Vitamin D Analogs Regulate the Vitamin D System and Cell Viability in Ovarian Cancer Cells

Background: Ovarian cancer (OC) is one of the most lethal cancers in women. The active form of vitamin D₃, 1,25-dihydroxyvitamin D₃ (1,25D₃, calcitriol) has anticancer activity in several cancers, including ovarian cancer, but the required pharmacological doses may cause hypercalcemia. We hypothesized that newly developed, low calcemic, vitamin D analogs (an1,25Ds) may be used as anticancer agents instead of calcitriol in ovarian cancer cells. Methods: We used two patient-derived high-grade serous ovarian cancer (HGSOC) cell lines with low (13781) and high (14433) mRNA expression levels of the gene encoding 1,25-dihydroxyvitamin D₃ 24-hydroxylase CYP24A1, one of the main target genes of calcitriol. We tested the effect of calcitriol and four structurally related series of an1,25Ds (PRI-1906, PRI-1907, PRI-5201, PRI-5202) on cell number, viability, the expression of CYP24A1, and the vitamin D receptor (VDR). Results: CYP24A1 mRNA expression increased in a concentration-dependent manner after treatment with all compounds. In both cell lines, after 4 h, PRI-5202 was the most potent analog (in 13781 cells: EC₅₀ = 2.98 ± 1.10 nmol/L, in 14433 cells: EC₅₀ = 0.92 ± 0.20 nmol/L), while PRI-1907 was the least active one (in 13781 cells: EC₅₀ = n/d, in 14433 cells: EC₅₀ = n/d). This difference among the analogs disappeared after 5 days of treatment. The 13781 cells were more sensitive to the an1,25Ds compared with 14433 cells. The an1,25Ds increased nuclear VDR levels and reduced cell viability, but only in the 13781 cell line. Conclusions: The an1,25Ds had different potencies in the HGSOC cell lines and their efficacy in increasing CYP24A1 expression was cell line- and chemical structure-dependent. Therefore, choosing sensitive cancer cell lines and further optimization of the analogs’ structure might lead to new treatment options against ovarian cancer.     

Development of In Vitro and In Vivo Evaluation Systems for Vitamin D Derivatives and Their Application to Drug Discovery

We have developed an in vitro system to easily examine the affinity for vitamin D receptor (VDR) and CYP24A1-mediated metabolism as two methods of assessing vitamin D derivatives. Vitamin D derivatives with high VDR affinity and resistance to CYP24A1-mediated metabolism could be good therapeutic agents. This system can effectively select vitamin D derivatives with these useful properties. We have also developed an in vivo system including a Cyp27b1-gene-deficient rat (a type I rickets model), a Vdr-gene-deficient rat (a type II rickets model), and a rat with a mutant Vdr (R270L) (another type II rickets model) using a genome editing method. For Cyp27b1-gene-deficient and Vdr mutant (R270L) rats, amelioration of rickets symptoms can be used as an index of the efficacy of vitamin D derivatives. Vdr-gene-deficient rats can be used to assess the activities of vitamin D derivatives specialized for actions not mediated by VDR. One of our original vitamin D derivatives, which displays high affinity VDR binding and resistance to CYP24A1-dependent metabolism, has shown good therapeutic effects in Vdr (R270L) rats, although further analysis is needed.     

Expression of Vitamin D Receptor (VDR) Positively Correlates with Survival of Urothelial Bladder Cancer Patients

Vitamin D3 shows tumoristatic and anticancer effects by acting through the vitamin D receptor (VDR), while hydroxylation of 25-hydroxyvitamin D3 at position 1α by CYP27B1 is an essential step in its activation. The expression of both the VDR and CYP27B1 has been found in many normal and cancer tissues, but there is a lack of information about its expression in human bladder cancers. The aim of the present research was to examine whether the expression of the VDR and CYP27B1 in bladder cancer was related to the prognostic markers and disease outcome. We analyzed VDR and CYP27B1 in samples of tumor and normal tissues obtained from 71 urinary bladder cancer patients. The highest VDR immunostaining was found in normal epithelium and was significantly lower in bladder cancer cells (p < 0.001 with Mann–Whitney U test). VDR expression was lowest in more advanced (pT2b–pT4) (p = 0.005 with Mann–Whitney U test) and metastasizing cancers (p < 0.05 and p = 0.004 with Mann–Whitney U test for nuclear and cytoplasmic VDR immunostaining, respectively). The lack of cytoplasmic and nuclear VDR was also related to shorter overall survival (for cytoplasmic VDR immunolocalization 13.3 vs. 55.3 months of survival, HR = 1.92, p = 0.04 and for nuclear VDR immunostaining 13.5 vs. 55.3 months of survival, HR = 2.47, p = 0.002 with Mantel-Cox test). In cases with the lack of high cytoplasmic VDR staining the non-classic differentiations (NDs) was observed in higher percentage of tumor area. CYP27B1 expression was lower in cancer cells than in normal epithelial cells (p = 0.03 with Mann–Whitney U test), but its expression did not correlate with tumor stage (pT), metastasizing, grade, mitotic activity or overall survival. In conclusion, expression of the VDR and CYP27B1 are deregulated in urothelial bladder cancers. Although our results showing a relationship between the decreased VDR expression and prognostic markers and survival time indicate potential usefulness of VDR as a new indicator of a poorer prognosis, further studies are needed in different patient cohorts by independent groups to validate this hypothesis. We also suggest that vitamin D-based therapies may represent an adjuvant strategy in treatment for bladder cancers expressing VDR.     

