Análise da estabilidade genética de células-tronco mesenquimais humanas

Human multipotent mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have become an important and attractive therapeutic tool since they are easily isolated and cultured, have in vitro expansion potential, substantial plasticity and secrete bioactive molecules that exert trophic effects. The human umbilical cord as a cell source for cell therapy will help to avoid several ethical, political, religious and technical issues. One of the main issues with SC lines from different sources, mainly those of embryonic origin, is the possibility of chromosomal alterations and genomic instability during in vitro expansion. Cells isolated from one umbilical cord exhibited a rare balanced paracentric inversion, likely a cytogenetic constitutional alteration, karyotype: 46,XY,inv(3)(p13p25~26). Important genes related to cancer predisposition and others involved in DNA repair are located in 3p25~26. Titanium is an excellent biomaterial for bone-implant integration; however, the use can result in the generation of particulate debris that can accumulate in the tissues adjacent to the prosthesis, in the local bone marrow, in the lymph nodes, liver and spleen. Subsequently may elicit important biological responses that aren´t well studied. In this work, we have studied the genetic stability of MSC isolated from the umbilical cord vein during in vitro expansion, after the cryopreservation, and under different concentrations and time of exposition to titanium microparticles. Cells were isolated, in vitro expanded, demonstrated capacity for osteogenic, adipogenic and chondrogenic differentiation and were evaluated using flow cytometry, so they met the minimum requirements for characterization as MSCs. The cells were expanded under different concentrations and time of exposition to titanium microparticles. The genetic stability of MSCs was assessed by cytogenetic analysis, fluorescence in situ hybridization (FISH) and analysis of micronucleus and other nuclear alterations (CBMN). The cells were able to internalize the titanium microparticles, but MSCs preserve their morphology, differentiation capacity and surface marker expression profiles. Furthermore, there was an increase in the genomic instability after long time of in vitro expansion, and this instability was greater when cells were exposed to high doses of titanium microparticles that induced oxidative stress. It is necessary always assess the risks/ benefits of using titanium in tissue therapy involving MSCs, considering the biosafety of the use of bone regeneration using titanium and MSCs. Even without using titanium, it is important that the therapeutic use of such cells is based on analyzes that ensure quality, security and cellular stability, with the standardization of quality control programs appropriate. In conclusion, it is suggested that cytogenetic analysis, FISH analysis and the micronucleus and other nuclear alterations are carried out in CTMH before implanting in a patient

Transcriptoma diferencial entre células-tronco mesenquimais humanas jovens e senescentes

Human mesenchymal stem cells (MSC) are powerful sources for cell therapy in regenerative medicine. The long time cultivation can result in replicative senescence or can be related to the emergence of chromosomal alterations responsible for the acquisition of tumorigenesis features in vitro. In this study, for the first time, the expression profile of MSC with a paracentric chromosomal inversion (MSC/inv) was compared to normal karyotype (MSC/n) in early and late passages. Furthermore, we compared the transcriptome of each MSC in early passages with late passages. MSC used in this study were obtained from the umbilical vein of three donors, two MSC/n and one MSC/inv. After their cryopreservation, they have been expanded in vitro until reached senescence. Total RNA was extracted using the RNeasy mini kit (Qiagen) and marked with the GeneChip ® 3 IVT Express Kit (Affymetrix Inc.). Subsequently, the fragmented aRNA was hybridized on the microarranjo Affymetrix Human Genome U133 Plus 2.0 arrays (Affymetrix Inc.). The statistical analysis of differential gene expression was performed between groups MSC by the Partek Genomic Suite software, version 6.4 (Partek Inc.). Was considered statistically significant differences in expression to p-value Bonferroni correction ˂.01. Only signals with fold change ˃ 3.0 were included in the list of differentially expressed. Differences in gene expression data obtained from microarrays were confirmed by Real Time RT-PCR. For the interpretation of biological expression data were used: IPA (Ingenuity Systems) for analysis enrichment functions, the STRING 9.0 for construction of network interactions; Cytoscape 2.8 to the network visualization and analysis bottlenecks with the aid of the GraphPad Prism 5.0 software. BiNGO Cytoscape pluggin was used to access overrepresentation of Gene Ontology categories in Biological Networks. The comparison between senescent and young at each group of MSC has shown that there is a difference in the expression parttern, being higher in the senescent MSC/inv group. The results also showed difference in expression profiles between the MSC/inv versus MSC/n, being greater when they are senescent. New networks were identified for genes related to the response of two of MSC over cultivation time. Were also identified genes that can coordinate functional categories over represented at networks, such as CXCL12, SFRP1, xvi EGF, SPP1, MMP1 e THBS1. The biological interpretation of these data suggests that the population of MSC/inv has different constitutional characteristics, related to their potential for differentiation, proliferation and response to stimuli, responsible for a distinct process of replicative senescence in MSC/inv compared to MSC/n. The genes identified in this study are candidates for biomarkers of cellular senescence in MSC, but their functional relevance in this process should be evaluated in additional in vitro and/or in vivo assays

