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Purpose Skin ageing is marked by structural and functional changes in epidermis and dermis, which result clinically in wrinkles, loss of elasticity, and rough-textured appearance. In this context, different dermal fillers have been used to overcome these negative effects associated with skin ageing, such as hyaluronic acid (HA) and poly-L-lactic acid (PLLA). Despite their low immunogenicity, these materials can cause an inflammatory reaction after application. Materials and Methods Considering high demand of HA and PLLA as filler material, this study aimed to evaluate their in vitro and in vivo effects. For the in vitro study, human dermal fibroblast cell cultures were supplemented with HA or PLLA for 24, 48, and 72 h. The following parameters were assayed: 1) cell proliferation, 2) cell viability, and 3) quantification of type I collagen by ELISA. For the in vivo study, HA or PLLA was injected in the dermis of Wistar rats and the tissues were collected after 15, 30, and 60 days for histologic evaluation and for quantification of type I collagen by Western blotting. The quantitative data were statistically analyzed using an ANOVA two-way. The significance level was set at 5%. Results At 72 h, high cell proliferation was observed for HA compared to control (p

Objective: To evaluate the cytotoxicity of stainless-steel orthodontic bands and their influence on the expression of the antioxidant genes in human gingival fibroblasts. Materials and Methods: Ten bands of each brand (Dentsply-Sirona, Dentaurum, TP Orthodontics, and Morelli) were conditioned in 0.2 g/mL culture medium at 37°C for 14 days, and the corresponding conditioned media were applied over the fibroblasts. Cell viability was assessed after 24, 48, and 72 hours of exposure to the conditioned media by trypan blue exclusion assay. Expression of the antioxidant defense genes peroxiredoxin 1 (PRDX1), superoxide dismutase 1 (SOD1), and glutathione peroxidase 1 (GPX1) were evaluated by quantitative polymerase chain reaction after 24 hours of exposure. These parameters were compared to those of the cells not exposed to the conditioned media of the bands (control). Results: All bands promoted a reduction in the number of viable cells in the periods of 48 and 72 hours (P < .01). Analysis of gene expression showed a significant increase in the levels of PRDX1 transcripts caused by the conditioned media of the Dentsply-Sirona, TP Orthodontics, and Morelli bands (P < .01) as well as induction of SOD1 by the conditioned media of the Dentaurum and Morelli (P < .01). Expression of GPX1 was not influenced by the conditioned media. Conclusions: The orthodontic bands showed toxicity to fibroblasts and increased the expression of PRDX1 and SOD1 antioxidant genes, indicating induction of oxidative stress in the cells.

Introdução: Evidências indicam que o metabolismo orienta o destino das células-tronco hematopoéticas entre a auto renovação e a diferenciação. Particularmente para células eritróides, que perdem núcleo e organelas em estágios terminais, a via glicolítica deve ser crucial durante todo o processo de desenvolvimento. As principais proteínas envolvidas nessa via, como o transportador de glicose 1 (Glut1), a enzima glicolítica lactato desidrogenase (LDH) e a enzima reguladora metabólica piruvato desidrogenase quinase 1 (PDK1), podem ser úteis para a avaliação da eritropoese. Objetivos: Verificar a expressão das proteínas do metabolismo da glicose durante todo o processo de diferenciação eritróide na medula óssea. Material e métodos: Produtos de biópsia de medula óssea de 31 doadores saudáveis foram recebidos dos arquivos do departamento de anatomia patológica de instituição parceira, com aprovação do Comitê de Ética Institucional. Tais amostras foram processadas e incubadas com anticorpos primários para análise por imunohistoquímica. Os anticorpos utilizados foram específicos para Glut1, LDH, PDK1, hemoglobina, E-caderina e CD68. Este material foi também corado com hematoxilina-eosina. Resultados: A coloração imunohistoquímica mostrou que Glut1 foi expresso nas células eritróides durante todo o processo de diferenciação, mas LDH e PDK1 foram detectadas preferencialmente em células eritróides imaturas. No total, essas proteínas favoreceriam alta atividade glicolítica nos estágios iniciais, proporcionando equilíbrio energético e redox. Essa atividade diminuiria em direção aos estágios terminais, acompanhando o declínio na demanda de energia. Conclusão: Glut1 seria um marcador eritróide imunohistoquímico e de citometria de fluxo em todas as fases de maturação, enquanto LDH e PDK1 seriam adequados para distinguir células eritropoéticas imaturas.

The present study aimed to compare the expression of p63/p40 with smooth muscle actin (SMA) and vimentin (VIM) by myoepithelial cells in minor salivary gland tumors. Fifty-two formalin-fixed paraffin-embedded samples of minor salivary gland tumors derived from intercalated duct (pleomorphic adenoma [PA], adenoid cystic carcinoma [ACC], epithelial-myoepithelial carcinoma [EMC], polymorphous adenocarcinoma [PAC], and secretory carcinoma [SC]) and 3 samples of minor salivary gland tumors derived from excretory duct (mucoepidermoid carcinoma [MEC]) were evaluated by means of immunohistochemistry. The data were analyzed qualitatively. The results indicated that p63 and p40 expression were detected in myoepithelial cells present in PA, ACC, and EMC. However, both proteins were also observed in squamous areas of PA and all cases of MEC. SMA were noticed in some myoepithelial cells of PA, ACC, and EMC. Expression of SMA was negative in the other salivary gland tumors evaluated. VIM was constantly expressed by myoepithelial cells in PA, ACC, and EMC. VIM was also observed in cells of PAC and SC, but not in squamous areas of PA and MEC. In conclusion, p63 expression is almost comparable with VIM in detecting myoepithelial cells, an immunolabeling pattern not followed by p40, and consequently, caution has to be taken during the interpretation of salivary gland tumor exhibiting an p63/p40 phenotype in order to avoid a misdiagnosis.

Background and objective Soluble epoxide hydrolase (sEH) is an enzyme in the arachidonate cascade which converts epoxy fatty acids (EpFAs), such as epoxyeicosatrienoic acids (EETs) produced by cytochrome P450 enzymes, to dihydroxy-eicosatrienoic acids. In the last 20 years with the development of inhibitors to sEH it has been possible to increase the levels of EETs and other EpFAs in in vivo models. Recently, studies have shown that EETs play a key role in blocking inflammation in a bone resorption process, but the mechanism is not clear. In the current study we used the sEH inhibitor (1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea [TPPU]) to investigate the immunomodulatory effects in a mouse periodontitis model. Material and methods Mice were infected on days 0, 2, and 4 with Aggregatibacter actinomycetemcomitans and divided into groups (n = 6) that were treated orally, daily for 15 days, with 1 mg/kg of TPPU. Then, the mice were killed and their jaws were analyzed for bone resorption using morphometry. Immunoinflammatory markers in the gingival tissue were analyzed by microarray PCR or western blotting. Results Infected mice treated with TPPU showed lower bone resorption than infected mice without treatment. Interestingly, infected mice showed increased expression of sEH; however, mice treated with TPPU had a reduction in expression of sEH. Besides, several proinflammatory cytokines and molecular markers were downregulated in the gingival tissue in the group treated with 1 mg/kg of TPPU. Conclusion The sEH inhibitor, TPPU, showed immunomodulatory effects, decreasing bone resorption and inflammatory responses in a bone resorption mouse model.

