MART-10, a 1α,25(OH)2D3 Analog, Potently Represses Metastasis of ER+ Breast Cancer Cells with VEGF-A Overexpression.

BACKGROUND: Breast cancer ranks second in the list of cancer-related deaths for women. Even under multidisciplinary treatment, 25-50% of patients with breast cancer still ultimately develop metastasis, leading to poor prognosis. In addition to inducing angiogenesis, vascular endothelial growth factor-A (VEGF-A) is believed to directly increase cancer cell metastatic potential and overexpression of VEGF-A is associated with higher invasiveness of breast cancer. 1α,25(OH)2D3, the active form of vitamin D, and its analogs have been widely applied as anticancer agents in the past. MATERIAL AND METHODS: Western blot, migration and invasion assays, enzyme-linked immunosorbent assay, and immunofluorescent stain were applied in this study. RESULT: VEGF-A increased cell migration and invasion in estrogen receptor-positive (ER+) breast cancer MCF-7 cells. VEGF-A induced an autocrine loop in MCF-7 cells as VEGF-A treatment increased both VEGF-A expression and secretion. The expression of VEGF receptor type 2 (VEGFR2) and neuropilin 1 was also up-regulated by VEGF-A in MCF-7 cells. In addition, F-actin synthesis and LIM domain kinase 1 (LIMK-1) phosphorylation were increased by VEGF-A. VEGF-A also increased ß-catenin expression and nuclear translocation of both ß-catenin and nuclear factor-ĸB (NF-ĸB), indicating increased ß-catenin and NF-ĸB activity. 1α,25(OH)2D3 and MART-10, an analog of 1α,25(OH)2D3, effectively repressed VEGF-A-induced MCF-7 cell migration and invasion and other VEGF-A-induced effects on MCF-7 cells, with MART-10 being more potent than 1α,25(OH)2D3 Conclusion: MART-10 can be deemed as a promising agent for prevention and treatment of metastasis of ER+ breast cancer with VEGF-A overexpression.

1α,25(OH)2D3 Analog, MART-10, Inhibits Neuroendocrine Tumor Cell Metastasis After VEGF-A Stimulation.

AIMS: Pancreatic neuroendocrine tumors (PanNETs) are usually diagnosed in an advanced stage. Most patients with PanNETs die of metastasis. Vascular endothelial growth factor-A (VEGF-A) is a strong stimulator of angiogenesis and tumor metastasis. We aimed to investigate the effect of MART-10 [19-nor-2α-(3-hydroxypropyl)-1α,25(OH)2D3], a 1α,25-dihydroxy-vitamin D3 (1α,25(OH)2D3) analog, on PanNET cell metastasis after VEGF-A stimulation. MATERIALS AND METHODS: Migration and invasion assays, western blot, and immunofluorescent staining were applied in this study. RESULTS: VEGF-A increased PanNET cell migration and invasion, which was attenuated by 1α,25(OH)2D3 and MART-10. VEGF-A treatment stimulated epithelial-mesenchymal transition (EMT) of PanNET cells. During this process, expression of snail family transcriptional repressor 1 and 2, and fibronectin was up-regulated. 1α,25(OH)2D3 and MART-10 counteracted VEGF-A-induced EMT. In addition, expression of neuropilin 1, a key protein in VEGF-A signaling, was down-regulated by 1α,25(OH)2D3 and MART-10. Furthermore, synthesis of F-actin was increased by VEGF-A and reduced by 1α,25(OH)2D3 and MART-10. CONCLUSION: Our data indicate that MART-10 could be deemed a promising drug for PanNET treatment.

ASSESSMENT OF SERUM 25-HYDROXYVITAMIN D CONCENTRATIONS IN TWO COLLECTIONS OF CAPTIVE GORILLAS (GORILLA GORILLA GORILLA).

