Illuminating the function of the orphan transporter, SLC22A10, in humans and other primates.

SLC22A10 is an orphan transporter with unknown substrates and function. The goal of this study is to elucidate its substrate specificity and functional characteristics. In contrast to orthologs from great apes, human SLC22A10, tagged with green fluorescent protein, is not expressed on the plasma membrane. Cells expressing great ape SLC22A10 orthologs exhibit significant accumulation of estradiol-17ß-glucuronide, unlike those expressing human SLC22A10. Sequence alignments reveal a proline at position 220 in humans, which is a leucine in great apes. Replacing proline with leucine in SLC22A10-P220L restores plasma membrane localization and uptake function. Neanderthal and Denisovan genomes show proline at position 220, akin to modern humans, indicating functional loss during hominin evolution. Human SLC22A10 is a unitary pseudogene due to a fixed missense mutation, P220, while in great apes, its orthologs transport sex steroid conjugates. Characterizing SLC22A10 across species sheds light on its biological role, influencing organism development and steroid homeostasis.

Effect of Antioxidants in Medicinal Products on Intestinal Drug Transporters.

The presence of mutagenic and carcinogenic N-nitrosamine impurities in medicinal products poses a safety risk. While incorporating antioxidants in formulations is a potential mitigation strategy, concerns arise regarding their interference with drug absorption by inhibiting intestinal drug transporters. Our study screened thirty antioxidants for inhibitory effects on key intestinal transporters-OATP2B1, P-gp, and BCRP in HEK-293 cells (OATP2B1) or membrane vesicles (P-gp, BCRP) using 3H-estrone sulfate, 3H-N-methyl quinidine, and 3H-CCK8 as substrates, respectively. The screen identified that butylated hydroxyanisole (BHA) and carnosic acid inhibited all three transporters (OATP2B1, P-gp, and BCRP), while ascorbyl palmitate (AP) inhibited OATP2B1 by more than 50%. BHA had IC50 values of 71 ± 20 µM, 206 ± 14 µM, and 182 ± 49 µM for OATP2B1, BCRP, and P-gp, respectively. AP exhibited IC50 values of 23 ± 10 µM for OATP2B1. The potency of AP and BHA was tested with valsartan, an OATP2B1 substrate, and revealed IC50 values of 26 ± 17 µM and 19 ± 11 µM, respectively, in HEK-293-OATP2B1 cells. Comparing IC50 values of AP and BHA with estimated intestinal concentrations suggests an unlikely inhibition of intestinal transporters at clinical concentrations of drugs formulated with antioxidants.

Cardiac contraction and relaxation are regulated by distinct subcellular cAMP pools.

Cells interpret a variety of signals through G-protein-coupled receptors (GPCRs) and stimulate the generation of second messengers such as cyclic adenosine monophosphate (cAMP). A long-standing puzzle is deciphering how GPCRs elicit different physiological responses despite generating similar levels of cAMP. We previously showed that some GPCRs generate cAMP from both the plasma membrane and the Golgi apparatus. Here we demonstrate that cardiomyocytes distinguish between subcellular cAMP inputs to elicit different physiological outputs. We show that generating cAMP from the Golgi leads to the regulation of a specific protein kinase A (PKA) target that increases the rate of cardiomyocyte relaxation. In contrast, cAMP generation from the plasma membrane activates a different PKA target that increases contractile force. We further validated the physiological consequences of these observations in intact zebrafish and mice. Thus, we demonstrate that the same GPCR acting through the same second messenger regulates cardiac contraction and relaxation dependent on its subcellular location.

The Clinical Pharmacogenetics Implementation Consortium Guideline for SLCO1B1, ABCG2, and CYP2C9 genotypes and Statin-Associated Musculoskeletal Symptoms.

