Cigarette smoke sustains immunosuppressive microenvironment inducing M2 macrophage polarization and viability in lung cancer settings.

BACKGROUND: It is amply demonstrated that cigarette smoke (CS) has a high impact on lung tumor progression worsening lung cancer patient prognosis and response to therapies. Alteration of immune cell types and functions in smokers' lungs have been strictly related with smoke detrimental effects. However, the role of CS in dictating an inflammatory or immunosuppressive lung microenvironment still needs to be elucidated. Here, we investigated the effect of in vitro exposure to cigarette smoke extract (CSE) focusing on macrophages. METHODS: Immortalized murine macrophages RAW 264.7 cells were cultured in the presence of CS extract and their polarization has been assessed by Real-time PCR and cytofluorimetric analysis, viability has been assessed by SRB assay and 3D-cultures and activation by exposure to Poly(I:C). Moreover, interaction with Lewis lung carcinoma (LLC1) murine cell models in the presence of CS extract were analyzed by confocal microscopy. RESULTS: Obtained results indicate that CS induces macrophages polarization towards the M2 phenotype and M2-phenotype macrophages are resistant to the CS toxic activity. Moreover, CS impairs TLR3-mediated M2-M1 phenotype shift thus contributing to the M2 enrichment in lung smokers. CONCLUSIONS: These findings indicate that, in lung cancer microenvironment of smokers, CS can contribute to the M2-phenotype macrophages prevalence by different mechanisms, ultimately, driving an anti-inflammatory, likely immunosuppressive, microenvironment in lung cancer smokers.

Preliminary experience on the use of sucrosomial iron in hemodialysis: focus on safety, hemoglobin maintenance and oxidative stress.

PURPOSE: Iron is usually administered in hemodialysis patients by parenteral route, as oral absorption is poor due to high hepcidin levels. However, administrations of intravenous iron and iron overload are associated with high oxidative stress and systemic inflammation that can affect patient survival. With this study, we evaluated an alternative type of oral iron for the treatment of anemia in hemodialysis patients. The formulation consists in ferric pyrophosphate covered by phospholipids plus sucrose ester of fatty acid matrix, named sucrosomial iron, whose absorption is not influenced by hepcidin. METHODS: Twenty-four (24) patients undergoing chronic hemodialysis switched iron supplementation from intravenous (ferric gluconate 62.5 mg weekly) to oral (sucrosomial iron, 90 mg weekly in 3 administrations of 30 mg) route for 3 months. Classical anemia, iron metabolism, inflammation and nutritional biomarkers were monitored, as well as biomarkers of oxidative stress, such as protein-bound di-tyrosines, protein carbonylation, advanced oxidation protein products and protein thiols. RESULTS: Over the 3 months, hemoglobin values remained stable, as the values of hematocrit and mean corpuscular volume. In parallel, other anemia parameters dropped, including ferritin, transferrin saturation and serum iron. On the other side, nutritional biomarkers, such as total proteins and transferrin, increased significantly during the time frame. We also observed a significant decrease in white blood cells as well as a non-significant reduction in C-reactive protein and some oxidative stress biomarkers, such as protein carbonyls and di-tyrosines. CONCLUSION: Our study demonstrates that a therapy with sucrosomial iron in hemodialysis patients is safe and can maintain stable hemoglobin levels in a three-month period with a possible beneficial effect on oxidative stress parameters. However, the reduction of ferritin and transferrin saturation suggests that a weekly dosage of 90 mg is not sufficient in hemodialysis patients in the long time to maintain hemoglobin.

Antioxidants in smokers.