Análise da estabilidade genética de células-tronco mesenquimais humanas

Human multipotent mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have become an important and attractive therapeutic tool since they are easily isolated and cultured, have in vitro expansion potential, substantial plasticity and secrete bioactive molecules that exert trophic effects. The human umbilical cord as a cell source for cell therapy will help to avoid several ethical, political, religious and technical issues. One of the main issues with SC lines from different sources, mainly those of embryonic origin, is the possibility of chromosomal alterations and genomic instability during in vitro expansion. Cells isolated from one umbilical cord exhibited a rare balanced paracentric inversion, likely a cytogenetic constitutional alteration, karyotype: 46,XY,inv(3)(p13p25~26). Important genes related to cancer predisposition and others involved in DNA repair are located in 3p25~26. Titanium is an excellent biomaterial for bone-implant integration; however, the use can result in the generation of particulate debris that can accumulate in the tissues adjacent to the prosthesis, in the local bone marrow, in the lymph nodes, liver and spleen. Subsequently may elicit important biological responses that aren´t well studied. In this work, we have studied the genetic stability of MSC isolated from the umbilical cord vein during in vitro expansion, after the cryopreservation, and under different concentrations and time of exposition to titanium microparticles. Cells were isolated, in vitro expanded, demonstrated capacity for osteogenic, adipogenic and chondrogenic differentiation and were evaluated using flow cytometry, so they met the minimum requirements for characterization as MSCs. The cells were expanded under different concentrations and time of exposition to titanium microparticles. The genetic stability of MSCs was assessed by cytogenetic analysis, fluorescence in situ hybridization (FISH) and analysis of micronucleus and other nuclear alterations (CBMN). The cells were able to internalize the titanium microparticles, but MSCs preserve their morphology, differentiation capacity and surface marker expression profiles. Furthermore, there was an increase in the genomic instability after long time of in vitro expansion, and this instability was greater when cells were exposed to high doses of titanium microparticles that induced oxidative stress. It is necessary always assess the risks/ benefits of using titanium in tissue therapy involving MSCs, considering the biosafety of the use of bone regeneration using titanium and MSCs. Even without using titanium, it is important that the therapeutic use of such cells is based on analyzes that ensure quality, security and cellular stability, with the standardization of quality control programs appropriate. In conclusion, it is suggested that cytogenetic analysis, FISH analysis and the micronucleus and other nuclear alterations are carried out in CTMH before implanting in a patient

Transcriptoma diferencial entre células-tronco mesenquimais humanas jovens e senescentes

