Epigenetic Mechanisms Histone Deacetylase-Dependent Regulate the Glioblastoma Angiogenic Matrisome and Disrupt Endothelial Cell Behavior In Vitro.

Glioblastoma (GBM) is the most aggressive brain tumor and different efforts have been employed in the search for new drugs and therapeutic protocols for GBM. Epitranscriptomics has shed light on new druggable Epigenetic therapies specifically designed to modulate GBM biology and behavior such as Histone Deacetylase inhibitors (iHDAC). Although the effects of iHDAC on GBM have been largely explored, there is a lack of information on the underlaying mechanisms HDAC-dependent that modulate the repertoire of GBM secreted molecules focusing on the set of Extracellular Matrix (ECM) associated proteins, the Matrisome, that may impact the surrounding tumor microenvironment. To acquire a better comprehension of the impacts of HDAC activity on the GBM Matrisome, we studied the alterations on the Matrisome-associated ECM regulators, Core Matrisome ECM glycoproteins, ECM-affiliated proteins and Proteoglycans upon HDAC inhibition in vitro as well as their relationship with glioma pathophysiological/clinical features and angiogenesis. For this, U87MG GBM cells were treated for with iHDAC or vehicle (control) and the whole secretome was processed by Mass Spectrometry NANOLC-MS/MS. In silico analyses revealed that proteins associated to the Angiogenic Matrisome (AngioMatrix), including Decorin, ADAM10, ADAM12 and ADAM15 were differentially regulated in iHDAC versus control secretome. Interestingly, genes coding for the Matrisome proteins differentially regulated were found mutated in patients and were correlated to glioma pathophysiological/clinical features. In vitro functional assays, using HBMEC endothelial cells exposed to the secretome of control or iHDAC treated GBM cells, coupled to 2D and 3D GBM cell culture system, showed impaired migratory capacity of endothelial cells and disrupted tubulogenesis in a Fibronectin and VEGF independent fashion. Collectively, our study provides understanding of epigenetic mechanisms HDAC-dependent to key Matrisomal proteins that may contribute to identify new druggable Epigenetic therapies or gliomagenesis biomarkers with relevant implications to improve therapeutic protocols for this malignancy.

A proteomic approach to compare saliva from individuals with and without oral leukoplakia.

BACKGROUND: Oral leukoplakia is the most common potentially malignant disorder in the oral cavity and can precede carcinoma. This study aimed to identify possible oral leukoplakia salivary biomarkers. METHODS: Unstimulated saliva was collected from participants and protein concentration was determined. Proteins were then precipitated with cold acetone and separated using 2DE over a pH range of 3-10. Spot demarcation and matching were performed and protein identification was done through MS analysis. Oral leukoplakia tissues were submitted to immunohistochemistry analysis for keratin 10 (CK10). A complementary analysis of oral leukoplakias that were not included previously was performed in addition. RESULTS: 226±10 spots were identified in oral leukoplakia 2DE gels, and 262±12 spots were identified in volunteers. Twenty-two spots were highly abundant in oral leukoplakias or not detected in the control group, such as apolipoprotein A1, alpha amylase, cystatins, keratin 10, and lysozyme precursor. All were identified. All oral leukoplakia cases were immunopositive for CK10, mainly in the superficial epithelial layers. CONCLUSIONS: The 2DE salivary protein profiles of individuals with and without oral leukoplakia were observably different. CK10 appears to be an interesting protein and should be further studied in oral carcinogenesis. SIGNIFICANCE: MS-based proteomics enables large-scale analysis of proteins. Proteomics can provide detailed descriptions of proteomes of cells and tissues, including body fluids, and appears as a powerful tool to study human disorders. Saliva is readily accessible through non invasive collection and can mirror diverse disease states. Saliva from both diseased and healthy subjects can be analyzed through 2DE and differences between groups could be found. Routine immunohistochemistry analysis confirmed one of these findings, with CK10 being positive tissues from individuals with oral leukoplakia. Therefore, the present study allows insights into development of an important potential oral cancer precursor, named oral leukoplakia. However, the results can be extrapolated and tested in other precancer states, such as proliferative verrucous leukoplakia, patients at risk of oral cancer due to lifestyle behavior and/or cancer history in the family or even those who are under surveillance after a treated primary oral cancer.

Proteomic analysis reveals differentially secreted proteins in the urine from patients with clear cell renal cell carcinoma.

