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Made available in DSpace on 2021-06-25T10:58:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Gastric cancer is the fifth most common malignancy worldwide. Serjania marginata Casar. (SM) displays anti-inflammatory properties and has been used to treat gastrointestinal disorders. In the current study, we examined whether the hydroethanolic extract of SM leaves exerted cytotoxic, mutagenic, and protective effects in non-tumor gastric epithelium cells (MNP01) and gastric adenocarcinoma cells (ACP02) in vitro and analyzed whether its action was selective. Initially, cell viability (MTT assay), cell cycle kinetics (flow cytometry), and cell proliferation (total protein content) were analyzed. In addition, genomic instability (cytokinesis-block micronucleus cytome assay), anti/pro-oxidant status (CM-H2DCFDA probe), and transcriptional expression (RT-qPCR) of genes related to cell cycle, cell death, and antioxidant defense were also evaluated. The SM extract was cytotoxic toward MNP01 and ACP02 cells at concentrations greater than 300 and 100 μg·ml−1, respectively, and decreased protein content only toward ACP02 cells at 200 μg ml−1. In ACP02 cells, the SM extract at 100 μg·ml−1 associated with doxorubicin (DXR; 0.2 μg ml−1) clearly promoted cell cycle arrest at the G2/M phase. The extract alone was not mutagenic to either cell type and reversed DXR-induced DNA damage and H2O2-induced oxidative stress in MNP01 cells. The gene expression experiments showed that SM hydroethanolic extract exerts an antioxidant response via NFE2L2 activation in non-tumor gastric cells, and cell cycle arrest (G2/M) in ACP02 gastric cancer cells via the TP53 pathway. The selective action of SM indicates that it is a promising therapeutic agent to treat gastric diseases and merits further studies. Department of General Biology Center of Biological Sciences State University of Londrina (UEL) Natural Products Laboratory Anhanguera-Uniderp University Experimental Campus of São Vicente São Paulo State University (UNESP) Epigenetics and Genomic Instability Laboratory Instituto de Investigaciones Biológicas Clemente Estable Department of Biological Sciences School of Pharmaceutical Sciences São Paulo State University (UNESP) Experimental Campus of São Vicente São Paulo State University (UNESP) Department of Biological Sciences School of Pharmaceutical Sciences São Paulo State University (UNESP)

Made available in DSpace on 2020-12-12T01:46:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-03-01 Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão Fridericia platyphylla (Cham.) L.G. Lohmann (FP) has cytotoxic, anti-inflammatory, and analgesic properties. We aimed to characterize the cytotoxic and antiproliferative effects of FP extract on normal (GAS) and tumor-derived (ACP02 and HepG2) cell lines. The effective concentrations (EC50s) by tetrazolium bromide assay (MTT) were 56.16, 43.68, and 42.57 µg mL−1 and 69.38, 41.73, and 52.39 µg mL−1 by neutral red assay for GAS, ACP02, and HepG2 cells, respectively. The extract decreased nuclear division indices, which was not reflected in cell proliferation curves. Flow cytometric analyses showed that even 30 µg mL−1 extract (shown to be noncytotoxic by MTT assay) increased the sub-G1 population, indicating cell death due to apoptosis and necrosis. A cytokinesis-block micronucleus cytome assay showed that 30 µg mL−1 of the extract increased the frequency of nuclear buds in tumor cells. Real-time quantitative polymerase chain reaction showed CCND1 upregulation in doxorubicin-treated GAS cells and BCL-XL, BIRC5, and MET downregulation in 5 or 30 µg mL−1 in FP extract-treated ACP02 cells. In conclusion, FP extract modulated apoptosis- and cell cycle-related genes and presented selective cytotoxicity toward tumor cells that deserves further investigation by testing other cell types. Our results demonstrated that even medicinal plants exert adverse effects depending