Growth-inhibition of human-melanoma cells by vitamin-d analogs

The antiproliferative activity of 1,25(OH)(2)-vitamin D-3, and four vitamin D analogs was assessed in RPMI-7951, a human melanoma cell line which expresses the vitamin D receptor. Proliferation assays consisted of a [H-3]-thymidine incorporation assay, and a 6-day growth study. The affinity of vitamin D analogs for vitamin D receptor relative to 125(OH)(2)-vitamin D-3 was determined with a hydroxyapatite-based competitive binding assay. For the proliferation assays, cells were treated with 10(-8) M 1,25(OH)(2)-vitamin D-3, 1,25(OH)(2)-16-ene-23-yne-vitamin D-3 (Ro 23-7553), 1,25(OH)(2)-16-ene-23-yne-26,27-hexafluoro-vitamin D-3 (Ro 24-5531), 1,25(OH),-16,23Z-diene-26,27-hexafluoro-vitamin D-3 (Ro 25-5317), and 1 alpha-fluoro-25(OH)- 16-ene-23-yne-hexafluoro-vitamin D-3 (Ro 24-5583). 1,25(OH)(2)-vitamin D-3 and the four analogs all significantly inhibited melanoma cell growth (P<0.05). Competitive binding of the vitamin D analogs to vitamin D receptor ranged from 51% to 72% that of 1,25(OH)(2)-vitamin D-3, suggesting a receptor-mediated response. These results demonstrate that analogs of 1,25(OH)(2)-vitamin D-3 are potent antiproliferative agents in human melanoma cells in vitro.     

Influence of 1 alpha,25-dihydroxyvitamin D-3 on growth regulation through epidermal growth factor receptor in human breast cancer cell lines

We investigated the relationship between epidermal growth factor (EGF) dependent cell growth and antiproliferative effects of 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) in hormone responsive breast cancer cell lines in vitro. MCF-7 breast cancer cells and GMC-M, which is a serum-independent, hormone receptor-positive subtype derived from MCF-7, were used in this study. EGF stimulated the growth Of both cell lines, and 1,25(OH)(2)D-3 inhibited the EGF-stimulated cell growth in a dose dependent fashion. But treatment with 1,25(OH)(2)D-3 did not change the EGF receptor (EGFR) level significantly in either cell line. GMC-M had a higher level of EGFR and was more sensitive to EGF than MCF-7. These results suggest that other mechanisms of action, which are different from EGFR modulation, concern with the growth inhibitory effect of 1,25(OH)(2)D-3, and that 1,25(OH)(2)D-3 will be a new effective treatment for breast cancer irrespective of EGFR.     

The vitamin D3 analog, ILX-23-7553, enhances the response to Adriamycin and irradiation in MCF-7 breast tumor cells

Ionizing radiation and the anthracycline antibiotic, Adriamycin, generally fail to promote a primary apoptotic response in experimental breast tumor cell lines. Similarly, the primary response of breast tumor cells to vitamin D3 (1,25(OH)2D3) and vitamin D3 analogs such as EB 1089 is growth inhibition. Previous studies have demonstrated that pretreatment of MCF-7 breast tumor cells with vitamin D3 or EB 1089 can increase sensitivity to both Adriamycin and irradiation. PURPOSE: The capacity of the vitamin D3 analog, ILX 23-7553, to enhance the antiproliferative and cytotoxic effects of Adriamycin or irradiation and to promote apoptosis in MCF-7 breast tumor cells was assessed in the present study. RESULTS: Pretreatment of MCF-7 cells with ILX 23-7553 followed by Adriamycin or irradiation decreased viable cell numbers by 97% and 93%, respectively. Cell numbers were reduced by 56%, 74% and 75% by ILX 23-7553, Adriamycin and irradiation alone. Pretreatment with ILX 23-7553 also shifted the dose response curve for clonogenic survival, increasing sensitivity to Adriamycin 2.5-fold and sensitivity to radiation fourfold. In addition, ILX 23-7553 pretreatment conferred sensitivity to Adriamycin- or irradiation-induced DNA fragmentation and resulted in morphological changes indicative of apoptotic cell death in MCF-7 cells. Statistical analysis demonstrated that ILX 23-7553 interacts additively and not synergistically with both Adriamycin and irradiation. CONCLUSIONS: ILX 23-7553 enhances the effects of Adriamycin and irradiation in MCF-7 breast tumor cells by decreasing viable cell numbers, reducing clonogenic survival and inducing apoptotic cell death. Current studies are focused on elucidating the mechanisms underlying the induction of apoptosis as well as understanding the nature of the interactions between ILX 23-7553 and Adriamycin or irradiation.     