OBJECTIVE: The aim of this study was to evaluate the differentially secreted protein profile in the urine from patients with clear cell renal cell carcinoma (ccRCC) using mass spectrometry-based methods. Urine composition can reflect kidney physiology and can be used to detect markers for renal diseases. Moreover, characterization of the secretome is likely to assist in the investigation of new drugs for biological targets and diagnose the ccRCC at an early stage. METHODS AND MATERIALS: Urine samples from patients were divided according to Fuhrman degree (FI-IV), which was associated with the cellular differentiation as good prognosis (GP) and poor prognosis (PP). Healthy individuals were used as the control group (CG). We used both qualitative and quantitative mass spectrometry-based analyses that involved the following approaches: 1-dimensional gel electrophoresis combined with liquid chromatography mass spectrometry in tandem (1DE LC-MS/MS), in-solution digestion combined with label-free 1-dimensional LC-MS(E) (1D LC-MS(E)), and bidimensional gel electrophoresis combined with matrix-assisted laser desorption/ionization time of flight in tandem (2DE MALDI-TOF/TOF) or combined with LC-MS/MS. RESULTS: All the strategies allowed the identification of 354 proteins from the CG, GP, and PP groups. Qualitative experiments using 1DE LC-MS/MS analysis detected different protein profiles, and 224 proteins were identified in all groups. The label-free MS(E) quantitative analysis identified 113 proteins and generated novel information on secreted protein profiles, including 49 up-secreted proteins in the urine from patients with ccRCC and 40 down-secreted proteins related to the CG. Proteins such as kininogen-1, uromodulin, apolipoprotein D, polyubiquitin, and CD59 glycoprotein were down secreted according to the groups CG>GP>PP. In contrast, apolipoprotein A, fibrinogen, and haptoglobin were up secreted in patient groups. The same expression profile observed for kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin was corroborated by 2DE LC-MS/MS or 2DE MALDI-TOF/TOF analyses. These 2 strategies also showed 13 differentially secreted proteins among the 3 groups. CONCLUSIONS: The proteins kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin presented similar quantitative protein profiles according to MS(E) and 2DE approaches. The latter proteins were up secreted and the former ones were down-regulated. The strategies used proved to be valuable in identifying proteins that were differentially secreted in urine from patients with RCC.

Proteomics analysis of tissue samples from patients with squamous cell carcinoma of the penis and positive to human papillomavirus.

PURPOSE: The aim of this study was to identify possible protein biomarkers and/or candidates for therapeutic targets in tissues of patients with SCCP, infected by HPV, applying one dimensional electrophoresis (1DE), followed by direct mass spectrometry (MS) analysis. MATERIALS AND METHODS: Tissues from 10 HPV positive patients with SCCP and from 10 patients with HPV negative non-tumorous penile foreskins were analyzed applying 1D electrophoresis, followed by analysis with direct mass spectrometry (MS). RESULTS: Sixty-three different proteins were identified in the first group and 50 in the second group. Recognition was possible for 28 proteins exclusively detected in Group 1 and 21 proteins presented only in Group 2. CONCLUSION: Some proteins in the first group are directly involved in the development of other types of cancer, and therefore, suitable for analysis. Complement C3 protein is a strong candidate for evaluating SCCP patients.

Proteomics analysis of tissue samples from patients with squamous cell carcinoma of the penis and positive to human papillomavirus

ABSTRACTPurpose:The aim of this study was to identify possible protein biomarkers and/or candidates for therapeutic targets in tissues of patients with SCCP, infected by HPV, applying one dimensional electrophoresis (1DE), followed by direct mass spectrometry (MS) analysis.Materials and Methods:Tissues from 10 HPV positive patients with SCCP and from 10 patients with HPV negative non-tumorous penile foreskins were analyzed applying 1D electrophoresis, followed by analysis with direct mass spectrometry (MS).Results:Sixty-three different proteins were identified in the first group and 50 in the second group. Recognition was possible for 28 proteins exclusively detected in Group 1 and 21 proteins presented only in Group 2.Conclusion:Some proteins in the first group are directly involved in the development of other types of cancer, and therefore, suitable for analysis. Complement C3 protein is a strong candidate for evaluating SCCP patients.

Comparative proteomic analysis of whole saliva from chronic periodontitis patients.

Chronic periodontal disease is a chronic inflammatory process affecting tooth supporting tissues in the presence of pathogenic bacterial biofilm. There is some evidence for changes in the protein composition of whole saliva from chronic periodontitis patients, but there have been no studies using a proteomic approach. Hence, the aim of this study was to compare the protein profiles of unstimulated whole saliva from patients with periodontitis and healthy subjects by two complementary approaches (2D-gel electrophoresis and liquid chromatography). Protein spots of interest were analyzed by MALDI-TOF-TOF, and the data was complemented by an ESI-Q-TOF experiment. The analyses revealed that subjects with periodontal disease have increased amounts of blood proteins (serum albumin and hemoglobin) and immunoglobulin, and they have a lower abundance of cystatin compared to the control group. A higher number of protein spots were observed in the periodontitis group, of which most were identified as alpha-amylase. This higher number of alpha-amylase variants seems to be caused by hydrolysis by cysteine proteases under such inflammatory conditions. This approach gives novel insights into alterations of salivary protein in presence of periodontal inflammation and may contribute to the improvement of periodontal diagnosis.

