Kinetic studies of 25-hydroxy-19-nor-vitamin D3 and 1 alpha,25-dihydroxy-19-nor-vitamin D3 hydroxylation by CYP27B1 and CYP24A1.
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.
Urushino
Naoko
N
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto, Japan.
Nakabayashi
Sachie
S
Arai
Midori A
MA
Kittaka
Atsushi
A
Chen
Tai C
TC
Yamamoto
Keiko
K
Hayashi
Keiko
K
Kato
Shigeaki
S
Ohta
Miho
M
Kamakura
Masaki
M
Ikushiro
Shinichi
S
Sakaki
Toshiyuki
T
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000378:metabolism; D002248:Carbon Monoxide / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002762:Cholecalciferol / Q000378:metabolism* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008297:Male; D008958:Models, Molecular; D010957:Plasmids / Q000235:genetics; D011467:Prostate / Q000166:cytology / Q000378:metabolism; D013250:Steroid Hydroxylases; D013950:Thymus Gland / Q000378:metabolism; D065668:Vitamin D3 24-Hydroxylase
C114865:1-hydroxy-19-norvitamin D3; C083611:1,25-dihydroxy-19-norprevitamin D3; D002762:Cholecalciferol; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C585549:CYP24A1 protein, human; D065668:Vitamin D3 24-Hydroxylase; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D002117:Calcitriol