RESUMO
This study aimed to investigate the antitumor and immunomodulatory properties of the flavonoid apigenin (5,7,4'-trihydroxyflavone), which was extracted from Croton betulaster Mull, in glioma cell culture using the high-proliferative rat C6 glioma cell line as a model. Apigenin was found to have the ability to reduce the viability and proliferation of C6 cells in a time-dependent and dose-dependent manner, with an IC50 of 22.8 µmol/l, 40 times lower than that of temozolomide (1000 µmol/l), after 72 h of apigenin treatment. Even after C6 cells were treated with apigenin for 48 h, high proportions of C6 cells entered apoptosis (39.56%) and autophagy (22%) as shown by flow cytometry using annexin V/propidium iodide and acridine orange staining, respectively. In addition, the flavonoid apigenin induced cell accumulation in the G0/G1 phase of the cell cycle and inhibited glioma cell migration efficiently. Moreover, apigenin induced astroglial differentiation and morphological changes in C6 cells, characterized by increased expression of glial fibrillary acidic protein and decreased expression of nestin protein, a typical marker of neuronal precursors. The immunomodulating effects of apigenin were also characterized by a change in the inflammatory profile as evidenced by a significant decrease in interleukin-10 and tumor necrosis factor production and increased nitric oxide levels. Because apigenin can induce differentiation, apoptosis, and autophagy, can alter the profile of cytokines involved in regulating the immune response, and can reduce the survival, growth, proliferation, and migration of C6 cells, this flavonoid may be considered a potential antitumor drug for the adjuvant treatment of malignant gliomas.
Assuntos
Apigenina/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Diferenciação Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Proteína Glial Fibrilar Ácida/biossíntese , Glioma/imunologia , Glioma/patologia , Interleucina-10/biossíntese , Nestina/biossíntese , Óxido Nítrico/biossíntese , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Chagas disease, caused by Trypanosoma cruzi infection, is a leading cause of heart failure in Latin American countries. In a previous study, we showed beneficial effects of granulocyte colony-stimulating factor (G-CSF) administration in the heart function of mice with chronic T. cruzi infection. Presently, we investigated the mechanisms by which this cytokine exerts its beneficial effects. Mice chronically infected with T. cruzi were treated with human recombinant G-CSF (3 courses of 200 µg/kg/d for 5 d). Inflammation and fibrosis were reduced in the hearts of G-CSF-treated mice, compared with the hearts of vehicle-treated mice, which correlated with decreased syndecan-4, intercellular adhesion molecule-1, and galectin-3 expressions. Marked reductions in interferon-γ and tumor necrosis factor-α and increased interleukin-10 and transforming growth factor-ß were found after G-CSF administration. Because the therapy did not induce a Th1 to Th2 immune response deviation, we investigated the role of regulatory T (Treg) cells. A significant increase in CD3(+)Foxp3(+) cells was observed in the hearts of G-CSF-treated mice. In addition, a reduction of parasitism was observed after G-CSF treatment. Our results indicate a role of Treg cells in the immunosuppression induced by G-CSF treatment and reinforces its potential therapeutic use for patients with Chagas disease.
Assuntos
Cardiomiopatia Chagásica/tratamento farmacológico , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Imunomodulação , Miocardite/tratamento farmacológico , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Complexo CD3/genética , Complexo CD3/metabolismo , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/metabolismo , Citocinas/genética , Citocinas/metabolismo , Fibrose/tratamento farmacológico , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Galectina 3/genética , Galectina 3/metabolismo , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/imunologia , Coração/parasitologia , Humanos , Injeções Intraperitoneais , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/imunologia , Miocárdio/metabolismo , Miocárdio/patologia , Carga Parasitária , Sindecana-4/genética , Sindecana-4/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Transcrição Gênica , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/patogenicidadeRESUMO
BACKGROUND: Apoptosis is a highly controlled process of cell death that can be induced by periodontopathogens. The present study aimed to investigate the expression of Fas and Bcl-2 proteins by CD3+ T cells in vitro under stimulation by total Porphyromonas gingivalis antigens and purified recombinant P. gingivalis HmuY protein. RESULTS: CD3+ T cells derived from CP patients and stimulated with HmuY expressed higher levels of Bcl-2 compared to identical cells stimulated with P. gingivalis crude extract or cells derived from NP control subjects (p = 0.043). CONCLUSION: The authors hypothesize that P. gingivalis HmuY plays a role in the pathogenesis of chronic periodontitis, possibly by reducing or delaying apoptosis in T cells through a pathway involving the Bcl-2 protein.
