Action of low calcemic 1alpha,25-dihydroxyvitamin D3 analogue EB1089 in head and neck squamous cell carcinoma

BACKGROUND: 1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and its analogues inhibit growth of various types of cancer cells. Although the therapeutic potential of 1,25(OH)(2)D(3) is limited by its tendency to induce hypercalcemia, analogues such as EB1089 are potent inhibitors of cell growth and exhibit reduced calcemic effects. We analyzed the antiproliferative and calcemic effects of EB1089 in tissue culture and animal models of head and neck squamous cell carcinoma (SCC) to investigate its potential as a chemotherapeutic/chemopreventive agent. METHODS: The effects of 1,25(OH)(2)D(3) and EB1089 on cell growth and expression of p21(WAF1/CIP1) and p27(KIP1), which encode cyclin-dependent kinase inhibitors, and a novel target, gadd45alpha, a growth-arrest and DNA-damage gene, were monitored in cultured murine AT-84 SCC cells. The effects of these agents on AT-84 cell growth in vitro and on growth of AT-84 tumors in syngeneic C3H mice were monitored; treatment started at the time of tumor implantation (early tumor model) or after 12 days (late tumor model). Weight and serum calcium levels were also monitored in these animals. All P values were two-sided. RESULTS: Both 1,25(OH)(2)D(3) and EB1089 arrested proliferation of AT-84 cells in G(0)/G(1) phase, inhibited p21(WAF1/CIP1) expression, and induced expression of p27(KIP1) protein. 1,25(OH)(2)D(3) also enhanced the expression of gadd45alpha, apparently by a p53-independent mechanism. There was a statistically significant decrease in tumor growth for 1,25(OH)(2)D(3)-treated mice (P<.001 for early tumor model) and EB1089-treated mice (P<.001 and P =.001 for early and late tumor models, respectively). Unlike 1,25(OH)(2)D(3), EB1089 did not induce cachexia or hypercalcemia. The effects of 1,25(OH)(2)D(3) and EB1089 on expression of p21(WAF1/CIP1) and GADD45alpha were similar in tumors and in vitro. CONCLUSIONS: EB1089 completely inhibited growth of AT-84 SCC cells at nanomolar concentrations, reduced tumor growth, and did not have calcemic effects. Our results support continued investigation of EB1089 as a chemopreventive/chemotherapeutic agent for head and neck SCC.     

The G gamma / T-15 transgenic mouse model of androgen-independent prostate cancer: target cells of carcinogenesis and the effect of the vitamin D analogue EB 1089.

Transgenic mouse models of prostate cancer provide unique opportunities to understand the molecular events in prostate carcinogenesis and for the preclinical testing of new therapies. We studied the G gamma T-15 transgenic mouse line, which contains the human fetal globin promoter linked to SV40 T antigen (Tag) and which develops androgen-independent prostate cancer. Using the immunohistochemistry of normal mouse prostates before tumor formation, we showed that the target cells of carcinogenesis in G gamma T-15 mice are located in the basal epithelial layer. We tested the efficacy of the 1,25(OH)(2)D(3) analogue, EB 1089, to chemoprevent prostate cancer in these transgenic mice. Compared with treatment with placebo, treatment with EB 1089 at three different time points before the onset of prostate tumors in mice did not prevent or delay tumor onset. However, EB 1089 significantly inhibited prostate tumor growth. At the highest dose, EB 1089 inhibited prostate tumor growth by 60% (P = 0.0003) and the growth in the number of metastases, although this dose also caused significant hypercalcemia and weight loss. We conducted several in vitro experiments to explore why EB 1089 did not prevent the occurrence of the primary tumors. EB 1089 significantly inhibited the growth of a Tag-expressing human prostate epithelial cell line, BPH-1, and an androgen-insensitive subline of LNCaP cells [which was not inhibited by 1,25(OH)(2)D(3)]. Thus, neither Tag expression nor androgen insensitivity explain the absence of chemopreventive effect. Conversely, neither 1,25(OH)(2)D(3) nor EB 1089 inhibited the growth of the normal rat prostate basal epithelial cell line NRP-152. It is likely that EB 1089 was not effective in delaying the growth of the primary tumor in G gamma T-15 transgenic mice because the target cells of carcinogenesis in these mice are located in the basal epithelial layer. We conclude that G gamma T-15 transgenic mice are a useful model for testing vitamin D-based therapies in androgen-insensitive prostate cancer but are not suitable for studies of vitamin D-based chemoprevention. The superiority of EB 1089 over 1,25(OH)(2)D(3) in the growth suppression of androgen-insensitive prostate cancer cells supports the use of EB 1089 in androgen-insensitive prostate cancer.     

