Selective vitamin D receptor modulators and their effects on colorectal tumor growth.

The active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is an endocrine hormone whose classic role is the maintenance of calcium homeostasis. It is well documented that 1,25(OH)(2)D(3) also has anti-tumor effects on a number of cancers and cancer cell lines including breast, colorectal, gastric, liver, ovarian, prostate, and non-melanoma skin cancers. Included in the anti-tumor activities of 1,25(OH)(2)D(3) are its ability to cause antiproliferation, prodifferentation and decrease angiogenesis. Furthermore, through regulation of the plaminogen activator (PA) system and a class of proteolytic enzymes called matrix metalloproteinases (MMPs), 1,25(OH)(2)D(3) reduces the invasive spread of tumor cells. Because of the calcemic limitations of using 1,25(OH)(2)D(3) as a therapy, we have tested the effects of a novel Gemini vitamin D analogue, Deuterated Gemini (DG), on mouse colorectal cancer. We demonstrated that DG is more potent in reducing tumor volume and mass, compared to control and 1,25(OH)(2)D(3). DG significantly prevented (100% reduction, p<0.05) the invasive spread of colorectal tumor cells into the surrounding muscle, and had no effect on serum calcium levels. Thus, DG acts as a selective vitamin D receptor modulator (SVDRM) by enhancing select anti-tumor characteristic 1,25(OH)(2)D(3) activities, without inducing hypercalcemia. Thus, DG shows promise in the development of colorectal cancer therapies.

Vitamin D and cancer.

The correlation between decreased morbidity and mortality of cancer and exposure to sunlight is known. The many biological functions of vitamin D that contribute to cancer prevention have only recently begun to be appreciated. Once activated 1,25-dihydroxyvitamin D [1,25(OH)2D3] functions as a potent inhibitor of normal and cancer cellular proliferation. Vitamin D deficiency in mice led to a 60% increase in colon tumor growth, compared to vitamin D-sufficient mice. The ligand binding domain of the Vitamin D receptor was shown to accommodate a class of 1,25(OH)2D3-analogs that possess an additional side-arm. These novel Gemini analogs were evaluated in vitro and in vivo. Select Gemini analogs were 100 times or more effective in inhibiting colon tumor growth in mice, compared to their parent compound. Correcting vitamin D deficiency may decrease the risk of developing colon cancer, while the novel Gemini 1,25(OH)2D3-analogs have the potential for therapeutic application in human colon cancer.

Translational study of vitamin D differentiation therapy of myeloid leukemia: effects of the combination with a p38 MAPK inhibitor and an antioxidant.

Human myeloid leukemia cell lines are induced to terminal differentiation into monocyte lineage by 1,25-dihydroxyvitamin D3 (1,25D3) or its analogs (deltanoids). However, translation of these findings to the clinic is limited by calcemic effects of deltanoids. Strategies to overcome this problem include combination of deltanoids with other compounds to induce differentiation at lower, noncalcemic, deltanoid concentrations. We previously showed that either carnosic acid, an antioxidant, or SB202190, a p38 MAPK inhibitor, increase the potency of 1,25D3 in the HL60 cell line. Here, we report that simultaneous addition of both these agents further increases differentiation potency of deltanoids in this cell line and in freshly obtained leukemic cells ex vivo. Activity of MAPK pathways showed that increased differentiation was associated with enhanced activity of JNK pathway in all responding cell subtypes. Our studies suggest that patients with CML or AML subtypes M2 and M4, but not M1, M3 or M4eo, are particularly suitable for this combination therapy. We conclude that the established cell line HL60 presents a good model for some, but not all, subtypes of myeloid leukemia, and that the JNK pathway plays an important role in monocytic differentiation of human leukemic cells ex vivo, as well as in vitro.

Synergistic induction of HL60 cell differentiation by ketoconazole and 1-desoxy analogues of vitamin D3.

