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The identification of factors that respond to anti-angiogenic therapy would represent a significant advance in the therapeutic management of metastatic-colorectal-cancer (mCRC) patients. We previously reported the relevance of VEGF-A and some components of the renin–angiotensin-aldosterone system (RAAS) in the response to anti-angiogenic therapy in cancer patients. Therefore, this prospective study aims to evaluate the prognostic value of basal plasma levels of VEGF-A and angiotensin-converting enzyme (ACE) in 73 mCRC patients who were to receive bevacizumab-based therapies as a first-line treatment. We found that high basal VEGF-A plasma levels were significantly associated with worse overall survival (OS) and progression-free survival (FPS). On the other hand, low ACE levels were significantly associated with poor OS. Importantly, a simple scoring system combining the basal plasma levels of VEGF-A and ACE efficiently stratified mCRC patients, according to OS, into high-risk or low-risk groups, prior to their treatment with bevacizumab. In conclusion, our study supports that VEGF-A and ACE may be potential biomarkers for selecting those mCRC patients who will most benefit from receiving chemotherapy plus bevacizumab treatment in first-line therapy. Additionally, our data reinforce the notion of a close association between the RAAS and the anti-angiogenic response in cancer.

Simple Summary Liquid biopsy is a noninvasive approach that provides tumor molecular profiling. On the other hand, the vast majority of pancreatic tumors are pancreatic ductal adenocarcinomas (PDAC), which are characterized by pronounced inflammation. Therefore, we hypothesized that the combination of biomarkers of systemic inflammation, such as the neutrophil-to-lymphocyte-ratio (NLR) and platelet-to-lymphocyte ratio (PLR), with liquid biopsy-based biomarkers may increase their clinical usefulness. Our study shows that combining NLR, PLR, and the standard PDAC marker CA19-9 with circulating cell-free DNA and circulating RAS-mutated DNA outperforms traditional clinical tools for the clinical management of metastatic PDAC patients. Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a highly inflammatory microenvironment and liquid biopsy has emerged as a promising tool for the noninvasive analysis of this tumor. In this study, plasma was obtained from 58 metastatic PDAC patients, and neutrophil–lymphocyte ratio (NLR), platelet–lymphocyte ratio (PLR), circulating cell-free DNA (cfDNA) concentration, and circulating RAS mutation were determined. We found that NLR was significantly associated with both overall survival (OS) and progression-free survival. Remarkably, NLR was an independent risk factor for poor OS. Moreover, NLR and PLR positively correlated, and combination of both inflammatory markers significantly improved the prognostic stratification of metastatic PDAC patients. NLR also showed a positive correlation with cfDNA levels and RAS mutant allelic fraction (MAF). Besides, we found that neutrophil activation contributed to cfDNA content in the plasma of metastatic PDAC patients. Finally, a multi-parameter prognosis model was designed by combining NLR, PLR, cfDNA levels, RAS mutation, RAS MAF, and CA19-9, which performs as a promising tool to predict the prognosis of metastatic PDAC patients. In conclusion, our study supports the idea that the use of systemic inflammatory markers along with circulating tumor-specific markers may constitute a valuable tool for the clinical management of metastatic PDAC patients.

Cancer stem cells (CSCs) are involved in the resistance of estrogen (ER)-positive breast tumors against endocrine therapy. On the other hand, nitric oxide (NO) plays a relevant role in CSC biology, although there are no studies addressing how this important signaling molecule may contribute to resistance to antihormonal therapy in ER+ breast cancer. Therefore, we explored whether targeting NO in ER+ breast cancer cells impacts CSC subpopulation and sensitivity to hormonal therapy with tamoxifen. NO was targeted in ER+ breast cancer cells by specific NO depletion and NOS2 silencing and mammosphere formation capacity, stem cell markers and tamoxifen sensitivity were analyzed. An orthotopic breast tumor model in mice was also performed to analyze the efficacy of NO-targeted therapy plus tamoxifen. Kaplan-Meier curves were made to analyze the association of NOS2 gene expression with survival of ER+ breast cancer patients treated with tamoxifen. Our results show that targeting NO inhibited mamosphere formation, CSC markers expression and increased the antitumoral efficacy of tamoxifen in ER+ breast cancer cells, whereas tamoxifen-resistant cells displayed higher expression levels of NOS2 and Notch-1 compared with parental cells. Notably, NO-targeted therapy plus tamoxifen was more effective than either treatment alone in an orthotopic breast tumor model in immunodeficient mice. Furthermore, low NOS2 expression was significantly associated with a higher metastasis-free survival in ER+ breast cancer patients treated with tamoxifen. In conclusion, our data support that NO-targeted therapy in ER+ breast cancer may contribute to increase the efficacy of antihormonal therapy avoiding the development of resistance to these treatments.

Nitric oxide (NO) has been highlighted as an important agent in tumor processes. However, a complete understanding of the mechanisms by which this simple diatomic molecule contributes in tumorigenesis is lacking. Evidence is rapidly accumulating that metabolic reprogramming is a major new aspect of NO biology and this review is aimed to summarize recent research progress on this novel feature that expands the complex and multifaceted role of NO in cancer. Therefore, we discuss how NO may influence glucose and glutamine utilization by tumor cells, and its participation in the regulation of mitochondrial function and dynamics, that is an important mechanism through which cancer cells reprogram their metabolism to meet the biosynthetic needs of rapid proliferation. Finally, we also discuss the NO-related metabolic rewiring involved in the modification of the tumor microenvironment to support cancer invasion and the escape from immune system-mediated recognition. Protein S-nitrosylation appears as a common mechanism by which NO signaling reprograms metabolism. Hence, future research is needed on dysregulated S-nitrosylation/denitrosylation in cancer to comprehend the NO-induced metabolic changes in tumor cells and the role of NO in the metabolic crosstalk within tumor microenvironment.

Newly emerged proteomic methodologies, particularly data‐independent acquisition (DIA) analysis–related approaches, would improve current gene expression–based classifications of colorectal cancer (CRC). Therefore, this study was aimed to identify protein expression signatures using SWATH‐MS DIA and targeted data extraction, to aid in the classification of molecular subtypes of CRC and advance in the diagnosis and development of new drugs. For this purpose, 40 human CRC samples and 7 samples of healthy tissue were subjected to proteomic and bioinformatic analysis. The proteomic analysis identified three different molecular CRC subtypes: P1, P2 and P3. Significantly, P3 subtype showed high agreement with the mesenchymal/stem‐like subtype defined by gene expression signatures and characterized by poor prognosis and survival. The P3 subtype was characterized by decreased expression of ribosomal proteins, the spliceosome, and histone deacetylase 2, as well as increased expression of osteopontin, SERPINA 1 and SERPINA 3, and proteins involved in wound healing, acute inflammation and complement pathway. This was also confirmed by immunodetection and gene expression analyses. Our results show that these tumours are characterized by altered expression of proteins involved in biological processes associated with immune evasion and metastasis, suggesting new therapeutic options in the treatment of this aggressive type of CRC.