Combined effect of vitamin D3 analogs and paclitaxel on the growth of MCF-7 breast cancer cells in vivo

Vitamin D3 analogs and paclitaxel (Taxol) are able to inhibit the in vitro growth of a variety of malignant cells including breast cancer cells. These two compounds decrease growth by different mechanisms and they have non-overlapping toxicities. We examined the abilities of three vitamin D3 compounds to inhibit growth of a human mammary cancer (MCF-7) in BNX triple immunodeficient mice either alone or with Taxol. Vitamin D3 analogs were 1,25(OH)2D3 (code name, Compound C), 1,25(OH)2-16-ene-23-yne-19-n or-26,27-F6-D3 (Compound LH), and 24a,26a,27a,-trihomo-22,24-diene-1,25(OH)2D3 (EB1089). At the doses chosen, the antitumor effect of vitamin D3 analogs alone was greater than that of Taxol alone, and an additive effect was observed when a vitamin D3 analog and Taxol were administered together. EB1089 was the most potent compound, and the EB1089 plus Taxol was the most active combination, decreasing the tumor mass nearly 4-fold compared to controls. Weight-gain in each of the experimental cohorts at the end of the study was less than the control group, but the gain was significantly less in only two experimental groups (those receiving either EB1089 or Compound C plus Taxol). None of the animals became hypercalcemic, and their complete blood counts, serum electrolyte analyses, and liver and renal functions were all fairly similar and within the normal range. In summary, this combination of a vitamin D3 analog and Taxol has the potential to be a therapy for breast cancer.     

Androgen receptor and vitamin D receptor in human ovarian cancer: growth stimulation and inhibition by ligands

The data suggest that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and androgens are essential for regulation of growth and differentiation in, e.g., human reproductive tissues. We investigated the possible cross-talk between 1,25(OH)2D3 and androgens in the human ovarian cancer cell line OVCAR-3. Our data demonstrate that 1,25(OH)2D3 and androgen (dihydrotestosterone, DHT) regulate the growth of OVCAR-3 cells. Nine days' treatment of OVCAR-3 cells with 100 nM DHT resulted in 48% stimulation of growth, whereas growth inhibition (73%) was observed after treatment with 100 nM 1,25(OH)2D3. The combination of 1,25(OH)2D3 and DHT showed that 1,25(OH)2D3 clearly reduces the growth-stimulatory effect of DHT on OVCAR-3 cells. Moreover, Western blot analysis revealed that these cells contain receptors for 1,25(OH)2D3 (VDR) and androgen (AR). Expression of VDR and AR was up-regulated by their cognate ligands. Up-regulation of AR by 1,25(OH)2D3 and of VDR by DHT provides evidence of cross-talk between 2 signaling pathways in OVCAR-3 cells. We also studied the immuno-histochemical distribution of VDRs and ARs in rat ovaries and human ovarian cancer cases. In rat ovaries, VDRs were observed mainly in granulosa and theca cells and ARs in granulosa cells and surface epithelium. In the human ovarian cancer cases studied, 43% were VDR-positive and 64% AR-positive. Combining the results suggests that the growth of ovarian tissue might be regulated by 1,25(OH)2D3 and androgen.     

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.     