BACKGROUND: The goal of differentiation therapy is to induce cancer cells to stop proliferating and to express characteristics of normal cells. Vitamin D analogues, such as the deltanoids, are being evaluated as differentiation agents in the treatment of several human cancers (e.g., myeloid leukemias); however, these compounds have a tendency to produce hypercalcemia in patients receiving therapy. A combination of a differentiation-inducing deltanoid with a compound that blocks entry of calcium into cells (e.g., ketoconazole) may offer a new approach to differentiation therapy and address the problem of hypercalcemia. We investigated whether various ketoconazole-deltanoid combinations would alter cellular differentiation or intracellular calcium homeostasis in comparison with deltanoids used alone. METHODS: Cultured human leukemia HL60 cells were treated with ketoconazole-deltanoid combinations. Markers of differentiation (expression of CD11b and CD14 antigens and of non-specific esterase) were measured by flow cytometry and cytochemistry; cell cycle distribution was measured by flow cytometry of propidium iodide-stained cells. Expression of differentiation-related genes was assessed by northern blotting and immunoblotting, and changes in intracellular calcium homeostasis were monitored by fluorescence analysis of fura-2-containing cells. RESULTS: Ketoconazole strongly potentiated the differentiating activity of the deltanoids, which exhibited low potency when used alone. Ketoconazole-deltanoid combinations had little effect on HL60 cell-cycle distribution, although the cells did stop proliferating and they differentiated. Ketoconazole-deltanoid combinations produced only minor changes in intracellular calcium homeostasis compared with changes produced by 1,25-dihydroxyvitamin D3, either alone or in combination with ketoconazole. CONCLUSION: These results suggest that ketoconazole may be useful in combination with vitamin D analogues in the differentiation therapy for myeloid leukemias.

19-nor-hexafluoride analogue of vitamin D3: a novel class of potent inhibitors of proliferation of human breast cell lines.

Breast cancer cells express vitamin D3 receptors and 1,25-dihydroxyvitamin D3 suppressed growth of these cells. We have synthesized six novel vitamin D3 analogues to identify those with expanded capacity to inhibit the proliferative ability of breast cancer cells. These analogues incorporated many of the structural motifs shown previously to have antiproliferative activity in several cell types. Six breast cancer cell lines were used as targets. Dose-response studies showed that each of the analogues had antiproliferative activities, and LH [1,25-(OH)2-16-ene-23-yne-26,27-F6-19-nor D3] was the most potent analogue, suppressing at 10(-11) M greater than 50% clonal proliferation (ED50) of the MCF-7 and SK-BR-3 breast cancer cells, increasing the proportion of MCF-7 cells in the G0-G1 phase, and decreasing those in the S phase of the cell cycle. Pulse-exposure studies showed that a 3-day exposure to LH (10(-7) M) in liquid culture was adequate to achieve a 50% inhibition of MCF-7 clonal growth in soft agar in the absence of the analogue, suggesting that the growth inhibition mediated by LH is irreversible. The cyclin-dependent kinase inhibitor known as p27Kip1 helps regulate the cell cycle and can mediate growth arrest in response to extracellular growth inhibitors. The analogue LH (10(-7) M) induced elevated expression of p27Kip1 in MCF-7 and SK-BR-3 cells. Taken together, these results indicate that LH is an extremely potent vitamin D3 analogue markedly inhibiting clonal growth of MCF-7 and SK-BR-3 cells with concomitant cell cycle arrest at G0-G1 and increased expression of p27Kip1. Compound LH is worthy of in vivo analysis for possible future clinical trials.

Structure-function studies on analogues of 1 alpha,25-dihydroxyvitamin D3: differential effects on leukemic cell growth, differentiation, and intestinal calcium absorption.