Cigarette smoke (CS) is likely the most common preventable cause of human morbidity and mortality worldwide. Consequently, inexpensive interventional strategies for preventing CS-related diseases would positively impact health systems. Inhaled CS is a powerful inflammatory stimulus and produces a shift in the normal balance between antioxidants and oxidants, inducing oxidative stress in both the respiratory system and throughout the body. This enduring and systemic pro-oxidative state within the body is reflected by increased levels of oxidative stress and inflammation biomarkers seen in smokers. Smokers might benefit from consuming antioxidant supplements, or a diet rich in fruit and vegetables, which can reduce the CS-related oxidative stress. This review provides an overview of the plasma profile of antioxidants observable in smokers and examines the heterogeneous literature to elucidate and discuss the effectiveness of interventional strategies based on antioxidant supplements or an antioxidant-rich diet to improve the health of smokers. An antioxidant-rich diet can provide an easy-to-implement and cost-effective preventative strategy to reduce the risk of CS-related diseases, thus being one of the simplest ways for smokers to stay in good health for as long as possible. The health benefits attributable to the intake of antioxidants have been observed predominantly when these have been consumed within their natural food matrices in an optimal antioxidant-rich diet, while these preventive effects are rarely achieved with the intake of individual antioxidants, even at high doses.

In vitro copper oxide nanoparticle toxicity on intestinal barrier.

The use of CuO nanoparticles (NPs) has increased greatly and their potential effects on human health need to be investigated. Differentiated Caco-2 cells were treated from the apical (Ap) and the basolateral (Bl) compartment with different concentrations (0, 10, 50 and 100 µg/mL) of commercial or sonochemically synthesized (sono) CuO NPs. Sono NPs were prepared in ethanol (CuOe) or in water (CuOw), obtaining CuO NPs differing in size and shape. The effects on the Caco-2 cell barrier were assessed via transepithelial electrical resistance (TEER) evaluation just before and after 1, 2 and 24 hours of exposure and through the analysis of cytokine release and biomarkers of oxidative damage to proteins after 24 hours. Sono CuOe and CuOw NPs induced a TEER decrease with a dose-dependent pattern after Bl exposure. Conversely, TEER values were not affected by the Ap exposure to commercial CuO NPs and, concerning the Bl exposure, only the lowest concentration tested (10 µg/mL) caused a TEER decrease after 24 hours of exposure. An increased release of interleukin-8 was induced by sono CuO NPs after the Ap exposure to 100 µg/mL and by sono and commercial CuO after the Bl exposure to all the concentrations. No effects of commercial and sono CuO NPs on interleukin-6 (with the only exception of 100 µg/mL Bl commercial CuO) and tumor necrosis factor-α release were observed. Ap treatment with commercial and CuOw NPs was able to induce significant alterations on specific biomarkers of protein oxidative damage (protein sulfhydryl group oxidation and protein carbonylation).

Cigarette smoke and glutathione: Focus on in vitro cell models.

Cigarette smoke (CS) is one of the most important preventable risk factors for the development of respiratory diseases, cardiovascular diseases, stroke, and various types of cancer. Due to its high intracellular concentration and central role in maintaining the cellular redox state, glutathione (GSH) is one of the key players in several enzymatic and non-enzymatic reactions necessary for protecting cells against CS-induced oxidative stress. A plethora of in vitro cell models have been used over the years to assess the effects of CS on intracellular GSH and its disulphide forms, i.e. glutathione disulphide (GSSG) and S-glutathionylated proteins. In this review, we described the effects of cell exposure to CS on cellular GSH and formation of its oxidized forms and adducts (GSH-conjugates). We also discussed the limitations and relevance of in vitro cell models of exposure to CS and critically assessed the congruence between smokers and in vitro cell models. What emerges clearly is that results obtained in vitro should be interpreted with extreme caution, bearing in mind the limitations of the specific cell model used. Despite this, in vitro cell models remain important tools in the assessment of CS-induced oxidative damage.

Cytotoxic and proinflammatory responses induced by ZnO nanoparticles in in vitro intestinal barrier.

