RESUMO
The amphibian family Leptodactylidae is divided into three sub-families: Leiuperinae, Leptodactylinae, and Paratelmatobiinae. Host-defense peptides (HDPs) present in the skins of frogs belonging to the Leptodactylinae have been studied extensively, but information is limited regarding peptides from Leiuperinae species. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Tungara frog Engystomops pustulosus (Leiuperinae) collected in Trinidad led to the isolation and structural characterization of previously undescribed pustulosin-1 (FWKADVKEIG KKLAAKLAEELAKKLGEQ), [Q28E] pustulosin-1 (pustulosin-2), and pustulosin-3 (DWKETAKELLKKIGAKVAQVISDKLNPAPQ). The primary structures of these peptides do not resemble those of previously described frog skin HDPs. In addition, the secretions contained tigerinin-1EP (GCKTYLIEPPVCT) with structural similarity to the tigerinins previously identified in skin secretions from frogs from the family Dicroglossidae. Pustulosin-1 and -3 adopted extended α-helical conformations in 25% trifluoroethanol-water and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). Pustulosin-1 and -3 displayed cytotoxic activity against a range of human tumor-derived cell lines (A549, MDA-MB-231, and HT29), but their therapeutic potential for development into anti-cancer agents is limited by their comparable cytotoxic activity against non-neoplastic human umbilical vein endothelial cells. The peptides also displayed weak antimicrobial activity against Escherichia coli (MIC = 125 µM) but were inactive against Staphylococcus aureus. Tigerinin-1EP was inactive against both the tumor-derived cells and bacteria.
Assuntos
Antineoplásicos , Neoplasias , Animais , Humanos , Peptídeos Catiônicos Antimicrobianos/química , Células Endoteliais/metabolismo , Proteínas de Anfíbios/química , Anuros/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/metabolismo , Neoplasias/metabolismo , Pele/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
The host-defense peptide ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH2 ), first isolated from the Caribbean frog Leptodactylus nesiotus, inhibited growth of clinically relevant Gram-positive and Gram-negative bacteria as well as a strain of the major emerging yeast pathogen Candida parapsilosis. Increasing cationicity while maintaining amphipathicity by the substitution Asp4 âLys increased potency against the microorganisms by between 4- and 16-fold (MIC ≤3 µM) compared with the naturally occurring peptide. The substitution Ala18 âLys and the double substitution Asp4 âLys and Ala18 âLys had less effects on potency. The [D4K] analog also showed 2.5- to 4-fold greater cytotoxic potency against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells (LC50 values in the range of 12-20 µM) compared with ocellatin-3N but was less hemolytic to mouse erythrocytes. However, the peptide showed no selectivity for tumor-derived cells [LC50 = 20 µM for human umbilical vein endothelial cells (HUVECs)]. Ocellatin-3N and [D4K]ocellatin-3N stimulated the release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥1 nM, and [A18K]ocellatin-3N, at concentrations ≥0.1 nM. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 µM, indicating that plasma membrane integrity had been preserved. The three peptides produced an increase in intracellular [Ca2+ ] in BRIN-BD11 cells when incubated at a concentration of 1 µM. In view of its high insulinotropic potency and relatively low hemolytic activity, the [A18K] ocellatin analog may represent a template for the design of agents with therapeutic potential for the treatment of patients with type 2 diabetes.
Assuntos
Anti-Infecciosos , Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Diabetes Mellitus Tipo 2 , Neoplasias Pulmonares , Camundongos , Animais , Humanos , Peptídeos Catiônicos Antimicrobianos/química , Lisina , Antibacterianos/química , Diabetes Mellitus Tipo 2/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Células Endoteliais/metabolismo , Proteínas de Anfíbios/farmacologia , Bactérias Gram-Positivas , Bactérias Gram-Negativas , Neoplasias Pulmonares/metabolismo , Insulina/metabolismo , Antineoplásicos/farmacologia , Anuros/metabolismo , Pele/metabolismoRESUMO
Triple-negative breast cancer (TNBC) is a type of breast malignancy characterized by a high proliferative rate and metastatic potential leading to treatment failure, relapse, and poor prognosis. Therefore, efforts are continuously being devoted to understanding its biology and identifying new potential targets. Programmed death-ligand 1 (PD-L1) is an immunosuppressive protein that inactivates T cells by binding to the inhibitory receptor programmed death-1 (PD-1). PD-L1 overexpression in cancer cells contributes to immune evasion and, subsequently, poor survival and prognosis in several cancers, including breast cancer. Apart from its inhibitory impact on T cells, this ligand is believed to have an intrinsic role in cancer cells. This study was performed to clarify the PD-1 independent role of PD-L1 in TNBC MDA-MB-231 cells by knocking out the PD-L1 using three designs of CRISPR-Cas9 lentiviral particles. Our study revealed that PD-L1 knockout significantly inhibited MDA-MB-231 cell proliferation and colony formation in vitro and tumor growth in the chick embryo chorioallantoic membrane (CAM) model in vivo. PD-L1 knockout also decreased the migration and invasion of MDA-MB-231 cells in vitro. We have shown that PD-L1 knockout MDA-MB-231 cells have low levels of p-Akt and p-ERK in addition to some of their downstream proteins, c-Fos, c-Myc, p21, survivin, and COX-2. Furthermore, PD-L1 knockout significantly decreased the expression of Snail and RhoA. This study shows the intrinsic role of PD-L1 in TNBC independently of its binding to PD-1 receptors on T cells. It may pave the way for developing novel therapeutic strategies using PD-L1 inhibitors alone and in combination to treat TNBC more effectively.
