Vitamin D Compounds PRI-2191 and PRI-2205 Enhance Anastrozole Activity in Human Breast Cancer Models.

1,25-Dihydroxycholecalciferol, the hormonally active vitamin D3 metabolite, is known to exhibit therapeutic effects against breast cancer, mainly by lowering the expression of estrogen receptors and aromatase activity. Previously, the safety of the vitamin D active metabolite (24R)-1,24-dihydroxycholecalciferol (PRI-2191) and 1,25(OH)2D3 analog PRI-2205 was tested, and the in vitro activity of these analogs against different cancer cell lines was studied. We determined the effect of the two vitamin D compounds on anastrozole (An) activity against breast cancer based on antiproliferative activity, ELISA, flow cytometry, enzyme inhibition potency, PCR, and xenograft study. Both the vitamin D active metabolite and synthetic analog regulated the growth of not only estrogen receptor-positive cells (T47D and MCF-7, in vitro and in vivo), but also hormone-independent cancer cells such as SKBR-3 (HER-2-positive) and MDA-MB-231 (triple-negative), despite their relatively low VDR expression. Combined with An, PRI-2191 and PRI-2205 significantly inhibited the tumor growth of MCF-7 cells. Potentiation of the antitumor activity in combined treatment of MCF-7 tumor-bearing mice is related to the reduced activity of aromatase by both An (enzyme inhibition) and vitamin D compounds (switched off/decreased aromatase gene expression, decreased expression of other genes related to estrogen signaling) and by regulation of the expression of the estrogen receptor ERα and VDR.

Differential Response of Lung Cancer Cells, with Various Driver Mutations, to Plant Polyphenol Resveratrol and Vitamin D Active Metabolite PRI-2191.

Plant polyphenols and vitamins D exhibit chemopreventive and therapeutic anticancer effects. We first evaluated the biological effects of the plant polyphenol resveratrol (RESV) and vitamin D active metabolite PRI-2191 on lung cancer cells having different genetic backgrounds. RESV and PRI-2191 showed divergent responses depending on the genetic profile of cells. Antiproliferative activity of PRI-2191 was noticeable in EGFRmut cells, while RESV showed the highest antiproliferative and caspase-3-inducing activity in KRASmut cells. RESV upregulated p53 expression in wtp53 cells, while downregulated it in mutp53 cells with simultaneous upregulation of p21 expression in both cases. The effect of PRI-2191 on the induction of CYP24A1 expression was enhanced by RESV in two KRASmut cell lines. The effect of RESV combined with PRI-2191 on cytokine production was pronounced and modulated. RESV cooperated with PRI-2191 in regulating the expression of IL-8 in EGFRmut cells, while OPN in KRASmut cells and PD-L1 in both cell subtypes. We hypothesize that the differences in response to RESV and PRI-2191 between EGFRmut and KRASmut cell lines result from the differences in epigenetic modifications since both cell subtypes are associated with the divergent smoking history that can induce epigenetic alterations.

Tacalcitol increases the sensitivity of colorectal cancer cells to 5-fluorouracil by downregulating the thymidylate synthase.

5-Fluorouracil (5-FU) is an anticancer drug that is most frequently used to treat colorectal cancer (CRC) patients, but unfortunately it shows limited efficacy. We recently demonstrated that vitamin D analogs (VDAs), particularly tacalcitol (coded as PRI-2191), potentiate its anticancer activity in an in vivo mouse and human CRC model. The purpose of this study was to explain the mechanism underlying the enhancement of 5-FU efficacy by PRI-2191 towards human HT-29 CRC cells. We showed that PRI-2191 induces the CDKN1A (gene encoding p21Waf1/Cip1) expression directly through vitamin D receptor (VDR) in a p53-independent manner and thus decreases the thymidylate synthase expression both at the mRNA and protein level. It is the main mechanism by which PRI-2191 improves the anticancer efficacy of 5-FU towards HT-29 cells. Additionally, we indicated that the VDR also participates in 5-FU mechanism of action. 5-FU significantly increased TYMS (gene encoding thymidylate synthase (TS)) and BIRC5 (gene encoding survivin) level in HT-29 cells with silenced VDR. Furthermore, PRI-2191 induced E-cadherin and ZO-1 expression and thus reduced the level of BIRC5 in HT-29 cells. The induction of E-cadherin expression may also contribute to the reduction of c-Myc level and consequently the downregulation of TS. Our results also indicate that calcium-sensing receptor (CaSR) plays a role in the activity of PRI-2191 but has no influence on the 5-FU mechanism of action. In conclusion, we suggest that both VDR and CaSR might be useful as molecular markers for predicting treatment outcomes and identifying the CRC patient subgroups who might benefit from 5-FU-based chemotherapy combined with vitamin D analog.

