Viola Tamási

Changes in miRNA expression in solid tumors: an miRNA profiling in melanomas.

In this review, we briefly described microRNA biogenesis, function and the principal approaches for studying the function of microRNAs (miRNA) in solid cancers. There are currently hundreds of confirmed miRNAs in humans, and computational predictions suggest that the total count might be more than thousand. The regulatory nature of miRNAs combined with the large number of presumptive target genes suggests that they are essential regulators of a wide range of cellular processes. To illustrate the importance of miRNA-mediated regulation in solid cancer some confirmed interactions were collected. Their relevance is described in detail in melanomas from the aspect of diagnosis, the potential application of miRNAs as biomarkers and as potential therapeutic tools.

A novel pregnane X receptor and S14‐mediated lipogenic pathway in human hepatocyte

The pregnane X receptor (PXR) initially isolated as a nuclear receptor regulating xenobiotic and drug metabolism and elimination, seems to play an endobiotic role by affecting lipid homeostasis. In mice, PXR affects lipid homeostasis and increases hepatic deposit of triglycerides. In this study, we show that, in human hepatocyte, PXR activation induces an increase of de novo lipogenesis through the up‐regulation of S14. S14 was first identified as a thyroid‐responsive gene and is known to transduce hormone‐related and nutrient‐related signals to genes involved in lipogenesis through a molecular mechanism not yet elucidated. We demonstrate that S14 is a novel transcriptional target of PXR. In addition, we report an increase of fatty acid synthase (FASN) and adenosine triphosphate citrate lyase genes expression after PXR activation in human hepatocyte, leading to an increase of fatty acids accumulation and de novo lipogenesis. RNA interference of the expression of S14 proportionally decreases the FASN induction, whereas S14 overexpression in human hepatic cells provokes an increase of fatty acids accumulation and lipogenesis. These results demonstrate for the first time that xenobiotic or drug‐activated PXR promote aberrant hepatic de novo lipogenesis via activation of the nonclassical S14 pathway. In addition, these data suggest that the up‐regulation of S14 by PXR may promote aberrant hepatic lipogenesis and hepatic steatosis in human hepatocytes. (HEPATOLOGY 2009.)

Meta-analysis of adrenocortical tumour genomics data: Novel pathogenic pathways revealed

Sporadic adrenocortical tumours are common, but their pathogenesis is poorly elucidated. In this study, we present a meta-analysis and review of gene expression microarray and comparative genome hybridization (CGH) studies performed to date on these tumours, including our own data. Data of whole genome microarray studies from altogether 164 tumours (97 benign, 67 malignant) and 18 normal tissues were reclassified and reanalysed. Significant gene sets and cytogenetic changes from publications without available genomic data were also examined including 269 benign, 215 malignant tumour and 30 normal tissues. In our experimental study, 11 tumour and four normal samples were analysed by parallel mRNA and CGH profiling. Data were examined by an integrative bioinformatics approach (GeneSpring, Gene Set Enrichment Analysis and Ingenuity Pathway Analysis softwares) searching for common gene expression changes and paralleling chromosome aberrations. Both meta-analysis of available mRNA and CGH profiling data and our experimental study revealed three major pathogenetic pathways: (1) cell cycle, (2) retinoic acid signalling (including lipopolysaccharide/Toll like receptor 4 pathway), (3) complement system and antigen presentation. These pathways include novel, previously undescribed pathomechanisms of adrenocortical tumours, and associated gene products may serve as diagnostic markers of malignancy and therapeutic targets.

Dehydroepiandrosterone Induces Human CYP2B6 through the Constitutive Androstane Receptor

Dehydroepiandrosterone (DHEA), the major precursor of androgens and estrogens, has several beneficial effects on the immune system, on memory function, and in modulating the effects of diabetes, obesity, and chemical carcinogenesis. Treatment of rats with DHEA influences expression of cytochrome P450 (P450) genes, including peroxisome proliferator-activated receptor α (PPARα)- and pregnane X receptor (PXR)-mediated induction of CYP4As and CYP3A23, and suppression of CYP2C11. DHEA treatment elevated the expression and activities of CYP3A4, CYP2C9, CYP2C19, and CYP2B6 in primary cultures of human hepatocytes. Induction of CYP3A4 in human hepatocytes was consistent with studies in rats, but induction of CYP2Cs was unexpected. The role of PXR in this response was studied in transient transfection assays. DHEA activated hPXR in a concentration-dependent manner. Because CYP2B6 induction by DHEA in human hepatocytes might involve either PXR or constitutive androstane receptor (CAR) activation, we performed experiments in primary hepatocytes from CAR knockout mice and observed that CAR was required for maximal induction of Cyp2b10 by DHEA. Furthermore, CAR-mediated Cyp2b10 induction by DHEA was inhibited by the inverse agonist of CAR, androstanol (5α-androstan-3α-ol). Further evidence for CAR activation was provided by cytoplasmic/nuclear transfer of CAR upon DHEA treatment. Elucidation of CAR activation and subsequent induction of CYP2B6 by DHEA presented an additional mechanism by which the sterol can modify the expression of P450s. The effect of DHEA on the activation of the xenosensors PPARα, PXR, and CAR, and the consequent potential for adverse drug/toxicant interactions should be considered in humans treated with this nutriceutical agent.