In recent years, an increasing number of studies have shown that there is an important connection between nitric oxide (NO) and the pathology of malignant diseases, but we are far from a complete comprehension of how this simple diatomic molecule contributes to tumorigenesis. The emerging identification of immune-mediated mechanisms regulated by NO may help to unravel the intricate and complex relationships between NO and cancer. Therefore, this review provides a summary of recent advances in our understanding of the immunomodulatory role of NO in cancer, and in particular the role of this pleiotropic signaling molecule as an immunosuppressive mediator in the tumor microenvironment. We will discuss the participation of NO in the different strategies used by tumors to escape from immune system-mediated recognition, including the acquisition of stem cell like capacities by tumor cells and the metabolic reprogramming of tumor infiltrating immune cells. Finally, we will also discuss different therapeutic strategies directed against NO for abating the immunosuppressive tumor microenvironment and to increase the efficacy of immunotherapy in cancer.

Initially discovered as a powerful regulator of vascular tone, nitric oxide (NO) has also been highlighted as a key factor in different diseases, including cancer. This molecule is aberrantly produced during tumor progression mainly through the enzymatic catabolism of arginine by three different proteins belonging to the nitric oxide synthase (NOS) family. The relevance of each isoenzyme as the main source of NO in carcinogenesis may be critically different depending on the tumor context. Thus, the endothelial NOS (eNOS) protein is the most relevant source of NO in different scenarios where the cancer stem cell (CSC) phenotype is enhanced, such as the mesenchymal poor-prognosis subtype colorectal tumors. Besides, eNOS upregulation has been shown in different Apc conditional mouse models where Apc-deficient cells maintain a crypt progenitor-like phenotype associated with early colorectal lesions. Since NO signaling is independent of how it was produced, NO scavengers represent the best approach to target this molecule produced from any source. In this regard, the NO scavenger carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (c-PTIO) has been shown to improve the efficacy of immunotherapy, to impair the CSC phenotype, and to reduce tumor growth in vivo. Therefore, the control of the aberrant production of NO may represent a key upstream hub for targeting different cancer-related events and different NO-related pathologies.

BACKGROUND Currently, there are no predictive biomarkers for anti-angiogenic strategies in cancer, but response to anti-angiogenic drugs is associated with development of hypertension secondary to treatment. Therefore, this study explored the clinical relevance of genetic polymorphisms in some components of the renin-angiotensin system (RAS). MATERIAL AND METHODS Genomic DNA was isolated from peripheral blood from 95 metastatic breast or colorectal cancer patients treated with bevacizumab, and AGTR1-A1166C (rs5186), AGT-M235T (rs699) SNPs and ACE I/D (rs4646994) polymorphisms were genotyped using RT-PCR. Circulating vascular endothelial grow factor and angiotensin converting enzyme (ACE) levels were analysed using ELISA kits. The antitumoral activity of bevacizumab was assayed in mice orthotopically xenografted with AGTR1-overexpressing breast cancer cells. RESULTS The ACE IN/IN genotype was associated with a higher rate of disease progression compared to DEL/IN and DEL/DEL genotypes (36% vs. 11·1% P < 0·05). Similarly, AGTR1-1166A/A genotype was also associated with a higher rate of disease progression compared to AGTR1-1166A/C and AGTR1-1166C/C genotypes (24·4% vs. 2·7% P < 0·01). ACE IN/IN genotype was also found to be associated with shorter time to treatment failure compared to ACE IN/DEL and ACE DEL/DEL genotypes (14 weeks vs. 41·71, P = 0·033), whereas circulating ACE levels were found to be associated with a better response to bevacizumab treatment. Besides, in vivo experiments showed a significantly higher antitumoral activity of bevacizumab in tumours derived from AGTR1-overexpressing breast cancer cells. CONCLUSIONS A higher activity of ACE-angiotensin-II-AGTR1 axis is associated with a better response to bevacizumab, supporting that the RAS can be an important source of potential predictive markers of response to anti-angiogenic drugs.

During the course of cholestatic liver diseases, the toxic effect of bile acids accumulation has been related to the decreased expression of endothelial nitric oxide synthase (NOS-3) and cellular oxidative stress increase. In the present study, we have investigated the relationship between these two biological events. In the human hepatocarcinoma cell line HepG2, cytotoxic response to GCDCA was characterized by the reduced activity of the respiratory complexes II. +. III, the increased expression and activation of the transcription factor Sp1, and a higher binding capacity of this at positions -1386, -632 and -104 of the NOS-3 promoter (pNOS-3). This was associated with a decreased promoter activity and a consequent reduction of NOS-3 expression. The use of antioxidants in GCDCA-treated cells caused a lower activation of Sp1 and the recovery of the pNOS-3 activity and NOS-3 expression and activity. Similarly, the specific inhibition of Sp1 resulted in the improvement of NOS-3 expression. Both, antioxidant treatment and Sp1 inhibition were associated with the reduction of cell death-related parameters. Bile duct ligation in rats confirmed in vitro results concerning the activation of Sp1 and the reduction of NOS-3 expression. Our results provide direct evidence for the involvement of Sp1 in the regulation of NOS-3 expression during cholestasis. Thus, the identification of Sp1 as a potential negative regulator of NOS-3 expression represents a new mechanism by which the accumulation of bile acids causes a cytotoxic effect through the oxidative stress increase, and provides a new potential target in cholestatic liver diseases. Fil: González Rubio, Sandra. Universidad de Córdoba; España Fil: López Sánchez, Laura. Universidad de Córdoba; España. Universidad Carlos III de Madrid. Instituto de Salud; España Fil: Muñoz Castañeda, Juan. Universidad de Córdoba; España Fil: Linares, Clara I.. Universidad de Córdoba; España Fil: Aguilar Melero, Patricia. Universidad de Córdoba; España Fil: Rodríguez Perálvarez, Manuel. Universidad de Córdoba; España. Universidad Carlos III de Madrid. Instituto de Salud; España Fil: Sánchez Sánchez, Rafael. Reina Sofia University Hospital; España Fil: Fernández Alvarez, Ana Julia. Biomedicine Institute of Valencia; España. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Casado, Marta. Universidad Carlos III de Madrid. Instituto de Salud; España Fil: Montero Álvarez, Jose L.. Universidad de Córdoba; España. Universidad Carlos III de Madrid. Instituto de Salud; España Fil: Rodríguez Ariza, Antonio. Universidad de Córdoba; España. Universidad Carlos III de Madrid. Instituto de Salud; España Fil: Muntané, Jordi. Universidad de Córdoba; España. Universidad Carlos III de Madrid. Instituto de Salud; España Fil: de la Mata, Manuel. Universidad de Córdoba; España. Universidad Carlos III de Madrid. Instituto de Salud; España. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ferrín, Gustavo. Universidad de Córdoba; España. Universidad Carlos III de Madrid. Instituto de Salud; España

We explored whether the proteomic analysis of exhaled breath condensate (EBC) may provide biomarkers for noninvasive screening for the early detection of lung cancer (LC). EBC was collected from 192 individuals [49 control (C), 49 risk factor-smoking (S), 46 chronic obstructive pulmonary disease (COPD) and 48 LC]. With the use of liquid chromatography and tandem mass spectrometry, 348 different proteins with a different pattern among the four groups were identified in EBC samples. Significantly more proteins were identified in the EBC from LC compared with other groups (C: 12.4 ± 1.3; S: 15.3 ± 1; COPD: 14 ± 1.6; LC: 24.2 ± 3.6; P = 0.0001). Furthermore, the average number of proteins identified per sample was significantly higher in LC patients, and receiver operating characteristic curve (ROC) analysis showed an area under the curve of 0.8, indicating diagnostic value. Proteins frequently detected in EBC, such as dermcidin and hornerin, along with others much less frequently detected, such as hemoglobin and histones, were identified. Cytokeratins (KRTs) were the most abundant proteins in EBC samples, and levels of KRT6A, KRT6B, and KRT6C isoforms were significantly higher in samples from LC patients ( P = 0.0031, 0.0011, and 0.0009, respectively). Moreover, the amount of most KRTs in EBC samples from LC patients showed a significant positive correlation with tumor size. Finally, we used a random forest algorithm to generate a robust model using EBC protein data for the diagnosis of patients with LC where the area under the ROC curve obtained indicated a good classification (82%). Thus this study demonstrates that the proteomic analysis of EBC samples is an appropriated approach to develop biomarkers for the diagnosis of lung cancer.