Imatinib inhibits the regrowth of human colon cancer cells after treatment with 5-FU and cooperates with vitamin D analogue PRI-2191 in the downregulation of expression of stemness-related genes in 5-FU refractory cells.

Conventional cytotoxic drugs preferentially eliminate differentiated cancer cells but spare relatively more resistant stem-like cancer cells capable to initiate recurrence. Due to cancer cell plasticity, the stem-like phenotype can be also acquired by cancer cells refractory to treatment with cytotoxic drugs. We investigated whether drugs inhibiting receptor tyrosine kinases could be used to target human colon cancer cells initiating cancer regrowth following conventional cytotoxic treatment. The moderately differentiated cell line HT-29 and poorly differentiated cell line HCT-116 were exposed to 5-fluorouracil (5-FU). Cells that resisted the exposure to 5-FU were subsequently treated with imatinib or sunitinib. Both drugs reduced clonogenicity of 5-FU-refractory cells under normoxic and hypoxic culture conditions. The expression of numerous stemness-related genes was upregulated in cancer cells following the exposure to 5-FU, and remained at a high level in 5-FU-refractory cells undergoing renewal under normoxia, but decreased spontaneously under hypoxia. Imatinib downregulated the expression of stemness-related genes in cells undergoing renewal under normoxia. A combination of imatinib with PRI-2191, an analogue of 1,25-dihydroxyvitamin D3, downregulated stemness-related genes in HCT-116/5-FU cells more efficiently than imatinib alone. A synthetic analogue of 1,25-dihydroxyvitamin D2 (PRI-1906) abolished the effect of imatinib on gene expression in HCT-116/5-FU cells undergoing renewal under normoxia. Sunitinib promoted shift of phenotype of HT-29/5-FU cells undergoing renewal toward stem-like one. It suggests that the phenotype shift toward stemness induced by sequential sunitinib treatment following 5-FU treatment could increase a risk of cancer recurrence. In contrast to sunitinib, imatinib could be used both to interfere with cancer regrowth after conventional chemotherapy and to downregulate the expression of stemness-related genes in residual colon cancer cells capable to initiate cancer recurrence. The findings suggest that imatinib could also be combined with vitamin D analogue PRI-2191 to prevent recurrence more efficiently than imatinib alone and to compensate for vitamin D deficiency resulting from imatinib treatment.

Mannose receptor C type 2 mediates 1,25(OH)2D3/vitamin D receptor-regulated collagen metabolism through collagen type 5, alpha 2 chain and matrix metalloproteinase 13 in murine MC3T3-E1 cells.