Serum 25-hydroxyvitamin D concentrations were assessed in subadult to adult captive lowland gorillas ( Gorilla gorilla gorilla) (n = 26) at two institutions with different husbandry and management practices. Serum 25-hydroxyvitamin D (25[OH]D) concentrations for gorillas managed predominantly indoors was low (14.2 ± 5.9 ng/ml), despite consuming commercial biscuits fortified with vitamin D3. Concentrations of 25(OH)D in gorillas with near daily outdoor access were significantly higher than gorillas managed indoors, although many individuals still had serum values below concentrations recommended for adult humans. Consideration should be given to assessing 25(OH)D concentrations in all captive gorillas and providing specific supplementation, particularly to juveniles without access to direct sunlight.

MART-10 represses cholangiocarcinoma cell growth and high vitamin D receptor expression indicates better prognosis for cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a devastating disease due to no effective treatments available. Since the non-mineral functions of vitamin D emerges, 1α,25(OH)2D3, the active form of vitamin D, has been applied in anti-cancer researches. In this study, we demonstrated that both the 1α,25(OH)2D3 analog, MART-10, and 1α,25(OH)2D3 possessed anti-growth effect on human CCA cells with MART-10 much more potent than 1α,25(OH)2D3. The growth inhibition of both drugs were mediated by induction of G0/G1 cell cycle arrest through upregulation of p27 and downregulation of CDK4, CDK6, and cyclin D3. Human neutrophil gelatinase associated lipocalin (NGAL) was found to be involved in 1α,25(OH)2D3 and MART-10 meditated growth inhibition for CCA as knockdown of NGAL decreased Ki-67 expression in SNU308 cells and rendered SNU308 cells less responsive to 1α,25(OH)2D3 and MART-10 treatment. Vitamin D receptor (VDR) knockdown partly abolished MART-10-induced inhibition of NGAL and cell growth in SNU308 cells. The xenograft animal study demonstrated MART-10 could effectively repressed CCA growth in vivo without inducing obvious side effects. The IHC examination of human CCA specimen for VDR revealed that higher VDR expression was linked with better prognosis. Collectively, our results suggest that MART-10 could be a promising regimen for CCA treatment.

Clinical and Environmental Correlates of Serum BDNF: A Descriptive Study with Plausible Implications for AD Research.

BACKGROUND: Brain derived neurotrophic factor (BDNF) may play a central role in the pathogenesis of Alzheimer's disease (AD) through neurotrophic effects on basal cholinergic neurons. Reduced serum levels of BDND are observed among AD patients and may predict AD risk. Nevertheless, knowledge about factors associated with its levels in blood is lacking. OBJECTIVE: To identify clinical and demographic correlates of serum BDNF levels. METHODS: BDNF was measured from serum collected between 1992-1996 and 1998-2001 in participants from the Original and Offspring cohorts of the Framingham Study, respectively. A cross-sectional analysis was done to evaluate the relationship between clinical measures and BDNF levels using standard linear regression and stepwise models. Analyses were conducted in the total sample and separately in each cohort, and were adjusted for age and sex. RESULTS: BDNF was measured in 3,689 participants (mean age 65 years, 56% women; 82% Offspring). Cigarette smoking and high total cholesterol were associated with elevated BDNF levels, and history of atrial fibrillation was associated with decreased levels. Elevated BDNF levels were related to greater physical activity and lower Tumor Necrosis Factor-α levels in Offspring. Stepwise models also revealed associations with statin use, alcohol consumption and Apolipoprotein Eε4 genotype. CONCLUSION: Serum BDNF correlates with various metabolic, inflammatory and life-style measures which in turn have been linked with risk of AD. Future studies of serum BDNF should adjust for these correlates and are needed to further explore the underlying interplay between BDNF and other factors in the pathophysiology of cognitive impairment and AD.

25(OH)D Is Effective to Repress Human Cholangiocarcinoma Cell Growth through the Conversion of 25(OH)D to 1α,25(OH)2D3.