Objectives 1) To assess the association of NETosis and NETosis-derived products with the activity of the disease and the development of cardiovascular disease in RA; 2) To evaluate the involvement of NETosis on the effects of biologic therapies such as anti-TNF alpha (Infliximab) and anti-IL6R drugs (Tocilizumab). Methods One hundred and six RA patients and 40 healthy donors were evaluated for the occurrence of NETosis. Carotid-intimae media thickness was analyzed as early atherosclerosis marker. Inflammatory and oxidative stress mediators were quantified in plasma and neutrophils. Two additional cohorts of 75 RA patients, treated either with Infliximab (n = 55) or Tocilizumab (n = 20) for six months, were evaluated. Results NETosis was found increased in RA patients, beside myeloperoxidase and neutrophil elastase protein levels. Cell-free nucleosomes plasma levels were elevated, and strongly correlated with the activity of the disease and the positivity for autoantibodies, alongside inflammatory and oxidative profiles in plasma and neutrophils. Moreover, ROC analyses showed that cell-free nucleosomes levels could identify RA patients showing early atherosclerosis with high specificity. RA patients treated either with IFX or TCZ for six months exhibited decreased generation of NETs. Concomitantly, clinical parameters and serum markers of inflammation were found reduced. Mechanistic in vitro analyses showed that inhibition of NETs extrusion by either DNase, IFX or TCZ, further abridged the endothelial dysfunction and the activation of immune cells, thus influencing the global activity of the vascular system. Conclusions NETosis-derived products may have diagnostic potential for disease activity and atherosclerosis, as well as for the assessment of therapeutic effectiveness in RA.

Objective— Antiphospholipid syndrome (APS) leukocytes exhibit an oxidative perturbation, directly linked to alterations in mitochondrial dynamics and metabolism. This disturbance is related to the patients’ prothrombotic status and can be prevented by in vitro treatment with coenzyme Q10. Our aim was to investigate short-term effects of in vivo ubiquinol (reduced coenzyme Q 10 [Q red ]) supplementation on markers related to inflammation and thrombosis in APS through a prospective, randomized, crossover, placebo-controlled trial. Approach and Results— Thirty-six patients with APS were randomized to receive Q red (200 mg/d) or placebo for 1 month. Thirty-three patients with APS completed the intervention, which increased plasma coenzyme Q 10 . Q red improved endothelial function and decreased monocyte expression of prothrombotic and proinflammatory mediators, inhibited phosphorylation of thrombosis-related protein kinases, and decreased peroxides and percentage of monocytes with depolarized mitochondria; mitochondrial size was increased, and mitochondrial biogenesis–related genes were upregulated. Q red ameliorated extruded neutrophil extracellular traps in neutrophils and downregulated peroxides, intracellular elastase, and myeloperoxidase. Nanostring microRNA profiling revealed 20 microRNAs reduced in APS monocytes, and 16 of them, with a preponderance of cardiovascular disease–related target mRNAs, were upregulated. Monocytes gene profiling showed differential expression of 29 atherosclerosis-related genes, 23 of them changed by Q red . Interaction networks of genes and microRNAs were identified. Correlation studies demonstrated co-ordinated effects of Q red on thrombosis and endothelial function–associated molecules. Conclusions— Our results highlight the potential of Q red to modulate the overexpression of inflammatory and thrombotic risk markers in APS. Because of the absence of clinically significant side effects and its potential therapeutic benefits, Q red might act as safe adjunct to standard therapies in APS. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT02218476

The complex chemistry and biochemistry of nitric oxide (NO) and their relation to both physiological and pathological processes is one of the main reasons of the considerable difficulties in understanding the precise roles of this small molecule in cancer biology. One of the modifications mediated by NO is the coupling of a nitroso group moiety to a reactive thiol group in specific cysteine residues in peptides or proteins resulting in S-nitrosothiol (SNO) formation, a process commonly known as S-nitrosylation. In many cases, an altered production of NO and/or impaired SNO homeostasis promotes an aberrant S-nitrosylation of proteins. Importantly, this alteration in SNO homeostasis and S-nitrosylation of proteins plays an important role in a broad range of diseases, including cancer. Recent research in cell metabolism of SNOs has led to the identification and characterization of enzyme-mediated processes (nitrosylases and denitrosylases) that are implicated in targeted S-nitrosylation or denitrosylation of proteins in physiological settings. In this chapter, we will review these studies that may help to unravel the complex interrelationships between NO and the molecular pathways in the initiation and progression of tumors and their response to therapy.

ObjectiveStatins may have beneficial vascular effects in systemic lupus erythematosus (SLE) beyond their cholesterol-lowering action, although the mechanisms involved are not completely understood. We investigated potential mechanisms involved in the efficacy of fluvastatin in preventing atherothrombosis in SLE.MethodsEighty-five patients with SLE and 62 healthy donors were included in the study. Selected patients (n=27) received 20 mg/day fluvastatin for 1 month. Blood samples were obtained before the start and at the end of treatment. Monocytes from five patients were treated in vitro with fluvastatin.ResultsIncreased prothrombotic and inflammatory variables were found in patients with SLE. SLE monocytes displayed altered mitochondrial membrane potential and increased oxidative stress. Correlation and association analyses demonstrated a complex interplay among autoimmunity, oxidative stress, inflammation and increased risk of atherothrombosis in SLE. Fluvastatin treatment of patients for 1 month reduced the SLE Disease Activity Index and lipid levels, oxidative status and vascular inflammation. Array studies on monocytes demonstrated differential expression in 799 genes after fluvastatin treatment. Novel target genes and pathways modulated by fluvastatin were uncovered, including gene networks involved in cholesterol and lipid metabolism, inflammation, oxidative stress and mitochondrial activity. Electron microscopy analysis showed increased density volume of mitochondria in monocytes from fluvastatin-treated patients, who also displayed higher expression of genes involved in mitochondrial biogenesis. In vitro treatment of SLE monocytes confirmed the results obtained in the in vivo study.ConclusionsOur overall data suggest that fluvastatin improves the impairment of a redox-sensitive pathway involved in processes that collectively orchestrate the pathophysiology of atherothrombosis in SLE.

