RESUMO
From the venom of the Bothrops pictus snake, an endemic species from Peru, we recently have described toxins that inhibited platelet aggregation and cancer cell migration. In this work, we characterize a novel P-III class snake venom metalloproteinase, called pictolysin-III (Pic-III). It is a 62 kDa proteinase that hydrolyzes dimethyl casein, azocasein, gelatin, fibrinogen, and fibrin. The cations Mg2+ and Ca2+ enhanced its enzymatic activity, whereas Zn2+ inhibited it. In addition, EDTA and marimastat were also effective inhibitors. The amino acid sequence deduced from cDNA shows a multidomain structure that includes a proprotein, metalloproteinase, disintegrin-like, and cysteine-rich domains. Additionally, Pic-III reduces the convulxin- and thrombin-stimulated platelet aggregation and in vivo, it has hemorrhagic activity (DHM = 0.3 µg). In epithelial cell lines (MDA-MB-231 and Caco-2) and RMF-621 fibroblast, it triggers morphological changes that are accompanied by a decrease in mitochondrial respiration, glycolysis, and ATP levels, and an increase in NAD(P)H, mitochondrial ROS, and cytokine secretion. Moreover, Pic-III sensitizes to the cytotoxic BH3 mimetic drug ABT-199 (Venetoclax) in MDA-MB-231 cells. To our knowledge, Pic-III is the first SVMP reported with action on mitochondrial bioenergetics and may offer novel opportunities for promising lead compounds that inhibit platelet aggregation or ECM-cancer-cell interactions.
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Glucocorticoids (GCs) are a central component of multi-drug treatment protocols against T and B acute lymphoblastic leukemia (ALL), which are used intensively during the remission induction to rapidly eliminate the leukemic blasts. The primary response to GCs predicts the overall response to treatment and clinical outcome. In this review, we have critically analyzed the available data on the effects of GCs on sensitive and resistant leukemic cells, in order to reveal the mechanisms of GC resistance and how these mechanisms may determine a poor outcome in ALL. Apart of the GC resistance, associated with a decreased expression of receptors to GCs, there are several additional mechanisms, triggered by alterations of different signaling pathways, which cause the metabolic reprogramming, with an enhanced level of glycolysis and oxidative phosphorylation, apoptosis resistance, and multidrug resistance. Due to all this, the GC-resistant ALL show a poor sensitivity to conventional chemotherapeutic protocols. We propose pharmacological strategies that can trigger alternative intracellular pathways to revert or overcome GC resistance. Specifically, we focused our search on drugs, which are already approved for treatment of other diseases and demonstrated anti-ALL effects in experimental pre-clinical models. Among them are some "truly" re-purposed drugs, which have different targets in ALL as compared to other diseases: cannabidiol, which targets mitochondria and causes the mitochondrial permeability transition-driven necrosis, tamoxifen, which induces autophagy and cell death, and reverts GC resistance through the mechanisms independent of nuclear estrogen receptors ("off-target effects"), antibiotic tigecycline, which inhibits mitochondrial respiration, causing energy crisis and cell death, and some anthelmintic drugs. Additionally, we have listed compounds that show a classical mechanism of action in ALL but are not used still in treatment protocols: the BH3 mimetic venetoclax, which inhibits the anti-apoptotic protein Bcl-2, the hypomethylating agent 5-azacytidine, which restores the expression of the pro-apoptotic BIM, and compounds targeting the PI3K-Akt-mTOR axis. Accordingly, these drugs may be considered for the inclusion into chemotherapeutic protocols for GC-resistant ALL treatments.
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The study of proteins and mechanisms involved in the apoptosis and new knowledge about cancer's biology are essential for planning new drugs. Tumor cells develop several strategies to gain proliferative advantages, including molecular alterations to evade from apoptosis. Failures in apoptosis could contribute to cancer pathogenesis, since these defects can cause the accumulation of dividing cells and do not remove genetic variants that have malignant potential. The apoptosis mechanism is composed by proteins that are members of BCL-2 and cysteine-protease families. BH3-only peptides are the "natural" intracellular ligands of BCL-2 family proteins. On the other hand, studies have proved that phenothiazine compounds influence the induction of cellular death. To understand the characteristics of phenothiazines and their effects on tumoral cells and organelles involved in the apoptosis, as well as evaluating their pharmacologic potential, we have carried out computational simulation with the purpose of relating the structures of the phenothiazines with their biological activity. Since the tridimensional (3D) structure of the target protein is known, we have employed the molecular docking approach to study the interactions between compounds and the protein's active site. Hereafter, the molecular dynamics technique was used to verify the temporal evolution of the BCL-2 complexes with phenothiazinic compounds and the BH3 peptide, the stability and the mobility of these molecules in the BCL-2 binding site. From these results, the calculation of binding free energy between the compounds and the biological target was carried out. Thus, it was possible to verify that thioridazine and trifluoperazine tend to increase the stability of the BCL-2 protein and can compete for the binding site with the BH3 peptide.