Cholangiocarcinoma (CCA) is a devastating disease without effective treatments. 1α,25(OH)2D3, the active form of Vitamin D, has emerged as a new anti-cancer regimen. However, the side effect of hypercalcemia impedes its systemic administration. 25(OH)D is biologically inert and needs hydroxylation by CYP27B1 to form 1α,25(OH)2D3, which is originally believed to only take place in kidneys. Recently, the extra-renal expression of CYP27B1 has been identified and in vitro conversion of 25(OH)D to 1α,25(OH)2D3 has been found in some cancer cells with CYP27B1 expression. In this study, CYP27B1 expression was demonstrated in CCA cells and human CCA specimens. 25(OH)D effectively represses SNU308 cells growth, which was strengthened or attenuated as CYP27B1 overexpression or knockdown. Lipocalcin-2 (LCN2) was also found to be repressed by 25(OH)D. After treatment with 800 ng/mL 25(OH)D, the intracellular 1α,25(OH)2D3 concentration was higher in SNU308 cells with CYP27B1 overexpression than wild type SNU308 cells. In a xenograft animal experiment, 25(OH)D, at a dose of 6 µg/kg or 20 µg/kg, significantly inhibited SNU308 cells' growth without inducing obvious side effects. Collectively, our results indicated that SNU308 cells were able to convert 25(OH)D to 1α,25(OH)2D3 and 25(OH)D CYP27B1 gene therapy could be deemed as a promising therapeutic direction for CCA.

MART-10, a newly synthesized vitamin D analog, represses metastatic potential of head and neck squamous carcinoma cells.

Even with multidisciplinary treatment, the prognosis and quality of life of patients diagnosed with head and neck squamous cell carcinoma (HNSCC) are still not satisfactory. Previously, 19-Nor-2α-(3-hydroxypropyl)-1α,25(OH)2D3 (MART-10), the new brand 1α,25(OH)2D3 analog, has been demonstrated to be an effective drug to inhibit HNSCC growth in vitro. Since most cancer patients die of metastasis, in this study, the antimetastatic effect of MART-10 on HNSCC was investigated. Our results reveal that both 1α,25(OH)2D3 and MART-10 effectively repressed the migration and invasion of HNSCC cells, with MART-10 being much more potent than 1α,25(OH)2D3. The antimetastatic effect of 1α,25(OH)2D3 and MART-10 was mediated by attenuation of epithelial-mesenchymal transition (EMT), which was supported by the finding that the expression of EMT-inducing transcriptional factors, Sail and Twist, was inhibited by 1α,25(OH)2D3 and MART-10. The upregulation of E-cadherin and downregulation of N-cadherin in FaDu cells induced by both drugs further confirmed the repression of EMT. In addition, 1α,25(OH)2D3 and MART-10 treatment inhibited intracellular MMP-9 expression and extracellular MMP activity in FaDu cells. Collectively, our results suggest that the less-calcemia 1α,25(OH)2D3 analog, MART-10, is a promising drug for HNSCC treatment. Further clinical studies are warranted.

Genomewide meta-analysis identifies loci associated with IGF-I and IGFBP-3 levels with impact on age-related traits.

The growth hormone/insulin-like growth factor (IGF) axis can be manipulated in animal models to promote longevity, and IGF-related proteins including IGF-I and IGF-binding protein-3 (IGFBP-3) have also been implicated in risk of human diseases including cardiovascular diseases, diabetes, and cancer. Through genomewide association study of up to 30 884 adults of European ancestry from 21 studies, we confirmed and extended the list of previously identified loci associated with circulating IGF-I and IGFBP-3 concentrations (IGF1, IGFBP3, GCKR, TNS3, GHSR, FOXO3, ASXL2, NUBP2/IGFALS, SORCS2, and CELSR2). Significant sex interactions, which were characterized by different genotype-phenotype associations between men and women, were found only for associations of IGFBP-3 concentrations with SNPs at the loci IGFBP3 and SORCS2. Analyses of SNPs, gene expression, and protein levels suggested that interplay between IGFBP3 and genes within the NUBP2 locus (IGFALS and HAGH) may affect circulating IGF-I and IGFBP-3 concentrations. The IGF-I-decreasing allele of SNP rs934073, which is an eQTL of ASXL2, was associated with lower adiposity and higher likelihood of survival beyond 90 years. The known longevity-associated variant rs2153960 (FOXO3) was observed to be a genomewide significant SNP for IGF-I concentrations. Bioinformatics analysis suggested enrichment of putative regulatory elements among these IGF-I- and IGFBP-3-associated loci, particularly of rs646776 at CELSR2. In conclusion, this study identified several loci associated with circulating IGF-I and IGFBP-3 concentrations and provides clues to the potential role of the IGF axis in mediating effects of known (FOXO3) and novel (ASXL2) longevity-associated loci.

