RESUMO
Cancer is a relevant health problem worldwide. In 2020, leukemias represented the 13th most commonly reported cancer cases worldwide but the 10th most likely to cause deaths. There has been a progressive increase in the efficacy of treatments for leukemias; however, these still generate important side effects, so it is imperative to search for new alternatives. Defensins are a group of antimicrobial peptides with activity against cancer cells. However, the cytotoxic mechanism of these peptides has been described mainly for animal defensins. This study shows that defensin γ-thionin (Capsicum chinense) is cytotoxic to the K562 leukemia cells with an IC50 = 290 µg/mL (50.26 µM) but not for human peripheral blood mononuclear cells. Results showed that γ-thionin did not affect the membrane potential; however, the peptide modified the mitochondrial membrane potential (ΔΨm) and the intracellular calcium release. In addition, γ-thionin induced apoptosis in K562 cells, but the activation of caspases 8 and 9 was not detected. Moreover, the activation of calpains was detected at one hour of treatment, suggesting that γ-thionin activates the caspase-independent apoptosis. Furthermore, the γ-thionin induced epigenetic modifications on histone 3 in K562 cells, increased global acetylation (~2-fold), and specific acetylation marks at lysine 9 (H3K9Ac) (~1.5-fold). In addition, γ-thionin increased the lysine 9 methylation (H3K9me) and dimethylation marks (H3K9me2) (~2-fold), as well as the trimethylation mark (H3K9me3) (~2-fold). To our knowledge, this is the first report of a defensin that triggers caspase-independent apoptosis in cancer cells via calpains and regulating chromatin remodelation, a novel property for a plant defensin.
Assuntos
Antineoplásicos , Capsicum , Leucemia Mielogênica Crônica BCR-ABL Positiva , Tioninas , Animais , Humanos , Tioninas/farmacologia , Células K562 , Capsicum/química , Peptídeos Antimicrobianos , Chile , Leucócitos Mononucleares/metabolismo , Lisina/farmacologia , Apoptose , Peptídeos/farmacologia , Antineoplásicos/farmacologia , Caspases/metabolismo , Defensinas/farmacologia , Epigênese GenéticaRESUMO
Angiogenesis is involved in wound repair and tissue maintenance but is associated with diverse diseases. Pro-angiogenic factors such as vascular endothelial growth factor (VEGF) regulate this process. Therefore, searching for treatments to inhibit or promote angiogenesis is attractive. Reports from our group showed that plant antimicrobial peptides (PAPs) PaDef from avocado and γ-thionin from habanero pepper are cytotoxic on cancer cells. However, their functions as angiogenic regulators are unknown. In this work, we evaluate the effect of PaDef and γ-thionin on the angiogenic processes of two different endothelial cell lines: bovine endothelial cells (BUVEC) and the human endothelial cell line EA.hy926. The results showed that VEGF (10 ng/mL) stimulated the BUVEC (40 ± 7 %) and EA.hy926 cell proliferation (30 ± 9 %); however, peptides (5-500 ng/mL) reverted this effect. Besides, VEGF increased the migration of BUVEC (20 ± 8 %) and EA.hy926 cells (50 ± 6 %), but both PAPs (5 ng/mL) inhibited the VEGF stimulus (100 %). Furthermore, DMOG 50 µM (an inhibitor of HIF-hydroxylase) was used in BUVEC and EA.hy926 cells to determine the effect of hypoxia on VEGF and peptide activities. The DMOG reverted the inhibitory action of both peptides (100 %), indicating that peptides act through a HIF-independent pathway. Also, the PAPs do not affect the tube formation but decrease it in EA.hy926 cells stimulated with VEGF (100 %). Additionally, docking assays showed a possible interaction between PAPs and the VEGF receptor. These results suggest that plant defensins PaDef and γ-thionin are potential angiogenic modulators of the VEGF activity on endothelial cells.
Assuntos
Tioninas , Fator A de Crescimento do Endotélio Vascular , Animais , Bovinos , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células Endoteliais/metabolismo , Tioninas/metabolismo , Tioninas/farmacologia , Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio Vascular/farmacologia , Proliferação de Células , Defensinas/farmacologia , Movimento CelularRESUMO
BACKGROUND: Butyrate is a histone deacetylase inhibitor that induces apoptosis and inhibits cell proliferation of colorectal cancer cells. To improve its anticancer activity, butyrate has been evaluated mixed with drugs and different molecules. Plant antimicrobial peptides are attractive anticancer alternative molecules because they show selective cytotoxic activity against different cancer cell lines. In this work, we explore if the plant defensin c-thionin (Capsicum chinense) can improve butyrate activity on Caco-2 cell line and we also determined the mechanism of death activated. RESULTS: The combined treatment of c-thionin (3.5 mM) and butyrate (50 mM) showed higher cytotoxicity on Caco-2 cells with respect to single treatments. Also, the combined treatment reduced cell proliferation and exhibited a higher rate of apoptosis than single treatments. Combined treatment induced caspases 8 and 9 activation to an extent comparable with that of butyrate while c-thionin did not activate caspases. Additionally, reactive oxygen species generation preceded the onset of apoptosis, and superoxide anion production was higher in cells treated with the combined treatment. CONCLUSIONS: The c-thionin from Habanero chili pepper improved the butyrate cytotoxicity on Caco-2 cells. This effect occurred through apoptosis induction associated with reactive oxygen species production. Therefore, the combination of butyrate with cytotoxic antimicrobial peptides could be an attractive strategy for cancer therapy.
