RESUMO
PURPOSE OF REVIEW: Claudins, components of tight cell junctions in epithelial and endothelial cells, have emerged as a therapeutic target in gastrointestinal (GI) malignancies, particularly claudin 18.2 (CLDN18.2). RECENT FINDINGS: Zolbetuximab, a chimeric anti-CLDN18.2 monoclonal antibody (mAb), is currently under FDA review and may emerge as the first claudin targeted therapy approved. Phase 3 trials show that zolbetuximab in combination with front-line fluoropyrimidine plus oxaliplatin improves survival in advanced CLDN18.2 positive (≥75% of tumor cells) gastric adenocarcinoma (GAC) patients. Many other therapies (mAbs; CART; bispecific; ADCs) are under investigation. SUMMARY: CLDN18.2 will be an important target in GAC. Early understanding of how to target CLDN18.2 based on the level of expression (high, moderate, low) will be the key to success in this area. Studying these as separate entities should be considered. Resistance patterns, loss of CLDN18.2 expression, role in the refractory setting, and if any role in localized disease are questions that remain. Other targets for claudin that target claudin six and four are under investigation. Their role in GI malignancies will soon be further clarified.
Assuntos
Claudinas , Neoplasias Gastrointestinais , Humanos , Claudinas/antagonistas & inibidores , Claudinas/metabolismo , Ensaios Clínicos Fase III como Assunto , Neoplasias Gastrointestinais/tratamento farmacológico , Terapia de Alvo MolecularRESUMO
Tight junctions (TJs) are an important component of cell connectivity; they maintain cell polarity, permeability and adhesion, and participate in the regulation of cell proliferation and differentiation. The claudin (CLDN) family is integral to TJs, and CLDN6 is an important member of this family. Abnormal expression of CLDN6 can destroy the integrity of TJs through various mechanisms and can serve multiple roles in the occurrence and development of tumours. CLDN6 is widely expressed in various tumours but rarely expressed in healthy adult tissues. The aim of this review is to critically examine the recent literature on CLDN6, including its structure, expression in different tumours, regulatory mechanisms and therapeutic prospects. Although some conclusions are controversial, in certain tumours, such as liver, ovarian, endometrial and oesophageal cancer, and atypical teratoid/rhabdoid tumours, research consistently shows that CLDN6 is expressed in tumour tissues but is not expressed or is expressed at low levels in surrounding tissues. In these tumours, CLDN6 has potential as a carcinoembryonic antigen and a therapeutic target.
Assuntos
Claudinas/genética , Claudinas/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Animais , Proliferação de Células/genética , Claudinas/antagonistas & inibidores , Claudinas/química , Resistencia a Medicamentos Antineoplásicos , Humanos , Junções Íntimas/fisiologiaRESUMO
Claudin-2 promotes breast cancer liver metastasis by enabling seeding and early cancer cell survival. We now demonstrate that Claudin-2 is functionally required for colorectal cancer liver metastasis and that Claudin-2 expression in primary colorectal cancers is associated with poor overall and liver metastasis-free survival. We have examined the role of Claudin-2, and other claudin family members, as potential prognostic biomarkers of the desmoplastic and replacement histopathological growth pattern associated with colorectal cancer liver metastases. Immunohistochemical analysis revealed higher Claudin-2 levels in replacement type metastases when compared to those with desmoplastic features. In contrast, Claudin-8 was highly expressed in desmoplastic colorectal cancer liver metastases. Similar observations were made following immunohistochemical staining of patient-derived xenografts (PDXs) that we have established, which faithfully retain the histopathology of desmoplastic or replacement type colorectal cancer liver metastases. We provide evidence that Claudin-2 status in patient-derived extracellular vesicles may serve as a relevant prognostic biomarker to predict whether colorectal cancer patients have developed replacement type liver metastases. Such a biomarker will be a valuable tool in designing optimal treatment strategies to better manage patients with colorectal cancer liver metastases.