Circulating vascular endothelial growth factor and the risk of cardiovascular events.

OBJECTIVE: To investigate the relation of circulating concentrations of vascular endothelial growth factor (VEGF) for the risk of developing cardiovascular disease (CVD) in a large community-based sample. METHODS: We prospectively assessed the relation of circulating VEGF concentrations with the incidence of CVD among 3041 Framingham Heart Study participants (mean age 63.4±11.1 years, 59% women). Multivariable Cox proportional hazards models were estimated adjusting for standard risk factors to VEGF quartiles to incident CVD. Restricted cubic splines were used to examine the linearity of the association. RESULTS: After a mean follow-up of 8.8 (±2.8) years, 527 individuals experienced a first CVD event. Compared with participants in the first VEGF quartile, individuals in the second VEGF quartile had a 34% increased risk for future CVD (HR 1.34, 95% CI 1.03 to 1.74; p value=0.03) and individuals in third quartile had a 59% higher risk (HR 1.59; 95% CI 1.23 to 2.05, p value=0.0003). Individuals in the highest VEGF quartile had a similar cardiovascular risk as compared with those in the lowest VEGF quartile (HR 1.18, 95% CI 0.91 to 1.53, p value=0.21). Evaluation of restricted cubic splines confirmed the nonlinear, inverted U-shaped relation of serum VEGF and CVD events (p<0.0001 for model fit, p=0.006 for non-linearity). CONCLUSIONS: Circulating VEGF concentrations exhibit a complex non-linear (inverted U-shaped) relation with the risk of developing CVD events, with the lowest risk experienced at the lower and upper end of the distribution. The underlying pathophysiological mechanisms remain to be elucidated.

1α,25(OH)2D3 Analog, MART-10, Inhibits Neuroendocrine Tumor Cell Growth Through Induction of G0/G1 Cell-cycle Arrest and Apoptosis.

BACKGROUND: Neuroendocrine tumors (NETs) are the second most common digestive malignancy. For advanced NETs, survival is not satisfactory. Vitamin D has emerged as a promising anticancer drug. MATERIALS AND METHODS: Cell proliferation assay, western blot, flow cytometry, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays were applied. RESULTS: We demonstrated that RIN-m cells, neuroendocrine tumor cells, expressed vitamin D receptor (VDR) and VDR expression increased with increasing exposure to 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] or MART-10, a 1α,25(OH)2D3 analog. MART-10 had anti-growth effect on RIN-m cells comparable to those of 1α,25(OH)2D3 The growth inhibition of both drugs was mediated by induction of cell-cycle arrest at G0/G1 phase and apoptosis. Western blot assay further revealed that this G0/G1 arrest was due to the up-regulation of p27 and down-regulation of cyclin dependent kinase 4 (CDK4), with MART-10 also reducing CDK6. Apoptosis induction was further supported by increased cleaved caspase-3 expression after treatment. CONCLUSION: MART-10 appears to be a promising regimen for NET treatment.

The Vitamin D Analog, MART-10, Attenuates Triple Negative Breast Cancer Cells Metastatic Potential.

