Hypoxia-inducible factor 1A inhibition overcomes castration resistance of prostate tumors.

Androgen deprivation therapy (ADT) is a cornerstone of prostate cancer (PCa) management. Although tumors initially regress, many progress to a hormone-independent state termed castration-resistant PCa (CRPC), for which treatment options are limited. We here report that the major luminal cell population in tumors of Pten(i)pe-/- mice, generated by luminal epithelial cell-specific deletion of the tumor suppressor PTEN after puberty, is castration-resistant and that the expression of inflammation and stemness markers is enhanced in persistent luminal cells. In addition, hypoxia-inducible factor 1 (HIF1) signaling, which we have previously demonstrated to be induced in luminal cells of Pten(i)pe-/- mice and to promote malignant progression, is further activated. Importantly, we show that genetic and pharmacological inhibition of HIF1A sensitizes Pten-deficient prostatic tumors to castration and provides durable therapeutic responses. Furthermore, HIF1A inhibition induces apoptotic signaling in human CRPC cell lines. Therefore, our data demonstrate that HIF1A in prostatic tumor cells is a critical factor that enables their survival after ADT, and identify it as a target for CRPC management.

Hypoxia-mediated stabilization of HIF1A in prostatic intraepithelial neoplasia promotes cell plasticity and malignant progression.

Prostate cancer (PCa) is a leading cause of cancer-related deaths. The slow evolution of precancerous lesions to malignant tumors provides a broad time frame for preventing PCa. To characterize prostatic intraepithelial neoplasia (PIN) progression, we conducted longitudinal studies on Pten(i)pe-/- mice that recapitulate prostate carcinogenesis in humans. We found that early PINs are hypoxic and that hypoxia-inducible factor 1 alpha (HIF1A) signaling is activated in luminal cells, thus enhancing malignant progression. Luminal HIF1A dampens immune surveillance and drives luminal plasticity, leading to the emergence of cells that overexpress Transglutaminase 2 (TGM2) and have impaired androgen signaling. Elevated TGM2 levels in patients with PCa are associated with shortened progression-free survival after prostatectomy. Last, we show that pharmacologically inhibiting HIF1A impairs cell proliferation and induces apoptosis in PINs. Therefore, our study demonstrates that HIF1A is a target for PCa prevention and that TGM2 is a promising prognostic biomarker of early relapse after prostatectomy.

Single-cell analyses unravel cell type-specific responses to a vitamin D analog in prostatic precancerous lesions.

Epidemiological data have linked vitamin D deficiency to the onset and severity of various cancers, including prostate cancer, and although in vitro studies have demonstrated anticancer activities for vitamin D, clinical trials provided conflicting results. To determine the impact of vitamin D signaling on prostatic precancerous lesions, we treated genetically engineered Pten(i)pe-/- mice harboring prostatic intraepithelial neoplasia (PIN) with Gemini-72, a vitamin D analog with reported anticancer activities. We show that this analog induces apoptosis in senescent PINs, normalizes extracellular matrix remodeling by stromal fibroblasts, and reduces the prostatic infiltration of immunosuppressive myeloid-derived suppressor cells. Moreover, single-cell RNA-sequencing analysis demonstrates that while a subset of luminal cells expressing Krt8, Krt4, and Tacstd2 (termed luminal-C cells) is lost by such a treatment, antiapoptotic pathways are induced in persistent luminal-C cells. Therefore, our findings delineate the distinct responses of PINs and the microenvironment to Gemini-72, and shed light on mechanisms that limit treatment's efficacy.

Ascorbate kills breast cancer cells by rewiring metabolism via redox imbalance and energy crisis.