ObjectiveTo identify shared and differential molecular pathways involved in the pathogenesis of atherosclerosis (AT) and cardiovascular disease (CVD) in systemic lupus erythematosus (SLE), primary antiphospholipid syndrome (APS) and APS associated with SLE (APS plus SLE).Methods129 patients (42 APS, 31 APS plus SLE and 56 SLE) and 61 healthy donors were included. Microarray expression profiling was performed in monocytes. RT-PCR of selected genes and western blot were used to validate microarray data. Clinical and inflammatory parameters were also analysed.ResultsCompared with controls, 555, 1224 and 518 genes were differentially expressed in monocytes from SLE, APS plus SLE and APS patients, respectively. Approximately 25–30% of differentially expressed genes were related to AT and CVD. Each disease displayed a specific AT/CVD/Inflammation-related gene signature. Compared with SLE, APS showed alterations in mitochondria biogenesis and function and oxidative stress. Besides the interferon signature, found in APS plus SLE and SLE patients, various genes mediating atherosclerotic/inflammatory signalling were also differentially expressed in APS plus SLE. IgG-anticardiolipin (aCL) titres independently predicted both atherosclerotic and thrombosis in APS plus SLE. Moreover, a significant correlation of IgG-aCL titres with mRNA levels of certain inflammatory molecules in monocytes was further noticed. In vitro treatment of monocytes with IgG-aCL promoted an increase in the expression of the genes most significantly changed in APS plus SLE versus healthy donors.ConclusionsGene expression profiling allows the segregation of APS, APS plus SLE and SLE, with specific signatures explaining the pro-atherosclerotic and pro-thrombotic alterations in these highly related autoimmune diseases.

Killer-cell immunoglobulin-like receptors (KIRs) regulate the killing function of natural killer cells, which play an important role in the antibody-dependent cell-mediated cytotoxicity response exerted by therapeutic monoclonal antibodies (mAbs). However, it is unknown whether the extensive genetic variability of KIR genes and/or their human leukocyte antigen (HLA) ligands might influence the response to these treatments. This study aimed to explore whether the variability in KIR/HLA genes may be associated with the variable response observed to mAbs based anti-epidermal growth factor receptor (EGFR) therapies. Thirty-nine patients treated with anti-EGFR mAbs (trastuzumab for advanced breast cancer, or cetuximab for advanced colorectal or advanced head and neck cancer) were included in the study. All the patients had progressed to mAbs therapy and were grouped into two categories taking into account time to treatment failure (TTF ≤6 and ≥10 months). KIR genotyping (16 genetic variability) was performed in genomic DNA from peripheral blood by PCR sequence-specific primer technique, and HLA ligand typing was performed for HLA-B and -C loci by reverse polymerase chain reaction sequence-specific oligonucleotide methodology. Subjects carrying the KIR/HLA ligand combinations KIR2DS1/HLAC2C2-C1C2 and KIR3DS1/HLABw4w4-w4w6 showed longer TTF than non-carriers counterparts (14.76 vs. 3.73 months, p

Many of nitric oxide (NO) actions are mediated through the coupling of a nitroso moiety to a reactive cysteine leading to the formation of a S-nitrosothiol (SNO), a process known as S-nitrosylation or S-nitrosation. In many cases this reversible post-translational modification is accompanied by altered protein function and aberrant S-nitrosylation of proteins, caused by altered production of NO and/or impaired SNO homeostasis, has been repeatedly reported in a variety of pathophysiological settings. A growing number of studies are directed to the identification and characterization of those proteins that undergo S-nitrosylation and the analysis of S-nitrosoproteomes under pathological conditions is beginning to be reported. The study of these S-nitrosoproteomes has been fueled by advances in proteomic technologies that are providing researchers with improved tools for exploring this post-translational modification. Here we review novel refinements and improvements to these methods, and some recent studies of the S-nitrosoproteome in disease.

MicroRNAs markedly affect the immune system and have a relevant role in CVD and autoimmune diseases. Yet, no study has analyzed their involvement in atherothrombosis related to APS and SLE patients. This study intended to: 1) identify and characterize microRNAs linked to CVD in APS and SLE; 2) assess the effects of specific autoantibodies. Six microRNAs, involved in atherothrombosis development, were quantified in purified leukocytes from 23 APS and 64 SLE patients, and 56 healthy donors. Levels of microRNAs in neutrophils were lower in APS and SLE than in healthy donors. Gene and protein expression of miRNA biogenesis-related molecules were also reduced. Accordingly, more than 75% of identified miRNAs by miRNA profiling were underexpressed. In monocytes, miR124a and -125a were low, while miR-146a and miR-155 appeared elevated. Altered microRNAs’ expression was linked to autoimmunity, thrombosis, early atherosclerosis and oxidative stress in both pathologies. In vitro treatment of neutrophils, monocytes and ECs with aPL-IgG or anti-dsDNA-IgG antibodies deregulated microRNAs expression and decreased miRNA biogenesis-related proteins. Monocyte transfections with pre-miR-124a and/or -125a caused reduction in atherothrombosis-related target molecules. In conclusion, microRNA biogenesis, significantly altered in neutrophils of APS and SLE patients, is associated to their atherothrombotic status, further modulated by specific autoantibodies.

// Araceli Valverde 1 , Jon Penarando 1, 2 , Amanda Canas 1 , Laura M. Lopez-Sanchez 1, 2 , Francisco Conde 1, 2 , Silvia Guil-Luna 1 , Vanessa Hernandez 1, 2 , Carlos Villar 3 , Cristina Morales-Estevez 1, 4 , Juan de la Haba-Rodriguez 1, 2, 4 , Enrique Aranda 1, 2, 4 , Antonio Rodriguez-Ariza 1, 2, 4 1 Instituto Maimonides de Investigacion Biomedica de Cordoba (IMIBIC), Cordoba, Spain 2 Centro de Investigacion Biomedica en Red de Cancer, Madrid, Spain 3 Unidad de Gestion Clinica de Anatomia Patologica, Hospital Universitario Reina Sofia, Cordoba, Spain 4 Unidad de Gestion Clinica de Oncologia, Hospital Universitario Reina Sofia, Cordoba, Spain Correspondence to: Antonio Rodriguez-Ariza, email: antonio.rodriguez.exts@juntadeandalucia.es Keywords: β-catenin, colorectal cancer, COX-2, EGFR, FOXM1 Received: July 20, 2016 Accepted: January 23, 2017 Published: February 21, 2017 ABSTRACT Here we showed that the addition of the COX-2 inhibitor celecoxib improved the antitumor efficacy in colorectal cancer (CRC) of the monoclonal anti-EGFR antibody cetuximab. The addition of celecoxib augmented the efficacy of cetuximab to inhibit cell proliferation and to induce apoptosis in CRC cells. Moreover, the combination of celecoxib and cetuximab was more effective than either treatment alone in reducing the tumor volume in a mouse xenograft model. The combined treatment enhanced the inhibition of EGFR signaling and altered the subcellular distribution of β-catenin. Moreover, knockdown of FOXM1 showed that this transcription factor participates in this enhanced antitumoral response. Besides, the combined treatment decreased β-catenin/FOXM1 interaction and reduced the cancer stem cell subpopulation in CRC cells, as indicated their diminished capacity to form colonospheres. Notably, the inmunodetection of FOXM1 in the nuclei of tumor cells in human colorectal adenocarcinomas was significantly associated with response of patients to cetuximab. In summary, our study shows that the addition of celecoxib enhances the antitumor efficacy of cetuximab in CRC due to impairment of EGFR-RAS-FOXM1-β-catenin signaling axis. Results also support that FOXM1 could be a predictive marker of response of mCRC patients to cetuximab therapy.