Statins reduce cholesterol, prevent cardiovascular disease, and are among the most commonly prescribed medications in the world. Statin-associated musculoskeletal symptoms (SAMS) impact statin adherence and ultimately can impede the long-term effectiveness of statin therapy. There are several identified pharmacogenetic variants that impact statin disposition and adverse events during statin therapy. SLCO1B1 encodes a transporter (SLCO1B1; alternative names include OATP1B1 or OATP-C) that facilitates the hepatic uptake of all statins. ABCG2 encodes an efflux transporter (BCRP) that modulates the absorption and disposition of rosuvastatin. CYP2C9 encodes a phase I drug metabolizing enzyme responsible for the oxidation of some statins. Genetic variation in each of these genes alters systemic exposure to statins (i.e., simvastatin, rosuvastatin, pravastatin, pitavastatin, atorvastatin, fluvastatin, lovastatin), which can increase the risk for SAMS. We summarize the literature supporting these associations and provide therapeutic recommendations for statins based on SLCO1B1, ABCG2, and CYP2C9 genotype with the goal of improving the overall safety, adherence, and effectiveness of statin therapy. This document replaces the 2012 and 2014 Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for SLCO1B1 and simvastatin-induced myopathy.

Genome-Wide Meta-analysis Identifies Genetic Variants Associated With Glycemic Response to Sulfonylureas.

OBJECTIVE: Sulfonylureas, the first available drugs for the management of type 2 diabetes, remain widely prescribed today. However, there exists significant variability in glycemic response to treatment. We aimed to establish heritability of sulfonylurea response and identify genetic variants and interacting treatments associated with HbA1c reduction. RESEARCH DESIGN AND METHODS: As an initiative of the Metformin Genetics Plus Consortium (MetGen Plus) and the DIabetes REsearCh on patient straTification (DIRECT) consortium, 5,485 White Europeans with type 2 diabetes treated with sulfonylureas were recruited from six referral centers in Europe and North America. We first estimated heritability using the generalized restricted maximum likelihood approach and then undertook genome-wide association studies of glycemic response to sulfonylureas measured as HbA1c reduction after 12 months of therapy followed by meta-analysis. These results were supported by acute glipizide challenge in humans who were naïve to type 2 diabetes medications, cis expression quantitative trait loci (eQTL), and functional validation in cellular models. Finally, we examined for possible drug-drug-gene interactions. RESULTS: After establishing that sulfonylurea response is heritable (mean ± SEM 37 ± 11%), we identified two independent loci near the GXYLT1 and SLCO1B1 genes associated with HbA1c reduction at a genome-wide scale (P < 5 × 10-8). The C allele at rs1234032, near GXYLT1, was associated with 0.14% (1.5 mmol/mol), P = 2.39 × 10-8), lower reduction in HbA1c. Similarly, the C allele was associated with higher glucose trough levels (ß = 1.61, P = 0.005) in healthy volunteers in the SUGAR-MGH given glipizide (N = 857). In 3,029 human whole blood samples, the C allele is a cis eQTL for increased expression of GXYLT1 (ß = 0.21, P = 2.04 × 10-58). The C allele of rs10770791, in an intronic region of SLCO1B1, was associated with 0.11% (1.2 mmol/mol) greater reduction in HbA1c (P = 4.80 × 10-8). In 1,183 human liver samples, the C allele at rs10770791 is a cis eQTL for reduced SLCO1B1 expression (P = 1.61 × 10-7), which, together with functional studies in cells expressing SLCO1B1, supports a key role for hepatic SLCO1B1 (encoding OATP1B1) in regulation of sulfonylurea transport. Further, a significant interaction between statin use and SLCO1B1 genotype was observed (P = 0.001). In statin nonusers, C allele homozygotes at rs10770791 had a large absolute reduction in HbA1c (0.48 ± 0.12% [5.2 ± 1.26 mmol/mol]), equivalent to that associated with initiation of a dipeptidyl peptidase 4 inhibitor. CONCLUSIONS: We have identified clinically important genetic effects at genome-wide levels of significance, and important drug-drug-gene interactions, which include commonly prescribed statins. With increasing availability of genetic data embedded in clinical records these findings will be important in prescribing glucose-lowering drugs.

