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1.
Mol Cell Endocrinol ; 382(2): 960-70, 2014 Feb 15.
Article in English | MEDLINE | ID: mdl-24291609

ABSTRACT

1α-Hydroxylation of 25-hydroxyvitamin D3 is believed to be essential for its biological effects. In this study, we evaluated the biological activity of 25(OH)D3 itself comparing with the effect of cell-derived 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). First, we measured the cell-derived 1α,25(OH)2D3 level in immortalized human prostate cell (PZ-HPV-7) using [(3)H]-25(OH)D3. The effects of the cell-derived 1α,25(OH)2D3 on vitamin D3 24-hydroxylase (CYP24A1) mRNA level and the cell growth inhibition were significantly lower than the effects of 25(OH)D3 itself added to cell culture. 25-Hydroxyvitamin D3 1α-hydroxylase (CYP27B1) gene knockdown had no significant effects on the 25(OH)D3-dependent effects, whereas vitamin D receptor (VDR) gene knockdown resulted in a significant decrease in the 25(OH)D3-dependent effects. These results strongly suggest that 25(OH)D3 can directly bind to VDR and exerts its biological functions. DNA microarray and real-time RT-PCR analyses suggest that semaphorin 3B, cystatin E/M, and cystatin D may be involved in the antiproliferative effect of 25(OH)D3.


Subject(s)
Calcifediol/pharmacology , Prostate/drug effects , RNA, Messenger/genetics , Receptors, Calcitriol/genetics , Steroid Hydroxylases/genetics , Cell Line, Transformed , Cell Proliferation/drug effects , Chromatography, High Pressure Liquid , Cystatin M/genetics , Cystatin M/metabolism , Cystatins/genetics , Cystatins/metabolism , Gene Expression Regulation , Humans , Male , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Prostate/cytology , Prostate/metabolism , Protein Binding , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Calcitriol/antagonists & inhibitors , Receptors, Calcitriol/metabolism , Semaphorins/genetics , Semaphorins/metabolism , Signal Transduction , Steroid Hydroxylases/antagonists & inhibitors , Steroid Hydroxylases/metabolism , Tritium , Vitamin D3 24-Hydroxylase
2.
J Mol Endocrinol ; 47(2): 209-18, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21693624

ABSTRACT

According to the prevailing paradigm, 1α-hydroxylation of 25-hydroxyvitamin D(3) (25(OH)D(3)) and its analogs is a pre-requisite step for their biological effects. We previously reported that 25-hydroxy-19-nor-vitamin D(3) (25(OH)-19-nor-D(3)) had anti-proliferative activity in a cell line, PZ-HPV-7, which was derived from human non-cancerous prostate tissue, and suggested that 25(OH)-19-nor-D(3) acted after 1α-hydroxylation by vitamin D 1α-hydroxylase (CYP27B1). However, metabolic studies of 25(OH)-19-nor-D(3) using recombinant CYP27B1 revealed that 25(OH)-19-nor-D(3) was rarely subjected to 1α-hydroxylation. Therefore, in this report, we attempted to clarify the mechanism of 25(OH)-19-nor-D(3) action in intact cells using PZ-HPV-7 prostate cells. After incubating the cells with 25(OH)-19-nor-D(3), eight metabolites of 24-hydroxylase (CYP24A1) were detected, whereas no products of CYP27B1 including 1α,25-dihydroxy-19-nor-vitamin D(3) (1α,25(OH)(2)-19-nor-D(3)) were found. Furthermore, the time-dependent nuclear translocation of vitamin D receptor (VDR) and the subsequent transactivation of cyp24A1 gene in the presence of 25(OH)-19-nor-D(3) were almost identical as those induced by 1α,25(OH)(2)-19-nor-D(3). These results strongly suggest that 25(OH)-19-nor-D(3) directly binds to VDR as a ligand and transports VDR into the nucleus to induce transcription of cyp24A1 gene. In addition, knock down of cyp27B1 gene did not affect the anti-proliferative activity of 25(OH)-19-nor-D(3), whereas knock down of VDR attenuated the inhibitory effect. Thus, our results clearly demonstrate that the anti-proliferative activity of 25(OH)-19-nor-D(3) is VDR dependent but 1α-hydroxylation independent, suggesting that 25(OH)D(3) analogs such as 25(OH)-19-nor-D(3) could be attractive candidates for anticancer therapy.


Subject(s)
Calcitriol/pharmacology , Blotting, Western , Cell Line , Cell Proliferation/drug effects , Cholecalciferol , Humans , Protein Transport/drug effects , Protein Transport/genetics , RNA, Small Interfering , Receptors, Calcitriol/genetics , Receptors, Calcitriol/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Steroid Hydroxylases/genetics , Steroid Hydroxylases/metabolism , Vitamin D3 24-Hydroxylase
3.
Anticancer Res ; 29(9): 3547-53, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19667147

