Liliána Z. Fehér

MicroRNA profile of polyunsaturated fatty acid treated glioma cells reveal apoptosis-specific expression changes

BackgroundPolyunsaturated fatty acids (PUFAs) such as γ-linolenic acid (GLA), arachidonic acid (AA) and docosahexaenoic acid (DHA) have cytotoxic action on glioma cells.ResultsWe evaluated the cytotoxic action of GLA, AA and DHA on glioma cells with specific reference to the expression of miRNAs. Relative expression of miRNAs were assessed by using high throughput nanocapillary real-time PCR. Most of the miRNA target genes that showed altered expression could be classified as apoptotic genes and were up-regulated by PUFA or temozolomide treatment, while similar treatments resulted in repression of the corresponding mRNAs, such as cox2, irs1, irs2, ccnd1, itgb3, bcl2, sirt1, tp53inp1 and k-ras.ConclusionsOur results highlight involvement of miRNAs in the induction of apoptosis in glioma cells by fatty acids and temozolomide.

A cell-microelectronic sensing technique for the screening of cytoprotective compounds.

In recent years, a new cell-based high throughput paradigm has emerged, which seeks to identify novel, pharmacologically active cytoprotective compounds. The essence of this approach is to create experimental models of cell injury relevant for a particular disease by establishing in vitro cell-based models, followed by high-throughput testing of compounds that affect the cellular response in a desired manner. Prior approaches typically used simple end-point analyses. To assess the cytoprotective effects of novel drug candidates in real-time, we have applied a cell-microelectronic sensing technique (RT-CES), which measures changes in the impedance of individual microelectronic wells that correlates linearly with cell index (reflecting cell number, adherence and cell growth), thereby allowing the continuous determination of cell viability during oxidative stress. In vitro cytotoxicity was elicited by hydrogen peroxide in myocytes (H9c2) and hepatocytes (Hep3B). Cells were post-treated at 30 min with various reference molecules and novel cytoprotective compounds. Cytoprotection detected in the RT-CES system correlated well with the results of two classical end-point-based methods (improvement in MTT and reduction of LDH release). The RT-CES method, when used as described in the current report, is suitable for the screening of molecular libraries to identify molecules or molecule combinations that attenuate oxidative stress-induced cell damage.

Impact of Venlafaxine on Gene Expression Profile in Lymphocytes of the Elderly with Major Depression – Evolution of Antidepressants and the Role of the “Neuro-Immune” System

Antidepressive drugs offer considerable symptomatic relief in mood disorders and, although commonly discovered by screening with single biological targets, most interact with multiple receptors and signaling pathways. Antidepressants require a treatment regimen of several weeks before clinical efficacy is achieved in patient populations. While the biochemical mechanisms underlying the delayed temporal profile remain unclear, molecular adaptations over time are likely involved. The selective serotonin and noradrenaline reuptake inhibitor, venlafaxine, offers a dual antidepressive action. Its pharmacological behavior, however, is unknown at the genetic level, and it is difficult to monitor in human brain samples. Because the hypothalamic-pituitary-adrenal axis is often severely disrupted in mood disorders, lymphocytes may serve as models of neuropsychiatric conditions. As such, we examined the role of venlafaxine on the gene expression profile of human lymphocytes. DNA microarray was used to measure the expression patterns of multiple genes in human lymphocytes from depressed patients treated with this mood stabilizer. In this self-controlled study, RNAs of control and treated samples were purified, converted into cDNA and labeled with either Cy3 or Cy5, mixed and hybridized to DNA microarrays containing human oligonucleotides corresponding to more than 8,000 genes. Genes that were differentially regulated in response to treatment were selected for follow up on the basis on novelty, gene identity, and level of over-expression/repression, and selected transcripts were profiled by real-time PCR (data have been normalized to β-actin). Using software analysis of the microarray data, a number of transcripts were differentially expressed between control and treated samples, of which only 57 were found to significantly vary with the “P” value of 0.05 or lower as a result of exposure to venlafaxine. Of these, 31 genes were more highly expressed and 26 transcripts were found to be significantly less abundant. Most selected genes were verified with QRT-PCR to alter. As such, independent verification using QRT-PCR demonstrated the reliability of the method. Genes implicated in ionic homeostasis were differentially expressed, as were genes associated with cell survival, neural plasticity, signal transduction, and metabolism. Understanding how gene expression is altered over a clinically relevant time course of administration of venlafaxine may provide insight into the development of antidepressant efficacy as well as the underlying pathology of mood disorders. These changes in lymphocytes are thought to occur in the brain, and a “neuro-immune system” is proposed by this study.

Polyunsaturated fatty acids synergize with lipid droplet binding thalidomide analogs to induce oxidative stress in cancer cells.