Differential detergent sensitivity of extracellular vesicle subpopulations.

Extracellular vesicles (including exosomes, microvesicles and apoptotic bodies) are currently attracting rapidly increasing attention from various fields of biology due to their ability to carry complex information and act as autocrine, paracrine and even endocrine intercellular messengers. In the present study we investigated the sensitivity of size-based subpopulations of extracellular vesicles to different concentrations of detergents including sodium dodecyl sulphate, Triton X-100, Tween 20 and deoxycholate. We determined the required detergent concentration that lysed each of the vesicle subpopulations secreted by Jurkat, THP-1, MiaPaCa and U937 human cell lines. We characterized the vesicles by tunable resistive pulse sensing, flow cytometry and transmission electron microscopy. Microvesicles and apoptotic bodies were found to be more sensitive to detergent lysis than exosomes. Furthermore, we found evidence that sodium dodecyl sulphate and Triton X-100 were more effective in vesicle lysis at low concentrations than deoxycholate or Tween 20. Taken together, our data suggest that a combination of differential detergent lysis with tunable resistive pulse sensing or flow cytometry may prove useful for simple and fast differentiation between exosomes and other extracellular vesicle subpopulations as well as between vesicular and non-vesicular structures.

Interethnic differences of CYP2C9 alleles in healthy Hungarian and Roma population samples: Relationship to worldwide allelic frequencies

CYP2C9 gene polymorphisms are widely studied in several ethnic groups, however they are less known in the Roma population. The aim of this work was to study the ethnic differences of the CYP2C9 allele distribution in a healthy Roma population in order to compare them with a healthy Hungarian population. A total of 535 Hungarian and 465 Roma volunteers were genotyped for the CYP2C9*2 (Arg144Cys) and CYP2C9*3 (Ile359Leu) allelic variants by PCR-RFLP assay. The frequencies of the CYP2C9*1, *2 and *3 alleles in the Hungarian population were 0.787, 0.125, and 0.088 and in Roma 0.727, 0.118, and 0.155, respectively. We found a significant difference in CYP2C9*3 prevalence between the Hungarian and Roma populations, which have therapeutic consequences (p<0.005). The distribution of *1/*1, *1/*2, *1/*3, *2/*2, *2/*3, and *3/*3 genotypes in Hungarians were 0.620, 0.195, 0.139, 0.021, 0.015, and 0.011, while in Roma were 0.533, 0.168, 0.219, 0.011, 0.047, and 0.022, respectively. A significant difference was found between the Hungarian and Roma populations regarding the *1/*1, *1/*3 and the *2/*3 (p<0.005) genotypes. This is the first study to investigate the polymorphisms of CYP2C9 gene in the two largest populations in Hungary, healthy Hungarians and Roma. The prevalence of variant CYP2C9 alleles in the Hungarian population is similar to that observed in other European populations. In contrast, the Roma population differs from Hungarians, from most of other Caucasian groups, and from Indians in the incidence of CYP2C9 common variants. The difference in allele distribution patterns between the two populations studied has therapeutic implications as it influences the optimization of therapies.

Modulation of Receptor Phosphorylation Contributes to Activation of Peroxisome Proliferator Activated Receptor α by Dehydroepiandrosterone and Other Peroxisome Proliferators

Dehydroepiandrosterone (DHEA), a C19 human adrenal steroid, activates peroxisome proliferator-activated receptor α (PPARα) in vivo but does not ligand-activate PPARα in transient transfection experiments. We demonstrate that DHEA regulates PPARα action by altering both the levels and phosphorylation status of the receptor. Human hepatoma cells (HepG2) were transiently transfected with the expression plasmid encoding PPARα and a plasmid containing two copies of fatty acyl coenzyme oxidase (FACO) peroxisome-proliferator activated receptor responsive element consensus oligonucleotide in a luciferase reporter gene. Nafenopin treatment increased reporter gene activity in this system, whereas DHEA treatment did not. Okadaic acid significantly decreased nafenopin-induced reporter activity in a concentration-dependent manner. Okadaic acid treatment of primary rat hepatocytes decreased both DHEA- and nafenopin-induced FACO activity in primary rat hepatocytes. DHEA induced both PPARα mRNA and protein levels, as well as PP2A message in primary rat hepatocytes. Western blot analysis showed that the serines at positions 12 and 21 were rapidly dephosphorylated upon treatment with DHEA and nafenopin. Results using specific protein phosphatase inhibitors suggested that protein phosphatase 2A (PP2A) is responsible for DHEA action, and protein phosphatase 1 might be involved in nafenopin induction. Mutation of serines at position 6, 12, and 21 to an uncharged alanine residue significantly increased transcriptional activity, whereas mutation to negative charged aspartate residues (mimicking receptor phosphorylation) decreased transcriptional activity. DHEA action involves induction of PPARα mRNA and protein levels as well as increased PPARα transcriptional activity through decreasing receptor phosphorylation at serines in the AF1 region.

