Regulation of steroid receptor expression by 1alpha-hydroxyvitamin D5 in hormone-responsive breast cancer cells.

BACKGROUND: Vitamin D analog, 1alpha-hydroxy-24-ethyl-cholecalciferol (1alpha(OH)D5), is a less toxic VDR agonist that suppresses proliferation of breast cancer cells in vitro and in vivo. The present study assessed 1alpha(OH)D5-mediated regulation of VDR, and its potential anti-estrogenic activity in BT-474 cells. MATERIALS AND METHODS: The mRNA and protein expression of steroid receptors were determined using RT-PCR and Western blot analyses, respectively. RESULTS: VDR mRNA was up-regulated (180% of control) by 1alpha(OH)D5 within seven hours, whereas the expression of VDR protein increased by two-fold in 24 hours. This increase was abolished in presence of either actinomycin D or cyclohexamide. Additionally, there was a four-fold decrease in ERalpha mRNA and 40% decrease in ERalpha protein after 28 and 48 hours following 1alpha(OH)D5 treatment, respectively. Down-regulation of some of the estrogen-inducible genes was observed. CONCLUSION: Although no VDR stabilization by 1alpha(OH)D5 was observed, there was an increased expression of the VDR followed by partial anti-estrogenic activity in hormone-responsive BT-474 cells.

Overexpression of ER and VDR is not sufficient to make ER-negative MDA-MB231 breast cancer cells responsive to 1alpha-hydroxyvitamin D5.

1alpha-hydroxyvitamin D(5) [1alpha(OH)D(5)] is an active vitamin D analog showing promising chemopreventive effect in breast carcinogenesis. We previously reported that estrogen receptor (ER)-positive breast cancer cells were sensitive, whereas ER-negative breast cancer cells were relatively resistant to their antiproliferative effects. In the present study, we used ER-negative MDA-MB231, ER-transfected MDA-MB231 (S30) and ER-positive BT474 cell lines to evaluate the possible association between ER status and cellular sensitivity to 1alpha(OH)D(5) treatment. Our results demonstrate that ER expression in ER-negative breast cancer cells (S30) did not increase the sensitivity to 1alpha(OH)D(5), whereas in ER-positive BT474 cells, the significant antiproliferative effect of 1alpha(OH)D(5) was correlated with the downregulation of ER and progesterone receptor expression. Further analysis indicated that both MDA-MB231 and S30 cells express low vitamin D receptor (VDR) at transcriptional level and protein level. However, transfection of VDR failed to restore the sensitivity to 1alpha(OH)D(5) in MDA-MB231 and S30 cells, although VDR direct target gene CYP24 was more responsive to 1alpha(OH)D(5) treatment in MDA-MB231 and S30 cells overexpressing VDR. In addition, nuclear receptor cofactors NCoR1 and SRC1 that could potentially affect VDR action were also low in both MDA-MB231 and S30 cells in comparison with ER-positive, vitamin D-sensitive BT474 cells. These results suggest that in addition to the increased ER and VDR expression, the intact VDR signaling machinery as present in ER-positive, vitamin D-sensitive cells is essential for the antiproliferative action of vitamin D, whereas the direct VDR target genes such as CYP24 can remain responsive to augmented VDR expression.

Growth inhibition of carcinogen-transformed MCF-12F breast epithelial cells and hormone-sensitive BT-474 breast cancer cells by 1alpha-hydroxyvitamin D5.

Several studies have established the active form of vitamin D(3) as an effective tumor-suppressing agent; however, its antitumor activity is achieved at doses that are hypercalcemic in vivo. Therefore, less calcemic vitamin D(3) analog, 1alpha-hydroxy-24-ethyl-cholecalciferol (1alpha[OH]D5), was evaluated for its potential use in breast cancer chemoprevention. Previously, 1alpha(OH)D5 showed anticarcinogenic activity in several in vivo and in vitro models. However, its effects on growth of normal tissue were not known. The present study was conducted to determine the effects of 1alpha(OH)D5 on the growth of normal mouse mammary gland and normal-like human breast epithelial MCF-12F cells and to compare these effects with carcinogen-transformed MCF-12F and breast cancer cells. No significant difference was observed in the growth or morphology of cultured mouse mammary gland and MCF-12F cells in the presence of 1alpha(OH)D5. However, the transformed MCF-12F cells underwent growth inhibition (40-60%, P < 0.05) upon 1alpha(OH)D5 treatment as determined by cell viability assays. Cell cycle analysis showed marked increase (50%) in G-1 phase for cells treated with 1alpha(OH)D5 compared with the controls. Moreover, the percentage of cells in the synthesis (S) phase of cell cycle was decreased by 70% in transformed MCF-12F, BT-474 and MCF-7 cells. The growth arrest was preceded by an increase in expression of cell cycle regulatory proteins p21(Waf-1) and p27(Kip-1). In addition, differential expression studies of parent and transformed MCF-12F cell lines using microarrays showed that prohibitin mRNA was increased 4-fold in the transformed cells. These results indicate that the growth inhibitory effect of 1alpha(OH)D5 was achieved in both carcinogen-transformed MCF-12F and breast cancer cells at a dose that was non-inhibitory in normal-like breast epithelial cells.