SIRT1 activation enhances HDAC inhibition-mediated upregulation of GADD45G by repressing the binding of NF-κB/STAT3 complex to its promoter in malignant lymphoid cells.

We explored the activity of SIRT1 activators (SRT501 and SRT2183) alone and in combination with panobinostat in a panel of malignant lymphoid cell lines in terms of biological and gene expression responses. SRT501 and SRT2183 induced growth arrest and apoptosis, concomitant with deacetylation of STAT3 and NF-κB, and reduction of c-Myc protein levels. PCR arrays revealed that SRT2183 leads to increased mRNA levels of pro-apoptosis and DNA-damage-response genes, accompanied by accumulation of phospho-H2A.X levels. Next, ChIP assays revealed that SRT2183 reduces the DNA-binding activity of both NF-κB and STAT3 to the promoter of GADD45G, which is one of the most upregulated genes following SRT2183 treatment. Combination of SRT2183 with panobinostat enhanced the anti-growth and anti-survival effects mediated by either compound alone. Quantitative-PCR confirmed that the panobinostat in combination with SRT2183, SRT501 or resveratrol leads to greater upregulation of GADD45G than any of the single agents. Panobinostat plus SRT2183 in combination showed greater inhibition of c-Myc protein levels and phosphorylation of H2A.X, and increased acetylation of p53. Furthermore, EMSA revealed that NF-κB binds directly to the GADD45G promoter, while STAT3 binds indirectly in complexes with NF-κB. In addition, the binding of NF-κB/STAT3 complexes to the GADD45G promoter is inhibited following panobinostat, SRT501 or resveratrol treatment. Moreover, the combination of panobinostat with SRT2183, SRT501 or resveratrol induces a greater binding repression than either agent alone. These data suggest that STAT3 is a corepressor with NF-κB of the GADD45G gene and provides in vitro proof-of-concept for the combination of HDACi with SIRT1 activators as a potential new therapeutic strategy in lymphoid malignancies.

The novel JAK inhibitor AZD1480 blocks STAT3 and FGFR3 signaling, resulting in suppression of human myeloma cell growth and survival.

IL-6 and downstream JAK-dependent signaling pathways have critical roles in the pathophysiology of multiple myeloma (MM). We investigated the effects of a novel small-molecule JAK inhibitor (AZD1480) on IL-6/JAK signal transduction and its biological consequences on the human myeloma-derived cell lines U266 and Kms.11. At low micromolar concentrations, AZD1480 blocks cell proliferation and induces apoptosis of myeloma cell lines. These biological responses to AZD1480 are associated with concomitant inhibition of phosphorylation of JAK2, STAT3 and MAPK signaling proteins. In addition, there is inhibition of expression of STAT3 target genes, particularly Cyclin D2. Examination of a wider variety of myeloma cells (RPMI 8226, OPM-2, NCI-H929, Kms.18, MM1.S and IM-9), as well as primary myeloma cells, showed that AZD1480 has broad efficacy. In contrast, viability of normal peripheral blood (PB) mononuclear cells and CD138(+) cells derived from healthy controls was not significantly inhibited. Importantly, AZD1480 induces cell death of Kms.11 cells grown in the presence of HS-5 bone marrow (BM)-derived stromal cells and inhibits tumor growth in a Kms.11 xenograft mouse model, accompanied with inhibition of phospho-FGFR3, phospho-JAK2, phospho-STAT3 and Cyclin D2 levels. In sum, AZD1480 blocks proliferation, survival, FGFR3 and JAK/STAT3 signaling in myeloma cells cultured alone or cocultured with BM stromal cells, and in vivo. Thus, AZD1480 represents a potential new therapeutic agent for patients with MM.

Computer-aided applications of nanoscale smart materials for biomedical applications.