Vitamin D plays an important role in maintaining skeletal development and bone homeostasis. Although vitamin D has been extensively researched, the direct effect of 1,25(OH)2D3 on osteoblasts is unclear. To explore the 1,25(OH)2D3 action on murine osteoblasts, we performed tandem mass tag experiments on MC3T3-E1 cells treated with and without 1,25(OH)2D3. Three up-regulated proteins (MRC2, WWTR1 and RASSF2) related to bone metabolism were confirmed in this study. 1,25(OH)2D3 up-regulated the expression of MRC2 through vitamin D receptor. MRC2 affects collagen metabolism in osteoblasts. Combined with bioinformatics and parallel reaction monitoring analysis, we inhibited the expression of MRC2 to explore the relationship between MRC2 and collagens. Then we found MRC2 down-regulated COL5A2 and up-regulated MMP13. This study provides a protein profile of 1,25(OH)2D3-treated murine osteoblasts, reveals a newly discovered signaling axis (1,25(OH)2D3/VDR/MRC2/COL5A2 and MMP13), and explains the effect of 1,25(OH)2D3 on bone metabolism from a new perspective.

Steroid hormone calcitriol and its analog tacalcitol inhibit miR-125b expression in a human breast cancer MCF-7 cell line.

MiR-125b belongs to the class of microRNAs, which are short endogenous non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. Recently, it was reported that miR-125b was found to promote migration and invasion of MCF-7 cells and was involved in chemotherapeutic resistance. Decreasing miR-125b expression would have potential therapeutic significance in preventing dissemination of breast cancer cells. The objective of this study was to evaluate miR-125b expression levels in MCF-7 cells following treatment with 1,25-dihydroxyvitamin D3 (calcitriol) and 1,24-dihydroxyvitamin D3 (tacalcitol), active metabolite and synthetic analog of vitamin D3, respectively. We found that treatment with both calcitriol and tacalcitol caused a decrease in miR-125b expression. In addition, treatment with calcitriol and tacalcitol resulted in an increase in the level of pro-apoptotic BAK1 protein encoded by the target gene of miR-125b. We are discussing the putative mechanism of inhibition of the miR-125b expression by vitamin D receptor (VDR) agonists and we suggest that calcitriol and tacalcitol may be used as a miR-125b inhibitor in breast cancer cells expressing VDR.

Differential and Overlapping Effects of 20,23(OH)2D3 and 1,25(OH)2D3 on Gene Expression in Human Epidermal Keratinocytes: Identification of AhR as an Alternative Receptor for 20,23(OH)2D3.

A novel pathway of vitamin D activation by CYP11A has previously been elucidated. To define the mechanism of action of its major dihydroxy-products, we tested the divergence and overlap between the gene expression profiles of human epidermal keratinocytes treated with either CYP11A1-derived 20,23(OH)2D3 or classical 1,25(OH)2D3. Both secosteroids have significant chemical similarity with the only differences being the positions of the hydroxyl groups. mRNA was isolated and examined by microarray analysis using Illumina's HumanWG-6 chip/arrays and subsequent bioinformatics analyses. Marked differences in the up- and downregulated genes were observed between 1,25(OH)2D3- and 20,23(OH)2D3-treated cells. Hierarchical clustering identified both distinct, opposite and common (overlapping) gene expression patterns. CYP24A1 was a common gene strongly activated by both compounds, a finding confirmed by qPCR. Ingenuity pathway analysis identified VDR/RXR signaling as the top canonical pathway induced by 1,25(OH)2D3. In contrast, the top canonical pathway induced by 20,23(OH)2D3 was AhR, with VDR/RXR being the second nuclear receptor signaling pathway identified. QPCR analyses validated the former finding by revealing that 20,23(OH)2D3 stimulated CYP1A1 and CYP1B1 gene expression, effects located downstream of AhR. Similar stimulation was observed with 20(OH)D3, the precursor to 20,23(OH)2D3, as well as with its downstream metabolite, 17,20,23(OH)3D3. Using a Human AhR Reporter Assay System we showed marked activation of AhR activity by 20,23(OH)2D3, with weaker stimulation by 20(OH)D3. Finally, molecular modeling using an AhR LBD model predicted vitamin D3 hydroxyderivatives to be good ligands for this receptor. Thus, our microarray, qPCR, functional studies and molecular modeling indicate that AhR is the major receptor target for 20,23(OH)2D3, opening an exciting area of investigation on the interaction of different vitamin D3-hydroxyderivatives with AhR and the subsequent downstream activation of signal transduction pathways in a cell-type-dependent manner.