Assuntos
Neoplasias de Mama Triplo Negativas , Embrião de Galinha , Animais , Humanos , Neoplasias de Mama Triplo Negativas/metabolismo , Antígeno B7-H1/metabolismo , Receptor de Morte Celular Programada 1/genética , Linhagem Celular Tumoral , Recidiva Local de NeoplasiaRESUMO
Inflammatory bowel disease, comprising Crohn's disease (CD) and ulcerative colitis (UC), is often debilitating. The disease etiology is multifactorial, involving genetic susceptibility, microbial dysregulation, abnormal immune activation, and environmental factors. Currently, available drug therapies are associated with adverse effects when used long-term. Therefore, the search for new drug candidates to treat IBD is imperative. The peroxisome proliferator-activated receptor-γ (PPARγ) is highly expressed in the colon. PPARγ plays a vital role in regulating colonic inflammation. 1,8-cineole, also known as eucalyptol, is a monoterpene oxide present in various aromatic plants which possess potent anti-inflammatory activity. Molecular docking and dynamics studies revealed that 1,8-cineole binds to PPARγ and if it were an agonist, that would explain the anti-inflammatory effects of 1,8-cineole. Therefore, we investigated the role of 1,8-cineole in colonic inflammation, using both in vivo and in vitro experimental approaches. Dextran sodium sulfate (DSS)-induced colitis was used as the in vivo model, and tumor necrosis factor-α (TNFα)-stimulated HT-29 cells as the in vitro model. 1,8-cineole treatment significantly decreased the inflammatory response in DSS-induced colitis mice. 1,8-cineole treatment also increased nuclear factor erythroid 2-related factor 2 (Nrf2) translocation into the nucleus to induce potent antioxidant effects. 1,8-cineole also increased colonic PPARγ protein expression. Similarly, 1,8-cineole decreased proinflammatory chemokine production and increased PPARγ protein expression in TNFα-stimulated HT-29 cells. 1,8-cineole also increased PPARγ promoter activity time-dependently. Because of its potent anti-inflammatory effects, 1,8-cineole may be valuable in treating IBD.
Assuntos
Colite Ulcerativa , Colite , Doenças Inflamatórias Intestinais , Animais , Camundongos , Anti-Inflamatórios/farmacologia , Colite/metabolismo , Colite Ulcerativa/metabolismo , Colo/patologia , Sulfato de Dextrana , Eucaliptol/farmacologia , Inflamação/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , PPAR gama/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Metabolic reprogramming has been recognized as an essential emerging cancer hallmark. Dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), has been reported to have anti-cancer effects by reversing tumor-associated glycolysis. This study was performed to explore the anti-cancer potential of DCA in lung cancer alone and in combination with chemo- and targeted therapies using two non-small cell lung cancer (NSCLC) cell lines, namely, A549 and LNM35. DCA markedly caused a concentration- and time-dependent decrease in the viability and colony growth of A549 and LNM35 cells in vitro. DCA also reduced the growth of tumor xenografts in both a chick embryo chorioallantoic membrane and nude mice models in vivo. Furthermore, DCA decreased the angiogenic capacity of human umbilical vein endothelial cells in vitro. On the other hand, DCA did not inhibit the in vitro cellular migration and invasion and the in vivo incidence and growth of axillary lymph nodes metastases in nude mice. Treatment with DCA did not show any toxicity in chick embryos and nude mice. Finally, we demonstrated that DCA significantly enhanced the anti-cancer effect of cisplatin in LNM35. In addition, the combination of DCA with gefitinib or erlotinib leads to additive effects on the inhibition of LNM35 colony growth after seven days of treatment and to synergistic effects on the inhibition of A549 colony growth after 14 days of treatment. Collectively, this study demonstrates that DCA is a safe and promising therapeutic agent for lung cancer.
