Endocrine Abstracts

To dissect mechanisms which result in 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) insensitivity we have compared the antiproliferative actions of 1,25(OH)₂D₃ in a range of malignant breast cell lines (ZR-75-1, T-47D, MCF-7, MCF-7Res and MDA-MB-231) and the non-malignant breast epithelial cells MCF-12A cells. All the malignant cell lines displayed greater resistance than MCF-12A cells to 1,25(OH)₂D₃. For example, the ED50 dose in MCF-12A cells was 5 nM whereas in MDA-MB-231 cells it was greater than 100nM; the other cancer cell lines displayed an intermediate spectrum of responses. Further we used quantitative real- time RT-PCR to demonstrate that increased 1,25(OH)₂D₃ resistance correlated with significantly reduced expression of both CYP27b, the gene that encodes the 25(OH)D₃ activating enzyme 25-hydroxyvitamin D-1 alpha-hydroxylase, and the vitamin D receptor (VDR), and elevated expression of CYP24, the gene that encodes the 1,25(OH)₂D₃-metabolising enzyme 25(OH)D₃-24-hydroxylase. Thus in MDA-MB-231 cells, relative to MCF-12A cells, there was a 0.36 and 0.12 fold reduction in CYP27b and VDR mRNA respectively (p<0.05) and a dramatic 74-fold elevation in CYP24 levels (p<0.001).
To further investigate the altered role of CYP27b we overexpressed it in MCF-7 cells. Quantitive real-time RT-PCR and Western blotting confirmed that CYP27b expression was significantly increased (28 fold and 14 fold respectively). Furthermore using proliferation and apoptosis studies we demonstrate that the stable overexpression of CYP27b in MCF-7 cells significantly elevated their antiproliferative response to the pro hormone 25(OH)D₃, but not to the active hormone 1,25(OH)₂D₃ itself.
We proposed that the progression of breast carcinoma is associated not only with changes in VDR expression but also with the pre-receptor control of ligand availability through the vitamin D hydroxylases, CYP27b and CYP24.