Assuntos
Biomarcadores Tumorais/fisiologia , Claudinas/fisiologia , Neoplasias Colorretais/secundário , Neoplasias Hepáticas/patologia , Animais , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Adesão Celular/genética , Adesão Celular/fisiologia , Claudinas/antagonistas & inibidores , Claudinas/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/fisiopatologia , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Células HT29 , Hepatócitos/patologia , Xenoenxertos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/fisiopatologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos SCID , Domínios PDZ/genética , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
OBJECTIVE: Lithium chloride (LiCl) was a mood stabilizer for bipolar affective disorders and it could activate Wnt/ß-catenin signaling pathway both in vivo and in vitro. Colon is one of a very susceptible tissues to Wnt signaling pathway, and so it would be very essential to explore the toxic effect of a high dose of LiCl on colon. METHODS: C57BL/6 mice were injected intraperitoneally with 200 mg/kg LiCl one dose a day for 5 days to activate Wnt signal pathway in intestines. H&E staining was used to assess the colonic tissues of mice treated with high dose of LiCl. The expression of inflammation-associated genes and tight junction-associated genes in colons was measured using qPCR, Western blot and immunostaining methods. The gut microbiome was tested through 16S rDNA gene analysis. RESULTS: The differentiation of enteroendocrine cells in colon was inhibited by treatment of 200 mg/kg LiCl. The F4/80 positive macrophages in colon were activated by high dose of LiCl, and migrated from the submucosa to the lamina propria. The expression of pro-inflammatory genes TNFα and IL-1ß was increased in the colon of high dose of LiCl treated mice. Clostridium_sp_k4410MGS_306 and Prevotellaceae_UCG_001 were specific and predominant for the high dose of LiCl treated mice. The expression of IgA coding genes, Pigr and Claudin-15 was significantly decreased in the colon tissues of the high dose of LiCl treated mice. CONCLUSION: 200 mg/kg LiCl might cause the inflammation in colon of mice through activating F4/80 positive macrophages and inhibiting the expression of IgA coding genes in plasma cells and the expression of Pigr and Claudin-15 in colonic epithelial cells, providing evidences for the toxic effects of high dose of LiCl on colon.
Assuntos
Claudinas/antagonistas & inibidores , Colite/induzido quimicamente , Colo/efeitos dos fármacos , Cloreto de Lítio/toxicidade , Macrófagos/efeitos dos fármacos , Receptores de Superfície Celular/antagonistas & inibidores , Animais , Antimaníacos/administração & dosagem , Antimaníacos/toxicidade , Claudinas/biossíntese , Colite/metabolismo , Colite/patologia , Colo/metabolismo , Colo/patologia , Disbiose/induzido quimicamente , Disbiose/metabolismo , Disbiose/patologia , Expressão Gênica , Cloreto de Lítio/administração & dosagem , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/biossíntese , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/fisiologiaRESUMO
Chimeric antigen receptor (CAR)-T cells have shown efficacy in patients with B cell malignancies. Yet, their application for solid tumors has challenges that include limited cancer-specific targets and nonpersistence of adoptively transferred CAR-T cells. Here, we introduce the developmentally regulated tight junction protein claudin 6 (CLDN6) as a CAR target in solid tumors and a strategy to overcome inefficient CAR-T cell stimulation in vivo. We demonstrate that a nanoparticulate RNA vaccine, designed for body-wide delivery of the CAR antigen into lymphoid compartments, stimulates adoptively transferred CAR-T cells. Presentation of the natively folded target on resident antigen-presenting cells promotes cognate and selective expansion of CAR-T cells. Improved engraftment of CAR-T cells and regression of large tumors in difficult-to-treat mouse models was achieved at subtherapeutic CAR-T cell doses.
Assuntos
Vacinas Anticâncer/uso terapêutico , Claudinas/antagonistas & inibidores , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/imunologia , Animais , Claudinas/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA/uso terapêutico , Linfócitos T/imunologia , Linfócitos T/transplante , Vacinas Sintéticas/uso terapêuticoRESUMO
Claudin (CLDN) proteins, a tetra-transmembrane family containing over 20 members, have been identified as key structural and functional components of intercellular seals, tight junctions (TJs). CLDNs are involved in the barrier and fence functions of TJs. Loosening the TJ barrier is one strategy for increasing drug absorption and delivery to the brain. Due to aberrant CLDN expression, the TJ fence function is frequently dysregulated in carcinogenesis. In addition, CLDN-1 is a co-receptor for the hepatitis C virus. Together these characteristics indicate CLDNs as promising targets for drug development, and CLDN binders are potential candidates for delivering drugs, treating cancer, and preventing viral infection. Before 2008, a receptor-binding fragment of Clostridium perfringens enterotoxin was the only CLDN binder available. Since then, several challenges regarding the generation of monoclonal antibodies against CLDNs have been surmounted, leading to breakthroughs in CLDN-targeted drug development. Here, we provide an overview of the recent progress in technology using created CLDN binders-anti-CLDN monoclonal antibodies.