on the extract concentrations used and tissues investigated. Laboratory of Mutagenesis Department of Biological Sciences Faculty of Pharmaceutical Sciences São Paulo State University (UNESP) Laboratory of Mutagenesis and Oncogenetics Department of General Biology Center of Biological Sciences State University of Londrina (UEL) Laboratory of Cytogenetics and Mutagenesis Department of Biology Faculty of Philosophy Sciences and Letters of Ribeirão Preto (FFCLRP) Laboratory of Advanced Studies in Phytomedicines Department of Chemistry Federal University of Maranhão (UFMA) Campus Litoral Paulista São Paulo State University (UNESP) Laboratory of Mutagenesis Department of Biological Sciences Faculty of Pharmaceutical Sciences São Paulo State University (UNESP) Campus Litoral Paulista São Paulo State University (UNESP) Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão: 00863/16

Made available in DSpace on 2018-12-11T17:03:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-12-01 Brazilian flora biodiversity has been widely investigated to identify effective and safe phytotherapeutic compounds. Among the investigated plant species, the Byrsonima genus exhibits promising biological activities. This study aimed at evaluating the cytotoxicity of B. correifolia, B. verbascifolia, B. fagifolia and B. intermedia extracts using different assays in two cell lines (primary gastric and HepG2 cells). The different extract concentrations effects on cell viability were assayed using the MTT, aquabluer, neutral red and LDH assays. Non-cytotoxic concentrations were selected to generate cell proliferation curves and to assess cell cycle kinetics by flow cytometry. Byrsonima extracts differentially affected cell viability depending on the metabolic cellular state and the biological parameter evaluated. B. fagifolia and B. intermedia extracts exhibited lower cytotoxic effects than B. correifolia and B. verbascifolia in all assays. The results obtained with LDH and flow cytometry assays were more reliable, suggesting that they can be useful in the screening for herbal medicine and to further characterize these extracts as phytotherapeutic compounds. Department of General Biology State University of Londrina, PR 445 Km 380, s/n - Campus Universitário Department of Biological Sciences Faculty of Pharmaceutical Sciences of Araraquara São Paulo State University “Júlio de Mesquita Filho” School of Arts Science and Humanities São Paulo University Institute of Biosciences UNESP- Paulista State University, Coastal Campus of São Vicente Instituto de Investigaciones Biológicas Clemente Estable Department of Biological Sciences Faculty of Pharmaceutical Sciences of Araraquara São Paulo State University “Júlio de Mesquita Filho” Institute of Biosciences UNESP- Paulista State University, Coastal Campus of São Vicente

Made available in DSpace on 2018-12-11T17:21:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-08-18 Pouteria ramiflora (Mart.) Radlk., popularly known as curriola, is commonly used in Brazil as medicinal plant to treat worm infections, dysentery, pain, inflammation, hyperlipidemia, and obesity. At present the safety of this extract when used therapeutically in human remains to be determined. Thus, the aim of this study was to examine cytotoxicity, antiproliferative, and antimutagenic actions of this extract. The hydroalcoholic extract from P. ramiflora leaves consisted of flavonoids identified and quantified as myricetin-3-O-β-D-galactopyranoside (13.55 mg/g) and myricetin-3-O-α-L-rhamnopyranoside (9.61 mg/g). The extract exhibited cytotoxicity at concentrations higher than 1.5 µg/ml in human hepatocarcinoma (HepG2)and 2.5 µg/ml in non-tumoral primary gastric (GAS) cells using the MTT assay, and at concentrations higher than 3 µg/ml in HepG2 and 3.5 µg/ml in GAS cells by the neutral red assay. The extract did not show antiproliferative effect as evidenced by the nuclear division index (NDI). However, in the presence of benzo[a]pyrene (BaP) (positive