D₃ and the synthetic vitamin D analogs, 1α‐hydroxyvitamin D₃ [1α(OH)D₃], 1α‐hydroxyvitamin D₂ [1α(OH)D₂] and 25‐hydroxyvitamin D₃ [25(OH)D₃] were appraised for their vitamin D receptor (VDR) associated‐potencies as cholesterol lowering agents in mice in vivo. These precursors are activated in vivo: 1α(OH)D₃ and 1α(OH)D₂ are transformed by liver CYP2R1 and CYP27A1 to active VDR ligands, 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 1α,25‐dihydroxyvitamin D₂ [1,25(OH)₂D₂]_, respectively. 1α(OH)D₂ may also be activated by CYP24A1 to 1α,24‐dihydroxyvitamin D₂ [1,24(OH)₂D₂], another active VDR ligand. 25(OH)D₃, the metabolite formed via CYP2R1 and or CYP27A1 in liver from vitamin D₃, is activated by CYP27B1 in the kidney to 1,25(OH)₂D₃. In C57BL/6 mice fed the high fat/high cholesterol Western diet for 3 weeks, vitamin D analogs were administered every other day intraperitoneally during the last week of the diet. The rank order for cholesterol lowering, achieved via mouse liver small heterodimer partner (Shp) inhibition and increased cholesterol 7α‐hydroxylase (Cyp7a1) expression, was: 1.75 nmol/kg 1α(OH)D₃ > 1248 nmol/kg 25(OH)D₃ (dose ratio of 0.0014) > > 1625 nmol/kg vitamin D₃. Except for 1.21 nmol/kg 1α(OH)D₂ that failed to lower liver and plasma cholesterol contents, a significant negative correlation was observed between the liver concentration of 1,25(OH)₂D₃ formed from the precursors and liver cholesterol levels. The composite results show that vitamin D analogs 1α(OH)D₃ and 25(OH)D₃ exhibit cholesterol lowering properties upon activation to 1,25(OH)₂D₃: 1α(OH)D₃ is rapidly activated by liver enzymes and 25(OH)D₃ is slowly activated by renal Cyp27b1 in mouse.     

Proliferation and Differentiation of Cultured Human Keratinocytes is Modulated by 1,25(OH)2D3 and Synthetic Vitamin D3 Analogues in a Cell Density-, Calcium- and Serum-Dependent Manner*

Abstract: The natural form of vitamin D₃, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) decreases proliferation and promotes terminal differentiation of cultured human epidermal keratinocytes. The purpose of this study was to find out to what extent the culture conditions determine the sensitivety of keratinocytes to 1,25(OH)₂D₃ and synthetic vitamin D analogues. Human keratinocytes were grown in microplates. Cell proliferation (MTT-assay) and differentiation (quantity of transglutaminase type I) were measured consecutively in the same monolayer. When vitamin D₃ analogues were added to 50–60% confluent keratinocytes grown in serum-free keratinocyte growth medium with 0.09 mM Ca²⁺, stimulation of the proliferation was either minimal or non-existent, while differentiation was unaffected or slightly inhibited. There was no difference in the sensitivity to 1,25(OH)₂D₃ and the vitamin D₃ analogues. When 1,25(OH)₂D₃ was added to less confluent keratinocytes (30%) a marked antiproliferative effect was observed. Addition of 3% charcoal stripped foetal calf serum further enhanced the antiproliferative effect of 1,25(OH)₂D₃, and a difference in the sensitivity of the vitamin D₃ analogues was noted. If, finally, the Ca²⁺ concentration was raised to 0.3 mM, 1,25(OH)₂D₃ and the vitamin D₃ analogues stimulated differentiation. Also, a biphasic effect on proliferation occurred: stimulation at low vitamin D concentrations and inhibition at higher concentrations. Furthermore, keratinocytes became more sensitive to the synthetic vitamin D₃ analogues than to 1,25(OH)₂D₃: KH1060>EB1089>GS1500≥EB1213>calcipotriol>1,25(OH)₂D₃. For all compounds tested differentiation occurred at concentrations 10 to 30 times lower than for proliferation. These results indicate that the sensitivity to vitamin D₃ analogues as well as the direction of the response to vitamin D₃ analogues is dependent on the keratinocyte density, the availability of serum and Ca²⁺ concentrations.