Human mesenchymal stem cells (MSC) are powerful sources for cell therapy in regenerative medicine. The long time cultivation can result in replicative senescence or can be related to the emergence of chromosomal alterations responsible for the acquisition of tumorigenesis features in vitro. In this study, for the first time, the expression profile of MSC with a paracentric chromosomal inversion (MSC/inv) was compared to normal karyotype (MSC/n) in early and late passages. Furthermore, we compared the transcriptome of each MSC in early passages with late passages. MSC used in this study were obtained from the umbilical vein of three donors, two MSC/n and one MSC/inv. After their cryopreservation, they have been expanded in vitro until reached senescence. Total RNA was extracted using the RNeasy mini kit (Qiagen) and marked with the GeneChip ® 3 IVT Express Kit (Affymetrix Inc.). Subsequently, the fragmented aRNA was hybridized on the microarranjo Affymetrix Human Genome U133 Plus 2.0 arrays (Affymetrix Inc.). The statistical analysis of differential gene expression was performed between groups MSC by the Partek Genomic Suite software, version 6.4 (Partek Inc.). Was considered statistically significant differences in expression to p-value Bonferroni correction ˂.01. Only signals with fold change ˃ 3.0 were included in the list of differentially expressed. Differences in gene expression data obtained from microarrays were confirmed by Real Time RT-PCR. For the interpretation of biological expression data were used: IPA (Ingenuity Systems) for analysis enrichment functions, the STRING 9.0 for construction of network interactions; Cytoscape 2.8 to the network visualization and analysis bottlenecks with the aid of the GraphPad Prism 5.0 software. BiNGO Cytoscape pluggin was used to access overrepresentation of Gene Ontology categories in Biological Networks. The comparison between senescent and young at each group of MSC has shown that there is a difference in the expression parttern, being higher in the senescent MSC/inv group. The results also showed difference in expression profiles between the MSC/inv versus MSC/n, being greater when they are senescent. New networks were identified for genes related to the response of two of MSC over cultivation time. Were also identified genes that can coordinate functional categories over represented at networks, such as CXCL12, SFRP1, xvi EGF, SPP1, MMP1 e THBS1. The biological interpretation of these data suggests that the population of MSC/inv has different constitutional characteristics, related to their potential for differentiation, proliferation and response to stimuli, responsible for a distinct process of replicative senescence in MSC/inv compared to MSC/n. The genes identified in this study are candidates for biomarkers of cellular senescence in MSC, but their functional relevance in this process should be evaluated in additional in vitro and/or in vivo assays

Desenvolvimento de nanosistemas farmacêuticos para terapia gênica

Gene therapy is one of the major challenges of the post-genomic research and it is based on the transfer of genetic material into a cell, tissue or organ in order to cure or improve the patient s clinical status. In general, gene therapy consists in the insertion of functional genes aiming substitute, complement or inhibit defective genes. The achievement of a foreigner DNA expression into a population of cells requires its transfer to the target. Therefore, a key issue is to create systems, vectors, able to transfer and protect the DNA until it reaches the target. The disadvantages related to the use of viral vectors have encouraged efforts to develop emulsions as non-viral vectors. In fact, they are easy to produce, present suitable stability and enable transfection. The aim of this work was to evaluate two different non-viral vectors, cationic liposomes and nanoemulsions, and the possibility of their use in gene therapy. For the two systems, cationic lipids and helper lipids were used. Nanoemulsions were prepared using sonication method and were composed of Captex® 355; Tween® 80; Spam® 80; cationic lipid, Stearylamine (SA) or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and water (Milli-Q®). These systems were characterized by average droplet size, Polidispersion Index (PI) and Zeta Potential. The stability of the systems; as well as the DNA compaction capacity; their cytotoxicity and the cytotoxicity of the isolated components; and their transfection capacity; were also evaluated. Liposomes were made by hydration film method and were composed of DOTAP; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), containing or not Rhodaminephosphatidylethanolamine (PE- Rhodamine) and the conjugate Hyaluronic Acid DOPE (HA-DOPE). These systems were also characterized as nanoemulsions. Stability of the systems and the influence of time, size of plasmid and presence or absence of endotoxin in the formation of lipoplexes were also analyzed. Besides, the ophthalmic biodistribution of PE-Rhodamine containing liposomes was studied after intravitreal injection. The obtained results show that these systems are promising non-viral vector for further utilization in gene therapy and that this field seems to be very important in the clinical practice in this century. However, from the possibility to the practice, there is still a long way