Nanotechnology has the potential to impact the treatment of many diseases that currently plague society: cancer, AIDS, dementia of various kinds and so on. Nanoscale smart materials, such as carbon nanotubes, C(60), dendrimers and cyclodextrins, hold great promise for use in the development of better diagnostics, drug delivery and the alteration of biological function. Although experimentation is being used to explore the potential offered by these materials, it is by its very nature expensive in terms of time, resources and expertise. Insight with respect to the behavior of these materials in the presence of biological entities can be obtained much more rapidly by molecular dynamics simulation. Furthermore, the results of simulation may be used to guide experimentation so that it is much more productive than it might be in the absence of such information. The interactions of several nanoscale structures with biological macromolecules can already be probed effectively using molecular dynamics simulation. The results obtained should form the basis for significant new developments in the treatment of disease.

Chromosomal gains and losses indicate oncogene and tumor suppressor gene candidates in salivary gland tumors.

The incidence of salivary gland tumor in Poland is growing in the last two decades. Simultaneously a progress in understanding the genetic mechanisms of formation of this tumor was achieved by detecting several genes like PLAG1 involved in its pathogenesis. In this study we perform a whole genome, CGH analysis with the aim to identify recurrent, chromosomal copy number changes possibly indicating novel tumor suppressor gene or oncogene loci. 29 salivary tumor samples: Cystadenolymphoma-warthin (15) and adenoma polymorphum (14) located in the parotid (27) and submandibular gland (2) were collected and CGH was performed. The established copy number profiles were compared in order to asses the smallest common region of gains and losses. The delineated regions were further analyzed with the UCSC Genome Browser on Human Mar. 2006 Assembly to asses their gene content. Altogether, salivary gland tumors presented a different aberration pattern than these reported for head and neck squamous cell carcinoma (HNSCC) but no significant differences were observed between Warthin and adenoma polymorphum tumors. Moreover, several potential tumor suppressor genes and oncogenes were identified in the smallest, common altered regions. We show a frequent deletion of the harakiri gene (12q24.2) in 12/29 tumors and TP53 gene (17p13.1) in 11/29 tumors as potential tumor suppressors in salivary gland cancers. Besides, we detected a frequent amplification of the 13q22.1-22.2 region in 13/29 cases harboring the KLF5 and KLF12 genes. KLF5 regulates the expression of survivin, an oncogene widely expressed in the majority of human cancers. The observed alterations may indicate important genetic events in the formation of salivary gland tumors. Especially the amplification in 13q may be a mechanism contributing to the expression of survivin and tumor progression.

Second primary tumors (SPT) of head and neck: distinguishing of "true" SPT from micrometastasis by LOH analysis of selected chromosome regions.

The reason of treatment failures in head and neck tumors is often connected with the appearance of second primary tumors (SPT). Three mechanisms of SPT development of clonal or non clonal secondary tumors were described: 1. via micrometastases (clonal); 2. from a common carcinogenic field - Second Field Tumors (SFT - partially clonal); 3. via independent events (from different carcinogenic fields - "true" SPT - not clonal). Assessing the clonality of diagnosed tumors carries important clinical implications including chemoprevention, radiotherapy and general patient management. In this study a set of 12 microsatellite markers was used to find similarities and/or differences in allelic imbalance patterns between 22 pairs of tumors (the first tumor designate as index and SPT). The aim of the study was to identify a potential clonal origin and progression within given pairs of tumors. The results indicate that within the tumors diagnosed by clinical examination as SPT at least two mechanisms mentioned above should be taken into account as 6/23 (26%) were clonally unrelated ("true" SPT) and 3/23 (13%) carried clonal genetic changes (formation by micrometastasis or SFT). In 14/23 (61%) cases the results were insufficient or ambiguous to determine the clonality status. The final results indicate the complexity of carcinogenesis in these tumors and thus stress that clinical diagnosis of second primary tumors should be considered carefully.

Tissue factor pathway inhibitor and thrombin activatable fibrinolytic inhibitor plasma levels following burn and septic injuries in rats.