ZnO nanoparticles (NPs) are widely used nowadays, thus the gastrointestinal exposure to ZnO NPs is likely to be relevant and the effects on the intestinal barrier should be investigated. Polarized Caco-2 cells were exposed from the apical (Ap) and basolateral (Bl) compartments to increasing concentrations (0, 10, 50 and 100 µg/mL) of sonochemical (sono) and commercial ZnO NPs. The transepithelial electrical resistance (TEER), cell viability, proinflammatory cytokine release and presence of protein oxidative damage were evaluated after exposure. TEER was not significantly affected by Ap exposure to either sono or commercial ZnO NPs at any tested concentrations. After Bl exposure to sono ZnO NPs (all the concentrations) and to 100 µg/mL of commercial ZnO NPs TEER was decreased (P < 0.05). Ap and Bl exposure to 100 µg/mL sono ZnO NPs and Ap exposure to 50 µg/mL commercial ZnO NPs induced a significant (P < 0.05) release of interleukin-6. A significant (P < 0.05) release of interleukin-8 was observed after Ap exposure to ZnO NPs at 100 µg/mL and after Bl exposure to sono ZnO NPs at 100 µg/mL. Ap or Bl exposure to sono or commercial ZnO NPs did not affect tumour necrosis factor-alpha secretion or protein sulphydryl oxidation. In conclusion, the ZnO NP exposure from the Ap compartment appeared almost safe, while the exposure through the basal compartment appeared to be more hazardous and the different NP size and crystallinity seem to affect the mode of action, but further studies are necessary to elucidate better these toxicity mechanisms.

Protein carbonylation in human bronchial epithelial cells exposed to cigarette smoke extract.

Cigarette smoke is a well-established exogenous risk factor containing toxic reactive molecules able to induce oxidative stress, which in turn contributes to smoking-related diseases, including cardiovascular, pulmonary, and oral cavity diseases. We investigated the effects of cigarette smoke extract on human bronchial epithelial cells. Cells were exposed to various concentrations (2.5-5-10-20%) of cigarette smoke extract for 1, 3, and 24 h. Carbonylation was assessed by 2,4-dinitrophenylhydrazine using both immunocytochemical and Western immunoblotting assays. Cigarette smoke induced increasing protein carbonylation in a concentration-dependent manner. The main carbonylated proteins were identified by means of two-dimensional electrophoresis coupled to MALDI-TOF mass spectrometry analysis and database search (redox proteomics). We demonstrated that exposure of bronchial cells to cigarette smoke extract induces carbonylation of a large number of proteins distributed throughout the cell. Proteins undergoing carbonylation are involved in primary metabolic processes, such as protein and lipid metabolism and metabolite and energy production as well as in fundamental cellular processes, such as cell cycle and chromosome segregation, thus confirming that reactive carbonyl species contained in cigarette smoke markedly alter cell homeostasis and functions.

Determination of protein thiolation index (PTI) as a biomarker of oxidative stress in human serum.

We have introduced protein thiolation index (PTI), i.e. the molar ratio of the sum of all low molecular mass thiols bound to plasma proteins to protein free cysteinyl residues, as a sensitive biomarker of oxidative stress. According to the original procedure its determination requires a rapid separation of plasma and a specific treatment of samples to stabilize thiols. Here we demonstrate that samples can be collected without use of any anticoagulant to prevent blood clotting and without any stabilization of thiols too. This simplification of the determination of PTI makes its analysis more feasible also in routine clinical laboratories.

Assessment of glutathione/glutathione disulphide ratio and S-glutathionylated proteins in human blood, solid tissues, and cultured cells.

Glutathione (GSH) is the major non-protein thiol in humans and other mammals, which is present in millimolar concentrations within cells, but at much lower concentrations in the blood plasma. GSH and GSH-related enzymes act both to prevent oxidative damage and to detoxify electrophiles. Under oxidative stress, two GSH molecules become linked by a disulphide bridge to form glutathione disulphide (GSSG). Therefore, assessment of the GSH/GSSG ratio may provide an estimation of cellular redox metabolism. Current evidence resulting from studies in human blood, solid tissues, and cultured cells suggests that GSH also plays a prominent role in protein redox regulation via S -glutathionylation, i.e., the conjugation of GSH to reactive protein cysteine residues. A number of methodologies that enable quantitative analysis of GSH/GSSG ratio and S-glutathionylated proteins (PSSG), as well as identification and visualization of PSSG in tissue sections or cultured cells are currently available. Here, we have considered the main methodologies applied for GSH, GSSG and PSSG detection in biological samples. This review paper provides an up-to-date critical overview of the application of the most relevant analytical, morphological, and proteomics approaches to detect and analyse GSH, GSSG and PSSG in mammalian samples as well as discusses their current limitations.

