Loss of hepatic Mboat7 leads to liver fibrosis.

OBJECTIVE: The rs641738C>T variant located near the membrane-bound O-acyltransferase domain containing 7 (MBOAT7) locus is associated with fibrosis in liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcohol-related liver disease, hepatitis B and C. We aim to understand the mechanism by which the rs641738C>T variant contributes to pathogenesis of NAFLD. DESIGN: Mice with hepatocyte-specific deletion of MBOAT7 (Mboat7Δhep) were generated and livers were characterised by histology, flow cytometry, qPCR, RNA sequencing and lipidomics. We analysed the association of rs641738C>T genotype with liver inflammation and fibrosis in 846 NAFLD patients and obtained genotype-specific liver lipidomes from 280 human biopsies. RESULTS: Allelic imbalance analysis of heterozygous human liver samples pointed to lower expression of the MBOAT7 transcript on the rs641738C>T haplotype. Mboat7Δhep mice showed spontaneous steatosis characterised by increased hepatic cholesterol ester content after 10 weeks. After 6 weeks on a high fat, methionine-low, choline-deficient diet, mice developed increased hepatic fibrosis as measured by picrosirius staining (p<0.05), hydroxyproline content (p<0.05) and transcriptomics, while the inflammatory cell populations and inflammatory mediators were minimally affected. In a human biopsied NAFLD cohort, MBOAT7 rs641738C>T was associated with fibrosis (p=0.004) independent of the presence of histological inflammation. Liver lipidomes of Mboat7Δhep mice and human rs641738TT carriers with fibrosis showed increased total lysophosphatidylinositol levels. The altered lysophosphatidylinositol and phosphatidylinositol subspecies in MBOAT7Δhep livers and human rs641738TT carriers were similar. CONCLUSION: Mboat7 deficiency in mice and human points to an inflammation-independent pathway of liver fibrosis that may be mediated by lipid signalling and a potentially targetable treatment option in NAFLD.

Correction: The anti-tumorigenic activity of A2M-A lesson from the naked mole-rat.

[This corrects the article DOI: 10.1371/journal.pone.0189514.].

Haplotypes composed of minor frequency single nucleotide polymorphisms of the TNF gene protect from progression into sepsis: A study using the new sepsis classification.

OBJECTIVES: Several articles have provided conflicting results regarding the role of single nucleotide polymorphisms (SNPs) in the promoter region of the TNF gene in susceptibility to sepsis. Former articles have been based on previous definitions of sepsis. This study investigated the influence of TNF haplotypes on the development of sepsis using the new Sepsis-3 definitions. METHODS: DNA was isolated from patients suffering from infection and systemic inflammatory response syndrome. Haplotyping was performed for six SNPs of TNF. The serum levels of tumour necrosis factor alpha (TNF-α) of these patients were measured using an enzyme immunosorbent assay. Patients were classified into infection and sepsis categories using the Sepsis-3 definitions. Associations between the TNF haplotypes and the clinical characteristics and serum TNF-α levels of the patients were examined. RESULTS: The most common TNF haplotype h1 was composed of major alleles of the studied SNPs. Carriage of haplotypes composed of minor frequency alleles was associated with a lower risk of developing sepsis (odds ratio 0.41, 95% confidence interval 0.19-0.88, p=0.022), but this did not affect the 28-day outcome. Serum TNF-α levels were significantly higher among patients homozygous for h1 haplotypes who developed sepsis compared to infection (p=0.032); a similar result was not observed for patients carrying other haplotypes. CONCLUSIONS: Haplotypes containing minor frequency SNP alleles of TNF protect against the development of sepsis without affecting the outcome.

The anti-tumorigenic activity of A2M-A lesson from the naked mole-rat.

Cancer resistance is a major cause for longevity of the naked mole-rat. Recent liver transcriptome analysis in this animal compared to wild-derived mice revealed higher expression of alpha2-macroglobulin (A2M) and cell adhesion molecules, which contribute to the naked mole-rat's cancer resistance. Notably, A2M is known to dramatically decrease with age in humans. We hypothesize that this might facilitate tumour development. Here we found that A2M modulates tumour cell adhesion, migration and growth by inhibition of tumour promoting signalling pathways, e.g. PI3K / AKT, SMAD and up-regulated PTEN via down-regulation of miR-21, in vitro and in tumour xenografts. A2M increases the expression of CD29 and CD44 but did not evoke EMT. Transcriptome analysis of A2M-treated tumour cells, xenografts and mouse liver demonstrated a multifaceted regulation of tumour promoting signalling pathways indicating a less tumorigenic environment mediated by A2M. By virtue of these multiple actions the naturally occurring A2M has strong potential as a novel therapeutic agent.