Assuntos
Humanos , Butiratos , Capsicum/química , Adenocarcinoma , Neoplasias do Colo , Ciclo Celular , Espécies Reativas de Oxigênio , Apoptose , Células CACO-2 , Defensinas , TioninasRESUMO
Many Fusarium species are able to cause severe infections in plants as well as in animals and humans. Therefore, the discovery of new antifungal agents is of paramount importance. CaThi belongs to the thionins, which are cationic peptides with low molecular weights (â¼5 kDa) that have toxic effects against various microorganisms. Herein, we study the mechanism of action of CaThi and its combinatory effect with fluconazole (FLC) against Fusarium solani. The mechanism of action of CaThi was studied by growth inhibition, viability, plasma membrane permeabilization, ROS induction, caspase activation, localization, and DNA binding capability, as assessed with Sytox green, DAB, FITC-VAD-FMK, CaThi-FITC, and gel shift assays. The combinatory effect of CaThi and FLC was assessed using a growth inhibition assay. Our results demonstrated that CaThi present a dose dependent activity and at the higher used concentration (50 µg mL-1 ) inhibits 83% of F. solani growth, prevents the formation of hyphae, permeabilizes membranes, induces endogenous H2 O2 , activates caspases, and localizes intracellularly. CaThi combined with FLC, at concentrations that alone do not inhibit F. solani, result in 100% death of F. solani when combined. The data presented in this study demonstrate that CaThi causes death of F. solani via apoptosis; an intracellular target may also be involved. Combined treatment using CaThi and FLC is a strong candidate for studies aimed at improved targeting of F. solani. This strategy is of particular interest because it minimizes selection of resistant microorganisms.
Assuntos
Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Fluconazol/farmacologia , Tioninas/farmacologia , Antifúngicos/química , Peptídeos Catiônicos Antimicrobianos/química , Capsicum/química , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Frutas/química , Fusarium/efeitos dos fármacos , Fusarium/patogenicidade , Humanos , Hifas/efeitos dos fármacos , Hifas/patogenicidade , Tioninas/químicaRESUMO
ß-Defensins are members of the antimicrobial peptide superfamily that are produced in various species from different kingdoms, including plants. Plant defensins exhibit primarily antifungal activities, unlike those from animals that exhibit a broad-spectrum antimicrobial action. Recently, immunomodulatory roles of mammal ß-defensins have been observed to regulate inflammation and activate the immune system. Similar roles for plant ß-defensins remain unknown. In addition, the regulation of the immune system by mammalian ß-defensins has been studied in humans and mice models, particularly in immune cells, but few studies have investigated these peptides in epithelial cells, which are in intimate contact with pathogens. The aim of this work was to evaluate the effect of the chemically synthesized ß-defensin γ-thionin from Capsicum chinense on the innate immune response of bovine mammary epithelial cells (bMECs) infected with Staphylococcus aureus, the primary pathogen responsible for bovine mastitis, which is capable of living within bMECs. Our results indicate that γ-thionin at 0.1 µg/ml was able to reduce the internalization of S. aureus into bMECs (â¼50%), and it also modulates the innate immune response of these cells by inducing the mRNA expression (â¼5-fold) and membrane abundance (â¼3-fold) of Toll-like receptor 2 (TLR2), as well as by inducing genes coding for the pro-inflammatory cytokines TNF-α and IL-1ß (â¼14 and 8-fold, respectively) before and after the bacterial infection. γ-Thionin also induces the expression of the mRNA of anti-inflammatory cytokine IL-10 (â¼12-fold). Interestingly, the reduction in bacterial internalization coincides with the production of other antimicrobial products by bMECs, such as NO before infection, and the secretion into the medium of the endogenous antimicrobial peptide DEFB1 after infection. The results from this work support the potential use of ß-defensins from plants as immunomodulators of the mammalian innate immune response.