Atividade antibacteriana das plantas copaifera duckei dwyer e pseudobombax marginatum e sua interação com a superfície celular bacteriana

A resistência microbiana a antimicrobianos tem favorecido a busca por substâncias bioativas provenientes de plantas usadas na medicina popular, com o intuito de se obter novos fármacos com atividade antimicrobiana. Neste estudo, foi proposta a investigação da atividade antibacteriana do óleo-resina de Copaifera duckei e de diferentes extratos da casca de Pseudobombax marginatum, e seus possíveis mecanismos de ação. O potencial inibitório antibacteriano foi avaliado utilizando-se os métodos de difusão e diluição em ágar, e a bioautografia. O mecanismo de ação foi analisado por microscopia eletrônica, no qual se observou alterações na ultraestrutura bacteriana, e por eletroforese em SDS-PAGE, que determinou ação sobre as proteínas das superfícies celulares. A análise química foi realizada pelas técnicas de Espectrometria de massas acoplada ao Cromatógrafo a gás- EM/CG (C. duckei) e Cromatografia Líquida de Alta Eficiência- CLAE (P. marginatum). Entre as bactérias estudadas, B. cereus foi a mais suscetível às plantas em estudo, com concentrações inibitórias mínimas (CIMs) correspondentes a 0,3125 mg/mL para o óleo-resina de copaíba, e 0,5 mg/mL para extrato hidroalcoólico (1:1) e 0,512 mg/mL para a fração butanólica da casca P. marginatum, nos quais pôde-se observar alterações na parede celular do B. cereus, com remoção da camada S, espessamento da parede celular e formação de diversos septos nos centros de divisão celular. A análise química por EM/CG mostrou compostos terpênicos no óleo-resina de C. duckei, tendo como composto majoritário o β-bisaboleno, e a análise por CLAE mostrou a presença de compostos derivados da catequina na casca do P. marginatum. Desta forma, as plantas em estudo mostram um potencial antibacteriano considerável, podendo contribuir tanto na terapia antimicrobiana como na área de alimentos, tendo como um de seus prováveis sítios de ação a parede celular bacteriana

Avaliação por ressonância magnética funcional e estimulação magnética transcraniana da intervenção única da terapia espelho em pacientes após acidente vascular cerebral isquêmico

Mirror therapy (MT) is being used as a rehabilitation tool in various diseases, including stroke. Although some studies have shown its effectiveness, little is known about neural mechanisms that underlie the rehabilitation process. Therefore, this study aimed at assessing cortical neuromodulation after a single MT intervention in ischemic stroke survivors, by means of by functional Magnetic Resonance Imaging (fMRI) and Transcranial Magnetic Stimulation (TMS). Fifteen patients participated in a single thirty minutes MT session. fMRI data was analyzed bilaterally in the following Regions of Interest (ROI): Supplementary Motor Area (SMA), Premotor cortex (PMC), Primary Motor cortex (M1), Primary Sensory cortex (S1) and Cerebellum. In each ROI, changes in the percentage of occupation and beta values were computed. Group fMRI data showed a significant decreased in the percentage of occupation in PMC and cerebellum, contralateral to the affected hand (p <0.05). Significant increase in beta values was observed in the following contralateral motor areas: SMA, Cerebellum, PMC and M1 (p<0,005). Moreover, a significant decrease was observed in the following ipsilateral motor areas: PMC and M1 (p <0,001). In S1 a bilateral significant decrease (p<0.0005) was observed.TMS consisted of the analysis of Motor Evoked Potential (MEP) of M1 hotspot. A significant increase in the amplitude of the MEP was observed after therapy in the group (p<0,0001) and individually in 4 patients (p <0.05). Altogether, our results imply that single MT intervention is already capable of promoting changes in neurobiological markers toward patterns observed in healthy subjects. Furthermore, the contralateral hemisphere motor areas changes are opposite to the ones in the ipsilateral side, suggesting an increase system homeostasis.