Assuntos
Reprogramação Celular/genética , Ácido Dicloroacético/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Células A549 , Animais , Glicólise/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Metástase Neoplásica , Piruvato Desidrogenase Quinase de Transferência de Acetil/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite the significant advances in targeted- and immuno-therapies, lung and breast cancer are at the top list of cancer incidence and mortality worldwide as of 2020. Combination therapy consisting of a mixture of different drugs taken at once is currently the main approach in cancer management. Natural compounds are extensively investigated for their promising anti-cancer potential. This study explored the anti-cancer potential of butein, a biologically active flavonoid, on two major solid tumors, namely, A549 lung and MDA-MB-231 breast cancer cells alone and in combination with another natural anti-cancer compound, frondoside-A. We demonstrated that butein decreases A549 and MDA-MB-231 cancer cell viability and colony growth in vitro in addition to tumor growth on chick embryo chorioallantoic membrane (CAM) in vivo without inducing any noticeable toxicity. Additionally, non-toxic concentrations of butein significantly reduced the migration and invasion of both cell lines, suggesting its potential anti-metastatic effect. We showed that butein anti-cancer effects are due, at least in part, to a potent inhibition of STAT3 phosphorylation, leading to PARP cleavage and consequently cell death. Moreover, we demonstrated that combining butein with frondoside-A leads to additive effects on inhibiting A549 and MDA-MB-231 cellular viability, induction of caspase 3/7 activity, inhibition of colony growth, and inhibition of cellular migration and invasion. This combination reached a synergistic effect on the inhibition of HUVECs migration in vitro. Collectively, this study provides sufficient rationale to further carry out animal studies to confirm the relevance of these compounds' combination in cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Movimento Celular , Chalconas/farmacologia , Células Endoteliais/patologia , Glicosídeos/farmacologia , Triterpenos/farmacologia , Animais , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , Sinergismo Farmacológico , Células Endoteliais/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Invasividade Neoplásica , Neovascularização Patológica/patologia , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Fator de Transcrição STAT3/metabolismo , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The aim of the study was to determine the in vitro immunomodulatory, cytotoxic, and insulin-releasing activities of seven phylloseptin-TR peptides and plasticin-TR, first isolated from the frog Phyllomedusa trinitatis. The most cationic peptides, phylloseptin-1.1TR and phylloseptin-3.1TR, showed greatest cytotoxic potency against A549, MDA-MB231, and HT-29 human tumor-derived cells and against mouse erythrocytes. Phylloseptin-4TR was the most hydrophobic and the most effective peptide at inhibiting production of the proinflammatory cytokines TNF-α and IL-1ß by mouse peritoneal cells but was without effect on production of the antiinflammatory cytokine IL-10. Phylloseptin-2.1TR and phylloseptin-3.3TR were the most effective at stimulating the production of IL-10. The noncytotoxic peptide, plasticin-TR, inhibited production of TNF-α and IL-1ß but was without effect on IL-10 production. The results of CD spectroscopy suggest that the different properties of plasticin-TR compared with the immunostimulatory activities of the previously characterized plasticin-L1 from Leptodactylus laticeps may arise from greater ability of plasticin-TR to oligomerize and adopt a stable helical conformation in a membrane-mimetic environment. All peptides stimulated release of insulin from BRIN-BD11 rat clonal ß cells with phylloseptin-3.2TR being the most potent and effective and phylloseptin-2.1TR the least effective suggesting that insulinotropic potency correlates inversely with helicity. The study has provided insight into structure-activity relationships among the phylloseptins. The combination of immunomodulatory and insulinotropic activities together with low cytotoxicity suggests that phylloseptin-3.3TR and plasticin-TR may represent templates for the development of agents for use in antiinflammatory and type 2 diabetes therapies.
Assuntos
Anti-Inflamatórios/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Anuros , Citotoxinas/farmacologia , Proteínas do Olho/farmacologia , Imunomodulação/efeitos dos fármacos , Insulina/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas do Olho/química , Proteínas do Olho/imunologia , Proteínas do Olho/isolamento & purificação , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/isolamento & purificação , RatosRESUMO
Aberrantly high levels of tyrosine-phosphorylated signal transducer and activator of transcription 3 (p-STAT3) are found constitutively in ~50% of human lung and breast cancers, acting as an oncogenic transcription factor. We previously demonstrated that Manuka honey (MH) inhibits p-STAT3 in breast cancer cells, but the exact mechanism remained unknown. Herein, we show that MH-mediated inhibition of p-STAT3 in breast (MDA-MB-231) and lung (A549) cancer cell lines is accompanied by decreased levels of gp130 and p-JAK2, two upstream components of the IL-6 receptor (IL-6R) signaling pathway. Using an ELISA-based assay, we demonstrate that MH binds directly to IL-6Rα, significantly inhibiting (~60%) its binding to the IL-6 ligand. Importantly, no evidence of MH binding to two other cytokine receptors, IL-11Rα and IL-8R, was found. Moreover, MH did not alter the levels of tyrosine-phosphorylated or total Src family kinases, which are also constitutively activated in cancer cells, suggesting that signaling via other growth factor receptors is unaffected by MH. Binding of five major MH flavonoids (luteolin, quercetin, galangin, pinocembrin, and chrysin) was also tested, and all but pinocembrin could demonstrably bind IL-6Rα, partially (30-35%) blocking IL-6 binding at the highest concentration (50 µM) used. In agreement, each flavonoid inhibited p-STAT3 in a dose-dependent manner, with estimated IC50 values in the 3.5-70 µM range. Finally, docking analysis confirmed the capacity of each flavonoid to bind in an energetically favorable configuration to IL-6Rα at a site predicted to interfere with ligand binding. Taken together, our findings identify IL-6Rα as a direct target of MH and its flavonoids, highlighting IL-6R blockade as a mechanism for the anti-tumor activity of MH, as well as a viable therapeutic target in IL-6-dependent cancers.