Assuntos
Autoanticorpos/metabolismo , Claudinas/antagonistas & inibidores , Claudinas/metabolismo , Desenvolvimento de Medicamentos/tendências , Preparações Farmacêuticas/metabolismo , Sequência de Aminoácidos , Animais , Autoanticorpos/genética , Claudinas/genética , HumanosRESUMO
Clostridium perfringens enterotoxin (CPE) causes food poisoning and antibiotic-associated diarrhea. It uses some claudin tight junction proteins (eg, claudin-4) as receptors to form Ca2+-permeable pores in the membrane, damaging epithelial cells in small intestine and colon. We demonstrate that only a subpopulation of colonic enterocytes which are characterized by apical dislocation of claudins are CPE-susceptible. CPE-mediated damage was enhanced if paracellular barrier was impaired by Ca2+ depletion, proinflammatory cytokine tumor necrosis factor α, or dedifferentiation. Microscopy, Ca2+ monitoring, and electrophysiological data showed that CPE-mediated cytotoxicity and barrier disruption was limited by extent of CPE-binding. The latter was restricted by accessibility of non-junctional claudin molecules such as claudin-4 at apical membranes. Focal-leaks detected in HT-29/B6 colonic monolayers were verified for native tissue using colon biopsies. These mechanistic findings indicate how CPE-mediated effects may turn from self-limiting diarrhea into severe clinical manifestation such as colonic necrosis-if intestinal barrier dysfunction, eg, during inflammation facilitates claudin accessibility.
Assuntos
Claudinas/antagonistas & inibidores , Infecções por Clostridium/patologia , Clostridium perfringens/patogenicidade , Colo/patologia , Enterotoxinas/toxicidade , Doenças Transmitidas por Alimentos/patologia , Junções Íntimas/patologia , Linhagem Celular , Enterócitos/patologia , Humanos , Mucosa Intestinal/patologia , PermeabilidadeRESUMO
The downregulation of tight junction protein CLDN6 promotes breast cancer cell migration and invasion; however, the exact mechanism underlying CLDN6 downregulation remains unclear. CLDN6 silence is associated with DNA methyltransferase 1 (DNMT1) mediated DNA methylation, and DNMT1 is regulated by the transforming growth factor beta (TGFß)/SMAD pathway. Therefore, we hypothesized that TGFß/SMAD pathway, specifically SMAD2, may play a critical role for CLDN6 downregulation through DNA methyltransferase 1 (DNMT1) mediated DNA methylation. To test this hypothesis, we blocked the SMAD2 pathway with SB431542 in two human breast cancer cell lines (MCF-7 and SKBR-3). Our results showed that treatment with SB431542 led to a decrease of DNMT1 expression and the binding activity for CLDN6 promoter. The methylation level of CLDN6 promoter was decreased, and simultaneously CLDN6 protein expression increased. Upregulation of CLDN6 inhibited epithelial to mesenchymal transition (EMT) and reduced the migration and invasion ability of both MCF-7 and SKBR-3 cells. Furthermore, knocked down of CLDN6 abolished SB431542 effects on suppression of EMT associated gene expression and inhibition of migration and invasion. Thus, we demonstrated that the downregulation of CLDN6 is regulated through promoter methylation by DNMT1, which depends on the SMAD2 pathway, and that CLDN6 is a key regulator in the SMAD2/DNMT1/CLDN6 pathway to inhibit EMT, migration and invasion of breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Claudinas/genética , Proteína Smad2/genética , Neoplasias da Mama/patologia , Movimento Celular/genética , Proliferação de Células/genética , Claudinas/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Transdução de Sinais/genéticaRESUMO
With further understanding of the biology of gastric and gastroesophageal adenocarcinomas, strides are being made to find effective treatments through novel trial designs. This article focuses on the ongoing trials of drugs targeting specific hallmarks of gastric and gastroesophageal cancers, including oncogene addiction proliferative pathways (fibroblast growth factor receptor 2 amplified tumors), stem cell inhibition, apoptotic induction through claudin inhibitors, and matrix metalloproteinase inhibition. In developing novel therapeutics in treatment of patients with gastroesophageal adenocarcinomas, parallel research efforts to refine target population and biomarkers are crucial, and targeting the tumor genomics and microenvironment may be key in improving overall survival.
