kh-1060 and Breast-Neoplasms

Vitamin D-related compounds can inhibit cancer cell growth, but the biologic mechanism of this inhibition remains to be determined. We investigated the possibility that these compounds interfere with the activity of insulin-like growth factors. Such activity can be suppressed or otherwise modulated by specific insulin-like growth factor-binding proteins.. The human breast cancer cell line MCF-7 was used in this study. The effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and two related compounds, EB1089 and KH1060, on cell proliferation were assessed by monitoring cell numbers and by measuring cellular incorporation of [3H]thymidine. Changes in the accumulation of insulin-like growth factor-binding proteins in cell-conditioned media (i.e., culture fluids) were assessed by means of standard protein blotting techniques; ligand blots were probed with [125I]insulin-like growth factor I, and immunoblots were probed with antibodies raised against specific binding proteins. Binding protein messenger RNA levels were determined by use of RNA blotting methods and complementary DNA probes.. At concentrations of 10(-8) M and 10(-9) M, EB1089 and KH1060 exhibited stronger antiproliferative activity than 1,25(OH)2D3. When each of the vitamin D-related compounds was used separately at a concentration of 10(-9) M, a 20- to 25-fold increase in the concentration of insulin-like growth factor-binding proteins in MCF-7 cell-conditioned media was observed; this binding capacity was increased nine-fold, ninefold, and threefold, respectively, in the presence of 10(-10) M EB1089, KH1060, and 1,25(OH)2D3. Immunoblotting experiments demonstrated that all three vitamin D-related compounds induced the accumulation of insulin-like growth factor-binding protein 5 in cell-conditioned media. The accumulation of this binding protein was associated with an increase in cellular expression of its messenger RNA. EB1089 and 1,25(OH)2D3 attenuated the growth-promoting activity of insulin-like growth factor I on MCF-7 cells; however, these compounds did not inhibit the growth-promoting activity of long R3 IGF-I, an insulin-like growth factor I analogue with greatly reduced affinity for insulin-like growth factor-binding proteins.. Our results indicate that vitamin D-related compounds stimulate production of insulin-like growth factor-binding protein 5, thereby indirectly suppressing cell proliferation.

Topics: Antineoplastic Agents; Blotting, Northern; Blotting, Western; Breast Neoplasms; Calcitriol; Cell Division; Female; Humans; Insulin-Like Growth Factor Binding Protein 5; Tumor Cells, Cultured

We have analyzed the effect of 1,25-dihydroxyvitamin D3 on the expression of the gene encoding apolipoprotein D (apoD), a protein component of the human plasma lipid transport system that is overproduced by a specific subset of breast carcinomas. Northern blot analysis revealed that 1,25-dihydroxyvitamin D3 strongly up-regulated apoD mRNA levels in T-47D human breast cancer cells in a time- and dose-dependent manner. The potency of this vitamin as an inducer of apoD expression was stronger than the effect observed for such steroid hormones as androgens and progesterone, described previously as hormonal up-regulators of apoD expression in these cells. A time course study demonstrated that the induction of apoD mRNA reached a level of 5-fold over the untreated cells after 48 h of incubation in the presence of 10(-7) M 1,25-dihydroxyvitamin D3. A dose-response analysis showed that a 10(-6) M concentration of this vitamin consistently induced a maximal accumulation of 7-fold over the control cells. Similar up-regulatory effects on the apoD gene expression were obtained by treatment of T-47D cells with 1,25-dihydroxyvitamin D3 analogues, including MC 903, which is relatively devoid of hypercalcemic side effects in clinical applications. Western blot analysis revealed that the inductive effect of 1,25-dihydroxyvitamin D3 was also reflected at the protein level as an increase of immunoreactive protein in the conditioned media of vitamin-treated cells. This increased expression of apoD was accompanied by an inhibition of cell growth and morphological changes in T-47D cells. By contrast, we did not detect any inductive effect of 1,25-dihydroxyvitamin D3 on apoD gene expression in MDA-MB-231 cells, which are refractory to the growth-inhibitory effects of this compound. On the basis of these results, we propose 1,25-dihydroxyvitamin D3 as an important regulator of the expression of the apoD gene in breast carcinomas. We also suggest that apoD may be of interest as a biochemical marker of the action of 1,25-dihydroxyvitamin D3 derivatives in current studies using these compounds as inhibitors of breast cancer cell growth or as chemotherapeutic agents in the prevention of breast cancer.