control), an enhanced cytostatic effect in the NDI and flow cytometry was noted. It is of interest that when the extract was co-incubated with BaP a significant decrease in DNA damage was observed indicating an antimutagenic action. This protective effect might be attributed to myricetin and gallic acid found in P. ramiflora extract. The low cytotoxicity action and protective effect observed in the present study encourage further studies regarding other biological effects of P. ramiflora, as well as its potential use as a chemopreventive agent. Departamento de Biologia Geral Centro de Ciências Biológicas Universidade Estadual de Londrina–UEL Instituto Federal de Educação Ciência e Tecnologia de Mato Grosso–IFMT Departamento de Química Orgânica Instituto de Química Universidade Estadual Paulista Júlio de Mesquita Filho–UNESP Departamento de Ciências Biológicas Faculdade de Ciências Farmacêuticas Universidade Estadual Paulista Júlio de Mesquita Filho–UNESP Campus Experimental do Litoral Paulista Universidade Estadual Paulista Júlio de Mesquita Filho–UNESP Instituto de Investigaciones Biológicas Clemente Estable–IIBCE. Montevideo Departamento de Química Orgânica Instituto de Química Universidade Estadual Paulista Júlio de Mesquita Filho–UNESP Departamento de Ciências Biológicas Faculdade de Ciências Farmacêuticas Universidade Estadual Paulista Júlio de Mesquita Filho–UNESP Campus Experimental do Litoral Paulista Universidade Estadual Paulista Júlio de Mesquita Filho–UNESP

Made available in DSpace on 2018-12-11T17:16:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-04-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Bauhinia holophylla (Bong.) Steud. (Fabaceae) is a plant used in Brazilian folk medicine to treat diabetes and inflammation. This study evaluated the phytochemical properties, cytotoxic, apoptotic, mutagenic/antimutagenic effects and alterations in gene expression (RNAm) in HepG2 cells treated with the B. holophylla extract. The phytochemical profile highlight the presence of flavonoids isorhamentin and quercetin derivates. The MTT assay was used to evaluate the cytotoxicity of different concentrations for different treatment times. Three concentrations (7.5, 15, 30 µg/mL) were chosen for assessment of apoptosis (AO/EB), mutagenicity (micronucleus), and cell cycle kinetics (flow cytometry). Thereafter, the concentration of 7.5 µg/mL was chosen to evaluate the protective effects against DNA damage induced by benzo[a]pyrene (B[a]P). At concentrations higher than 7.5 µg/mL (between 10 and 50 µg/mL), the extract was cytotoxic, induced apoptosis, and caused antiproliferative effects. However, it did not induce micronucleus and a reduction of apoptotic and micronucleated cells was observed in treatments that included the extract and B[a]P. The protective effect is attributable to the presence of flavonoids, described as antioxidants, inhibitors of DNA adduct and activators of detoxifying enzymes. The results of the present study such as absence of cytotoxic and mutagenic effects and protective effects against known carcinogens suggest that B. holophylla has potential for use soon as herbal medicine. Department of General Biology Biological Sciences Center State University of Londrina - UEL, Rod Celso Garcia Cid PR 445, Km 380, University Campus, P.O. Box 6001 Department of Biological Sciences Faculty of Pharmaceutical Sciences of Araraquara São Paulo State University - UNESP Campus Litoral Paulista São Paulo State University - UNESP Department of Biological Sciences Faculty of Sciences of Bauru São Paulo State University – UNESP Department of Botany Biosciences Institute São Paulo State University - UNESP Institute of Biological Investigation Clemente Estable Department of Biological Sciences Faculty of Pharmaceutical Sciences of Araraquara São Paulo State University - UNESP Campus Litoral Paulista São Paulo State University - UNESP Department of Biological Sciences Faculty of Sciences of Bauru São Paulo State University – UNESP Department of Botany Biosciences Institute São Paulo State University - UNESP FAPESP: 2009/16147-5 FAPESP: 2009/52237-9 FAPESP: 2012/01996-0