BackgroundCytoplasmic lipid-droplets are common inclusions of eukaryotic cells. Lipid-droplet binding thalidomide analogs (2,6-dialkylphenyl-4/5-amino-substituted-5,6,7-trifluorophthalimides) with potent anticancer activities were synthesized.ResultsCytotoxicity was detected in different cell lines including melanoma, leukemia, hepatocellular carcinoma, glioblastoma at micromolar concentrations. The synthesized analogs are non-toxic to adult animals up to 1 g/kg but are teratogenic to zebrafish embryos at micromolar concentrations with defects in the developing muscle. Treatment of tumor cells resulted in calcium release from the endoplasmic reticulum (ER), induction of reactive oxygen species (ROS), ER stress and cell death. Antioxidants could partially, while an intracellular calcium chelator almost completely diminish ROS production. Exogenous docosahexaenoic acid or eicosapentaenoic acid induced calcium release and ROS generation, and synergized with the analogs in vitro, while oleic acid had no such an effect. Gene expression analysis confirmed the induction of ER stress-mediated apoptosis pathway components, such as GADD153, ATF3, Luman/CREB3 and the ER-associated degradation-related HERPUD1 genes. Tumor suppressors, P53, LATS2 and ING3 were also up-regulated in various cell lines after drug treatment. Amino-phthalimides down-regulated the expression of CCL2, which is implicated in tumor metastasis and angiogenesis.ConclusionsBecause of the anticancer, anti-angiogenic action and the wide range of applicability of the immunomodulatory drugs, including thalidomide analogs, lipid droplet-binding members of this family could represent a new class of agents by affecting ER-membrane integrity and perturbations of ER homeostasis.

A second field metachronous Merkel cell carcinoma of the lip and the palatine tonsil confirmed by microarray-based comparative genomic hybridisation

Merkel cell carcinoma was diagnosed in a 79-year-old Caucasian woman. The tumour was localised to the upper lip and was in stage T2. After successful cryosurgery and a 7-year tumour-free period, a new tumour developed in her palatine tonsil. Histologically and immunohistochemically, this resembled the tumour in the lip. The regional lymph nodes were devoid of metastasis. The paraffin-embedded material of the two tumours and the unaffected lymphatic tissue were analysed with DNA microarrays for comparative genomic hybridisation to assess the genetic relationship of the tumours. In both tumours, regions on 2p and 10p were commonly over-represented, while 41 regions on chromosomes 1–4, 6, 8–9, 11 and 14–22 were commonly under-represented. Chromosomes 1, 3, 4, 16–18 and X were most frequently involved in the DNA losses. In gene copy numbers in the two tumours, 31 chromosome locations were found to be differently affected. The partly similar and partly different molecular patterns indicated a genetic relationship between the tumours and excluded the possibility that the tonsillar tumour was a metastasis. The findings suggest that a genetically altered field was the reason for the development of the tonsillar cancer; thus, it can be regarded pathogenetically as a second field tumour.

Amplification of thymosin beta 10 and AKAP13 genes in metastatic and aggressive papillary thyroid carcinomas

Papillary thyroid carcinoma (PTC) is the most common well-differentiated thyroid cancer. Although the great majority of the cases exhibit an indolent clinical course, some of them develop local invasion with distant metastasis, and a few cases transform into undifferentiated/anaplastic thyroid carcinoma with a rapidly lethal course. To identify gene copy number alterations predictive of metastatic potential or aggressive transformation, array-based comparative genomic hybridization (CGH-array) was performed in 43 PTC cases. Formalin-fixed and paraffin-embedded samples from primary tumours of 16 cases without metastasis, 14 cases with only regional lymph node metastasis, and 13 cases with distant metastasis, recurrence or extrathyroid extension were analysed. The CGH-array and confirmatory quantitative real-time PCR results identified the deletion of the EIF4EBP3 and TRAK2 gene loci, while amplification of thymosin beta 10 (TB10) and Tre-2 oncogene regions were observed as general markers for PTC. Although there have been several studies implicating TB10 as a specific marker based on gene expression data, our study is the first to report on genomic amplification. Although no significant difference could be detected between the good and bad prognosis cases in the A-kinase anchor protein 13 (AKAP13) gene region, it was discriminative markers for metastasis. Amplification in the AKAP13 region was demonstrated in 42.9% and 15.4% of the cases with local or with distant metastasis, respectively, while no amplification was detected in non-metastatic cases. AKAP13 and TB10 regions may represent potential new genomic markers for PTC and cancer progression.