Efeito in vitro da terapia fotodinâmica sobre bactérias orais em suspensão e na formação de biofilme : ensaio com azul de metileno e toluidina ativados por luz halógena

Photodynamic therapy (PDT) has been proposed as an alternative method for the treatment of biofilm-dependent oral diseases like dental caries. This therapy consists of simultaneous action of a visible light (L) and a photosensitizer (FS) in the presence of oxygen, which leads to production of different reactive oxygen species that can interact with the bacterial cell components, and promote cell death. This study aims to evaluate the antimicrobial action of PDT on oral bacteria in suspension, as well as the formation of mono and multi-species biofilms, in vitro, from a standard strain of Streptococcus mutans (ATCC 25175) and saliva samples, respectively. The dye methylene blue (MB) and toluidine blue (TB) were used at a concentration of 100 mg/ L and activated by halogen light (600 to 750 nm) from a modified hand held photopolymerizer (Ultralux ®, Dabi Atlante, Ribeirão Preto , São Paulo, Brazil.). Planktonic cultures were prepared and submitted to different experimental conditions: 1. PDT using TB 2. PDT using MB, 3. L+ FS- , 4. TB + L - ; 5. MB+ L-; 6. L- FS- (negative control) and 7. administration of 0.12% chlorhexidine digluconate (positive control) (Periogard ®, Colgate-Palmolive Company, New York, USA). The immediate and mediated action of PDT on bacterial suspensions, as well as its effect on biofilm formation were observed from the number of colony-forming units per milliliter (CFU/mL) and measures optical density (OD). The data were statistically analyzed using the Kruskal-Wallis and Mann-Whitney test for the significance level of 5%. According to the results, the PDT showed no antibacterial action on suspensions of S. mutans, regardless of the dye used. PDT with MB activated by halogen light was able to reduce 86.6% CFU/mL multi-species planktonic cultures, however, this reduction was not significant (p > 0.05). PDT showed antibacterial effect, mediate on multi-species planktonic cultures with TB (p < 0.001) and MB (p < 0.001), activated by halogen light. PDT was able to prevent the formation of multispecies biofilm, through the activation of TB by halogen light (p = 0.01). We conclude that activation of the dye toluidine blue and methylene blue, by halogen light (PDT) showed antimicrobial activity, compared to multi-species planktonic cultures prepared from saliva samples

Análise imunoistoquímica após terapia fotodinâmica com cloro-alumínio ftalocianina em tecidos gengivais de humanos