Calcitriol and Its Analogs Establish the Immunosuppressive Microenvironment That Drives Metastasis in 4T1 Mouse Mammary Gland Cancer.

In our previous study, calcitriol and its analogs PRI-2191 and PRI-2205 stimulated 4T1 mouse mammary gland cancer metastasis. Therefore, we aimed to analyze the inflammatory response in 4T1-bearing mice treated with these compounds. Gene expression analysis of the splenocytes and regional lymph nodes demonstrated prevalence of the T helper lymphocytes (Th2) response with an increased activity of regulatory T (Treg) lymphocytes in mice treated with these compounds. We also observed an increased number of mature granulocytes and B lymphocytes and a decreased number of TCD4⁺, TCD4⁺CD25⁺, and TCD8⁺, as well as natural killer (NK) CD335⁺, cells in the blood of mice treated with calcitriol and its analogs. Among the splenocytes, we observed a significant decrease in NK CD335⁺ cells and an increase in TCD8⁺ cells. Calcitriol and its analogs decreased the levels of interleukin (IL)-1ß and IL-10 and increased the level of interferon gamma (IFN-γ) in the plasma. In the tumor tissue, they caused an increase in the level of IL-10. Gene expression analysis of lung tissue demonstrated an increased level of osteopontin (Spp1) and transforming growth factor ß (TGF-ß) mRNA. The expression of Spp1 was also elevated in lymph nodes. Calcitriol and its analogs caused prevalence of tumor-conducive changes in the immune system of 4T1 tumor-bearing mice, despite the induction of some tumor-disadvantageous effects.

Vitamin D Analogues Tacalcitol and Calcipotriol Inhibit Proliferation and Migration of T98G Human Glioblastoma Cells.

The active form of vitamin D (1α,25-dihydroxyvitamin D) acts as a steroid hormone and binds to the vitamin D receptor. This receptor is expressed in most cell types including cells in the central nervous system (CNS). Vitamin D has several functions in the body including effects on brain development, neuroprotection and immunological regulation. It has been shown that vitamin D has antiproliferative activities in different cancer cell lines. Tacalcitol and calcipotriol are synthetic analogues of 1α,25-dihydroxyvitamin D with reduced effect on calcium metabolism. The aim of this study was to analyse the effects of tacalcitol and calcipotriol on cell viability, proliferation and migration in the human glioblastoma cell line T98G. Glioblastoma is the most lethal type of primary tumours in the CNS. Both analogues decreased cell viability and/or growth, dose-dependently, in concentrations between 1 nM and 10 µM. Manual counting indicated suppressive effects by the vitamin D analogues on proliferation. Treatment with tacalcitol strongly suppressed thymidine incorporation, indicating that the vitamin D analogues mainly inhibit proliferation. Also, effects on cell migration were measured with wound-healing assay. Both calcipotriol and tacalcitol reduced the migration rate of T98G cells compared to vehicle-treated cells. However, they had no effect on caspase-3 and -7 activities, suggesting that their mechanism of action does not involve induction of apoptosis. The current results indicate that the vitamin D analogues tacalcitol and calcipotriol strongly reduce proliferation and migration of human glioblastoma T98G cells, suggesting a potential role for this type of compounds in treatment of brain cancer.

Vitamin D derivatives potentiate the anticancer and anti-angiogenic activity of tyrosine kinase inhibitors in combination with cytostatic drugs in an A549 non-small cell lung cancer model.