Assuntos
Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Mel , Receptores de Interleucina-6/antagonistas & inibidores , Fator de Transcrição STAT3/antagonistas & inibidores , Antineoplásicos/química , Comunicação Autócrina/efeitos dos fármacos , Produtos Biológicos/química , Linhagem Celular Tumoral , Humanos , Janus Quinase 2/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Fator de Transcrição STAT3/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND/AIMS: Breast cancer is the most common cancer in women worldwide, and within this cancer type, triple-negative breast cancers have the worst prognosis. The identification of new genes associated with triple-negative breast cancer progression is crucial for developing more specific anti-cancer targeted therapies, which could lead to a better management of these patients. In this context, we have recently demonstrated that SMARCAD1, a DEAD/H box-containing helicase, is involved in breast cancer cell migration, invasion, and metastasis. The aim of this study was to investigate the impact of the stable knockdown of SMARCAD1 on human breast cancer cell progression. METHODS: Using two different designs of shRNA targeting SMARCAD1, we investigated the impact of the stable knockdown of SMARCAD1 on human breast cancer cell proliferation and colony growth in vitro and on tumour growth in chick embryo and nude mouse xenograft models in vivo using MDA-MB-231 (ER-/PR-/ HER2-) and T47D (ER+/PR+/-/HER2-) human breast cancer cell lines. RESULTS: We found that SMARCAD1 knockdown resulted in a significant decrease in breast cancer cell proliferation and colony formation, leading to the significant inhibition of tumour growth in both the chick embryo and nude mouse xenograft models. This inhibition was due, at least in part, to a decrease in IKKß expression. CONCLUSION: These results indicate that SMARCAD1 is involved in breast cancer progression and can be a promising target for breast cancer therapy.
Assuntos
Neoplasias da Mama/patologia , DNA Helicases/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Embrião de Galinha , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , Progressão da Doença , Feminino , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Camundongos , Camundongos Nus , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Peptidomic analysis of norepinephrine-stimulated skin secretions from Italian stream frog Rana italica led to the purification and characterization of two host-defense peptides differing by a single amino acid residue belonging to the brevinin-1 family (brevinin-1ITa and -1ITb), a peptide belonging to the temporin family (temporin-ITa) and a component identified as prokineticin Bv8. The secretions contained relatively high concentrations of the methionine-sulphoxide forms of brevinin-1ITa and -1ITb suggesting that these peptides may have a role as antioxidants in the skin of this montane frog. Brevinin-1ITa (IVPFLLGMVPKLVCLITKKC) displayed potent cytotoxicity against non-small cell lung adenocarcinoma A549 cells (LC50 = 18 µM), breast adenocarcinoma MDA-MB-231 cells (LC50 = 8 µM) and colorectal adenocarcinoma HT-29 cells (LC50 = 18 µM), but the peptide was also strongly hemolytic against mouse erythrocytes (LC50 = 7 µM). Temporin-ITa (VFLGAIAQALTSLLGKL.NH2 ) was between three and fivefold less potent against these cells. Brevinin-1ITa inhibited growth of both Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli as well as a strain of the opportunist yeast pathogen Candida parapsilosis, whereas temporin-ITa was active only against S. epidermidis and C. parapsilosis. Both peptides stimulated the release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥1 nM, but brevinin-1ITa was cytotoxic to the cells at concentrations ≥3 µM. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Assuntos
Proteínas de Anfíbios/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Pele/metabolismo , Proteínas de Anfíbios/farmacologia , Proteínas de Anfíbios/toxicidade , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/toxicidade , Anuros/metabolismo , Escherichia coli/efeitos dos fármacos , Células HT29 , Hemólise/efeitos dos fármacos , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Ranidae , Staphylococcus epidermidis/efeitos dos fármacosRESUMO