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The survival of patients with non-Hodgkin's lymphoma (NHL) has substantially improved with current treatments. Nevertheless, the appearance of drug-resistant cancer cells leads to patient relapse. It is therefore necessary to find new antitumor therapies that can completely eradicate transformed cells. Chemotherapy-resistant cancer cells are characterized by the overexpression of members of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein family, such as Bcl-XL, Bcl-2, and Mcl-1. We have recently shown that peptides derived from the BH3 domain of the pro-apoptotic Bax protein may antagonize the anti-apoptotic activity of the Bcl-2 family proteins, restore apoptosis, and induce chemosensitization of tumor cells. In this study, we investigated the feasibility of releasing this peptide into the tumor microenvironment using live attenuated Salmonella enterica, which has proven to be an ally in cancer therapy due to its high affinity for tumor tissue, its ability to activate the innate and adaptive antitumor immune responses, and its potential use as a delivery system of heterologous molecules. Thus, we expressed and released the cell-permeable Bax BH3 peptide from the surface of Salmonella enterica serovar Typhimurium SL3261 through the MisL autotransporter system. We demonstrated that this recombinant bacterium significantly decreased the viability and increased the apoptosis of Ramos cells, a human B NHL cell line. Indeed, the intravenous administration of this recombinant Salmonella enterica elicited antitumor activity and extended survival in a xenograft NHL murine model. This antitumor activity was mediated by apoptosis and an inflammatory response. Our approach may represent an eventual alternative to treat relapsing or refractory NHL.
Assuntos
Proteínas de Bactérias , Vacinas Anticâncer/imunologia , Sistemas de Liberação de Medicamentos , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/patologia , Proteínas de Membrana Transportadoras , Fragmentos de Peptídeos/imunologia , Proteínas Proto-Oncogênicas/imunologia , Salmonella enterica/imunologia , Proteína X Associada a bcl-2/imunologia , Animais , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/química , Vacinas Anticâncer/administração & dosagem , Linhagem Celular , Permeabilidade da Membrana Celular , Sobrevivência Celular , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Linfoma não Hodgkin/mortalidade , Linfoma não Hodgkin/terapia , Proteínas de Membrana Transportadoras/química , Camundongos , Modelos Moleculares , Oligonucleotídeos/química , Fragmentos de Peptídeos/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes , Salmonella enterica/genética , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genéticaRESUMO
ABSTRACT Background: Older patients with acute myeloid leukemia are particularly difficult to treat, as they have a high risk of comorbidities, poor performance status and less tolerability to chemotherapy, as well as a more aggressive disease biology, responsible for the resistance to treatment. There is a need to explore novel therapeutic agents that are more effective and tolerable. Venetoclax, a BCL-2 inhibitor is a promising agent, as BCL-2 overexpression is present in 84% of acute myeloid leukemia patients at diagnosis and 95% of patients at relapse and has been associated with leukemia cell survival, chemotherapy resistance and poor prognosis. Objective: To review the available data about venetoclax in acute myeloid leukemia and how it can influence the treatment in older patients. Methods: Using the Pubmed database, we selected 29 articles published within the last 15 years, considering preclinical and clinical trials and review studies that combined venetoclax with acute myeloid leukemia. Results: Venetoclax has demonstrated promising results in preclinical and clinical trials, especially in patients with poor prognosis and the IDH mutation, with an excellent side-effect profile. However, resistance seems to develop rapidly with venetoclax monotherapy, because of antiapoptotic escape mechanisms. Conclusions: While the results with the use of venetoclax seem encouraging, it is not likely that targeting a single pathway will result in long-term disease control. The solution includes the use of combined therapy to block resistance mechanisms and enhance apoptosis, by reducing MCL-1, increasing BIM or inhibiting the complex IV in the mitochondria.
Assuntos
Leucemia Mieloide Aguda , Genes bcl-2 , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Terapia de Alvo Molecular , Azacitidina/uso terapêutico , Decitabina/uso terapêuticoRESUMO
BACKGROUND: Older patients with acute myeloid leukemia are particularly difficult to treat, as they have a high risk of comorbidities, poor performance status and less tolerability to chemotherapy, as well as a more aggressive disease biology, responsible for the resistance to treatment. There is a need to explore novel therapeutic agents that are more effective and tolerable. Venetoclax, a BCL-2 inhibitor is a promising agent, as BCL-2 overexpression is present in 84% of acute myeloid leukemia patients at diagnosis and 95% of patients at relapse and has been associated with leukemia cell survival, chemotherapy resistance and poor prognosis. OBJECTIVE: To review the available data about venetoclax in acute myeloid leukemia and how it can influence the treatment in older patients. METHODS: Using the Pubmed database, we selected 29 articles published within the last 15 years, considering preclinical and clinical trials and review studies that combined venetoclax with acute myeloid leukemia. RESULTS: Venetoclax has demonstrated promising results in preclinical and clinical trials, especially in patients with poor prognosis and the IDH mutation, with an excellent side-effect profile. However, resistance seems to develop rapidly with venetoclax monotherapy, because of antiapoptotic escape mechanisms. CONCLUSIONS: While the results with the use of venetoclax seem encouraging, it is not likely that targeting a single pathway will result in long-term disease control. The solution includes the use of combined therapy to block resistance mechanisms and enhance apoptosis, by reducing MCL-1, increasing BIM or inhibiting the complex IV in the mitochondria.
