RESUMO
BACKGROUND: Cancer stem cells (CSCs) are at the origin of tumour initiation and progression in gastric adenocarcinoma (GC). However, markers of metastasis-initiating cells remain unidentified in GC. In this study, we characterized CD44 variants expressed in GC and evaluated the tumorigenic and metastatic properties of CD44v3+ cells and their clinical significance in GC patients. METHODS: Using GC cell lines and patient-derived xenografts, we evaluated CD44+ and CD44v3+ GC cells molecular signature and their tumorigenic, chemoresistance, invasive and metastatic properties, and expression in patients-derived tissues. RESULTS: CD44v3+ cells, which represented a subpopulation of CD44+ cells, were detected in advanced preneoplastic lesions and presented CSCs chemoresistance and tumorigenic properties in vitro and in vivo. Molecular and functional analyses revealed two subpopulations of gastric CSCs: CD44v3+ CSCs with an epithelial-mesenchymal transition (EMT)-like signature, and CD44+/v3- CSCs with an epithelial-like signature; both were tumorigenic but CD44v3+ cells showed higher invasive and metastatic properties in vivo. CD44v3+ cells detected in the primary tumours of GC patients were associated with a worse prognosis. CONCLUSION: CD44v3 is a marker of a subpopulation of CSCs with metastatic properties in GC. The identification of metastasis-initiating cells in GC represents a major advance for further development of anti-metastatic therapeutic strategies.
Assuntos
Carcinoma , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/patologia , Células-Tronco Neoplásicas/metabolismo , Carcinoma/patologia , Receptores de Hialuronatos , Transição Epitelial-MesenquimalRESUMO
Gastric carcinomas (GC) are heterogeneous tumors, composed of a subpopulation of cluster of differentiation-44 (CD44)+ tumorigenic and chemoresistant cancer stem cells (CSC). YAP1 and TAZ oncoproteins (Y/T) interact with TEA domain family member 1 (TEAD) transcription factors to promote cell survival and proliferation in multiple tissues. Their activity and role in GC remain unclear. This work aimed to analyze Y/T-TEAD activity and molecular signature in gastric CSC, and to assess the effect of verteporfin, a Food and Drug Administration-approved drug preventing Y/T-TEAD interaction, on gastric CSC tumorigenic properties. Y/T-TEAD molecular signature was investigated using bioinformatical (KmPlot database), transcriptomic and immunostaining analyses in patient-derived GC and cell lines. Verteporfin effects on Y/T-TEAD transcriptional activity, CSC proliferation and tumorigenic properties were evaluated using in vitro tumorsphere assays and mouse models of patient-derived GC xenografts. High expressions of YAP1, TAZ, TEAD1, TEAD4 and their target genes were associated with low overall survival in nonmetastatic human GC patients (n = 444). This Y/T-TEAD molecular signature was enriched in CD44+ patient-derived GC cells and in cells resistant to conventional chemotherapy. Verteporfin treatment inhibited Y/T-TEAD transcriptional activity, cell proliferation and CD44 expression, and decreased the pool of tumorsphere-forming CD44+ /aldehyde dehydrogenase (ALDH)high gastric CSC. Finally, verteporfin treatment inhibited GC tumor growth in vivo; the residual tumor cells exhibited reduced expressions of CD44 and ALDH1, and more importantly, they were unable to initiate new tumorspheres in vitro. All these data demonstrate that Y/T-TEAD activity controls gastric CSC tumorigenic properties. The repositioning of verteporfin targeting YAP1/TAZ-TEAD activity could be a promising CSC-based strategy for the treatment of GC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Nucleares/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Transativadores/genética , Fatores de Transcrição/genética , Verteporfina/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Humanos , Receptores de Hialuronatos/biossíntese , Receptores de Hialuronatos/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Nucleares/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Fatores de Transcrição de Domínio TEA , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAPRESUMO
Extracellular vesicles (EVs) have been shown to play an important role in intercellular communication as carriers of DNA, RNA and proteins. While the intercellular transfer of miRNA through EVs has been extensively studied, the stability of extracellular miRNA (ex-miRNA) once engulfed by a recipient cell remains to be determined. Here, we identify the ex-miRNA-directed phenotype to be transient due to the rapid decay of ex-miRNA. We demonstrate that the ex-miR-223-3p transferred from polymorphonuclear leukocytes to cancer cells were functional, as demonstrated by the decreased expression of its target FOXO1 and the occurrence of epithelial-mesenchymal transition reprogramming. We showed that the engulfed ex-miRNA, unlike endogenous miRNA, was unstable, enabling dynamic regulation and a return to a non-invasive phenotype within 8 h. This transient phenotype could be modulated by targeting XRN1/PACMAN exonuclease. Indeed, its silencing was associated with slower decay of ex-miR-223-3p and subsequently prolonged the invasive properties. In conclusion, we showed that the 'steady step' level of engulfed miRNA and its subsequent activity was dependent on the presence of a donor cell in the surroundings to constantly fuel the recipient cell with ex-miRNAs and of XRN1 exonuclease, which is involved in the decay of these imported miRNA.