The monoclonal antibody trastuzumab against HER2/neu, which is overexpressed in 15-20% of breast cancers, has clinical efficacy but many patients do not respond to initial treatment or develop resistance during treatment. Nitric oxide (NO) regulates cell signaling by targeting specific cysteine residues in proteins, forming S-nitrosothiols (SNO) in a process known as S-nitrosylation. We previously reported that molecular characteristics in breast cancer may dictate the tumor response to impaired SNO homeostasis. In the present study, we explored the role of SNO homeostasis in HER2 breast tumors. The antiproliferative action of trastuzumab in HER2-overexpressing BT-474 and SKBR-3 cells was suppressed when S-nitrosoglutathione reductase (GSNOR/ADH5) activity, which plays a key role in SNO homeostasis, was specifically inhibited with the pyrrole derivative compound N6022. Moreover, GSNOR inhibition restored the activation of survival signaling pathways involved in the resistance to anti-HER2 therapies (AKT, Src and c-Abl kinases and TrkA/NRTK1, TrkB/NRTK2, EphA1 and EphA3 receptors) and reduced the apoptotic effect of trastuzumab. Accordingly, GSNOR inhibition augmented the S-nitrosylation of apoptosis-related proteins, including Apaf-1, pSer73/63 c-Jun, calcineurin subunit α and HSF1. In agreement with in vitro data, immunohistochemical analyses of 51 breast tumors showed that HER2 expression was associated with lower expression of GSNOR protein. Moreover, gene expression analysis confirmed that high ADH5/GSNOR gene expression was associated with high patient survival rates in HER2 tumors. In conclusion, our data provide evidence of molecular mechanisms contributing to the progression of HER2+ breast cancers and could facilitate the development of therapeutic options to counteract resistance to anti-HER2 therapies.

Statins have been successfully used in patients with hypercholesterolemia and cardiovascular diseases, but there is increasing evidence that they exert effects by much exceeding the lowering of cholesterol levels. Statins have antiatherosclerotic, antiinflammatory, antioxidant, immunomodulatory and antithrombotic effects. These "pleiotropic" effects stem from their inhibition of prenylation of the small GTP-binding proteins Ras and Rho, and to the disruption, or depletion, of cholesterol rich membrane micro-domains (membrane rafts). Through these pathways statins modulate immune responses by altering cytokine levels and by affecting the function of cells involved in both innate and adaptive responses. Anti-inflammatory and immunosuppressory properties of statins provide the rationale for their potential application in conditions in which the inflammation and immune response represent key pathogenic mechanisms, such as antiphospholipid syndrome, rheumatoid arthritis and systemic lupus erythematosus. Reduction of atherosclerosis progression in autoimmunity is also a very important effect. Statins pathways of action in systemic autoimmune diseases, and their potential therapeutic use are discussed in this review. The inhibition of mevalonate pathway by statins impairs modification of Ras and Rho GTPases, which play key roles in signaling pathways related to tumor formation, metastasis and cell death. There is experimental and clinical evidence that statins may improve the therapeutic outcome of anticancer drugs. Thus, this review will also discuss recent insights into the molecular mechanisms underlying the anticancer effects of statins and their assessment as promising candidates for inclusion into current therapeutic regimens for the treatment of malignant diseases.

Lung cancer currently ranks as the neoplasia with the highest global mortality rate. Although some improvements have been introduced in recent years, new advances in diagnosis are required in order to increase survival rates. New mildly invasive endoscopy-based diagnostic techniques include the collection of bronchoalveolar lavage fluid (BALF), which is discarded after using a portion of the fluid for standard pathological procedures. BALF proteomic analysis can contribute to clinical practice with more sensitive biomarkers, and can complement cytohistological studies by aiding in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. The range of quantitative proteomics methodologies used for biomarker discovery is currently being broadened with the introduction of data-independent acquisition (DIA) analysis-related approaches that address the massive quantitation of the components of a proteome. Here we report for the first time a DIA-based quantitative proteomics study using BALF as the source for the discovery of potential lung cancer biomarkers. The results have been encouraging in terms of the number of identified and quantified proteins. A panel of candidate protein biomarkers for adenocarcinoma in BALF is reported; this points to the activation of the complement network as being strongly over-represented and suggests this pathway as a potential target for lung cancer research. In addition, the results reported for haptoglobin, complement C4-A, and glutathione S-transferase pi are consistent with previous studies, which indicates that these proteins deserve further consideration as potential lung cancer biomarkers in BALF. Our study demonstrates that the analysis of BALF proteins by liquid chromatography–tandem mass spectrometry (LC–MS/MS), combining a simple sample pre-treatment and SWATH DIA MS, is a useful method for the discovery of potential lung cancer biomarkers. Significance Bronchoalveolar lavage fluid (BALF) analysis can contribute to clinical practice with more sensitive biomarkers, thus complementing cytohistological studies in order to aid in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. Here we report a panel of candidate protein biomarkers for adenocarcinoma in BALF. Forty-four proteins showed a fold-change higher than 3.75 among adenocarcinoma patients compared with controls. This report is the first DIA-based quantitative proteomics study to use bronchoalveolar lavage fluid (BALF) as a matrix for discovering potential biomarkers. The results are encouraging in terms of the number of identified and quantified proteins, demonstrating that the analysis of BALF proteins by a SWATH approach is a useful method for the discovery of potential biomarkers of pulmonary diseases.

Antiphospholipid syndrome (APS) is a disorder characterized by the association of arterial or venous thrombosis and/or pregnancy morbidity with the presence of antiphospholipid antibodies (anticardiolipin antibodies, lupus anticoagulant antibodies, and/or anti-β2-glycoprotein I antibodies). Several studies have contributed to uncovering the basis of antiphospholipid antibody pathogenicity, including the targeted cellular components, affected systems, involved receptors, intracellular pathways used, and the effector molecules that are altered in the process. Therapy for thrombosis traditionally has been based on long-term oral anticoagulation; however, bleeding complications and recurrence despite high-intensity anticoagulation can occur. Based on all the data obtained, new potential therapeutic agents have been proposed. Statins have a variety of direct effects on gene expression and the function of cells of both the innate and adaptive immune systems, many of which are related to blockade of GTPase isoprenylation. In APS, statins have multiple profound effects on monocyte, lymphocyte, and endothelial cell activities, all of which may contribute to thrombosis prevention in APS patients. Nevertheless, larger randomized trials are needed to validate the role of statins in the treatment of this autoimmune disease.

The cooperation of two classes of mutations in hematopoietic cells is hypothesized in a multistep pathogenesis model of acute myeloid leukemia (AML). Class I mutations confer a proliferative and/or survival advantage, whereas Class II mutations block hematopoietic differentiation and impair apoptosis in AML cells. In addition to these two classes of mutations, a relevant role for angiogenesis in the pathophysiology of AML has been recently proposed. The recognition that the vascular endothelial growth factor (VEGF) pathway is a key regulator of angiogenesis has led to the development of several VEGF-targeted approaches. These include neutralizing antibodies, VEGF traps or selective tyrosine kinase inhibitors for VEGFRs. Other drugs that indirectly affect VEGF pathway, such as statins or arsenic trioxide, also have been shown to possess antiangiogenic activity in leukemias. The benefits of these VEGF targeted agents and their current stage of development as novel anti-antiangiogenic therapies in AML are discussed in this review.

