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.

Correlação entre a infecção genital pelo vírus do papiloma humano, a resposta imune e os achados colpocitológicos em mulheres grávidas e não grávidas

The genital HPV infection is very common between men and women worldwide, affecting particularly young women, constituting a serious public health problem in less developed regions, favored by the poor living conditions of population. The cytology and colposcopy have notorious importance in the diagnosis of precursor lesions of cervical cancer and therefore its prevention. However, even with such diagnostic tools, the number of women who develop cervical cancer is still high. This study aims to assess the prevalence of genital tract infection by HPV in pregnant and nonpregnant women, evaluating the profile of the immune response presented by the women of these two groups in order to establish correlations among profile of immune response, presence of virus and occurrence of lesions of the uterine cervix. We analyzed specimens obtained from the cervix of 221 patients, 91 pregnant and 130 non-pregnant, aged 14-72 years. The women were subjected to colposcopic and cytologic evaluation detect possible changes in the cervix and then samples were collected in order to perform HPV detection by PCR and real-time PCR for detection of mRNA of pro-inflammatory and anti-inflammatory cytokines. In the present study, the overall prevalence of HPV genital infection was 28.1%; of which 31.9% were pregnant patients and 25.4% in non-pregnant women. Young women under 30 years and those with low educational level education showed a higher risk of HPV infection. Colposcopy showed better correlation with detection of HPV DNA by PCR, when compared to cytology. Generally, HPV infected patients, pregnant or not, exhibited reduced mRNA expression of both pro-inflammatory (IFN-γ, TNF-α) and anti-inflammatory (IL -10) cytokines, when compared to patients not infected by HPV. Nonpregnant patients infected presented increase mRNA expression of IL-17 in patients without injury, whereas those with lesion showed higher mRNA expression of TGF-β. Pregnant women without injury infected exhibited increased mRNA expression of TGF-β. There was no difference in HPV prevalence between pregnant and nonpregnant women. There was a reduction of pro-inflammatory cytokines, except IL-17, in all women infected by HPV. Moreover, we observed an increase of TGF-β in HPV-infected women who are pregnant or not. The results suggest that, in women in this study, HPV infection promoted changes in the profile of cytokines necessary for activation of effective immune response, possibly favoring viral persistence

Prevalência da infecção por Chlamydia Trachomatis em mulheres atendidas no Programa de rastreamento do câncer de colo do útero

Genital infection with Chlamydia trachomatis is now recognized as one of the most prevalent sexually transmitted infections (STDs). Despite major advances in laboratory diagnosis techniques, primarily the character of asymptomatic chlamydial infection in both men and in women constitutes the basis for the formation of reservoirs that perpetuate transmission and acquisition of this and other STDs. The asymptomatic in women favors the rise of infection to the upper genital tract, causing injuries that can result in infertility. An examination of population screening for early detection and treatment of asymptomatic infections is the key step in combating this major public health problem. The present study aimed to evaluate the prevalence of infection by C. trachomatis in sexually active women attended the screening program for cervical cancer of the uterus in health facilities in municipalities in different regions of the State of Rio Grande do Norte, and identify factors that may contribute to the spread of this pathogen and its relationship with the lesions of the uterine cervix. It is a cross-sectional study aimed at detecting the presence of genital tract infection by C. trachomatis either in isolated form or in association with human papilloma virus (HPV) infection in asymptomatic women. Were included in this study, a total sample of 1,134 women aged 13-76, mean 34.4 years, from March 2008 to September 2012. Specimens containing exfoliated cells of the epithelium of the uterine cervix were analyzed by examining Pap cytology for the detection of possible injuries, and the polymerase chain reaction (PCR) for detection of plasmid DNA from C. trachomatis and HPV. Infection with C. trachomatis was detected with overall prevalence rate of 8.1% in the isolated form and 2.8% in co-infection with HPV. The infection was detected in 7.4% of women with normal cytology 11.5% of those with atypical cells of undetermined significance (ASC-US) and 16.7% of those with low-grade squamous intraepithelial lesion (LSIL). We observed an association between C. trachomatis and incidence of low-grade squamous intraepithelial lesion (LSIL). The genital tract infection by C. trachomatis alone was associated with education level, ethnicity and parity, revealing that women with higher education, those of non-white ethnicity and those who had three or more pregnancies were more likely to acquire infection. Levels very close to statistical significance were observed for chronological age, age at first sexual intercourse and first pregnancy. There was no association with marital status, number of sexual partners. Co-infection with C. trachomatis and HPV was detected in 2.3% of women with normal cytology, who had 5.1% in ASC-US and 10.4% in those with LSIL. No association was found between infection C. trachomatis and increased risk of HPV infection, but women with simultaneous infection by both pathogens showed greater risk for LSIL. Co-infection was more prevalent among single women, who had in the first sexual intercourse under 18 years and those who had two or more sexual partners over a lifetime

Estratégias de intervenção utilizadas por enfermeiros da ESF do município de Natal/RN no controle do câncer do colo de útero