Simian virus 40-, but not human papillomavirus-, transformation of prostatic epithelial cells results in loss of growth-inhibition by 1,25-dihydroxyvitamin D-3

In addition to its well known calcemic actions, 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D] exhibits differentiating and antiproliferative effects in several types of cancer cells. 1,25(OH)(2)D receptors (VDR) as well as 1,25(OH)(2)D-mediated growth-inhibition have been demonstrated in human prostate cancer cell lines. In order to further develop model systems for the study of 1,25(OH)(2)D action and to elucidate the mechanism of growth-inhibition, we studied several human prostate cell lines immortalized with either simian virus 40 (SV40) or human papillomavirus type 18 (HPV). The SV40-transformed cell lines P69SV40-T and P153SV40-T were not growth-inhibited by 1,25(OH)(2)D at concentrations as high as 100 nM, whereas the HPV-transformed cells PZ-HPV-7 and CA-HPV-10 were growth-inhibited. All cell lines expressed VDR, and VDR mRNA was demonstrated by Northern blot analysis. All cells exhibited induction of 24-hydroxylase mRNA, a 1,25(OH)(2)D responsive gene, after 1,25(OH)(2)D treatment. In an attempt to understand the apparent dissociation of 1,25(OH)(2)D actions in the SV40-transformed cells, we turned to the human prostate cancer cell line DU 145. These cells, like the SV40-transformed cells, are not growth-inhibited but demonstrate induction of 24-hydroxylase mRNA after 1,25(OH)(2)D treatment. DU 145 cells contain a mutated retinoblastoma gene (Rb) which contributes to their uncontrolled growth, analogous to the disruption of Rb by SV40 and HPV. We compared DU,145 cells to DU 145 cells transfected with normal Rb (DU 145/Rb). Similar to DU 145, DU 145/Rb cells were not growth-inhibited by 1,25(OH)(2)D, while 24-hydroxylase mRNA was induced. These results suggest that divergent pathways mediate the growth-inhibitory effect of 1,25(OH)(2)D and its induction of 24-hydroxylase. It also appears that the antiproliferative effect of 1,25(OH)(2)D is mediated by an Rb-independent mechanism.     

Effect of the vitamin D3 analog ILX 23-7553 on apoptosis and sensitivity to fractionated radiation in breast tumor cells and normal human fibroblasts

PURPOSE: Previous work from this laboratory has demonstrated that the vitamin D(3) analogs EB 1089 and ILX 23-7553 enhance the response of breast tumor cells to ionizing radiation and promote radiation-induced apoptotic cell death. The current studies were designed to more closely simulate clinical radiotherapy in the treatment of breast cancer by examining the utility of ILX 23-7553 as an adjunct to fractionated ionizing radiation. The potential toxicity to normal tissue of the combination of ILX 23-7553 and fractionated radiation was assessed in a model of BJ human fibroblasts in culture. METHODS: MCF-7 cells and human fibroblasts were treated with fractionated radiation alone (5x2 Gy over 3 days), ILX 23-7553 alone (50 n M) or ILX 23-7553 followed by 5x2 Gy. Viable cell numbers were determined by trypan blue exclusion and apoptosis by the TUNEL assay. A statistical model of additivity was utilized to assess the nature of the interaction between ILX 23-7553 and fractionated radiation. RESULTS: Radiation and ILX 23-7553 each alone reduced viable cell numbers by 72+/-3.1% and 62+/-4.8%, respectively. Pretreatment with ILX 23-7553 followed by 5x2 Gy reduced viable cell numbers by 93.2+/-0.7%. The interaction between ILX 23-7553 and fractionated radiation appeared to be additive despite the fact that the combination of ILX 23-7553 and fractionated radiation also promoted a twofold increase in apoptotic cell death. ILX 23-7553 failed to enhance the response to radiation or to promote apoptosis in BJ human foreskin fibroblasts. CONCLUSIONS: ILX 23-7553 enhanced the antiproliferative and apoptotic effects of fractionated ionizing radiation in MCF-7 breast cancer cells. These effects appeared to be selective in that similar responses were not observed in a model of normal human fibroblasts. Vitamin D(3) analogs such as ILX 23-7553 may prove to have utility in combination with conventional radiotherapy of breast cancer as well as other malignancies which are sensitive to vitamin D(3).     