Functional and structural analysis of rare SLC2A2 variants associated with Fanconi-Bickel syndrome and metabolic traits.

Deleterious variants in SLC2A2 cause Fanconi-Bickel Syndrome (FBS), a glycogen storage disorder, whereas less common variants in SLC2A2 associate with numerous metabolic diseases. Phenotypic heterogeneity in FBS has been observed, but its causes remain unknown. Our goal was to functionally characterize rare SLC2A2 variants found in FBS and metabolic disease-associated variants to understand the impact of these variants on GLUT2 activity and expression and establish genotype-phenotype correlations. Complementary RNA-injected Xenopus laevis oocytes were used to study mutant transporter activity and membrane expression. GLUT2 homology models were constructed for mutation analysis using GLUT1, GLUT3, and XylE as templates. Seventeen FBS variants were characterized. Only c.457_462delCTTATA (p.Leu153_Ile154del) exhibited residual glucose uptake. Functional characterization revealed that only half of the variants were expressed on the plasma membrane. Most less common variants (except c.593 C>A (p.Thr198Lys) and c.1087 G>T (p.Ala363Ser)) exhibited similar GLUT2 transport activity as the wild type. Structural analysis of GLUT2 revealed that variants affect substrate-binding, steric hindrance, or overall transporter structure. The mutant transporter that is associated with a milder FBS phenotype, p.Leu153_Ile154del, retained transport activity. These results improve our overall understanding of the underlying causes of FBS and impact of GLUT2 function on various clinical phenotypes ranging from rare to common disease.

Organic Anion Transporter Polypeptide 1B1 Polymorphism Modulates the Extent of Drug-Drug Interaction and Associated Biomarker Levels in Healthy Volunteers.

Understanding transporter-mediated drug-drug interactions is an integral part of risk assessment in drug development. Recent studies support the use of hexadecanedioate (HDA), tetradecanedioate (TDA), coproporphyrin (CP)-I, and CP-III as clinical biomarkers for evaluating organic anion-transporting polypeptide (OATP)1B1 (SLCO1B1) inhibition. The current study investigated the effect of OATP1B1 genotype c.521T>C (OATP1B1-Val174Ala) on the extent of interaction between cyclosporin A (CsA) and pravastatin, and associated endogenous biomarkers of the transporter (HDA, TDA, CP-I, and CP-III), in 20 healthy volunteers. The results show that the levels of each clinical biomarker and pravastatin were significantly increased in plasma samples of the volunteers following administration of pravastatin plus CsA compared with pravastatin plus placebo. The overall fold change in the area under the concentration-time curve (AUC) and maximum plasma concentration (Cmax ) was similar among the four biomarkers (1.8-2.5-fold, paired t-test P value < 0.05) in individuals who were homozygotes or heterozygotes of the major allele, c.521T. However, the fold change in AUC and Cmax for HDA and TDA was significantly abolished in the subjects who were c.521-CC, whereas the respective fold change in AUC and Cmax for pravastatin and CP-I and CP-III were slightly weaker in individuals who were c.521-CC compared with c.521-TT/TC genotypes. In addition, this study provides the first evidence that SLCO1B1 c.521T>C genotype is significantly associated with CP-I but not CP-III levels. Overall, these results suggest that OATP1B1 genotype can modulate the effects of CsA on biomarker levels; the extent of modulation differs among the biomarkers.

Genome-Wide Association and Functional Studies Reveal Novel Pharmacological Mechanisms for Allopurinol.