The aim of this study was to evaluate the effects of caffeic acid phenethyl ester (CAPE) on osteoblast-like cell cultures (SAOS-2).SAOS-2 were exposed to CAPE at 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM. Non-exposed cultures were used as control. The following parameters were assayed: 1) cell viability at 1, 3, and 7 days; 2) alkaline phosphatase (ALP) activity at 5 and 10 days; 3) matrix mineralization at 14 days; and 4) Runt-related transcription factor 2 (RUNX2), ALP, osteopontin (SPP1), and osteocalcin (BGLAP) gene expression at 5 and 10 days. The data were analyzed by ANOVA two-way or Kruskal-Wallis (α = 5%).At day 1, cell viability was similar among all groups (p0.05). At days 3 and 7, cultures exposed to CAPE at 10 μM exhibited a significant reduction in cell viability compared with the others groups (p0.05). At day 5, ALP activity was similar among all experimental groups; at day 10, however, the stain intensity was higher in cultures exposed to CAPE at 100 nM and 10 nM in comparison with the other groups (p0.05). At days 5 and 10, RUNX2, ALP, SPP1, and BGLAP gene expression was greater in cultures exposed to CAPE in comparison with the control (p0.05). At day 14, matrix mineralization was similar in cultures exposed to CAPE at 1 nM and 10 nM (p0.05), but superior to those ones observed in the other experimental groups (p0.05).CAPE at low concentrations can positively module the osteogenesis in vitro.

Made available in DSpace on 2018-11-26T15:45:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Polymorphous adenocarcinoma (PAC) is a malignant epithelial neoplasm that affects almost exclusively the minor salivary glands, generally described as having a relatively good prognosis. Aberrant nuclear factor erythroid 2 (NF-E2)-related factor (Nrf2) activation in tumor cells has been associated with induction of antioxidant enzymes, such as peroxiredoxin I (Prx I) and increased matrix metalloproteinase (MMP) expression. In this context, the aim of the present study was to evaluate the expression of Nrf2 and correlate it with Prx I and MMP-2 secretion in PAC. Thirty-one cases of PAC from oral biopsies were selected and immunohistochemically analyzed for Nrf2 and Prx I. MMP-2 quantification was performed on primary cell cultures derived from PAC. Oral squamous cell carcinoma (OSCC) cell cultures were used as control. A high immunoexpression of Nrf2 was observed in both the cytoplasm and the nucleus of neoplastic cells from PAC. Nuclear staining for Nrf2 suggested its activation in the majority of the PAC cells, which was confirmed by the high expression of its target gene, Prx I. Quantification of MMP-2 secretion showed lower levels in PAC cell cultures when compared to OSCC cell cultures (p < 0.05). In conclusion, although Nrf2 overexpression has been frequently associated with high-grade malignancies, such relationship is not infallible and, in fact, the opposite may occur in low-grade tumors, such as PAC of minor salivary glands. Sao Leopoldo Mandic Inst & Res Ctr, Dept Oral Pathol, Rua Jose Rocha Junqueira 13, BR-13045755 Campinas, SP, Brazil State Univ Sao Paulo, Aracatuba, SP, Brazil Univ Fed Minas Gerais, Belo Horizonte, MG, Brazil State Univ Sao Paulo, Aracatuba, SP, Brazil FAPESP: 2015/12418-5 CNPq: 304031/2014-3

Multiple myeloma (MM) is characterised by intense protein folding and, consequently endoplasmic reticulum (ER) stress. The prostaglandin 15d-PGJ2 is able to raise oxidative stress levels within the cell and potentially trigger cell death. The aim of this study was to evaluate the antineoplastic effect of 15d-PGJ2 on MM in vitro and in vivo via ER and oxidative stress pathways. MM.1R and MM.1S cell lines were treated with 15d-PGJ2 at 1-10μM and evaluated with regard to proliferation, mRNA expression of PRDX1, PRDX4, GRP78, GRP94, CHOP, BCL-2 and BAX. Stress data was validated via oxidized glutathione assays. MM.1R cells were inoculated into NOD/SCID mice, which were subsequently treated daily with 15d-PGJ2 at 4mg/kg or vehicle (control), with tumour volume being monitored for 14days. 15d-PGJ2 reduced cell proliferation, induced cell death and apoptosis at 5μM and 10μM and Stress-related genes were upregulated at the same doses. Oxidized glutathione levels were also increased. 15d-PGJ2 at 4mg/kg in vivo halted tumour growth. In conclusion, 15d-PGJ2 induced myeloma cell death via ER stress in vitro. 15d-PGJ2 in vivo also inhibited tumour growth.

Objectives and background Sodium ascorbyl phosphate (SAP) is a hydrophilic and stable L-ascorbic acid derivative, being converted by the cell phosphatases into free ascorbic acid (AA), which allows its sustained release in the medium. AA participates in the maintenance and healing of the periodontium. It presents a regulatory role of the osteoblastic activity, stimulating the deposition of collagen extracellular matrix followed by the induction of genes associated with the osteoblastic phenotype. It also acts in the elimination of reactive oxygen species, abundantly produced by defense cells in periodontal disease. The aim of this study was to evaluate the effect of SAP on osteoblast viability and differentiation. Methods Mouse preosteoblastic cells of the MC3T3-E1 strain were used. Cell viability was assessed by the trypan blue dye exclusion assay and the expression of genes related to osteoblast differentiation by quantitative PCR. Collagen I secretion was evaluated by ELISA, and mineralized matrix formation was assayed by Alizarin red S staining. Results The results showed that SAP at concentrations from 50 to 500 µmol/L does not influence preosteoblast cell viability, but stimulates their differentiation, observed by the induction of RUNX2, COL1A1, and BGLAP2; by the higher secreted levels of collagen I; and also by the increase in the mineralization of the extracellular matrix in cells exposed to this agent at 200 or 400 µmol/L, compared with those not exposed. Conclusion By its stability and capacity to induce preosteoblastic cell differentiation, our results indicate that the incorporation of SAP into local release devices, membranes/scaffolds or biomaterials, could favor bone tissue formation and therefore periodontal healing.

Background: The use of biomaterials is commonplace in dentistry for bone regeneration. The aim of this study was to evaluate the performance of a new alloplastic material for bone repair in critical defects and to evaluate the extent of the inflammatory process. Methods: Forty-five New Zealand rabbits were divided into five groups according to evaluation time (7, 14, 30, 60, 120 days), totaling 180 sites with six-millimeter diameter defects in their tibiae. The defects were filled with alloplastic material consisting of poly (lactide-co-caprolactone), beta-tricalcium phosphate, hydroxyapatite and nano-hydroxyapatite (BTPHP) in three different presentations: paste, block, and membrane. Comparisons were established with reference materials, such as Bio-ossTM, Bio-oss CollagenTM, and Bio-gideTM, respectively. The samples were HE-stained and evaluated for inflammatory infiltrate (scored for intensity from 0 to 3) and the presence of newly formed bone at the periphery of the defects. Results: Greater bone formation was observed for the alloplastic material and equivalent inflammatory intensity for both materials, regardless of evaluation time. At 30 days, part of the synthetic biomaterial, regardless of the presentation, was resorbed. Conclusions: We concluded that this novel alloplastic material showed osteoconductive potential, biocompatibility, low inflammatory response, and gradual resorption, thus an alternative strategy for guided bone regeneration.