Cholestatic liver injury following extra- or intrahepatic bile duct obstruction causes nonparenchymal cell proliferation and matrix deposition leading to end-stage liver disease and cirrhosis. In cholestatic conditions, nitric oxide (NO) is mainly produced by a hepatocyte-inducible NO synthase (iNOS) as a result of enhanced inflow of endotoxins to the liver and also by accumulation of bile salts in hepatocytes and subsequent hepatocellular injury. This study was aimed to investigate the role of NO and S-nitrosothiol (SNO) homeostasis in the development of hepatocellular injury during cholestasis induced by bile duct ligation (BDL) in rats. Male Wistar rats (200-250 g) were divided into four groups (n=10 each), including sham-operated (SO), bile duct-ligated (BDL), tauroursodeoxycholic acid (TUDCA, 50 mg/kg) and S-methylisothiourea (SMT, 25 mg/kg) treated. After 7 days, BDL rats showed elevated serum levels of gamma-glutamiltranspeptidase, aspartate aminotransferase, alanine aminotransferase, LDH, and bilirubin, bile duct proliferation and fibrosis, compared with the SO group. TUDCA treatment did not significantly alter these parameters, but the iNOS inhibitor SMT ameliorated hepatocellular injury, as shown by lower levels of circulating hepatic enzymes and bilirubin, and a decreased grade of bile duct proliferation and fibrosis. Both TUDCA and SMT treatments reversed Mrp2 canalicular pump expression to control levels. However, only SMT treatment significantly lowered the increased levels of plasma NO and S-nitrosation (S-nitrosylation) of liver proteins in BDL rats. Moreover, BDL resulted in a reduction of the S-nitrosoglutathione reductase (GSNOR/Adh5) enzymatic activity and a downregulation of the GSNOR/Adh5 mRNA expression that was reverted by SMT, but not TUDCA, treatment. A total of 25 liver proteins, including S-adenosyl methionine synthetase, betaine-homocysteine S-methyltransferase, Hsp90 and protein disulfide isomerase, were found to be S-nitrosated in BDL rats. In conclusion, the inhibition of NO production during induced cholestasis ameliorates hepatocellular injury. This effect is in part mediated by the improvement of cell proficiency in maintaining SNO homeostasis.

Nitric oxide (NO) participates in the cell death induced by d-Galactosamine (d-GalN) in hepatocytes, and NO-derived reactive oxygen intermediates are critical contributors to protein modification and hepatocellular injury. It is anticipated that S-nitrosation of proteins will participate in the mechanisms leading to cell death in d-GalN-treated human hepatocytes. In the present study, d-GalN-induced cell death was related to augmented levels of NO production and S-nitrosothiol (SNO) content. The biotin switch assay confirmed that d-GalN increased the levels of S-nitrosated proteins in human hepatocytes. S-nitrosocysteine (CSNO) enhanced protein S-nitrosation and altered cell death parameters that were related to S-nitrosation of the executioner caspase-3. Fifteen S-nitrosated proteins participating in metabolism, antioxidative defense and cellular homeostasis were identified in human hepatocytes treated with CSNO. Among them, seven were also identified in d-GalN-treated hepatocytes. The results here reported underline the importance of the alteration of SNO homeostasis during d-GalN-induced cell death in human hepatocytes.

The main goal of this study was to analyze, using proteomic techniques, changes in protein expression of acute myeloid leukemia (AML) cells that could give insights into a better early prognosis for tumor pathophysiology. Proteomic analysis of different subtypes of AML cells was carried out using 2-DE and MALDI-TOF PMF analysis. Proteins identified as more significantly altered between the different AMLs belonged to the group of suppressor genes, metabolic enzymes, antioxidants, structural proteins and signal transduction mediators. Among them, seven identified proteins were found significantly altered in almost all the AML blast cells analyzed in relation to normal mononuclear blood cells: alpha-enolase, RhoGDI2, annexin A10, catalase, peroxiredoxin 2, tromomyosin 3, and lipocortin 1 (annexin 1). These differentially expressed proteins are known to play important roles in cellular functions such as glycolysis, tumor suppression, apoptosis, angiogenesis and metastasis, and they might contribute to the adverse evolution of the disease. Proteomic analysis has identified for the first time novel proteins that may either help to form a differential prognosis or be used as markers for disease outcome, thus providing potential new targets for rational pathogenesis-based therapies of AML.

Biochemical measurements in the sentinel clam Chamaelea gallina have been used as biomarkers of marine pollution. In this study, S9, cytosolic fractions (CF), and microsomal fractions (MF) prepared from unexposed clams and clams exposed to model pollutants were used to activate 2–aminoanthracene (2-AA) and 2–acetylaminofluorene (AAF) to mutagens in Salmonella typhimurium strain BA149, which overexpresses O-acetyltransferase. Arylamine activation was similar with subcellular fractions from unexposed and Aroclor 1254-exposed clams, but decreased with fractions from As(III)- and Cu(II)-exposed clams. Bioactivation of arylamines by CF was higher than by MF, and higher with NADH than with NADPH as the reducing agent. -Naphthoflavone inhibited AAF activation by CF and MF, but increased 2-AA activation nearly twofold. In contrast to the results with arylamine activation, benzo[a]pyrene hydroxylase (BPH) activity increased twofold in fractions from Aroclor 1254- and Cu(II)-exposed clams. Activation of 2-AA was also evaluated using S9 fractions from clams sampled at littoral sites with different pollutant levels. Compared to a reference site, lower 2-AA bioactivation and higher BPH activity were found in clams containing high levels of copper and organic contaminants, although the differences were not statistically significant. While these findings agree with the results of the model Cu(II) exposure, the effects of other pollutants cannot be ruled out. The results of the study demonstrate that arylamine activation by clams is not preferentially catalyzed by microsomal monooxygenases but by unknown cytosolic system(s), and that bioactivation of 2–AA and AAF appears to occur by different pathways. Environ. Mol. Mutagen. 41:55– 63, 2003. © 2003 Wiley-Liss, Inc.

PCB uptake and clearance by clams, Chamaelea gallina , were studied in specially designed flow-through channels. After 8 weeks exposure to 10 ppb Aroclor 1254 in water, clams were depurated for 10 weeks, in the same exposure channel or after transfer to clean systems. Accumulation of the 20 congeners studied depended on its initial abundance and physicochemical properties. A linear relationship was found between log bioconcentration factor and log octanol/water partition coefficient of each form. Clearance of each PCB depended also on its initial load and solubility, being faster in clams transferred to clean systems. Exposure significantly enhanced catalase and 6-P-gluconate dehydrogenase activities, but not other antioxidative enzymes. Superoxide dismutase, low during the exposure phase, increased seven-fold during depuration. Aroclor-treated clams had higher GSH levels than controls, but decreased to 15–35% after 2 days clearance, rose to 150% after 12 days, and declined to low levels by the end of the experience. Biotransformation of PCBs to quinones and redox cycling-promoted oxidative stress might explain the increased antioxidative defenses. The biochemical changes observed at the beginning of clearance could be attributed to clam handling, by adaptation to and recovery from hypoxic/anoxic stress.

Fourteen biochemical pollution biomarkers were analyzed in the clam Chamaelea gallina sampled at seven South-Spanish littoral sites at different times in 1999. They included enzymes that regenerate reduced nicotinamide adenine dinucleotide phosphate (NADPH) or maintain the cytosol-reduced (glucose-6-P- and isocitrate dehydrogenases, glutathione reductase), that decompose reactive oxygen species (catalase, superoxide dismutase, glutathione peroxidase), or detoxify glutathione-reactive electrophiles (soluble and microsomal glutathione transferases, glyoxalases II and I). The levels of reduced glutathione, malondialdehyde, and metallothionein and the glutathione redox status were also determined. Clams from Huelva sites with higher contaminant loads and metallothionein levels showed higher antioxidant and glutathione-related activities and a decreased glutathione level, lower malondialdehyde content, and a less oxidized glutathione status. This suggests that clams chronically exposed to contaminants released by Huelva and Guadalquivir Estuaries are better protected from oxidative stress than reference animals. Most biomarkers showed six-month cyclicity with up to threefold amplitudes, further supporting the inverse relationship between antioxidant defenses and oxidative damages. Thus the lower antioxidant defenses in June fit to maximum oxidative damages, whereas the high antioxidant defenses found in March and September through October agree with lower lipid damages and less oxidized glutathione status. The effect of environmental or endogenous factors on this cyclic response is discussed.