The hormonally active form of vitamin D, 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3], is an efficient stimulator of intestinal calcium absorption (ICA) and bone calcium mobilization (BCM) in humans and experimental animals and, as well, has been shown to be effective in inducing differentiation and inhibiting proliferation of leukemia cells. Thus, it has been proposed that analogues of 1,25(OH)2D3 could be synthesized which might allow for separation of biological functions, i.e., promote a differentiation of leukemia cells without a significant stimulation of ICA or BCM, both biological effects which can cause hypercalcemia in humans. Here we report the results of an evaluation of four analogues of the previously studied (Zhou et al., Blood, 74:82-92, 1989) 1 alpha,25-dihydroxy-16-ene-23-yne-vitamin D3 [1,25(OH)2-16-ene-23-yne-D3]; these analogues allowed evaluation of the consequences of (a) the presence or absence of six deuterium atoms on carbons 26 and 27 of the side chain and (b) the deletion or substitution by a fluorine atom of the 1 alpha-hydroxyl group on the A-ring. The 1,25(OH)2-16-ene-23-yne-D3 analogue was found to be 7-fold more potent than the parent 1,25(OH)2D3 with respect to (a) inhibition of clonal proliferation of HL-60 cells as well as (b) induction of differentiation of HL-60 promyelocytes. Variants of this analogue which possessed the six deuterium atoms on carbons 26 and 27 were slightly less active than the 1,25(OH)2-16-ene-yne-D3. However, replacement of the 1 alpha-hydroxyl group by a 1-fluoro group, or the absence of the 1-hydroxyl group, resulted in analogues that were somewhat less effective than the parent 1,25(OH)2D3 in achieving these biological responses but more potent as inhibitors of the renal mitochondrial 25-OH-D3-1 alpha-hydroxylase, the site of endogenous production of 1,25(OH)2D3. ICA and BCM were assessed in vivo in vitamin D-deficient chickens, and each of the analogues was markedly less potent than the standard 1,25(OH)2D3. The analogue 1,25(OH)2-16-ene-23-yne-D3 had 2% of the ICA and 3% of the BCM activity of the parent 1,25(OH)2D3. Absence of the 1 alpha-hydroxyl group or substitution of the 1-fluoro group for the 1-hydroxyl group significantly diminished both the ICA and BCM activity in comparison to 1,25(OH)2-16-ene-23-yne-D3. Receptor binding studies indicated that 1,25(OH)2-16-ene-23-yne-D3 competed about 75% as effectively as 1,25(OH)2D3 for 1,25(OH)2D3 receptors present in both chick intestinal cells and HL-60 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

19-nor-26,27-bishomo-vitamin D3 analogs: a unique class of potent inhibitors of proliferation of prostate, breast, and hematopoietic cancer cells.

Vitamin D3 [1,25-dihydroxyvitamin-D3 (1,25(OH)2D3)] modulates the proliferation and differentiation of many cell types. Analogs of 1,25(OH)2D3 that have greater potency may become adjuvant therapy for breast and prostate cancers, myelodysplastic syndrome, acute myelogenous leukemia in remission and other cell types, especially in the setting of low disease burden. A new class of analogs of 1,25(OH)2D3 has been synthesized that has a novel 19-nor motif, as well as incorporating many structural elements previously shown to increase potency. These analogs were examined for their effects on prostate cancer cell lines (PC-3, LNCaP, and DU 145), a human breast cell line (MCF-7), and an acute myeloid leukemia cell line (HL-60). Dose-response clonogenic studies showed that each of these analogs had more potent antiproliferative activities against the cancer cells than 1,25(OH)2D3, and 1,25-(OH)2-16,23Z-diene-26,27-bishomo-19-nor-D3 (Ro 27-2014) was the most potent analog [10-fold increased activity compared to 1,25(OH)2D3]. Further studies were performed using Ro 27-2014. Pulse-exposure studies showed that a 5-day pulse-exposure to Ro 27-2014 (10(-7) M) in liquid culture was adequate to achieve a 50% inhibition of MCF-7 clonal growth in soft agar in the absence of the analog, suggesting that the growth inhibition mediated by the analog was irreversible. Cell cycle analyses using MCF-7 cells showed that Ro 27-2014 (10(-7) M for 4 days) induced a significant increase in the number of cells in G0-G1 (72.8+/-8.9% versus 49.9+/-3.5% in control cells), with a concomitant decrease in the percent of cells in S phase (13.1+/-6.2% versus 35.8+/-3.5% in control cells). The chief toxicity of vitamin D3 compounds is hypercalcemia, and therefore, we examined calcemic activity of Ro 27-2014 in mice and found it not to induce hypercalcemia at doses of 0.05 microg i.p. three times per week. In contrast, the same dose of a 19-nor vitamin D3 compound with 6 fluorines on the side chain (1,25-(OH)2-16-ene-23-yne-26,27-F6-19-nor-D3), although also having potent anticancer activity, caused severe hypercalcemia (18 mg/dl). In summary, 19-nor vitamin D3 compounds with desaturation and lengthening of their side chains result in a series of compounds with a good therapeutic index, having potent anticancer activity and low toxicity.

5,6-trans-16-ene-vitamin D3: a new class of potent inhibitors of proliferation of prostate, breast, and myeloid leukemic cells.