Burn and septic injuries induce profound changes in coagulation status. This study examined the changes in plasma tissue factor pathway inhibitor (TFPI) and thrombin activatable fibrinolytic inhibitor (TAFI) levels in a rat model of burn and septic injuries. Rats underwent 30% TBSA cutaneous scald burn injury and septic insult was induced by caecal ligation and puncture (CLP). CLP was superimposed on burn injury to mimic the clinical model of sepsis complicating burn injury. Rats were pretreated with Cprofloxacin orally to colonize their gut with Enterococcus faecalis. TFPI and TAFI plasma levels were measured using functional activity assay kit with a chromogenic method at 24 and 72 hours following the injuries. TFPI levels decreased significantly at 24 hours in burn, CLP, and burn+CLP groups, followed by incomplete rebound recovery at 72 hours in all three groups. On the other hand, TAFI levels increased significantly at 24- and 72-hour time points in all three groups. These results suggest that burn, septic, and their combined injuries perturb coagulation cascade and thrombotic process toward the procoagulant pathway by impairing fibrinolysis.

Biomarker profiling of plasma from acute coronary syndrome patients. Application of ProteinChip Array analysis.

AIM: Acute coronary syndrome (ACS) is one of the leading causes of death in the world and remains a complex pathophysiologic process involving inflammatory, hemostatic and vascular processes. The purpose of this study was to identify unique proteomic biomarkers present in patients with ACS using a newly developed proteomic profiling technique, surface enhanced laser desorption/ionization (SELDI). METHODS: Citrated plasma samples obtained from clinically confirmed cases of ACS (n=100) and age matched controls (n=25) were profiled using SELDI-time of flight (TOF)-mass spectrometry (Ciphergen Biosystems, Freemont, CA, USA). A strong anion exchange (SAX) ProteinChip Array was used to profile these samples. In addition to spectra profiles, protein density plots were be obtained from the generated molecular profile. RESULTS: The SELDI profile in the molecular weight (MW) range of 0-150 kDa revealed a prominent 66.3 kDa albumin peak along with several distinct components at 28 kDa, 13.7 kDa and 6.5 kDa. Additional minor molecular components were also noted in the lower MW range (<6 kDa). There was a cluster of peaks between 10 and 12 kDa that were unique to the patients with ACS; about 1/3 of the ACS patients exhibited these peaks as evident in the ProteinChip Array spectrum. None of the age-matched controls exhibited the peaks in this MW range, nor did the normal human plasma pool that was used as an additional control. The relative intensity of these novel molecular components in the range of 10-12 kDa represent unique proteins/peptides which are generated in specific pathologic states associated with ACS. CONCLUSIONS: These observations suggest that patients with ACS have a unique cluster of molecular components that are present in their SELDI profile. It might be possible to use these patterns to identify high-risk patients who may be more susceptible to the development of unstable plaque, which may eventually lead to myocardial infarction. Identification and characterization of these molecular components will also help in the understanding of the pathogenesis of ACS. These unique peaks may represent pathologic proteins, novel inflammatory mediators or protease cleavage products. Further studies need to be done to better characterize and identify these molecular components and their pathologic role in ACS.

Differential regulation of thrombin activatable fibrinolytic inhibitor by low molecular weight heparins. Pharmacologic implications.

AIM: Thrombin activatable fibrinolytic inhibitor (TAFI) is activated via cleavage by thrombin thrombomodulin in complex, and can be regulated by anticoagulant drugs such as the heparins. Low molecular weight heparins (LMWHs) have different antithrombin/anti-Xa profiles and therefore vary in the degree to which they inhibit TAFI. The purpose of this study was to determine the differential regulation of TAFI by LMWHs. METHODS: Dalteparin, enoxaparin, tinzaparin, parnaparin and heparin were supplemented to normal human pooled plasma at different concentrations (0-2.5 U). A chromogenic based assay (Pentapharm Inc., Basil, Switzerland) was used to measure activatable TAFI in each set of samples. RESULTS: Heparin clearly had the highest degree of TAFI inhibition with an IC50 of 0.10 U, which correlates with its coagulation profile. Dalteparin, Tinzaparin, Parnaparin had similar IC50s, 0.6-0.8 U/ml respectively, while enoxaparin had a higher IC50 (>1.0 U/ml). These results strongly correlate with the anti-IIa inhibition of each agent but not with the anti-Xa. However, it is interesting to note that these drugs are administered according to anti-Xa units not anti-IIa. CONCLUSIONS: These results suggest that each LMWH may inhibit TAFI to a different extent that is not dependent on the anti-Xa potency. Indiscriminate inhibition of TAFI may cause bleeding, while suboptimal inhibition may result in thrombosis. Because of the compositional difference, heparin and LMWHs may produce differential inhibition of TAFI and therefore result in product dependent modulation of hemostatic process which may or may not be related to their antithrombin effects.

