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Introduction: Breast cancer (BC) is the leading cause of death among women, primarily due to its potential for metastasis. As BC progresses, the extracellular matrix (ECM) produces more type-I collagen, resulting in increased stiffness. This alteration influences cellular behaviors such as migration, invasion, and metastasis. Specifically, cancer cells undergo changes in gene expression that initially promote an epithelial-to-mesenchymal transition (EMT) and subsequently, a transition from a mesenchymal to an amoeboid (MAT) migration mode. In this way, cancer cells can migrate more easily through the stiffer microenvironment. Despite their importance, understanding MATs remains challenging due to the difficulty of replicating in vitro the conditions for cell migration that are observed in vivo. Methods: To address this challenge, we developed a three-dimensional (3D) growth system that replicates the different matrix properties observed during the progression of a breast tumor. We used this model to study the migration and invasion of the Triple-Negative BC (TNBC) cell line MDA-MB-231, which is particularly subject to metastasis. Results: Our results indicate that denser collagen matrices present a reduction in porosity, collagen fiber size, and collagen fiber orientation, which are associated with the transition of cells to a rounder morphology with bleb-like protrusions. We quantified how this transition is associated with a more persistent migration, an enhanced invasion capacity, and a reduced secretion of matrix metalloproteinases. Discussion: Our findings suggest that the proposed 3D growth conditions (especially those with high collagen concentrations) mimic key features of MATs, providing a new platform to study the physiology of migratory transitions and their role in BC progression.
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Although significant progress has been made in the implementation of new breast cancer treatments over the last three decades, this neoplasm annually continues to show high worldwide rates of morbidity and mortality. In consequence, the search for novel therapies with greater effectiveness and specificity has not come to a stop. Among the alternative therapeutic targets, the human gonadotropin-releasing hormone type I and type II (hGnRH-I and hGnRH-II, respectively) and its receptor, the human gonadotropin-releasing hormone receptor type I (hGnRHR-I), have shown to be powerful therapeutic targets to decrease the adverse effects of this disease. In the present review, we describe how the administration of GnRH analogs is able to reduce circulating concentrations of estrogen in premenopausal women through their action on the hypothalamus-pituitary-ovarian axis, consequently reducing the growth of breast tumors and disease recurrence. Also, it has been mentioned that, regardless of the suppression of synthesis and secretion of ovarian steroids, GnRH agonists exert direct anticancer action, such as the reduction of tumor growth and cell invasion. In addition, we discuss the effects on breast cancer of the hGnRH-I and hGnRH-II agonist and antagonist, non-peptide GnRH antagonists, and cytotoxic analogs of GnRH and their implication as novel adjuvant therapies as antitumor agents for reducing the adverse effects of breast cancer. In conclusion, we suggest that the hGnRH/hGnRHR system is a promising target for pharmaceutical development in the treatment of breast cancer, especially for the treatment of advanced states of this disease.
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Messenger RNA 3'-end polyadenylation is an important regulator of gene expression in eukaryotic cells. In our search for new ways of treating parasitic infectious diseases, we looked at whether or not alterations in polyadenylation might control the survival of Entamoeba histolytica (the agent of amoebiasis in humans). We used molecular biology and computational tools to characterize the mRNA cleavage factor EhCFIm25, which is essential for polyadenylation in E. histolytica. By using a strategy based on the systematic evolution of ligands by exponential enrichment, we identified single-stranded RNA aptamers that target EhCFIm25. The results of RNA-protein binding assays showed that EhCFIm25 binds to the GUUG motif in vitro, which differs from the UGUA motif bound by the homologous human protein. Accordingly, docking experiments and molecular dynamic simulations confirmed that interaction with GUUG stabilizes EhCFIm25. Incubating E. histolytica trophozoites with selected aptamers inhibited parasite proliferation and rapidly led to cell death. Overall, our data indicate that targeting EhCFIm25 is an effective way of limiting the growth of E. histolytica in vitro. The present study is the first to have highlighted the potential value of RNA aptamers for controlling this human pathogen.