The 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is the physiologically active form of vitamin D3 that inhibits proliferation and induces differentiation of a variety of malignant cells. We evaluated a newly synthesized vitamin D3 analogue [1,25(OH)2-16-ene-5,6-trans-D3 (Ro 25-4020)] that has a novel 5,6-trans motif. Dose-response studies showed that 1,25(OH)2-16-ene-5,6-trans-D3 had 10-100-fold greater antiproliferative activities than 1,25(OH)2D3 when measuring clonal growth of breast (MCF-7) and prostate (LNCaP) cancer cell lines as well as a myeloid leukemia cell line (HL-60). Because the chief toxicity of vitamin D3 is hypercalcemia, we examined the calcemic activity of 1,25(OH)2-16-ene-5,6-trans-D3 in mice. Remarkably, 1,25(OH)2-16-ene-5,6-trans-D3 was at least 40-fold less calcemic as compared with 1,25(OH)2D3 and 1,25(OH)2-16-ene-D3 (Ro 24-2637). To explore the mechanism by which the 1,25(OH)2-16-ene-5,6-trans-D3 analogue mediated its antiproliferative activity, several studies were performed. Pulse-exposure studies showed that a 4-day pulse exposure to 1,25(OH)2-16-ene-5,6-trans-D3 (10(-7) M) in liquid culture was adequate to achieve a 40% inhibition of MCF-7 clonal growth in the absence of the analogue, suggesting that the growth inhibition mediated by 1,25(OH)2-16-ene-5,6-trans-D3 was at least in part irreversible. Cell cycle studies showed that 1,25(OH)2-16-ene-5,6-trans-D3 increased the proportion of MCF-7 cells in the G0-G1 phase and decreased those in the S phase. Furthermore, 1,25(OH)2-16-ene-5,6-trans-D3 induced an elevated expression of the cyclin-dependent kinase inhibitors, p21waf1 and p27kip1. In addition, 1,25(OH)2-16-ene-5,6-trans-D3 almost completely inhibited telomerase activity, as measured by telomeric repeat amplification protocol assay and human telomerase reverse transcriptase mRNA. For each of the growth-related parameters that were examined, the vitamin D3 analogue was more active than 1,25(OH)2D3. In contrast, 1,25(OH)2D3 was more calcemic than 1,25(OH)2-16-ene-5,6-trans-D3. In summary, 1,25(OH)2-16-ene-5,6-trans-D3, having a novel 5,6-trans motif, strongly inhibited clonal proliferation and reduced telomerase activity with low calcemic activity, suggesting further testing in in vivo cancer models. This analogue may gain a therapeutic niche for selected malignancies.

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.

cis-diamminedichloroplatinum(II) cross-linking of the human myeloid cell nuclear differentiation antigen to DNA in HL-60 cells following 1,25-dihydroxy vitamin D3-induced monocyte differentiation.

Protein-DNA interactions of the human myeloid cell nuclear differentiation antigen (MNDA) were examined in vivo in proliferating HL-60 promyelocytic leukemia cells and following induction of differentiation by 1,25-dehydroxyvitamin D3. Intact cells were treated with the reversible cross-linking agent cis-diamminedichloroplatinum(II) and the MNDA levels in the isolated protein-DNA complexes were determined. Less than 1% of the total intracellular level of MNDA was cross-linked to DNA in the noninduced proliferating HL-60 cells. Once the cells were induced to differentiate into monocytes, the amount of antigen cross-linked to the DNA increased to over 5% of the total intracellular level. The increased efficiency of cross-linking the MNDA to DNA was specific for monocyte-induced HL-60 differentiation, achieved with three inducers, and was not observed in association with granulocyte-induced differentiation. On a molar basis the phorbol ester (12-O-tetradecanoylphorbol-13-acetate) was the most effective inducer of monocyte differentiation, followed by 1,25-dihydroxy-16-ene-23-ynecholecalciferol which was more effective than 1,25-dihydroxycholecalciferol. A cesium chloride gradient analysis of the nucleic acid-protein fraction isolation from cis-diamminedichloroplatinum(II)-treated, monocyte-induced HL-60 cells documented the authenticity of the association between the MNDA and DNA. The results indicate that a significant level of chromatin reorganization may accompany monocyte-induced differentiation that leads to much higher levels of MNDA-DNA cross-linking to DNA. The expression of the MNDA is restricted to human myeloid cells and the present results indicate that a fraction of this low abundance nuclear protein is specifically located near the DNA [within cis-diamminedichloroplatinum(II) cross-linking distance] and that this association may be modulated specifically during monocyte differentiation.