Numerous in vitro and in vivo studies have demonstrated that calcitriol [1,25(OH)2D3] and different vitamin D analogs possess antineoplastic activity, regulating proliferation, differentiation and apoptosis, as well as angiogenesis. Vitamin D compounds have been shown to exert synergistic effects when used in combination with different agents used in anticancer therapies in different cancer models. The aim of this study was to evaluate the mechanisms of the cooperation of the vitamin D compounds [1,24(OH)2D3 (PRI­2191) and 1,25(OH)2D3] with tyrosine kinase inhibitors (imatinib and sunitinib) together with cytostatics (cisplatin and docetaxel) in an A549 non-small cell lung cancer model. The cytotoxic effects of the test compounds used in different combinations were evaluated on A549 lung cancer cells, as well as on human lung microvascular endothelial cells (HLMECs). The effects of such combinations on the cell cycle and cell death were also determined. In addition, changes in the expression of proteins involved in cell cycle regulation, angiogenesis and the action of vitamin D were analyzed. Moreover, the effects of 1,24(OH)2D3 on the anticancer activity of sunitinib and sunitinib in combination with docetaxel were examined in an A549 lung cancer model in vivo. Experiments aiming at evaluating the cytotoxicity of the combinations of the test agents revealed that imatinib and sunitinib together with cisplatin or docetaxel exerted potent anti-proliferative effects in vitro on A549 lung cancer cells and in HLMECs; however, 1,24(OH)2D3 and 1,25(OH)2D3 enhanced the cytotoxic effects only in the endothelial cells. Among the test agents, sunitinib and cisplatin decreased the secretion of vascular endothelial growth factor (VEGF)­A from the A549 lung cancer cells. The decrease in the VEGF­A level following incubation with cisplatin correlated with a higher p53 protein expression, while no such correlation was observed following treatment of the A549 cells with sunitinib. Sunitinib together with docetaxel and 1,24(OH)2D3 exhibited a more potent anticancer activity in the A549 lung cancer model compared to double combinations and to treatment with the compounds alone. The observed anticancer activity may be the result of the influence of the test agents on the process of tumor angiogenesis, for example, through the downregulation of VEGF­A expression in tumor and also on the induction of cell death inside the tumor.

Unfavorable effect of calcitriol and its low-calcemic analogs on metastasis of 4T1 mouse mammary gland cancer.

Low vitamin D status is considered as a risk factor for breast cancer and has prognostic significance. Furthermore, vitamin D deficiency increases after adjuvant cancer therapy, which alters bone metabolism increasing the risk of osteoporosis. It is now postulated that vitamin D supplementation in breast cancer treatment delays the recurrence of cancer thereby extending survival. We evaluated the impact of calcitriol and its low-calcemic analogs, PRI­2191 and PRI­2205, on the tumor growth, angiogenesis, and metastasis of 4T1 mouse mammary gland cancer. Gene expression analysis related to cancer invasion/metastasis, real­time PCR, ELISA, western blotting, and histochemical studies were performed. In vitro studies were conducted to compare the effects of calcitriol and its analogs on 4T1 and 67NR cell proliferation and expression of selected proteins. Calcitriol and its analogs increased lung metastasis without influencing the growth of primary tumor. The levels of plasma 17ß-estradiol and transforming growth factor ß (TGFß) were found to be elevated after treatment. Moreover, the results showed that tumor blood perfusion improved and osteopontin (OPN) levels increased, whereas vascular endothelial growth factor (VEGF) and TGFß levels decreased in tumors from treated mice. All the studied treatments resulted in increased collagen content in the tumor tissue in the early step of tumor progression, and calcitriol caused an increase in collagen content in lung tissue. In addition, in vitro proliferation of 4T1 tumor cells was not found to be affected by calcitriol or its analogs in contrast to non-metastatic 67NR cells. Calcitriol and its analogs enhanced the metastatic potential of 4T1 mouse mammary gland cancer by inducing the secretion of OPN probably via host cells. In addition, OPN tumor overexpression prevailed over the decreasing tumor TGFß level and blood vessel normalization via tumor VEGF deprivation induced by calcitriol and its analogs. Moreover, the increased plasma TGFß and 17ß-estradiol levels contributed to the facilitation of metastatic process.

Protective effects of tacalcitol against oxidative damage in human epidermal melanocytes.