Candida parapsilosis is an increasingly important human pathogen. However, little is known about its potential to cause disease. The aims of the present study were to analyse the production of acid proteinases by clinical isolates of C. parapsilosis in the presence of different keratinous substrates from human sources (stratum corneum, nail and hair) and to verify the capability of yeast cells to adhere and grow as biofilm on these substrates. By scanning electron microscopy, it was observed that all C. parapsilosis sensu stricto isolates adhered to the keratinous substrates. For the isolate recovered from onychomycosis, the cell population attached to stratum corneum and hair keratin consisted mainly of blastoconidia. Differently, on nail keratin, pseudohyphae production was observed. Overall, there was a loose association between yeast cells and keratinous substrates. However, on stratum corneum, flocculent extracellular material was seen evolving cells from the onychomycosis isolate by forming a biofilm-like structure. The isolates recovered from onychomycosis and cutaneous lesion produced higher amount of acid proteinases in medium supplemented with nail keratin and stratum corneum keratin, respectively, than that in salt medium (absence of keratin). Furthermore, no differences were observed in the amount of acid proteinases produced by the isolate recovered from tracheal secretion in the media tested (absence and presence of keratin substrates). The information derived from this study will further our understanding of acid proteinase production by C. parapsilosis isolates and provide an insight into pathogenic mechanisms in C. parapsilosis particularly from isolates recovered from superficial mycoses.

Candida parapsilosis is found frequently as commensal organism on epithelial tissues, and is also an increasing cause of nosocomial infection. Scanning electron microscope (SEM) observations were used to analyse the capability of C. parapsilosis cells to adhere and grow as biofilm on human natural substrates and to compare the adherence pattern of isolates exhibiting distinct phenotypes. Cells from the crepe phenotype are predominantly elongated and form pseudohyphae whereas cells from the smooth phenotype are yeast-shaped, either in liquid cultures or on human nail and hair surfaces. The electron micrographs revealed that C. parapsilosis cells from the smooth phenotype adhered in higher number to both surfaces compared to the observed for the crepe phenotype. SEM analysis of human hair surface revealed that cells from the smooth phenotype appear as clumped blastoconidia of uniform morphology embedded in a flocculent extracellular material forming biofilm. The extracellular material and biofilm were seeing in a less extension in the crepe phenotype. A distinct adherence pattern was observed when human nail was used as substrate. Here C. parapsilosis cells seem to be linked to surface structures of human nail plate. Fibrillar extracellular material was observed connecting neighbouring cells as well as nail surface.

Made available in DSpace on 2018-12-11T16:59:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-12-24 Ethnopharmacological relevance The Brazilian Cerrado is an important source of natural products, such as Myrcia bella Cambess (MB, also known as mercurinho). MB leaves are popularly used for the treatment of diabetes and gastrointestinal disorders; however, only its hypoglycemic activity has been experimentally described. Aim of the study Because MB is used to treat gastrointestinal disorders, the present study characterized biological activities of hydroalcoholic MB extract in human normal and tumor gastric cells. Materials and methods Cytotoxic, antiproliferative, genotoxic and protective effects were evaluated, as well as the effects of the MB extract