Regarding breast cancer treatment, triple negative breast cancer (TNBC) is a difficult issue. Most TNBC patients die of cancer metastasis. Thus, to develop a new regimen to attenuate TNBC metastatic potential is urgently needed. MART-10 (19-nor-2α-(3-hydroxypropyl)-1α,25(OH)2D3), the newly-synthesized 1α,25(OH)2D3 analog, has been shown to be much more potent in cancer growth inhibition than 1α,25(OH)2D3 and be active in vivo without inducing obvious side effect. In this study, we demonstrated that both 1α,25(OH)2D3 and MART-10 could effectively repress TNBC cells migration and invasion with MART-10 more effective. MART-10 and 1α,25(OH)2D3 induced cadherin switching (upregulation of E-cadherin and downregulation of N-cadherin) and downregulated P-cadherin expression in MDA-MB-231 cells. The EMT(epithelial mesenchymal transition) process in MDA-MB-231 cells was repressed by MART-10 through inhibiting Zeb1, Zeb2, Slug, and Twist expression. LCN2, one kind of breast cancer metastasis stimulator, was also found for the first time to be repressed by 1α,25(OH)2D3 and MART-10 in breast cancer cells. Matrix metalloproteinase-9 (MMP-9) activity was also downregulated by MART-10. Furthermore, F-actin synthesis in MDA-MB-231 cells was attenuated as exposure to 1α,25(OH)2D3 and MART-10. Based on our result, we conclude that MART-10 could effectively inhibit TNBC cells metastatic potential and deserves further investigation as a new regimen to treat TNBC.

Metabolism and Action of 25-Hydroxy-19-nor-Vitamin D3 in Human Prostate Cells.

Since the discovery of 1α,25(OH)2D3 in the early 1970s, it has been widely accepted that this metabolite is responsible for the biological actions of vitamin D. Likewise, we have assumed that 25(OH)-19-nor-D3-dependent growth inhibition of human prostate PZ-HPV-7 cells was the result of its subsequent conversion to 1α,25(OH)2-19-nor-D3, catalyzed by CYP27B1 within the prostate cells. However, further in vitro studies in a reconstituted system using recombinant CYP27B1 revealed that 25(OH)-19-nor-D3 was hardly converted to 1α,25(OH)2-19-nor-D3 by the enzyme. The kinetic analysis of 1α-hydroxylation of 25(OH)D3 and 25(OH)-19-nor-D3 demonstrated that the k(cat)/K(m) for 25(OH)-19-nor-D3 is less than 0.1% of that for 25(OH)D3. When 25(OH)-19-nor-D3 was added to cultured PZ-HPV-7 cells, eight metabolites were detected, while no 1α,25(OH)2-19-nor-D3 was found. In addition, the time course of VDR translocation into the nucleus induced by 100 nM 25(OH)-19-nor-D3, and the subsequent transactivation of CYP24A1 gene were almost identical to those induced by 1 nM 1α,25(OH)2-19-nor-D3. These results strongly suggest that 25(OH)-19-nor-D3 binds directly to VDR as a ligand to transport VDR into the nucleus to induce CYP24A1 gene transactivation. Furthermore, knockdown of CYP27B1 gene did not affect the antiproliferative activity of 25(OH)-19-nor-D3, whereas VDR knockdown attenuated the effect, suggesting that the antiproliferative activity of 25(OH)-19-nor-D3 is VDR dependent but CYP27B1 independent. Finally, our recent studies using the same cell line demonstrate that 25(OH)D3 can act as a VDR agonist to induce gene transactivation. These findings suggest that vitamin D analogs without 1α-hydroxyl group could be developed as drugs for osteoporosis or cancer treatment.

MART-10, the new brand of 1α,25(OH)2D3 analog, is a potent anti-angiogenic agent in vivo and in vitro.

