RESUMO
Out of the 14 avian ß-defensins identified in the Gallus gallus genome, only 3 are present in the chicken egg, including the egg-specific avian ß-defensin 11 (Gga-AvBD11). Given its specific localization and its established antibacterial activity, Gga-AvBD11 appears to play a protective role in embryonic development. Gga-AvBD11 is an atypical double-sized defensin, predicted to possess 2 motifs related to ß-defensins and 6 disulfide bridges. The 3-dimensional NMR structure of the purified Gga-AvBD11 is a compact fold composed of 2 packed ß-defensin domains. This fold is the archetype of a structural family, dubbed herein as avian-double-ß-defensins (Av-DBD). We speculate that AvBD11 emanated from a monodomain gene ancestor and that similar events might have occurred in arthropods, leading to another structural family of less compact DBDs. We show that Gga-AvBD11 displays antimicrobial activities against gram-positive and gram-negative bacterial pathogens, the avian protozoan Eimeria tenella, and avian influenza virus. Gga-AvBD11 also shows cytotoxic and antiinvasive activities, suggesting that it may not only be involved in innate protection of the chicken embryo, but also in the (re)modeling of embryonic tissues. Finally, the contribution of either of the 2 Gga-AvBD11 domains to these biological activities was assessed, using chemically synthesized peptides. Our results point to a critical importance of the cationic N-terminal domain in mediating antibacterial, antiparasitic, and antiinvasive activities, with the C-terminal domain potentiating the 2 latter activities. Strikingly, antiviral activity in infected chicken cells, accompanied by marked cytotoxicity, requires the full-length protein.
Assuntos
Proteínas Aviárias/genética , Embrião de Galinha/imunologia , Galinhas/fisiologia , Desenvolvimento Embrionário/imunologia , beta-Defensinas/genética , Sequência de Aminoácidos , Animais , Proteínas Aviárias/ultraestrutura , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Infecções Bacterianas/veterinária , Bioensaio , Embrião de Galinha/crescimento & desenvolvimento , Embrião de Galinha/microbiologia , Embrião de Galinha/parasitologia , Coccidiose/imunologia , Coccidiose/parasitologia , Coccidiose/veterinária , Eimeria tenella/imunologia , Evolução Molecular , Genoma , Imunidade Inata/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Aviária/imunologia , Influenza Aviária/virologia , Ressonância Magnética Nuclear Biomolecular , Filogenia , Domínios Proteicos/genética , Domínios Proteicos/imunologiaRESUMO
Inflammation, oxidative stress, and protease/protease inhibitor imbalance with excessive production of proteases are factors associated with pathogenesis of the chronic obstructive pulmonary disease (COPD). In this study, we report that kallikrein-related peptidase 5 (KLK5) is a crucial protease involved in extracellular matrix (ECM) remodeling and bronchial epithelial repair after injury. First, we showed that KLK5 degrades the basal layer formed by culture of primary bronchial epithelial cells from COPD or non-COPD patients. Also, exogenous KLK5 acted differently on BEAS-2B cells already engaged in epithelial-to-mesenchymal transition (EMT) or on 16HBE 14o- cells harboring epithelial characteristics. Indeed, by inducing EMT, KLK5 reduced BEAS-2B cell adherence to the ECM. This effect, neutralized by tissue factor pathway inhibitor 2, a kunitz-type serine protease inhibitor, was due to a direct proteolytic activity of KLK5 on E-cadherin, ß-catenin, fibronectin, and α5ß1 integrin. Thus, KLK5 may strengthen EMT mechanisms and promote the migration of cells by activating the mitogen-activated protein kinase signaling pathway required for this function. In contrast, knockdown of endogenous KLK5 in 16HBE14o- cells, accelerated wound healing repair after injury, and exogenous KLK5 addition delayed the closure repair. These data suggest that among proteases, KLK5 could play a critical role in airway remodeling events associated with COPD during exposure of the pulmonary epithelium to inhaled irritants or smoking and the inflammation process.
Assuntos
Remodelação das Vias Aéreas , Brônquios/patologia , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal , Calicreínas/metabolismo , Neoplasias Pulmonares/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Idoso , Antígenos CD/genética , Antígenos CD/metabolismo , Brônquios/metabolismo , Caderinas/genética , Caderinas/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Humanos , Calicreínas/genética , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/metabolismo , Transdução de SinaisRESUMO
The protease activities are tightly regulated by inhibitors and dysregulation contribute to pathological processes such as cancer and inflammatory disorders. Tissue factor pathway inhibitor 2 (TFPI-2) is a serine proteases inhibitor, that mainly inhibits plasmin. This protease activated matrix metalloproteases (MMPs) and degraded extracellular matrix. Other serine proteases are implicated in these mechanisms like kallikreins (KLKs). In this study, we identified for the first time that TFPI-2 is a potent inhibitor of KLK5 and 12. Computer modeling showed that the first Kunitz domain of TFPI-2 could interact with residues of KLK12 near the catalytic triad. Furthermore, like plasmin, KLK12 was able to activate proMMP-1 and -3, with no effect on proMMP-9. Thus, the inhibition of KLK12 by TFPI-2 greatly reduced the cascade activation of these MMPs and the cleavage of cysteine-rich 61, a matrix signaling protein. Moreover, when TFPI-2 bound to extracellular matrix, its classical localisation, the KLK12 inhibition was retained. Finally, TFPI-2 was downregulated in human non-small-cell lung tumour tissue as compared with non-affected lung tissue. These data suggest that TFPI-2 is a potent inhibitor of KLK12 and could regulate matrix remodeling and cancer progression mediated by KLK12.