CYP24A1 Expression Inversely Correlates with Melanoma Progression: Clinic-Pathological Studies

The major role of 24-hydroxylase (CYP24A1) is to maintain 1,25-dihydroxyvitamin D3 (1,25(OH)₂D3) homeostasis. Recently, it has been discovered that CYP24A1 also catalyses the hydroxylation of 20(OH)D3, producing dihydroxy-derivatives that show very effective antitumorigenic activities. Previously we showed a negative correlation of vitamin D receptor (VDR) and CYP27B1 expression with progression, aggressiveness and overall or disease-free survivals of skin melanomas. Therefore, we analyzed CYP24A1 expression in relation to clinicopathomorphological features of nevi, skin melanomas and metastases. In melanocytic tumors, the level of CYP24A1 was higher than in the normal epidermis. The statistically highest mean CYP24A1 level was found in nevi and early stage melanomas. With melanoma progression, CYP24A1 levels decreased and in advanced stages were comparable to the normal epidermis and metastases. Furthermore, the CYP24A1 expression positively correlated with VDR and CYP27B1, and negatively correlated with mitotic activity. Lower CYP24A1 levels correlated with the presence of ulceration, necrosis, nodular type and amelanotic phenotypes. Moreover, a lack of detectable CYP24A1 expression was related to shorter overall and disease-free survival. In conclusion, the local vitamin D endocrine system affects melanoma behavior and an elevated level of CYP24A1 appears to have an important impact on the formation of melanocytic nevi and melanomagenesis, or progression, at early stages of tumor development.     

Vitamin D3 Treatment Alters Thyroid Functional Morphology in Orchidectomized Rat Model of Osteoporosis

Vitamin D plays an essential role in prevention and treatment of osteoporosis. Thyroid hormones, in addition to vitamin D, significantly contribute to regulation of bone remodeling cycle and health. There is currently no data about a possible connection between vitamin D treatment and the thyroid in the context of osteoporosis. Middle-aged Wistar rats were divided into: sham operated (SO), orchidectomized (Orx), and cholecalciferol-treated orchidectomized (Orx + Vit. D₃; 5 µg/kg b.m./day during three weeks) groups (n = 6/group). Concentration of 25(OH)D in serum of the Orx + Vit. D₃ group increased 4 and 3.2 times (p < 0.0001) respectively, compared to Orx and SO group. T_4, TSH, and calcitonin in serum remained unaltered. Vit. D₃ treatment induced changes in thyroid functional morphology that indicate increased utilization of stored colloid and release of thyroid hormones in comparison with hormone synthesis, to maintain hormonal balance. Increased expression of nuclear VDR (p < 0.05) points to direct, TSH independent action of Vit. D on thyrocytes. Strong CYP24A1 immunostaining in C cells suggests its prominent expression in response to Vit. D in this cell subpopulation in orchidectomized rat model of osteoporosis. The indirect effect of Vit. D on bone, through fine regulation of thyroid function, is small.     