Adenoid cystic carcinoma (AdCC) and polymorphous adenocarcinoma (PAC) are included among the most common salivary gland cancers. They share clinical and histological characteristics, making their diagnosis challenging in specific cases. MicroRNAs (miRNA) are short, non-coding RNA sequences of 19-25 nucleotides in length that are involved in post-transcriptional protein expression. They have been shown to play important roles in neoplastic and non-neoplastic processes and have been suggested as diagnostic and prognostic markers.This study, using quantitative RT-PCR, investigated miR-150, miR-455-3p and miR-375 expression, in order to identify a possible molecular distinction between AdCC and PAC.miRNA-150 and miRNA-375 expression was significantly decreased in AdCC and PAC compared with salivary gland tissue controls, whilst miRNA-455-3p showed significantly increased expression in AdCC when compared to PAC, (P 0.05). miR-150, miR-357 and miR-455-3p expression in AdCC, PAC and control was not associated with age, gender nor with anatomic site (major and minor salivary glands) (P 0.05).MiR-455-3p could be used as a complimentary tool in the diagnosis of challenging AdCC cases.

In tumorigenesis, not only genetic and epigenetic alterations but also stromal cells and soluble factors in the tumor microenvironment can affect the progression of different neoplasms. In salivary gland tumors derived from the intercalated duct, the myoepithelial (Myo) component modulates the neoplastic phenotype positively. Indeed, in carcinoma ex-pleomorphic adenoma (CXPA) where the malignant component is represented only by epithelial cells, the Myo cells present a role in the progression of this neoplasia. Nevertheless, great interest has been taken in the mechanisms of cellular communication mediated by microvesicles (MV). These structures are secreted by different cells and may induce epigenetic modifications. In malignant neoplasia, MV may affect different stages of carcinogenesis, which comprise the modulation of the extracellular matrix components, including metalloproteinases (MMPs). MMPs 2 and 9 exert an essential role in the epithelial-mesenchymal transition, consequently increasing the potential of migration and invasion of plethora tumors, including CXPA. The present in vitro study aimed to characterize MV derived from malignant neoplastic epithelial cell cultures (CAL27 cells) and their effect on the expression of MMPs in benign Myo cell cultures. Malignant epithelial cells (CAL 27 cell line) were cultured until they reached confluence. Then, the culture medium was collected and the MV were extracted. The Myo cultures, derived from pleomorphic adenomas explants, were exposed to MV from CAL 27 for 24 h. After this period, the secretion of MMPs 2, 9 and their inhibitors, TIMP-1 and TIMP-2, was assayed by means of ELISA. The Myo cultures not exposed to MV were used as control. The results indicated that Myo exposed to MV exhibited a significant increase of MMP-2 and MMP-9 expression (p0.05). In conclusion, the data suggest that MV can modulate MMP and their inhibitors’ expression in Myo cell, which may promote extracellular matrix degradation and CXPA invasion. Citation Format: Gabriel Bernardo Rena, Lucas Novaes Teixeira, Vera Cavalcanti Araújo, Ana Paula Dias Demasi, Elizabeth Ferreira Martinez. Effect of microvesicles on metalloproteinases secretion by myoepithelial cells [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A34.

Endoglin, known to be expressed in proliferating vessels, is of worth when evaluating microvessel density as a prognostic factor in many types of malignancies, including some subtypes of leukemia cells. In childhood acute lymphoblastic leukemia, endoglin is associated with adverse outcome. In bone marrow, endoglin identifies the repopulating hematopoietic stem cells. Mast cells are a component of normal tissue and play an important role in the regulation of several processes, including inflammation and neoplasia. The aim of this study was to evaluate the use of endoglin as a biological marker of mast cells compared with the gold standard stains. We studied 15 specimens of neurofibroma, 9 of mastocytosis, and 6 of fibrous scar tissue through immunohistochemistry (for endoglin and mast cell tryptase) and histochemical staining using toluidine blue. Quantitative analysis of the cells was performed by counting 5 hotspots. The validity of endoglin as a mast cell marker was assessed by intraclass correlation coefficient. The Kruskal-Wallis test was used to compare mast cell count for each marker. A strong endoglin expression was found in the cytoplasmic granules of mast cells within the 3 groups. Similar results were observed with mast cell tryptase as well as toluidine blue. The intraclass correlation coefficient revealed that endoglin is a highly reliable biomarker of mast cells when compared with mast cell tryptase and toluidine blue. In conclusion, endoglin may assist in the diagnosis and pathogenesis study of various processes associated with mast cells. An endoglin-neutralizing treatment for solid cancers and leukemia could also affect mastocytes and the immunologic system.

Myoepithelial cells have been implicated in the regulation of the transition from in situ to invasive neoplasia in salivary gland tumors. Considering the importance of the microenvironment of the tumor, the present in vitro study therefore analyzed the morphological and phenotypic changes undergone by benign myoepithelial cells from pleomorphic adenoma (PA) stimulated by tumor-conditioned medium. The benign myoepithelial cells were obtained from PA and were cultured with fibronectin extracellular matrix protein, supplemented with tumor-conditioned medium, which was harvested from breast ductal adenocarcinoma AU-565 and melanoma Hs 852.T cells. The morphological alterations were assessed by immunofluorescence analysis using vimentin antibody. The α-smooth muscle actin (α-SMA) and fibroblast growth factor (FGF)-2 proteins were analyzed by indirect immunofluorescence and quantitative polymerase chain reaction (qPCR). No morphological changes were observed in the myoepithelial cells cultured in fibronectin protein under stimulation from either tumor-conditioned medium. The immunofluorescence results, which were supported by qPCR analysis, revealed that only α-SMA was upregulated in the fibronectin substratum, with or without tumor-conditioned medium obtained from breast ductal adenocarcinoma and melanoma cells. No significant difference in FGF-2 mRNA expression was detected when the cells were cultured either in the tumor-conditioned medium or in the fibronectin substratum. The tumor-conditioned medium harvested from breast ductal adenocarcinoma and melanoma did not affect myoepithelial cell differentiation and function, which was reflected by the fact that there was no observed increase in α-SMA and FGF-2 expression, respectively.

Aim The regulation of Wnt-β-catenin signalling, which is crucial for osteoblast differentiation and for bone resorption, is driven by critical inhibitors such as sclerostin (SOST) and dickkopf-related protein 1 (DKK1). As such, the aim of this study was to evaluate the involvement of SOST and DKK1 in human chronic periodontitis. Material and Methods Gingival biopsies and serum were sampled from systemically healthy non-periodontitis (n = 15) and chronic periodontitis subjects (n = 15). The mRNA and protein levels of SOST, DKK1 and TNF-α in periodontal tissues were measured by qPCR and by enzyme-linked immunosorbent assay (ELISA) respectively. Serum levels of SOST, DKK1 and TNF-α were assessed by ELISA. Results The mRNA and protein levels of SOST, DKK1 and TNF-α were significantly increased in the gingival tissues of the chronic periodontitis when compared to the non-periodontitis group (p

B cell malignancies are classified according to the postulated differentiation stage of the originating cell. During differentiation, structural and molecular changes occur to support massive processing of immunoglobulin in the endoplasmic reticulum (ER) of plasma cells at the final stage. When overloaded, the ER generates unfolded proteins and hydrogen peroxide (H2O2), which may cause cell death. Peroxiredoxins (Prxs) I and IV belong to a family of proteins able to catalyze peroxide detoxification. Here, we investigated a potential association of these enzymes with immunoglobulin production in B cell neoplasms. Our results demonstrated that the expression of Prx IV was induced as cells became competent to synthesize immunoglobulin light chains, as observed by immunohistochemistry in tissue sections of B cell neoplasms and also by qPCR and Western blotting analyses in malignant B cell lines. Prx I was frequently highly expressed, indicating additional regulatory processes besides ER activity. Results obtained exclusively with myeloma cells have shown that expression of Prxs I and IV, both at mRNA and protein levels, was associated with light chain secretion quantified by ELISA. We suggest that Prxs I and IV may provide survival advantages for terminally differentiated neoplastic B cells by the elimination of H2O2 and, in the case of Prx IV, by the conversion of this toxic in a functional agent driving oxidative protein folding in the ER. In this sense, multiple myeloma and lymphomas demonstrated to synthesize immunoglobulin chains may benefit from strategic therapies targeting the adaptive pathway to ER stress, including inhibition of Prxs I and IV activity.