Allopurinol, which lowers uric acid (UA) concentration, is increasingly being recognized for its benefits in cardiovascular and renal disease. However, response to allopurinol is variable. We gathered samples from 4,446 multiethnic subjects for a genome-wide association study of allopurinol response. Consistent with previous studies, we observed that the Q141K variant in ABCG2 (rs2231142), which encodes the efflux pump breast cancer resistance protein (BCRP), associated with worse response to allopurinol. However, for the first time this association reached genome-wide level significance (P = 8.06 × 10-11 ). Additionally, we identified a novel association with a variant in GREM2 (rs1934341, P = 3.22 × 10-6 ). In vitro studies identified oxypurinol, the active metabolite of allopurinol, as an inhibitor of the UA transporter GLUT9, suggesting that oxypurinol may modulate UA reabsorption. These results provide strong evidence for a role of BCRP Q141K in allopurinol response, and suggest that allopurinol may have additional hypouricemic effects beyond xanthine oxidase inhibition.

Organic cation transporter 3 (Oct3) is a distinct catecholamines clearance route in adipocytes mediating the beiging of white adipose tissue.

Beiging of white adipose tissue (WAT) is a particularly appealing target for therapeutics in the treatment of metabolic diseases through norepinephrine (NE)-mediated signaling pathways. Although previous studies report NE clearance mechanisms via SLC6A2 on sympathetic neurons or proinflammatory macrophages in adipose tissues (ATs), the low catecholamine clearance capacity of SLC6A2 may limit the cleaning efficiency. Here, we report that mouse organic cation transporter 3 (Oct3; Slc22a3) is highly expressed in WAT and displays the greatest uptake rate of NE as a selective non-neural route of NE clearance in white adipocytes, which differs from other known routes such as adjacent neurons or macrophages. We further show that adipocytes express high levels of NE degradation enzymes Maoa, Maob, and Comt, providing the molecular basis on NE clearance by adipocytes together with its reuptake transporter Oct3. Under NE administration, ablation of Oct3 induces higher body temperature, thermogenesis, and lipolysis compared with littermate controls. After prolonged cold challenge, inguinal WAT (ingWAT) in adipose-specific Oct3-deficient mice shows much stronger browning characteristics and significantly elevated expression of thermogenic and mitochondrial biogenesis genes than in littermate controls, and this response involves enhanced ß-adrenergic receptor (ß-AR)/protein kinase A (PKA)/cyclic adenosine monophosphate (cAMP)-responsive element binding protein (Creb) pathway activation. Glycolytic genes are reprogrammed to significantly higher levels to compensate for the loss of ATP production in adipose-specific Oct3 knockout (KO) mice, indicating the fundamental role of glucose metabolism during beiging. Inhibition of ß-AR largely abolishes the higher lipolytic and thermogenic activities in Oct3-deficient ingWAT, indicating the NE overload in the vicinity of adipocytes in Oct3 KO adipocytes. Of note, reduced functional alleles in human OCT3 are also identified to be associated with increased basal metabolic rate (BMR). Collectively, our results demonstrate that Oct3 governs ß-AR activity as a NE recycling transporter in white adipocytes, offering potential therapeutic applications for metabolic disorders.

Transporters in Drug Development: 2018 ITC Recommendations for Transporters of Emerging Clinical Importance.

This white paper provides updated International Transporter Consortium (ITC) recommendations on transporters that are important in drug development following the 3rd ITC workshop. New additions include prospective evaluation of organic cation transporter 1 (OCT1) and retrospective evaluation of organic anion transporting polypeptide (OATP)2B1 because of their important roles in drug absorption, disposition, and effects. For the first time, the ITC underscores the importance of transporters involved in drug-induced vitamin deficiency (THTR2) and those involved in the disposition of biomarkers of organ function (OAT2 and bile acid transporters).

Computational Discovery and Experimental Validation of Inhibitors of the Human Intestinal Transporter OATP2B1.