BACKGROUND: Angiogenesis is the hall marker for cancer growth and metastasis. Thus, anti-angiogenesis emerges as a new way to treat cancer. 1α,25(OH)2D3 is recently getting popular due to the non-mineral functions, which have been applied fore cancer treatment. The newly-synthesized 1α,25(OH)2D3 analog, MART-10, has been proved to be much more potent than 1α,25(OH)2D3 regarding inhibiting cancer cells growth and metastasis without inducing hypercalcemia in vivo. In this study, we aimed to investigate the effect of MART-10 and 1α,25(OH)2D3 on angiogenesis in vitro and in vivo. METHODS AND RESULTS: MART-10 and 1α,25(OH)2D3 were able to repress VEGFA-induced human umbilical vein endothelial cells (HUVECs) migration, invasion and tube formation, but not proliferation, with MART-10 much more potent than 1α,25(OH)2D3. The Chick Chorioallantoic Membrane (CAM) assay and matrigeal angiogenesis assay further confirmed the in vivo more potent anti-angiogenesis effect of MART-10. MART-10 inhibited the VEGFA-induced HUVECs angiogenesis process through downregulation of Akt and Erk 1/2 phosphorylation. The VEGFA-VEGFR2 (VEGF receptor 2) axis is the main signal transducing pathway to stimulate angiogenesis. A positive autocrine manner was found for the first time in HUVECs as treated by VEGFA, which induced VEGFA expression and secretion, and VEGFR2 expression. MART-10 and 1α,25(OH)2D3 were demonstrated to be able to repress this positive autocrine manner, thus inhibiting angiogenesis. CONCLUSIONS: MART-10 and 1α,25(OH)2D3 both are effective anti-angiogenesis agents. Given MART-10 is much more potent than 1α,25(OH)2D3 and active in vivo without obvious side effect, MART-10 should be deemed as a promising anti-cancer agent.

MART-10, the vitamin D analog, is a potent drug to inhibit anaplastic thyroid cancer cell metastatic potential.

The survival rate of anaplastic thyroid cancer (ATC) is still very poor due to its fast growth and high metastatic potential. Currently, no effective treatment is available. The active form of vitamin D3, 1α,25(OH)2D3, has been shown to have a anti-metastatic effect in pre-clinical studies, however induction of hypercalcemia hampered its clinical application. The new class of less-calcemic vitamin D analog, 19-nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3 (MART-10), is much more potent than 1α,25(OH)2D3 to repress cancer growth and metastasis in a variety of cancers. In this study, we demonstrated that both 1α,25(OH)2D3 and MART-10 could effectively inhibit the migration and invasion of ATC cells, 8305C and 8505C, with MART-10 much more potent than 1α,25(OH)2D3. The anti-metastatic effect of 1α,25(OH)2D3 and MART-10 on ATC cells is mediated by reversal of cadherin switch (upregulation of E-cadherin and downregulation of N-cadherin), which led to the attenuation of EMT process, and decrease of F-actin formation. We further showed that the expressions of Slug, the EMT-related transcriptional factor, and MMP-9 were inhibited by 1α,25(OH)2D3 and MART-10 in 8505C cells, but not in 8303C cells. Since metastasis is the important cause of ATC-related death, our results strongly encourage the further in vivo study of MART-10 application against ATC.

Hepatocellular carcinoma cells express 25(OH)D-1α-hydroxylase and are able to convert 25(OH)D to 1α,25(OH)2D, leading to the 25(OH)D-induced growth inhibition.

Hepatocellular carcinoma (HCC) is the most diagnosed liver cancer without effective treatments available for advanced HCC. Vitamin D is getting popular due to its anti-cancer characteristics. However, the clinical application of 1α,25(OH)2D, the active form of vitamin, is hampered by its hypercalcemia side effect. 1α,25(OH)2D is converted from 25(OH)D, the index of serum vitamin D status, by CYP27B1, which is originally found in kidneys but recently detected in non-renal tissues. 25(OH)D has been shown to repress some cancers expressing CYP27B1 due to the local conversion of 25(OH)D to 1α,25(OH)2D, which works in a intra-, auto-, or paracrine manner and thus minimizes the risk of hypercalcemia. In this study, we found CYP27B1 expression in human hepatocyte, HCC, and HepG2 cells. As we treated HepG2 cells with 25(OH)D, the 1α,25(OH)2D target gene CYP24A1 expression was increased and was further upregulated as CYP27B1 transfection or downregulated as CYP27B1 knockdown. Other 1α,25(OH)2D target genes in HepG2 cells, p21 and p27 were also stimulated by 25(OH)D after CYP27B1 transfection. Further, 25(OH)D could inhibit HepG2 cells growth, which was potentiated by CYP27B1 transfection. Collectively, we showed for the first time that HCC expressed CYP27B1 and was able to covert 25(OH)D to 1α,25(OH)2D in vitro, thus responsive to 25(OH)D treatment. Our data justifies the application of 25(OH)D and CYP27B1 gene transfection therapy in further HCC treatment studies.

