Small-molecule inhibitor - tyrphostin AG1296 regulates proliferation, survival and migration of rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is the most commonly occurring malignant soft tissue tumor in children. Despite improving its treatment methods, the current outcome in the advanced stages of this tumor is not satisfactory. RMS cells are characterized by abnormal cellular signaling due to the changes in the activity of the tyrosine kinases. Thus, substances blocking the mitogenic signal transmitted by receptors with tyrosine kinase activity raise hopes for inhibition of the uncontrolled cell growth. In this study, we examined the anticancer activity of tyrphostin AG1296, a tyrosine kinase inhibitor that binds to the intracellular domain of the PDGF (platelet-derived growth factor) receptor in human RMS alveolar and embryonal cell lines. We have discovered that tyrphostin AG1296 completely inhibited cell proliferation and effectively inhibited cell viability. Tyrphostin AG1296 induced apoptosis of the RMS cells and significantly inhibited their migration. Additionally, investigated inhibitor slightly inhibited expression of AKT and phosphorylation of ERK in alveolar RMS cells. Importantly, the inhibitor exerted also potent effects on the nanomechanical properties and cytoskeleton organization of RMS cells. To conclude, tyrphostin AG1296 is a promising compound in the treatment of alveolar RMS. Undoubtedly, a better knowledge of receptor pathomechanism of tyrosine kinases may contribute to developing new, more effective ways of RMS treatment.

Comparing surface properties of melanoma cells using time of flight secondary ions mass spectrometry.

Various techniques have been already reported to differentiate between normal (non-malignant) and cancerous cells based on their physico-chemical properties. This is relatively simple when studied cancerous cells originate from distant stages of cancer progression. Here, studies on chemical properties of two closely related human melanoma cell lines are presented: WM115 melanoma cells were taken from the vertical growth phase while WM266-4 from the skin metastatic site of the same patient. Their chemical properties were studied by two techniques, namely time-of-flight secondary ion mass spectra (ToF SIMS) and photothermal microspectroscopy (PTMS), used to record mass and photothermal spectra of cells, respectively. In our approach, independently of the spectra type, its full range, i.e. masses and wavenumbers within the range 0-500 kDa and 500-4000 cm-1, underwent a similar methodology for principal component analysis (PCA). PCA outcome shows results groupped depending on the sample type (either WM115 or WM266-4 cells). The results are independent of the method applied to study chemical properties of melanoma cells, indicating that cancer-related changes are large enough to be identified with these techniques and to differentiate between cells originating from vertical growth phase and skin metastatis.

Data on step-by-step atomic force microscopy monitoring of changes occurring in single melanoma cells undergoing ToF SIMS specialized sample preparation protocol.

Data included in this article are associated with the research article entitled 'Protocol of single cells preparation for time-of-flight secondary ion mass spectrometry' (Bobrowska et al., 2016 in press) [1]. This data file contains topography images of single melanoma cells recorded using atomic force microscopy (AFM). Single cells cultured on glass surface were subjected to the proposed sample preparation protocol applied to prepare biological samples for time-of-flight secondary ion mass spectrometry (ToF SIMS) measurements. AFM images were collected step-by-step for the single cell, after each step of the proposed preparation protocol. It consists of four main parts: (i) paraformaldehyde fixation, (ii) salt removal, (iii) dehydrating, and (iv) sample drying. In total 13 steps are required, starting from imaging of a living cell in a culture medium and ending up at images of a dried cell in the air. The protocol was applied to melanoma cells from two cell lines, namely, WM115 melanoma cells originated from primary melanoma site and WM266-4 ones being the metastasis of WM115 cells to skin.

The systemic inflammatory response in coronary artery bypass grafting: what is the role of the very low ejection fraction (EF < or = 30%)?