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Disruption of the physiologic balance between cell proliferation and cell death is an important step of cancer development. Increased resistance to apoptosis is a key oncogenic mechanism in several hematological malignancies and, in many cases, especially in lymphoid neoplasias, has been attributed to the upregulation of BCL-2. The BCL-2 protein is the founding member of the BCL-2 family of apoptosis regulators and was the first apoptosis modulator to be associated with cancer. The recognition of the important role played by BCL-2 for cancer development and resistance to treatment made it a relevant target for therapy for many diseases, including solid tumors and hematological neoplasias. Among the different strategies that have been developed to inhibit BCL-2, BH3-mimetics have emerged as a novel class of compounds with favorable results in different clinical settings, including chronic lymphocytic leukemia (CLL). In April 2016, the first inhibitor of BCL-2, venetoclax, was approved by the US Food and Drug Administration for the treatment of patients with CLL who have 17p deletion and had received at least one prior therapy. This review focuses on the relevance of BCL-2 for apoptosis modulation at the mitochondrial level, its potential as therapeutic target for hematological malignancies, and the results obtained with selective inhibitors belonging to the BH3-mimetics, especially venetoclax used in monotherapy or in combination with other agents.
Assuntos
Neoplasias Hematológicas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/uso terapêutico , Neoplasias Hematológicas/patologia , Humanos , Proteínas de Transporte da Membrana Mitocondrial/uso terapêuticoRESUMO
Endostatin (ES) is an antiangiogenic protein that exhibits antitumor activity in animal models. However, the activity observed in animals was not observed in human clinical trials. ES-BAX is a fusion protein composed of two functional domains: ES, which presents specificity and is internalized by activated endothelial cells and the proapoptotic BH3 domain of the protein BAX, a peptide inductor of cellular death when internalized. We have previously shown (Chura-Chambi et al., Cell Death Dis, 5, e1371, 2014) that ES-BAX presents improved antitumor activity in relation to wild-type ES. Secondary and tertiary structures of ES-BAX are similar to ES, as indicated by homology-modeling studies and molecular dynamics simulations. Tryptophan intrinsic fluorescence and circular dichroism spectroscopy corroborate these data. 15 N HSQC NMR indicates that ES-BAX is structured, but some ES residues have suffered chemical shift perturbations, suggesting that the BH3 peptide interacts with some parts of the ES protein. ES and ES-BAX present similar stability to thermal denaturation. The production of stable hybrid proteins can be a new approach to the development of therapeutic agents presenting specificity for tumoral endothelium and improved antitumor effect.
Assuntos
Antineoplásicos/química , Endostatinas/química , Proteínas Recombinantes de Fusão/química , Proteína X Associada a bcl-2/química , Endostatinas/genética , Humanos , Espectroscopia de Ressonância Magnética , Domínios Proteicos , Proteínas Recombinantes de Fusão/genética , Proteína X Associada a bcl-2/genéticaRESUMO
ABSTRACT Cancerous cells develop resistance to cell death by over expression of anti-apoptotic proteins, which are specific to interact with pro-apoptotic and BH3-only proteins of Bcl-2 family. Delineating crucial residues mediating the heterodimer complexes (anti-apoptotic proteins - pro-apoptotic/BH3-only proteins) is indispensable to develop specific antagonists to anti-apoptotic proteins. In these backgrounds, we have herein reported crucial residues of hBaxBH3 and hBcl-B (an anti-apoptotic protein specifically interacts with human Bax but does not interact with human Bak) for hetero dimerization of the polypeptides and as well validated the structural determinants of the polypeptides through variety of virtual 'alanine mutants' and 'switch mutants' by using an array of computational methods. Residues such as D53, S60, E61, K64, E69 and D71 of hBaxBH3 and R45, H50, F53, F54, Y57, M71, S74, V75, R86, V88, T89, F93 and F159 of hBcl-B were found to be crucial residues of the polypeptides for intermolecular interaction leading hetero dimerization. Moreover, 'pharmacophoric residues' for the hBaxBH3 and hBcl-B have also been figured out and rationalized.