19-Norvitamin D analogs for breast cancer therapy.

The active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3 or calcitriol), is known to inhibit the proliferation and invasiveness of many types of cancer cells, including breast, colon, pancreatic, prostate, and liver cancer cells. These findings support the use of 1α,25(OH)2D3 for the treatment of these types of cancer. However, 1α,25(OH)2D3 can cause hypercalcemia, so analogs of 1α,25(OH)2D3 that are less calcemic but exhibit more potent anti-tumor activity would be good candidates as therapeutic agents. Therefore, a series of 19-norvitamin D analogs, in which the methylidene group on C19 is replaced with 2 hydrogen atoms, have been synthesized by several laboratories. In our laboratory, we have designed and synthesized a series of 2α-functional group substituted 19-norvitamin D3 analogs and examined their anti-proliferative activity. Among them, 2α- and 2ß-(3-hydroxypropyl)-1α,25-dihydroxy-19-norvitamin D3 (MART-10 and MART-11) were found to be the most promising. Here, we review the rationale and approaches for the synthesis of different 19-norvitamin D analogs, and the pre-clinical studies using these analogs in breast cancer cells, in particular, we chose MART-10 for its potential application to the prevention and treatment of breast cancer.

Circulating brain-derived neurotrophic factor concentrations and the risk of cardiovascular disease in the community.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is a pleiotropic peptide involved in maintaining endothelial integrity. It is unknown if circulating BDNF levels are associated with risk of cardiovascular disease (CVD). METHODS AND RESULTS: We prospectively investigated the association of circulating BDNF levels with cardiovascular events and mortality in 3687 participants (mean age 65 years, 2068 women) from the Framingham Heart Study (FHS). Using a common nonsynonomous single nucleotide polymorphism (SNP) in the BDNF gene (rs6265), we then performed a Mendelian randomization experiment in the CARDIoGRAM (Coronary ARtery DIsease Genome-Wide Replication And Meta-Analysis) consortium (>22,000 coronary artery disease [CAD] cases, >60,000 controls) to investigate whether SNP rs6265 was associated with CAD in CARDIoGRAM and, if so, whether the effect estimate differed from that predicted based on FHS data. On follow-up (median 8.9 years), 467 individuals (261 women) in FHS experienced a CVD event, and 835 (430 women) died. In multivariable-adjusted Cox regression, serum BDNF was associated inversely with CVD risk (hazard ratio [HR] per 1-SD increase 0.88, 95% CI 0.80 to 0.97, P=0.01) and with mortality (HR 0.87, 95% CI 0.80 to 0.93, P=0.0002). SNP rs6265 was associated with BDNF concentrations (0.772 ng/mL increase per minor allele copy) in FHS. In CARDIoGRAM, SNP rs6265 was associated with CAD (odds ratio 0.957, 95% CI 0.923 to 0.992), a magnitude consistent with the predicted effect (HR per minor allele copy 0.99, 95% CI 0.98 to 1.0; P=0.06 for difference between predicted and observed effect). CONCLUSION: Higher serum BDNF is associated with a decreased risk of CVD and mortality. Mendelian randomization suggests a causal protective role of BDNF in the pathogenesis of CVD.

Synthesis and metabolic studies of 1α,2α,25-, 1α,4α,25- and 1α,4ß,25-trihydroxyvitamin D3.