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Antiprotozoários/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Entamoeba histolytica/crescimento & desenvolvimento , Fatores de Poliadenilação e Clivagem de mRNA/antagonistas & inibidores , Fatores de Poliadenilação e Clivagem de mRNA/química , Motivos de Aminoácidos , Antiprotozoários/química , Aptâmeros de Nucleotídeos/química , Sítios de Ligação , Biologia Computacional/métodos , Entamoeba histolytica/efeitos dos fármacos , Entamoeba histolytica/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , RNA/farmacologia , Técnica de Seleção de Aptâmeros , Trofozoítos/efeitos dos fármacos , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/metabolismoRESUMO
MicroRNAs (miRNAs) are small non-coding RNAs that function as negative regulators of gene expression. Recent evidences suggested that host cells miRNAs are involved in the progression of infectious diseases, but its role in amoebiasis remains largely unknown. Here, we reported an unexplored role for miRNAs of human epithelial colon cells during the apoptosis induced by Entamoeba histolytica. We demonstrated for the first time that SW-480 colon cells change their miRNAs profile in response to parasite exposure. Our data showed that virulent E. histolytica trophozoites induced apoptosis of SW-480 colon cells after 45 min interaction, which was associated to caspases-3 and -9 activation. Comprehensive profiling of 667 miRNAs using Taqman Low-Density Arrays showed that 6 and 15 miRNAs were significantly (FC > 1.5; p < 0.05) modulated in SW-480 cells after 45 and 75 min interaction with parasites, respectively. Remarkably, no significant regulation of the 6-miRNAs signature (miR-526b-5p, miR-150, miR-643, miR-615-5p, miR-525, and miR-409-3p) was found when SW-480 cells were exposed to non-virulent Entamoeba dispar. Moreover, we confirmed that miR-150, miR-643, miR-615-5p, and miR-525 exhibited similar regulation in SW-480 and Caco2 colon cells after 45 min interaction with trophozoites. Exhaustive bioinformatic analysis of the six-miRNAs signature revealed intricate miRNAs-mRNAs co-regulation networks in which the anti-apoptotic XIAP, API5, BCL2, and AKT1 genes were the major targets of the set of six-miRNAs. Of these, we focused in the study of functional relationships between miR-643, upregulated at 45 min interaction, and its predicted target X-linked inhibitor of apoptosis protein (XIAP). Interestingly, interplay of amoeba with SW-480 cells resulted in downregulation of XIAP consistent with apoptosis activation. More importantly, loss of function studies using antagomiRs showed that forced inhibition of miR-643 leads to restoration of XIAP levels and suppression of both apoptosis and caspases-3 and -9 activation. Congruently, mechanistic studies using luciferase reporter assays confirmed that miR-643 exerts a postranscripcional negative regulation of XIAP by targeting its 3'-UTR indicating that it's a downstream effector. In summary, we provide novel lines of evidence suggesting that early-branched eukaryote E. histolytica may promote apoptosis of human colon cells by modulating, in part, the host microRNome which highlight an unexpected role for miRNA-643/XIAP axis in the host cellular response to parasites infection.
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Apoptose , Entamoeba histolytica/crescimento & desenvolvimento , Células Epiteliais/parasitologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , MicroRNAs/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Linhagem Celular , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Fatores de TempoRESUMO
The 25 kDa subunit of the Clevage Factor Im (CFIm25) is an essential factor for messenger RNA polyadenylation in human cells. Therefore, here we investigated whether the homologous protein of Entamoeba histolytica, the protozoan responsible for human amoebiasis, might be considered as a biochemical target for parasite control. Trophozoites were cultured with bacterial double-stranded RNA molecules targeting the EhCFIm25 gene, and inhibition of mRNA and protein expression was confirmed by RT-PCR and Western blot assays, respectively. EhCFIm25 silencing was associated with a significant acceleration of cell proliferation and cell death. Moreover, trophozoites appeared as larger and multinucleated cells. These morphological changes were accompanied by a reduced mobility, and erythrophagocytosis was significantly diminished. Lastly, the knockdown of EhCFIm25 affected the poly(A) site selection in two reporter genes and revealed that EhCFIm25 stimulates the utilization of downstream poly(A) sites in E. histolytica mRNA. Overall, our data confirm that targeting the polyadenylation process represents an interesting strategy for controlling parasites, including E. histolytica. To our best knowledge, the present study is the first to have revealed the relevance of the cleavage factor CFIm25 as a biochemical target in parasites.
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Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Genes de Protozoários/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Morte Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Entamoeba histolytica/patogenicidade , Entamebíase/parasitologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Genes Reporter , Humanos , Fagocitose , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Trofozoítos/citologia , Trofozoítos/metabolismo , Fatores de Virulência/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
The pathogenic amoeba Entamoeba histolytica is able to migrate within various compartments of the human body. The present article reviews progress in understanding the mechanisms of cell motility in E. histolytica during human intestinal invasion and, in particular, how the three-dimensional characteristics of the environment regulate the parasite's behaviour. The amoeboid mode of migration that applies to E. histolytica's displacements on two-dimensional surfaces is also expected to apply to the three-dimensional environment in the human intestine although several unknown, distinct modalities may be involved. Recent advances in the field of tissue engineering have provided clues on how the construction of a human colon model could help us to understand the host's intestinal physiology and its changes following amoebic infection.