Growth inhibition of HT-29 human colon cancer cells by analogues of 1,25-dihydroxyvitamin D3.

The use of 1,25-dihydroxyvitamin D3 as an antiproliferative agent in the treatment of cancer is limited by its hypercalcemic effects. Analogues with equivalent or greater antiproliferative activities but smaller hypercalcemic effects have been developed. The antiproliferative effects of 1,25-dihydroxyvitamin D3 and four analogues were studied in HT-29 and SW620 human colon cancer cell lines, moderate and low expressors of the vitamin D receptor, respectively. HT-29 is a primary, moderately differentiated, cell line, while SW620 is metastatic and poorly differentiated. Growth curve studies, proliferation assays, and clonogenic assays were used to assess the antiproliferative effects of 1,25-dihydroxyvitamin D3, 1,25-dihydroxy-16-ene-23-yne-D3, 1,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-D3, 1,25-dihydroxy-16,23E-diene-26,27-hexafluoro-D3, and 1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-D3. Growth of HT-29 cells was significantly inhibited by all four analogues at 10(-8) M (P < 0.05). Analogues 1,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-D3, 1,25-dihydroxy-16,23E-diene-26,27-hexafluoro-D3, and 1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-D3 were 2 times as potent as analogue 1,25-dihydroxy-16-ene-23-yne-D3 and 1,25-dihydroxyvitamin D3. SW620 cells did not show any growth inhibition with any of the compounds tested. The affinities of the three most potent analogues for the vitamin D receptor were similar to that of 1,25-dihydroxyvitamin D3, while that of analogue 1,25-dihydroxy-16-ene-23-yne-D3 was lower. These results demonstrate that, as in leukemic cells, analogues of 1,25-dihydroxyvitamin D3 are potent antiproliferative agents in colon cancer cells and this activity is most likely mediated through the vitamin D receptor.

20-epi-vitamin D3 analogues: a novel class of potent inhibitors of proliferation and inducers of differentiation of human breast cancer cell lines.

We have studied the in vitro biological activities and mechanism of action of 1,25-dihydroxyvitamin D3 (1,25D3) and four potent 1,25D3 analogues [20-epi-22oxa-24a,26a,27a-tri-homo-1,25(OH)2D3 (KH 1060); 20-epi-1,25(OH)2D3; 1,25(OH)2-16ene-D3; and 1,25(OH)2-16ene-23yne-D3] on proliferation and differentiation of estrogen receptor-negative (MDA-MB-436, BT-20, SK-BR-3, and MDA-MB-231), estrogen receptor-weakly positive (BT474), and estrogen receptor-positive (MCF-7) breast cancer cell lines. Dose-response studies showed that KH 1060 was the most potent analogue, because it was able to induce differentiation in all seven breast cancer cell lines (measured by lipid staining) and to suppress more than 50% clonal proliferation (ED50) at 10(-10) M in all cell lines, except MDA-MB-436 and BT-20. To explore how these compounds mediated antiproliferative actions, their effects on the cell cycle, on expression of bcl-2 and p53, and on apoptosis were assessed. Five of six cell lines have a mutant p53 gene, whereas MCF-7 has wild-type p53. Immunohistochemical staining showed that the p53 protein was predominantly localized in the nucleus in each of the breast cancer cell lines except for MCF-7, which expressed the protein predominantly in the cytoplasm. After incubation with KH 1060 (3 days; 10(-7) M), expression of bcl-2 protein as determined by immunohistochemical localization was markedly decreased in BT-474, MCF-7, and MDA-MB-231; these same cells were profoundly inhibited in their clonal proliferation and arrested in the G0/G1 phase of the cell cycle when cultured with KH 1060. In contrast, BT-20 and MDA-MB-436 cells that were refractory to the antiproliferative effect of KH 1060 (ED50 < 10(-6) M) had no down-regulation of their bcl-2 expression and no cell cycle changes after exposure to KH 1060. MCF-7 showed morphological changes and DNA fragmentation, indicative of apoptosis after 48 h incubation with KH 1060 (10(-6) M), during which time p53 protein accumulated in the nucleus and decreased in the cytoplasm. In contrast, no apoptosis was detected in three other breast lines (MDA-MB-231, SK-BR-3, and BT-474) that had a mutated p53. In conclusion, the data indicate that KH 1060 is an extremely potent 1,25D3 analogue inducing differentiation of all six breast cancer lines and potently inhibiting clonal growth of four of them with concomitant decreased bcl-2 and cell cycle arrest at G0/G1.(ABSTRACT TRUNCATED AT 400 WORDS)

1 alpha,25-Dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol (Ro24-5531), a new deltanoid (vitamin D analogue) for prevention of breast cancer in the rat.