1,25-dihydroxyvitamin D3 and retonic acid analogues induce differentiation in breast cancer cells with function- and cell-specific additive effects

Vitamin D₃ derivatives and retinoids can induce cell cycle arrest, differentiation and cell death in many cell lines. These compounds can act cooperatively in some of their functions and may be of potential use either individually or in combination in the treatment of breast cancer. The effects of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), all-trans retinoic acid (ATRA) and several analogues were evaluated on malignant phenotypic traits of breast cancer cell lines MCF-7, T-47D and MDA-MB-231. Both 1,25(OH)₂D₃ and ATRA caused a decrease in anchorage independent colony formation in MCF-7 and T-47D cells in a dose-dependent manner. The effects of 1,25(OH)₂D₃ 10^–10 and 10^–9M were synergistic with ATRA 10^–8M in T-47D cells but were antagonistic in both MCF-7 and in T-47D cells at most concentrations. Both 1,25(OH)₂D₃ and ATRA individually induced an accumulation of MCF-7 cells in the G₁ phase of the cell cycle and an associated increase in p21^WAF1/Cip1, p27^Kipl and a dephosphorylation of Rb but the effects were not additive. Both compounds inhibited the invasive capacity of MDA-MB-231 cells. 1,25(OH)₂D₃ but not ATRA caused an increase in E-cadherin levels in MDA-MB-231 cells. These two functions were not additive. The compounds 1,25(OH)₂D₃, a noncalcemic analogue 1,25(OH)₂-16-ene-23-yne-D₃, ATRA, AGN195183, an RAR-specific agonist, and AGN190168 (tazarotene), an RAR-selective agonist, induced differentiation as determined by measurements of lipid droplet formation. The individual effects of 1,25(OH)₂-16-ene-23-yne-D₃ combined with ATRA or with tazarotene at 10^–9M each were additive in MCF-7 and MDA-MB-231 cells on lipid formation. The data demonstrate that both 1,25(OH)₂D₃, ATRA, and selected analogues induce a more differentiated phenotype in breast cancer cells with additive effects that are function- and cell-specific.     

1,25-Dihydroxyvitamin D3 and all-trans-retinoic acid sensitize breast cancer cells to chemotherapy-induced cell death

We investigated the capacity of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and all-trans-retinoic acid (ATRA) to sensitize three breast cancer cell lines to the cell killing effects of paclitaxel (Taxol) and Adriamycin, two chemotherapeutic agents commonly used in the treatment of breast cancer. In tissue culture colony assays, 1,25(OH)2D3 and ATRA were synergistic in inhibiting the clonogenicity of MCF-7 and T-47D cells that expressed estrogen receptor; vitamin D receptor; retinoic acid receptors (RARs) alpha, beta, and gamma; and retinoid X receptors alpha, beta, and gamma but were not additive in MDA-MB-231 cells that lacked expression of estrogen receptor, RARalpha, and RARbeta. The hormones used individually or in combination induced up to 40-50% cell death by a trypan blue exclusion assay in a dose-dependent manner up to concentrations of 10(-7) M in MCF-7 and T-47D cells, more modestly in MDA-MB-231 cells, and not at all in MCF-10 and MCF-12 nontransformed mammary epithelial cells. Pretreating the cancer cell lines with 1,25(OH)2D3 and ATRA individually or in combination for 3 days prior to a 1-h incubation with paclitaxel or Adriamycin decreased the ED50 for inhibition of colony formation or for cell death by trypan blue by up to 2 logs for paclitaxel and up to 1 log for Adriamycin in all three cell lines but had no effect on chemotherapy-induced MCF-12 cell death. The effects of the hormones were synergistic with those of the chemotherapy agents in all of the breast cancer cell lines, generally at the higher concentrations. Cell death took place by apoptosis. To determine one potential reason for the greater potentiation of the effects of paclitaxel than those of Adriamycin, we determined the effects of preincubation of MCF-7 cells on paclitaxel-induced phosphorylation of Bcl-2. Pretreatment of MCF-7 cells with either 1,25(OH)2D3 or ATRA increased the phosphorylation of Bcl-2 by variable concentrations of paclitaxel. These data suggest that pretreatment of breast cancer with 1,25(OH)2D3 or ATRA lowers the threshold for cell killing by chemotherapy agents and may provide a novel treatment option for this disease.     