Although photodynamic therapy have been used as a useful tool over the past 30 years in oncology, few clinical trials have been conducted in dentistry. Photodynamic therapy (PDT) uses non - toxic photosensitizers and selective which are administered in target cells followed by local application of visible light, producing reactive oxygen species capable of causing cell death by apoptosis or necrosis, injured the local vasculature, and exert important effects on the im mune system. New generations of photosensitizing agents, such as nanoparticulate phthalocyanines, has shown excellent results in antitumor and antibacterial activity . In this context, the present work constitutes the first clinical protocol of local appli cation of nanoemulsion chloro - aluminum phthalocyanine (AlClFc) followed by irradiation in human gingiva, and analyzed descriptively and comparatively , by means of immunohistochemistry , the expression of RANK , RANKL , OPG and VEGF in a split - mouth model . Eight healthy volunteers with clinical indication for extraction were included in the study . Seven days before the extraction, was injected in the gingiva of participants, 5 μ M of nanoemulsion AlClFc followed by irra diation with diode laser (660nm , 7 J/cm2 ), the contralateral side was used as control. Tissue specimens were removed seven days after the TFD is performed. Tissues sample were divided into two groups (test and con trol groups) for histological and immunohistochemical analysis. Patients were monitored at days, 0, 7, 14 and 30 to assess adverse effects of the therapy. Vascular alterations were seen in gingival samples that received PDT. Areas of edema and vascular con gestion, and intense vascularization were viewed . Additionally, dystrophic calcification in subepithelial region were observed in the test group. The results showed a similar pattern of immunostaining scores of RANK, RANKL and VEGF between the test and co ntrol groups, with no statistically significant difference (p = 0.317, p = 0.777, p = 0 .814, respectively). RANK and RANKL exhibited weak or absent immunostaining in most specimens analyzed. There was n o immunostaining for OPG. VEGF showed moderate to stro ng immunostaining in specimens from the test group. In addition, the clinical study showed that therapy was well tolerated by all patients. Adverse effects were short - time and completely reversible. Taken together, the results presented in this study showe d that PDT mediated by nanoemulsion containing AlClPc is safe for clinical application in gingival tissue and suggests that a strong immunostaining for VEGF after therapy .

Desenvolvimento de nanosistemas farmacêuticos para terapia gênica

Gene therapy is one of the major challenges of the post-genomic research and it is based on the transfer of genetic material into a cell, tissue or organ in order to cure or improve the patient s clinical status. In general, gene therapy consists in the insertion of functional genes aiming substitute, complement or inhibit defective genes. The achievement of a foreigner DNA expression into a population of cells requires its transfer to the target. Therefore, a key issue is to create systems, vectors, able to transfer and protect the DNA until it reaches the target. The disadvantages related to the use of viral vectors have encouraged efforts to develop emulsions as non-viral vectors. In fact, they are easy to produce, present suitable stability and enable transfection. The aim of this work was to evaluate two different non-viral vectors, cationic liposomes and nanoemulsions, and the possibility of their use in gene therapy. For the two systems, cationic lipids and helper lipids were used. Nanoemulsions were prepared using sonication method and were composed of Captex® 355; Tween® 80; Spam® 80; cationic lipid, Stearylamine (SA) or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and water (Milli-Q®). These systems were characterized by average droplet size, Polidispersion Index (PI) and Zeta Potential. The stability of the systems; as well as the DNA compaction capacity; their cytotoxicity and the cytotoxicity of the isolated components; and their transfection capacity; were also evaluated. Liposomes were made by hydration film method and were composed of DOTAP; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), containing or not Rhodaminephosphatidylethanolamine (PE- Rhodamine) and the conjugate Hyaluronic Acid DOPE (HA-DOPE). These systems were also characterized as nanoemulsions. Stability of the systems and the influence of time, size of plasmid and presence or absence of endotoxin in the formation of lipoplexes were also analyzed. Besides, the ophthalmic biodistribution of PE-Rhodamine containing liposomes was studied after intravitreal injection. The obtained results show that these systems are promising non-viral vector for further utilization in gene therapy and that this field seems to be very important in the clinical practice in this century. However, from the possibility to the practice, there is still a long way

Fenômeno vivido por familiares de pacientes internados em unidade de terapia intensiva

COMASSETTO, Isabel, ENDERS, Bertha Cruz. Fenômeno vivido por familiares de pacientes internados em Unidade de Terapia Intensiva. Revista Gaúcha de Enfermagem., Porto Alegre(RS), v.30,n., p.46-53. Mar. 2009. Disponivel em: < http://www.seer.ufrgs.br/index.php/RevistaGauchadeEnfermagem/search/results>.