1α，25-二羟基维生素D3对人卵巢癌细胞株表皮生长因子受体的调节

目的：研究1α,25-二羟基维生素D3（1,25VD3）对人卵巢癌细胞株表皮生长因子受体（EGFR）表达的调节作用。方法：用1,25VD3或其类似物EB1089处理人卵巢癌细胞株OVCAK3、2008和CAOV3,应用Westernblot、NorthernBlot和mRNA稳定性分析等方法检测EGFR表达的变化。结果：1,25VD3或EB1089作用后三种人卵巢癌细胞中EGFR的蛋白和mRNA表达水平均下降。用1,25VD3和放线菌素D、RNA酶抑制剂处理OVCAR3细胞后,Northernblotting分析结果显示,1,25VD3作用4h后没有改变EGFRmRNA的半衰期。结论：1,25VD3在mRNA转录水平下调人卵巢癌细胞株EGFR的表达。     

Comparative effects of 1,25(OH)2D3 and EB1089 on cell cycle kinetics and apoptosis in MCF-7 breast cancer cells

1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], the active metabolite of vitamin D, inhibits breast cancer cell growth both in vivo and in vitro. In addition to its anti-proliferative effects, 1,25(OH)₂D₃ induces morphological and biochemical markers of apoptosis in MCF-7 cells. In the studies reported here, we compared the effects of 1,25(OH)₂D₃ and EB1089, a low calcemic vitamin D analog, on cell cycle kinetics and apoptosis in MCF-7 cells. Both vitamin D compounds reduced viable MCF-7 cell number in a time and dose dependent manner, with EB1089 approximately 50 fold more potent than 1,25(OH)₂D₃. Flow cytometric analysis indicated that both agents induced cell cycle arrest in G₀/G₁ which was associated with accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein. MCF-7 cells treated with either 1,25(OH)₂D₃ or EB1089 for 48 h exhibited characteristics of apoptosis, including cytoplasmic condensation, pyknotic nuclei, condensed chromatin and DNA fragmentation. Cells treated with either agent exhibited up regulation of proteins associated with mammary gland regression (clusterin and cathepsin B) and down regulation of the anti-apoptotic protein bcl-2. These studies demonstrate that, despite its lower calcemic activity in vivo, the vitamin D analog EB1089 induces effects that are indistinguishable from those of 1,25(OH)₂D₃ on cell number, cell cycle and indices of apoptosis in MCF-7 cells in vitro. In addition, since both agents rapidly down regulate estrogen receptor, disruption of estrogen dependent signalling may play a role in the induction of apoptosis by vitamin D compounds in MCF-7 cells.     

Inhibition of prostate cancer metastasis in vivo: a comparison of 1,23-dihydroxyvitamin D (calcitriol) and EB1089.

