Alena Fučíková

Proteomic insights into chronic anthracycline cardiotoxicity.

Chronic anthracycline cardiotoxicity is a feared complication of cancer chemotherapy. However, despite several decades of primarily hypothesis-driven research, the molecular basis of this phenomenon remains poorly understood. The aim of this study was to obtain integrative molecular insights into chronic anthracycline cardiotoxicity and the resulting heart failure. Cardiotoxicity was induced in rabbits (daunorubicin 3mg/kg, weekly, 10weeks) and changes in the left ventricular proteome were analyzed by 2D-DIGE. The protein spots with significant changes (p<0.01, >1.5-fold) were identified using MALDI-TOF/TOF. Key data were corroborated by immunohistochemistry, qRT-PCR and enzyme activity determination and compared with functional, morphological and biochemical data. The most important alterations were found in mitochondria - especially in proteins crucial for oxidative phosphorylation, energy channeling, antioxidant defense and mitochondrial stress. Furthermore, the intermediate filament desmin, which interacts with mitochondria, was determined to be distinctly up-regulated and disorganized in its expression pattern. Interestingly, the latter changes reflected the intensity of toxic damage in whole hearts as well as in individual cells. In addition, a marked drop in myosin light chain isoforms, activation of proteolytic machinery (including the proteasome system), increased abundance of chaperones and proteins involved in chaperone-mediated autophagy, membrane repair as well as apoptosis were found. In addition, dramatic changes in proteins of basement membrane and extracellular matrix were documented. In conclusion, for the first time, the complex proteomic signature of chronic anthracycline cardiotoxicity was revealed which enhances our understanding of the basis for this phenomenon and it may enhance efforts in targeting its reduction.

Targeted Proteomics Driven Verification of Biomarker Candidates Associated with Breast Cancer Aggressiveness

Breast cancer is the most common and molecularly relatively well characterized malignant disease in women, however, its progression to metastatic cancer remains lethal for 78% of patients 5years after diagnosis. Novel markers could identify the high risk patients and their verification using quantitative methods is essential to overcome genetic, inter-tumor and intra-tumor variability and translate novel findings into cancer diagnosis and treatment. We recently identified 13 proteins associated with estrogen receptor, tumor grade and lymph node status, the key factors of breast cancer aggressiveness, using untargeted proteomics. Here we verified these findings in the same set of 96 tumors using targeted proteomics based on selected reaction monitoring with mTRAQ labeling (mTRAQ-SRM), transcriptomics and immunohistochemistry and validated in 5 independent sets of 715 patients using transcriptomics. We confirmed: (i) positive association of anterior gradient protein 2 homolog (AGR2) and periostin (POSTN) and negative association of annexin A1 (ANXA1) with estrogen receptor status; (ii) positive association of stathmin (STMN1), cofilin-1 (COF1), plasminogen activator inhibitor 1 RNA-binding protein (PAIRBP1) and negative associations of thrombospondin-2 (TSP2) and POSTN levels with tumor grade; and (iii) positive association of POSTN, alpha-actinin-4 (ACTN4) and STMN1 with lymph node status. This study highlights a panel of gene products that can contribute to breast cancer aggressiveness and metastasis, the understanding of which is important for development of more precise breast cancer treatment.

Plasma concentration of fibronectin is decreased in patients with hypertrophic cardiomyopathy.

OBJECTIVES To confirm the initial iTRAQ-based proteomic findings related to the plasma fibronectin level and hypertrophic cardiomyopathy using an antibody-based assay. METHODS The iTRAQ technique was used for the discovery proteomic analysis of the pooled plasma samples. The ELISA was used for verification of fibronectin plasma concentration in individual samples. Additional five related plasma proteins were assessed: matrix metalloproteinase 2, matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 1, tissue inhibitor of metalloproteinase 2 and brain natriuretic peptide. RESULTS The plasma fibronectin level in patients with hypertrophic cardiomyopathy was significantly lower in comparison to the healthy subjects [215.5±47.3μg/ml, n=17 vs. 376.7±134.8μg/ml, n=17; p<0.0001]. CONCLUSION In this study we present and confirm our initial proteomic findings and we suggest fibronectin as a potential indicator of an extracellular matrix remodeling related to the hypertrophic cardiomyopathy.

Effect of selenium-enriched defatted rape seeds on tissue cadmium and essential elements utilization in rats

The effect of selenium-enriched defatted rapeseeds (DRS) added to the diet on Se uptake by rat organisms (whole blood, liver, kidney) was investigated in model conditions. Additionally, the response of other essential trace and major mineral elements (Cu, Ca, Fe, K, Mg, Mn, P, S, and Zn) in rat organisms on selenium and/or DRS addition was assessed. The experimental diets for the individual experimental groups were prepared as follows: group DRS 0 (control diet), group DRS 30 (30% of soybean meal in the diet replaced with DRS), group DRS60 (60% of soybean meal in the diet replaced with DRS), and group DRS100 (100% of soybean meal in the diet replaced with DRS), either unenriched or naturally fortified with Se. Whereas Se content in the blood and liver of the animals remained unchanged, increased Se levels were observed in kidney of the selenized DRS100 group compared to the others. Moreover, the results showed decreasing Cd content in the rat liver with increasing Se content in the diet, confirming antagonism of these elements. Concerning the essential elements, the application of the selenized DRS did not result in any serious imbalance in the utilization of these elements. In contrast, Se addition seems to be helpful for the improved utilization of essential elements such as P, S, and Zn, compared to the DRS-containing diet without Se fortification.