Biological Evaluation of Double Point Modified Analogues of 1,25-Dihydroxyvitamin D2 as Potential Anti-Leukemic Agents

Structurally similar double-point modified analogues of 1,25-dihydroxyvitamin D₂ (1,25D₂) were screened in vitro for their pro-differentiating activity against the promyeloid cell line HL60. Their affinities towards human full length vitamin D receptor (VDR) and metabolic stability against human vitamin D 24-hydroxylase (CYP24A1) were also tested. The analogues (PRI-1730, PRI-1731, PRI-1732, PRI-1733 and PRI-1734) contained 5,6-trans modification of the A-ring and of the triene system, additional hydroxyl or unsaturation at C-22 in the side chain and reversed absolute configuration (24-epi) at C-24 of 1,25D₂. As presented in this paper, introduction of selected structural modifications simultaneously in two distinct parts of the vitamin D molecule resulted in a divergent group of analogues. Analogues showed lower VDR affinity in comparison to that of the parent hormones, 1,25D₂ and 1,25D₃, and they caused effective HL60 cell differentiation only at high concentrations of 100 nM and above. Unexpectedly, introducing of a 5,6-trans modification combined with C-22 hydroxyl and 24-epi configuration switched off entirely the cell differentiation activity of the analogue (PRI-1734). However, this analogue remained a moderate substrate for CYP24A1, as it was metabolized at 22%, compared to 35% for 1,25D₂. Other analogues from this series were either less (12% for PRI-1731 and PRI-1733) or more (52% for PRI-1732) resistant to the enzymatic deactivation. Although the inactive analogue PRI-1734 failed to show VDR antagonism, when tested in HL60 cells, its structure might be a good starting point for our design of a vitamin D antagonist.     

Alzheimer’s Disease-Associated SNP rs708727 in SLC41A1 May Increase Risk for Parkinson’s Disease: Report from Enlarged Slovak Study

SLC41A1 (A1) SNPs rs11240569 and rs823156 are associated with altered risk for Parkinson’s disease (PD), predominantly in Asian populations, and rs708727 has been linked to Alzheimer’s disease (AD). In this study, we have examined a potential association of the three aforementioned SNPs and of rs9438393, rs56152218, and rs61822602 (all three lying in the A1 promoter region) with PD in the Slovak population. Out of the six tested SNPs, we have identified only rs708727 as being associated with an increased risk for PD onset in Slovaks. The minor allele (A) in rs708727 is associated with PD in dominant and completely over-dominant genetic models (OR_D = 1.36 (1.05–1.77), p = 0.02, and OR_COD = 1.34 (1.04–1.72), p = 0.02). Furthermore, the genotypic triplet GG_(rs708727) + AG_(rs823156) + CC_(rs61822602) might be clinically relevant despite showing a medium (h ≥ 0.5) size difference (h = 0.522) between the PD and the control populations. RandomForest modeling has identified the power of the tested SNPs for discriminating between PD-patients and the controls to be essentially zero. The identified association of rs708727 with PD in the Slovak population leads us to hypothesize that this A1 polymorphism, which is involved in the epigenetic regulation of the expression of the AD-linked gene PM20D1, is also involved in the pathoetiology of PD (or universally in neurodegeneration) through the same or similar mechanism as in AD.     

Variants of the Low Oxygen Sensors EGLN1 and HIF-1AN Associated with Acute Mountain Sickness

Two low oxygen sensors, Egl nine homolog 1 (EGLN1) and hypoxia-inducible factor 1-α inhibitor (HIF-1AN), play pivotal roles in the regulation of HIF-1α, and high altitude adaption may be involved in the pathology of acute mountain sickness (AMS). Here, we aimed to analyze single nucleotide polymorphisms (SNPs) in the untranslated regions of the EGLN1 and HIF-1AN genes and SNPs chosen from a genome-wide adaptation study of the Han Chinese population. To assess the association between EGLN1 and HIF-1AN SNPs and AMS in a Han Chinese population, a case–control study was performed including 190 patients and 190 controls. In total, thirteen SNPs were genotyped using the MassARRAY^® MALDI-TOF system. Multiple genetic models were tested; The Akaike’s information criterion (AIC) and Bayesian information criterion (BIC) values indicated that the dominant model may serve as the best-fit model for rs12406290 and rs2153364 of significant difference. However, these data were not significant after Bonferroni correction. No significant association was noted between AMS and rs12757362, rs1339894, rs1361384, rs2009873, rs2739513 or rs2486729 before and after Bonferroni correction. Further haplotype analyses indicated the presence of two blocks in EGLN1; one block consists of rs12406290-rs2153364, located upstream of the EGLN1 gene. Carriers of the “GG” haplotype of rs12406290-rs2153364 exhibited an increased risk of AMS after adjustments for age and smoking status. However, no significant association was observed among HIF-1AN 3''-untranslated region (3''-UTR) polymorphisms, haplotype and AMS. Our study indicates that variants in the EGLN1 5''-UTR influence the susceptibility to AMS in a Han Chinese population.     