Verification and characterization of an alternative low density lipoprotein receptor-related protein 1 splice variant.

BACKGROUND: Low density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a ubiquitously expressed multi-ligand endocytosis receptor implicated in a wide range of signalling, among others in tumour biology. Tumour-associated genomic mutations of the LRP1 gene are described, but nothing is known about cancer-associated expression of LRP1 splice variants Therefore, the focus of this study was on an annotated truncated LRP1 splice variant (BC072015.1; NCBI GenBank), referred to as smLRP1, which was initially identified in prostate and lung carcinoma. METHODS: Using PCR and quantitative PCR, the expression of LRP1 and smLRP1 in different human tissues and tumour cell lines was screened and compared on tumour biopsies of head and neck squamous cell carcinoma (HNSCC). Using a recently developed anti-smLRP1 antibody, the expression of the putative LRP1 protein isoform in tumour cell lines in Western blot and immunofluorescence staining was further investigated. RESULTS: The alternative transcript smLRP1 is ubiquitously expressed in 12 human cell lines of different origin and 22 tissues which is similar to LRP1. A shift in expression of smLRP1 relative to LRP1 towards smLRP1 was observed in most tumour cell lines compared to healthy tissue. The expression of LRP1 as well as smLRP1 is decreased in HNSCC cell lines in comparison to healthy mucosa. In vitro results were checked using primary HNSCC. Furthermore, the expression of the protein isoform smLRP1 (32 kDa) was confirmed in human tumour cell lines. CONCLUSIONS: Similar to LRP1, the truncated splice variant smLRP1 is ubiquitously expressed in healthy human tissues, but altered in tumours pointing to a potential role of smLRP1 in cancer. Comparative results suggest a shift in expression in favour of smLRP1 in tumour cells that warrant further evaluation. The protein isoform is suggested to be secreted.

Analysis of cultivable microbiota and diet intake pattern of the long-lived naked mole-rat.

BACKGROUND: A variety of microbial communities exist throughout the human and animal body. Genetics, environmental factors and long-term dietary habit contribute to shaping the composition of the gut microbiota. For this reason the study of the gut microbiota of a mammal exhibiting an extraordinary life span is of great importance. The naked mole-rat (Heterocephalus glaber) is a eusocial mammal known for its longevity and cancer resistance. METHODS: Here we analyzed its gut microbiota by cultivating the bacteria under aerobic and anaerobic conditions and identifying their species by mass spectrometry. RESULTS: Altogether, 29 species of microbes were identified, predominantly belonging to Firmicutes, and Bacteroidetes. The most frequent species were Bacillus megaterium (45.2 %), followed by Bacteroides thetaiotaomicron (19.4 %), Bacteroides ovatus, Staphylococcus sciuri and Paenibacillus spp., each with a frequency of 16.1 %. CONCLUSION: Overall, the gut of the naked mole-rat is colonized by diverse, but low numbers of cultivable microbes compared with humans and mice. The primary food plants of the rodents are rich in polyphenols and related compounds, possessing anti-microbial, anti-inflammatory, anti-oxidative as well as anti-cancer activity which may contribute to their exceptionally healthy life.

Low sulfide levels and a high degree of cystathionine ß-synthase (CBS) activation by S-adenosylmethionine (SAM) in the long-lived naked mole-rat.

Hydrogen sulfide (H2S) is a gaseous signalling molecule involved in many physiological and pathological processes. There is increasing evidence that H2S is implicated in aging and lifespan control in the diet-induced longevity models. However, blood sulfide concentration of naturally long-lived species is not known. Here we measured blood sulfide in the long-lived naked mole-rat and five other mammalian species considerably differing in lifespan and found a negative correlation between blood sulfide and maximum longevity residual. In addition, we show that the naked mole-rat cystathionine ß-synthase (CBS), an enzyme whose activity in the liver significantly contributes to systemic sulfide levels, has lower activity in the liver and is activated to a higher degree by S-adenosylmethionine compared to other species. These results add complexity to the understanding of the role of H2S in aging and call for detailed research on naked mole-rat transsulfuration.

Polymorphisms of cystathionine beta-synthase gene are associated with susceptibility to sepsis.

