Gemini Vitamin Research Articles

Gemini Vitamin D Analogues Inhibit Estrogen Receptor–Positive and Estrogen Receptor–Negative Mammary Tumorigenesis without Hypercalcemic Toxicity

Numerous preclinical, epidemiologic, and clinical studies have suggested the benefits of vitamin D and its analogues for the prevention and treatment of cancer. However, the hypercalcemic effects have limited the use of 1alpha,25(OH)(2)D(3), the hormonally active form of vitamin D. To identify vitamin D analogues with better efficacy and low toxicity, we have tested >60 novel Gemini vitamin D analogues with a unique structure of two side chains for growth inhibition of breast cancer cells. Our initial studies found that some Gemini analogues are 5-15 times more active than 1alpha,25(OH)(2)D(3) in growth inhibition assay. In vivo experiments were designed to study the inhibitory effect of selected Gemini vitamin D analogues against mammary carcinogenesis by using (a) an N-methyl-N-nitrosourea-induced estrogen receptor (ER)-positive mammary tumor model and (b) an MCF10DCIS.com xenograft model of ER-negative mammary tumors. Among vitamin D analogues we tested, Gemini 0072 [1alpha,25-dihydroxy-20S-21(3-trideuteromethyl-3-hydroxy-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-19-nor-cholecalciferol] and Gemini 0097 [1alpha,25-dihydroxy-20R-21(3-trideuteromethyl-3-hydroxy-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-19-nor-cholecalciferol] administration inhibited by 60% the NMU-induced mammary tumor burden compared with the NMU-treated control group, but these compounds were devoid of hypercalcemia toxicity. In an ER-negative xenograft model, Gemini 0097 significantly suppressed tumor growth without hypercalcemia toxicity. We found that the inhibitory effect of Gemini 0097 was associated with an increased level of cyclin-dependent kinase inhibitor p21 and the insulin-like growth factor binding protein 3 in both ER-positive and ER-negative mammary tumors. Our results suggest that Gemini vitamin D analogues may be potent agents for the prevention and treatment of both ER-positive and ER-negative breast cancer without hypercalcemia toxicity.

Novel Gemini vitamin D 3 analogs have potent antitumor activity

The active form of vitamin D 3, 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3], modulates proliferation and induces differentiation of many cancer cells. A new class of analogs of vitamin D 3 has been synthesized, having two side-chains attached to carbon-20 (Gemini) and deuterium substituted on one side-chain. We have examined six of these analogs for their ability to inhibit growth of myeloid leukemia (HL-60), prostate (LNCaP, PC-3, DU145), lung (H520), colon (HT-29), and breast (MCF-7) cancer cell lines. Dose–response clonogenic studies showed that all six analogs had greater antiproliferative activities against cancer cells than 1,25(OH) 2D 3. Although they had similar potency, the most active of these analogs was BXL-01-0120. BXL-01-0120 was 529-fold more potent than 1,25(OH) 2D 3 in causing 50% clonal growth inhibition (ED 50) of HL-60 cells. Pulse-exposure studies demonstrated that exposure to BXL-01-120 (10 −9 M, 48 h) resulted in 85% clonal inhibition of HL-60 growth. BXL-01-0120 (10 −11 M, 4 days) induced the differentiation marker, CD11b. Also, morphologically differentiation was more prominent compared to 1,25(OH) 2D 3. Annexin V assay showed that BXL-01-0120 (10 −10 M, 4 days) induced significantly ( p < 0.05) more apoptosis than 1,25(OH) 2D 3. In summary, these analogs have a unique structure resulting in extremely potent inhibition of clonal proliferation of various types of cancer cells, especially HL-60 cells.