Protein Carbonylation in Human Smokers and Mammalian Models of Exposure to Cigarette Smoke: Focus on Redox Proteomic Studies.

SIGNIFICANCE: Oxidative stress is one mechanism whereby tobacco smoking affects human health, as reflected by increased levels of several biomarkers of oxidative stress/damage isolated from tissues and biological fluids of active and passive smokers. Many investigations of cigarette smoke (CS)-induced oxidative stress/damage have been carried out in mammalian animal and cellular models of exposure to CS. Animal models allow the investigation of many parameters that are similar to those measured in human smokers. In vitro cell models may provide new information on molecular and functional differences between cells of smokers and nonsmokers. Recent Advances: Over the past decade or so, a growing number of researches highlighted that CS induces protein carbonylation in different tissues and body fluids of smokers as well as in in vivo and in vitro models of exposure to CS. CRITICAL ISSUES: We review recent findings on protein carbonylation in smokers and models thereof, focusing on redox proteomic studies. We also discuss the relevance and limitations of these models of exposure to CS and critically assess the congruence between the smoker's condition and laboratory models. FUTURE DIRECTIONS: The identification of protein targets is crucial for understanding the mechanism(s) by which carbonylated proteins accumulate and potentially affect cellular functions. Recent progress in redox proteomics allows the enrichment, identification, and characterization of specific oxidative protein modifications, including carbonylation. Therefore, redox proteomics can be a powerful tool to gain new insights into the onset and/or progression of CS-related diseases and to develop strategies to prevent and/or treat them. Antioxid. Redox Signal. 26, 406-426.

Dietary flavonoids advance timing of moult but do not affect redox status of juvenile blackbirds (Turdus merula).

Flavonoids are the most abundant plant polyphenols, widely occurring in fruits and berries, and show a strong antioxidant activity in vitro Studies of avian species feeding on berries suggest that dietary flavonoids have health-promoting effects and may enhance the expression of melanin-based plumage traits. These effects are probably mediated by the antioxidant activity of flavonoids. However, the effect of dietary flavonoids on oxidative status has never been investigated in any bird species. We analysed the effects of dietary flavonoids on blood non-enzymatic antioxidants and protein oxidative damage of juvenile European blackbirds (Turdus merula). In addition, we analysed the effects of flavonoid-enriched diet on body condition and on the timing of moult from juvenile to adult plumage. Dietary flavonoids did not significantly affect redox status but significantly advanced the onset of moult, hastening plumage development. Moulting birds showed higher protein oxidative damage compared with those that had not yet started moulting. The probability of initiating moult after 40 days of dietary treatment was higher for birds with low circulating levels of oxidizing agents and high glutathione concentration. The metabolization of flavonoids could have altered their redox potential, resulting in no net effects on redox status. However, flavonoid consumption before and during moult may contribute to enhance plumage development. Moreover, our findings suggest that moulting feathers may result in redox imbalance. Given their effect on moult and growth of melanin-rich feathers, fruit flavonoids may have contributed to shape plant fruiting time in relation to fruit consumption preferences by birds.

Pitfalls in the analysis of the physiological antioxidant glutathione (GSH) and its disulfide (GSSG) in biological samples: An elephant in the room.

Glutathione (GSH) is the most abundant low-molecular-mass thiol within cells and one of the major antioxidant compounds in body fluids. Under pro-oxidant conditions, two GSH molecules donate one electron each and are converted into glutathione disulfide (GSSG). The GSH/GSSG molar ratio is considered a powerful index of oxidative stress and disease risk. Despite high interest in GSH/GSSG titration as measures of thiol redox balance, no broad agreement has yet been reached as to the best pre-analytical and analytical methods for the quantitation of these molecules in biological samples. Consequently, measured concentrations of GSH and GSSG and calculated GSH/GSSG molar ratios vary widely among laboratories. Here, we describe in detail the main analytical and pre-analytical problems related to the artificial oxidation of the sulfhydryl (SH) group of GSH that occur during sample manipulation. We underline how this aspect has been neglected for long time after its first description more than fifty years ago. Finally, selected reliable procedures and methods to measure GSH and GSSG in biological samples are discussed.