Human organic anion transporters (OATPs) are vital for the uptake and efflux of drugs and endogenous compounds. Current identification of inhibitors of these transporters is based on experimental screening. Virtual screening remains a challenge due to a lack of experimental three-dimensional protein structures. Here, we describe a workflow to identify inhibitors of the OATP2B1 transporter in the DrugBank library of over 5,000 drugs and druglike molecules. OATP member 2B1 transporter is highly expressed in the intestine, where it participates in oral absorption of drugs. Predictions from a Random forest classifier, prioritized by docking against multiple comparative protein structure models of OATP2B1, indicated that 33 of the 5,000 compounds were putative inhibitors of OATP2B1. Ten predicted inhibitors that are prescription drugs were tested experimentally in cells overexpressing the OATP2B1 transporter. Three of these ten were validated as potent inhibitors of estrone-3-sulfate uptake (defined as more than 50% inhibition at 20 µM) and tested in multiple concentrations to determine exact IC50. The IC50 values of bicalutamide, ticagrelor, and meloxicam suggest that they might inhibit intestinal OATP2B1 at clinically relevant concentrations and therefore modulate the absorption of other concomitantly administered drugs.

Targeted disruption of organic cation transporter 3 attenuates the pharmacologic response to metformin.

Metformin, the most widely prescribed antidiabetic drug, requires transporters to enter tissues involved in its pharmacologic action, including liver, kidney, and peripheral tissues. Organic cation transporter 3 (OCT3, SLC22A3), expressed ubiquitously, transports metformin, but its in vivo role in metformin response is not known. Using Oct3 knockout mice, the role of the transporter in metformin pharmacokinetics and pharmacodynamics was determined. After an intravenous dose of metformin, a 2-fold decrease in the apparent volume of distribution and clearance was observed in knockout compared with wild-type mice (P < 0.001), indicating an important role of OCT3 in tissue distribution and elimination of the drug. After oral doses, a significantly lower bioavailability was observed in knockout compared with wild-type mice (0.27 versus 0.58, P < 0.001). Importantly, metformin's effect on the plasma glucose concentration-time curve was reduced in knockout compared with wild-type mice (12 versus 30% reduction, respectively, P < 0.05) along with its accumulation in skeletal muscle and adipose tissue (P < 0.05). Furthermore, the effect of metformin on phosphorylation of AMP activated protein kinase, and expression of glucose transporter type 4 was absent in the adipose tissue of Oct3(-/-) mice. Additional analysis revealed that an OCT3 3' untranslated region variant was associated with reduced activity in luciferase assays and reduced response to metformin in 57 healthy volunteers. These findings suggest that OCT3 plays an important role in the absorption and elimination of metformin and that the transporter is a critical determinant of metformin bioavailability, clearance, and pharmacologic action.

Impact of polymorphisms in drug pathway genes on disease-free survival in adults with acute myeloid leukemia.

Acute myeloid leukemia (AML) is a clinically heterogeneous disease, with a 5-year disease-free survival (DFS) ranging from under 10% to over 70% for distinct groups of patients. At our institution, cytarabine, etoposide and busulfan are used in first or second remission patients treated with a two-step approach to autologous stem cell transplantation (ASCT). In this study, we tested the hypothesis that polymorphisms in the pharmacokinetic and pharmacodynamic pathway genes of these drugs are associated with DFS in AML patients. A total of 1659 variants in 42 genes were analyzed for their association with DFS using a Cox-proportional hazards model. One hundred and fifty-four genetically European patients were used for the primary analysis. An intronic single nucleotide polymorphism (SNP) in ABCC3 (rs4148405) was associated with a significantly shorter DFS (hazard ratios (HR)=3.2, P=5.6 × 10(-6)) in our primary cohort. In addition, a SNP in the GSTM1-GSTM5 locus, rs3754446, was significantly associated with a shorter DFS in all patients (HR=1.8, P=0.001 for 154 European ancestry; HR=1.7, P=0.028 for 125 non-European patients). Thus, for the first time, genetic variants in drug pathway genes are shown to be associated with DFS in AML patients treated with chemotherapy-based autologous ASCT.