BACKGROUND: Ultrasmall superparamagnetic iron oxide nanoparticles (USPIO) are being developed for several biomedical applications including drug delivery and imaging. However, little is known about their possible adverse effects on thrombosis and cardiac oxidative and DNA damage. METHODS: Presently, we investigated the acute (1 h) effect of intravenously (i.v.) administered USPIO in mice (0.4, 2 and 10 µg/kg). Diesel exhaust particles (DEP; 400 µg/kg) were used as positive control. RESULTS: USPIO induced a prothrombotic effect in pial arterioles and venules in vivo and increased the plasma plasminogen activator inhibitor-1 (PAI-1). Both thrombogenicity and PAI-1 concentration were increased by DEP. The direct addition of USPIO (0.008, 0.04 and 0.2 µg/ml) to untreated mouse blood dose-dependently induced in vitro platelet aggregation. USPIO caused a shortening of activated partial thromboplastin time (aPTT) and prothrombin time (PT). Similarly, DEP administration (1 µg/ml) triggered platelet aggregation in vitro in whole blood. DEP also reduced PT and aPTT. The plasma levels of creatine phosphokinase-MB isoenzyme (CK-MB), lactate dehydrogenase (LDH) and troponin-I were increased by USPIO. DEP induced a significant increase of CK-MB, LDH and troponin I levels in plasma. The cardiac levels of markers of oxidative stress including lipid peroxidation, reactive oxygen species and superoxide dismutase activity were increased by USPIO. Moreover, USPIO caused DNA damage in the heart. Likewise, DEP increased the markers of oxidative stress and induced DNA damage in the heart. CONCLUSION: We conclude that acute i.v. administration of USPIO caused thrombosis and cardiac oxidative stress and DNA damage. These findings provide novel insight into the pathophysiological effects of USPIO on cardiovascular homeostasis, and highlight the need for a thorough evaluation of their toxicity.
Assuntos
Doença das Coronárias/induzido quimicamente , Dano ao DNA , Nanopartículas de Magnetita/toxicidade , Mutagênicos/toxicidade , Oxidantes/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Trombose/induzido quimicamente , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Ensaio Cometa , Doença das Coronárias/metabolismo , Relação Dose-Resposta a Droga , Coração/efeitos dos fármacos , Injeções Intravenosas , Fenômenos Magnéticos , Nanopartículas de Magnetita/administração & dosagem , Nanopartículas de Magnetita/química , Camundongos Endogâmicos BALB C , Mutagênicos/química , Miocárdio/metabolismo , Oxidantes/administração & dosagem , Oxidantes/química , Tempo de Tromboplastina Parcial , Tamanho da Partícula , Inibidor 1 de Ativador de Plasminogênio/agonistas , Inibidor 1 de Ativador de Plasminogênio/sangue , Agregação Plaquetária/efeitos dos fármacos , Tempo de Protrombina , Trombose/metabolismo , Testes de Toxicidade AgudaRESUMO
Epstein-Barr virus (EBV; human herpesvirus 4) is an oncogenic herpesvirus implicated in the pathogenesis of several human malignancies. A number of recent studies indicate that EBV can manipulate the local microenvironment by excreting viral and cellular components in nanovesicles called exosomes. In this study, we investigated the impact of EBV-derived exosomes on apoptosis of recipient cells and the molecular pathway involved in this process. Exosomes from EBV-infected but not from non-infected cells induced apoptosis in a number of different cell types, including B-cells, T-cells and epithelial cells. However, this phenomenon was not universal and the Burkitt's lymphoma-derived B-cell line BJAB was found to be resistant to apoptosis. Exosomes from both type I and type III EBV latently infected cells induced apoptosis in a dose- and time-dependent manner. Moreover, cells exposed to EBV exosomes did not form colonies in soft agar assays. We further show that fluorescently labelled exosomes derived from EBV-infected cells are taken up by non-infected cells and induce apoptosis via the extrinsic pathway. Inhibition of caspase-3/7/8 blocks EBV exosome-mediated apoptosis. Furthermore, our data indicate that EBV exosomes trigger apoptosis through the Fas ligand (FasL)-mediated extrinsic pathway, as FasL was present in EBV exosomal fractions and anti-FasL antibodies could block EBV exosome-mediated apoptosis. Together, these data support the notion that EBV hijacks the exosome pathway to excrete viral and cellular components that can modulate its microenvironment.