The steroid hormone 1,25-dihydroxyvitamin D [1,25(OH)2D, also known as calcitriol] is known to inhibit the proliferation and to promote the differentiation of human prostate cancer cells. Additionally, we showed that 1,25(OH)2D markedly inhibits the invasiveness of human prostate cancer cells in vitro (G. G. Schwartz et al., Cancer Epidemiol. Biomark. Prev., 6: 727-732, 1997). These properties support the use of 1,25(OH)2D as differentiation therapy in prostate cancer. However, the use of 1,25(OH)2D in vivo is limited by the risk of hypercalcemia. We therefore compared the effects of 1,25(OH)2D and of EB1089, an analogue of 1,25(OH)2D with reduced calcemic effects, in an in vivo model of androgen-insensitive metastatic prostate cancer, the rat Dunning MAT LyLu prostate cancer model. Tumor growth and metastasis were studied using Copenhagen rats given s.c. injections of MAT LyLu cells. Fifty male rats were divided into five groups of 10 rats each. Four experimental groups received i.p. injections of low and high doses of 1,25(OH)2D and EB1089 (0.5 and 1.0 microg/kg, low and high, respectively). A control group received injections of vehicle only. Tumor volumes were measured three times per week. Rats were weighed weekly. The number of metastases to the lungs and the extent of hypercalcemia were evaluated. Compared with controls, tumor volumes were significantly smaller in all experimental groups. Similarly, the number of lung metastases (number of foci/lung) was reduced markedly by both 1,25(OH)2D and EB1089. Control rats developed 22.7 (+/- 1.98 SE) tumor foci per lung. Rats treated with 1,25(OH)2D and with EB1089 (1.0 microg/kg) developed 10.4 (+/- 2.81) and 7.70 (+/- 1.29) tumor foci, respectively (P < 0.001 and P < 0.0001, respectively; drug versus control). Compared with controls (10.79 +/- 0.1 mg/dl), serum calcium levels were significantly elevated in both 1,25(OH)2D and EB1089-treated rats (P < 0.01). However, EB1089 was significantly less calcemic than 1,25(OH)2D (12.59 +/- 0.21 mg/dl versus 14.47 +/- 0.46 mg/dl; 1.0 microg/kg; P < 0.001). Rats treated with 1,25(OH)2D showed marked weight loss: 20.0 +/- 1.9% and 26.3 +/- 1.7% of their initial weight (low and high doses, respectively, P < 0.001). Weight loss was significantly lower in rats treated with EB1089 at the high dose 8.4 (+/- 2.9) %. Moreover, rats treated with low-dose EB1089 gained 5.2 (+/- 3.7) % of their initial weight. In conclusion, 1,25(OH)2D and EB1089 showed marked and equivalent inhibition of prostate cancer metastasis in vivo. EB1089 was significantly less calcemic than 1,25(OH)2D and did not induce severe weight loss. This is the first report of a vitamin D analogue that significantly inhibits prostate cancer metastasis in vivo and that does so without producing cachexia or unacceptable hypercalcemia.     

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.     

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.     

Vitamin D receptor and p21/WAF1 are targets of genistein and 1,25-dihydroxyvitamin D3 in human prostate cancer cells

We investigated mechanisms by which genistein and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] act synergistically to inhibit the growth of the human prostate cancer cell line LNCaP. We demonstrate that 1,25(OH)(2)D(3) and genistein cooperate to up-regulate the vitamin D receptor protein by increasing the stability of the vitamin D receptor. Genistein and 1,25(OH)(2)D(3) also cooperate to up-regulate the levels of p21/WAF1 (p21). Small interfering RNA-mediated knockdown of p21 expression showed that p21 is essential for significant growth inhibition of LNCaP cells in response to either compound or their combination. We conclude that one mechanism of synergism between genistein and 1,25(OH)(2)D(3) is through genistein modulation of vitamin D signaling.     

Antiproliferative effect of a vitamin D3 analog, EB1089, on HL-60 cells by the induction of TGF-beta receptor

EB1089 is a novel 1,25(OH)₂D₃ analog that has more potent antitumor properties with reduced hypercalcemic effects than 1,25(OH)₂D₃. We investigated the role of transforming growth factor-β1 (TGF-β1) in the growth inhibition of human acute myeloid leukemia cell line, HL-60, by EB1089. Clonal growth of HL-60 cells was inhibited in a dose-dependent manner by EB1089. Although TGF-β1 alone slightly inhibited proliferation of HL-60 cells, the addition of TGF-β1 into culture treated with 10⁻⁸ M of EB1089 showed a significant synergistic antiproliferative effect in a dose-dependent manner. EB1089 up-regulated the expression of TGF-β receptor type I (TGF-β RI), type II (TGF-β RII) and TGF-β1. Antiproliferative effect of EB1089 was partially reversed by TGF-β1 neutralizing antibody (anti-TGF-β1). Vitamin D receptor (VDR) expression was increased by TGF-β1, suggesting synergistic action of TGF-β1 and EB1089. Combined treatment of EB1089 and TGF-β1 resulted in an increased expression of cyclin-dependent kinase inhibitor (CDKI), p27 protein, compared to either ligand alone. Up-regulation of p27 protein expression by either TGF-β1 or EB1089 was reduced by anti-TGF-β1. These findings suggest that TGF-β1 is involved in the antiproliferative effect of EB1089 on HL-60 cells.     