Evaluation of Norepinephrine Transporter Expression and Metaiodobenzylguanidine Avidity in Neuroblastoma: A Report from the Children's Oncology Group.

Purpose. (123)I-metaiodobenzylguanidine (MIBG) is used for the diagnostic evaluation of neuroblastoma. We evaluated the relationship between norepinephrine transporter (NET) expression and clinical MIBG uptake. Methods. Quantitative reverse transcription PCR (N = 82) and immunohistochemistry (IHC; N = 61) were performed for neuroblastoma NET mRNA and protein expression and correlated with MIBG avidity on diagnostic scans. The correlation of NET expression with clinical features was also performed. Results. Median NET mRNA expression level for the 19 MIBG avid patients was 12.9% (range 1.6-73.7%) versus 5.9% (range 0.6-110.0%) for the 8 nonavid patients (P = 0.31). Median percent NET protein expression was 50% (range 0-100%) in MIBG avid patients compared to 10% (range 0-80%) in nonavid patients (P = 0.027). MYCN amplified tumors had lower NET protein expression compared to nonamplified tumors (10% versus 50%; P = 0.0002). Conclusions. NET protein expression in neuroblastoma correlates with MIBG avidity. MYCN amplified tumors have lower NET protein expression.

Interaction of sirolimus and everolimus with hepatic and intestinal organic anion-transporting polypeptide transporters.

The goal of this study was to assess the interaction of the mTOR inhibitors (ImTORs) sirolimus and everolimus with the human organic anion-transporting polypeptides (OATPs) expressed in hepatocytes and enterocytes by conducting uptake experiments using (i) transfected HEK293T cells, (ii) the hepatocyte-like HepaRG cell line and (iii) the enterocyte-like Caco-2 cell line. Sirolimus and everolimus inhibited in a dose-dependent manner the uptake of [³H]-estrone sulphate by OATP1A2 and OATP1B1 and that of mycophenolic acid 7-O-glucuronide (MPAG) by OATP1B3. ImTOR apparent 50% inhibitory concentrations (IC50) for OATPs were 11.9 µM (OATP1A2), 9.8 µM (OATP1B1) and 1.3 µM (OATP1B3) for sirolimus and 4.2 µM (OATP1A2), 4.1 µM (OATP1B1) and 4.3 µM (OATP1B3) for everolimus. No transport of sirolimus or everolimus by OATP1A2, OATP1B1 or OATP1B3 was observed in HEK-transfected cells and the OAT/OATP/MRP chemical inhibitor probenecid did not significantly decrease the uptake of sirolimus and everolimus in HepaRG and Caco-2 cells, but tended to increase their intracellular accumulation presumably through efflux inhibition. In conclusion, our data suggest that the major OATP transporters expressed in the liver and the intestine do not contribute to the pharmacokinetics of sirolimus and everolimus. However, ImTORs are inhibitors of these transporters.

Organic cation transporters modulate the uptake and cytotoxicity of picoplatin, a third-generation platinum analogue.

Picoplatin, a third-generation platinum agent, is efficacious against lung cancers that are otherwise resistant or become refractory during platinum treatment. This effort was aimed at the determination of the influence of organic cation transporters 1, 2, and 3 (OCT1, OCT2, and OCT3) and their genetic variants on cellular uptake of picoplatin and on the individual components of the ensuing cytotoxicity such as DNA adduct formation. The effect of OCT1 on picoplatin pharmacokinetics and antitumor efficacy was determined using OCT knockout mice and HEK293 xenografts stably expressing OCT1. The uptake and DNA adduct formation of picoplatin were found to be significantly enhanced by the expression of the OCTs. Expression of OCT1 and OCT2, but not OCT3, significantly enhanced picoplatin cytotoxicity, which was reduced in the presence of an OCT inhibitor. Common reduced functional variants of OCT1 and OCT2 led to reduction in uptake and DNA adduct formation of picoplatin in comparison with the reference OCT1 and OCT2. Pharmacokinetic parameters of picoplatin in Oct1(-/-) and Oct1(+/+) mice were not significantly different, suggesting that the transporters do not influence the disposition of the drug. In contrast, the volume of OCT1-expressing xenografts in mice was significantly reduced by picoplatin treatment, suggesting that OCT1 may enhance the antitumor efficacy of picoplatin. These studies provide a basis for follow-up clinical studies that would seek to examine the relationship between the anticancer efficacy of picoplatin and expression levels of OCTs and their genetic variants in tumors. Mol Cancer Ther; 9(4); 1058-69. (c)2010 AACR.