ABSTRACT

The high incidence of prostate cancer and lack of an effective, long-term treatment for metastatic disease highlights the need for more potent non-calcemic vitamin D analogs as potential alternative or combinational prostate cancer therapies. Among the analogs, 19-nor-1alpha,25-dihydroxyvitamin D2 (19-nor-1alpha,25(OH)2D2) known as paricalcitol or Zempler, has less calcemic effects and an equipotential activity as 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) in several in vivo and in vitro systems. It was recently demonstrated that a modified analog of paricalcitol, 19-nor-2alpha-(3-hydroxypropyl)-1alpha,25-dihydroxyvitamin D3 (MART-10) compared to 1alpha,25(OH)2D3 was more effective in inhibiting proliferation of an immortalized normal prostate cell line (PZ-HPV-7) (1,000-fold) and invasion of PC-3 prostate cancer cells (10-fold). In this study, the effects of MART-10 and 1alpha,25(OH)2D3 on proliferation, vitamin D receptor transactivation, vitamin D-binding protein (DBP) binding, CYP24A1 (24-OHase) substrate hydroxylation kinetics, and induction of CYP24A1 gene expression were compared in an androgen-dependent prostate cancer cell model, LNCaP. The results demonstrated that MART-10 was 1,000-fold more active than 1alpha,25(OH)2D3 in inhibiting LNCaP cell proliferation. MART-10 was more active than 1alpha,25(OH)2D3 in up-regulating a vitamin D receptor-responsive Luciferase construct and inducing CYP24A1 gene expression in LNCaP prostate cancer cells. In addition, MART-10 has a lower affinity for DBP and less substrate degradation by CYP24A1 compared to 1alpha,25(OH)2D3, indicating that MART-10 has more bioavailability and a longer half-life. Thus, these data suggest that MART-10 may be a potential candidate as a therapeutic agent for prostate cancer, especially for patients who fail in conventional therapies.


Subject(s)
Androgens/physiology , Cholecalciferol/analogs & derivatives , Neoplasms, Hormone-Dependent/drug therapy , Prostatic Neoplasms/drug therapy , Base Sequence , Cell Line, Tumor , Cholecalciferol/therapeutic use , DNA Primers , Humans , Male , Neoplasms, Hormone-Dependent/pathology , Polymerase Chain Reaction , Prostatic Neoplasms/pathology , RNA, Messenger/genetics , Steroid Hydroxylases/genetics , Vitamin D3 24-Hydroxylase
4.
Drug Metab Dispos ; 35(9): 1482-8, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17553915

ABSTRACT

Our previous study demonstrated that 25-hydroxy-19-nor-vitamin D(3) [25(OH)-19-nor-D(3)] inhibited the proliferation of immortalized noncancerous PZ-HPV-7 prostate cells similar to 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25(OH)(2)D(3)], suggesting that 25(OH)-19-nor-D(3) might be converted to 1 alpha,25-dihydroxy-19-nor-vitamin D(3) [1 alpha,25(OH)(2)-19-nor-D(3)] by CYP27B1 before exerting its antiproliferative activity. Using an in vitro cell-free model to study the kinetics of CYP27B1-dependent 1 alpha-hydroxylation of 25(OH)-19-nor-D(3) and 25-hydroxyvitamin D(3) [25(OH)D(3)] and CYP24A1-dependent hydroxylation of 1 alpha,25(OH)-19-nor-D(3) and 1 alpha,25(OH)(2)D(3), we found that k(cat)/K(m) for 1 alpha-hydroxylation of 25(OH)-19-nor-D(3) was less than 0.1% of that for 25(OH)D(3), and the k(cat)/K(m) value for 24-hydroxylation was not significantly different between 1 alpha,25(OH)(2)-19-nor-D(3) and 1 alpha,25(OH)(2)D(3). The data suggest a much slower formation and a similar rate of degradation of 1 alpha,25(OH)(2)-19-nor-D(3) compared with 1 alpha,25(OH)(2)D(3). We then analyzed the metabolites of 25(OH)D(3) and 25(OH)-19-nor-D(3) in PZ-HPV-7 cells by high-performance liquid chromatography. We found that a peak that comigrated with 1 alpha,25(OH)(2)D(3) was detected in cells incubated with 25(OH)D(3), whereas no 1 alpha,25(OH)(2)-19-nor-D(3) was detected in cells incubated with 25(OH)-19-nor-D(3). Thus, the present results do not support our previous hypothesis that 25(OH)-19-nor-D(3) is converted to 1 alpha,25(OH)(2)-19-nor-D(3) by CYP27B1 in prostate cells to inhibit cell proliferation. We hypothesize that 25(OH)-19-nor-D(3) by itself may have a novel mechanism to activate vitamin D receptor or it is metabolized in prostate cells to an unknown metabolite with antiproliferative activity without 1 alpha-hydroxylation. Thus, the results suggest that 25(OH)-19-nor-D(3) has potential as an attractive agent for prostate cancer therapy.


Subject(s)
25-Hydroxyvitamin D3 1-alpha-Hydroxylase/metabolism , Calcitriol/analogs & derivatives , Cholecalciferol/metabolism , Cytochrome P-450 Enzyme System/metabolism , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/genetics , Animals , Calcitriol/metabolism , Carbon Monoxide/metabolism , Cattle , Cell Line , Cell Proliferation/drug effects , Cholecalciferol/pharmacology , Chromatography, High Pressure Liquid , Cytochrome P-450 Enzyme System/genetics , Escherichia coli/metabolism , Humans , Hydroxylation , Kinetics , Male , Models, Molecular , Plasmids/genetics , Prostate/cytology , Prostate/metabolism , Steroid Hydroxylases , Thymus Gland/metabolism , Vitamin D3 24-Hydroxylase
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