AIM: Patients with depressed left ventricular function are more susceptible to develop postoperative complications after cardiac surgery. The aim of the present study was to examine the effect of severe left ventricular dysfunction on the activation of systemic inflammatory reaction during and after coronary artery bypass grafting (CABG). METHODS: Clinical prospective study; 32 selected patients underwent CABG; 16 patients had depressed left ventricular function before the operation (low ejection fraction [EF] <30%)--Low EF group (study group). Sixteen patients had normal left ventricular function (normal EF, >50%)--Normal EF group (control group). The levels of inflammatory mediators TNF-alpha, IL-6, IL-8 and IL-10 were measured preoperatively, during and after cardiopulmonary bypass (CPB) and 24 hours postoperatively. RESULTS: Higher levels of almost all of inflammatory mediators were detected in patients with depressed left ventricular function compared with patients of normal EF group. IL-6 levels were found statistically significant higher in Low EF group before the induction of anesthesia (P=0.039) and after the administration of protamine (P=0.02). IL-8 levels were found statistically significant higher in Low EF group before the induction of anesthesia (P=0.05), 30 min after the start of CPB (P=0.02), after the administration of protamine (P=0.015) and 24 hours after the end of the operation (P=0.05). No statistically significant differences were demonstrated between the 2 groups of study relative to TNF-alpha and IL-10. CONCLUSION: A greater activation of systemic inflammatory reaction occurred in patients with depressed left ventricular function than in patients with normal cardiac function when they underwent CABG with extracorporeal circulation.

Rheological properties of erythrocytes in patients with high risk of cardiovascular disease.

Rheological properties of erythrocytes from patients with high risk of cardiovascular disease (CVD) were analyzed in relation to individual patient risk factors as well as to the medication. Additionally, comparative statistical analysis was performed considering plasma concentration of the selected mediators of vascular endothelium: 6-keto-prostaglandin F(1alpha) (PGF(1alpha)), sVCAM-1 and E-selectin adhesion molecules and interleukin-6 (IL-6). It was found that antihypertensive therapy with angiotensin-converting enzyme inhibitor (ACEI) is accompanied by improvement of RBC rheology: the increase of deformability and the decrease of aggregability. This improvement is probably mediated by endothelial prostacyclin and nitric oxide which are generated by ACEI. A correlation was observed between RBC deformability/aggregability and the patient's hematocrit level, what implicates that the hematocrit level should be explicitly taken into consideration when investigating rheological properties of erythrocytes. A strong relationship was also found between the plasma concentration of sVCAM-1 and patient's age.

Probing molecular interaction between concanavalin A and mannose ligands by means of SFM.

Recently, the scanning force microscope (SFM) has been widely used for direct monitoring of specific interactions between biologically active molecules. Such studies have employed the SFM liquid-cell setup, which allows measurements to be made in the native environment with force resolution down to a tenth of a picoNewton. In this study, the ligand-receptor strength of monoclonal anti-human prostatic acid phosphatase and prostatic acid phosphatase, representing an antigen-antibody system with a single type of interaction, was determined. Then, the interaction force occurring between concanavalin A and the carbohydrate component of the glycoproteins arylsulfatase A and carboxypeptidase Y was measured. High mannose-type glycans were sought on the human prostate carcinoma cell surface. Application of an analysis based on the Poisson distribution of the number of bonds formed in all these measured systems allowed the strength of the molecular interaction to be calculated. The values of the force acting between two single molecules were 530+/-25, 790+/-32, and 940+/-39 pN between prostatic acid phosphatase and monoclonal anti-human prostatic acid phosphatase, between concanavalin A and arylsulfatase A, and between concanavalin A and carboxypeptidase Y, respectively. The value calculated from data collected for the force between concanavalin A and mannose-containing ligands present on the surface of human prostate carcinoma cells was smaller, 116+/-17 pN. The different values of the binding force between concanavalin A and mannose-containing ligands were attributed to the structural changes of the carbohydrate components.

Ventilator-associated pneumonia and atelectasis: evaluation through bronchoalveolar lavage fluid analysis.