CCAAT/enhancer-binding protein delta: a molecular target of 1,25-dihydroxyvitamin D3 in androgen-responsive prostate cancer LNCaP cells

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, inhibits the proliferation of prostate cancer cells. However, the molecular mechanisms by which 1,25(OH)2D3 inhibits the proliferation of these cells remain to be fully elucidated. In this study, we used microarray technology to identify target genes of 1,25(OH)2D3 in androgen-responsive prostate cancer LNCaP cells. 1,25(OH)2D3 up-regulated CCAAT/enhancer-binding protein delta (C/EBPdelta) by approximately 5-fold in these cells. Knockdown of C/EBPdelta expression by RNA interference showed that C/EBPdelta is essential for the significant growth inhibition of LNCaP cells in response to 1,25(OH)2D3 treatment. Moreover, we found that 1,25(OH)2D3 induced C/EBPdelta in other cancer cells, including the estrogen receptor (ER)-expressing MCF-7 and T47D breast cancer cells that are sensitive to the growth inhibitory effects of 1,25(OH)2D3. On the other hand, 1,25(OH)2D3 was not able to induce C/EBPdelta in either androgen receptor-negative PC-3 and DU145 or ER-negative breast cancer MDA-MB-231 cells that were relatively resistant to growth inhibition by 1,25(OH)2D3. Furthermore, forced expression of C/EBPdelta in prostate cancer LNCaP as well as breast cancer MCF-7 and T47D cells dramatically reduced their clonal growth. Taken together, forced expression of C/EBPdelta in cancer cells may be a promising therapeutic strategy.     

Vitamin-D3 derivatives and breast-tumor cell growth: Effect on intracellular calcium and apoptosis

Vitamin-D₃ derivatives are now well-recognized growth inhibitors of numerous tumoral cells and in particular breast-cancer cells. However, the mechanisms by which they operate are not well established. Among the wide range of physiological and biological functions of vitamin-D₃ derivatives, the best described include their action on calcium homeostasis. In this study, we sought to establish whether the effects of vitamin-D₃ derivatives on breast-cancer cell growth may be in part related to intracellular calcium modulation and induction of apoptosis. To address these questions, we used, in addition to 1,25(OH)₂D₃, the active metabolite of vitamin D₃, a non-calcemic 1,25(OH)₂D₃ derivative: Ro 23-7553 [16-ene-23-yne-1,25(OH)₂D₃], which in our hands was more potent than the parent compound in inhibiting breast-cancer cell growth. We showed that the efficiency of both compounds in growth inhibition was higher in the estradiol-receptor-positive-breast-tumor MCF-7 cells than in the estradiol-receptor-negative MDA-MB 231 cells. In MCF-7 cells in particular, important modifications of intracellular calcium related to the emptying of intracellular pools were observed. The depletion of Ca⁺⁺ from intracellular stores was followed by the induction of apoptosis. Such a phenomenon was never observed in MDA-MB 231 cells. Our results suggest that the action of vitamin-D₃ derivatives on the depletion of calcium stores, which was more significant in MCF-7 than in MDA-MB 231 cells, may induce apoptosis in the former cells and account for the high efficiency of vitamin-D₃ derivatives on growth inhibition of MCF-7 breast-tumor cells. © 1995 Wiley-Liss, Inc.     