Abstract 2097: Gemini vitamin D analogs inhibit estrogen receptor positive and estrogen receptor negative mammary tumorigenesis without hypercalcemic toxicity

Abstract Numerous preclinical, epidemiological and clinical studies with vitamin D and analogs have suggested the benefits of vitamin D and analogs for prevention and treatment of cancer. However, the hypercalcemic effects have limited the use of 1α,25(OH)2D3, the hormonally active form of vitamin D, and many of its classical synthetic analogs as potent agents for cancer prevention and treatment. To identify vitamin D analogs with better efficacy and low toxicity, we have tested more than 60 novel Gemini vitamin D analogs, which have a unique structure of two six-carbon chains with a C-20-normal and a C-20-epi side chain. Our initial studies found that some Gemini analogs are many-fold more active than 1α,25(OH)2D3 in growth inhibition assay. We further examined the inhibitory effect of selected Gemini vitamin D analogs against mammary carcinogenesis in vivo by using N-methyl-N-nitrosourea (NMU)-induced mammary tumor model which is ER-positive. Among Gemini vitamin D analogs we tested, Gemini 0072 and Gemini 0097 treated groups showed approximately 60% suppressive activity in NMU-induced mammary tumor growth compared to the control group. In a dose dependent experiment, Gemini 0097 significantly reduced the average tumor burden per rat without affecting the body weight. At all doses tested, Gemini 0097 did not exert any calcemic toxicity. In addition, we determined the inhibitory effect of Gemini 0097 in MCF10DCIS xenograft model of ER-negative mammary tumors. Gemini 0097 significantly inhibited MCF10DCIS xenograft tumor growth without calcemic toxicity. We analyzed the tumor samples and found that the inhibitory effect of Gemini 0097 in vivo was through (a) increasing cyclin dependent kinase inhibitor, p21 and (b) inducing insulin-like growth factor binding protein 3 (IGFBP3) in both ER-positive and ER-negative mammary tumors. Our results suggest that Gemini vitamin D analogs may be potent agents for the prevention and treatment of both ER positive and ER negative breast cancer without calcemic toxicity.

Activation of Bone Morphogenetic Protein Signaling by a Gemini Vitamin D3 Analogue Is Mediated by Ras/Protein Kinase Cα

Bone morphogenetic proteins (BMP) are members of the transforming growth factor-beta superfamily, and they play an important role for embryonic development, for bone and cartilage formation, and during carcinogenesis. We have previously shown that the novel Gemini vitamin D(3) analogue, Ro-438-3582 [Ro3582; 1 alpha,25-dihydroxy-20S,21(3-hydroxy-3-methylbutyl)-23-yne-26,27-hexafluorocholecalciferol], inhibited cell proliferation and activated the BMP/Smad signaling pathway in MCF10AT1 breast epithelial cells. In this report, we investigated the upstream signaling pathways responsible for the activation of BMP/Smad signaling by Ro3582. Among seven different serine/threonine kinase inhibitors that we tested, protein kinase C (PKC) inhibitors blocked the effects of Ro3582 on the phosphorylation of Smad1/5, mRNA synthesis for BMP-2 and BMP-6, and cell growth in MCF10AT1 cells. Overexpression of PKC alpha, but not PKC epsilon, PKC delta or PKC zeta isoforms, increased Ro3582-induced phosphorylation of Smad1/5, suggesting that PKC alpha mediates the activation of Smad signaling and inhibition of cell proliferation. Interestingly, the activation of Smad signaling by Ro3582 was shown in Ha-ras-transfected MCF10AT1 cells, but not in the parent cell line (MCF10A without Ras). Inhibiting Ras activity blocked the translocation of PKC alpha to the plasma membrane and the phosphorylation of Smad1/5 induced by Ro3582, indicating that Ras is necessary for the activation of PKC alpha and Smad signaling. In conclusion, Ro3582 inhibits cell proliferation and activates BMP/Smad signaling via a Ras and PKC alpha pathway in breast epithelial cells.

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

The active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is an endocrine hormone whose classic role is the maintenance of calcium homeostasis. It is well documented that 1,25(OH)2D3 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)2D3 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)2D3 reduces the invasive spread of tumor cells. Because of the calcemic limitations of using 1,25(OH)2D3 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)2D3. 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)2D3 activities, without inducing hypercalcemia. Thus, DG shows promise in the development of colorectal cancer therapies.