Three different A-ring perhydroxylated trihydroxyvitamin D3 metabolites were synthesized from their appropriate A-ring precursors and CD-ring for their potential therapeutic applications. We first chemically synthesized 1α,2α,25-trihydroxyvitamin D3 [1α,2α,25(OH)3D3] to study its VDR binding affinity because this metabolite is a product of recombinant human CYP3A4 catalysis when 2α-(3-hydroxypropoxy)-1α,25-dihydroxyvitamin D3 (O2C3), a more potent vitamin D receptor (VDR) binder than 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], is used as the substrate. We found that this metabolite retained 27.3% of the VDR binding affinity compared to 1α,25(OH)2D3. The kcat/Km value of CYP24A1 for 1α,2α,25(OH)3D3 is 60% of that for 1α,25(OH)2D3. Since the biological activity and the metabolic fate of a naturally occurring C4-hydroxylated vitamin D2 metabolite found in the serum of rats treated with pharmacological doses of vitamin D2 have never been described, we next synthesized 1α,4α,25-trihydroxyvitamin D3 and its diastereoisomer, 1α,4ß,25-trihydroxyvitamin D3, to study their metabolism and biological activities. Both 4-hydroxylated isomers showed weaker VDR binding affinity than 1α,25(OH)2D3. Although either 4-hydroxylated isomer can be metabolized by CYP24A1 almost at the same level as 1α,25(OH)2D3, their metabolic patterns catalyzed by uridine 5'-diphosphoglucuronosyltransferase (UGT) are different; only the 4α-hydroxylated analog can be metabolized by UGT to produce a glucuronate conjugate. The results provide important information for the synthesis of new novel chemotherapeutic vitamin D analogs which would be less subjective to degradation and therefore more bioavailable than 1α,25(OH)2D3. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

The vitamin D analog, MART-10, represses metastasis potential via downregulation of epithelial-mesenchymal transition in pancreatic cancer cells.

Pancreatic cancer (PDA) is a devastating disease and there is no effective treatment available at present. To develop new regiments against PDA is urgently needed. Previously we have shown that vitamin D analog, MART-10 (19-nor-2α-(3-hydroxypropyl)-1α,25(OH)2D3), exerted potent antiproliferative effect on PDA in vitro and in vivo without causing hypercalcemia. Since metastasis is the major cause of PDA-related death, we therefore investigate the anti-metastasis effect of MART-10 on PDA. Our results showed that both 1α,25(OH)2D3 and MART-10 repressed migration and invasion of BxPC-3 and PANC cells with MART-10 much more potent than 1α,25(OH)2D3. 1α,25(OH)2D3 and MART-10 inhibited epithelial-mesenchymal transition (EMT) in pancreatic cancer cells through downregulation of Snail, Slug, and Vimentin expression in BxPC-3 and PANC cells. MART-10 further blocked cadherin switch (from E-cadherin to N-cadherin) in BxPC-3 cells. The F-actin synthesis in the cytoplasm of BxPC-3 cells was also repressed by 1α,25(OH)2D3 and MART-10 as determined by immunofluorescence stain. Both 1α,25(OH)2D3 and MART-10 decreased MMP-2 and -9 secretion in BxPC-3 cells as determined by western blot and zymography. Collectively, MART-10 should be deemed as a promising regimen against PDA.

Chemopreventive and chemotherapeutic effect of dietary supplementation of vitamin D on cholangiocarcinoma in a Chemical-Induced animal model.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer. Vitamin D, a pro-hormone, is getting popular due to its hormone-like functions after converted to its active form, 1α,25(OH)2D3. Here, we show that dietary supplementation with 6 IU/g of vitamin D greatly suppressed ICC initiation and progression without apparent toxicity in a chemically induced rat model. Microarray analysis of rat ICC tissues showed vitamin D supplementation modulated the expressions of several unique genes, including lipocalin 2 (Lcn2), confirmed by RT-qPCR and immunohistochemical (IHC) staining. Further, 53 of 80 human ICC specimens (66%) exhibited high LCN2 expression and LCN2 knockdown in SNU308 cells decreased cell growth and migration, suggesting LCN2 be an oncogene in human ICC. As human ICC SNU1079 cells were treated by 1α,25(OH)2D3, LCN2 expression and cell proliferation were attenuated. The downregulation of LCN2 expression was blunted when vitamin D receptor (VDR) was knocked down, implicating that the in vivo Lcn2 downregulation is a direct consequence of vitamin D supplementation Our results support the prevailing concept that vitamin D status is negatively associated with cancer incidence and mortality and suggest LCN2 may be a potential target against ICC. Further studies of application of vitamin D or its analog against ICC are warranted.