Synergistic and antagonistic properties of selected cultures of lactic acid and propionic acid bacteria. Part II. Proteolytic activity.

The objective of this work was to study the dynamics of proteolytic activity for selected cultures of propionic acid and lactic acid bacteria used in the rennet cheese production. The studies were carried out in two model systems, with CH-N-19, Nizostar and their associated cultures in two media differing in the nitrogen compound structure. The addition of coagulating enzyme to the medium accelerated the proteolytic processes occurring upon the contribution of the examined cultures but did not influence the changes in the aminopeptidase specificity towards the substrates used (Leu-pNA for LAB and Pro-pNA for PAB).

Synergistic and antagonistic properties of selected cultures of lactic acid and propionic acid bacteria. Part I. Fermentation activity.

The objective of this work was to study the dynamics of fermentation activity of selected cultures of lactic acid and propionic acid bacteria used in the cheese production. At the same time, it was decided to follow their interactions. The studies were conducted in two model systems with CH-N-19, Nizostar, and their associated cultures in two media. On a basis of the evaluation of physiological and fermentation activities, the antagonistic properties between the examined culture systems were not found and the differences observed only resulted from specific traits of the examined microorganisms.

Effects of phosphorolytic and cell wall-degrading enzymes on the performance of growing broilers fed wheat-based diets containing different calcium levels.

A study was conducted to determine the cumulative effects of phosphorolytic enzymes, cell wall-degrading enzymes, and citric acid and Ca levels on feed intake, BW gain (BWG), feed conversion, intestinal viscosity, and toe ash of broilers (d 1 to 21) fed wheat-based diets. Broilers were fed the following six diets at either 0.59, 0.69, or 0.79% Ca: 1) a negative control (NC) diet, 0.17% available P; 2) NC + 750 phytase units/kg diet; (3) phytase + 3,156 units of acid phosphatase/kg diet; 4) phytase + acid phosphatase + 1,900 units of pectinase/g diet; 5) phytase + acid phosphatase + pectinase + 3% citric acid; and (6) NC plus 0.24% available P. The 18 dietary treatments were fed to four pen replicates of eight birds each. Phytase addition at the low Ca level increased BWG, improved feed intake and conversion and toe ash, and reduced intestinal viscosity and ileal length. Subsequent addition of acid phosphatase, at 0.69% Ca, resulted in increases in BWG, 42%; feed intake 32%; feed conversion 7.5%; and toe ash, 63% over the NC diet. Pectinase addition produced further improvements in 21-d BWG and feed intake at 0.59 and 0.79% Ca, increased toe ash in chicks fed 0.79% Ca, and reduced intestinal viscosity. Supplementation of wheat-based 0.17% available P diets with phytase and acid phosphatase and with appropriate concentrations of pectinase, citric acid, and Ca significantly improved BWG, feed intake and conversion and intestinal viscosity over the 0.41% available P diets. Bone mineralization of chicks fed phytase + acid phosphatase and 0.69% Ca and those fed phytase + acid phosphatase + pectinase + citric acid and 0.59% Ca was similar to that of chicks fed the 0.41% available P diets.

DNA copy number losses are more frequent in primary larynx tumors with lymph node metastases than in tumors without metastases.

Comparative genomic hybridization was performed on 38 primary laryngeal carcinomas divided into two groups according to the metastatic phenotype. DNA copy number changes were detected in 22 of the 38 cases (57.9%). Gains were most frequently observed at 3q, 8q, and 9q, and losses were found in decreasing order at 18q, 3p, and 4. The mean value of losses was 2.5 times as high in metastasizing primary tumors (23/38) as in nonmetastasizing tumors. The most frequent losses in metastasizing tumors were at 18q, 3p, and 5q.