Topics: Antineoplastic Agents; Apolipoproteins; Apolipoproteins D; Breast Neoplasms; Calcitriol; Carcinoma; Cycloheximide; Dactinomycin; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Growth Inhibitors; Humans; Protein Synthesis Inhibitors; RNA, Messenger; RNA, Neoplasm; Time Factors; Tumor Cells, Cultured

The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and four novel synthetic analogues (EB1089, KH1060, KH1230 and CB1093) on IGF-I-stimulated growth of MCF-7 human breast cancer cells have been determined. A significant time- and dose-dependent inhibition of IGF-I-stimulated cell growth was seen with EB1089, such that after 7 days of treatment with 10(-8) M EB1089, the mitogenic effect of IGF-I (30 ng/ml) was negated. Comparison with 1,25(OH)2D3 showed the synthetic analogues to be more potent. The anti-oestrogen ICI 182,780 similarly inhibited IGF-I-stimulated growth of these cells and in combination with EB1089 exerted additional inhibitory effects. Retinoids (all-trans-retinoic acid or the isomer 9-cis-retinoic acid) were less effective in limiting MCF-7 cell responsiveness to IGF-I but, in combination with EB1089, a co-operative effect was achieved. Using radioligand-binding techniques, we observed that 1,25(OH)2D3 and EB1089 down-regulated the levels of 125I-IGF-I binding to MCF-7 cell membranes. Scatchard analysis showed that EB1089 decreased maximal binding approximately 2-fold. Vitamin D derivatives were also demonstrated to reduce IGF-I receptor expression in MCF-7 cells by Western analysis. Our findings demonstrate that vitamin D derivatives limit responsiveness of MCF-7 cells to the mitogenic effects of IGF-I, which may be mediated by reduction of IGF-I receptor expression.

Topics: Antineoplastic Agents; Breast Neoplasms; Calcitriol; Dose-Response Relationship, Drug; Female; Humans; Immunosuppressive Agents; Insulin-Like Growth Factor I; Mitosis; Time Factors; Tumor Cells, Cultured

The mechanisms involved in the antiproliferative action of calcitriol (1 alpha, 25(OH)2D3) were investigated using human breast carcinoma epithelial cells (the MCF-7 cell line). Calcitriol and KH1060, a synthetic analog, inhibited cell growth in a time-and dose-dependent way. The substances similarly stimulated total TGF-beta secretion after 24 hours, and Northern blot analyses showed that mRNA levels for TGF-beta 1 were increased, as well. When MCF-7 cells were co-incubated with calcitriol and a neutralizing anti TGF-beta 1, beta 2, beta 3 antibody, growth inhibition was completely abrogated. With KH1060, the antibody could only partly block growth inhibition. This study shows that TGF-beta is involved in the growth response to calcitriol and KH1060 in MCF-7 cells.

Topics: Adenocarcinoma; Breast Neoplasms; Calcitriol; Dose-Response Relationship, Drug; Female; Humans; Immunosuppressive Agents; Transforming Growth Factor beta; Tumor Cells, Cultured

The hormone 1,25-dihydroxyvitamin D3 (VD) has a potential use as an anti-tumor agent, but its clinical applications are restricted by its strong calcemic activity. This has led to the development of VD analogues with selectively increased growth-inhibitory activity. One of the most potent analogues is KH1060, which is a 20-epi-22-oxa-derivative of VD. In human breast cancer MCF-7 cells, we studied the growth-inhibitory activities of a set of 8 analogues that cover conservative structural changes from 20-epi-VD (MC1288) to KH1060. In the same cellular system, we analyzed the potential of these 8 analogues to stimulate reporter gene activity driven by a recently discovered novel-type VD response element. We found that this VD response element is more appropriate than classical VD response elements to correlate anti-proliferative effects of VD analogues with their gene-regulatory properties.