Atividades biológicas de polissacarídeos sulfatados extraídos da alga vermelha gracilaria caudata

In recent years, sulfated polysaccharides (SP) from marine algae have emerged as an important class of natural biopolymers with potential pharmacology applications. Among these, SP isolated from the cell walls of red algae have been study due to their anticoagulant,antithrombotic and anti-inflammatory activities. In the present study, three sulfated polysaccharides fractions denominated F1.5v, F2.0v and F3.0v were obtained from seaweed G. caudate by proteolysis followed to acetone fractionation. Gel electrophoresis using 0.05 M 1,3-diaminopropane-acetate buffer, pH 9,0, stained with 0.1% toluidine blue, showed the presence of SP in all fractions. The chemical analysis demonstrated that all the fractions are composed mainly of galactose. These compounds were evaluated in anticoagulant, antioxidant and antiproliferative activities. In anticoagulant activity evaluated through aPTT and PT tests, no one fractions presented anticoagulant activity at tested concentrations (0.1 mg/mL; 1.0 mg/mL; 2.0 mg/mL).The antioxidant activities of the three fractions were evaluated by the following in vitro systems: Total antioxidant capacity, superoxide and hydroxyl radical scavenging, ferrous chelating activity and reducing power. The fractions were found to have different levels of antioxidant activity in the systems tested. F1.5v shows the highest activity, especially in the ferrous chelating system, with 70% of ferrous inhibiting at 1.0 mg.mL-1. Finally, all the fractions showed dose-dependent antiproliferative activity against HeLa cells. The fractions F1.5v and F2.0v presented the highest antiproliferative activity at 2.0 mg/mL with 42.7% and 37.0% of inhibition, respectively. Ours results suggests that the sulfated polysaccharides from seaweed G. caudata are promising compounds in antioxidant and/or antitumor therapy

Purificação, caracterização e avaliação da atividade antiproliferativa de um inibidor de quimotripsina tipo kunitz de semente de Eryhrina velutina

A chymotrypsin inhibitor was purified from Erythrina velutina seeds by ammonium sulphate fractionation, affinities chromatographies on Trypsin-Sepharose, Quimotrypsin-Sepharose and reversed phase C-18 FPLC/AKTA system. The inhibitor, named EvCI, shown molecular mass of 17 kDa, as determined by SDSPAGE. 2D-PAGE showed four isoinhibitors with pI values of 4,42, 4,63, 4,83 and 5,06, with molecular mass of 17 kDa each. The aminoacid sequence of EvCI was determined by MALDI-TOF-MS and showed a high similarity with other Kunitz-type inhibitor of Erythrina variegata. EvCI competitively inhibited chymotrypsin, with Ki of 4 x10-8 M, but did not inhibited trypsin, pancreatic elastase, bromelain and papain. The inhibitory activity of EvCI was stable over wide pH and temperature ranges. In the presence of DTT 100 mM for 120 min, EvCI lost 50 % of activity. Cytotoxicity was studied in HeLa, MDA, HepG2, K562 and PC3 cells after 72-h incubation period. EvCl inhibited HeLa cells growth with an IC50 value of 50 μg/ml. Subsequent studies in HeLa cells analysis of cell death by annexin V/PI double-staining and cell cycle, using flow cytometry. The results provide evidence for a cytostatic activity of EvCl and support further studies on potential application of this inhibitors as an antiproliferative agent in combined therapy against cervical cancer

Bioprospecção de polissacarídeos sulfatados de macroalgas marinhas do litoral do Rio Grande do Norte: caracterização de uma heterofucana extraída da alga marron sargassum filipendula que induz apoptose em células hela