Recurrent DNA copy number losses associated with metastasis of larynx carcinoma.

Squamous cell carcinomas of the head and neck show frequent and complex chromosome aberrations, but little is known about the changes that occur during the metastatic process. To compare the accumulation of changes in primary and metastatic tumors we analyzed 19 pairs of primary larynx cancer tumors and their metastases. The most frequent changes were found at 3p, 3q, 5p, 9, and 13. Losses at 13, 8p, and 9q were more frequent in metastases than in primary tumors.

Genotoxicity of inhalation anesthetics halothane and isoflurane in human lymphocytes studied in vitro using the comet assay.

The alkaline single cell gel electrophoresis (comet) assay was applied to study genotoxic properties of two inhalation anesthetics-halothane and isoflurane-in human peripheral blood lymphocytes (PBL). The cells were exposed in vitro to either halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) or isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether) at concentrations 0.1-10 mM in DMSO. The anesthetics-induced DNA strand breaks as well as alkali-labile sites were measured as total comet length (i.e., increase of a DNA migration). Both analysed drugs were capable of increasing DNA migration in a dose-dependent manner. In experiments conducted at two different electrophoretic conditions (0. 56 and 0.78 V/cm), halothane was able to increase DNA migration to a higher extent than isoflurane. The comet assay detects DNA strand breaks induced directly by genotoxic agents as well as DNA degradation due to cell death. For this reason a contribution of toxicity in the observed effects was examined. We tested whether the exposed PBL were able to repair halothane- and isoflurane-induced DNA damage. The treated cells were incubated in a drug-free medium at 37 degrees C for 120 min to allow processing of the induced DNA damage. PBL exposed to isoflurane at 1 mM were able to complete repair within 60 min whereas for halothane a similar result was obtained at a concentration lower by one order of magnitude: the cells exposed to halothane at 1 mM removed the damage within 120 min only partly. We conclude that the increase of DNA migration induced in PBL by isoflurane at 1 mM and by halothane at 0.1 mM was not a result of cell death-associated DNA degradation but was caused by genotoxic action of the drugs. The DNA damage detected after the exposure to halothane at 1 mM was in part a result of DNA fragmentation due to cell death.

[Chromosome damage in the course of laryngeal squamous cell carcinoma]. / Uszkodzenia chromosomów w przebiegu plaskonablonkowego raka krtani.

The aim of the article is a review of own cytogenic studies on laryngeal cancer confronted with the literature data. Spontaneous and bleomycin-induced chromosome instability was analysed in peripheral blood lymphocytes in relation to genetic risk of cancer incidence and progression. Comparative genome hybridization (CGH) was applied to demonstrate gains and losses of DNA copy number in tumour and non-tumour laryngeal mucosa. The profiles of imbalances of DNA copy number were shown to differ between metastazing and non-metastazing tumours. Preliminary data indicate a frequent loss of Y chromosome in tumour cells. The loss of heterozygosity at chromosome p53 locus (17p) has been shown to be more frequent than at chromosome locus coding 16 gene (9p). Altogether, the experiments have proven that a dynamics of chromosome aberrations is highest at the stage of metastasis.

[Sex-dependence of larynx susceptibility to tobacco smoke carcinogens. The role of androgen hormones]. / Plec a podatnosc krtani na dzialanie czynników kancerogennych obecnych w dymie tytoniowym. Rola hormonów androgennych.

According to epidemiological data concerning laryngeal cancer there is almost ninefold higher morbidity in male population than in females. The aim of this study was to analyse on molecular level the genotoxic effects arising under the same exposure to tobacco smoke in males and females. In biopsies received from laryngectomy material we determined levels of aromatic and alkylated DNA adducts in tumours and in adjacent non-tumour tissue. It was established that levels of DNA adducts found in male DNA samples exceeded those in female DNA samples 1.5-3.8 times, which was recognised as a molecular evidence for epidemiological differences. Next, testosterone and sex hormones binding globulin (SHBG) were determined in male subjects blood serum. The levels of testosterone and SHBG very weakly correlated with DNA adduct levels even when subjects were separated into age groups. It is concluded that levels of testosterone and SHBG are not the proper markers of individual susceptibility to genotoxicity of tobacco smoke carcinogens.