Objective— Previous studies have suggested a relationship between anticyclic citrullinated protein (CCP) levels and development of cardiovascular disease in rheumatoid arthritis (RA). However, a limited number of studies have demonstrated an involvement of anti-CCPs in those processes. This study was aimed to define the specific role of these auto-antibodies in the pro-oxidative, inflammatory, and proatherogenic profile observed in leukocytes from RA patients. Approach and Results— Seventy-five RA patients and 31 healthy donors were enrolled. Carotid intima media thickness was evaluated as atherosclerosis marker. Several procoagulant and inflammatory factors, leukocyte activation, and oxidative stress markers were analyzed in plasma and leukocyte subsets. Anti-CCPs were purified from plasma of RA patients, and in vitro treatment of healthy leukocytes was conducted. High titers of anti-CCPs were associated to altered expression of prothrombotic and inflammatory markers, high oxidative stress, and pathological carotid intima media thickness in RA patients. Notably, gene expression analysis showed that lymphocytes were major players in altered inflammatory profile, monocytes were responsible for the protrombotic and atherogenic status, and neutrophils mainly displayed a pro-oxidative feature. In vitro treatment with purified anti-CCPs fully recapitulated that pathogenic profile, promoting the activation of leukocytes. Conclusions— Anti-CCPs are key players in the inflammatory and proatherogenic status of RA patients. The effects are specific of the immune cell targeted, promoting overexpression of thrombotic, inflammatory, and pro-oxidative markers in monocytes; pro-oxidative status in neutrophils; and proinflammatory profile in lymphocytes. Targeting these autoantibodies would be an excellent strategy to prevent the development of cardiovascular disease in RA.

The induction of polyploidy is considered the reproductive end of cells, but there is evidence that polyploid giant cancer cells (PGCCs) contribute to cell repopulation during tumor relapse. However, the role of these cells in the development, progression and response to therapy in colon cancer remains undefined. Therefore, the main objective of this study was to investigate the generation of PGCCs in colon cancer cells and identify mechanisms of formation. Treatment of HCT-116 and Caco-2 colon cancer cells with the hypoxia mimic CoCl2 induced the formation of cells with larger cell and nuclear size (PGCCs), while the cells with normal morphology were selectively eliminated. Cytometric analysis showed that CoCl2 treatment induced G2 cell cycle arrest and the generation of a polyploid cell subpopulation with increased cellular DNA content. Polyploidy of hypoxia-induced PGCCs was confirmed by FISH analysis. Furthermore, CoCl2 treatment effectively induced the stabilization of HIF-1α, the differential expression of a truncated form of p53 (p47) and decreased levels of cyclin D1, indicating molecular mechanisms associated with cell cycle arrest at G2. Generation of PGCCs also contributed to expansion of a cell subpopulation with cancer stem cells (CSCs) characteristics, as indicated by colonosphere formation assays, and enhanced chemoresistance to 5-fluorouracil and oxaliplatin. In conclusion, the pharmacological induction of hypoxia in colon cancer cells causes the formation of PGCCs, the expansion of a cell subpopulation with CSC characteristics and chemoresistance. The molecular mechanisms involved, including the stabilization of HIF-1 α, the involvement of p53/p47 isoform and cell cycle arrest at G2, suggest novel targets to prevent tumor relapse and treatment failure in colon cancer.

Mutagenic activation of arylamines by mollusc S9 fractions was evaluated as a biomarker for marine pollution. Two bivalve species were used as bioindicators, the common mussel (Mytilus edulis) and the striped venus (Chameleo gallina). A strain of Salmonella typhimurium overproducing O-acetyltransferase was used as indicator of mutagenicity. Mussels from an area of the North Atlantic Spanish zone that was exposed to an accidental crude oil spill were compared to bivalves from a reference area. C. gallina samples were from low polluted and highly polluted areas of the South Atlantic Spanish littoral. The promutagen 2-aminoanthracene (2-AA) was activated to mutagenic derivative(s) by S9 fractions from both C. gallina and M. edulis. Animals from contaminated sites showed higher arylamine activation capabilities than reference animals. This was further correlated with the mutagenic activities of corresponding cyclopentone-dichloromethane animal extracts. 2-AA activation by mollusc S9 was potentiated by alpha-naphthoflavone (ANF), known to inhibit PAH-inducible CYP1A cytochromes from vertebrates, but inhibited by methimazole (MZ), a substrate of the flavin monooxygenase (FMO) system. 2-AA-activating enzymes were mainly cytosolic; this localization clearly suggests that such activity could be attributed to soluble enzymes, different from the CYP1A or FMO systems. In conclusion, mutagenic activation of arylamines by mollusc S9, using as indicator a strain of Salmonella typhimurium that overproduces O-acetyltransferase, could be a reliable biomarker for marine pollution.

This paper examines the relationship in Escherichia coli between the in vivo content of 8-oxoguanine (8-oxoG) in chromosomal DNA and deficiencies of various key antioxidant defences. The structural genes for catalases (katG and katE), cytosolic superoxide dismutases (sodA and sodB) or formamidopyrimidine-DNA glycosylase (fpg) were inactivated to obtain bacterial strains lacking the scavenger enzymes for H2O2 or O2.- or the DNA repair protein for 8-oxoG. Wild-type bacteria showed 5-fold increased sensitivity to both lethality and mutagenesis by H2O2 in K medium (1% casamino acids and 1% glucose), as compared with nutrient broth. This higher sensitivity was associated with increased chromosomal oxidative damage, estimated as the 8-oxodG content, and with a marked decrease in both catalase and SOD activities. Bacteria lacking both cytosolic SODs (sodA sodB mutant) displayed increased 8-oxodG content in chromosomal DNA (2.8-fold that of the wild-type) when grown under standard aerated conditions. Comparatively, no significant difference in 8-oxodG content was observed in cells grown without aeration. Bacteria totally devoid of catalase activity (katG katE mutant) showed wild-type contents of 8-oxodG in chromosomal DNA when grown under aerated conditions. Nevertheless, the protective role of catalase in preventing formation of 8-oxodG in chromosomal DNA became evident under oxidative stress conditions: growth under hyperoxygenation and, particularly, following H2O2 exposure. Catalase deficiency resulted in a dramatic decrease in viability after H2O2 exposure. A deficiency of Fpg protein also sensitized E.coli to H2O2 lethality, though to lesser extent than a deficiency of catalase activity. However, the scavenger enzyme and the DNA repair protein protected equally against 8-oxoG formed in vivo upon H2O2 treatment.

