In the present study, we have demonstrated 1α-hydroxylase protein expression in 15 out of 19 (79%) analyzed breast cancer specimens, in 10 apparently normal breast biopsies from breast cancer patients and also in MCF-7 cells. The observed somewhat reduced overall 1α-hydroxylase mRNA expression level as well as expression of VDR protein and mRNA in the tumors were apparently consistent with the immunohistochemical results, also indicating that representative mRNA was isolated. Of the analyzed tumors, 95% stained for VDR, in agreement with the 80% to 90% observed in earlier studies [
37,
38].
Previous studies have demonstrated 1α-hydroxylation of the prohormone 25(OH)D
3 and inhibition of cell proliferation in cultured prostate cancer cells expressing 1α-hydroxylase [
24,
26]. 1α-hydroxylase is also expressed and active in colorectal cancer [
20‐
23] and in ovarian cancer [
40]. The non-calcemic prohormone 25(OH)D
3, which exhibits very low activity
in vitro and
in vivo in the absence of 1α-hydroxylase [
41,
42], has been considered a future preventive and/or therapeutic option. A problem is rapid 24-hydroxylation and subsequent degradation of 25(OH)D
3 and of locally synthesized 1,25(OH)
2D
3. Use of more specific 24-hydroxylase inhibitors [
43] than liarozole and ketoconazole [
34,
44] may present future therapeutic options. We reasoned that an alternative to 25(OH)D
3 could be a non-1α-hydroxylated vitamin D analogue [
27], with a relative resistance to 24-hydroxylation by the 24-hydroxylase. The activity of 1α-hydroxylase in the kidney is tightly regulated by PTH and 1,25(OH)
2D
3 and even large increases in serum 25(OH)D
3 will not produce hypercalcemia [
25]. Similarily, local production of a VDR binding analogue by hydroxylation would not be expected to cause the systemic effect of hypercalcemia. Here we have shown that the non-1α-hydroxylated prodrug of EB1089 (EB1285) could activate transcription in MCF-7 cells, which express the 1α-hydroxylase enzyme. The activation of transcription was ketoconazole-sensitive, strongly suggesting that the observed effect was due to 1α-hydroxylation. Thus, a vitamin D analogue could constitute a substrate for the 1α-hydroxylase enzyme; however, EB1285 exhibited low transcription activation potential compared to EB1089 in MCF-7 cells. This may indicate low 1α-hydroxylase enzyme activity in the cells and/or possibly inefficient hydroxylation due to steric hindrance between substrate and enzyme. Design of novel non-1α-hydroxylated vitamin D analogues for the prevention or treatment of proliferative disorders in which 1α-hydroxylase is expressed or induced is warranted.