Functional genetic variation in the basal promoter of the organic cation/carnitine transporters OCTN1 (SLC22A4) and OCTN2 (SLC22A5).

The organic cation/ergothioneine transporter OCTN1 (SLC22A4) and the high-affinity carnitine transporter OCTN2 (SLC22A5), play an important role in the disposition of xenobiotics and endogenous compounds. Here, we analyzed the sequence of the proximal promoter regions of OCTN1 and OCTN2 in four ethnic groups and determined the effects of the identified genetic variants on transcriptional activities and mRNA expression. Six variants were found in the proximal promoter of OCTN1, one of which showed high allele frequency ranging from 13 to 34% in samples from individuals with ancestries in Africa, Europe, China, and Mexico. OCTN1 haplotypes had similar activities as the reference in luciferase reporter assays. For OCTN2, three of the seven variants identified in the proximal promoter showed allele frequencies greater than 29.5% in all populations, with the exception of -207C>G (rs2631367) that was monomorphic in Asian Americans. OCTN2 haplotypes containing -207G, present in all populations, were associated with a gain of function in luciferase reporter assays. Consistent with reporter assays, OCTN2 mRNA expression levels in lymphoblastoid cell lines (LCLs) from gene expression analysis were greater in samples carrying a marker for -207G. This SNP seems to contribute to racial differences in OCTN2 mRNA expression levels in LCLs. Our study with healthy subjects (n = 16) homozygous for either -207C or -207G, showed no appreciable effect of this SNP on carnitine disposition. However, there were significant effects of gender on carnitine plasma levels (p < 0.01). Further in vivo studies of OCTN2 promoter variants on carnitine disposition and variation in drug response are warranted.

Inhibition of Vitamin D3 metabolism enhances VDR signalling in androgen-independent prostate cancer cells.

Induction of growth arrest and differentiation by 1alpha,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) occurs in non-malignant cell types but is often reduced in cancer cells. For example, androgen-independent prostate cancer cells, DU-145 and PC-3, are relatively insensitive to the anti-proliferative action of 1,25-(OH)(2)D(3). This appears to be due to increased 1,25-(OH)(2)D(3)-metabolism, as a result of CYP24 enzyme-induction, which in turn leads to decreased anti-proliferative efficacy. In the in vitro rat kidney mitochondria assay, the 2-(4-hydroxybenzyl)-6-methoxy-3,4-dihydro-2H-naphthalen-1-one (4) was found to be a potent inhibitor of Vitamin D(3) metabolising enzymes (IC(50) 3.5 microM), and was shown to be a more potent inhibitor than the broad spectrum P450 inhibitor ketoconazole (IC(50) 20 microM). The combination of the inhibitor and 1,25-(OH)(2)D(3) caused a greater inhibition of proliferation in DU-145 cells than when treated with both agents alone. Examination of the regulation of VDR target gene mRNA in DU-145 cells revealed that co-treatment of 1,25-(OH)(2)D(3) plus inhibitor of Vitamin D(3) metabolising enzymes co-ordinately upregulated CYP24, p21(waf1/cip1) and GADD45alpha.