OBJECTIVE: Surfactant offers protection against alveolar collapse and contributes to the local defense mechanism, but it is unclear if surfactant alterations have a role in the development of atelectasis or ventilator-associated pneumonia (VAP). The present study was undertaken to monitor surfactant, as well as biochemical BAL fluid alterations, during the course of VAP and atelectasis in mechanically ventilated patients without primary cardiopulmonary disease, to elucidate the pathogenesis and to differentiate these two entities. DESIGN. Prospective controlled study. SETTING: 14-bed general ICU of a 750-bed University Hospital. PATIENTS: Sixty-one ventilated patients, without primary cardiopulmonary disease-normal initial chest X-ray, satisfactory oxygenation (PaO(2)/FiO(2)>300 mmHg), and expected time of ventilation exceeding 2 weeks-were initially enrolled. Twelve of them developed VAP and eight lobar or segmental atelectasis during the 2-week study period. INTERVENTIONS: An initial BAL was performed in all patients within 48 h from admission. Patients who developed VAP or atelectasis were subjected to a second and third BAL during and after the resolution of VAP or atelectasis, respectively. MEASUREMENTS AND RESULTS: VAP and atelectasis resulted in a significant increase of total protein and markers of inflammation, such as PAF and neutrophils, which partially remitted after their resolution. Large surfactant aggregates, which contribute to surface tension decrease, were significantly reduced during both entities and remained low even after their resolution. CONCLUSIONS: BAL alterations during VAP and atelectasis suggest increased alveolar-capillary permeability, severe surfactant abnormalities, and signs of local inflammatory reaction. These alterations are associated with the observed deteriorated gas exchange and lung mechanics and could predispose to further lung injury in ventilated patients.

The effect of chitosan on stiffness and glycolytic activity of human bladder cells.

The cell's cytoskeleton together with the cell membrane and numerous accessory proteins determines the mechanical properties of cell. Any factors influencing cell organization and structure can cause alterations in mechanical properties of cell (its ability for deformation and adhesion). The determination of the local elastic properties of cells in their culture conditions has opened the possibility for the measurement of the influence of different factors on the mechanical properties of the living cells. The effect of the chitosan on the stiffness of the non-malignant transitional epithelial cells of ureter (HCV 29) and the transitional cell cancer of urine bladder (T24) was determined using scanning force microscopy. The investigations were performed in the culture medium (RPMI 1640) containing 10% fetal calf serum in the presence of the microcrystalline chitosan of the three different deacetylation degrees. In parallel, the effect of chitosan on production of lactate and ATP level was determined. The results showed the strong correlation between the decrease of the energy production and the increase in Young's modulus values obtained for the cancer cells treated with chitosan.

Differential determination of phospholipase A(2) and PAF-acetylhydrolase in biological fluids using fluorescent substrates.

The purpose of the present study was the development and evaluation of a fluorimetric method for the screening and differential determination of phospholipase A(2) and PAF-acetylhydrolase in bronchoalveolar lavage (BAL) fluid and serum. Phospholipase A(2) was determined using C(12)-NBD-PC in the presence of Ca(2+), from the slope of the fluorescence enhancement due to the formation of C(12)-NBD-fatty acid. PAF-acetylhydrolase was determined using C(6)-NBD-PC, from the slope of the curve due to C(6)-NBD-fatty acid formation in the absence of Ca(2+). The results were confirmed after TLC analysis. The method's selectivity was evaluated by comparing to radiometric measurements. Light scattering did not interfere and inner filter effects was not observed under our experimental conditions. The effects of pH, temperature, and Ca(2+) were investigated. Protein caused an increase in the background fluorescence of both NBD-PCs. The standard curves of both NBD-fatty acids exhibited the same slope. Linearity extended at least up to 4. 5 nmoles per ml of reaction mixture at the normal pH 7.4. The fluorescence of the NBD-fatty acids remained stable for increasing concentrations of BAL fluid and serum and for BSA up to 100 microg/ml of reaction mixture. Porcine pancreatic PLase A(2) showed preference for C(12)-NBD-PC in the presence of Ca(2+), while without Ca(2+), serum PAF-AcH hydrolyzed only C(6)-NBD-PC. The method is highly sensitive, accurate, and reproducible and can be applied for the differential determination of phospholipase A(2) and PAF-acetylhydrolase activities in BAL fluid and serum.