BACKGROUND: Oxidative damage may lead to the dysfunction of melanocytes (MCs) and is one of the causative mechanisms involved in the pathogenesis of vitiligo. OBJECTIVES: This study was designed to investigate the protective effects of the vitamin D3 analog tacalcitol on oxidative damage induced by hydrogen peroxide (H2 O2 ) in human epidermal MCs. METHODS: Human epidermal MCs were cultured and identified by l-DOPA staining and HMB-45 immunohistochemical staining. The model of oxidative damage induced by H2 O2 was established, and the cells were treated with tacalcitol. The viability of MCs was determined using an MTS assay. Morphological changes in cell dendrites were observed by microscopy, and the rate of change of dendrites was calculated. The reactive oxygen species (ROS) level in MCs was determined using immunofluorescence microscopy. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels in MCs were determined using the WST-1 and TBA methods, respectively. RESULTS: In comparison with the control group, the viability of MCs and SOD activity were significantly decreased in the H2 O2 group (P < 0.05) and significantly increased in the tacalcitol group (P < 0.05). In comparison with the control group, the rate of change of cell dendrites and levels of ROS and MDA were significantly increased in the H2 O2 group (P < 0.05) and significantly decreased in the tacalcitol group (P < 0.05). CONCLUSIONS: Tacalcitol can reduce oxidative damage induced by H2 O2 in MCs by inhibiting intracellular ROS overproduction, increasing SOD activity, and decreasing the level of MDA, thereby reducing cell apoptosis.

Synthesis and Biological Evaluation of Vitamin D3 Metabolite 20S,23S-Dihydroxyvitamin D3 and Its 23R Epimer.

The vitamin D3 metabolite, 20S,23S-dihydroxyvitamin D3, was chemically synthesized for the first time and identified to be the same as the enzymatically produced metabolite. The C23 absolute configurations of both 20S,23S/R-dihydroxyvitamin D3 epimers were unambiguously assigned by NMR and Mosher ester analysis. Their kinetics of CYP27B1 metabolism were investigated during the production of their 1α-hydroxylated derivatives. Bioactivities of these products were compared in terms of vitamin D3 receptor activation, anti-inflammatory, and antiproliferative activities.

Differential interference of vitamin D analogs PRI-1906, PRI-2191, and PRI-2205 with the renewal of human colon cancer cells refractory to treatment with 5-fluorouracil.

This study was aimed to determine whether hypocalcemic analogs of active forms of vitamins D modulate expression of genes related to stem-like phenotype in colon cancer cell lines HT-29 and HCT-116 undergoing renewal after the treatment with 5-fluorouracil (5-FU). Both lines express vitamin D receptor, but differ in differentiation stage and vitamin D sensitivity. Cells that resisted the 5-FU exposure were treated with synthetic analog of 1,25-dihydroxyvitamin D2 (PRI-1906) and analogs of 1,25-dihydroxyvitamin D3 (PRI-2191 and PRI-2205). Proliferative activity was more profoundly affected by vitamin D analogs in HT-29/5-FU than in HCT-116/5-FU cells. In HT-29/5-FU cells, analogs PRI-1906 and PRI-2191 downregulated the expression of genes related to survival, re-growth, and invasiveness during renewal, while PRI-2205 increased expression of genes related to differentiation only. In HCT-116/5-FU cells, PRI-2191 decreased the expression of stemness- and angiogenesis-related genes, whereas PRI-1906 augmented their expression. The effects in HCT-116/5-FU cells were observed at higher concentrations of the analogs than those used for HT-29/5-FU cells. Out of the series of analogs studied, PRI-2191 might be used to counteract the renewal of both moderately and poorly differentiated cancer cells following conventional treatment.

Synthesis and Biological Activity of 2-Methylene Analogues of Calcitriol and Related Compounds.