Assuntos
Capsicum/química , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Tioninas/farmacologia , Sequência de Aminoácidos , Animais , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Regulação da Expressão Gênica/imunologia , Imunidade Inata , Fatores Imunológicos/isolamento & purificação , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/microbiologia , Extratos Vegetais/química , Cultura Primária de Células , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Transdução de Sinais , Staphylococcus aureus/fisiologia , Tioninas/isolamento & purificação , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , beta-Defensinas/biossíntese , beta-Defensinas/imunologia , beta-Defensinas/metabolismoRESUMO
BACKGROUND: Thionins are a family of plant antimicrobial peptides (AMPs), which participate in plant defense system against pathogens. Here we describe some aspects of the CaThi thionin-like action mechanism, previously isolated from Capsicum annuum fruits. Thionin-like peptide was submitted to antimicrobial activity assays against Candida species for IC50 determination and synergism with fluconazole evaluation. Viability and plasma membrane permeabilization assays, induction of intracellular ROS production analysis and CaThi localization in yeast cells were also investigated. RESULTS: CaThi had strong antimicrobial activity against six tested pathogenic Candida species, with IC50 ranging from 10 to 40 µg.mL(-1). CaThi antimicrobial activity on Candida species was candidacidal. Moreover, CaThi caused plasma membrane permeabilization in all yeasts tested and induces oxidative stresses only in Candida tropicalis. CaThi was intracellularly localized in C. albicans and C. tropicalis, however localized in nuclei in C. tropicalis, suggesting a possible nuclear target. CaThi performed synergistically with fluconazole inhibiting all tested yeasts, reaching 100% inhibition in C. parapsilosis. The inhibiting concentrations for the synergic pair ranged from 1.3 to 4.0 times below CaThi IC50 and from zero to 2.0 times below fluconazole IC50. CONCLUSION: The results reported herein may ultimately contribute to future efforts aiming to employ this plant-derived AMP as a new therapeutic substance against yeasts.
Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Capsicum/química , Fluconazol/farmacologia , Tioninas/farmacologia , Candida/crescimento & desenvolvimento , Sinergismo Farmacológico , Frutas/química , Testes de Sensibilidade MicrobianaRESUMO
Antimicrobial peptides (AMPs) are part of the innate immune defense mechanism of many organisms and are promising candidates to treat infections caused by pathogenic bacteria to animals and humans. AMPs also display anticancer activities because of their ability to inactivate a wide range of cancer cells. Cancer remains a cause of high morbidity and mortality worldwide. Therefore, the development of methods for its control is desirable. Attractive alternatives include plant AMP thionins, defensins, and cyclotides, which have anticancer activities. Here, we provide an overview of plant AMPs anticancer activities, with an emphasis on their mode of action, their selectivity, and their efficacy.
Assuntos
Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Antineoplásicos/uso terapêutico , Imunidade Inata/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Anti-Infecciosos/uso terapêutico , Ciclotídeos/uso terapêutico , Defensinas/uso terapêutico , Humanos , Imunidade Inata/imunologia , Neoplasias/patologia , Plantas/química , Tioninas/uso terapêuticoAssuntos
Humanos , Masculino , Feminino , Educação Alimentar e Nutricional , Tioninas/análise , Tecnologia de Alimentos , Fabaceae , Faculdades de Medicina , Ensino , Bioquímica , LaboratóriosRESUMO
Plants defend themselves against pathogens with production of antimicrobial peptides (AMPs). Herein we describe the discovery of a new antifungal and antibacterial peptide from fruits of Capsicum annuum that showed similarity to an already well characterized family of plant AMPs, thionins. Other fraction composed of two peptides, in which the major peptide also showed similarity to thionins. Among the obtained fractions, fraction 1, which is composed of a single peptide of 7 kDa, was sequenced by Edman method and its comparative sequence analysis in database (nr) showed similarity to thionin-like peptides. Tests against microorganisms, fraction 1 presented inhibitory activity to the cells of yeast Saccharomyces cerevisiae, Candida albicans, and Candida tropicalis and caused growth reduction to the bacteria species Escherichia coli and Pseudomonas aeruginosa. Fraction 3 caused inhibitory activity only for C. albicans and C. tropicalis. This fraction was composed of two peptides of â¼7 and 10 kDa, and the main protein band correspondent to the 7 kDa peptide, also showed similarity to thionins. This plasma membrane permeabilization assay demonstrates that the peptides present in the fractions 1 and 3 induced changes in the membranes of all yeast strains, leading to their permeabilization. Fraction 1 was capable of inhibiting acidification of the medium of glucose-induced S. cerevisiae cells 78% after an incubation time of 30 min, and opposite result was obtained for C. albicans. Experiments demonstrate that the fraction 1 and 3 were toxic and induced changes in the membranes of all yeast strains, leading to their permeabilization.