Ramóna Madácsi

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.

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.

Quantification of punctate iron sources using magnetic resonance phase

Iron‐mediated tissue damage is present in cerebrovascular and neurodegenerative diseases and neurotrauma. Brain microbleeds are often present in these maladies and are assuming increasing clinical importance. Because brain microbleeds present a source of pathologic iron to the brain, the noninvasive quantification of this iron pool is potentially valuable. Past efforts to quantify brain iron have focused on content estimation within distributed brain regions. In addition, conventional approaches using “magnitude” images have met significant limitations. In this study, a technique is presented to quantify the iron content of punctate samples using phase images. Samples are modeled as magnetic dipoles and phase shifts due to local dipole field perturbations are mathematically related to sample iron content and radius using easily recognized geometric features in phase images. Phantoms containing samples of a chitosan‐ferric oxyhydroxide composite (which serves as a mimic for hemosiderin) were scanned with a susceptibility‐weighted imaging sequence at 11.7 T. Plots relating sample iron content and radius to phase image features were compared to theoretical predictions. The primary result is the validation of the technique by the excellent agreement between theory and the iron content plot. This research is a potential first step toward quantification of punctate brain iron sources such as brain microbleeds. Magn Reson Med, 2010.

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.

Lipid droplet binding thalidomide analogs activate endoplasmic reticulum stress and suppress hepatocellular carcinoma in a chemically induced transgenic mouse model

BackgroundHepatocellular carcinoma (HCC) is the most frequent and aggressive primary tumor of the liver and it has limited treatment options.ResultsIn this study, we report the in vitro and in vivo effects of two novel amino-trifluoro-phtalimide analogs, Ac-915 and Ac-2010. Both compounds bind lipid droplets and endoplasmic reticulum membrane, and interact with several proteins with chaperone functions (HSP60, HSP70, HSP90, and protein disulfide isomerase) as determined by affinity chromatography and resonant waveguide optical biosensor technology. Both compounds inhibited protein disulfide isomerase activity and induced cell death of different HCC cells at sub or low micromolar ranges detected by classical biochemical end-point assay as well as with real-time label-free measurements. Besides cell proliferation inhibiton, analogs also inhibited cell migration even at 250 nM. Relative biodistribution of the analogs was analysed in native tissue sections of different organs after administration of drugs, and by using fluorescent confocal microscopy based on the inherent blue fluorescence of the compounds. The analogs mainly accumulated in the liver. The effects of Ac-915 and Ac-2010 were also demonstrated on the advanced stages of hepatocarcinogenesis in a transgenic mouse model of N-nitrosodiethylamine (DEN)-induced HCC. Significantly less tumor development was found in the livers of the Ac-915- or Ac-2010-treated groups compared with control mice, characterized by less liver tumor incidence, fewer tumors and smaller tumor size.ConclusionThese results imply that these amino-trifluoro-phthalimide analogs could serve potent clinical candidates against HCC alone or in combination with dietary polyunsaturated fatty acids.

Synthesis and Cytoprotective Characterization of 8-Hydroxyquinoline Betti Products

The 8-hydroxyquinoline pharmacophore scaffold has been shown to possess a range of activities as metal chelation, enzyme inhibition, cytotoxicity, and cytoprotection. Based on our previous findings we set out to optimize the scaffold for cytoprotective activity for its potential application in central nervous system related diseases. A 48-membered Betti-library was constructed by the utilization of formic acid mediated industrial-compatible coupling with sets of aromatic primary amines such as anilines, oxazoles, pyridines, and pyrimidines, with (hetero)aromatic aldehydes and 8-hydroxiquinoline derivatives. After column chromatography and re-crystallization, the corresponding analogues were obtained in yields of 13–90%. The synthesized analogs were optimized with the utilization of a cytoprotection assay with chemically induced oxidative stress, and the most active compounds were further tested in orthogonal assays, a real time cell viability method, a fluorescence-activated cell sorting (FACS)-based assay measuring mitochondrial membrane potential changes, and gene expression analysis. The best candidates showed potent, nanomolar activity in all test systems and support the need for future studies in animal models of central nervous system (CNS) disorders.