Sepsis is the systemic inflammatory host response to infection. Cystathionine beta-synthase (CBS)-dependent homocysteine (Hcy) pathway was demonstrated to affect disease severity and mortality in patients with severe sepsis/septic shock. Independent studies identified a single-nucleotide polymorphism (SNP, rs6586282, hg19 chr21:g.44478497C>T) in intron 14 of the CBS-coding gene (CBS) associated with Hcy plasma levels. We aimed to describe the association of this SNP and variants of a splice donor-affecting variable-number tandem repeat (VNTR, NG_008938.1:g.22763_22793[16_22]) 243 bp downstream of rs6586282 with severe human sepsis. We analyzed the VNTR structure and genotyped variants of rs6586282 and a neighboring SNP (rs34758144, hg19 chr21:g.44478582G>A) in two case-control studies including patients with severe sepsis/septic shock from Germany (n=168) and Greece (n=237). In both studies, we consistently observed an association of CBS VNTR alleles with sepsis susceptibility. Risk linearly increased with number of tandem repeats (per allele odds ratio in the adjusted analysis 1.34; 95% confidence interval (CI)=1.17-1.55; P<0.001). Association had also been shown for rs34758144 whose risk allele is in linkage disequilibrium with one long VNTR allele (19 repeat). In contrast, we observed no evidence for an effect on 28-day survival in patients with severe sepsis/septic shock (per allele hazard ratio in the adjusted analysis for VNTR 1.10; 95% CI=0.95-1.28; P=0.20). In a minigene approach, we demonstrated alternative splicing in distinct VNTR alleles, which, however, was independent of the number of tandem units. In conclusion, there is no ordinary conjunction between human CBS and severe sepsis/septic shock, but CBS genotypes are involved in disease susceptibility.

Analysis of Alpha-2 Macroglobulin from the Long-Lived and Cancer-Resistant Naked Mole-Rat and Human Plasma.

BACKGROUND: The naked mole-rat (NMR) is a long-lived and cancer resistant species. Identification of potential anti-cancer and age related mechanisms is of great interest and makes this species eminent to investigate anti-cancer strategies and understand aging mechanisms. Since it is known that the NMR expresses higher liver mRNA-levels of alpha 2-macroglobulin than mice, nothing is known about its structure, functionality or expression level in the NMR compared to the human A2M. RESULTS: Here we show a comprehensive analysis of NMR- and human plasma-A2M, showing a different prediction in glycosylation of NMR-A2M, which results in a higher molecular weight compared to human A2M. Additionally, we found a higher concentration of A2M (8.3±0.44 mg/mL vs. and 4.4±0.20 mg/mL) and a lower total plasma protein content (38.7±1.79 mg/mL vs. 61.7±3.20 mg/mL) in NMR compared to human. NMR-A2M can be transformed by methylamine and trypsin resulting in a conformational change similar to human A2M. NMR-A2M is detectable by a polyclonal antibody against human A2M. Determination of tryptic and anti-tryptic activity of NMR and human plasma revealed a higher anti-tryptic activity of the NMR plasma. On the other hand, less proteolytic activity was found in NMR plasma compared to human plasma. CONCLUSION: We found transformed NMR-A2M binding to its specific receptor LRP1. We could demonstrate lower protein expression of LRP1 in the NMR liver tissue compared to human but higher expression of A2M. This was accompanied by a higher EpCAM protein expression as central adhesion molecule in cancer progression. NMR-plasma was capable to increase the adhesion in human fibroblast in vitro most probably by increasing CD29 protein expression. This is the first report, demonstrating similarities as well as distinct differences between A2M in NMR and human plasma. This might be directly linked to the intriguing phenotype of the NMR and suggests that A2M might probably play an important role in anti-cancer and the anti-aging mechanisms in the NMR.

Mutation in CYP27A1 identified in family with coronary artery disease.