Gene expression profiling changes induced by a novel Gemini Vitamin D derivative during the progression of breast cancer

We investigated gene expression changes induced by a novel Gemini Vitamin D 3 analog, RO-438-3582 (1α,25-dihydroxy-20 S-21(3-hydroxy-3-methyl-butyl)-23-yne-26,27-hexafluoro-cholecalciferol, Ro3582), in a unique human breast MCF10 model. We used two breast epithelial cell lines from this model, namely MCF10AT1 ( Ha-ras oncogene transfected MCF10A, early premalignant) and MCF10CA1a (fully malignant and metastatic derived from the MCF10AT1 line). We analyzed gene expression changes induced by Ro3582 using GeneChip technology, quantitative RT-PCR, Western blot analysis, or a gene transcription assay. Interestingly, we found distinct gene expression profile differences between Ro3582-induced response of the early premalignant MCF10AT1 and the malignant and metastatic MCF10CA1a cell lines. Moreover, while the Gemini Vitamin D 3 analog Ro3582 modulated the expression of several Vitamin D target genes such as the 24-hydroxylase, CD14, osteocalcin, and osteopontin in both cell lines, Ro3582 regulated many genes involved in cell proliferation and apoptosis, cell adhesion, invasion, angiogenesis as well as cell signaling pathways, such as the BMP and TGF-β systems, differently in the two cell lines. The Gemini Vitamin D 3 analog Ro3582 induced more significant gene changes in the early premalignant MCF10AT1 cells than in the malignant metastatic MCF10CA1a cells, suggesting that Gemini Vitamin D 3 analogs may be more effective in preventing the progression of an early stage of breast carcinogenesis than in treating late stage breast cancer.

A Novel Vitamin D Derivative Activates Bone Morphogenetic Protein Signaling in MCF10 Breast Epithelial Cells

We investigated the action of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], a novel Gemini vitamin D(3) analog Ro-438-3582 [1alpha,25-dihydroxy-20S-21(3-hydroxy-3-methyl-butyl)-23-yne-26,27-hexafluorocholecalciferol (Ro3582)], and a classic vitamin D(3) analog Ro-26-2198 [1alpha,25-dihydroxy-16,23(Z)-diene-26,27-hexafluoro-19-nor-cholecalciferol (Ro2198)] in modulating the transforming growth factor-beta (TGF-beta)/bone morphogenetic protein (BMP) system in MCF10 immortalized breast epithelial cells. We found that 1alpha,25(OH)(2)D(3), Ro3582, and Ro2198 all enhanced BMP/Smad signaling by increasing the phosphorylation of receptor-regulated Smads. Ro3582 was more active than Ro2198, but both were considerably more active than 1alpha,25(OH)(2)D(3.) Ro3582 enhanced BMP/Smad signaling by 1) inducing the phosphorylation of receptor-regulated Smads (Smad1/5), 2) increasing the accumulation of phosphorylated Smad1/5 in the nucleus, and 3) activating BMP-mediated transcription in MCF10 breast epithelial cells. Furthermore, Ro3582 induced the synthesis of BMP-2 and BMP-6 mRNA and protein, and the expression of Smad6 mRNA in MCF10 breast epithelial cells was inhibited by Ro3582. The induction of phospho-Smad1/5 by Ro3582 was inhibited by treatment with the BMP antagonist Noggin, whereas neutralizing antibody to TGF-beta did not block the induction of phospho-Smad1/5 by Ro3582. Treatment with Noggin also blocked the effect of Ro3582 on nuclear accumulation of phospho-Smad1/5 and the induction of BMP-2 and BMP-6 mRNA synthesis. These results indicate that the activation of BMP/Smad signaling by the Gemini vitamin D(3) analog Ro3582 may be through the production of BMP ligands, including BMP-2 and BMP-6, and/or down-regulation of the inhibitory Smad6. This is the first report to show that 1alpha,25(OH)(2)D(3) and its derivatives activate BMP/Smad-specific signaling in human breast epithelial cells.