Assuntos
Transição Epitelial-Mesenquimal/genética , Exorribonucleases/metabolismo , MicroRNAs/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias/genética , Estabilidade de RNA , Linhagem Celular Tumoral , Exossomos/metabolismo , Humanos , Invasividade Neoplásica , Neoplasias/enzimologia , Neoplasias/metabolismo , Neoplasias/patologia , Neutrófilos/metabolismoRESUMO
MicroRNAs (miRNAs) are a recently discovered category of small RNA molecules that regulate gene expression at the post-transcriptional level. Accumulating evidence indicates that miRNAs are aberrantly expressed in a variety of human cancers, thus being oncogenic. The inhibition of oncogenic miRNAs (defined as the blocking of miRNAs' production or function) would find application in the therapy of different types of cancer in which these miRNAs are implicated. In this work, we describe the design and synthesis of new small-molecule RNA ligands with the aim of inhibiting Dicer-mediated processing of oncogenic miRNAs. One of the synthesized compound (4b) composed of the aminoglycoside neomycin conjugated to an artificial nucleobase and to amino acid histidine is able to selectively decrease miR-372 levels in gastric adenocarcinoma (AGS) cells and to restore the expression of the target LATS2 protein. This activity led to the inhibition of proliferation of these cells. The study of the interactions of 4b with pre-miR-372 allowed for the elucidation of the molecular mechanism of the conjugate, thus leading to new perspectives for the design of future inhibitors.
Assuntos
Aminoácidos/química , Carcinogênese , MicroRNAs/biossíntese , Neomicina/química , Neomicina/farmacologia , Purinas/química , Pirimidinas/química , Adenocarcinoma/patologia , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , MicroRNAs/genética , Simulação de Acoplamento Molecular , Neomicina/metabolismo , Conformação de Ácido Nucleico , Neoplasias Gástricas/patologiaRESUMO
MicroRNAs (miRNAs) are a recently discovered category of small RNA molecules that regulate gene expression at the post-transcriptional level. Accumulating evidence indicates that miRNAs are aberrantly expressed in a variety of human cancers and that the inhibition of these oncogenic miRNAs could find application in the therapy of different types of cancer. Herein, we describe the synthesis and biological evaluation of new small-molecule drugs that target oncogenic miRNAs production. In particular, we chose to target two miRNAs (i.e., miRNA-372 and -373) implicated in various types of cancer, such as gastric cancer. Their precursors (pre-miRNAs) are overexpressed in cancer cells and lead to mature miRNAs after cleavage of their stem-loop structure by the enzyme Dicer in the cytoplasm. Some of the newly synthesized conjugates can inhibit Dicer processing of the targeted pre-miRNAs in vitro with increased efficacy relative to our previous results (D.D. Vo et al., ACS Chem. Biol. 2014, 9, 711-721) and, more importantly, to inhibit proliferations of adenocarcinoma gastric cancer (AGS) cells overexpressing these miRNAs, thus representing promising leads for future drug development.