Assuntos
Adenocarcinoma , Antineoplásicos/uso terapêutico , Neoplasias Esofágicas , Proteínas de Neoplasias , Neoplasias Gástricas , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Claudinas/antagonistas & inibidores , Claudinas/genética , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Gelatinases/antagonistas & inibidores , Gelatinases/genética , Gelatinases/metabolismo , Amplificação de Genes , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
Targeted therapy and immunotherapy have revolutionized treatment of various cancers in the past decade. Despite targeted therapy with trastuzumab in Her2-positive gastric cancer patients, survival has been dismal, mostly due to disease progression and toxicity related to the treatments. One area of active development is looking for ideal monoclonal antibodies (IMAB) specific to the proteins only on the tumor and hence avoiding unnecessary side effects. Claudin proteins with isoform 2 are one such protein, specific for several cancers, particularly gastric cancer and its metastases, leading to the development of anti-claudin 18.2 specific antibody, claudiximab. This review will highlight the latest development of claudiximab as first in class IMAB for the treatment of gastric cancer.
Assuntos
Adenocarcinoma/terapia , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Claudinas/imunologia , Terapia de Alvo Molecular , Neoplasias Gástricas/terapia , Adenocarcinoma/química , Adenocarcinoma/imunologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/imunologia , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/efeitos adversos , Antineoplásicos Imunológicos/imunologia , Biomarcadores Tumorais , Claudinas/análise , Claudinas/antagonistas & inibidores , Ensaios Clínicos como Assunto , Relação Dose-Resposta Imunológica , Neoplasias Esofágicas/química , Neoplasias Esofágicas/imunologia , Neoplasias Esofágicas/terapia , Feminino , Previsões , Humanos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Masculino , Isoformas de Proteínas/análise , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/imunologia , Neoplasias Gástricas/química , Neoplasias Gástricas/imunologia , Resultado do TratamentoRESUMO
Non-small cell lung cancer (NSCLC) has a 5-y survival rate of â¼16%, with most deaths associated with uncontrolled metastasis. We screened for stem cell identity-related genes preferentially expressed in a panel of cell lines with high versus low metastatic potential, derived from NSCLC tumors of Kras(LA1/+);P53(R172HΔG/+) (KP) mice. The Musashi-2 (MSI2) protein, a regulator of mRNA translation, was consistently elevated in metastasis-competent cell lines. MSI2 was overexpressed in 123 human NSCLC tumor specimens versus normal lung, whereas higher expression was associated with disease progression in an independent set of matched normal/primary tumor/lymph node specimens. Depletion of MSI2 in multiple independent metastatic murine and human NSCLC cell lines reduced invasion and metastatic potential, independent of an effect on proliferation. MSI2 depletion significantly induced expression of proteins associated with epithelial identity, including tight junction proteins [claudin 3 (CLDN3), claudin 5 (CLDN5), and claudin 7 (CLDN7)] and down-regulated direct translational targets associated with epithelial-mesenchymal transition, including the TGF-ß receptor 1 (TGFßR1), the small mothers against decapentaplegic homolog 3 (SMAD3), and the zinc finger proteins SNAI1 (SNAIL) and SNAI2 (SLUG). Overexpression of TGFßRI reversed the loss of invasion associated with MSI2 depletion, whereas overexpression of CLDN7 inhibited MSI2-dependent invasion. Unexpectedly, MSI2 depletion reduced E-cadherin expression, reflecting a mixed epithelial-mesenchymal phenotype. Based on this work, we propose that MSI2 provides essential support for TGFßR1/SMAD3 signaling and contributes to invasive adenocarcinoma of the lung and may serve as a predictive biomarker of NSCLC aggressiveness.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Claudinas/antagonistas & inibidores , Neoplasias Pulmonares/patologia , Proteínas de Ligação a RNA/fisiologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular Tumoral , Claudinas/fisiologia , Humanos , Camundongos , Metástase NeoplásicaRESUMO
STUDY QUESTION: Are Sertoli cell tight junctions (TJs) disrupted in men undergoing hormonal contraception? SUMMARY ANSWER: Localization of the key Sertoli cell TJ protein, claudin-11, was markedly disrupted by 8 weeks of gonadotropin suppression, the degree of which was related to the extent of adluminal germ cell suppression. WHAT IS KNOWN ALREADY: Sertoli cell TJs are vital components of the blood-testis barrier (BTB) that sequester developing adluminal meiotic germ cells and spermatids from the vascular compartment. Claudin-11 knockout mice are infertile; additionally claudin-11 is spatially disrupted in chronically gonadotropin-suppressed rats coincident with a loss of BTB function, and claudin-11 is disorganized in various human testicular disorders. These data support the Sertoli cell TJ as a potential site of hormonal contraceptive action. STUDY DESIGN, SIZE, DURATION: BTB proteins were assessed by immunohistochemistry (n = 16 samples) and mRNA (n = 18 samples) expression levels in available archived testis tissue from a previous study of 22 men who had undergone 8 weeks of gonadotropin suppression and for whom meiotic and post-meiotic germ cell numbers were available. The gonadotropin suppression regimens were (i) testosterone enanthate (TE) plus the GnRH antagonist, acyline (A); (ii) TE + the progestin, levonorgestrel, (LNG); (iii) TE + LNG + A or (iv) TE + LNG + the 5α-reductase inhibitor, dutasteride (D). A control group consisted of seven additional men, with three archived samples available for this study. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Immunohistochemical localization of claudin-11 (TJ) and other junctional type markers [ZO-1 (cytoplasmic plaque), ß-catenin (adherens junction), connexin-43 (gap junction), vinculin (ectoplasmic specialization) and ß-actin (cytoskeleton)] and quantitative PCR was conducted using matched frozen testis tissue. MAIN RESULTS AND THE ROLE OF CHANCE: Claudin-11 formed a continuous staining pattern at the BTB in control men. Regardless of gonadotropin suppression treatment, claudin-11 localization was markedly disrupted and was broadly associated with the extent of meiotic/post-meiotic germ cell suppression; claudin-11 staining was (i) punctate (i.e. 'spotty' appearance) at the basal aspect of tubules when the average numbers of adluminal germ cells were <15% of control, (ii) presented as short fragments with cytoplasmic extensions when numbers were 15-25% of control or (iii) remained continuous when numbers were >40% of control. Changes in localization of connexin-43 and vinculin were also observed (smaller effects than for claudin-11) but ZO-1, ß-catenin and ß-actin did not differ, compared with control. LIMITATIONS, REASONS FOR CAUTION: Claudin-11 was the only Sertoli cell TJ protein investigated, but it is considered to be the most pivotal of constituent proteins given its known implication in infertility and BTB function. We were limited to testis samples which had been gonadotropin-suppressed for 8 weeks, shorter than the 74-day spermatogenic wave, which may account for the heterogeneity in claudin-11 and germ cell response observed among the men. Longer suppression (12-24 weeks) is known to suppress germ cells further and claudin-11 disruption may be more uniform, although we could not access such samples. WIDER IMPLICATIONS OF THE FINDINGS: These findings are important for our understanding of the sites of action of male hormonal contraception, because they suggest that BTB function could be ablated following long-term hormone suppression treatment. STUDY FUNDING/COMPETING INTERESTS: National Health and Medical Research Council (Australia) Program Grants 241000 and 494802; Research Fellowship 1022327 (to R.I.M.) and the Victorian Government's Operational Infrastructure Support Program. None of the authors have any conflicts to disclose. TRIAL REGISTRATION NUMBER: Not applicable.
Assuntos
Claudinas/antagonistas & inibidores , Anticoncepcionais Masculinos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Células de Sertoli/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Inibidores de 5-alfa Redutase/farmacologia , Adulto , Androgênios/farmacologia , Barreira Hematotesticular/citologia , Barreira Hematotesticular/efeitos dos fármacos , Barreira Hematotesticular/metabolismo , Claudinas/genética , Claudinas/metabolismo , Dutasterida/farmacologia , Humanos , Imuno-Histoquímica , Levanogestrel/farmacologia , Masculino , Pessoa de Meia-Idade , Oligopeptídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Reprodutibilidade dos Testes , Células de Sertoli/citologia , Espermatogênese/efeitos dos fármacos , Testosterona/análogos & derivados , Testosterona/farmacologia , Adulto JovemRESUMO
Human osteosarcoma (OS) represents one of the most common primary sarcomas often originating in the metaphyses of long bones. However, its underlying molecular pathogenesis is still only vaguely understood. Several tight junction proteins were shown to be associated with and involved in tumorigenesis. This study is aimed to evaluate the role of Claudin 8 (CLDN8) in human OS. Lentivirus-based short hairpin RNA targeting CLDN8 specifically depleted its endogenous expression in U2OS and SW1353 OS cells, with a reduction by 97.7% and 89.3%, respectively, in contrast to control. Depletion of CLDN8 led to a significant diminution in cell viability and proliferation. To test the mechanism by which CLDN8 modulates cell proliferation, the flow cytometry assay and apoptosis assay were performed and confirmed that G1-S transition was blocked and a strong proapoptotic effect was induced in U2OS cells by CLDN8 knockdown. These data demonstrate that CLDN8 plays an essential role in OS proliferation in vitro, which will provide a new opportunity for discovering and identifying novel effective treatment strategies.