Peroxisome proliferator-activated receptor-gamma (PPARγ) regulates both glucose metabolism and bone mass. Evidence suggests that the therapeutic modulation of PPARγ with synthetic agonists activity may elicit undesirable effects on bone. However, there is no information regarding its natural agonist 15d-PGJ2, besides its excellent anti-inflammatory action. In the present study the effects of 15d-PGJ2 on osteoblastic cells were determined. Osteoblastic cells (MC3T3) were cultured in an osteogenic medium in the presence of 1, 3 or 10 μM of 15d-PGJ2 during 21 days and alizarin and Von Kossa staining were employed. The protein expression (type-I collagen, osteonectin, osteopontin, RANKL, osteoprotegerin, HDAC-9c and PPAR-γ) was evaluated after 3 days in the presence of 15d-PGJ2 by western blotting and indirect immunofluorescence methods. The production of mineralized extracellular matrix was observed by transmission electron microscopy. After 72 h of culture, the mRNA was extracted for RT-qPCR analysis of RUNX expression. In the presence of all 3 tested 15d-PGJ2 doses, alizarin red and Von kossa staining were positive demonstrating the ability to the osteoblast differentiation. Type-I collagen and osteonectin proteins expression were up-regulated (p < 0.05) after 72 h in the presence of the smaller doses of 15d-PGJ2. In contrast, osteopontin, RANKL and OPG expression did not significantly alter. In the presence of 15d-PGJ2 it was possible to visualize mineralized nodules in the extracellular matrix confirmed with the increased RUNX mRNA expression. 15d-PGJ2 at small doses increased the osteoblast activity and the bone-related proteins expression.

The aim of this study was to evaluate peroxiredoxin I (Prx I) participation in the cellular antioxidant response to low-dose X-rays through the analysis of its expression in buccal mucosa cells from patients of different ages following panoramic dental radiography. Of the 50 patients included in this study, oral mucosa cells from six adults were collected for the immunofluorescence cytological analysis. The other 44 patients, 11 patients aged below 20 years; 22 patients aged between 20 and 50 years; and 11 patients aged above 50 years, were submitted to panoramic dental radiography, and oral mucosa cells were collected for the gene expression analysis before and 1 hour after exposure. The results demonstrated Prx I expression in the cytoplasm of oral mucosa cells either before or after radiation exposure. The quantitative analysis showed that in oral mucosa cells from patients aged below 50 years the mRNA levels of PRDX1 were significantly increased after radiation exposure. On the other hand, the cells from patients aged above 50 years presented significantly lower PRDX1 transcript levels after radiation exposition. Panoramic radiography leads to increased Prx I expression in buccal mucosa cells, probably as an adaptive response to eliminate X-ray-induced ROS, except in cells from elderly people. Even low doses of radiation employed for dental purposes are capable to provoke stress to cells, which was demonstrated via the induction of the antioxidant gene PRDX1. In elderly patients, such mechanism was demonstrated to be impaired.

There is controversial evidence regarding the levels of antioxidant molecules in type 2 diabetes periodontitis patients. Thus, the aim of the present study was to evaluate the gene expression of antioxidant enzymes in the gingival tissue of poorly and well-controlled type 2 diabetic subjects with chronic periodontitis (CP).Gingival biopsies were harvested from systemically and periodontally healthy subjects (n=12), systemically healthy subjects with CP (n=15), well-controlled (n=8) and poorly controlled (n=14) diabetic subjects with CP. The messenger RNA (mRNA) levels of peroxiredoxin (PRDX) 1 and 2, catalase (CAT), glutathione peroxidase (GPX1) and superoxide dismutase (SOD) 1 and 2 were measured by quantitative polymerase chain reaction (qPCR).The results showed that PRDX1 and GPX1 were up-regulated by periodontitis (p0.05), independently of the glycaemic status, whilst PRDX2 and SOD2 genes were slightly influenced by periodontitis, but significantly induced when periodontitis was associated with DM, especially under a poor glycaemic control (p0.05). Moreover, CAT and SOD1 expressions were not significantly influenced by any of these inflammatory disorders (p0.05).In conclusion, both PRDX1 and GPX1 were overexpressed in CP whilst PRDX2 and SOD2 were up-regulated especially in the poorly controlled diabetic group with CP.

Specific cytokines and the costimulatory protein CD40 play role in inducing immunoglobulin (Ig)A production by B cells in the humoral immune response. However, to date, the role of these mediators was not investigated in chronic periodontitis. Therefore, the aim of this study was to assess the local levels of interleukin (IL)-21, IL-21 receptor (IL-21R), IL-4, IL-10 and CD40 ligand (CD40L) on chronic periodontitis subjects and their relationship with the salivary levels of IgA. Gingival biopsies and un-stimulated saliva were collected from chronic periodontitis (n = 15) and periodontally healthy (n = 15) subjects. The mRNA levels of IL-4, IL-10, IL-21, IL-21R, CD40L in the gingival biopsies were evaluated by quantitative real-time polymerase chain reaction. The salivary levels of IgA and the levels of IL-4 and IL-10 in the gingival biopsies were analyzed by ELISA. The mean levels of IgA were significantly higher in the chronic periodontitis compared to periodontally healthy group (P < 0.05). The mRNA levels for IL-21 was higher (P < 0.05) in the chronic periodontitis when compared to the healthy group. However, the expression of IL-21R and CD40L did not differ between groups. The IL-10 was significantly elevated at mRNA and protein levels in chronic periodontitis when compared to periodontally healthy group (P < 0.05). Conversely, the mRNA levels as well as the protein amount of IL-4 were significantly lower (P < 0.05) in chronic periodontitis than healthy ones. In conclusion, the upregulation of IL-21 and IL-10 and downregulation of IL-4 in periodontitis tissues may be collectively involved in the increased levels of salivary IgA in chronic periodontitis subjects.