Bleomycin-induced DNA damage and its removal in lymphocytes of breast cancer patients studied by comet assay.

The studies concerned the response to bleomycin treatment in peripheral blood lymphocytes (PBL) of breast cancer (BC) subjects. The level of BLM-induced DNA strand breaks was evaluated using alkaline comet assay followed by visual scoring. The sensitivity to genotoxic exposure as well as the time-course of damage removal were estimated and analysed in comparison to control (healthy) subjects. Despite high inter-individual variability, the differences between the BC and non-cancer groups still proved to be statistically significant. Lymphocytes of the BC subjects appeared to be more sensitive to BLM exposure as shown by higher level of DNA damage. The DNA repair capacity was weaker in PBL obtained from BC patients than that in lymphocytes of controls.

The efficacy of an enzymic cocktail and a fungal mycelium in dephosphorylating corn-soybean meal-based feeds fed to growing turkeys.

A study was conducted to determine the efficacy of phytase, an enzymic cocktail, and a waste Aspergillus niger mycelium to hydrolyze phytate present in corn-soybean meal diets. One hundred turkey poults were assigned to dietary treatments for 2 wk (days 7 to 21). Dietary treatments included: 1) NRC (1994) diet (NRC), with recommended concentration of 0.6% available P (aP) and 1.2% Ca; 2) Phytase diet (PHYT), 1,000 units phytase/kg diet, 0.16% aP, and 0.84% Ca; 3) cocktail diet (COC), 1,000 units of phytase/kg diet plus acid phosphatase (100 units/g of diet), acid protease (42 units/g of diet), pectinase (2.94%), 0.16% aP, and 0.84% Ca; 4) Fungal mycelium diet (MYC), 5% mycelium, 0.16% aP, and 0.84% Ca; and 5) a positive control diet (CTRL+), 0.42% aP, and 0.84% Ca. Turkeys fed the PHYT diet consumed less feed and gained less weight but retained more P than poults fed the CTRL+ or NRC diets. Poults fed the COC diet performed as well as poults fed CTRL+ or NRC diets but retained more P (77%) and Ca (68%). Poults fed the MYC diet retained 79% P, gained the most weight, and were more efficient than poults fed any other dietary treatment. In vitro P release from experimental diets correlated well (R = 0.906) with P retention as observed in the feeding trial. Compared with the diet containing phytase as the sole supplemental enzyme, both the enzymic cocktail and fungal mycelium enhanced performance, bone mineralization, and retention of P and Ca in growing turkeys.

Relationship between citric acid production and accumulation of phytate-degrading enzymes in Aspergillus niger mycelia.

The ability of eight strains of Aspergillus niger to produce citric acid by the solid surface method were found to correlate with their capabilities to synthesize intracellular enzymes which degrade phytates (phytase and acid phosphatase). Another high correlation was observed between phytase and acid phosphatase activities bound to the cell walls of mycelia.

A comparison of some methods for the determination of methoxyl groups in commercial pectin preparations.

The results of methoxyl groups determination in pectin preparations were compared. The methods of direct (I, II) and indirect (III, IV) methanol determination connected with enzymatic (II, IV) or alkaline (I, III) pectin demethylation were applied. Higher values were obtained using indirect methanol methods than in the case of a direct methanol determination. The difference between these groups of methods was statistically significant. The precision of all the methods was high. The degree of methylation (DM) was calculated based on methanol content; moreover in the methods III and IV it was also determined from the ratio of methylated carboxyl groups to a total of acidic groups. From the difference (%) between these two ways of calculating DM the purity of pectin can be determined. The method based on enzymatic demethylation and direct methanol analysis is recommended as a precise and selective one.