The coast of Rio Grande do Norte has more than 100 species of seaweed, mostly unexplored regarding their pharmacological potential. The sulfated polysaccharides (PS) are by far the more seaweed compounds studied, these present a range of biological properties, such as anticoagulant activity, anti-inflammatory, antitumor and antioxidant properties. In this study, we extract sulfated polysaccharide rich-extracts of eleven algae from the coast of Rio Grande do Norte (Dictyota cervicornis; Dictiopterys delicatula; Dictyota menstruallis; Dictyota mertensis; Sargassum filipendula; Spatoglossum schröederi; Gracilaria caudata; Caulerpa cupresoides; Caulerpa prolifera; Caulerpa sertularioides e Codim isthmocladum), and these were evaluated for the potential anticoagulant, antioxidant and antiproliferative. All polysaccharide extracts showed activity for anticoagulant, antioxidant and/or antiproliferative activity, especially D. delicatula and S. filipendula, which showed the most prominent pharmacological potential, thereby being chosen to have their sulfated polysaccharides extracted. By fractionating method were obtained six fractions rich in sulfated polysaccharides to the algae D. delicatula (DD-0,5V, DD-0, 7V, DD-1,0v, DD-1,3v, DD-1,5v and DD-2,0) and five fractions to the alga S. filipendula (SF-0,5V, SF-0,7V, SF-1,0v, SF-1,5v and SF-2,0v). For the anticoagulant assay only the fractions of D. delicatula showed activity, with emphasis on DD-1, 5v that presented the most prominent activity, with APTT ratio similar to clexane® at 0.1 mg/mL. When evaluated the antioxidant potential, all fractions showed potential in all tests (total antioxidant capacity, hydroxyl and superoxide radicals scavenging, ferrous chelation and reducing power), however, the ability to chelate iron ions appears as the main mechanism antioxidant of sulfated polysaccharides from seaweed. In antiproliferative assay, all heterofucanas showed dose-dependent activity for the inhibition of cell proliferation of HeLa, however, with the exception of SF-0,7V, SF- 1,0v and SF-1,5v, all fractions showed antiproliferative activity against MC3T3, a normal cell line. The heterofucana SF-1,5V had its antiproliferative mechanism of action evaluated. This heterofucan induces apoptosis in HeLa cells by a pathway caspase independent, promoting the release of apoptosis Inducing Factor (AIF) in the cytosol, which in turn induces chromatin condensation and DNA fragmentation into 50Kb fragments. These results are significant in that they provide a mechanistic framework for further exploring the use of SF-1.5v as a novel chemotherapeutics against human cervical cancer.

Modulação da expressão da proteína XPA em resposta ao tratamento com azul de metileno fotossensibilizado

Reactive oxygen species (ROS) are produced by aerobic metabolism and react with biomolecules, such as lipids, proteins and DNA. In high concentration, they lead to oxidative stress. Among ROS, singlet oxygen (1O2) is one of the main ROS involved in oxidative stress and is one of the most reactive forms of molecular oxygen. The exposure of some dyes, such as methylene blue (MB) to light (MB+VL), is able to generate 1O2 and it is the principle involved in photodynamic therapy (PDT). 1O2 e other ROS have caused toxic and carcinogenic effects and have been associated with ageing, neurodegenerative diseases and cancer. Oxidative DNA damage is mainly repaired by base excision repair (BER) pathway. However, recent studies have observed the involvement of nucleotide excision repair (NER) factors in the repair of this type of injury. One of these factors is the Xeroderma Pigmentosum Complementation Group A (XPA) protein, which acts with other proteins in DNA damage recognition and in the recruitment of other repair factors. Moreover, oxidative agents such as 1O2 can induce gene expression. In this context, this study aimed at evaluating the response of XPA-deficient cells after treatment with photosensitized MB. For this purpose, we analyzed the cell viability and occurrence of oxidative DNA damage in cells lines proficient and deficient in XPA after treatment with MB+VL, and evaluated the expression of this enzyme in proficient and complemented cells. Our results indicate an increased resistance to treatment of complemented cells and a higher level of oxidative damage in the deficient cell lines. Furthermore, the treatment was able to modulate the XPA expression up to 24 hours later. These results indicate a direct evidence for the involvement of NER enzymes in the repair of oxidative damage. Besides, a better understanding of the effects of PDT on the induction of gene expression could be provided