Coronary artery disease (CAD) is a leading cause of death worldwide. Myocardial infarction is the most severe outcome of CAD. Despite extensive efforts, the genetics of CAD is poorly understood. We aimed to identify the genetic cause of CAD in a pedigree with several affected individuals. Exome sequencing led to identification of a mutation in CYP27A1 that causes p.Arg225His in the encoded protein sterol 27-hydroxylase as the likely cause of CAD in the pedigree. The enzyme is multifunctional, and several of its functions including its functions in vitamin D metabolism and reverse cholesterol transport (RCT) are relevant to the CAD phenotype. Measurements of vitamin D levels suggested that the mutation does not affect CAD by affecting this parameter. We suggest that the mutation may cause CAD by affecting RCT. Screening of all coding regions of the CYP27A1 in 100 additional patients led to finding four variations (p.Arg14Gly, p.Arg26Lys, p.Ala27Arg, and p.Val86Met) in seven patients that may contribute to their CAD status. CYP27A1 is the known causative gene of cerebrotendinous xanthomatosis, a disorder which is sometimes accompanied by early onset atherosclerosis. This and the observation of potentially harmful variations in unrelated CAD patients provide additional evidence for the suggested causative role of the p.Arg225His mutation in CAD.

Alternative 5' untranslated regions are involved in expression regulation of human heme oxygenase-1.

The single nucleotide polymorphism rs2071746 and a (GT)n microsatellite within the human gene encoding heme oxygenase-1 (HMOX1) are associated with incidence or outcome in a variety of diseases. Most of these associations involve either release of heme or oxidative stress. Both polymorphisms are localized in the promoter region, but previously reported correlations with heme oxygenase-1 expression remain not coherent. This ambiguity suggests a more complex organization of the 5' gene region which we sought to investigate more fully. We evaluated the 5' end of HMOX1 and found a novel first exon 1a placing the two previously reported polymorphisms in intronic or exonic positions within the 5' untranslated region respectively. Expression of exon 1a can be induced in HepG2 hepatoma cells by hemin and is a repressor of heme oxygenase-1 translation as shown by luciferase reporter assays. Moreover, minigene approaches revealed that the quantitative outcome of alternative splicing within the 5' untranslated region is affected by the (GT)n microsatellite. This data supporting an extended HMOX1 gene model and provide further insights into expression regulation of heme oxygenase-1. Alternative splicing within the HMOX1 5' untranslated region contributes to translational regulation and is a mechanistic feature involved in the interplay between genetic variations, heme oxygenase-1 expression and disease outcome.

Association studies of the copy-number variable ß-defensin cluster on 8p23.1 in adenocarcinoma and chronic pancreatitis.

BACKGROUND: Human ß-defensins are a family of antimicrobial peptides located at the mucosal surface. Both sequence multi-site variations (MSV) and copy-number variants (CNV) of the defensin-encoding genes are associated with increased risk for various diseases, including cancer and inflammatory conditions such as psoriasis and acute pancreatitis. In a case-control study, we investigated the association between MSV in DEFB104 as well as defensin gene (DEF) cluster copy number (CN), and pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP). RESULTS: Two groups of PDAC (N=70) and CP (N=60) patients were compared to matched healthy control groups CARLA1 (N=232) and CARLA2 (N=160), respectively. Four DEFB104 MSV were haplotyped by PCR, cloning and sequencing. DEF cluster CN was determined by multiplex ligation-dependent probe amplification.Neither the PDAC nor the CP cohorts show significant differences in the DEFB104 haplotype distribution compared to the respective control groups CARLA1 and CARLA2, respectively.The diploid DEF cluster CN exhibit a significantly different distribution between PDAC and CARLA1 (Fisher's exact test P=0.027), but not between CP and CARLA2 (P=0.867). CONCLUSION: Different DEF cluster b CN distribution between PDAC patients and healthy controls indicate a potential protective effect of higher CNs against the disease.

Polymorphisms in the 3'-untranslated region of the CDH1 gene are a risk factor for primary gastric diffuse large B-cell lymphoma.

BACKGROUND: Primary gastric B-cell lymphomas arise from mucosa-associated lymphatic tissue (MALT) in patients with chronic Helicobacter pylori infection. We investigated whether germline variants in the CDH1 gene, coding for E-cadherin, genetically predispose patients to primary gastric B-cell lymphoma. DESIGN AND METHODS: Single marker analyses of the CDH1 gene were conducted in patients with primary gastric B-cell lymphoma (n=144), in patients with primary gastric high-grade lymphoma (n=61), and in healthy blood donors (n=361). Twelve single nucleotide polymorphisms were genotyped by TaqMan(®) technology. Allelic imbalance was tested by pyrosequencing and clone direct sequencing of heterozygote genomic and cDNA. Mutation detection was conducted around the poly-A signal of the CDH1 3'-untranslated region. The influence of the 3'-untranslated region on protein translation was determined by a luciferase reporter assay. RESULTS: Single marker analyses identified two single nucleotide polymorphisms in strong linkage disequilibrium located in the CDH1 3'-untranslated region. One of them was significantly associated with primary gastric diffuse large B-cell lymphomas after correction for multiple testing and this association was confirmed in an independent sample set. Patients homozygous for the rare T allele (rs1801026) had a 4.9-fold increased risk (95% CI: 1.5-15.9) of developing primary gastric diffuse large B-cell lymphoma. Allelic imbalance and reporter gene assays indicated a putative influence on mRNA stability and/or translational efficacy. CONCLUSIONS: We identified variants in CDH1 as the first potential genetic risk factors for the development of primary gastric diffuse large B-cell lymphomas. One of the potentially causative variants affects allelic CDH1 expression. These findings support the hypothesis that besides somatic alterations of B-cells, germline variants in the CDH1 gene contribute to a predisposition to the development of primary gastric diffuse large B-cell lymphomas.