Estudo do mecanismo cinético de ação da álcool desidrogenase de Thermoanaerobium brockii sobre o sistema isopropanol/acetona

Neste trabalho, o mecanismo cinético de ação da álcool desidrogenase de Thermoanaerobium brockii (TBADH) atuando sobre o sistema isopropanol/acetona foi estudado, visando tanto a elucidação do mecanismo cinético da TBADH quanto explicar o sistema acoplado de reciclagem da coenzima utilizando altas concentrações de isopropanol, 30 (por cento v/v), como co-solvente e co-substrato no meio reacional e com somente uma enzima atuando nas duas reações. Para tanto, realizamos estudos de velocidade inicial de produtos, estudos de inibição por produto e estudos de inibição por formação de complexo "dead-end", nos quais pirazol foi utilizado como inibidor. Os estudos de velocidade inicial na ausência de produtos, forneceram gráficos de Lineweaver-Burk tanto com efeito de inclinação, indicando que o mecanismo cinético era do tipo sequencial. A análise do significado cinético do complexo ternário central, E-coenzima-substrato, sugeriu que o mecanismo cinético era mais provavelmente, do tipo Theorell_Chance Bi-Bi. Nos estudos de inibição por produto objetivemos, gráficos de duplos recíprocos que apresentaram quatro perfis de inibição do tipo competitiva linear (entre NADP e NADPH e entre isopropanol e acetona) e quatro perfis de inibição do tipo mista linear (entre NADP e acetona e entre NADPH e isopropanol), desta maneira os mecanismos cinéticos dos tipos sequencial ordenado Bi-Bi, em estado estacionário, e sequencial ao acaso Bi-Bi, em estado estacionário foram totalmente descartado. Através do estudo de inibição por formação de complexo "dead-end, o mecanismo cinético do tipo sequencial ao acso Bi-Bi, em rápido equilíbrio, foi descartado, ficando caracterizado que no mecanismo cinético proposto a coenzima atua como o primeiro substrato a se ligar à forma livre de enzima. Ambos os parâmetros cinéticos e termodinâmicos da reação foram estimados, pelos ajustes das respectivas equações de velocidade aos dados experimentais obtidos, utilizando-se um programa de computador de regressão não linear e um prorama de computador baseado no método de Hooke-Jeeves, método de procura direta comaceleração em distância, ambos desenvolvidos em nosso laboratório. O ajuste da equação de velocidade inicial para a inibição por formação de complexo "dead-end", forneceu uma estimativa da constante de dissociação...

The kinetic mechanism of the redox reaction isopropanoVacetone catalyzed by Thermoanaerobium broe/ai alcohol dehydrogenase (TBADH) was studied aiming both elucidation of the kinetic mechanism and gaining insight into the coupled-substrate approach that has been extensively used for NADPH regeneration, by using high concentrations of isopropanol, 30% (v/v), as a co-solvent and co-substrate in preparative-scale reductions of carbonyl compounds. For this purpose, initial velocity studies in the absence of products, product inhibition studies and dead-end inhibition studies, using pyrazole , were performed. Initial velocity studies in the absence of products in the forward (isopropanol oxidation) and reverse (acetone reduction) reactions showed that all the double reciprocal plots were of the intersecting type, indicating that the kinetic mechanism of TBADH is of the sequential type, involving ternary or central complexes not necessarily significant kinetically. Kinetic evaluation of the significance of the central complexes, E-coenzymes-substrates, suggested that the mechanism is most probably of the Theorell-Chance Bi-Bi type The patterns obtained in product inhibition studies consisted in four linear competitive inhibitions (between NADP and NADPH, as well as, between isopropanol and acetone) and four linear mixed type inhibitions (between NADP and acetone, as well as, between NADPH and isopropanol). With these studies both the steady-state sequential ordered Bi-Bi mechanism and the steady-state random sequential Hi-Hi mechanism could be ruled out. Dead-end inhibition studies, using pyrazole as inhibitor, showed that NADP is the first substrate to bind to the enzyme followed by the binding of isopropanol, then the rapid equilibrium random sequential Bi-Bi mechanism was also ruled out. Both the kinetic and thermodynamic parameters of the reaction were estimated with a non-linear regression analysis computer program and a computer program based on the Hooke-Jeeves direct search method, both developed in our laboratory. The fitting of the dead-end inhibition equation, with formation of a TBADHNADP-pyrazole complex to the experimental data obtained in the presence of the deadend inhibitor gave estimates of the pyrazole dissociation constant of the E-NADPpyrazole complex, as well as, of the NADP dissociation constant of the same complex. The values obtained for these dissociation constants, when compared to those obtained with horse liver alcohol dehydrogenase, enable us to suggest a model for the structure of the ternary complex TBADH-NADP-pyrazole, in which the interaction of pyrazole occurs only by a coordinate bond formed between one of the heterocyclic nitrogen atoms of the inhibitor with a zinc atom, probably present at the active site of the enzyme. The Theorell-Chance Bi-Bi kinetic mechanism, proposed in this study, satisfactorily explains the recycling process of the coenzyme NADPH, using high concentrations of isopropanol 30 % (v/v), as a co-solvent and co-substrate in preparative-scale enantioselective reactions reduction. Since the coenzyme is the first substrate to bind the free form of the enzyme in a sequential and compulsory order, the interaction of isopropanol with the free form of the enzyme is not possible. Thus preventing TBADH to be saturated with isopropanol. In this way, free TBADH will always be available to catalyze the reduction of the oxidized substrate and the NADP formed in this reaction, continuously reduced to NADPH by isopropanol oxidation concomitantly catalyzed by the same enzyme.