Cytochromes P450 Catalyze the Reduction of α,β-Unsaturated Aldehydes

The metabolism of α,β-unsaturated aldehydes, e.g., 4-hydroxynonenal, involves oxidation to carboxylic acids, reduction to alcohols, and glutathionylation to eventually form mercapturide conjugates. Recently, we demonstrated that P450s can oxidize aldehydes to carboxylic acids, a reaction previously thought to involve aldehyde dehydrogenase. When recombinant cytochrome P450 3A4 was incubated with 4-hydroxynonenal, O(2), and NADPH, several products were produced, including 1,4-dihydroxynonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), and an unknown metabolite. Several P450s catalyzed the reduction reaction in the order (human) P450 2B6 ≅ P450 3A4 > P450 1A2 > P450 2J2 > (mouse) P450 2c29. Other P450s did not catalyze the reduction reaction (human P450 2E1 and rabbit P450 2B4). Metabolism by isolated rat hepatocytes showed that HNA formation was inhibited by cyanamide, while DHN formation was not affected. Troleandomycin increased HNA production 1.6-fold while inhibiting DHN formation, suggesting that P450 3A11 is a major enzyme involved in rat hepatic clearance of 4-HNE. A fluorescent assay was developed using 9-anthracenealdehyde to measure both reactions. Feeding mice a diet containing t-butylated hydroxyanisole increased the level of both activities with hepatic microsomal fractions but not proportionally. Miconazole (0.5 mM) was a potent inhibitor of these microsomal reduction reactions, while phenytoin and α-naphthoflavone (both at 0.5 mM) were partial inhibitors, suggesting the role of multiple P450 enzymes. The oxidative metabolism of these aldehydes was inhibited >90% in an Ar or CO atmosphere, while the reductive reactions were not greatly affected. These results suggest that P450s are significant catalysts of the reduction of α,β-unsaturated aldehydes in the liver.

Role of xenobiotic metabolism in cancer: involvement of transcriptional and miRNA regulation of P450s

Cytochrome P450 enzymes (P450s) are important targets in cancer, due to their role in xenobiotic metabolism. Since P450s are the “bridges” between the environment and our body, their function can be linked in many ways to carcinogenesis: they activate dietary and environmental components to ultimate carcinogens (i), the cancer tissue maintains its drug resistance with altered expression of P450s (ii), P450s metabolize (sometimes activate) drugs used for cancer treatment (iii) and they are potential targets for anticancer therapy (iiii). These highly polymorphic enzymes are regulated at multiple molecular levels. Regulation is as important as genetic difference in the existing individual variability in P450 activity. In this review, examples of the transcriptional (DNA methylation, histone modification, modulation by xenosensors) and post-transcriptional (miRNA) regulation will be presented and thereby introduce potential molecular targets at which the metabolism of anticancer drugs, the elimination of cancerogenes or the progress of carcinogenesis could be affected.

Transcriptional Activation of PPARα by Phenobarbital in the Absence of CAR and PXR

The nuclear receptors CAR (constitutive androstane receptor) and PXR (pregnane X receptor) mediate the effects of phenobarbital on gene transcription. To investigate the relative contribution of these nuclear receptors to the expression of specific genes we studied the effect of phenobarbital in livers of wild type, CAR(-/-), PXR(-/-) and CAR/PXR(-/-) knockout mice. Spotted Steroltalk v1 cDNA arrays were applied containing probes for genes involved in drug metabolism, sterol biosynthesis, steroid synthesis/transport and heme synthesis. In the absence of CAR and PXR, phenobarbital unexpectedly induced mRNAs of several nuclear receptors, including PPARalpha and its target genes Cyp4a10 and Cyp4a14. Interestingly, in primary cultures of hepatocytes isolated from CAR/PXR(-/-) knockout mice, phenobarbital increased HNF-4alpha levels. In further experiments in these hepatocyte cultures we provide evidence that phenobarbital directly induces transcription of the PPARalpha gene via its HNF-4alpha response element, and indirectly by lack of inhibitory crosstalk of AMPK, CAR and PXR with HNF-4alpha. Our results provide further insight into CAR and PXR-independent effects of phenobarbital and the crosstalk between different nuclear receptor signaling pathways.