Soares A B, Demasi A P D, Altemani A & de Araujo V C (2011) Histopathology58, 377–382 Increased mucin 1 expression in recurrence and malignant transformation of salivary gland pleomorphic adenoma Aims: Pleomorphic adenoma (PA) is the most common salivary gland tumour with a tendency to recur (RPA) and a risk of malignant transformation. Mucin 1 (MUC-1) plays a role in the progression of many tumours and may be a marker to predict RPA. The aim of this study was to evaluate MUC-1 expression in different phases of the adenoma to carcinoma sequence. Methods and results: Twenty-one cases of PA, 18 cases of RPA, three cases of RPA with focal transformation (TRPA) and 11 cases of carcinoma ex-pleomorphic adenoma (CXPA) were analysed immunohistochemically for MUC1 expression using an antibody to MUC1/DF3. MUC1 reactivity in RPA was stronger than that observed in PA and, in all the different carcinoma groups, MUC-1 expression was significantly higher in carcinoma than in RPA and PA. Conclusion: This study has confirmed that MUC-1 is related to the recurrence of PA and that this molecule is associated with malignant transformation of PA with carcinoma cells overexpressing MUC-1.

Carcinoma ex pleomorphic adenoma is a rare salivary gland malignancy. It constitutes an important model for the study of carcinogenesis, as it can display the tumor in different stages of progression, from benign pleomorphic adenoma to frankly invasive carcinoma. Growth signaling pathways undergo continuous activation in human tumors, commonly as a consequence of the overexpression of ligands and receptors such as platelet-derived growth factor and platelet-derived growth factor receptor. Hydrogen peroxide is produced after platelet-derived growth factor receptor activation, and it is essential for the sequential phosphorylation cascade that drives cell proliferation and migration. By their ability to degrade hydrogen peroxide, peroxiredoxins are involved in growth factor signaling regulation and in the oxidative stress response. To verify the potential association of peroxiredoxin I, platelet-derived growth factor-A, and platelet-derived growth factor receptor-alpha with carcinoma ex pleomorphic adenoma progression, we investigated the expression of these molecules in carcinoma ex pleomorphic adenoma showing different degrees of invasion. The peroxiredoxin I, platelet-derived growth factor-A, and platelet-derived growth factor receptor-alpha proteins were present in remnant pleomorphic adenoma to only a small extent, but, collectively, they were highly expressed as soon as the malignant phenotype was achieved and remained at elevated concentrations during progression to the advanced stages of carcinoma ex pleomorphic adenoma. In addition, their locations overlapped significantly, strengthening their connection to this growth-signaling pathway. Our results indicate that carcinoma ex pleomorphic adenoma cells acquire at least 2 significant advantages relative to their normal counterparts: resistance to oxidative stress-induced apoptosis, conferred by high peroxiredoxin I concentrations, and sustained growth, reflecting platelet-derived growth factor-A and platelet-derived growth factor receptor-alpha overexpression.

Cysteine plays structural roles in proteins and can also participate in electron transfer reactions, when some structural folds provide appropriated environments for stabilization of its sulfhydryl group in the anionic form, called thiolate (RS(-)). In contrast, sulfhydryl group of free cysteine has a relatively high pK(a) (8,5) and as a consequence is relatively inert for redox reaction in physiological conditions. Thiolate is considerable more powerful as nucleophilic agent than its protonated form, therefore, reactive cysteine are present mainly in its anionic form in proteins. In this review, we describe several processes in which reactive cysteine in proteins take part, showing a high degree of redox chemistry versatility.

We investigated the changes in the oxidative stress response of yeast cells suffering mitochondrial dysfunction that could impair their viability. First, we demonstrated that cells with this dysfunction rely exclusively on cytosolic thioredoxin peroxidase I (cTPxI) and its reductant sulfiredoxin, among other antioxidant enzymes tested, to protect them against H2O2-induced death. This cTPxI-dependent protection could be related to its dual functions, as peroxidase and as molecular chaperone, suggested by mixtures of low and high molecular weight oligomeric structures of cTPxI observed in cells challenged with H2O2. We found that cTPxI deficiency leads to increased basal sulfhydryl levels and transcriptional activation of most of the H2O2-responsive genes, interpreted as an attempt by the cells to improve their antioxidant defense. On the other hand, mitochondrial dysfunction, specifically the electron transport blockage, provoked a huge depletion of sulfhydryl groups after H2O2 treatment and reduced the H2O2-mediated activation of some genes otherwise observed, impairing cell defense and viability. The transcription factors Yap1 and Skn7 are crucial for the antioxidant response of cells under inhibited electron flow condition and probably act in the same pathway of cTPxI to protect cells affected by this disorder. Yap1 cellular distribution was not affected by cTpxI deficiency and by mitochondrial dysfunction, in spite of the observed expression alterations of several Yap1-target genes, indicating alternative mechanisms of Yap1 activation/deactivation. Therefore, we propose that cTPxI is specifically important in the protection of yeast with mitochondrial dysfunction due to its functional versatility as an antioxidant, chaperone and modulator of gene expression.

During tumor invasion, benign myoepithelial cells of carcinoma ex-pleomorphic adenoma (CXPA) surround malignant epithelial cells and disappear. The mechanisms involved in the death and disappearance of these myoepithelial cells were investigated via analysis of the expression of regulatory proteins for apoptosis, autophagy and cellular senescence in an in situ in vitro model. Protein expression relating to apoptosis (Bax, Bcl-2, Survivin), autophagy (Beclin-1, LC3B) and cellular senescence (p21, p16) was evaluated using indirect immunofluorescence. β-galactosidase expression was assessed via histochemistry. Biopsies of CXPA (ex vivo) allowed immunhistochemical evaluation of p21 and p16, whilst LC3B, p21 and p16 protein expression was analyzed by western blotting. In the in vitro model, the myoepithelial cells were positive for LC3B (cytoplasm) and p21 (nucleus), whilst in vivo positivity for p21 and p16 was observed. In vitro, β-galactosidase activity increased in the myoepithelial cells over time. Western blotting analysis revealed an increased LC3B, p16 and p21 expression in the myoepithelial cells with previous contact with the malignant cells when compared with those without contact. The investigation of behavior of benign myoepithelial cells in ductal areas of CXAP revealed that the myoepithelial cells are involved in the autophagy-senescence phenotype that subsequently leads to their disappearance.

AIM: To evaluate the staining of esthetic orthodontic brackets by plaque disclosing solutions. METHODS: Two types of brackets manufactured by GAC/DENTSPLY(r) were evaluated: ceramic (n=30) and polycarbonate (n=30). The brackets were divided into 6 groups. Two control groups (n=6) were immersed in absolute ethanol: GI - ceramic brackets and GII - polycarbonate brackets. Four experimental groups (n=12) were immersed in different plaque disclosing solutions: GIII (ceramic brackets) and GIV (polycarbonate brackets) were immersed in Replak(r); GV (ceramic brackets) and GVI (polycarbonate brackets) were immersed in Replasul "S"(r). Relative quantitative analysis of the influence of plaque disclosing tablets on bracket staining was performed using reflectance spectrophotometry of stain deposition. Exploratory analysis of the data was performed using Analysis of Variance (ANOVA) in a 2x2 factorial setup (bracket x immersion) with additional treatments (controls). RESULTS: The results demonstrated that the ceramic brackets presented the highest amount of staining when Replasul "S"(r) was used (pd"0.05). However, when Replak(r) was used, no statistically significant difference was found in comparison with the control group (p>0.05). For polycarbonate brackets, staining was detected for both disclosing solutions (p>0.05). CONCLUSIONS: The disclosing solutions caused stain formation on polycarbonate brackets and, under the tested conditions, use of Replak(r) on ceramic brackets did not cause staining.

