Browsing by Author "Suboticki, Tijana (55933499100)"

Increased angiogenesis in BCR-ABL1 negative myeloproliferative neoplasms (MPNs) has been recognized, but its connection with clinical and molecular markers needs to be defined. The aims of study were to (1) assess bone marrow (BM) angiogenesis measured by microvessel density (MVD) using CD34 and CD105 antibodies; (2) analyze correlation of MVD with plasma angiogenic factors including vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8; (3) examine the association of MVD with clinicopathological and molecular markers. We examined 90 de novo MPN patients (30 polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET)) and 10 age-matched controls. MVD was analyzed by immunohistochemistry “hot spot” method, angiogenic factors by immunoassay and JAK2V617F, and CALR mutations by DNA sequencing and allelic PCR. MVD was significantly increased in MPNs compared to controls (PMF > PV > ET). Correlation between MVD and plasma angiogenic factors was found in MPNs. MVD was significantly increased in patients with JAK2V617F mutation and correlated with JAK2 mutant allele burden (CD34-MVD: ρ = 0.491, p < 0.001; CD105-MVD: ρ = 0.276, p = 0.02) but not with CALR mutation. MVD correlated with leukocyte count, serum lactate dehydrogenase, hepatomegaly, and splenomegaly. BM fibrosis was significantly associated with CD34-MVD, CD105-MVD, interleukin-8, and JAK2 mutant allele burden. JAK2 homozygote status had positive predictive value (100%) for BM fibrosis. Patients with prefibrotic PMF had significantly higher MVD than patients with ET, and we could recommend MVD to be additional histopathological marker to distinguish these two entities. This study also highlights the strong correlation of MVD with plasma angiogenic factors, JAK2 mutant allele burden, and BM fibrosis in MPNs. © 2016, Springer-Verlag Berlin Heidelberg.

Epithelial to mesenchymal transition (EMT) is a feature of several types of human cancer, including oral squamous cell carcinoma (OSCC). In the present study, tumor and margin cell cultures obtained from patients with OSCC were used to determine the expression patterns of certain EMT-associatedmarkers,includingvimentin,α-smoothmuscle actin, SLUG and SNAIL. In addition, other EMT-associated features, including clonal, proliferative and migratory potential were compared between the two cell types. Cell cultures were generated from tumor and margin tissue samples from 6 patients and cultured up to the fifth passage. EMT marker expression was assessed by reverse transcription-quantitative PCR. Cell proliferation, colony formation and scratch wound healing assays were conducted to characterize the two cell types in terms of proliferation rates, clonality and motility. All of the studied markers were expressed in tumor and margin cells. Although no significant differences were noted with regard to the aforementioned markers, their expression tended to be higher in margin cultures than in tumor cultures. The expressions of the EMT markers were also higher in the fifth passage compared with those noted at the first with a few exceptions. The rates of proliferation and cell migration were decreased during passages, while the number of colonies was increased in both types of cell culture. Tumor and margin cells indicated certain similarities with regard to EMT transition characteristics. © 2020 Spandidos Publications. All rights reserved.

Epithelial to mesenchymal transition (EMT) is a feature of several types of human cancer, including oral squamous cell carcinoma (OSCC). In the present study, tumor and margin cell cultures obtained from patients with OSCC were used to determine the expression patterns of certain EMT-associatedmarkers,includingvimentin,α-smoothmuscle actin, SLUG and SNAIL. In addition, other EMT-associated features, including clonal, proliferative and migratory potential were compared between the two cell types. Cell cultures were generated from tumor and margin tissue samples from 6 patients and cultured up to the fifth passage. EMT marker expression was assessed by reverse transcription-quantitative PCR. Cell proliferation, colony formation and scratch wound healing assays were conducted to characterize the two cell types in terms of proliferation rates, clonality and motility. All of the studied markers were expressed in tumor and margin cells. Although no significant differences were noted with regard to the aforementioned markers, their expression tended to be higher in margin cultures than in tumor cultures. The expressions of the EMT markers were also higher in the fifth passage compared with those noted at the first with a few exceptions. The rates of proliferation and cell migration were decreased during passages, while the number of colonies was increased in both types of cell culture. Tumor and margin cells indicated certain similarities with regard to EMT transition characteristics. © 2020 Spandidos Publications. All rights reserved.