Identification of dityrosine cross-linked sites in oxidized human serum albumin.

Reactive oxygen species (ROS) can oxidize virtually all cellular components. In proteins cysteine, methionine, tryptophan, and tyrosine residues are most prone to oxidation and their oxidized forms are thus considered as biomarkers of oxidative protein damages. Ultraviolet radiation and some endogenous ROS can produce tyrosine radicals reacting with other tyrosine residues yielding intra- or intermolecular cross-links in proteins. These 3,3'-dityrosines can be quantified by their characteristic fluorescence, but analytical methods to identify the modification sites in proteins are still missing. Although mass spectrometry (MS) is routinely used to map other post-translational modifications, the analysis of dityrosines is challenged by simultaneous fragmentations of both cross-linked peptide chains producing complex tandem mass spectra. Additionally, the fragmentation patterns differ from linear peptides. Here, we studied the fragmentation behavior of dityrosine cross-linked peptides obtained by incubating three peptides (AAVYHHFISDGVR, TEVSSNHVLIYLDK, and LVAYYTLIGASGQR) with horseradish peroxidase in the presence of hydrogen peroxide. Homo- and hetero-dimerization via dityrosine was monitored by fluorescence spectroscopy and MS. The fragmentation characteristics of dityrosine-linked peptides were studied on an ESI-LTQ-Orbitrap-MS using collision induced dissociation, which allowed localizing the cross-linked positions and provided generic rules to identify this oxidative modification. When human serum albumin oxidized with 50-fold molar excess of HOCl in phosphate buffer saline was analyzed by nanoRPC-ESI-MS/MS, an automatic database search considering all possible (in-silico generated) tyrosine-containing peptides as dynamic modifications revealed four different types of oxidatively modified tyrosine residues including dityrosines linking ten different Tyr residues. The automatic database search was confirmed by manual interpretation of each tandem mass spectrum.

Plasma protein thiolation index (PTI) as a biomarker of thiol-specific oxidative stress in haemodialyzed patients.

The role of oxidative stress in patients with end stage renal disease (ESRD), which occurs at significantly higher levels than in the general population, is often underestimated in clinical practice. Emerging evidence highlights the strong correlation of oxidative stress with chronic inflammation and cardiovascular disease, which are highly prevalent in most patients on maintenance haemodialysis (HD) and are a major risk factor for mortality in this population. In this study, total plasma thiols and plasma S-thiolated proteins were measured in patients with ESRD, before and after a regular HD session, and compared to age-matched healthy subjects. We found a significant decrease in the level of total plasma thiols and, conversely, a significant increase in the level of S-thiolated proteins in these patients. In most patients, post-HD plasma level of total thiols did not differ from the one in healthy subjects, whereas plasma level of S-thiolated proteins was lower in HD patients than in age-matched healthy controls. This suggests that a single HD session restores plasma thiol redox status and re-establishes the antioxidant capacity of plasma thiols. Additionally, we determined protein thiolation index (PTI), i.e., the molar ratio between the sum of all low molecular mass thiols bound to S-thiolated plasma proteins and protein free cysteinyl residues. Patients with ESRD had a significantly higher PTI compared to age-matched healthy subjects and HD was associated with a decrease in PTI to normal, or lower than normal, levels. Although this study is limited in size, our results suggest that PTI is a useful indicator of thiol-specific oxidative stress in patients with ESRD on maintenance HD. This study also emphasizes that PTI determination is a cheap and simple tool suitable for large-scale clinical studies that could be used for routine screening of thiol-specific oxidative stress.

Cigarette smoke induces alterations in the drug-binding properties of human serum albumin.