Background Pleomorphic adenoma (PA) is the most common salivary gland tumour. Although classified as benign, it has a tendency to recur (recurrent pleomorphic adenomas (RPA)), as well as the ability to undergo malignant transformation. It has been suggested that mutations in various families of growth factors and growth factor receptions are involved in the autonomous growth of tumour cells. The aim of the present study was to investigate the participation of platelet-derived growth factor (PDGF)-A, PDGF-B, PDGF-Rα, fibroblast growth factor (FGF)-2, Flg and BEK in PA, RPA and recurrent pleomorphic adenoma with malignant transformation (TRPA). Methods 18 cases of PA, 16 cases of RPA and two cases of RPA with focal malignant transformation (TRPA) were analysed for growth factor expression utilising immunohistochemical techniques via tissue microarray. Results There was a significant difference in PDGF-A, PDGF-B, PDGF-Rα, FGF-2, Flg and BEK expression in all groups. When comparing non-recurrent with recurrent tumours, PDGF-A, PDGF-B, PDGF-Rα, FGF-2, Flg and BEK reactivity in RPA was stronger than that observed in PA. All proteins were highly expressed in TRPA. Conclusions This research suggests that PDGF-A, PDGF-B, PDGF-Rα, FGF-2, BEK and Flg can be related to the recurrence of PA. In addition, this study shows that TRPA cells overexpress all growth factors, which has been reported in association with the malignant transformation.

Pleomorphic adenoma (PA) is the most common salivary gland tumour with a tendency to recur (RPA) and a risk of malignant transformation. Mucin 1 (MUC-1) plays a role in the progression of many tumours and may be a marker to predict RPA. The aim of this study was to evaluate MUC-1 expression in different phases of the adenoma to carcinoma sequence.Twenty-one cases of PA, 18 cases of RPA, three cases of RPA with focal transformation (TRPA) and 11 cases of carcinoma ex-pleomorphic adenoma (CXPA) were analysed immunohistochemically for MUC1 expression using an antibody to MUC1/DF3. MUC1 reactivity in RPA was stronger than that observed in PA and, in all the different carcinoma groups, MUC-1 expression was significantly higher in carcinoma than in RPA and PA.This study has confirmed that MUC-1 is related to the recurrence of PA and that this molecule is associated with malignant transformation of PA with carcinoma cells overexpressing MUC-1.

Aims To broaden understanding of phenomena involved in progression from classical adenoid cystic carcinomas (ACCs) to tumours with high-grade transformation (ACC-HGT) Methods Expression of proteins linked to cellular metabolism as well as the microvascular density (MVD) in conventional and transformed areas were analysed. Findings were compared with ordinary ACCs. In seven cases of ACC-HGT and in 18 ACCs the expressions of GLUT1, mitochondrial antigen (MTA), CD34 (for assessing MVD), α-SMA and P63 (for detection of myoepithelial cells) and Ki-67 (for evaluation of proliferation index) were examined. Results The transformed component corresponded to adenocarcinomas with frequent (four cases) or scarce/absent (three cases) gland differentiation. In the latter, Ki-67 index was higher, two patients presented lymphatic metastasis and one died of disease. In the former, there was one long-term survivor and one with liver metastasis. Conventional areas of both ACC-HGT and ACC were negative for GLUT1 in most cases (83.3% and 81.3%, respectively) and exhibited low or no expression of MTA (100% and 66.7% of cases, respectively). In contrast, the HGT component presented increased expression of both proteins (GLUT1+ in 50% of cases; MTA+ in 100%). However, the degree of GLUT1 expression did not correlate with clinical outcome. MVD did not differ significantly between conventional and transformed components. Conclusions Transformation of classical ACC into ACC-HGT encompasses adenocarcinomas with variable degrees of differentiation and seems to lead to metabolic changes without reflection in tumour vasculature. Despite the tumours9 higher GLUT1 expression, this protein has no utility as a prognostic marker.

Increasing emphasis has been placed on the role of myoepithelial cells, the contractile components of secretory glands, in the in situ to invasive carcinoma transition. These cells are placed at the interface between luminal epithelial cells and the stromal compartment, which favors their cross-talk with all other cell types comprising the tumor micro-environment. To obtain some clues about this cross-talk and also to better understand our previous immunoprofile study of myoepithelial cells in salivary gland carcinoma ex-pleomorphic adenoma (CXPA), we investigated FGF-2 expression in CXPA in situ structures as well as in cells cultured under conditions attempting to simulate the cellular interactions of this tumor stage. We have observed by immunohistochemistry that myoepithelial cells of CXPA in situ structures overexpress FGF-2. In addition, our results supported by qPCR and Western blotting, demonstrated that the expression of FGF-2 in the benign myoepithelial cells was in fact increased by stimulation with the conditioned medium from malignant cells. Low molecular weight FGF-2, known to be primarily released from the cells to exert its biological activity through receptors, was the predominant FGF-2 form detected in the benign myoepithelial cells. Specific FGF-2 receptors were found in the malignant epithelial but not in the benign myo-epithelial cells of CXPA, indicating a paracrine role for benign myoepithelial cell-derived FGF-2. Abnormal paracrine myo-epithelial/epithelial cell interactions and also myoepithelial/ stromal cell interactions could favor tumor growth, invasion and metastasis.

Mucoepidermoid carcinoma (MEC), the most common primary salivary malignancy, shows great variability in clinical behaviour, thus demanding investigation to identify of prognostic markers. Since Warburg's studies, unrestricted cell growth during tumorigenesis has been linked to altered metabolism, implying hypoxic stimulation of glycolysis and diminished contribution of mitochondrial oxidative phosphorylation to cellular ATP supply. Hypothesizing that the study of MEC metabolic status could lead to the discovery of prognostic markers, we investigated by immunohistochemistry the expression of glucose transporter 1 (Glut-1), mitochondrial antigen and peroxiredoxin I (Prx I) in samples of MEC from different histological grades. Our results showed that mitochondrial antigen and Prx I were expressed in the majority of the MEC cases independent of the histological grade. In contrast Glut-1 expression increased significantly as the tumours became more aggressive. These results suggested that oxidative phosphorylation may contribute to ATP supply in all stages of MEC progression, and that the relative contribution of glycolysis over mitochondria for cellular ATP supply increases during MEC progression, favouring growth under low oxygen concentration. In addition, the observed high Prx I protein levels could provide protection to tumour cells against reactive oxygen species generated as a consequence of mitochondrial function and hypoxia-reoxygenation cycling. Altogether our findings suggest that upregulation of Glut-1 and Prx I constitute successful adaptive strategies of MEC cells conferring a growth advantage over normal salivary gland cells in the unstable oxygenation tumour environment.

Background: Oncocytic lesions, particularly frequent in the salivary glands, are characterized by cells with an atypical accumulation of mitochondria. This accumulation has been recognized as a compensatory mechanism to intrinsic functional defects of these organelles, resulting in energy production impairment and increased generation of reactive oxygen species (ROS), including hydrogen peroxide (H2O2). Peroxiredoxin I (Prx I) is a H2O2 scavenging protein and the expression of its yeast homolog was reported to be influenced by mitochondrial function. Methods: In this study, we evaluated Prx I expression in oncocytic lesions of salivary glands by immunohistochemistry. Results: Our results showed that Prx I is overexpressed in oncocytes regardless of the salivary gland lesion where they appear. Conclusions: These results suggest that Prx I expression in oncocytes is related to its ability to decompose mitochondrial-derived H2O2 and that it could provide to the cells a protective role in an environment that, by continuously producing potential DNA-damaging ROS, predisposes to genome instability and cellular transformation.