Plasma glycogen phosphorylase BB is associated with pulmonary artery wedge pressure and left ventricle mass index in patients with hypertrophic cardiomyopathy.

Radek Pudil*, Martina Vašatová, Juraj Lenčo, Miloš Tichý, Vı́t Řeháček, Alena Fučı́ková, Jan M. Horáček, Jan Vojáček, Miloslav Pleskot, Jiřı́ Stulı́k and Vladimı́r Palička 1 Faculty of Medicine Hradec Kralove, First Department of Medicine, Charles University Prague, Hradec Kralove, Czech Republic 2 Faculty of Medicine Hradec Kralove, Institute of Clinical Biochemistry and Diagnostics, Charles University Prague, Hradec Kralove, Czech Republic 3 Faculty of Military Health Sciences, Department of Molecular Pathology, University of Defence, Hradec Kralove, Czech Republic

Proteomic Methods of Detection and Quantification of Protein Toxins

Biological toxins are a heterogeneous group of compounds that share commonalities with biological and chemical agents. Among them, protein toxins represent a considerable, diverse set. They cover a broad range of molecular weights from less than 1000 Da to more than 150 kDa. This review aims to compare conventional detection methods of protein toxins such as in vitro bioassays with proteomic methods, including immunoassays and mass spectrometry-based techniques and their combination. Special emphasis is given to toxins falling into a group of selected agents, according to the Centers for Disease Control and Prevention, such as Staphylococcal enterotoxins, Bacillus anthracis toxins, Clostridium botulinum toxins, Clostridium perfringens epsilon toxin, ricin from Ricinus communis, Abrin from Abrus precatorius or control of trade in dual-use items in the European Union, including lesser known protein toxins such as Viscumin from Viscum album. The analysis of protein toxins and monitoring for biological threats, i.e., the deliberate spread of infectious microorganisms or toxins through water, food, or the air, requires rapid and reliable methods for the early identification of these agents.

The Response of Macro- and Micronutrient Nutrient Status and Biochemical Processes in Rats Fed on a Diet with Selenium-Enriched Defatted Rapeseed and/or Vitamin E Supplementation

The response of nutrient status and biochemical processes in (i) Wistar and (ii) spontaneously hypertensive (SHR) rats upon dietary intake of selenium- (Se-) enriched defatted rapeseed (DRS) and/or vitamin E fortification was examined to assess the health benefit of DRS in animal nutrition. Twenty-four individuals of each type of rat were used: The control group was fed with an untreated diet (Diet A). In Diets B and C, soybean meal was replaced with defatted DRS, which comprised 14% of the total diet. The selenized DRS application resulted in ~3-fold increase of Se content in the diet. Diet C was also fortified with the addition of vitamin E, increasing the natural content by 30%. The Se content of the blood and kidneys tended to increase in the DRS groups, where the changes were significant (P < 0.05) only in the case of SHR rats. The iodine (I) content and the proportion of iodide in rat livers indicated a lower transformation rate of iodide into organoiodine compounds compared to the control. Slight and ambiguous alterations in the antioxidative response of the rat were observed in the DRS groups, but the addition of vitamin E to the diet helped to moderate these effects.

Vascular Endothelial Growth Factor Is Associated with the Morphologic and Functional Parameters in Patients with Hypertrophic Cardiomyopathy

Background. Hypertrophic cardiomyopathy (HCM) is mostly autosomal dominant disease of the myocardium, which is characterized by myocardial hypertrophy. Vascular endothelial growth factor (VEGF) is involved in myocyte function, growth, and survival. The aim of study was to analyze the clinical significance of VEGF in structural and functional changes in patient with HCM. Methods. In a group of 21 patients with nonobstructive HCM, we assessed serum VEGF and analyzed its association with morphological and functional parameters. Compared to healthy controls, serum VEGF was increased: 199 (IQR: 120.4–260.8) ng/L versus 20 (IQR: 14.8–37.7) ng/L, P < 0.001. VEGF levels were associated with left atrium diameter (r = 0.51, P = 0.01), left ventricle ejection fraction (r = −0.56, P = 0.01), fractional shortening (r = −0.54, P = 0.02), left ventricular mass (r = 0.61, P = 0.03), LV mass index (r = 0.46, P = 0.04), vena cava inferior diameter (r = 0.65, P = 0.01), and peak gradient of tricuspid regurgitation (r = 0.46, P = 0.03). Conclusions. Increased VEGF level is associated with structural and functional parameters in patients with HCM and serves as a potential tool for diagnostic process of these patients.