on gene expression. Results The MB extract induced cytotoxicity in tumor cells at lower concentrations compared with normal cells as assessed by the MTT assay. Moreover, the MB extract induced necrosis based on acridine orange/ethidium bromide staining. An antiproliferative effect was evidenced through an arrest in the G2/M phase detected by flow cytometry and a decrease in the nuclear division index using the cytokinesis-block micronucleus cytome assay. Cells treated with MB extract combined with doxorubicin (DXR) showed increased NUBDs, which may be related to the gene amplification of CCND1. Antimutagenic effects were also observed and may be associated with the antioxidant activities detected using the CM-H2DCFDA probe. Conclusions Our findings showed the following: (a) high concentrations of MB induced cytotoxicity and cell death by necrosis; (b) its antiproliferative effect was associated with G2/M arrest; and (c) its antioxidant activity could be responsible for the observed antimutagenic effects and for protective effects against gastrointestinal disorders previously described to MB. Although these effects are not specific to normal or tumor cells, they provide a panel of biological activities for further exploration. Department of Biological Sciences Faculty of Pharmaceutical Sciences São Paulo State University (UNESP) Department of General Biology Center of Biological Sciences State University of Londrina (UEL) Experimental Campus of São Vicente São Paulo State University (UNESP) Instituto de Investigaciones Biológicas Clemente Estable Department of Biological Sciences Faculty of Sciences São Paulo State University (UNESP) Department of Botany Institute of Bioscience São Paulo State University (UNESP) Department of Biological Sciences Faculty of Pharmaceutical Sciences São Paulo State University (UNESP) Experimental Campus of São Vicente São Paulo State University (UNESP) Department of Biological Sciences Faculty of Sciences São Paulo State University (UNESP) Department of Botany Institute of Bioscience São Paulo State University (UNESP)

Immunosuppressive drugs are used to suppress immune system activity in transplant patients and reduce the risk of organ rejection. The present study evaluated the potential cytotoxic, genotoxic and mutagenic of the immunosuppressive drugs cyclosporine (CsA) and tacrolimus (FK-506) on normal human fibroblasts ( MRC-5 cells). Based on plasma concentrations of the immunosuppressive drugs, which were obtained from the records of kidney transplant patients at the Kidney Institute of Londrina, Brazil, 11 concentrations of each immunosuppressive were chosen to evaluate cell viability using the MTT assay. From these results, CsA and FK-506 concentrations of 135, 300, 675, and 1520 ng/ml and 8, 16, 24, and 32 ng/ml, respectively, were evaluated using (i) the comet assay, (ii) the nuclear division index (NDI), (iii) the micronucleus test (CBMN) and (iv) cell proliferation curves generated by quantifying cell numbers and protein levels. In this study, 1520 to 3420 ng/ml CsA decreased cell viability after 48 h of exposure. Genotoxic effects were observed only with a concentration of 1520 ng/ml after 3 h of exposure and with concentrations of 675 and 1520 ng/ml after 24 h of exposure. Mutagenic effects were observed only for the concentration of 1520 ng/ml. FK-506 decreased cell viability after 72 h of exposure for concentrations up to 20 ng/ml; genotoxic effects were observed with concentrations up to 8 ng/ml for both treatment times (3 and 24 h) and mutagenic effects were observed with concentrations of 24 and 32 ng/ml after 24 h of treatment. The cell proliferation curves demonstrated the absence of cytostatic effects of these drugs, and these data were confirmed by the NDI analysis. Our results suggest that concentrations lower than 300 ng/ml of CsA and 16 ng/ml of FK-506 are safe for use, as they did not induce genotoxic and mutagenic damage or affect MRC-5 cell viability and proliferation.