Assuntos
Aminoglicosídeos/química , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Neoplasias Gástricas/química , Evolução Biológica , Sistemas de Liberação de Medicamentos , Humanos , MicroRNAs/química , Modelos Moleculares , Nebramicina/análogos & derivados , Nebramicina/química , Neomicina/química , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Relação Estrutura-AtividadeRESUMO
MicroRNAs, small non-coding RNAs expressed by eukaryotic cells, play pivotal roles in shaping cell differentiation and organism development. Deregulated microRNA expression is associated with several types of diseases including cancers, immune disorders and infection. Acting at the post-transcriptional level, miRNAs have expanded our understanding of the control of gene expression in regulatory networks involved in the adaptation to environmental situations such as biotic stress. It is increasingly clear that miRNAs are an important part of the host response to microbes. This review presents the current state of knowledge about the role of miRNAs in the response to both bacterial pathogens and commensal bacteria in human cells or animal experimental models. Some microRNAs, including miR-146, miR-155, miR-125, let-7 and miR-21, are commonly affected during bacterial infection and contribute to immune responses protecting the organism against overwhelmed inflammation. Cell-specific relationships between miRNAs and their targets are also engaged in the alterations induced by virulent bacteria in the proliferation/differentiation/apoptosis pathways of their host cells. In a separate role, miRNA modulation also represents a mechanism through which commensal bacteria impact the regulation of the barrier function and intestinal homeostasis.
Assuntos
Bactérias/imunologia , Infecções Bacterianas/imunologia , Interações Hospedeiro-Patógeno , MicroRNAs/metabolismo , Animais , Humanos , Evasão da Resposta Imune , ImunidadeRESUMO
UNLABELLED: We performed a review of public microarray data that revealed a significant down-regulation of Rnd3 expression in hepatocellular carcinoma (HCC), as compared to nontumor liver. Rnd3/RhoE is an atypical RhoGTPase family member because it is always under its active GTP-bound conformation and not sensitive to classical regulators. Rnd3 down-regulation was validated by quantitative real-time polymerase chain reaction in 120 independent tumors. Moreover, Rnd3 down-expression was confirmed using immunohistochemistry on tumor sections and western blotting on human tumor and cell-line extracts. Rnd3 expression was significantly lower in invasive tumors with satellite nodules. Overexpression and silencing of Rnd3 in Hep3B cells led to decreased and increased three-dimensional cell motility, respectively. The short interfering RNA-mediated down-regulation of Rnd3 expression induced a loss of E-cadherin at cell-cell junctions that was linked to epithelial-mesenchymal transition through the up-regulation of the zinc finger E-box binding homeobox protein, ZEB2, and the down-regulation of miR-200b and miR-200c. Rnd3 knockdown mediated tumor hepatocyte invasion in a matrix-metalloproteinase-independent, and Rac1-dependent manner. CONCLUSION: Rnd3 down-regulation provides an invasive advantage to tumor hepatocytes, suggesting that RND3 might represent a metastasis suppressor gene in HCC.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteínas rho de Ligação ao GTP/fisiologia , Caderinas/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Transição Epitelial-Mesenquimal , Proteínas de Homeodomínio/fisiologia , Humanos , Invasividade Neoplásica , Proteínas Repressoras/fisiologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Amphiphilic block copolymers are molecules composed of hydrophilic and hydrophobic segments having the capacity to spontaneously self-assemble into a variety of supramolecular structures like micelles and vesicles. Here, we propose an original way to self-assemble amphiphilic block copolymers into a supported bilayer membrane for defined coating of nanoparticles. The heart of the method rests on a change of the amphiphilicity of the copolymer that can be turned off and on by varying the polarity of the solvent. In this condition, the assembly process can take advantage of specific molecular interactions in both organic solvent and water. While the concept potentially could be applied to any type of charged substrates, we focus our interest on the design of a new type of polymer assembly mimicking the virus morphology. A capsid-like shell of glycoprotein-mimic amphiphilic block copolymer was self-assembled around a positively charged complex of siRNA and polyethyleneimine. The process requires two steps. Block copolymers first interact with the complexes dispersed in DMSO through electrostatic interactions. Next, the increase of the water content in the medium triggers the hydrophobic effect and the concomitant self-assembly of free block copolymer molecules into a bilayer membrane at the complex surface. The higher gene silencing activity of the copolymer-modified complexes over the complexes alone shows the potential of this new type of nanoconstructs for biological applications, especially for the delivery of therapeutic biomolecules.