Assuntos
Proteínas de Bactérias/metabolismo , Complexo CD3/análise , Interações Hospedeiro-Patógeno , Porphyromonas gingivalis/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Linfócitos T/microbiologia , Receptor fas/biossíntese , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Periodontite/microbiologia , Porphyromonas gingivalis/isolamento & purificação , Proteínas Proto-Oncogênicas c-bcl-2/genética , Linfócitos T/química , Adulto Jovem , Receptor fas/genéticaRESUMO
BACKGROUND: Periodontitis is a progressive inflammatory process, and its pathogenesis is related to the presence of a dysbiotic subgingival biofilm that elicits the immune response. Porphyromonas gingivalis is a keystone pathogen, and its Lys-gingipain (Kgp) virulence factor is involved in the pathogen-host interaction through the production of cytokines by host cells, but the specific mechanisms of this interaction have not been elucidated. The present study evaluated the in vitro production of interferon-gamma (IFN-γ), interleukin (IL)-6, and IL-1ß cytokines in response to antigenic stimulation of peripheral blood mononuclear cells (PBMCs) with novel Kgp synthetic peptides. METHODS: Our previous in silico study predicted 16 immunogenic peptides from Kgp protein. Nine peptides derived from different regions of the protein were chemically synthesized. The synthetic peptides Kgp12, 17, and 18 were selected based on the immunoglobulin G immunoreactivity in the serum of patients with periodontitis (P) and individuals without periodontitis (WP), and they were used in in vitro stimulation of PBMC derived from groups P and WP. Enzyme-linked immunosorbent assay and microsphere-based flow cytometric assay were used to verify the levels of the cytokines produced in PBMC cultures after 48 hours. RESULTS: Kgp12, 17, and 18 peptides induced lower production of IFN-γ. Kgp12 induced higher levels of IFN-γ in WP than in P individuals. Kgp12 induced higher production of IL-6 and IL-1ß compared with the other stimuli. CONCLUSION: The novel Kgp synthetic peptides tested herein are immunogenic peptides (epitopes) since they induced the production of cytokines by PBMC and therefore may be useful tools in evaluating the pathogen-host interaction.
Assuntos
Interferon gama , Interleucina-6 , Citocinas , Cisteína Endopeptidases Gingipaínas , Humanos , Interleucina-1beta , Leucócitos Mononucleares , PeptídeosRESUMO
OBJECTIVE: Modulation of cell-mediated immunity by microorganisms in periodontal diseases has been widely studied; however, the proliferative activity and/or programmed death of mononuclear cells under periodontopathogenic stimuli are not yet well understood. The aim of this study was to investigate in vitro proliferation and death of peripheral blood mononuclear cells (PBMC) upon stimulation with Porphyromonas gingivalis (Pg) antigens. DESIGN: In 19 patients with chronic periodontitis (CP) and 16 controls without periodontitis (NP) the following clinical parameters were evaluated: bleeding on probing, probing depth, and clinical attachment level. PBMC were cultured under Pg stimuli and apoptosis/necrosis and proliferation assays were carried out for 18 and 48 h, respectively. Fluorescence of labelled cells was determined using flow cytometry. RESULTS: PBMC of CP and NP subjects exhibited a lower proliferative response to Pg LPS (p<0.05) and HmuY protein (p<0.001) compared with non-stimulated cells. Early apoptosis was induced by Pg LPS (p<0.01) and Pg extract (p<0.05), whilst all antigens induced late apoptosis (Pg LPS: p<0.001; Pg extract: p<0.001; HmuY: p<0.01) and necrosis (Pg LPS: p<0.01; Pg extract: p<0.001; HmuY: p<0.001). Pg LPS induced higher late apoptosis than HmuY (p<0.05). Only Pg LPS-induced necrosis tended to be higher in CP compared with NP. CONCLUSIONS: The inhibitory effect of cell proliferation caused by Pg LPS and HmuY protein is not observed when these antigens comprise Pg extract. Despite induced apoptosis, some still unknown mechanism determines the inflammatory outcome in cell death stimulated by HmuY.