A produção de enzimas hidrolíticas por espécies do gênero Candida é de fundamental importância, uma vez que são responsáveis pela aquisição de nutrientes, adaptação e invasão a vários tecidos do hospedeiro. Devido a escassez de estudos relativos a produção de proteases por isolados clínicos de Candida parapsilosis, neste trabalho foi avaliado a cinética de crescimento, produção de proteases ácidas, produção de proteases queratinolíticas e análise da interação de isolados clínicos de C. parapsilosis (lesão ungueal e de pele e secreção traqueal) em presença de substratos queratínicos. Os isolados foram cultivados por 10 dias em Meio de Sais (MS) e MS acrescido de cabelo, pele e unha (pH 5,0 e 8,0). Os isolados apresentaram cinéticas de crescimento similares tanto em pH 5,0 quanto no 8,0, no entanto, no pH 5,0 pode ser observado diferenças entre as taxas de crescimento nos diferentes cultivos (MSP>MSU≥MSC>MS). Para detecção da atividade proteolítica foram utilizados os sobrenadantes de cultivo provenientes dos cultivos em pH 5,0 e no ensaio enzimático foi utilizado hemoglobina e BSA. Já para análise da atividade queratinolítica foram utilizados os sobrenadantes provenientes dos cultivos em pH 5,0 e 8,0 e os substratos para os ensaios foram queratina azure e estrato córneo. Os resultados mostraram, pela primeira vez na literatura, que todos os isolados produziram proteases ácidas e proteases queratinolíticas quando cultivados nos diferentes meios de cultivo. A suplementação dos meios de cultivo com substratos queratínicos induziu tanto a produção de proteases ácidas quanto de proteases queratinolíticas pelos isolados de lesão de pele e unha (P0,05). As eletromicrografias revelaram que todos os isolados apresentaram interação celular aos substratos queratínicos. Os resultados obtidos indicam que os isolados de C. parapsilosis de lesão de pele e unha, em presença de substratos queratínicos, são induzidos de forma diferenciada a expressarem proteases ácidas e queratinolíticas e, que tecidos queratinizados podem ser colonizados por isolados de C. parapsilosis. The production of hydrolytic enzymes by Candida species is of fundamental importance, since they are responsible for nutrient acquisition, adaptation and invasion of various host tissues. Because of the paucity of studies on protease production by clinical isolates of Candida parapsilosis, this study evaluated the kinetics of growth, production of acid proteases, production of keratinolytic protease and analysis the interaction of clinical isolates of C. parapsilosis (nail and skin lesions and tracheal secretion) in presence of keratin substrates. The isolates were cultured for 10 days in medium salts (MS) and MS supplemented with hair, skin and nail (pH 5.0 and 8.0). The isolates showed similar growth kinetics in both pH 5.0 and in 8.0, however, at pH 5.0 can be observed differences in growth rates in different cultures (MSP> MSU ≥ MSC> MS). For detection of proteolytic activity were used for culture supernatants from the cultures at pH 5.0 and in enzyme assay was used hemoglobin and BSA. As for keratinolytic activity analysis were used supernatants from the cultures at pH 5.0 and 8.0 and substrates for the assays were keratin azure and stratum corneum. The results showed for the first time in literature, that all isolates produced acid proteases and keratinolytic proteases when grown in different culture media. Supplementation of media with keratin substrates induced the production of acid proteases and keratinolytic proteases for isolates from skin and nail lesions (P> 0.05). In the case of keratinolytic proteases of cultivation pH and substrates used in enzymatic assays also affected the activities of isolated lesions (P> 0.05). In the case of keratinolytic proteases of cultivation pH and substrates used in enzymatic assays also affected the activities of isolated lesions, and the isolated lesion of nail was significantly more protease producer than the other strains tested (P 0.05). The electron micrographs revealed that all isolates showed cell interaction with substrates keratin. The results indicate that the isolates of C. parapsilosis skin lesions and nail, in presence of substrates keratin, are induced to differentially express acidic and keratinolytic protease, and keratinized tissues can be colonized by isolates of C. parapsilosis.

Identification of Candida isolates obtained from oral cavity of elderly healthy individuals revealed the predominance of non-albicans Candida species (88.9%) compared to Candida albicans (11%). CHROMagar Candida differential medium and PCR revealed the presence of Candida tropicalis (33.3%), Candida glabrata (27.8%), and Candida krusei (16.7%). We investigated the presence of virulence attributes in a total of 18 isolates, including acid protease and phospholipase production, hemolytic activity, and biofilm production. Extracellular protease was found in five isolates (27.8%) whereas extracellular phospholipase was found in three isolates (17%). All isolates showed hemolytic activity. About 56% of the isolates were weakly positive for biofilm formation (score +) whereas a minority (5.6%) of them showed strong biofilm formation (score 4+). Susceptibility in vitro of the isolates to fluconazole was carried out by microdilution method. Fluconazole showed a strong inhibition against most buccal isolates. The resistant isolates were 2 C. tropicalis, 2 C. glabrata, and 1 C. krusei.