Assuntos
Neoplasias Ósseas/patologia , Proliferação de Células , Claudinas/metabolismo , Osteossarcoma/patologia , Apoptose , Western Blotting , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Ciclo Celular , Claudinas/antagonistas & inibidores , Claudinas/genética , Humanos , Lentivirus/genética , Osteossarcoma/genética , Osteossarcoma/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, mainly due to its high rates of postoperative recurrence and metastasis. Moreover, there is no widely accepted prognostic marker of recurrence. Therefore, the aim of the present study was to determine whether such a marker could be provided by a microRNA (miRNA), since recent evidence indicates that miRNAs are important contributors to the metastatic phenotype. In the present study, we showed that miR-99b was expressed at high levels in tissues of patients with HCC and in cell lines derived from HCCs. Elevated levels of miR-99b predicted poor overall survival as well as disease-free survival of patients with HCC. Moreover, miR-99b expression levels correlated with capsule formation and microvascular invasion, which are required for postoperative recurrence. Overexpression or knockdown of miR-99b expression increased or inhibited, respectively, the metastasis of HCC cells in vitro. Furthermore, using a dualluciferase assay, we demonstrated that miR-99b inhibited the expression of claudin 11 (CLDN11), a component of tight junction strands by directly targeting the 3'-untranslated region of CLDN11 mRNA. In addition, CLDN11 expression was increased or decreased when miR-99b expression was inhibited or elevated in the HCC cells, respectively. Moreover, the expression of miR-99b was inversely correlated with CLDN11 mRNA or CLDN11 levels in the HCC tissues. These findings suggest that a high level of miR-99b expression is an independent prognostic factor and correlates with poor survival of patients with HCC. Therefore, inhibition of miR-99b expression may serve as a therapeutic approach for inhibiting the metastatic phenotype of HCC.
Assuntos
Carcinoma Hepatocelular/genética , Claudinas/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Claudinas/antagonistas & inibidores , Intervalo Livre de Doença , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/patologia , Masculino , MicroRNAs/biossínteseRESUMO
Interleukin-18 (IL-18) was recently reported to have a pro-tumor effect in various cancers. Increased IL-18 levels in the serum of cancer patients correlated with malignancy, and IL-18 acts a crucial factor for cell migration in gastric cancer and melanoma. Claudins, which are the most important tight junction proteins, are also linked with cancer progression and metastasis. However, the relationship between claudins and IL-18 is not well-understood. Here, we show that the migratory ability of MCF-7 cells was reduced when endogenous IL-18 expression was inhibited with IL-18 siRNA. Moreover, exogenous IL-18 enhanced breast cancer cell migration and suppressed the expression of the tight junction proteins claudin-1, claudin-3, claudin-4, and claudin-12 in MCF-7 cells. Knockdown of claudin-3, claudin-4, and claudin-12, but not claudin-1, increased breast cancer migration with maximal effects observed in claudin-12 siRNA-transfected cells. To investigate whether the mitogen-activated protein kinase (MAPK) signaling pathway is involved in IL-18-induced cell migration and claudin-12 expression, cells were pretreated with SB203580 (an inhibitor of p38 MAPK) or PD98059 (an inhibitor of ERK1/2) prior to the addition of IL-18. Although pretreatment of MCF-7 cells with SB203580 blocked both the enhanced cell migration and the decreased claudin-12 expression, PD98059 only blocked cell migration and did not affect claudin-12 expression. In addition, exogenous IL-18 induced rapid phosphorylation of p38 MAPK. These results suggest that IL-18 is an important factor inducing breast cancer cell migration through down-regulation of claudin-12 and activation of the p38 MAPK pathway.