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Disenteria Amebiana/parasitologia , Entamoeba histolytica/fisiologia , Intestinos/fisiopatologia , Intestinos/parasitologia , Animais , Colo/parasitologia , Colo/fisiopatologia , Entamoeba histolytica/patogenicidade , Humanos , Movimento , Técnicas de Cultura de Órgãos , Engenharia TecidualRESUMO
Small GTPases are signalling molecules that regulate important cellular processes. GTPases are deactivated by GTPase-activating proteins (GAPs). While human GAPs have been intensively studied, no GAP has yet been characterized in Entamoeba histolytica. In this study, we identified and characterized a novel nucleocytoplasmic RhoGAP in E. histolytica termed EhRhoGAPnc. In silico analyses of the domain structure revealed a previously undescribed peptide region within the carboxy-terminal region of EhRhoGAPnc capable of interacting with phosphatidic acid and phosphatidylinositol 3,5-bisphosphate. The full structural GAP domain showed increase GAP activity compared with the minimum region able to display GAP activity, as analysed both by experimental assays and molecular dynamics simulations. Furthermore, we identified amino acid residues that promote interactions between EhRhoGAPnc and its target GTPases EhRacC and EhRacD. Immunofluorescence studies revealed that EhRhoGAPnc colocalized with EhRacC and EhRacD during uroid formation but not during erythrophagocytosis. Interestingly, during erythrophagocytosis of red blood cells, EhRhoGAPnc colocalized with phosphatidic acid and phosphatidylinositol 3,5-bisphosphate. Overexpression of EhRhoGAPnc in E. histolytica led to inhibition of actin adhesion plate formation, migration, adhesion of E. histolytica to MDCK cells and consequently to an impairment of the cytopathic activity.
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Entamoeba histolytica/patogenicidade , Proteínas Ativadoras de GTPase/fisiologia , Proteínas de Protozoários/fisiologia , Proteínas rac de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Núcleo Celular/metabolismo , Sequência Conservada , Citoplasma/metabolismo , Entamoeba histolytica/enzimologia , Eritrócitos/parasitologia , Proteínas Ativadoras de GTPase/química , Humanos , Simulação de Dinâmica Molecular , Fagocitose , Transporte Proteico , Proteínas de Protozoários/químicaRESUMO
Entamoeba histolytica is the intestinal parasite responsible for human amoebiasis that is a leading cause of death in developing countries. In this protozoan, heterogeneity in DNA content, polyploidy and genome plasticity have been associated to alterations in mechanisms controlling DNA replication and cell division. Studying the function of the transcription factor EhPC4, we unexpectedly found that it is functionally related to DNA replication, and multinucleation. Site-directed mutagenesis on the FRFPKG motif revealed that the K127 residue is required for efficient EhPC4 DNA-binding activity. Remarkably, overexpression of EhPC4 significantly increased cell proliferation, DNA replication and DNA content of trophozoites. A dramatically increase in cell size resulting in the formation of giant multinucleated trophozoites (polykaryon) was also found. Multinucleation event was associated to cytokinesis failure leading to abortion of ongoing cell division. Consistently, genome-wide profiling of EhPC4 overexpressing trophozoites revealed the up-regulation of genes involved in carbohydrates and nucleic acids metabolism, chromosome segregation and cytokinesis. Forced overexpression of one of these genes, EhNUDC (nuclear movement protein), led to alterations in cytokinesis and partially recapitulated the multinucleation phenotype. These data indicate for the first time that EhPC4 is associated with events related to polyploidy and genome stability in E. histolytica.
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Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Poliploidia , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Proliferação de Células , Segregação de Cromossomos , Sequência Conservada , Citocinese , DNA/genética , DNA/metabolismo , Replicação do DNA , Evolução Molecular , Expressão Gênica , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/químicaRESUMO
Adhesion to cells, cytotoxicity and proteolysis are functions required for virulence and pathogenicity of Entamoeba histolytica. However, there was no correlation between these in vitro functions and the early elimination of non-pathogenic E. dispar and non-virulent E. histolytica (nvEh) in experimental amoebic liver abscesses developed in hamsters. Thus, additional functions may be involved in amoebic pathogenicity and virulence. In the present study, an integral experimental assessment, including innovative technologies for analyses of amoebal pathophysiology, cell biology, biochemistry and transcriptomics, was carried out to elucidate whether other cellular processes are involved in amoebal pathogenicity and virulence. In comparison with virulent E. histolytica, the data indicated that the main reasons for the early clearance of nvEh from hamster liver are decreased intracellular H2 O2 detoxification rate and deficient heat shock protein expression, whereas for E. dispar, it is a relatively lower capacity for O2 reduction. Therefore, maintenance of an intracellular hypoxic environment combined with the induction of an adequate parasite response to oxidative stress are essential requirements for Entamoeba survival in the liver, and therefore for pathogenicity.