In an attempt to prepare vitamin D analogues that are superagonists, (20R)- and (20S)-isomers of 1α-hydroxy-2-methylenevitamin D3 and 1α,25-dihydroxy-2-methylenevitamin D3 have been synthesized. To prepare the desired A-ring dienyne fragment, two different approaches were used, both starting from the (-)-quinic acid. The obtained derivative was subsequently coupled with the C,D-ring enol triflates derived from the corresponding Grundmann ketones, using the Sonogashira reaction. Moreover, (20R)- and (20S)-1α,25-dihydroxy-2-methylenevitamin D3 compounds with an (5E)-configuration were prepared by iodine catalyzed isomerization. All four 2-methylene analogues of the native hormone were characterized by high in vitro activity. As expected, the 25-desoxy analogues were much less potent. Among the synthesized compounds, two of them, 1α,25-dihydroxy-2-methylenevitamin D3 and its C-20 epimer, were found to be almost as active as 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD) on bone but more active in intestine.

Vitamin D decreases the secretion of matrix metalloproteinase-2 and matrix metalloproteinase-9 in fibroblasts derived from Taiwanese patients with chronic rhinosinusitis with nasal polyposis.

Vitamin D and its derivatives have modulatory effects in immunological and inflammatory responses. Such properties suggest that they might have an impact on chronic inflammatory airway diseases, including nasal polyposis. The aim of this study was to understand the role of vitamin D in chronic rhinosinusitis with nasal polyps (CRSwNP) by investigating its effect on the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9 in nasal polyp-derived fibroblasts. Two primary fibroblast cultures were established from nasal polyp tissues obtained during surgery. The nasal polyp-derived fibroblasts were stimulated with tumor necrosis factor-α (TNF-α; 10 ng/mL) for 24 hours, followed by replacement with media alone or with vitamin D derivatives (calcitriol or tacalcitol; 10µM) and incubated for another 24 hours. After the treatments, the levels of MMP-2 and MMP-9 secreted were evaluated by both enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. ELISA results revealed that TNF-α could substantially stimulate the secretion of MMP-2 (p < 0.01) and MMP-9 (p < 0.001) in nasal polyp-derived fibroblasts. More importantly, such stimulatory effect was significantly suppressed by adding calcitriol (p ≤ 0.01 for MMP-2 and p < 0.001 for MMP-9) or tacalcitol (p < 0.005 for both MMP-2 and MMP-9). The ELISA results were also confirmed by Western blot analysis. The inhibitory effect of vitamin D derivatives on MMP-2 and MMP-9 secretion could potentiate their application in pharmacotherapy of Taiwanese CRSwNP patients.

Phosphonate analogues of 1α, 25 dihydroxyvitamin D3 are promising candidates for antitumoural therapies.

The active metabolite of vitamin D, 1α, 25 dihydroxyvitamin D3 (calcitriol) is classically known to regulate calcium and phosphate homeostasis and bone mineralization. In addition, calcitriol has also been documented to act as a potent anticancer agent in multiple cell culture and animal models of cancer. However, major side effects, such as hypercalcemia, hinder broad-spectrum therapeutic uses of calcitriol in cancer chemotherapy. Synthesis of calcitriol analogues with the same or increased antiproliferative and pro-differentiating activities, and with reduced undesired effects on calcium and bone metabolism, is getting significant attention towards rational therapeutics to treat cancer. In this regard, phosphonate analogues have been shown to display a certain degree of dissociation between the vitamin D activity in vitro and undesired hypercalcemia in vivo. However, few phosphonates have been described in the literature and fewer of them tested for antitumoral effects. Our group has synthesized a novel vitamin D analogue (EM1) bearing an alkynylphosphonate moiety that combines the low calcemic properties of phosphonates with the decreased metabolic inactivation due to the presence of a triple bond between C-23 and C-24. Biological assays demonstrated that this analogue has potent antiproliferative effects in a wide panel of tumour cell lines, even in those resistant to calcitriol treatment. Importantly, EM1 does not show toxic effects in animals, even administered at high doses and for extended periods of time. In the current review we discuss the effects and the potential application in cancer of vitamin D and its derivatives, with an emphasis on phosphonate analogues.