The idea to use megadoses of ascorbate (vitamin C) for cancer treatment has recently been revived. Despite clear efficacy in animal experimentation, our understanding of the cellular and molecular mechanisms of this treatment is still limited and suggests a combined oxidative and metabolic mechanism behind the selective cytotoxicity of ascorbate towards cancerous cells. To gain more insight into the cellular effects of high doses of ascorbate, we performed a detailed analysis of metabolic changes and cell survival of both luminal and basal-like breast cancer cells treated with ascorbate and revealed a distinctive metabolic shift virtually reversing the Warburg effect and triggering a severe disruption of redox homeostasis. High doses of ascorbate were cytotoxic against MCF7 and MDA-MB231 cells representing luminal and basal-like breast cancer phenotypes. Cell death was dependent on ascorbate-induced oxidative stress and accumulation of ROS, DNA damage, and depletion of essential intracellular co-factors including NAD+/NADH, associated with a multifaceted metabolic rewiring. This included a sharp disruption of glycolysis at the triose phosphate level, a rapid drop in ATP levels, and redirection of metabolites toward lipid droplet accumulation and increased metabolites and enzymatic activity in the pentose phosphate pathway (PPP). High doses of ascorbate also inhibited the TCA cycle and increased oxygen consumption. Together the severe disruptions of the intracellular metabolic homeostasis on multiple levels "redox crisis and energetic catastrophe" consequently trigger a rapid irreversible cell death.

A Multitarget Gold(I) Complex Induces Cytotoxicity Related to Aneuploidy in HCT-116 Colorectal Carcinoma Cells.

A novel alkynyl phosphane gold(I) complex (trimethylphosphane)(3-(1,3-dimethylxanthine-7-yl)prop-1-yn-1-yl)gold(I) 1 displayed mutiple biological activites including selective proliferation inhibitory, anti-metastatic, and anti-angiogenic effects. The complex also induced effects related to aneuploidy in HCT-116 colon carcinoma cells, which might be mainly ascribed to the dysfunction of mitochondrial bioenergetics and downregulation of glycolysis. Induction of aneuploidy beyond a critical level can provide an effective strategy to target cancer, in particular colorectal tumours with a low tolerance of aneuploidy, and could be of relevance for 1 and other metallodrugs.

p53-Dependent Anti-Proliferative and Pro-Apoptotic Effects of a Gold(I) N-Heterocyclic Carbene (NHC) Complex in Colorectal Cancer Cells.

The tumor suppressor p53 has a diverse mutational profile in human malignancies, which is known to influence the potency of various chemotherapeutics, such as platins and anti-metabolites. However, the impact of the mutations in the TP53 gene (coding for p53) on the anti-cancer efficacy of gold complexes remains incompletely understood. We therefore investigated the anti-tumor properties of a gold(I) N-heterocyclic carbene (NHC) complex-termed MC3-in human colorectal cancer (CRC) cell lines encompassing three different p53 variations: HCT116 wild-type (WT), HCT116 p53-/-, and HT-29 (mutant; R273H). MC3 treatment induced intracellular reactive oxygen species (ROS) levels, and p21 expression, leading to cell cycle arrest in all cell lines, regardless of their p53 status. The pro-apoptotic response, however, was found to occur in a p53-dependent manner, with WT p53 harboring cells showing the highest responsiveness. Additionally, p73, which was speculated to substitute p53 in p53-deficient cells, was found to be markedly reduced with MC3 treatment in all the cell lines and knocking down its levels did not impact MC3's anti-tumor effects in HCT116 p53-/- cells. Collectively, our results suggest that this small molecule has anti-cancer properties in the context of deficient or mutant p53 and may therefore have chemotherapeutic potential for clinical application.

In vitro metabolic activation of vitamin D3 by using a multi-compartment microfluidic liver-kidney organ on chip platform.