24-Oxo metabolites of vitamin D3 analogues: disassociation of their prominent antileukemic effects from their lack of calcium modulation

The seco-steroid hormone, 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] inhibits proliferation and induces differentiation of malignant cells including those of the hematopoietic system. The 24-oxo metabolite of 1,25(OH)(2)D(3) also has prominent antiproliferative activities against various cancer cells. We chemically synthesized five novel 24-oxo vitamin D(3) analogues and evaluated their abilities both to inhibit clonal growth and induce differentiation of myeloid leukemia cells and to cause hypercalcemia. The 1alpha,25-dihydroxy-16-ene-D(3) [1,25(OH)(2)-16-ene-D(3)] and 1alpha,25-dihydroxy-16-ene-19-nor-D(3) [1,25(OH)(2)-16-ene-19-nor-D(3)] and their 24-oxo metabolites showed greater potency than 1,25(OH)(2)D(3) in their abilities to inhibit clonal proliferation of HL-60, NB4, and U937 leukemic cell lines as measured by methylcellulose soft-gel assay. Their inhibition of clonal growth was irreversible as analyzed by pulse exposure studies. The synthetic analogues also had greater potency than 1,25(OH)(2)D(3) to induce differentiation of HL-60 and NB4 cells as measured by generation of superoxide, nonspecific esterase production, and induction of CD11b and CD14 cell surface antigens and to increase the proportion of these cells in the G(0)-G(1) phase of the cell cycle. For most assays, the 24-oxo metabolite was slightly more potent than the unmodified analogue, and 50% activity was usually found in the nanomolar range. These analogues and their 24-oxo metabolites also inhibited fresh leukemic cell clonal proliferation. Expression of p27(KIP1), a cyclin-dependent kinase inhibitor that plays an important role in blocking the cell cycle, was found by Western blot analysis to be induced by the analogues and their 24-oxo metabolites in both HL-60 and U937 cells, suggesting a possible mechanism by which these analogues inhibit leukemic growth. Notably, the calcemic activity tested by injections of 1alpha,25-dihydroxy-16-ene-24-oxo-19-nor-D(3) in mice was at least 12-fold less than 1alpha,25(OH)(2)-16-ene-19-nor-D(3). Taken together, chemically synthesized 24-oxo metabolites of 1alpha,25(OH)(2)-16-ene-D(3) and 1alpha,25(OH)(2)-16-ene-19-nor-D(3) irreversibly inhibited proliferation and induced differentiation of acute myeloid leukemia cells with minimal toxicity; these compounds may have a role in the maintenance phase of therapy for acute myeloid leukemia.     

Vitamin D analogues suppress IGF-I signalling and promote apoptosis in breast cancer cells

Survival factors are known to promote cell viability, and factor deprivation can be a potent apoptotic signal. Insulin-like growth factors are potent mitogens and inhibitors of apoptosis for many normal and neoplastic cells with insulin-like growth factor-I (IGF-I) being the most effective in many breast cancer cell lines. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its analogues inhibit IGF-I-stimulated growth of MCF-7 human breast cancer cells. The aim of this study was to determine the relationship between inhibition of IGF-I responsiveness and induction of apoptosis by vitamin D analogues in breast cancer cells. Vitamin D analogues EB1089 and CB1093 inhibited autonomous and IGF-I-stimulated growth of MCF-7 and T47D cells and autonomous growth of IGF-I-insensitive Hs578T cells. In MCF-7 cells, IGF-I alone (4 nM) protected against apoptosis mediated by serum deprivation. Co-treatment with vitamin D analogues prevented the anti-apoptotic effects of IGF-I. In T47D cells, IGF-I treatment provided only partial protection against apoptosis induced by serum deprivation and co-incubation of serum-deprived cells with 100 nM CB1093 and IGF-I abrogated this partial protection. In Hs578T cells, addition of IGF-I did not prevent apoptosis induced by serum deprivation. However, treatment with CB1093 attenuated the protective effect of the serum in these cells. Our findings suggest that vitamin D analogues inhibit IGF-I signalling pathways to promote apoptosis in breast cancer cells.