Carcinoma ex-pleomorphic adenoma (CXPA) is an aggressive salivary gland malignancy, usually derived from a long-standing or a recurrent benign tumor, the pleomorphic adenoma (PA). In the context of dynamic reciprocity, changes in the composition and structure of extracellular matrix proteins and cell surface receptors have been frequently associated with dysfunctional adhesion and invasive behavior of tumor cells. It is not fully understood if these changes are involved in the conversion of PA to CXPA. In this study, different progression stages of CXPA were investigated regarding the expression of the major extracellular matrix proteins, collagen type I, and of E-cadherin and beta-catenin, the components of adherens junctions. By immunohistochemical analysis, we have demonstrated that direct contact of tumor cells with fibrillar type I collagen, particularly near the invasive front and in invasive areas prevailing small nests of CXPA cells, could be associated with reduced expression of the E-cadherin and beta-catenin adhesion molecules and with invasive behavior of epithelial, but not of CXPA with myoepithelial component. Our results also suggested that this association could depend on the organization of collagen molecules, being prevented by high-order polymeric structures. These findings could implicate the local microenvironment in the transition from the premalignant PA to invasive CXPA.

Background: Plasmablastic lymphoma (PBL) and multiple myeloma (MM) are B cell-derived malignancies that share many morphologic and immunophenotypic traits, making the differential diagnosis particularly complicated. We have recently demonstrated that peroxiredoxin I (PrdxI) is expressed in plasma cells but not in B lymphocytes, suggesting that its expression is development-associated. Aim: To analyze PrdxI expression in PBL and in MM in order to study its utilization as an additional diagnostic molecular tool. Methods and results: Eight cases of PBL and nine of MM were studied by immunohistochemistry. We have demonstrated that PrdxI expression is closely connected with the immunoglobulin production capacity of the cells, which means high in MM, but absent in PBL cases, except one, wherein few cells were stained. Conclusions: We hypothesize PrdxI as a component of the unfolded protein response (UPR), an adaptive pathway essential for plasma cell differentiation. As we have not detected immunoglobulin in our PBL cases, we suggest that UPR was not activated in the cells, accounting for the impediment of the developmental process, and for the inhibition of PrdxI expression observed. PrdxI could be considered an additional plasma cell functional marker and could also be speculated as a therapeutic target in the treatment of MM.

Intravascular papillary endothelial hyperplasia (IPEH) is a benign, nonneoplastic, vascular lesion. The main significance of the lesion lies in the fact that it may be mistaken for angiosarcoma. Oral lesions are uncommon and the present paper reports 2 cases of oral IPEH, in different sites. Histologically, the tissue was characterized by papillary fronds lined by proliferating endothelium. Immunohistochemically (IHC), the lesion was positive for CD34, smooth muscle actin (SMA), type I and IV collagen, vimentin, and laminin, but it was negative for CD105. Local excision was the treatment of choice. No recurrence was observed during a 1-year and 6-month follow-up period, respectively. The clinical, histological, and immunohistochemical characteristics are discussed.

To investigate whether salivary carcinomas with and without myoepithelial differentiation could present differences regarding degree of angiogenesis, we compared tumor vascularization between adenoid cystic (31 cases) and epithelial-myoepithelial carcinomas (14) versus mucoepidermoid (37) carcinoma. The expression of peroxiredoxin I was also studied to verify the potential relationship between cellular metabolism and microvascular density. Microvascular density for CD34 and CD105 were significantly lower in carcinomas with myoepithelial differentiation. However, no correlation was found between degree of angiogenesis and amounts of myoepithelial cells. High-grade peroxiredoxin I expression was found in 73.7% of mucoepidermoid carcinomas, whereas 85.1% of carcinomas with myoepithelial differentiation presented low-grade expression. In conclusion, carcinomas with myoepithelial differentiation, regardless of the amounts of myoepithelial cells, are associated to a significantly lower vascular density. The reasons for this lower angiogenic activity remain to be determined but could be related to metabolic characteristics of the cancer cells.

The participation of reactive oxygen species (ROS) in the immune response, both as pathogen killers and as mediators of signaling pathways, is well established. However, little is known about the enzymes responsible for ROS elimination in immune cells. Peroxiredoxin I (PrdxI) is a multifunctional enzyme that exhibits thioredoxin-dependent peroxidase activity. It has been described as a major hydrogen peroxide (H(2)O(2))-inducible protein in mouse peritoneal macrophages. In order to characterize its participation in the antioxidant defense of inflammatory/immune cells in greater detail, we evaluated its expression at sites of the oral cavity affected by inflammatory disorders induced by different agents (infectious, chemical, mechanical or tumor). In this study we demonstrated, by immunohistochemistry, that PrdxI is expressed in plasma cells, but not in B lymphocytes, regardless of the inflammation-inducing agent. We suggest that PrdxI induction could be considered a crucial part of the cellular adaptive response to the B-cell differentiation process to cope with the additional H(2)O(2) associated with massive disulfide bond formation during immunoglobulin folding in the endoplasmic reticulum of plasma cells. PrdxI could diminish the tissue damage that accompanies inflammation.

Orientador : Luis Eduardo Soares Netto Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Tiorredoxina peroxidase citoplasmática I (cTPxI) é uma enzima antioxidante de Saccharomyces cerevisiae pertencente à família das peroxirredoxinas, conservada desde bactérias até humanos, capaz de reduzir hidroperóxidos às custas de um substrato contendo tio!. Nossos estudos a respeito de sua função em leveduras demonstraram que CTPX1 é altamente expresso em células expostas a diferentes concentrações de peróxido de hidrogênio. Resultados posteriores revelaram que cTPxI é essencial para a defesa antioxidante de células com deficiências respiratórias, demonstrado pela elevada sensibilidade ao H2O2 de células mutantes em CTPX1 (l1ctpxI) com disfunções mitocondriais. Além disso, mostramos que a remoção de DNA mitocondrial ou a deleção do gene COXI0, necessário para a montagem de cito cromo c oxidase, induzem a formação de HzOz. Independentemente de como e onde a função mitocondrial é perturbada, a produção endógena de EROs parece ser necessária para o aumento da sensibilidade ao estresse oxidativo de células Dctpxl Este fenômeno foi demonstrado por testes de viabilidade e por determinações dos níveis de grupos sulfidrila protéica e não protéica. Em acordo, a expressão de CTPX1 é bastante elevada nas condições em que a proteína correspondente é essencial para a defesa antioxidante. Finalmente, obtivemos evidências de que cTPxI especificamente protege células com deficiência respiratória contra EROs, uma vez que deleções de genes que codificam outras peroxidases não promoveram alterações de sensibilidade de células com mitocôndrias funcionalmente defeituosas ao H2O2. Portanto, cTPxI parece ser especialmente importante na proteção de células expostas simultaneamente a fontes de EROs endógenas e exógenas. Deficiências respiratórias são freqüentemente associadas ao processo de envelhecimento, doenças neurodegenerativas e câncer em células humanas. De acordo com nossos resultados, peroxirredoxinas, especialmente aquelas que apresentam elevada similaridade a cTPxI de levedura, poderiam exercer papel decisivo no destino, sobrevivência ou morte, de células afetadas por estas desordens Abstract: Cytosolic thioredoxin peroxidase I (cTPxI) is a Saccharomyces cerevisiae antioxidant enzyme that belongs to peroxiredoxins family, conserved from bacteria to humans, able to reduce hydroperoxides in the presence of a thiol substrate. Our study concerning its role in yeast cells demonstrated that CTPXI is highly expressed in cells exposed to different H2O2 concentrations. Further results revealed that cTPx I is essential for the antioxidant defense of respiratory deficient cells, demonstrated by high sensitivity to H2O2 of ctpxI mutant cells with dysfunctional mitochondria. In addition, we have shown that lack of mitochondrial DNA or COX10 deletion, gene required for cytochrome c oxidase assembly, lead to H2O2 increased generation. Regardless where or how mitochondrial function is perturbed, endogenous ROS production seems to be necessary to increase sensitivity of ctpxI cells to oxidative stress. This phenomenon was demonstrated by viability tests and by sulfhydryl content determinations. Accordingly, C1PX1 expression was high in conditions where the corresponding protein was essential to antioxidant defense. Finally, we have obtained evidences suggesting that cTPxI specifically protects respiratory deficient cells against ROS, since deletion of genes encoding other peroxidases did not alter the sensitivity of cells with mitochondrial dysfunction to HzOz. Therefore, cTPxI seems to be remarkably important in the protection of cells exposed simultaneously to ROS generated by endogenous and exogenous sources. Respiratory deficiencies are frequently associated with cancer, neurodegenerative diseases and aging in human cells. Our results suggest that peroxiredoxins, specially those with high similarity to yeast cTPxI, could exert decisive role in the destiny, survival or death, of cells affected by these disorders Doutorado Bioquímica Doutor em Biologia Funcional e Molecular