Organ-on-chip platforms provide models that allow the representation of human physiological processes in cell-based miniaturized systems. Potential pre-clinical applications include drug testing and toxicity studies. Here we describe the use of a multi-compartment micro-fluidic chip to recapitulate hepatic vitamin D metabolism (vitamin D to 25-hydroxyvitamin D) and renal bio-activation (25-hydroxyvitamin D to 1,25-dihydroxyvitamin D) in humans. In contrast to cultivation in conventional tissue culture settings, on-chip cultivation of HepG2 and RPTEC cells in interconnected chambers, used to mimic the liver and kidneys, respectively, resulted in the enhanced expression of vitamin D metabolizing enzymes (CYP2R1, CYP27B1 and CYP24A1). Pump-driven flow of vitamin D3-containing medium through the microfluidic chip produced eluate containing vitamin D3 metabolites. LC-MSMS showed a strong accumulation of 25-hydroxyvitamin D. The chip eluate induced the expression of differentiation markers in HL-60 (acute myeloid leukemia) cells, assessed by qPCR and FACS analysis, in a manner similar to treatment with reference standards indicating the presence of fully activated 1,25 dihydroxyvitamin D, although the latter was not detected in the eluate by LC-MSMS. Interestingly, 25-hydroxyvitamin D by itself led to weak activation of HL-60 cells suggesting that 25-hydroxyvitamin D is also an active metabolite. Our experiments demonstrate that complex metabolic interactions can be reconstructed outside the human body using dedicated organ-on-chip platforms. We therefore propose that such systems may be used to mimic the in vivo metabolism of various micronutrients and xenobiotics.

Activation of pro-survival metabolic networks by 1,25(OH)2D3 does not hamper the sensitivity of breast cancer cells to chemotherapeutics.

BACKGROUND: We have previously identified 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the bioactive form of vitamin D3, as a potent regulator of energy-utilization and nutrient-sensing pathways in prostate cancer cells. In the current study, we investigated the effects of 1,25(OH)2D3 on breast cancer (BCa) cell metabolism using cell lines representing distinct molecular subtypes, luminal (MCF-7 and T-47D), and triple-negative BCa (MDA-MB-231, MDA-MB-468, and HCC-1143). METHODS: 1,25(OH)2D3's effect on BCa cell metabolism was evaluated by employing a combination of real-time measurements of glycolysis/oxygen consumption rates using a biosensor chip system, GC/MS-based metabolomics, gene expression analysis, and assessment of overall energy levels. The influence of treatment on energy-related signaling molecules was investigated by immunoblotting. RESULTS: We show that 1,25(OH)2D3 significantly induces the expression and activity of the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (G6PD) in all BCa cell lines, however differentially influences glycolytic and respiratory rates in the same cells. Although 1,25(OH)2D3 treatment was found to induce seemingly anti-oxidant responses in MCF-7 cells, such as increased intracellular serine levels, and reduce the expression of its putative target gene thioredoxin-interacting protein (TXNIP), intracellular reactive oxygen species levels were found to be elevated. Serine accumulation in 1,25(OH)2D3-treated cells was not found to hamper the efficacy of chemotherapeutics, including 5-fluorouracil. Detailed analyses of the nature of TXNIP's regulation by 1,25(OH)2D3 included genetic and pharmacological inhibition of signaling molecules and metabolic enzymes including AMP-activated protein kinase and G6PD, as well as by studying the ITCH (E3 ubiquitin ligase)-TXNIP interaction. While these investigations demonstrated minimal involvement of such pathways in the observed non-canonical regulation of TXNIP, inhibition of estrogen receptor (ER) signaling by tamoxifen mirrored the reduction of TXNIP levels by 1,25(OH)2D3, demonstrating that the latter's negative regulation of ER expression is a potential mechanism of TXNIP modulation. CONCLUSIONS: Altogether, we propose that regulation of energy metabolism contributes to 1,25(OH)2D3's anti-cancer effects and that combining 1,25(OH)2D3 with drugs targeting metabolic networks in tumor cells may lead to synergistic effects.

Expression of TXNIP in Cancer Cells and Regulation by 1,25(OH)2D3: Is It Really the Vitamin D3 Upregulated Protein?