Assuntos
Apoptose/fisiologia , Exossomos/química , Proteína Ligante Fas/metabolismo , Herpesvirus Humano 4/fisiologia , Linfoma de Burkitt/virologia , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Proteína Ligante Fas/genética , Regulação da Expressão Gênica/fisiologia , Células HEK293 , HumanosRESUMO
Pseudhymenochirin-1Pb (Ps-1Pb; IKIPSFFRNILKKVGKEAVSLIAGALKQS) and pseudhymenochirin-2Pa (Ps-2Pa; GIFPIFAKLLGKVIKVASSLISKGRTE) are amphibian peptides with broad spectrum antimicrobial activities and cytotoxicity against mammalian cells. In the membrane-mimetic solvent 50% (v/v) trifluoroethanol-H2O, both peptides adopt a well-defined α-helical conformation that extends over almost all the sequence and incorporates a flexible bend. Both peptides significantly (p < 0.05) stimulate the rate of release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥ 0.1 nM but produce loss of integrity of the plasma membrane at concentrations ≥ 1 µM. Increasing cationicity by the substitution Glu(17) â l-Lys in Ps-1Pb and Glu(27) â l-Lys in Ps-2Pa generates analogues with increased cytotoxicity and reduced insulin-releasing potency. In contrast, the analogues [R8r]Ps-1Pb and [K8k,K19k]Ps-2Pa, incorporating d-amino acid residues to destabilize the α-helical domains, retain potent insulin-releasing activity but are nontoxic to BRIN-BD11 cells at concentrations of 3 µM. [R8r]Ps-1Pb produces a significant increase in insulin release rate at 0.3 nM and [K8k,K19k]Ps-2Pa at 0.01 nM. Both analogues show low hemolytic activity (IC50 > 100 µM) but retain broad-spectrum antimicrobial activity and remain cytotoxic to a range of human tumor cell lines, albeit with lower potency than the naturally occurring peptides. These analogues show potential for development into agents for type 2 diabetes therapy.
Assuntos
Proteínas de Anfíbios/isolamento & purificação , Proteínas de Anfíbios/farmacologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Insulina/metabolismo , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Animais , Peptídeos Catiônicos Antimicrobianos/química , Linhagem Celular Tumoral , Humanos , Secreção de Insulina , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
BACKGROUND: In the present study, we investigated the effect of Salinomycin on the survival of three human breast cancer cell lines MCF-7, T47D and MDA-MB-231 grown in adherent culture conditions. METHODS: Cell viability was measured by Cell Titer-Glo and Trypan blue exclusion assay. Apoptosis was determined by caspase 3/7 activation, PARP cleavage and Annexin V staining. Cell cycle distribution was assessed by propidium iodide flow cytometry. Senescence was confirmed by measuring the senescence-associated ß-galactosidase activity. Changes in protein expression and histone hyperacetylation was determined by western blot and confirmed by immunofluorescence assay. RESULTS: Salinomycin was able to inhibit the growth of the three cell lines in time- and concentration-dependent manners. We showed that depending on the concentrations used, Salinomycin elicits different effects on the MDA-MB-231 cells. High concentrations of Salinomycin induced a G2 arrest, downregulation of survivin and triggered apoptosis. Interestingly, treatment with low concentrations of Salinomycin induced a transient G1 arrest at earlier time point and G2 arrest at later point and senescence associated with enlarged cellmorphology, upregulation of p21 protein, increase in histone H3 and H4 hyperacetylation and expression of SA-ß-Gal activity. Furthermore, we found that Salinomycin was able to potentiate the killing of the MCF-7 and MDA-MB-231 cells, by the chemotherapeutic agents, 4-Hydroxytamoxifen and frondo side A, respectively. CONCLUSION: Our data are the first to link senescence and histone modifications to Salinomycin. SIGNIFICANCE: This study provides a new insight to better understand the mechanism of action of Salinomycin, at least in breast cancer cells.
Assuntos
Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas do Citoesqueleto/metabolismo , Histonas/metabolismo , Piranos/farmacologia , Acetilação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Feminino , Fase G2/efeitos dos fármacos , Glicosídeos/farmacologia , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/fisiologia , Survivina , Triterpenos/farmacologiaRESUMO
BACKGROUND: Pistacia lentiscus (Anacardiaceae) is a flowering plant traditionally used in the treatment of various skin, respiratory, and gastrointestinal disorders. The aim of this study was to assess whether Pistacia lentiscus oil has any short term toxic effects in vivo and in vitro. METHODS: Pistacia lentiscus oil (100µl) was administered orally into mice for 5 days. RESULTS: Measurements of body weight did not show any weight loss. Serum concentration of LDH did not show any significant statistical difference when compared to control mice. Similarly, blood, kidney or liver function tests showed no toxicity with Pistacia lentiscus oil when compared to the control group. Examination of gastrointestinal tissues sections revealed similar structural features with no difference in cell proliferation. In