Cancer of the cervix (cervical cancer) is the second most prevalent cancer among Brazilian women. The high rates of cervical cancer in Brazil justify the implementation of effective strategies to control this, which include actions to promote health, primary prevention, early detection, screening, treatment and palliative care. Despite the existence of the National Programme for Control of the CCU there was no reduction in the incidence and mortality of this disease in Brazil. The Family Health Strategy (FHS) has the potential to facilitate such control and, in this context, one should consider that nurses play a central role. The study aimed to know the general intervention strategies used by nurses FHS of Natal / RN in CCU control, and how specific: analyzing the knowledge of these nurses on the CCU, the actions developed in the ESF for the control of CCU and identify the difficulties faced by them to perform it. This is a descriptive exploratory quantitative developed through a structured interview guide with 106 nurses who have experience in controlling the CCU in FHS teams of Natal / RN. Data analysis was performed using descriptive statistic s. The results pointed to actions taken in the FHS to control the CCU, collection of cervical cancer screening, health education activities, nursing consultation, referral of suspected cases for medical monitoring and active women with abnormal test result . The actions that were not mentioned by the nurses included: forming groups of prevention and health promotion; expand coverage of exams and office hours of consultations, establishment of alternatives to end the pent-up demand in the health units, participation in treatment or rehabilitation process users with the CCU; interventions for pain management, alliances and partnerships with schools, in dustry and the use of protocols. This study can be seen that the practice nurses partially shares to the CCU in Natal / RN. The participants of this study, when asked about the CCU, specifically for signs and symptoms of disease and risk factors in general showed important gaps. Difficulties such as lack of materials for collection of Pap smear; inadequate physical space in the Health Units; pent-up demand in the service, delay in arrival of the test results; obstacles in the actions of referral and counter-referral and cultural factors make the CCU control is compromised. It is believed in this research contributed to a reflection on the importance of the role of nurses in the development of the ESF control actions CCU, pointing out the factors that affect these. It is important to involve all nurses who comprise the ESF as knowledgeable of the risk factors, signs and symptoms, and existing tools for the early detection of cervical cancer in the pursuit of quality improvement actions to promote women`s health, contributing in planning future interventions that may reduce mortality from this disease in Natal / RN.

Análise molecular dos éxons 8 a 11 do gene da p53 em amostras de câncer de colo de útero no Rio Grande do Norte

Mutations on TP53 gene are common in human cancer but not in cervical cancer where they are rarely found and the inactivation and degradation of p53 protein are attributed to the action of E6 viral oncogene from high risk human papillomavirus (HPV). Analysis of cervical cancer cell lines suggests that HPV negative samples shows mutation on TP53, but clinical approaches didn t confirmed this hypothesis. However, in most TP53 mutations studies on cervical cancer, only the exons 5 to 8 were analyzed. Approximately 90% of mutations described are on this region. Recent studies on several cancer suggests that mutation frequency in the other exons must be considered. The aim of this work was to verify whether mutations on coding and non-coding regions occur in cancer tissue from cervical cancer in patients from Rio Grande do Norte using Denaturing Gradient Gel Electrophoresis (DGGE) as screening tool. Exons 8 to 11 were analyzed including some introns from 80 tumor samples and 8 peripheral blood samples from healthy women. DNA were submitted to PCR using primers with GC clamp on the end of one of them. The results were observed for each region after DGGE and silver staining. It was observed no amplified fragment with different migration profile from those obtained from DNA of peripheral blood. These results agree with those from literature where TP53 mutations in cervical cancer have been described in a very low frequency

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.

Indução de morte celular em células de adenocarcinoma cervical humano (HeLa) pela lectina da esponja Cinachyrella apion (CaL)

Cancer is a term used to represent a set of more than 100 diseases, including malignant tumors from different locations. The malignancies are the second leading cause of death in the population, representing approximately 17% of deaths of known cause. Strategies that induce differentiation have had limited success in the treatment of established cancers. In this work, a lectin purified from the marine sponge Cinachyrella apion (CaL) was evaluated due to its hemolytic, cytotoxic and antiproliferative properties, besides the ability to induce cell death via apoptosis in tumor cells. The antiproliferative activity of CaL was tested against cell lines, with the highest inhibition of tumor growth for HeLa, reducing cell growth at a dose dependent manner, with a concentration of 10 μg/mL. The hemolytic activity and toxicity against peripheral blood cells were tested using the concentration of IC50 for both trials and twice the IC50 for analysis in flow cytometry, indicating that CaL is not toxic to these cells. To assess the mechanism of cell death caused by CaL in HeLa cells, we performed flow cytometry and western blotting. The results showed the lectin probably induces cell death by apoptosis activation by pro-apoptotic protein Bax, promoting mitochondrial membrane permeabilization, cell cycle arrest in S phase, with accumulation of cells of approximately 57% in this phase, and acting as both dependent and/or independent of caspases pathway. These results suggest that CaL has the potential to be used as drug treatment against cancer.

Indução de morte celular em células de adenocarcinoma cervical humano (HeLa) pela lectina da esponja Cinachyrella apion (CaL)

Cancer is a term used to represent a set of more than 100 diseases, including malignant tumors from different locations. The malignancies are the second leading cause of death in the population, representing approximately 17% of deaths of known cause. Strategies that induce differentiation have had limited success in the treatment of established cancers. In this work, a lectin purified from the marine sponge Cinachyrella apion (CaL) was evaluated due to its hemolytic, cytotoxic and antiproliferative properties, besides the ability to induce cell death via apoptosis in tumor cells. The antiproliferative activity of CaL was tested against cell lines, with the highest inhibition of tumor growth for HeLa, reducing cell growth at a dose dependent manner, with a concentration of 10 μg/mL. The hemolytic activity and toxicity against peripheral blood cells were tested using the concentration of IC50 for both trials and twice the IC50 for analysis in flow cytometry, indicating that CaL is not toxic to these cells. To assess the mechanism of cell death caused by CaL in HeLa cells, we performed flow cytometry and western blotting. The results showed the lectin probably induces cell death by apoptosis activation by pro-apoptotic protein Bax, promoting mitochondrial membrane permeabilization, cell cycle arrest in S phase, with accumulation of cells of approximately 57% in this phase, and acting as both dependent and/or independent of caspases pathway. These results suggest that CaL has the potential to be used as drug treatment against cancer.