Thioredoxin-interacting protein (TXNIP) was originally identified in HL-60 cells as the vitamin D3 upregulated protein 1, and is now known to be involved in diverse cellular processes, such as maintenance of glucose homeostasis, redox balance, and apoptosis. Besides the initial characterization, little is known about if and how 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induces TXNIP expression. We therefore screened multiple cancerous cell lines of different tissue origins, and observed induction, repression, or no change in TXNIP expression in response to 1,25(OH)2D3. In-depth analyses on HL-60 cells revealed a rapid and transient increase in TXNIP mRNA levels by 1,25(OH)2D3 (3-24 h), followed by a clear reduction at later time points. Furthermore, a strong induction in protein levels was observed only after 96 h of 1,25(OH)2D3 treatment. Induction of TXNIP expression by 1,25(OH)2D3 was found to be dependent on the availability of glucose in the culture medium, as well as the presence of a functional glucose transport system, indicating an inter-dependence of 1,25(OH)2D3 actions and glucose-sensing mechanisms. Moreover, the inhibition of de novo protein synthesis by cycloheximide reduced TXNIP half-life in 24 h, but not in 96 h-1,25(OH)2D3-treated HL-60 cells, demonstrating a possible influence of 1,25(OH)2D3 on TXNIP stability in long-term treatment.

Vitamin D as a Novel Regulator of Tumor Metabolism: Insights on Potential Mechanisms and Implications for Anti-Cancer Therapy.

1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the bioactive form of vitamin D, has been shown to possess significant anti-tumor potential. While most studies so far have focused on the ability of this molecule to influence the proliferation and apoptosis of cancer cells, more recent data indicate that 1,25(OH)2D3 also impacts energy utilization in tumor cells. In this article, we summarize and review the evidence that demonstrates the targeting of metabolic aberrations in cancers by 1,25(OH)2D3, and highlight potential mechanisms through which these effects may be executed. We shed light on the ability of this molecule to regulate metabolism-related tumor suppressors and oncogenes, energy- and nutrient-sensing pathways, as well as cell death and survival mechanisms such as autophagy.

1,25(OH)2D3 disrupts glucose metabolism in prostate cancer cells leading to a truncation of the TCA cycle and inhibition of TXNIP expression.

Prostate cell metabolism exhibits distinct profiles pre- and post-malignancy. The malignant metabolic shift converts prostate cells from "citrate-producing" to "citrate-oxidizing" cells, thereby enhancing glucose metabolism, a phenotype that contrasts classical tumoral Warburg metabolism. An on-line biosensor chip system (BIONAS 2500) was used to monitor metabolic changes (glycolysis and respiration) in response to the putative anti-cancer nutraceutical 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], in different prostate cancer (PCa) cell lines (LNCaP, VCaP, DU145 and PC3). LNCaP cells exhibited profound metabolic responsiveness to the treatment and thus extensive analysis of metabolism-modulating effects of 1,25(OH)2D3 were performed, including mRNA expression analysis of key metabolic genes (e.g. GLUT1 and PDHK1), analysis of TCA cycle metabolites, glucose uptake/consumption measurements, ATP production, and mitochondrial biogenesis/activity. Altogether, data demonstrate a vivid disruption of glucose metabolism by 1,25(OH)2D3, illustrated by a decreased glucose uptake and an accumulation of citrate/isocitrate due to TCA cycle truncation. Depletion of glycolytic intermediates led to a consistent decrease in TXNIP expression in response to 1,25(OH)2D3, an effect that coincided with the activation of AMPK signaling and a reduction in c-MYC expression. Reduction in TXNIP levels in response to 1,25(OH)2D3 was rescued by an AMPK signaling inhibitor and mimicked by a MYC inhibitor highlighting the possible involvement of both pathways in mediating 1,25(OH)2D3's metabolic effects in PCa cells. Furthermore, pharmacological and genetic modulation of the androgen receptor showed similar and disparate effects on metabolic parameters compared to 1,25(OH)2D3 treatment, highlighting the AR-independent nature of 1,25(OH)2D3's metabolism-modulating effects.