Topics: Amino Acid Sequence; Antineoplastic Agents; Binding Sites; Breast Neoplasms; Calcitriol; Calcium; Cell Division; Humans; Molecular Sequence Data; Molecular Structure; Promoter Regions, Genetic; Receptors, Calcitriol; Signal Transduction; Tumor Cells, Cultured

The hormone 1,25-dihydroxyvitamin D3 (VD) has the potential for clinical use in several diseases, such as cancer, osteoporosis, and psoriasis. The action of VD is mediated by primary responding genes that contain in their promoter region a binding site for the transcription factor VDR. Most of the known VD response elements are formed by a direct repeat of two hexameric core binding motifs spaced by three nucleotides (DR3) bound by a heterodimer of VDR and the retinoid X receptor (RXR). Various VD analogues have been developed in order to optimize the therapeutic profile of VD. This report presents a novel experimental system that may help in the understanding of the structural basis for the high potency of a VD analogue like KH1060, which is a 20-epi-22-oxa-derivative of VD. In human breast cancer cells, MCF-7, the half-maximal gene activation values for KH1060 and seven of its structural precursors were determined on a DR3-type VD response element. These eight analogues cover conservative structural changes from 20-epi-VD (MC1288) to KH1060. With a modified version of the limited protease digestion assay the functional affinity of the analogues to VDR was measured. The functional receptor affinity of the eight analogues was found to be directly proportional to their potency in VDR-RXR-mediated gene activity.

Topics: Binding Sites; Breast Neoplasms; Calcitriol; Dose-Response Relationship, Drug; Endopeptidases; Gene Expression; Humans; Protein Conformation; Receptors, Calcitriol; Receptors, Retinoic Acid; Recombinant Proteins; Retinoid X Receptors; Structure-Activity Relationship; Transcription Factors; Transfection; Tumor Cells, Cultured

Breast cancer is a devastating disease. New approach in therapy are needed. In this chapter, we describe our efforts to identify novel vitamin D3 analogs which may have a potent antiproliferative effect on breast cancer without causing hypercalcemia.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Calcitriol; Cell Differentiation; Cell Division; Cell Nucleus; Humans; Immunohistochemistry; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has potential to be used as an antitumor agent, but its clinical application is restricted by the strong calcemic activity. Therefore, new vitamin D3 analogues are developed with increased growth inhibitory and reduced calcemic activity. In the present study, we have examined the antiproliferative effects of four novel vitamin D3 analogues (CB966, EB1089, KH1060, and 22-oxa-calcitriol) on breast cancer cells, either alone or in combination with the antiestrogen tamoxifen. The estrogen-dependent ZR-75-1 and estrogen-responsive MCF-7 cell lines were used as a model. It was shown that, with EB1089 and KH1060, the same growth inhibitory effect as 1,25-(OH)2D3 could be reached at up to 100-fold lower concentrations, whereas CD966 and 22-oxa-calcitriol were nearly equipotent with 1,25-(OH)2D3. The growth inhibition by the vitamin D3 compounds could be augmented by combined treatment with tamoxifen. At the maximal effective concentrations of the vitamin D3 compounds, the effect of combined treatment was addictive (MCF-7 cells) or less than additive (ZR-75-1 cells). Tamoxifen increased the sensitivity of the cells to the vitamin D3 compounds 2- to 4000-fold, which was expressed by a shift to lower median effective concentration values. Thereby, the vitamin D3 compounds may be used at even lower dosages in combination therapy with tamoxifen. A major problem of tamoxifen therapy is the development of tamoxifen resistance. We have observed that tamoxifen-resistant clones of ZR-75-1 cells retain their response to the vitamin D3 compounds. Regulation of the growth-related oncogene c-myc (mRNA level) and the estrogen receptor (protein level) were studied but appeared not to be related to the antiproliferative action of the vitamin D3 compounds. Together, our data point to a potential benefit of combination therapy with 1,25-(OH)2D3 or vitamin D3 analogues and tamoxifen for the treatment of breast cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Calcitriol; Cell Division; Drug Resistance; Drug Screening Assays, Antitumor; Estradiol; Humans; Proto-Oncogene Proteins c-myc; Receptors, Calcitriol; Tamoxifen; Tumor Cells, Cultured