Vitamin D receptors in breast cancer cells

Summary 1,25-(OH)₂-Vitamin D₃, the active metabolite of vitamin D, is a secosteroid hormone with known differentiating activity in leukemic cells. Studies have demonstrated the presence of vitamin D receptors (VDR) in a wide range of tissues and cell types. Antiproliferative activity of 1,25-(OH)₂-vitamin D₃ has been documented in osteosarcoma, melanoma, colon carcinoma, and breast carcinoma cells. This study was designed to analyze vitamin D receptor level in breast cancer cells as a marker of differentiation and as a predictor of growth inhibition by 1,25-(OH)₂-vitamin D₃.VDR messenger RNA was found to be present in relatively high levels in well-differentiated cells and in low levels in poorly differentiated cells. All cell lines had detectable VDR mRNA. Radiolabeled ligand binding assay showed a similar pattern. MCF-7 and T47D cells, which express VDR at moderate levels, showed significant growth inhibition by 10^–9 M 1,25-(OH)₂-vitamin D₃ (p < 0.05). MDA-MB-231 cells, which have very low levels of VDR, demonstrated no growth inhibition by 1,25-(OH)₂-vitamin D₃ at concentrations up to 10^–6 M. Based on these results it can be stated that VDR expression is lost with de-differentiation and that receptor is essential for the antiproliferative response to 1,25-(OH)₂-vitamin D₃.     

Vitamin D receptor (VDR) localization in human promyelocytic leukemia cells

Although vitamin D analogs are known to induce the differentiation of the HL-60 promyelocytic leukemia cells, the effect of vitamin D analogs on the distribution of vitamin D receptor (VDR) in these cells is not well studied. This report showed, by confocal microscopy, that VDR mainly resided in the cytoplasm in the absence of VDR ligands. When cells were treated with 19-nor-1alpha,25-(OH)(2)D(2) or 1,25(OH)(2)D(3), VDR moved from the cytoplasm into the nucleus in a time-dependent manner. VDR could be observed in the nucleus as early as 6 h after drug treatment and was still observed in the nucleus 3 days after one single addition of 100 nM 19-nor-1alpha,25-(OH)(2)D(2) or 1,25(OH)(2)D(3). The VDR protein level was significantly increased by 19-nor-1alpha,25-(OH)(2)D(2) or 1,25(OH)(2)D(3) in a dose-dependent manner, while the VDR mRNA level was not affected by either compound. These results suggest that binding of vitamin D analogs to VDR induced receptor translocation into the nucleus, which stabilizes the receptor, resulting in an accumulation of the VDR protein.     

Colonic epithelial cell proliferation decreases with increasing levels of serum 25-hydroxy vitamin D.

Epidemiological evidence suggests a potential role for vitamin D in colon cancer prevention. Vitamin D, absorbed from the intestine or derived from solar ultraviolet light, is metabolized in the liver to 25-hydroxyvitamin D (25-OH D(3)). Previous studies examining effects of vitamin D upon carcinogenesis have focused upon the active metabolite 1,25-dihydroxyvitamin D [1,25-(OH)(2) D(3)], which interacts with nuclear vitamin D receptors in several organs. Until recently, the metabolism of 25-OH D(3) to 1,25-(OH)(2) D(3) was believed to occur only in the kidney, but more recent studies have shown that 25-OH D(3) conversion to 1,25-(OH)(2) D(3) can occur in other tissues. We examined the association between fasting levels of 25-OH D(3), 1,25-(OH)(2) D(3), and BsmI polymorphism of the vitamin D receptor (VDR) gene with indices of colonic epithelial cell proliferation and differentiation in a chemoprevention study, after giving vitamin D or calcium and taking rectal biopsies that were incubated with bromodeoxyuridine. Vitamin D receptor polymorphism was determined by genotyping of the 3' BsmI polymorphism in intron eight of the VDR gene. No significant changes in cell proliferation or in differentiation were found in subjects between study start and end. However, fasting serum levels of 25-OH D(3) showed a highly significant decrease with whole crypt labeling index and the size of the proliferative compartment (phi h). There was no correlation between serum levels of 1,25-(OH)(2) D(3) and the proliferative parameters. Calcium supplementation induced a significant effect upon the relationship between serum 25-OH D(3) and rectal epithelial cell labeling index and phi h when studied by covariance analysis without a relationship with 1,25-(OH)(2) D(3) levels. VDR genotype did not influence the effects of serum 25-OH D(3) or serum 1,25-(OH)(2) D(3) levels upon proliferation. These data suggest that there might be a local effect of 25-OH D(3) on colonic epithelial cells through conversion of 25-OH D(3) to 1,25-(OH)(2) D(3). Subsequent studies have demonstrated the presence of 1alpha-hydroxylase mRNA in normal colorectal epithelium and in colorectal cancer. Thus, vitamin D may have an important role in determining the effects of calcium on colorectal epithelial proliferation and may explain some of the discrepancies found previously in studies that examine the direct role of calcium on the colorectal epithelium.