We have used the vitamin D analogue, 1 alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalcifero l (Ro24-5531), for inhibition of mammary carcinogenesis induced by N-nitroso-N-methylurea (NMU) in Sprague-Dawley rats. Rats were first treated with a single dose of either 15 or 50 mg/kg body weight NMU and then fed Ro24-5531 (2.5 or 1.25 nmol/kg of diet) for 5-7 months. Ro24-5531 significantly extended tumor latency and lessened tumor incidence as well as tumor number in rats treated with the lower dose of NMU. In rats treated with the higher dose of NMU, Ro24-5531 was fed in combination with tamoxifen; in these experiments, Ro24-5531 significantly enhanced the ability of tamoxifen to reduce total tumor burden, as well as to increase the probability that an animal would be tumor free at the end of the experiment. In vitro, Ro24-5531 was 10-100 times more potent than 1,25-dihydroxyvitamin D3 for inhibition of proliferation of human breast cancer cell lines as well as primary cultures of cells from 2 patients with acute myelogenous leukemia. When fed chronically, Ro24-5531 did not elevate serum calcium in the present studies. We propose the new term, "deltanoids," for the set of molecules composed of vitamin D and its synthetic analogues, in a manner similar to the naming of "retinoids" for the corresponding set of molecules related to vitamin A.

Synergistic growth inhibition of prostate cancer cells by 1 alpha,25 Dihydroxyvitamin D(3) and its 19-nor-hexafluoride analogs in combination with either sodium butyrate or trichostatin A.

Prostate cancer is a major cause of male cancer death. In vitro and in vivo data support a role for 1 alpha,25 Dihydroxyvitamin D(3) (1 alpha,25(OH)(2)D(3)) in regulating the growth and differentiation of the normal prostate gland yet prostate cancer cells appear significantly less sensitive to this action. Vitamin D(3) receptor (VDR) content or mutational status do not correlate clearly with the antiproliferative effects of 1 alpha,25(OH)(2)D(3) and therefore it is unclear why prostate cancer cell lines are significantly less sensitive to this action. We hypothesized that the antiproliferative responses of prostate cancer cells to 1 alpha,25(OH)(2)D(3) are suppressed by a process involving histone deacetylation. Sodium butyrate (NaB) and trichostatin A (TSA) are inhibitors of histone deacetylase (HDAC) activity. Low doses of NaB or TSA (300 microM and 15 nM respectively), which alone were relatively inactive, synergized with 1 alpha,25(OH)(2)D(3) in liquid and semi-solid agar to inhibit the growth of LNCaP, PC-3 and DU-145 prostate cancer cells. Still greater synergy was observed between vitamin D(3) hexafluoride analogs and either NaB or TSA. The mechanism appeared to involve neither the cyclin-dependent kinase inhibitor, p21((waf1/cip1)) nor cell cycle arrest, but rather induction of apoptosis. These data suggest that cells dysregulate the normal pro-apoptotic signals of 1 alpha,25(OH)(2)D(3) during prostate cancer development by a mechanism involving histone deacetylation. Combination therapy with potent vitamin D(3) analogs and clinically approved HDAC inhibitors may overcome this lesion and improve the treatment of both androgen-dependent and independent prostate cancer.

Colon cancer and solar ultraviolet B radiation and prevention and treatment of colon cancer in mice with vitamin D and its Gemini analogs.