Elasticity of normal and cancerous human bladder cells studied by scanning force microscopy.

Scanning force microscopy was used for the determination of the elastic properties of living cells in their culture conditions. The studies were carried out on human epithelial cells. Two similar lines of normal cells (Hu609 and HCV29) and three cancerous ones (Hu456, T24, BC3726) were measured using the scanning force microscope in order to collect the force versus indentation curves. The BC3726 line originates from the HCV29 cell line which was transformed by the v-ras oncogene. To evaluate their elastic properties, Young's modulus values were determined. The present study has shown that normal cells have a Young's modulus of about one order of magnitude higher than cancerous ones. Such a change might be attributed to a difference in the organisation of cell cytoskeletons and requires further studies.

Bronchoalveolar lavage alterations in pulmonary embolism.

The objective of this study was to determine quantitative and qualitative surfactant alterations, proteins, and platelet activating factor (PAF) in bronchoalveolar lavage (BAL) fluid from patients with pulmonary thromboembolism (PTE) with respect to ventilated patients without PTE. Patients with PTE underwent BAL at the most affected lung area on the first and tenth days of PTE diagnosis. Total proteins and albumin, total lipids, individual phospholipid classes, PAF and PAF-acetylhydrolase (PAF-AcH) activity were determined in BAL fluid. Total proteins and albumin were found to be increased in both successive samples of patients with PTE when compared with the control group (p < 0.001 and p < 0.05, respectively). Total phospholipids, though, were elevated on the first day, but they decreased on the tenth day, in comparison with the control groups (p < 0.05). Alterations in the percentage of individual phospholipid classes were observed in both successive samples of BAL fluid when compared with those in the control subjects. PAF and PAF-AcH were detected in high levels on the first day (p < 0.001), which were reduced on the tenth day (p < 0.05). An inverse correlation between PAF levels and PaO2/FIO2 ratio was observed. Finally, the percentage of macrophages decreased and the percentage of neutrophils increased during the course of PTE. In conclusion, pulmonary embolism is associated with alterations in lung surfactant and inflammation in lung tissue, expressed by an increase in PAF and in neutrophils.

Isolation of a phospholipid inhibitor of platelet activating factor-induced activity from perfused rat liver: identification as phosphatidylglycerol.

An endogenous inhibitor of platelet activating factor action was isolated from perfused rat liver. It was purified by thin-layer chromatography and high-performance liquid chromatography and subjected to chemical modifications in order to identify its structure. On the basis of its fast atom bombardment-mass spectrum it was characterized as phosphatidylglycerol composed mainly of 16:0/18:1 and 16:0/20:2 fatty acyl chains ([M+H]+ at m/z 749 and 775, respectively) and very minor levels of 18:0/18:1 and 18:0/20:2. The purified compound exhibited inhibition on rabbit platelet aggregation induced by 5 x 10(-10) M platelet activating factor (PAF) at an EC50 value near 2.5 x 10(-6) M and on the serotonin secretion at an EC50 7 x 10(-6) M. Other phospholipids isolated from the liver preparations, such as phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin (diphosphatidylglycerol), and phosphatidic acid, exhibited no inhibitory activity in the concentration range from 1 x 10(-4) to 1 x 10(-7) M nor did they induce any aggregation, or lysis, of the platelets. Of importance, phosphatidylglycerol could inhibit thrombin- and ADP-induced aggregation of rabbit platelets. These results suggested a possible site of inhibition common to the signal transduction pathway of these agonists. Preliminary binding experiments showed a noncompetitive type of inhibition on PAF binding to intact rabbit platelets.