Statistical inference of allelic imbalance from transcriptome data.

Next-generation sequencing and the availability of high-density genotyping arrays have facilitated an analysis of somatic and meiotic mutations at unprecedented level, but drawing sensible conclusions about the functional relevance of the detected variants still remains a formidable challenge. In this context, the study of allelic imbalance in intermediate RNA phenotypes may prove a useful means to elucidate the likely effects of DNA variants of unknown significance. We developed a statistical framework for the assessment of allelic imbalance in next-generation transcriptome sequencing (RNA-seq) data that requires neither an expression reference nor the underlying nuclear genotype(s), and that allows for allele miscalls. Using extensive simulation as well as publicly available whole-transcriptome data from European-descent individuals in HapMap, we explored the power of our approach in terms of both genotype inference and allelic imbalance assessment under a wide range of practically relevant scenarios. In so doing, we verified a superior performance of our methodology, particularly at low sequencing coverage, compared to the more simplistic approach of completely ignoring allele miscalls. Because the proposed framework can be used to assess somatic mutations and allelic imbalance in one and the same set of RNA-seq data, it will be particularly useful for the analysis of somatic genetic variation in cancer studies.

Posttranslational modification of human glyoxalase 1 indicates redox-dependent regulation.

BACKGROUND: Glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) are ubiquitously expressed cytosolic enzymes that catalyze the conversion of toxic alpha-oxo-aldehydes into the corresponding alpha-hydroxy acids using L-glutathione (GSH) as a cofactor. Human Glo1 exists in various isoforms; however, the nature of its modifications and their distinct functional assignment is mostly unknown. METHODOLOGY/PRINCIPAL FINDINGS: We characterized native Glo1 purified from human erythrocytes by mass spectrometry. The enzyme was found to undergo four so far unidentified posttranslational modifications: (i) removal of the N-terminal methionine 1, (ii) N-terminal acetylation at alanine 2, (iii) a vicinal disulfide bridge between cysteine residues 19 and 20, and (iv) a mixed disulfide with glutathione on cysteine 139. Glutathionylation of Glo1 was confirmed by immunological methods. Both, N-acetylation and the oxidation state of Cys(19/20), did not impact enzyme activity. In contrast, glutathionylation strongly inhibited Glo1 activity in vitro. The discussed mechanism for enzyme inhibition by glutathionylation was validated by molecular dynamics simulation. CONCLUSION/SIGNIFICANCE: It is shown for the first time that Glo1 activity directly can be regulated by an oxidative posttranslational modification that was found in the native enzyme, i.e., glutathionylation. Inhibition of Glo1 by chemical reaction with its co-factor and the role of its intramolecular disulfides are expected to be important factors within the context of redox-dependent regulation of glucose metabolism in cells.

Haplotyping and copy number estimation of the highly polymorphic human beta-defensin locus on 8p23 by 454 amplicon sequencing.