Albumin is the most abundant plasma protein and serves as a transport and depot protein for numerous endogenous and exogenous compounds. Earlier we had shown that cigarette smoke induces carbonylation of human serum albumin (HSA) and alters its redox state. Here, the effect of whole-phase cigarette smoke on HSA ligand-binding properties was evaluated by equilibrium dialysis and size-exclusion HPLC or tryptophan fluorescence. The binding of salicylic acid and naproxen to cigarette smoke-oxidized HSA resulted to be impaired, unlike that of curcumin and genistein, chosen as representative ligands. Binding of the hydrophobic fluorescent probe 4,4'-bis(1-anilino-8-naphtalenesulfonic acid) (bis-ANS), intrinsic tryptophan fluorescence, and susceptibility to enzymatic proteolysis revealed slight changes in albumin conformation. These findings suggest that cigarette smoke-induced modifications of HSA may affect the binding, transport and bioavailability of specific ligands in smokers.

Pathophysiology of tobacco smoke exposure: recent insights from comparative and redox proteomics.

First-hand and second-hand tobacco smoke are causally linked to a huge number of deaths and are responsible for a broad spectrum of pathologies such as cancer, cardiovascular, respiratory, and eye diseases as well as adverse effects on female reproductive function. Cigarette smoke is a complex mixture of thousands of different chemical species, which exert their negative effects on macromolecules and biochemical pathways, both directly and indirectly. Many compounds can act as oxidants, pro-inflammatory agents, carcinogens, or a combination of these. The redox behavior of cigarette smoke has many implications for smoke related diseases. Reactive oxygen and nitrogen species (both radicals and non-radicals), reactive carbonyl compounds, and other species may induce oxidative damage in almost all the biological macromolecules, compromising their structure and/or function. Different quantitative and redox proteomic approaches have been applied in vitro and in vivo to evaluate, respectively, changes in protein expression and specific oxidative protein modifications induced by exposure to cigarette smoke and are overviewed in this review. Many gel-based and gel-free proteomic techniques have already been used successfully to obtain clues about smoke effects on different proteins in cell cultures, animal models, and humans. The further implementation with other sensitive screening techniques could be useful to integrate the comprehension of cigarette smoke effects on human health. In particular, the redox proteomic approach may also help identify biomarkers of exposure to tobacco smoke useful for preventing these effects or potentially predictive of the onset and/or progression of smoking-induced diseases as well as potential targets for therapeutic strategies.

New insights in extracellular matrix remodeling and collagen turnover related pathways in cultured human tenocytes after ciprofloxacin administration.

We characterized the effect of ciprofloxacin (CPX) in cultured human tenocytes by morphological and molecular methods. Collagen type I and III mRNA and protein levels were unaffected, but lysyl hydroxylase 2b mRNA levels progressively decreased after CPX administration. MMP-1 protein levels significantly increased after 20 µg/ml CPX administration but remained unmodified at the higher dose, whilst MMP-2 activity was unchanged. Tissue inhibitor of MMP (TIMP-1) gene expression decreased after CPX treatment, whilst TIMP-2 and transforming growth factor-ß1 gene expression, the cytoskeleton arrangement, and cytochrome c expression remained unmodified. Secreted Protein Acidic and Rich in Cysteine mRNA and protein levels remained almost unchanged, whilst N-cadherin mRNA levels resulted significantly down-regulated and connexin 43 gene expression tended to decrease after CPX administration. The CPX-induced decreased ability to cross-link collagen and decreased TIMP-1 levels, possibly leading to higher activity of MMPs in ECM degradation, together with the down-regulation of N-cadherin and connexin 43 are consistent with a reduced ability to maintain tissue homeostasis, possibly making the tendon more susceptible to rupture.

Protein carbonylation in human endothelial cells exposed to cigarette smoke extract.