Purpose: A common feature of malignancies is increased reactive oxygen species (ROS) and reactive nitrogen species (RNS). We analyzed the influence of oxidative and nitrosative stress on the activation of AKT/mTOR signaling pathway in myeloproliferative neoplasms (MPN). Methods: Oxidative stress-induced gene expression in circulatory CD34+ cells of MPN patients was studied by microarray analysis. Biomarkers of oxidative and nitrosative stress were determined using spectrophotometry in plasma and erythrocyte lysate. The levels of nitrotyrosine, inducible NO synthase (iNOS) and AKT/mTOR/p70S6K phosphorylation were determined by immunocytochemistry and immunoblotting in granulocytes of MPN patients. Results: Antioxidants superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPx1) gene expression were increased in circulatory CD34+ cells, while SOD1 and GPx enzymes were reduced in the erythrocytes of MPN. Plasma malonyl-dialdehyde and protein carbonyl levels were elevated in MPN. The total antioxidant capacity in plasma and erythrocyte catalase (CT) activities was the most prominent in primary myelofibrosis (PMF) with JAK2V617F heterozygosity. The total nitrite / nitrate (NOx) level was augmented in the plasma of PMF patients (p<0.001), while nitrotyrosine and iNOS were generally increased in the granulocytes of MPN patients. Activation of AKT / mTOR signaling was the most significant in PMF (p<0.01), but demonstrated JAK2V617F dependence and consequent p70S6K phosphorylation in the granulocytes of essential thrombocytemia (ET) and polycythemia vera (PV) patients. Hydrogen peroxide stimulated mTOR pathway, iNOS and nitrotyrosine quantities, the last one prevented by the antioxidant n-acetyl-cysteine (NAC) in the granulocytes of MPN. Conclusion: Our study showed increased levels of oxidative and nitrosative stress parameters in MPN with JAK2V617F dependence. The ROS enhanced the constitutive activation of AKT / mTOR signaling and nitrosative parameters in MPN. © 2018 Zerbinis Publications. All Rights Reserved.

Purpose: A common feature of malignancies is increased reactive oxygen species (ROS) and reactive nitrogen species (RNS). We analyzed the influence of oxidative and nitrosative stress on the activation of AKT/mTOR signaling pathway in myeloproliferative neoplasms (MPN). Methods: Oxidative stress-induced gene expression in circulatory CD34+ cells of MPN patients was studied by microarray analysis. Biomarkers of oxidative and nitrosative stress were determined using spectrophotometry in plasma and erythrocyte lysate. The levels of nitrotyrosine, inducible NO synthase (iNOS) and AKT/mTOR/p70S6K phosphorylation were determined by immunocytochemistry and immunoblotting in granulocytes of MPN patients. Results: Antioxidants superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPx1) gene expression were increased in circulatory CD34+ cells, while SOD1 and GPx enzymes were reduced in the erythrocytes of MPN. Plasma malonyl-dialdehyde and protein carbonyl levels were elevated in MPN. The total antioxidant capacity in plasma and erythrocyte catalase (CT) activities was the most prominent in primary myelofibrosis (PMF) with JAK2V617F heterozygosity. The total nitrite / nitrate (NOx) level was augmented in the plasma of PMF patients (p<0.001), while nitrotyrosine and iNOS were generally increased in the granulocytes of MPN patients. Activation of AKT / mTOR signaling was the most significant in PMF (p<0.01), but demonstrated JAK2V617F dependence and consequent p70S6K phosphorylation in the granulocytes of essential thrombocytemia (ET) and polycythemia vera (PV) patients. Hydrogen peroxide stimulated mTOR pathway, iNOS and nitrotyrosine quantities, the last one prevented by the antioxidant n-acetyl-cysteine (NAC) in the granulocytes of MPN. Conclusion: Our study showed increased levels of oxidative and nitrosative stress parameters in MPN with JAK2V617F dependence. The ROS enhanced the constitutive activation of AKT / mTOR signaling and nitrosative parameters in MPN. © 2018 Zerbinis Publications. All Rights Reserved.