BACKGROUND: The beta-defensin gene cluster (DEFB) at chromosome 8p23.1 is one of the most copy number (CN) variable regions of the human genome. Whereas individual DEFB CNs have been suggested as independent genetic risk factors for several diseases (e.g. psoriasis and Crohn's disease), the role of multisite sequence variations (MSV) is less well understood and to date has only been reported for prostate cancer. Simultaneous assessment of MSVs and CNs can be achieved by PCR, cloning and Sanger sequencing, however, these methods are labour and cost intensive as well as prone to methodological bias introduced by bacterial cloning. Here, we demonstrate that amplicon sequencing of pooled individual PCR products by the 454 technology allows in-depth determination of MSV haplotypes and estimation of DEFB CNs in parallel. RESULTS: Six PCR products spread over approximately 87 kb of DEFB and harbouring 24 known MSVs were amplified from 11 DNA samples, pooled and sequenced on a Roche 454 GS FLX sequencer. From approximately 142,000 reads, approximately 120,000 haplotype calls (HC) were inferred that identified 22 haplotypes ranging from 2 to 7 per amplicon. In addition to the 24 known MSVs, two additional sequence variations were detected. Minimal CNs were estimated from the ratio of HCs and compared to absolute CNs determined by alternative methods. Concordance in CNs was found for 7 samples, the CNs differed by one in 2 samples and the estimated minimal CN was half of the absolute in one sample. For 7 samples and 2 amplicons, the 454 haplotyping results were compared to those by cloning/Sanger sequencing. Intrinsic problems related to chimera formation during PCR and differences between haplotyping by 454 and cloning/Sanger sequencing are discussed. CONCLUSION: Deep amplicon sequencing using the 454 technology yield thousands of HCs per amplicon for an affordable price and may represent an effective method for parallel haplotyping and CN estimation in small to medium-sized cohorts. The obtained haplotypes represent a valuable resource to facilitate further studies of the biomedical impact of highly CN variable loci such as the beta-defensin locus.

Alpha2-macroglobulin inhibits the malignant properties of astrocytoma cells by impeding beta-catenin signaling.

Targets that could improve the treatment of brain tumors remain important to define. This study of a transformation-associated isoform of alpha2-macroglobulin (A2M*) and its interaction with the low-density lipoprotein receptor-related protein-1 (LRP1) suggests a new mechanism for abrogating the malignant potential of astrocytoma cells. LRP1 bound A2M* found to be associated with an inhibition of tumor cell proliferation, migration, invasion, spheroid formation, and anchorage-independent growth. Transcriptional studies implicated effects on the Wnt/beta-catenin signaling pathway. Notably, LRP1 antibodies could phenocopy the effects of A2M*. Our findings suggest a pathway of tumor suppression in astrocytoma that might be tractable to therapeutic exploitation.

Curcumin inhibits glyoxalase 1: a possible link to its anti-inflammatory and anti-tumor activity.

BACKGROUND: Glyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumin's potency as an Glo1 inhibitor. METHODOLOGY/PRINCIPAL FINDINGS: Cultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (K(i) = 5.1+/-1.4 microM). Applying a whole blood assay, IC(50) values of pro-inflammatory cytokine release (TNF-alpha, IL-6, IL-8, IL-1beta) were found to be positively correlated with the K(i)-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1. CONCLUSIONS/SIGNIFICANCE: The results described herein provide new insights into curcumin's biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumin's potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent.

Carbonyl reductase 1 is a predominant doxorubicin reductase in the human liver.

A first step in the enzymatic disposition of the antineoplastic drug doxorubicin (DOX) is the reduction to doxorubicinol (DOX-OL). Because DOX-OL is less antineoplastic but more cardiotoxic than the parent compound, the individual rate of this reaction may affect the antitumor effect and the risk of DOX-induced heart failure. Using purified enzymes and human tissues we determined enzymes generating DOX-OL and interindividual differences in their activities. Human tissues express at least two DOX-reducing enzymes. High-clearance organs (kidney, liver, and the gastrointestinal tract) express an enzyme with an apparent Km of approximately 140 microM. Of six enzymes found to reduce DOX, Km values in this range are exhibited by carbonyl reductase 1 (CBR1) and aldo-keto reductase (AKR) 1C3. CBR1 is expressed in these three organs at higher levels than AKR1C3, whereas AKR1C3 has higher catalytic efficiency. However, inhibition constants for DOX reduction with 4-amino-1-tert-butyl-3-(2-hydroxyphenyl)pyrazolo[3,4-d]pyrimidine (an inhibitor that can discriminate between CBR1 and AKR1C3) were identical for CBR1 and human liver cytosol, but not for AKR1C3. These results suggest that CBR1 is a predominant hepatic DOX reductase. In cytosols from 80 human livers, the expression level of CBR1 and the activity of DOX reduction varied >70- and 22-fold, respectively, but showed no association with CBR1 gene variants found in these samples. Instead, the interindividual differences in CBR1 expression and activity may be mediated by environmental factors acting via recently identified xenobiotic response elements in the CBR1 promoter. The variability in the CBR1 expression may affect outcomes of therapies with DOX, as well as with other CBR1 substrates.