Kinetic studies of 25-hydroxy-19-nor-vitamin D₃ and 1α,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-D₃] inhibited the proliferation of immortalized noncancerous PZ-HPV-7 prostate cells similar to 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D ₃], suggesting that 25(OH)-19-nor-D₃ might be converted to 1α,25-dihydroxy-19-nor-vitamin D₃ [1α,25(OH) ₂-19-nor-D₃] by CYP27B1 before exerting its antiproliferative activity. Using an in vitro cell-free model to study the kinetics of CYP27B1-dependent 1α-hydroxylation of 25(OH)-19-nor-D ₃ and 25-hydroxyvitamin D₃ [25(OH)D₃] and CYP24A1-dependent hydroxylation of 1α,25(OH)-19-nor-D₃ and 1α,25(OH)₂D₃, we found that k_cat/K _m for 1α-hydroxylation of 25(OH)-19-nor-D₃ was less than 0.1% of that for 25(OH)D₃, and the k_cat/K_m value for 24-hydroxylation was not significantly different between 1α,25(OH)₂-19-nor-D₃ and 1α,25(OH) ₂D₃. The data suggest a much slower formation and a similar rate of degradation of 1α,25(OH)₂-19-nor-D₃ compared with 1α,25(OH)₂D₃. We then analyzed the metabolites of 25(OH)D₃ and 25(OH)-19-nor-D₃ in PZ-HPV-7 cells by high-performance liquid chromatography. We found that a peak that comigrated with 1α,25(OH)₂D₃ was detected in cells incubated with 25(OH)D₃, whereas no 1α,25(OH) ₂-19-nor-D₃ was detected in cells incubated with 25(OH)-19-nor-D₃. Thus, the present results do not support our previous hypothesis that 25(OH)-19-nor-D₃ is converted to 1α,25(OH)₂-19-nor-D₃ by CYP27B1 in prostate cells to inhibit cell proliferation. We hypothesize that 25(OH)-19-nor-D₃ 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 1α-hydroxylation. Thus, the results suggest that 25(OH)-19-nor-D₃ has potential as an attractive agent for prostate cancer therapy.