Assuntos
Polietilenoimina/química , RNA Interferente Pequeno/química , Eletrólitos/química , Interações Hidrofóbicas e Hidrofílicas , Polietilenoimina/síntese química , EstereoisomerismoRESUMO
The replication of the genomic RNA of the hepatitis C virus (HCV) of positive polarity involves the synthesis of a replication intermediate of negative polarity by the viral RNA-dependent RNA polymerase (NS5B). In vitro and likely in vivo, the NS5B initiates RNA synthesis without primers. This de novo mechanism needs specific interactions between the polymerase and viral RNA elements. Cis-acting elements involved in the initiation of (-) RNA synthesis have been identified in the 3' non-coding region and in the NS5B coding region of the HCV RNA. However, the detailed contribution of sequences and/or structures of (-) RNA involved in the initiation of (+) RNA synthesis has been less studied. In this report, we identified an RNA element localized between nucleotides 177 and 222 from the 3'-end of the (-) RNA that is necessary for efficient initiation of RNA synthesis by the recombinant NS5B. By site-directed mutagenesis experiments, we demonstrate that the structure rather than the primary sequence of this domain is important for RNA synthesis. We also demonstrate that the intact structure of this RNA element is also needed for efficient RNA synthesis when the viral NS5B functions in association with other viral and cellular proteins in cultured hepatic cells.
Assuntos
Hepacivirus/genética , RNA Viral/biossíntese , RNA Viral/química , Sequências Reguladoras de Ácido Ribonucleico , Sequência de Bases , Linhagem Celular , Hepacivirus/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Conformação de Ácido NucleicoRESUMO
The intracellular transport of drugs and therapeutics represents one of the most exciting and challenging areas at the interface of chemistry, biology, and medicine. Most of the effort in this field so far has been devoted to the development of peptide-based delivery systems that can translocate therapeutic agents into their intracellular targets. More recently, the use of bioinspired non-natural foldamers has resulted in the successful delivery of cargo molecules, which possess a wide range of sizes and physicochemical properties across the cell membrane. We report herein the synthesis of aromatic amide foldamers and their biological evaluation as cell-penetrating agents. By using a well-established synthetic route, a series of fluorescein-labeled cationic aryl amide conjugates has been constructed, and their cellular uptake into various human cell lines has been analyzed by flow cytometry and fluorescence microscopy. The assays revealed that longer oligomers achieve greater cellular translocation, with octamer Q8 proving to be a remarkable vehicle for all three cell lines. Biological studies have also indicated that these helices are biocompatible, thus showing promise in their application as cell-penetrating agents and as vehicles to deliver biologically active molecules into cells.
Assuntos
Amidas/metabolismo , Membrana Celular/química , Peptídeos/metabolismo , Amidas/síntese química , Amidas/química , Sobrevivência Celular/fisiologia , Endocitose/fisiologia , Citometria de Fluxo , Células HeLa , Humanos , Células Jurkat , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Peptídeos/síntese química , Peptídeos/química , Estrutura Secundária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We developed a functional selection system based on randomized genetic elements (GE) to identify potential regulators of hepatitis C virus (HCV) RNA translation, a process initiated by an internal ribosomal entry site (IRES). A retroviral HCV GE library was introduced into HepG2 cells, stably expressing the Herpes simplex virus thymidine kinase (HSV-TK) under the control of the HCV IRES. Cells that expressed transduced GEs inhibiting HSV-TK were selected via their resistance to ganciclovir. Six major GEs were rescued by PCR on the selected cell DNA and identified as HCV elements. We validated our strategy by further studying the activity of one of them, GE4, encoding the 5' end of the viral NS5A gene. GE4 inhibited HCV IRES-, but not cap-dependent, reporter translation in human hepatic cell lines and inhibited HCV infection at a post-entry step, decreasing by 85% the number of viral RNA copies. This method can be applied to the identification of gene expression regulators.