Functional Analyses of Four CYP1A1 Missense Mutations Present in Patients with Atypical Femoral Fractures

Osteoporosis is the most common metabolic bone disorder and nitrogen-containing bisphosphonates (BP) are a first line treatment for it. Yet, atypical femoral fractures (AFF), a rare adverse effect, may appear after prolonged BP administration. Given the low incidence of AFF, an underlying genetic cause that increases the susceptibility to these fractures is suspected. Previous studies uncovered rare CYP1A1 mutations in osteoporosis patients who suffered AFF after long-term BP treatment. CYP1A1 is involved in drug metabolism and steroid catabolism, making it an interesting candidate. However, a functional validation for the AFF-associated CYP1A1 mutations was lacking. Here we tested the enzymatic activity of four such CYP1A1 variants, by transfecting them into Saos-2 cells. We also tested the effect of commonly used BPs on the enzymatic activity of the CYP1A1 forms. We demonstrated that the p.Arg98Trp and p.Arg136His CYP1A1 variants have a significant negative effect on enzymatic activity. Moreover, all the BP treatments decreased CYP1A1 activity, although no specific interaction with CYP1A1 variants was found. Our results provide functional support to the hypothesis that an additive effect between CYP1A1 heterozygous mutations p.Arg98Trp and p.Arg136His, other rare mutations and long-term BP exposure might generate susceptibility to AFF.     

Influence of Insulin Receptor Single Nucleotide Polymorphisms on Glycaemic Control and Formation of Anti-Insulin Antibodies in Diabetes Mellitus

Single nucleotide polymorphisms (SNPs) in insulin and insulin receptor genes may influence the interaction between the two molecules, as may anti-insulin antibodies (IAs), commonly found in patients with type 1 diabetes mellitus (T1D) or type 2 diabetes mellitus (T2D) treated with exogenous insulin. We examined the impact of two SNPs in the human insulin gene (INS), rs3842752 and rs689, and two in the insulin receptor gene (INSR) rs2245649 and rs2229429, on disease susceptibility, glycaemic control, and IAs formation in 100 T1D patients and 101 T2D patients treated with insulin. 79 individuals without diabetes were typed as healthy controls. The minor alleles of rs3842752 and rs689 in INS protected against T1D (OR: 0.50, p = 0.01 and OR: 0.44; p = 0.002, respectively). The minor alleles of both rs2245649 and rs2229429 in INSR were risk factors for poor glycaemic control (HbA1c ≥ 80 mmol/mol) in T1D (OR: 5.35, p = 0.009 and OR: 3.10, p = 0.01, respectively). Surprisingly, the minor alleles of rs2245649 and rs2229429 in INSR associated strongly with the absence of IAs in T1D (OR = 0.28, p = 0.008 and OR = 0.30, p = 0.002, respectively). In conclusion, the minor alleles of the investigated INS SNPs protect against T1D, and the minor alleles of the investigated INSR SNPs are associated with poor glycaemic control and the absence of IAs in T1D.     

SLCO1B1 c.388A>G Polymorphism Is Associated with HDL-C Levels in Response to Atorvastatin in Chilean Individuals

The use of statins as the preferred lipid-lowering therapy has clearly demonstrated its efficacy in the treatment of hypercholesterolemia, reducing also the risk of coronary events and cardiovascular disease mortality. In this study, we assessed single nucleotide polymorphisms (SNPs) in the SLCO1B1 gene and their effect on atorvastatin response. We included 129 Chilean hypercholesterolemic patients undergoing 10 mg/day of atorvastatin therapy during 4 weeks. Lipid profile was determined before and after drug administration. Genotyping of SLCO1B1 rs4149056 (c.521T>C) SNP was performed with allele-specific polymerase chain reaction, whilst polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for genotyping the SLCO1B1 rs2306283 (c.388A>G) variant. After statin therapy, concentrations of TC, LDL-C and TG had a decrease from baseline (p < 0.05). Also, HDL-C levels increased (p < 0.05). Minor allele frequencies for the rs2306283 and rs4149056 variants were 0.547 and 0.136, respectively. LDL-C response to atorvastatin was not associated with the SLCO1B1 rs4149056 nor the rs2306283 polymorphisms (p > 0.05). However, the latter SNP was associated with HDL-C variability after atorvastatin medication (p = 0.02). This study indicates that LDL-C reduction following atorvastatin therapy is not influenced by the SNPs evaluated. In addition, the polymorphism rs2306283 at the SLCO1B1 gene determines greater HDL-C concentrations in response to atorvastatin medication in Chilean hypercholesterolemic subjects.     