Many of nitric oxide (NO) actions are mediated through the coupling of a nitroso moiety to a reactive cysteine leading to the formation of a S-nitrosothiol (SNO), a process known as S-nitrosylation or S-nitrosation. In many cases this reversible post-translational modification is accompanied by altered protein function and aberrant S-nitrosylation of proteins, caused by altered production of NO and/or impaired SNO homeostasis, has been repeatedly reported in a variety of pathophysiological settings. A growing number of studies are directed to the identification and characterization of those proteins that undergo S-nitrosylation and the analysis of S-nitrosoproteomes under pathological conditions is beginning to be reported. The study of these S-nitrosoproteomes has been fueled by advances in proteomic technologies that are providing researchers with improved tools for exploring this post-translational modification. Here we review novel refinements and improvements to these methods, and some recent studies of the S-nitrosoproteome in disease.

The exact mechanisms underlying the role of oxidative stress in the pathogenesis and the prothrombotic or proinflammatory status of antiphospholipid syndrome (APS) remain unknown. Here, we investigate the role of oxidative stress and mitochondrial dysfunction in the proatherothrombotic status of APS patients induced by IgG-antiphospholipid antibodies and the beneficial effects of supplementing cells with coenzyme Q10 (CoQ10). A significant increase in relevant prothrombotic and inflammatory parameters in 43 APS patients was found compared with 38 healthy donors. Increased peroxide production, nuclear abundance of Nrf2, antioxidant enzymatic activity, decreased intracellular glutathione, and altered mitochondrial membrane potential were found in monocytes and neutrophils from APS patients. Accelerated atherosclerosis in APS patients was found associated with their inflammatory or oxidative status. CoQ10 preincubation of healthy monocytes before IgG-antiphospholipid antibody treatment decreased oxidative stress, the percentage of cells with altered mitochondrial membrane potential, and the induced expression of tissue factor, VEGF, and Flt1. In addition, CoQ10 significantly improved the ultrastructural preservation of mitochondria and prevented IgG-APS–induced fission mediated by Drp-1 and Fis-1 proteins. In conclusion, the oxidative perturbation in APS patient leukocytes, which is directly related to an inflammatory and pro-atherothrombotic status, relies on alterations in mitochondrial dynamics and metabolism that may be prevented, reverted, or both by treatment with CoQ10.

Wnt/b-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/b-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, b-catenin nuclear translocation, up-regulation of genes related to the Wnt/b-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes c-myc and p53 were observed. While in the other protocol there was a Wnt/b-catenin inactivation. Hepatocytes with nuclear translocation of b-catenin also had abnormal cellular proliferation, and expressed membrane proteins involved in hepatocellular carcinoma, metastatic behavior and cancer stem cells. Further, these cells had also increased auto-renewal capability as shown in spheroids formation assay. Comparison of both differentiation protocols by 2D-DIGE proteomic analysis revealed differential expression of 11 proteins with altered expression in hepatocellular carcinoma. Cathepsin B and D, adenine phosphoribosyltransferase, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or lactate dehydrogenase b-chain were up-regulated only with the protocol associated with Wnt signaling activation while other proteins involved in tumor suppression, such as transgelin or tropomyosin b-chain were downregulated in this protocol. In conclusion, our results suggest that activation of the Wnt/b-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype

Nitric oxide (NO) can modulate cell function by the coupling of a nitroso moiety to a reactive cysteine in target proteins leading to the formation of a S-nitrosothiol (SNO), a process commonly known as S-nitrosylation. Aberrant S-nitrosylation of proteins, caused by altered production of NO and/or impaired SNO homeostasis, constitutes a mechanism that has been recently postulated in numerous pathophysiological settings. The thiol microenvironment, cellular redox environment, and activity of transnitrosylases and denitrosylases have been proposed as determinant factors for the specificity of S-nitrosylation. A number of methodological approaches have recently been developed for the proteomic identification of S-nitrosylated proteins and/or the identification of specific sites of nitrosylation. This review will consider novel aspects of SNO homeostasis and S-nitrosylation, the latest proteomic methods for the identification of S-nitrosylated cysteines in proteins, and how these novel technologies will impact our current knowledge of the role of deregulated S-nitrosylation in disease.

Autoimmune diseases (AIDs) have been associated with accelerated atherosclerosis (AT) leading to increased cardio- and cerebrovascular disease risk. Traditional risk factors, as well as systemic inflammation mediators, including cytokines, chemokines, proteases, autoantibodies, adhesion receptors, and others, have been implicated in the development of these vascular pathologies. Yet, the characteristics of vasculopathies may significantly differ depending on the underlying disease. In recent years, many new genes and signalling pathways involved in autoimmunity with often overlapping patterns between different disease entities have been further detected. Epigenetics, the control of gene packaging and expression independent of alterations in the DNA sequence, is providing new directions linking genetics and environmental factors. Epigenetic regulatory mechanisms comprise DNA methylation, histone modifications, and microRNA activity, all of which act upon gene and protein expression levels. Recent findings have contributed to our understanding of how epigenetic modifications could influence AID development, not only showing differences between AID patients and healthy controls, but also showing how one disease differs from another and even how the expression of key proteins involved in the development of each disease is regulated.

Metabolic activation of known promutagens by liver S9 fractions of Mugil sp. (grey mullet) from two zones of the South Atlantic Spanish littoral was determined and related to their pollution levels. Sediments from the putative contaminated area contained high concentrations of PAHs, PCBs and pesticides, and animals from the polluted site exhibited higher concentrations of metals than those from the reference area. Hepatic S9 fractions of mullets from the polluted site showed 5.1-, 18.6- and 42.8-fold higher capability to activate benzo(a)pyrene, 2-acetyl-aminofluorene and 2-aminoanthracene, respectively, than those from reference animals. Cadmium, a highly toxic metal, was one of the pollutants detected in the contaminated area. Gilthead seabream (Sparus aurata) were exposed under controlled conditions to different Cd concentrations in order to investigate the effects of Cd on fish promutagen activation capability. A clear dose-response relationship was observed between Cd concentration, EROD activity and metabolic activation of 2-aminoanthracene and benzo(a)pyrene. Our data indicate that the enhanced promutagen activation by fish S9 fractions accompanying induction of EROD activity is a sensitive and reliable index of pollution in aquatic environments.

The involvement of bone marrow hematopoietic stem cells (BMHSC) mobilization during liver regeneration from hepatectomized patients is under debate. The main aim of this study was to investigate the role of BMHSC mobilization after hepatic resection in 33 patients with liver disease.Mobilization of CD34(+) BMHSC after 72 h of surgery was found in peripheral blood of some, but not all, of the hepatectomized patients. These CD34(+) cells co-expressed other stem cells markers. The patients without BMHSC mobilization showed high levels of circulating and liver tissue BMHSC (CD34(+) cells) previous to surgery. Therefore, two types of patients: "mobilizers" and "non-mobilizers" were distinguished based on the values of CD34(+) cells before and after surgery. Changes in cytokines involved in the hepatic regeneration (HGF and TGF-β), and in BMHSC mobilization process (SCF, SDF-1, IL-12, or MMP-2), were detected in both groups. In addition, a higher activation previous to surgery of the SDF-1/CXCR4 axis in liver tissue was observed in non mobilizers patients compared to mobilizer patients.BMHSC mobilization seems to be associated with variations in the levels of cytokines and proteolytic enzymes involved in hepatic regeneration and bone marrow matrix degradation. Hepatectomy may be an insufficient stimulus for BMSHC mobilization. The pre-hepatectomy higher levels CD34(+) cells in peripheral blood and liver, associated to the activation of hepatic SDF-1/CXCR4 axis, suggest a BMHSC mobilization process previous to surgery in non mobilizer patients.