this context, pharmacological dilutions of Pistacia lentiscus oil (10(-6) - 10(-3)) did not affect the viability (cell death and proliferation) of mouse gastric stem cells, human colorectal cancer cells HT29, human hepatoma cells HepG2. However, it appears that at the dose and time point studied, Pistacia lentiscus oil treatment has targeted various cytochrome P450s and has specifically inhibited the activities and the expression of CYP2E1, CYP3A4, CYP1A1 and CYP1A2 differentially in different tissues. Our results also demonstrate that there is no appreciable effect of Pistacia lentiscus oil on the GSH-dependent redox homoeostasis and detoxification mechanism in the tissues. CONCLUSION: These data suggest a good safety profile of short term oral use of Pistacia lentiscus oil as a monotherapy in the treatment of various skin, respiratory, and gastrointestinal disorders. However, due to its inhibitory effect of various cytochrome P450s and mainly CYP3A4, this might have implications on the bioavailability and metabolism of drugs taken in combination with Pistacia lentiscus oil. More attention is needed when Pistacia lentiscus oil is intended to be uses in combination with other pharmacological agents in order to avoid potential drug-drug interaction leading to toxicity. This study will help in safer use of Pistacia lentiscus oil for therapeutic purpose.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Preparações Farmacêuticas/metabolismo , Pistacia/química , Óleos de Plantas/administração & dosagem , Óleos de Plantas/farmacologia , Administração Oral , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Feminino , Frutas/química , Células HT29 , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos , Especificidade de Órgãos/efeitos dos fármacos , Óleos de Plantas/química , Relação Estrutura-AtividadeRESUMO
Background:Pancreatic cancer is a leading cause of cancer-related mortality worldwide with increasing global incidence. We previously reported the anticancer effect of Rhus coriaria ethanolic extract (RCE) in triple negative breast and colon cancer cells. Herein, we investigated the anticancer effect of RCE on human pancreatic cancer cells. Methods: Cell viability was measured using Cell Titer-Glo and staining of viable and dead cells based on differential permeability to two DNA binding dyes. Cell cycle distribution and annexin V staining was carried out in Muse cell analyzer. Protein level was determined by Western blot. Tumor growth was assessed by in ovo chick embryo chorioallantoic membrane assay. Results: We found that RCE significantly inhibited the viability and colony growth of pancreatic cancer cells (Panc-1, Mia-PaCa-2, S2-013, AsPC-1). The antiproliferative effects of RCE in pancreatic cancer cells (Panc-1 and Mia-PaCa-2) were mediated through induction of G1 cell cycle arrest, Beclin-1-independent autophagy, and apoptosis. RCE activated both the extrinsic and intrinsic pathways of apoptosis and regulated the Bax/Bcl-2 apoptotic switch. Mechanistically, we found that RCE inhibited the AKT/mTOR pathway, downstream of which, inactivation of the cell cycle regulator p70S6K and downregulation of the antiapoptotic protein survivin was observed. Additionally, we found that RCE-induced autophagy preceded apoptosis. Further, we confirmed the anticancer effect of RCE in a chick embryo xenograft model and found that RCE inhibited the growth of pancreatic cancer xenografts without affecting embryo survival. Conclusion: Collectively, our findings demonstrate that Rhus coriaria exerts potent anti-pancreatic cancer activity though cell cycle impairment, autophagy, and apoptosis, and is hence a promising source of anticancer phytochemicals.
RESUMO
Five host-defense peptides (figainin 2PL, hylin PL, raniseptin PL, plasticin PL, and peptide YL) were isolated from norepinephrine-stimulated skin secretions of the banana tree dwelling frog Boana platanera (Hylidae; Hylinae) collected in Trinidad. Raniseptin PL (GVFDTVKKIGKAVGKFALGVAKNYLNS.NH2) and figainin 2PL (FLGTVLKLGKAIAKTVVPMLTNAMQPKQ. NH2) showed potent and rapid bactericidal activity against a range of clinically relevant Gram-positive and Gram-negative ESKAPE + pathogens and Clostridioides difficile. The peptides also showed potent cytotoxic activity (LC50 values < 30 µM) against A549, MDA-MB-231 and HT29 human tumor-derived cell lines but appreciably lower hemolytic activity against mouse erythrocytes (LC50 = 262 ± 14 µM for raniseptin PL and 157 ± 16 µM for figainin 2PL). Hylin PL (FLGLIPALAGAIGNLIK.NH2) showed relatively weak activity against microorganisms but was more hemolytic. The glycine-leucine-rich peptide with structural similarity to the plasticins (GLLSTVGGLVGGLLNNLGL.NH2) and the non-cytotoxic peptide YL (YVPGVIESLL.NH2) lacked antimicrobial and cytotoxic activities. Hylin PL, raniseptinPL and peptide YL stimulated the rate of release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥100 nM. Peptide YL was the most effective (2.3-fold increase compared with basal rate at 1 µM concentration) and may represent a template for the design of a new class of incretin-based anti-diabetic drugs.