Assuntos
Regulação Viral da Expressão Gênica , Hepacivirus/genética , Biossíntese de Proteínas , Proteínas não Estruturais Virais/genética , Regiões 5' não Traduzidas/química , Linhagem Celular , Clonagem Molecular , Biblioteca Gênica , Humanos , RNA Viral/química , Sequências Reguladoras de Ácido RibonucleicoRESUMO
BACKGROUND & AIMS: Gastric carcinoma is related mostly to CagA+-Helicobacter pylori infection, which disrupts the gastric mucosa turnover and elicits an epithelial-mesenchymal transition (EMT) and preneoplastic transdifferentiation. The tumor suppressor Hippo pathway controls stem cell homeostasis; its core, constituted by the large tumor suppressor 2 (LATS2) kinase and its substrate Yes-associated protein 1 (YAP1), was investigated in this context. METHODS: Hippo, EMT, and intestinal metaplasia marker expression were investigated by transcriptomic and immunostaining analyses in human gastric AGS and MKN74 and nongastric immortalized RPE1 and HMLE epithelial cell lines challenged by H pylori, and on gastric tissues of infected patients and mice. LATS2 and YAP1 were silenced using small interfering RNAs. A transcriptional enhanced associated domain (TEAD) reporter assay was used. Cell proliferation and invasion were evaluated. RESULTS: LATS2 and YAP1 appear co-overexpressed in the infected mucosa, especially in gastritis and intestinal metaplasia. H pylori via CagA stimulates LATS2 and YAP1 in a coordinated biphasic pattern, characterized by an early transient YAP1 nuclear accumulation and stimulated YAP1/TEAD transcription, followed by nuclear LATS2 up-regulation leading to YAP1 phosphorylation and targeting for degradation. LATS2 and YAP1 reciprocally positively regulate each other's expression. Loss-of-function experiments showed that LATS2 restricts H pylori-induced EMT marker expression, invasion, and intestinal metaplasia, supporting a role of LATS2 in maintaining the epithelial phenotype of gastric cells and constraining H pylori-induced preneoplastic changes. CONCLUSIONS: H pylori infection engages a number of signaling cascades that alienate mucosa homeostasis, including the Hippo LATS2/YAP1/TEAD pathway. In the host-pathogen conflict, which generates an inflammatory environment and perturbations of the epithelial turnover and differentiation, Hippo signaling appears as a protective pathway, limiting the loss of gastric epithelial cell identity that precedes gastric carcinoma development.
Assuntos
Transição Epitelial-Mesenquimal/imunologia , Mucosa Gástrica/patologia , Infecções por Helicobacter/patologia , Lesões Pré-Cancerosas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Proteínas de Ciclo Celular/metabolismo , Feminino , Mucosa Gástrica/microbiologia , Regulação Neoplásica da Expressão Gênica/imunologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Masculino , Metaplasia/genética , Metaplasia/microbiologia , Metaplasia/patologia , Camundongos , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/imunologia , Fatores de Proteção , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/patologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas de Sinalização YAPRESUMO
Helicobacter pylori infection, the main risk factor for gastric cancer (GC), leads to an epithelial-mesenchymal transition (EMT) of gastric epithelium contributing to gastric cancer stem cell (CSC) emergence. The Hippo pathway effectors yes-associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ) control cancer initiation and progression in many cancers including GC. Here, we investigated the role of TAZ in the early steps of H. pylori-mediated gastric carcinogenesis. TAZ implication in EMT, invasion, and CSC-related tumorigenic properties were evaluated in three gastric epithelial cell lines infected by H. pylori. We showed that H. pylori infection increased TAZ nuclear expression and transcriptional enhancer TEA domain (TEAD) transcription factors transcriptional activity. Nuclear TAZ and zinc finger E-box-binding homeobox 1 (ZEB1) were co-overexpressed in cells harboring a mesenchymal phenotype in vitro, and in areas of regenerative hyperplasia in gastric mucosa of H. pylori-infected patients and experimentally infected mice, as well as at the invasive front of gastric carcinoma. TAZ silencing reduced ZEB1 expression and EMT phenotype, and strongly inhibited invasion and tumorsphere formation induced by H. pylori. In conclusion, TAZ activation in response to H. pylori infection contributes to H. pylori-induced EMT, invasion, and CSC-like tumorigenic properties. TAZ overexpression in H. pylori-induced pre-neoplastic lesions and in GC could therefore constitute a biomarker of early transformation in gastric carcinogenesis.