It has been recognized that people who live at higher latitudes and who are vitamin D deficient are at higher risk of dying from many common cancers including colon cancer. To evaluate the role of vitamin D deficiency on colon tumor growth, Balb/c adult male mice were fed either a vitamin D sufficient or vitamin D deficient diet for 10 weeks. Mice were arranged into groups of six and each animal received subcutaneously 10(4) MC-26 cells in the posterior trunk. The tumor size was recorded daily. By day 9 there was a significant difference in tumor volume between the vitamin D sufficient and vitamin D deficient mice. By day 18 the vitamin D deficient animals had a tumor size that was 56% larger compared to the animals that were vitamin D sufficient. To determine whether treatment with active vitamin D analogs could further decrease colon tumor growth in a vitamin D sufficient state, groups of mice were treated with the novel 19-nor-Gemini compounds. The mice were fed a low calcium diet. Twenty-four hours after tumor implantation, the mice received, three times weekly, one of the vitamin D analogs or the vehicle. The group that received Gemini 1,25-dihydroxy-21(3-hydroxy-3-trifluoromethyl-4-trifluoro-butynyl)-19-nor-20S-cholecalciferol (3) showed a dose-dependent decrease in tumor volume. On day 19, at the dose level of 0.02microg molar equivalents (E), the tumor volume was reduced by 41% when compared to the control group. At the same time point, the hexadeuterated analog 1,25-dihydroxy-21(3-hydroxy-3-trifluoromethyl-4-trifluoro-butynyl)-26,27-hexadeutero-19-nor-20S-cholecalciferol (4), administered at the 10-fold lower dose of 0.002microgE, showed a 52% reduction in tumor volume (p<0.05), compared to the control group. Animals that received 1,25(OH)(2)D(3) at 0.002 and 0.02microg showed a trend in tumor volume reduction at the highest dose but the changes were not statistically significant. An evaluation of serum calcium concentrations revealed that the calcium levels were normal in all groups, except the group receiving 0.02microgE of 4. The results from these studies demonstrate that vitamin D deficiency may accelerate colon cancer growth and that novel Gemini analogs of 1,25(OH)(2)D(3) may be an effective new approach for colon cancer treatment.

Increased sensitivity to a vitamin D3 analog in HL-60 myeloid leukemic cells resistant to all-trans retinoic acid.

All-trans retinoic acid (ATRA) has been demonstrated to induce the in vitro differentiation of human myeloid leukemic cells by several investigators. In addition, ATRA has been reported to induce complete remission in patients with acute promyelocytic leukemia. However, the majority of the patients relapse after several months of ATRA therapy. Since one possible cause of relapse in these patients is drug resistance, it is of interest to investigate the antileukemic activity of other agents that can be used in combination with ATRA in order to overcome this problem. Vitamin D3 analogs such as 1,25-dihydroxy-delta 16-23-yne-cholecalciferol (16-23-D3) are also capable of inducing differentiation of myeloid leukemic cells. In this study we have isolated a clone of HL-60 leukemic cells that is resistant to ATRA. We have observed that the resistant cell line (HL-60/RA) was more sensitive to the antileukemic action of 16-23-D3 than the parental cell line with respect to the inhibition of cell growth and DNA synthesis, the induction of differentiation and the loss of cell clonogenicity.

Growth inhibitory effects of 1,25-dihydroxyvitamin D3 and its synthetic analogue, 1alpha,25-dihydroxy-16-ene-23yne-26,27-hexafluoro-19-nor-cholecalcifero l (Ro 25-6760), on a human colon cancer xenograft.

The effect of 1,25-dihydroxyvitamin D3 and its synthetic analogue, 1alpha,25-dihydroxy-16-ene-23yne-26,27-hexafluoro-19-n or-cholecalciferol (Ro 25-6760), have been evaluated both in vitro and in vivo in human colorectal cancer cell lines expressing high (HT-29) and low (SW-620) levels of vitamin D receptor. 1,25-Dihydroxyvitamin D3 caused significant dose-dependent growth inhibition of HT-29 cells at concentrations ranging from 10(-11) to 10(-6). The antiproliferative effect of Ro 25-6760 on HT-29 cells was also dose-dependent with cell counts on day 6, ranging from 98% of control at 10(-11) M to 14% of control at 10(-6) M. However, 1,25-dihydroxyvitamin D3 and Ro 25-6760 did not have any growth inhibitory effect on SW-620 at all concentrations. In mice with HT-29 tumor xenografts, administration of vitamin D at 0.1 and 0.2 microg/injection i.p. three times/week did not cause any significant tumor growth delay, whereas synthetic analogue Ro 25-6760, at both concentrations, caused a significant tumor growth inhibition in comparison with the control arm. In 30% of mice treated by R0 25-6760 the tumors disappeared on average after the second injection, and tumor growth did not resume after drug withdrawal. However, both 1,25-dihydroxyvitamin D3 or Ro 25-6760 had no growth inhibitory effect at all applied concentrations in mice with the SW-620 tumor xenografts. The mechanism for this impressive growth inhibition is not yet elucidated and warrants further investigation.