Differences in p53 status significantly influence the cellular response and cell survival to 1,25-dihydroxyvitamin D3-metformin cotreatment in colorectal cancer cells.

Mutations in the tumor suppressor p53 are highly prevalent in cancers and are known to influence the sensitivity of cells to various chemotherapeutics including the anti-cancer candidates 1,25-dihydrovitamin D3 [1,25D3] and metformin. Previous studies have demonstrated additive/synergistic anti-cancer effects of the 1,25D3-metformin combination in different models, however, the influence of p53 status on the efficacy of this regimen has not been investigated. The CRC colorectal cancer (CRC) cell lines HCT116 wild-type (wt), HCT116 p53-/-, and HT-29 (mutant; R273H) were employed, covering three different p53 variations. Synergistic effects of the combination were confirmed in all cell lines using MTT assay. Detailed evaluation of the combination's effects was performed, including on-line measurements of cellular metabolism (glycolysis/respiration) using a biosensor chip system, analyses of mitochondrial activity (membrane potential and ATP/ROS production), mRNA expression analysis of WNT/ß-catenin pathway players, and a comprehensive proteomic screen using immunoblotting and ELISA microarrays. AMPK signaling was found to be more strongly induced in response to all treatments in HCT116 wt cells compared to other cell lines, an observation that was coupled to a stronger accumulation of intracellular ROS in response to metformin/combination, and finally an induction in autophagy, depicted by an increase in LC3II:LC3I ratio in combination-treated cells compared to mono-treatments. An induction in apoptotic signaling was observed in the other cell lines in response to the combination, illustrated by a decrease in expression of pro-survival Bcl2 family members. P53 status impacts cellular responses to the combination but does not hamper its anti-proliferative synergy.

Effects of 1,25(OH)2D3 on Cancer Cells and Potential Applications in Combination with Established and Putative Anti-Cancer Agents.

The diverse effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the bio-active form of vitamin D, on cancer cell metabolism and proliferation has made it an interesting candidate as a supporting therapeutic option in cancer treatment. An important strategy in cancer therapy is the use of combination chemotherapy to overcome drug resistance associated with numerous anti-cancer agents and to provide better means of avoiding undesirable side effects. This complex strategy is widely adopted by oncologists and several established "cocktails" of chemotherapeutics are routinely administered to cancer patients. Among the principles followed in designing such treatment regimens is the use of drugs with different mechanisms of action to overcome the issue of tumor heterogeneity and to evade resistance. In light of the profound and diverse effects of 1,25(OH)2D3 reported by in vitro and in vivo studies, we discuss how these effects could support the use of this molecule in combination with "classical" cytotoxic drugs, such as platins and anti-metabolites, for the treatment of solid and hematological tumors. We also examine recent evidence supporting synergistic activities with other promising anti-cancer drug candidates, and postulate mechanisms through which 1,25(OH)2D3 may help evade chemoresistance.

A multi-target caffeine derived rhodium(i) N-heterocyclic carbene complex: evaluation of the mechanism of action.

A rhodium(i) and a ruthenium(ii) complex with a caffeine derived N-heterocyclic carbene (NHC) ligand were biologically investigated as organometallic conjugates consisting of a metal center and a naturally occurring moiety. While the ruthenium(ii) complex was largely inactive, the rhodium(i) NHC complex displayed selective cytotoxicity and significant anti-metastatic and in vivo anti-vascular activities and acted as both a mammalian and an E. coli thioredoxin reductase inhibitor. In HCT-116 cells it increased the reactive oxygen species level, leading to DNA damage, and it induced cell cycle arrest, decreased the mitochondrial membrane potential, and triggered apoptosis. This rhodium(i) NHC derivative thus represents a multi-target compound with promising anti-cancer potential.

Alkynyl gold(I) phosphane complexes: Evaluation of structure-activity-relationships for the phosphane ligands, effects on key signaling proteins and preliminary in-vivo studies with a nanoformulated complex.