The specific role of cytosolic thioredoxin peroxidase I (cTPx I), encoded by TSA1 (thiol-specific antioxidant), was investigated in the oxidative stress response of Saccharomyces cerevisiae. In most cases, deletion of TSA1 has showed only a slight effect on hydrogen peroxide sensitivity. However, when the functional state of the mitochondria was compromised, the necessity of TSA1 in cell protection against this oxidant was much more evident. All the procedures used to disrupt the mitochondrial respiratory chain promoted increases in the generation of H2O2 in cells, which could be related to their elevated sensitivity to oxidative stress. In fact, TSA1 is highly expressed when cells with respiratory deficiency are exposed to H2O2. In conclusion, our results indicate that cTPx I is a key component of the antioxidant defense in respiratory-deficient cells.

Multiple myeloma (MM) is an incurable malignancy characterized by the accumulation of terminally differentiated plasma cells in the bone marrow, usually accompanied by continuous monoclonal immunoglobulin production. This production requires proper folding and assembly of immunoglobulin chains, conducted by disulfide bonds formation in the endoplasmic reticulum (ER). Disulfide bonds are formed via electron transfer from thiol groups in a protein relay system, from protein disulfide isomerase (PDI) to ER oxidoreductase1 (ERO1) and finally to molecular oxygen, generating one molecule of hydrogen peroxide (H2O2) as secondary product for each disulfide bond formed. The high ER protein-folding demand usually placed on MM cells predisposes them to accumulation of unfolded proteins, which results in ER stress and also oxidative stress, both capable to induce cell death. Cellular defense systems for these specific forms of stress, known as the unfolded protein response (UPR) and oxidative stress response, have been explored as therapeutic targets for MM. 15-Deoxy-D12,14-prostaglandin J2 (15d-PGJ2) is a member of J2 series of PGs, characterized by the presence of an electrophilic carbonyl group in the cyclopentenone ring, which confers them biological properties that are different from those of other components of the PG family. These include anti-inflammatory, anti-proliferative and pro-apoptotic activities, depending on the concentration and cell type. Starting from the observation that15d-PGJ2 is emerging as the most potent antineoplastic agent of the J2 series of PGs, in the present study we investigated its effect on MM cells. We found that 15d-PGJ2 displayed superior cytotoxicity than dexamethasone in MM cell lines. Using flow cytometry, we demonstrated intracellular reactive oxygen species (ROS) formation in these cells after 15d-PGJ2 treatment, which was associated with an augmented ratio of oxidized to reduced glutathione (GSH), indicating that the cells were under oxidative stress. Due to its electrophilic property, it has been suggested that 15d-PGJ2 may directly react with GSH and/or other thiol compounds. Such reactions would deplete essential cellular antioxidants thereby leading to oxidative stress in MM cells. Results from qPCR analysis showed that 15d-PGJ2 induces transcription of UPR genes, including GRP78, GRP94, EDEM1 and also PRDX4. GRP78/BiP and GRP94 are ER-resident chaperones, members of the heat shock protein (HSP) 70 family and HSP 90 family, respectively. These chaperones were described to bind to immunoglobulin chains assisting their assembly. The ER degradation-enhancer mannosidase-like protein 1 (Edem1) is involved in the degradation of misfolded proteins to prevent their aggregation and cytotoxicity. PRDX4 encodes the ER-localized peroxiredoxin (Prx) IV, an enzyme not yet recognized as an UPR component, but recently demonstrated to use the H2O2 produced by ERO1 during disulfide bond formation to generate another pair of disulfide, with the additional benefit of reducing H2O2 to water. Increased transcription of these genes indicates an attempt to preserve ER-processing function and MM cells survival in response to accumulated misfolded proteins. In this regard, 15d-PGJ2-induced oxidative stress, by provoking oxidative modification of proteins, could have intensified the formation of misfolded proteins, thereby activating UPR signaling. In agreement with 15d-PGJ2-mediated accumulation of misfolded proteins, which are prevented from progressing further along the secretory pathway, our results from ELISA have shown a massive decrease in the concentration of light chain immunoglobulin secreted by MM cells in the presence of 15d-PGJ2, but not dexamethasone. These data suggest that 15d-PGJ2 favors ROS accumulation and protein misfolding, especially threatening the survival of cells that have a high protein-folding load and/or are susceptible to oxidative stress. In this sense, multiple myeloma and related disorders may benefit from 15d-PGJ2 therapy. Disclosures: Demasi: FAPESP: Research Funding. Napimoga:FAPESP: Research Funding.

SummaryIn order to investigate the role of myoepithelial cell and tumor microenvironment in salivary gland neoplasma, we have performed a study towards the effect of different extracellular matrix proteins (basement membrane matrix, type I collagen and fibronectin) on morphology and differentiation of benign myoepithelial cells from pleomorphic adenoma cultured with malignant cell culture medium from squamous cell carcinoma. We have also analyzed the expression of α-smooth muscle actin (α-SMA) and FGF-2 by immunofluorescence and qPCR. Our immunofluorescence results, supported by qPCR analysis, demonstrated that α-SMA and FGF-2 were upregulated in the benign myoepithelial cells from pleomorphic adenoma in all studied conditions on fibronectin substratum. However, the myoepithelial cells on fibronectin substratum did not alter their morphology under malignant conditioned medium stimulation and exhibited a stellate morphology and, occasionally focal adhesions with the substratum. In summary, our data demonstrated that the extracellular matrix exerts an important role in the morphology of the benign myoepithelial cells by the presence of focal adhesions and also inducing increase FGF-2 and α-SMA expression by these cells, especially in the fibronectin substratum.