Three synthetic vitamin D analogues induce prostate-specific acid phosphatase and prostate-specific antigen while inhibiting the growth of human prostate cancer cells in a vitamin D receptor-dependent fashion.

Numerous studies have indicated that the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 protects against the development of clinical prostate cancer (PC). Whether this hormone also has therapeutic potential for patients with advanced PC has not yet been evaluated. Several synthetic vitamin D analogues are now available that have reduced hypercalcemic effects and yet effectively induce differentiation in some cell types. For these reasons, these analogues may be safer and more effective for cancer therapy than the natural hormone. In the current study, 13 such analogues were screened for their abilities to inhibit the growth of PC cell lines. Three of the most consistently effective analogues (Ro 23-7553, Ro 24-5531, and Ro 25-6760) were then chosen for further analysis. Growth studies using clones of the JCA-1 cell line that were transfected with the vitamin D receptor cDNA indicate that the antiproliferative effects of these analogues require vitamin D receptor expression. Furthermore, these three analogues induce the secretion of prostate-specific acid phosphatase and prostate-specific antigen (two markers of the differentiated prostatic phenotype) in the cell line LNCaP. These in vitro studies suggest that Ro 23-7553, Ro 24-5531, and Ro 25-6760 should be further evaluated as therapeutic agents for the treatment of PC.

Vitamin D3-retinoid X receptor dimerization, DNA binding, and transactivation are differentially affected by analogs of 1,25-dihydroxyvitamin D3.

A number of analogs of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] have been synthesized that act as more potent inducers of cellular differentiation and inhibitors of cell growth than the natural ligand; at the same time, many of the analogs have reduced hypercalcemic properties. This combination makes these compounds attractive candidates for clinical use. The mechanism by which the analogs act, however, is unclear. Potentially, the analogs could be taken up more readily, be more slowly catabolized, or have higher binding affinities for the vitamin D receptor (VDR). Analogs of 1,25-(OH)2D3 could also differentially modulate one or more of the activities of VDR, namely dimerization, DNA binding, and/or transcriptional regulation. To directly examine this latter possibility, we used a sensitive assay for the kinetics of dimerization and DNA binding, surface plasmon resonance, and report here that three 1,25-(OH)2D3 analogs, 1,25-(OH)2-16-ene-23-yne-D3, 1,25-(OH)2-16-ene-23-yne-26,27-di home-D3, and 1,25-(OH)2-26,27-hexafluoro-16-ene-23-yne-D3, all confer distinct rate and equilibrium constants for VDR-retinoid X receptor heterodimerization and DNA binding to a specific vitamin D response element relative to the natural ligand. In response to the hexafluoro analog, the apparent Kd for DNA binding by VDR was significantly lower than that for 1,25-(OH)2D3, and correspondingly, in vivo transactivation from a responsive reporter was greater. Interestingly, solution heterodimerization was not affected by this analog. These results suggest that vitamin D analogs do indeed confer biological effects by acting directly and differentially at the level of VDR, and that specific vitamin D analogs can act on distinct receptor functions.

1,25-Dihydroxyvitamin D3 stimulates avian and mammalian cartilage growth in vitro.

We addressed the question of whether 1,25-dihydroxyvitamin D3 (1,25-(OH)2D) could directly stimulate cartilage growth in vitro. Pelvic leaflets from chick embryos and scapular growth plates from fetal pigs were organ cultured in serum-free medium in the presence and absence of 1,25-(OH)2D. After 3 days of incubation, 1,25-(OH)2D had increased the pelvic cartilage wet weight 42% and the dry weight 32% above the weight of cartilages incubated in medium alone. 1,25-(OH)2D (10(-9) M-10(-12) M) caused a dose-dependent increase in weight, with maximal increases at 10(-9) M. Furthermore, two deuterized derivatives of 1,25-(OH)2D, 26,27-D6-1,25-(OH)2D3 and 24,26,27-D8-1,25-(OH)2D3, stimulated pelvic cartilage growth in vitro. 26,27-D6-1,25-(OH)2D stimulated increases in growth plate weight above growth plates incubated in medium alone. 26,27-D6-1,25-(OH)2D3 appeared to be potent at lower concentrations than 1,25-(OH)2D on growth plate cartilage. Thus, 1,25-(OH)2D stimulated in vitro growth in two growing cartilage models, the avian pelvic cartilage and the mammalian scapular growth plate cartilage.