A Polymorphism at the Translation Start Site of the Vitamin D Receptor Gene Is Associated with the Response to Anti-Osteoporotic Therapy in Postmenopausal Women from Southern Italy

The present study investigated the effect of two single nucleotide polymorphisms (SNPs) of the vitamin D receptor (VDR) gene, rs1544410 A/G and rs2228570 C/T, in modulating bone mineral density (BMD) and the response to treatment with bisphosphonates or strontium ranelate in postmenopausal osteoporosis (PMO). Four hundred eighteen postmenopausal women from Southern Italy treated with bisphosphonates or strontium ranelate for three years were enrolled and stratified according to their genotype. Changes in BMD were expressed as the delta t-score (Δt-score). Allelic frequencies for rs1544410 A/GSNP were 11.2% AA, 50.0% GA and 38.8% GG; for rs2228570 C/TSNP were 54.8% CC, 39.5% TC and 5.7% TT. TT carriers showed a lower t-score than TC and CC (both p < 0.02) genotypes and were more responsive to the therapy when compared to both TC (p < 0.02) and CC (p < 0.05) carriers. Specifically, TT carriers receiving alendronate demonstrated a significant improvement of the Δt-score compared to TC and CC (both p < 0.0001) carriers. After adjustment for confounders, the Δt-score showed evidence of a statistically significant positive association with TT in all treatments considered. Therapy response was independent of rs1544410 A/G SNP; instead, rs2228570 C/TSNP was associated with a better response to antiresorptive treatment, thus suggesting that the therapy for PMO should be personalized.     

Risk-association of CYP11A1 polymorphisms and breast cancer among han chinese women in southern china

Exposure to endogenous sex hormones has been reported as a risk factor for breast cancer. The CYP11A1 gene encodes the key enzyme that catalyzes the initial and rate-limiting step in steroid hormone synthesis. In this study, the associations between single nucleotide polymorphisms (SNPs) in CYP11A1 and breast cancer susceptibility were examined. Six SNPs in CYP11A1 were genotyped using the MassARRAY IPLEX platform in 530 breast cancer patients and 546 healthy controls. Association analyses based on a χ(2) test and binary logistic regression were performed to determine the odds ratio (OR) and 95% confidence interval (95% CI) for each SNP. Two loci (rs2959008 and rs2279357) showed evidence of associations with breast cancer risk. The variant genotype C/T-C/C of rs2959008 was significantly associated with a decreased risk (age-adjusted OR, 0.75; 95% CI, 0.58-0.96; P = 0.023) compared with the wild-type TT. However, the homozygous TT variant of rs2279357 exhibited increased susceptibility to breast cancer (age-adjusted OR, 1.44; 95% CI, 1.05-1.98; P = 0.022). The locus rs2959003 also showed an appreciable effect, but no associations were observed for three other SNPs. Our results suggest that polymorphisms of CYP11A1 are related to breast cancer susceptibility in Han Chinese women of South China.     

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.     

Risk-Association of DNMT1 Gene Polymorphisms with Coronary Artery Disease in Chinese Han Population

Recently, a significant epigenetic component in the pathogenesis of Coronary Artery Disease (CAD) has been realized. Here, we evaluated the possible association of candidate Single Nucleotide Polymorphisms (SNPs) in the epigenetic-regulatory gene, DNA methyltransferase 1 (DNMT1), with CAD in Chinese Han population. Five tag SNPs (rs16999593, rs2336691, rs2228611, rs4804494, rs7253062) were analyzed by High Resolution Melt (HRM) method in 476 CAD patients and 478 controls. Overall, there were significant differences in the genotype and allele distributions of rs2228611 and rs2336691, between patients and controls. The minor A allele of rs2228611 was associated with a lower risk of CAD (p = 0.034); modest effect in the additive analysis but also marginal significance was found in the recessive model [OR_additive = 0.404 (0.184, 0.884), p = 0.023 and OR_recessive = 0.452 (0.213, 0.963), p = 0.040] after adjusting for confounders. While the rs2336691 A allele were associated with a higher risk of developing CAD (p = 0.037); borderline significant association in both additive and dominant models [OR_additive = 1.632 (1.030, 2.583), p = 0.037 and OR_dominant = 1.599 (1.020, 2.507), p = 0.040]. In conclusion, these data provide the first evidence that occurrence of CAD may be moderated by genetic variation in the gene involved in the epigenetic machinery.