Assuntos
Células Epiteliais/metabolismo , Mucosa Gástrica/patologia , Infecções por Helicobacter/metabolismo , Células-Tronco Neoplásicas/patologia , Animais , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/fisiologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/patologia , Helicobacter pylori/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Camundongos , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Cancer stem cells (CSCs) present chemo-resistance mechanisms contributing to tumour maintenance and recurrence, making their targeting of utmost importance in gastric cancer (GC) therapy. The Hippo pathway has been implicated in gastric CSC properties and was shown to be regulated by leukaemia inhibitory factor receptor (LIFR) and its ligand LIF in breast cancer. This study aimed to determine LIF's effect on CSC properties in GC cell lines and patient-derived xenograft (PDX) cells, which remains unexplored. LIF's treatment effect on CSC markers expression and tumoursphere formation was evaluated. The Hippo kinase inhibitor XMU-MP-1 and/or the JAK1 inhibitor Ruxolitinib were used to determine Hippo and canonical JAK/STAT pathway involvement in gastric CSCs' response to LIF. Results indicate that LIF decreased tumorigenic and chemo-resistant CSCs, in both GC cell lines and PDX cells. In addition, LIF increased activation of LATS1/2 Hippo kinases, thereby decreasing downstream YAP/TAZ nuclear accumulation and TEAD transcriptional activity. LIF's anti-CSC effect was reversed by XMU-MP-1 but not by Ruxolitinib treatment, highlighting the opposite effects of these two pathways downstream LIFR. In conclusion, LIF displays anti-CSC properties in GC, through Hippo kinases activation, and could in fine constitute a new CSCs-targeting strategy to help decrease relapse cases and bad prognosis in GC.
RESUMO
Gastric MALT lymphoma (GML) is directly caused by Helicobacter pylori infection but occurs only in a small number of infected subjects. Mechanisms underlying the initiation and progression of GML remain unclear. MicroRNAs (miRNAs) are small non-coding RNAs that are now considered as major players in inflammation and carcinogenesis, acting as oncogenes or tumor suppressors. Previous laboratory studies have shown in a GML mouse model that overexpression of a distinct set of five miRNAs (miR-21a, miR-135b, miR-142a, miR-150, miR-155) could play a critical role in the pathogenesis of GML. Our goal was to compare the miRNA expression profile obtained in the GML mouse model to that in human GML (11 cases of GML compared to 17 cases of gastritis control population). RTqPCR on the five dysregulated miRNAs in the GML mouse model and PCR array followed by RTqPCR confirmation showed that four miRNAs were up-regulated (miR-150, miR-155, miR-196a, miR-138) and two miRNAs down-regulated (miR-153, miR-7) in the stomachs of GML patients vs. gastritis control population. The analysis of their validated targets allowed us to postulate that these miRNAs (except miR-138) could act synergistically in a common signaling cascade promoting lymphomagenesis and could be involved in the pathogenesis of GML.
RESUMO
We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.
Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/patogenicidade , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Linhagem Celular , Linhagem Celular Tumoral , Hepacivirus/enzimologia , Hepacivirus/genética , Humanos , RNA Viral/efeitos dos fármacos , Técnica de Seleção de Aptâmeros , Transfecção , Proteínas não Estruturais Virais/genética , Vírion/metabolismo , Replicação ViralRESUMO
Although the application of sorafenib, a small inhibitor of tyrosine protein kinases, to cancer treatments remains a worldwide option in chemotherapy, novel strategies are needed to address the low water solubility (< 5 µM), toxicity, and side effects issues of this drug. In this context, the use of nanocarriers is currently investigated in order to overcome these drawbacks. In this contribution, we report a new type of sorafenib-based nanoparticles stabilized by hybrid nucleoside-lipids. The solid lipid nanoparticles (SLNs) showed negative or positive zeta potential values depending on the nucleoside-lipid charge. Transmission electron microscopy of sorafenib-loaded SLNs revealed parallelepiped nanoparticles of about 200 nm. Biological studies achieved on four different cell lines, including liver and breast cancers, revealed enhanced anticancer activities of Sorafenib-based SLNs compared to the free drug. Importantly, contrast phase microscopy images recorded after incubation of cancer cells in the presence of SLNs at high concentration in sorafenib (> 80 µM) revealed a total cancer cell death in all cases. These results highlight the potential of nucleoside-lipid-based SLNs as drug delivery systems.
RESUMO
MicroRNAs are key factors in the regulation of gene expression and their deregulation has been directly linked to various pathologies such as cancer. The use of small molecules to tackle the overexpression of oncogenic miRNAs has proved its efficacy and holds the promise for therapeutic applications. Here we describe the screening of a 640-compound library and the identification of polyamine derivatives interfering with in vitro Dicer-mediated processing of the oncogenic miR-372 precursor (pre-miR-372). The most active inhibitor is a spermine-amidine conjugate that binds to the pre-miR-372 with a KD of 0.15 µM, and inhibits its in vitro processing with a IC50 of 1.06 µM. The inhibition of miR-372 biogenesis was confirmed in gastric cancer cells overexpressing miR-372 and a specific inhibition of proliferation through de-repression of the tumor suppressor LATS2 protein, a miR-372 target, was observed. This compound modifies the expression of a small set of miRNAs and its selective biological activity has been confirmed in patient-derived ex vivo cultures of gastric carcinoma. Polyamine derivatives are promising starting materials for future studies about the inhibition of oncogenic miRNAs and, to the best of our knowledge, this is the first report about the application of functionalized polyamines as miRNAs interfering agents.
Assuntos
Antineoplásicos/farmacologia , MicroRNAs/metabolismo , Poliaminas/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Poliaminas/isolamento & purificação , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Supressoras de Tumor/biossínteseRESUMO
Helicobacter pylori infection is considered as an excellent model of chronic inflammation-induced tumor development. Our project focuses on gastric MALT lymphoma (GML) related to H. pylori infection and mediated by the chronic inflammatory process initiated by the infection. Recently, microRNAs (miRNAs) have emerged as a new class of gene regulators, which play key roles in inflammation and carcinogenesis acting as oncogenes or tumor suppressors. Their precise characterization in the development of inflammation and their contribution in regulating host cells responses to infection by H. pylori have been little explored. Our goal was to analyze the changes in miRNAs in a GML mouse model using BALB/c mice thymectomized at day 3 post-birth (d3Tx model) and to clarify their implication in GML pathogenesis. PCR array followed by RT-qPCR identified five miRNAs (miR-21a, miR-135b, miR-142a, miR-150, miR-155) overexpressed in the stomachs of GML-developing d3Tx mice infected by H. pylori. The analysis of their putative targets allowed us to identify TP53INP1, an anti-proliferative and pro-apoptotic protein, as a common target of 4 of the 5 up-regulated miRNAs. We postulate that these miRNAs may act in synergy to promote the development of GML. miR-142a was also overexpressed in mouse sera samples and therefore could serve as a diagnostic marker. In situ hybridization on gastric samples with miR-142a revealed a global up-regulation of this miRNA by the tumor microenvironment at the lymphoma stage. Dysregulation of miR-21a, miR-135b, miR-142a, miR-150, miR-155 could play a critical role in the pathogenesis of GML and might offer potential applications as therapeutic targets and novel biomarkers for this disease.