High-Density Real-Time PCR-Based in Vivo Toxicogenomic Screen to Predict Organ-Specific Toxicity

Toxicogenomics, based on the temporal effects of drugs on gene expression, is able to predict toxic effects earlier than traditional technologies by analyzing changes in genomic biomarkers that could precede subsequent protein translation and initiation of histological organ damage. In the present study our objective was to extend in vivo toxicogenomic screening from analyzing one or a few tissues to multiple organs, including heart, kidney, brain, liver and spleen. Nanocapillary quantitative real-time PCR (QRT-PCR) was used in the study, due to its higher throughput, sensitivity and reproducibility, and larger dynamic range compared to DNA microarray technologies. Based on previous data, 56 gene markers were selected coding for proteins with different functions, such as proteins for acute phase response, inflammation, oxidative stress, metabolic processes, heat-shock response, cell cycle/apoptosis regulation and enzymes which are involved in detoxification. Some of the marker genes are specific to certain organs, and some of them are general indicators of toxicity in multiple organs. Utility of the nanocapillary QRT-PCR platform was demonstrated by screening different references, as well as discovery of drug-like compounds for their gene expression profiles in different organs of treated mice in an acute experiment. For each compound, 896 QRT-PCR were done: four organs were used from each of the treated four animals to monitor the relative expression of 56 genes. Based on expression data of the discovery gene set of toxicology biomarkers the cardio- and nephrotoxicity of doxorubicin and sulfasalazin, the hepato- and nephrotoxicity of rotenone, dihydrocoumarin and aniline, and the liver toxicity of 2,4-diaminotoluene could be confirmed. The acute heart and kidney toxicity of the active metabolite SN-38 from its less toxic prodrug, irinotecan could be differentiated, and two novel gene markers for hormone replacement therapy were identified, namely fabp4 and pparg, which were down-regulated by estradiol treatment.

Curcumin and Its Analogue Induce Apoptosis in Leukemia Cells and Have Additive Effects with Bortezomib in Cellular and Xenograft Models

Combination therapy of bortezomib with other chemotherapeutics is an emerging treatment strategy. Since both curcumin and bortezomib inhibit NF-κB, we tested the effects of their combination on leukemia cells. To improve potency, a novel Mannich-type curcumin derivative, C-150, was synthesized. Curcumin and its analogue showed potent antiproliferative and apoptotic effects on the human leukemia cell line, HL60, with different potency but similar additive properties with bortezomib. Additive antiproliferative effects were correlated well with LPS-induced NF-κB inhibition results. Gene expression data on cell cycle and apoptosis related genes, obtained by high-throughput QPCR, showed that curcumin and its analogue act through similar signaling pathways. In correlation with in vitro results similar additive effect could be obsereved in SCID mice inoculated systemically with HL60 cells. C-150 in a liposomal formulation given intravenously in combination with bortezomib was more efficient than either of the drugs alone. As our novel curcumin analogue exerted anticancer effects in leukemic cells at submicromolar concentration in vitro and at 3 mg/kg dose in vivo, which was potentiated by bortezomib, it holds a great promise as a future therapeutic agent in the treatment of leukemia alone or in combination.

Gene and protein expression changes in response to normoxic perfusion in mouse hearts

INTRODUCTION Although crystalloid-perfused isolated heart models are widely used in cardiovascular research, there are several limitations of these techniques. Changes in cardiac gene expression pattern due to normoxic perfusion itself have not been studied, despite its potential importance to provide useful information on limitations of this model. Therefore, here we investigated the time-dependent effect of normoxic, normothermic perfusion on global gene expression at mRNA and protein levels. METHODS Hearts from male CFLP mice were perfused according to the Langendorff technique. We assessed relative gene expression changes by DNA microarray analysis of 8000 genes after 0, 60 and 120 min perfusion. RESULTS Twelve genes exhibited significant up-regulation and 27 showed repression in hearts perfused for 60 or 120 min as compared to 0 min controls. Expression changes of 17 selected genes were verified and an additional 19 genes were examined by real-time quantitative PCR. Genes with altered expression included those coding for Creatin kinase, Lactate dehydrogenase, Voltage-dependent anion channel 1, a Disintegrin and Metalloprotease domain 3, Integrin alpha 7, Long-chain acyl-CoA dehydrogenase, Casein kinase II, Ketohexokinase, Chloride ion current inducer protein, Matrix metalloproteinase 2 and 9, Superoxide dismutases and Nitric oxide synthases, etc. DISCUSSION Our results show that normoxic crystalloid perfusion itself results in time-dependent changes in cardiac gene expression which should be considered when designing ex vivo perfusion protocols in the mouse heart to mimic cardiac pathologies as many of these genes have been suspected to influence several cardiovascular diseases.

Aromatic sulfonamides containing a condensed piperidine moiety as potential oxidative stress-inducing anticancer agents.

A 30-membered piperidine ring-fused aromatic sulfonamide library was synthetized, including N-arylsulfonyl 1,2,3,4-tetrahydroquinolines, 1,2,3,4-tetrahydroisoquinolines and 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indoles. The compounds induced oxidative stress and glutathione depletion in HT168 melanoma and K562 leukemia cells and in micromolar concentrations exerted cytotoxic effects. Among the tested sulfonamides, compounds 21, 22, 23, 35 and 41 exhibited 100% cytotoxic effects with low (< 10 µM) EC50 values on K562 cells. The cytotoxicity of lead compound 22 was investigated in 24 different cancer cell lines, and it was found to be active against leukemia, melanoma, glioblastoma, and liver, breast and lung cancer cells, as confirmed by classical biochemical and holographic microscopic analyses.