Assuntos
Anuros , Pele , Animais , Pele/metabolismo , Humanos , Anuros/metabolismo , Camundongos , Linhagem Celular Tumoral , Proteínas de Anfíbios/farmacologia , Proteínas de Anfíbios/química , Proteínas de Anfíbios/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/química , Hemólise/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Sequência de Aminoácidos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Células HT29 , Musa/químicaRESUMO
Alyteserin-2a (ILGKLLSTAAGLLSNL.NH(2)) is a cationic, amphipathic α-helical cell-penetrating peptide, first isolated from skin secretions of the midwife toad Alytes obstetricans. Structure-activity relationships were investigated by synthesizing analogs of alyteserin-2a in which amino acids on the hydrophobic face of the helix were replaced by L-tryptophan and amino acids on the hydrophilic face were replaced by one or more L-lysine or D-lysine residues. The Trp-containing peptides display increased cytotoxic activity against non-small cell lung adenocarcinoma A549 cells (up to 11-fold), but hemolytic activity against human erythrocytes increases in parallel. The potency of the N15K analog against A549 cells (LC(50) = 13 µM) increases sixfold relative to alyteserin-2a and the therapeutic index (ratio of LC(50) for erythrocytes and tumor cells) increases twofold. Incorporation of a D-Lys(11) residue into the N15K analog generates a peptide that retains potency against A549 cells (LC(50) = 15 µM) but whose therapeutic index is 13-fold elevated relative to the native peptide. [G11k, N15K] alyteserin-2a is also active against human hepatocarcinoma HepG2 cells (LC(50) = 26 µM), breast adenocarcinoma MDA-MB-231 cells (LC(50) = 20 µM), and colorectal adenocarcinoma HT-29 cells (LC(50) = 28 µM). [G11k, N15K] alyteserin-2a, in concentrations as low as 1 µg/mL, significantly (P < 0.05) inhibits the release of the immune-suppressive cytokines IL-10 and TGF-ß from unstimulated and concanavalin A-stimulated peripheral blood mononuclear cells. The data suggest a strategy of increasing the cationicity while reducing the helicity of naturally occurring amphipathic α-helical peptides to generate analogs with improved cytotoxicity against tumor cells but decreased activity against non-neoplastic cells.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Anuros/metabolismo , Neoplasias/tratamento farmacológico , Pele/metabolismo , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Células HT29 , Humanos , Dados de Sequência Molecular , Pele/química , Relação Estrutura-AtividadeRESUMO
Lung cancer is the second most commonly diagnosed cancer and has the highest mortality rate worldwide despite the remarkable advances in its treatment. Origanum majorana Essential Oil (OMEO) has been shown to be effective against non-small cell lung cancer (NSCLC) cells, decreasing their viability and colony growth in vitro, as well as inhibiting tumor growth in chick embryo chorioallantoic membranes (CAM) and nude mice in vivo. OMEO is mainly composed of four monoterpenes, namely terpinen-4-ol, sabinene hydrate, α-terpinene, and γ-terpinene. In this study, we aimed to investigate the potential anticancer effects of these monoterpenes, either alone or in combination, on NSCLC. Our findings indicate that these four monoterpenes significantly decreased NSCLC cell viability in a concentration-dependent manner, reduced their colony growth in vitro, and also downregulated survivin expression in these cells. Moreover, different combined mixtures of these monoterpenes further enhanced their anticancer effects on cellular viability, with a terpinen-4-ol and sabinene hydrate combination being the most potent. We also found that terpinen-4-ol, in combination with sabinene hydrate, markedly enhanced the anticancer effect of the individual monoterpenes on NSCLC viability within a shorter treatment duration through, at least in part, survivin downregulation. Furthermore, this combination enhanced the inhibition of colony growth in vitro and the tumor growth of NSCLC cells xenografted onto chick embryo CAM in vivo. Altogether, our study highlights the potential of these monoterpenes for use in further pre-clinical investigations against various cancer hallmarks.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Óleos Voláteis , Origanum , Embrião de Galinha , Camundongos , Animais , Monoterpenos/farmacologia , Óleos Voláteis/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Survivina/metabolismo , Camundongos Nus , Neoplasias Pulmonares/tratamento farmacológicoRESUMO
Frogs from the extensive amphibian family Hylidae are a rich source of peptides with therapeutic potential. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Giant Gladiator Treefrog Boana boans (Hylidae: Hylinae) collected in Trinidad led to the isolation and structural characterization of five host-defense peptides with limited structural similarity to figainin 2 and picturin peptides from other frog species belonging to the genus Boana. In addition, the skin secretions contained high concentrations of tryptophyllin-BN (WRPFPFL) in both C-terminally α-amidated and non-amidated forms. Figainin 2BN (FLGVALKLGKVLG KALLPLASSLLHSQ) and picturin 1BN (GIFKDTLKKVVAAVLTTVADNIHPK) adopt α-helical conformations in trifluroethanol-water mixtures and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). The CD data also indicate contributions from turn structures. Both peptides and picturin 2BN (GLMDMLKKVGKVALT VAKSALLP) inhibited the growth of clinically relevant Gram-negative and Gram-positive bacteria with MIC values in the range 7.8-62.5 µM. Figainin 2BN was potently cytotoxic to A549, MDA-MB-231 and HT-29 human tumor-derived cells (LC50 = 7-14 µM) but displayed comparable potency against non-neoplastic HUVEC cells (LC50 = 15 µM) indicative of lack of selectivity for cancer cells.