Gold alkynyl complexes with phosphane ligands of the type (alkynyl)Au(I)(phosphane) represent a group of bioorganometallics, which has only recently been evaluated biologically in more detail. Structure-activity-relationship studies regarding the residues of the phosphane ligand (P(Ph)3, P(2-furyl)3, P(DAPTA)3, P(PTA)3, P(Et)3, P(Me)3) of complexes with an 4-ethynylanisole alkyne ligand revealed no strong differences concerning cytotoxicity. However, a relevant preference for the heteroatom free alkyl/aryl residues concerning inhibition of the target enzyme thioredoxin reductase was evident. Complex 1 with the triphenylphosphane ligand was selected for further studies, in which clear effects on cell morphology were monitored by time-lapse microscopy. Effects on cellular signaling were determined by ELISA microarrays and showed a significant induction of the phosphorylation of ERK1 (extracellular signal related kinase 1), ERK2 and HSP27 (heat shock protein 27) in HT-29 cells. Application of 1 in-vivo in a mouse xenograft model was found to be challenging due to the low solubility of the complex and required a formulation strategy based on a peanut oil nanoemulsion.

Genetic variation in vitamin D receptor gene (Fok1:rs2228570) is associated with risk of coronary artery disease.

OBJECTIVE: The Fok1 polymorphism (rs2228570) in vitamin D receptor gene appears to be the only polymorphism influencing size of translated protein. Investigations into its association with coronary artery disease (CAD) are sparse. METHODS: Male patients (n = 98) with verified CAD were recruited alongside age- and sex-matched controls (n = 55). Genotyping was performed by PCR-RFLP and plasma 25-Hydroxyvitamin D levels were assessed by HPLC-UV. RESULTS: The C-variant (mutant) was predominantly expressed in patients compared to controls (68.9% versus 55.5%; p = 0.025). The observed genotypes were not associated with 25-Hydroxyvitamin D levels. CONCLUSION: This study presents Fok1 polymorphism as a potential genetic marker for CAD.

Vitamin D receptor gene polymorphisms (TaqI and ApaI) in relation to 25-hydroxyvitamin D levels and coronary artery disease incidence.

CONTEXT/OBJECTIVE: Previous studies have illustrated the association of the ApaI and TaqI polymorphisms of the vitamin D receptor gene, located in non-coding and coding regions, respectively, with diseases such as cancer and cardiovascular disease; however, investigating such association in Egyptian patients with coronary artery disease (CAD) has never been formerly attempted. MATERIALS AND METHODS: Male patients (n = 137), 35-50 years of age, with verified CAD, were recruited alongside age- and sex-matched controls (n = 58). Genotyping and 25-hydroxyvitamin D [25(OH)D] measurement were performed by polymerase chain reaction RFLP and HPLC, respectively. RESULTS: Comparison of the genotypic distribution of both the TaqI and ApaI polymorphisms between patients and controls yielded insignificant results (p = 0.55 and 0.7, respectively). Comparison of the allelic distribution of both polymorphisms also yielded insignificant results. The TaqI polymorphism was not found to predict 25(OH)D levels, whereas the wild-type genotype of the ApaI polymorphism was associated with greater levels of 25(OH)D (p = 0.02), taking all subjects into consideration. DISCUSSION/CONCLUSION: This study presents the ApaI and TaqI polymorphisms as non-influencing players in the pathogenesis of CAD in Egyptian males and the ability of only the ApaI polymorphism to predict 25(OH)D levels, thus warranting further investigations of the triangular relationship between the polymorphisms, 25(OH)D and CAD incidence.

Investigating the cardio-protective abilities of supplemental L-arginine on parameters of endothelial function in a hypercholesterolemic animal model.