Assuntos
Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Claudinas/fisiologia , Interleucina-18/fisiologia , Sistema de Sinalização das MAP Quinases , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Movimento Celular/fisiologia , Claudina-1/antagonistas & inibidores , Claudina-1/genética , Claudina-1/fisiologia , Claudina-3/antagonistas & inibidores , Claudina-3/genética , Claudina-3/fisiologia , Claudina-4/antagonistas & inibidores , Claudina-4/genética , Claudina-4/fisiologia , Claudinas/antagonistas & inibidores , Claudinas/genética , Regulação para Baixo/efeitos dos fármacos , Feminino , Flavonoides/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Imidazóis/farmacologia , Interleucina-18/antagonistas & inibidores , Interleucina-18/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células MCF-7 , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/genética , Proteínas Recombinantes/farmacologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/fisiologiaRESUMO
Pancreatic cancer continues to be a leading cause of cancer-related death worldwide and there is an urgent need to develop novel diagnostic and therapeutic strategies to reduce the mortality of patients with this disease. In pancreatic cancer, some tight junction proteins, including claudins, are abnormally regulated and therefore are promising molecular targets for diagnosis, prognosis and therapy. Claudin-4 and -18 are overexpressed in human pancreatic cancer and its precursor lesions. Claudin-4 is a high affinity receptor of Clostridium perfringens enterotoxin (CPE). The cytotoxic effects of CPE and monoclonal antibodies against claudin-4 are useful as novel therapeutic tools for pancreatic cancer. Claudin-18 could be a putative marker and therapeutic target with prognostic implications for patients with pancreatic cancer. Claudin-1, -7, tricellulin and marvelD3 are involved in epithelial to mesenchymal transition (EMT) of pancreatic cancer cells and thus might be useful as biomarkers during disease. Protein kinase C is closely related to EMT of pancreatic cancer and regulates tight junctions of normal human pancreatic duct epithelial cells and the cancer cells. This review focuses on the regulation of tight junctions via protein kinase C during EMT in human pancreatic cancer for the purpose of developing new diagnostic and therapeutic modalities for pancreatic cancer.
Assuntos
Antineoplásicos/uso terapêutico , Desenho de Fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Terapia de Alvo Molecular , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas de Junções Íntimas/antagonistas & inibidores , Junções Íntimas/efeitos dos fármacos , Animais , Claudinas/antagonistas & inibidores , Claudinas/metabolismo , Humanos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/patologiaRESUMO
BACKGROUND: Claudins are known as tight junction proteins, and their expression pattern in gastric cancer is still controversial. The relationship between the expression patterns of tight junction proteins and tumor proliferation in early gastric cancer is still far from clear. AIMS: To investigate the expression patterns of claudin-18 and Ki-67 in early gastric cancer at the invasive front and surrounding normal gastric mucosa and to investigate the biological function of claudin-18 in the proliferation and invasion of cancer cells. METHODS: Seventy-five early gastric cancer lesions removed via endoscopic mucosal resection or endoscopic submucosal resection were evaluated. All gastric cancer lesions were diagnosed as differentiated adenocarcinoma using the Japanese Classification of Gastric Carcinoma. To assess epithelial proliferation, immunostaining with Ki-67 was performed, and the labeling index was calculated. To assess the expression of epithelial tight junction proteins, immunofluorescent staining of claudin-18 was performed. The immunoreactivity of claudin-18 was graded according to the number of stained cells. Correlation analysis was performed by Spearman's rank correlation coefficient. Transfection of claudin-18 small interfering RNA (siRNA) was accomplished in MKN74, a claudin-18-positive gastric cancer cell line, to investigate the effect of claudin-18 on proliferation and invasion of cancer cells. RESULTS: Claudin-18 was significantly down-regulated in gastric cancer compared to surrounding gastric normal mucosa or intestinal metaplasia. The Ki-67 labeling index of gastric cancer at the invasive front was inversely correlated with the claudin-18 level, but that at the mucosal lesion was not correlated. Claudin-18 knockdown significantly promoted the proliferation of MKN74 compared with control siRNA-transfected cells. MKN74 invasion increased significantly with claudin-18 siRNA transfection compared with control siRNA transfection. CONCLUSIONS: Down-regulation of claudin-18 is associated with the proliferative potential at the invasive front of gastric cancer, suggesting that it has a pivotal role in gastric cancer progression.