Synthetic strategy and biological activity of A-ring stereoisomers of 1,25- dihydroxyvitamin D3 and C2-modified analogues.

The hormonally active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1a), has a wide variety of biological activities and its major molecular target is considered to be the vitamin D receptor (VDR). The A-ring stereoisomers of 1a as well as its C2-modified analogues, which have different stereochemistry at the C1 and/or C3 hydroxy groups, are of interest since recent metabolic studies have shown that catabolism could occur through A-ring modification. In this review, a practical and versatile synthesis of the A-ring enyne precursors by the convergent method of Trost and coworkers, which is needed to construct all possible A-ring stereoisomers of 1,25-dihydroxyvitamin D3 (1a-d), and the C2-modified analogues (4a-d, 5a-d, 6a-d and 7a-d) is described. A strategy for the synthesis and evaluation of all possible A-ring stereoisomers of 1a and their A-ring modified analogues is important, and this will stimulate synthesis and biological studies into vitamin D.

Vitamin D analogs combined with 5-fluorouracil in human HT-29 colon cancer treatment.

In the present study, we evaluated the antitumor effect of two synthetic analogs of vitamin D, namely PRI-2191 [(24R)-1,24-dihydroxyvitamin D3] and PRI-2205 (5,6-trans calcipotriol), in combined human colon HT-29 cancer treatment with 5-fluorouracil (5-FU). Mice bearing HT-29 tumors transplanted subcutaneously or orthotopically were injected with vitamin D analogs and 5-FU in various schedules. A statistically significant inhibition of subcutaneous or orthotopic tumor growth was observed as a result of combined therapy. In HT-29 tumors and in cells from in vitro culture, we observed increased vitamin D receptor (VDR) expression after treatment with either PRI-2205 or 5-FU alone, or in combination. Moreover, PRI-2205 decreased the percentage of cells from intestinal tumors in G2/M and S stages and increased sub-G1. Increased VDR expression was also observed after combined treatment of mice with 5-FU and PRI-2191. Moreover, our docking studies showed that PRI-2205 has stronger affinity for VDR, DBP and CAR/RXR ligand binding domains than PRI-2191. PRI-2191 analog, used with 5-FU, increased the percentage of subcutaneous tumor cells in G0/G1 and decreased the percentage in G2/M, S and sub-G1 populations as compared to 5-FU alone. In in vitro studies, we observed increased expression of p21 and p-ERK1/2 diminution via use of both analogs as compared to use of 5-FU alone. Simultaneously, PRI-2191 antagonizes some pro-apoptotic activities of 5-FU in vitro. However, in spite of these disadvantageous effects in terms of apoptosis, the therapeutic effect expressed as tumor growth retardation by PRI-2191 is significant. Our results suggest that the mechanism of potentiation of 5-FU antitumor action by both analogs is realized via increased p21 expression and decreased p-ERK1/2 level which may lead to diminution of thymidylate synthase expression. Higher binding affinity for VDR, DBP, but also for CAR\RXR ligand binding domain of PRI-2205 may, in part, explain its very low toxicity with sustained anticancer activity.

Using yeast to model calcium-related diseases: example of the Hailey-Hailey disease.

Cross-complementation studies offer the possibility to overcome limitations imposed by the inherent complexity of multicellular organisms in the study of human diseases, by taking advantage of simpler model organisms like the budding yeast Saccharomyces cerevisiae. This review deals with, (1) the use of S. cerevisiae as a model organism to study human diseases, (2) yeast-based screening systems for the detection of disease modifiers, (3) Hailey-Hailey as an example of a calcium-related disease, and (4) the presentation of a yeast-based model to search for chemical modifiers of Hailey-Hailey disease. The preliminary experimental data presented and discussed here show that it is possible to use yeast as a model system for Hailey-Hailey disease and suggest that in all likelihood, yeast has the potential to reveal candidate drugs for the treatment of this disorder. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.