Cigarette smoke is a significant independent risk factor for vascular diseases and is a leading cause of structural and functional alterations of the vascular endothelium. In this study, we show protein carbonylation in the human umbilical vein endothelial cell line (ECV-304) exposed to whole-phase cigarette smoke extract. The main carbonylated proteins, including cytoskeletal proteins, glycolytic enzymes, xenobiotic metabolizing and antioxidant enzymes, and endoplasmic reticulum proteins, were identified by means of two-dimensional electrophoresis and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry (redox proteomics). Morphological analyses by fluorescence microscopy evidenced alterations in the microtubule cytoskeleton, especially at longer exposure time to cigarette smoke extract. Morphological analyses by transmission electron microscopy showed vacuolisation of the cytoplasm, alteration of mitochondria ultrastructure, and some enlargement of the perinuclear space. The possible role played by protein carbonylation caused by reactive species contained in cigarette smoke in the cigarette smoke-induced endothelial injury is discussed.

Pancreatic cancer cells retain the epithelial-related phenotype and modify mitotic spindle microtubules after the administration of ukrain in vitro.

The aim of this study is to characterize the phenotype of pancreatic ductal adenocarcinoma (PDAC) cells in relation to the expression of epithelial-to-mesenchymal transition (EMT) markers and determine whether ukrain, an anticancer drug based on the alkaloids extracted from greater celandine, modulates in vitro the malignant behavior of PDAC cells in order to extend our understanding of its therapeutic potential. Three cell lines (HPAF-II, HPAC, and PL45) were treated with ukrain (5, 10, and 20 µmol/l) for 48 h or left untreated (control). Cell proliferation was assessed by growth curves. Apoptosis was determined by Hoechst nuclear staining and by cytochrome c and caspase-8 expressions. The EMT markers E-cadherin, ß-catenin, and vimentin, as well as actin and tubulin cytoskeletons, were analyzed by immunofluorescence. Interphase and mitotic microtubules as well as abnormal mitotic figures were studied by fluorescence microscopy after tubulin immunolabeling. Ukrain strongly suppressed cell proliferation and induced apoptosis possibly through an extrinsic pathway as cytochrome c immunoreactivity suggested that the integrity of the mitochondria was not affected. Tubulin expression indicated an antiproliferative effect of ukrain on the basis of alterations in mitotic spindle microtubule dynamics, leading to abnormal mitosis. Membranous E-cadherin/ß-catenin immunoreactivity was similarly expressed in control-treated and ukrain-treated cells, although the drug upregulated E-cadherin in cell lysates. Our results suggest that ukrain exerts its chemotherapeutic action on PDAC cells targeting mitotic spindle microtubules, leading to abnormal mitosis and apoptosis, and favoring cell cohesiveness. The differentiated epithelial phenotype of HPAF-II, HPAC, and PL45 cell lines concomitant with a highly invasive potential suggests that further experiments will be necessary to definitively clarify the role of EMT in PDAC progression.

Protein thiolation index (PTI) as a biomarker of oxidative stress.

Several biomarkers of oxidative stress have been proposed and used in clinical research but so far unreliable or, at least, controversial results have been obtained. Given the high susceptibility of sulfhydryl groups to oxidation, we here suggest the use of a protein thiolation index (PTI), i.e., the molar ratio between the sum of all low molecular mass thiols bound to plasma proteins (forming, as a whole, S-thiolated proteins) and protein free cysteinyl residues, as a suitable biomarker of oxidative stress. While titration of free thiols can be performed by a simple spectrophotometric procedure, accurate quantification of S-thiolated proteins is problematic and current methods require, in most cases, application of time-consuming chromatographic techniques, making their application to large-scale clinical studies difficult. Here we report a new spectrophotometric method which relies on the specific determination of low molecular mass thiols released from S-thiolated proteins after dithiothreitol reduction. These amino acids can be titrated by conjugation with ninhydrin which, reacting with primary and secondary amine groups, yields a deep blue-purple color, which can be spectrophotometrically revealed. PTI showed an age dependency with a near linear increase during aging in humans. In addition, PTI was significantly higher in patients suffering from alkaptonuria with respect to healthy controls, suggesting that increased prooxidant conditions occur in the blood of these subjects.