Assuntos
Adenocarcinoma/patologia , Movimento Celular , Proliferação de Células , Claudinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Gástricas/patologia , Adenocarcinoma/metabolismo , Idoso , Apoptose , Western Blotting , Claudinas/antagonistas & inibidores , Claudinas/genética , Progressão da Doença , Regulação para Baixo , Feminino , Imunofluorescência , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Humanos , Técnicas Imunoenzimáticas , Masculino , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , RNA Interferente Pequeno/genética , Neoplasias Gástricas/metabolismo , Células Tumorais CultivadasRESUMO
Disruption or loss of tight junction structure and function is associated with tumor growth, invasion and metastasis in tumors of human epithelial origin. Since claudin is the most important backbone protein of tight junctions, the downregulation or loss of claudin expression is hypothesized to be important for tumor development and metastasis. In the current study, RNA interference (RNAi) was used to knock down the expression of claudin6 to investigate the effect of claudin6 downregulation on the malignant phenotype in the human breast epithelium cell line HBL100. The junctional function was investigated by measuring the transepithelial electrical resistance across the confluent epithelial cell layer. Manual cell counting and wound healing assays were performed to examine cell proliferation and migration. Changes in matrix metalloproteinase2 (MMP2) expression and activity were examined by reverse transcription polymerase chain reaction (RTPCR) and gelatin zymography. The expression of p38 mitogenactivated protein kinases (MAPKs) and phosphorylated p38 MAPK were measured by western blot analysis. Claudin6 knockdown resulted in significantly lower transepithelial electrical resistance (P<0.001), higher growth rate (P<0.001) and migratory ability (P<0.001) accompanied with an increased MMP2 expression and activity (P<0.001). Furthermore, a decreased expression of phosphorylated p38 MAPK (P<0.001) was detected in HBL100 cells. These observations support the hypothesis that a decreased expression of claudin6 contributes to the malignant progression of human breast cancer.
Assuntos
Mama/citologia , Movimento Celular , Proliferação de Células , Claudinas/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , RNA Interferente Pequeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Western Blotting , Mama/metabolismo , Células Cultivadas , Claudinas/antagonistas & inibidores , Claudinas/genética , Impedância Elétrica , Feminino , Humanos , Metaloproteinase 2 da Matriz/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cicatrização , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Twist1 is a transcription factor that is involved in epithelial-mesenchymal transition by suppressing intercellular adhesion. In this study, we aimed to determine the role of Twist1 in the regulation of claudin-4 expression and investigate its specific mechanisms and clinical implications using human esophageal carcinoma cell lines and tissues. As a result, up-regulation of Twist1 decreased both gene and protein expression levels of endogenous claudin-4 and the suppression was mediated by direct binding of Twist1 to the canonical E-box in the promoter region of claudin-4. In addition, there was a significant inverse correlation of claudin-4 with Twist1 in esophageal cancer tissues. High Twist1 and low claudin-4 expression was associated with the poorest prognosis and was more highly correlated with adverse outcome than any other subgroup with statistical significance (p=0.001). Our results indicate that Twist1 induces the repression of claudin-4 expression during the epithelial-mesenchymal transition in esophageal carcinoma.
Assuntos
Carcinoma de Células Escamosas/patologia , Claudinas/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Adolescente , Adulto , Idoso , Sequência de Bases , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Linhagem Celular Tumoral , Criança , Pré-Escolar , Claudina-4 , Claudinas/antagonistas & inibidores , Intervalo Livre de Doença , Regulação para Baixo , Elementos E-Box , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/mortalidade , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Nucleares/genética , Prognóstico , Regiões Promotoras Genéticas , Proteína 1 Relacionada a Twist/genética , Adulto JovemRESUMO
Tight junctions mediate the intercellular diffusion barrier found in epithelial tissues but they are not static complexes; instead there is rapid movement of individual proteins within the junctions. In addition some tight junction proteins are continuously being endocytosed and recycled back to the plasma membrane. Understanding the dynamic behaviour of tight junctions is important as they are altered in a range of pathological conditions including cancer and inflammatory bowel disease. In this study we investigate the effect of treating epithelial cells with a small molecule inhibitor (YM201636) of the lipid kinase PIKfyve, a protein which is involved in endocytic trafficking. We show that MDCK cells treated with YM201636 accumulate the tight junction protein claudin-1 intracellularly. In contrast YM201636 did not alter the localization of other junction proteins including ZO-1, occludin and E-cadherin. A biochemical trafficking assay was used to show that YM201636 inhibited the endocytic recycling of claudin-1, providing an explanation for the intracellular accumulation. Claudin-2 was also found to constantly recycle in confluent MDCK cells and treatment with YM201636 blocked this recycling and caused accumulation of intracellular claudin-2. However, claudin-4 showed negligible endocytosis and no detectable intracellular accumulation occurred following treatment with YM201636, suggesting that not all claudins show the same rate of endocytic trafficking. Finally, we show that, consistent with the defects in claudin trafficking, incubation with YM201636 delayed formation of the epithelial permeability barrier. Therefore, YM201636 treatment blocks the continuous recycling of claudin-1/claudin-2 and delays epithelial barrier formation.