Endothelial dysfunction is now widely recognized as an early marker of cardiovascular disease, making its treatment, or complete avoidance, an emerging, interesting therapeutic target. This study investigated the ability of the highly intriguing amino acid L-arginine to influence endothelial function. Its therapeutic potential is also compared to that of known cardiovascular medications, namely nitroglycerin [a nitric oxide (NO) donor] and enalapril [an angiotensin-converting enzyme (ACE) inhibitor]. Fifty male New Zealand rabbits were included in the study, divided into 5 equal groups: control, hypercholesterolemia (untreated), hypercholesterolemia (+L-arginine), hypercholesterolemia (+enalapril), and hypercholesterolemia (+nitroglycerin). Biochemical investigations included measurement of circulating NOx, malondialdehyde (MDA), and lipid profile markers, as well as dimethylarginine dimethylaminohydrolase (DDAH) and ACE activities. Furthermore, aortic ACE activity and blood platelet aggregation were estimated. A histopathological examination and intimal thickness measurement were also conducted. Compared to the untreated hypercholesterolemic group, all agents were capable of positively influencing MDA levels, platelet aggregation and intimal thickness; however, only the L-arginine group was capable of beneficially and significantly altering both NOx levels and serum and aortic ACE activities. No agents were capable of modulating serum DDAH activity inhibited by hypercholesterolemia. Based on the results of this study, L-arginine appears to be a novel cardio-protective agent, illustrated by its ability to ameliorate the deleterious effects of hypercholesterolemia on endothelial function, in a manner comparable to, and sometimes more potent than, commonly used cardiovascular medications.

Triangular relationship between single nucleotide polymorphisms in the CYP2R1 gene (rs10741657 and rs12794714), 25-hydroxyvitamin d levels, and coronary artery disease incidence.

OBJECTIVE: To investigate the relationship between the rs10741657 and rs12794714 polymorphisms in the CYP2R1 gene, 25(OH)D levels, and coronary artery disease (CAD) incidence. METHODS: In total, 134 male patients with verified CAD were recruited, alongside 109 age- and sex-matched controls. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism, using the corresponding restriction enzyme for each polymorphism, whereas 25(OH)D levels were analyzed by HPLC-UV. RESULTS: 25(OH)D levels were significantly lower in patients. The genotypic and allelic distributions of the rs10741657 polymorphism were significantly different between patients and controls, whereas insignificant results were obtained for the rs12794714 polymorphism. Furthermore, rs10741657, but not rs12794714, predicted 25(OH)D levels. CONCLUSION: The rs10741657 polymorphism is a novel genetic marker for CAD.

Interplay of vitamin D and nitric oxide in post-menopausal knee osteoarthritis.

AIM: Mounting evidence has presented nitric oxide (NO) and vitamin D (vitD) as having independently complex roles in osteoarthritis (OA). However, a mechanistic or an observational connection between them has never been investigated in the disease. This study investigates the correlation between circulating 25-hydroxyvitamin D [25(OH)D] and total NO as nitrate/nitrite (NO x ) in patients with knee OA. METHODS: The recruited subjects comprised 36 post-menopausal women with knee OA, ages 50-60 years, as well as 10 healthy males, 20-30 years of age. 25(OH)D and NO x levels were determined using high-performance liquid chromatography and spectrophotometrically using Griess reaction, respectively. RESULTS: The mean (SEM) 25(OH)D and NO x concentrations of OA patients were 25.0 (1.6) ng/mL and 32.45 (2.18) µM, respectively, and 35.4 (2.1) ng/mL and 25.49 (2.23) µM, respectively, for controls. Comparison of mean 25(OH)D and NO x concentrations of OA patients and controls yielded significant results (P = 0.001 and 0.034, respectively). NO notably decreased with decreasing 25(OH)D concentration in patients. However, significant results in terms of mean NO x concentration were observed in the comparison of normal and deficient vitD OA groups (P = 0.048). CONCLUSION: Results suggest that vitD increases NO production and inducible NO synthase expression in osteoarthritic chondrocytes possibly leading to a protective effect.