Assuntos
Glicoproteínas , Calicreínas , Carcinoma Pulmonar de Células não Pequenas , Humanos , Lipoproteínas , Neoplasias PulmonaresRESUMO
Human tissue kallikreins (KLKs) are 15 members of the serine protease family and are present in various healthy human tissues including airway tissues. Multiple studies have revealed their crucial role in the pathophysiology of a number of chronic, infectious and tumour lung diseases. KLK1, 3 and 14 are involved in asthma pathogenesis, and KLK1 could be also associated with the exacerbation of this inflammatory disease caused by rhinovirus. KLK5 was demonstrated as an influenza virus activating protease in humans, and KLK1 and 12 could also be involved in the activation and spread of these viruses. KLKs are associated with lung cancer, with up- or downregulation of expression depending on the KLK, cancer subtype, stage of tumour and also the microenvironment. Functional studies showed that KLK12 is a potent pro-angiogenic factor. Moreover, KLK6 promotes malignant-cell proliferation and KLK13 invasiveness. In contrast, KLK8 and KLK10 reduce proliferation and invasion of malignant cells. Considering the involvement of KLKs in various physiological and pathological processes, KLKs appear to be potential biomarkers and therapeutic targets for lung diseases.
Assuntos
Calicreínas/metabolismo , Pneumopatias/enzimologia , Proliferação de Células , Humanos , Pneumopatias/patologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologiaRESUMO
KLK12, a kallikrein peptidase, is thought to take part in the control of angiogenesis. Our analysis of the secretome of endothelial cells (ECs) that had been treated with KLK12 showed that KLK12 converts the extracellular matrix- or membrane-bound precursor of platelet-derived growth factor B (PDGF-B) into a soluble form. Both PDGF-B and vascular endothelial growth factor A (VEGF-A) take part in the induction of angiogenesis by KLK12 in a coculture model of angiogenesis that mimics endothelial tubule formation. We used a cellular approach to analyze the interplay between KLK12, PDGF-B, and VEGF-A and showed that release of PDGF-B by KLK12 leads to the fibroblast-mediated secretion of VEGF-A. This then stimulates EC differentiation and the formation of capillary tube-like structures. Thus, KLK12 favors the interaction of ECs and stromal cells. The released PDGF-B acts as a paracrine factor that modulates VEGF-A secretion by stromal cells, which ultimately leads to angiogenesis. Moreover, the genes encoding KLK12 and PDGFB are both expressed in ECs and up-regulated in tumor cells kept under hypoxic conditions, which is consistent with the physiological involvement of KLK12 in PDGF-B maturation.
Assuntos
Calicreínas/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Western Blotting , Linhagem Celular , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Imunofluorescência , Humanos , Calicreínas/farmacologia , Espectrometria de Massas , Proteínas Proto-Oncogênicas c-sis/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
The dysregulated expression of kallikrein-related peptidase 6 (KLK6) is involved in non-small cancer (NSCLC) cell growth. However, the mechanism that sustains KLK6 signaling remains unknown. We used an isogenic non-small cell lung cancer (NSCLC) cell model system to demonstrate that KLK6 promotes the proliferation of lung tumoral cells and restrains their apoptosis in vitro via ligand-dependent EGFR transactivation. KLK6 activated the ERK and Akt pathways and triggered the nuclear translocation of ß-catenin. The stimulating effects of KLK6 required its proteolytic activity and were dependent on the protease-activated receptor 2 (PAR2). These observations support the concept of a role for KLK6 in the oncogenesis of NSCLC.
Assuntos
Receptores ErbB/metabolismo , Calicreínas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Receptor PAR-2/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Sobrevivência Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inativação Gênica , Humanos , Ligantes , Proteínas Mutantes/metabolismo , Fosforilação , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/metabolismoRESUMO
Tissue factor pathway inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI-2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI-2 is frequently down-regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI-2 down-regulation in the National Cancer Institute (NCI)-H460 non-small cell lung cancer cell line using specific micro interfering micro-interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI-2 down-regulation enhanced cell adhesion to collagen IV and laminin via an increase in α(1) integrin on cell surface, and increased MMP expression (mainly MMP-1 and -3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI-2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP-1, -3 and -7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Neoplasias Pulmonares/fisiopatologia , Metaloproteinases da Matriz/metabolismo , Invasividade Neoplásica , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Colágeno Tipo IV/metabolismo , Regulação para Baixo , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina alfa1/biossíntese , Laminina/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Metaloproteinases da Matriz/biossíntese , Metaloproteinases da Matriz/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Interferência de RNA , Transdução de Sinais , Células Estromais , Transplante Heterólogo , Microambiente TumoralRESUMO
Overexpression of human papillomavirus (HPV E6 and HPV E7) oncogenes in human cervical cells results in the development of cancer, and E6 and E7 proteins are therefore targets for preventing cervical cancer progression. Here, we describe the silencing of E6 and E7 expression in cervical carcinoma cells by RNA interference. In order to increase the efficacy of the RNA interference, HPV pseudovirions coding for a short hairpin RNA (shRNA) sequence were produced. The results indicated the degradation of E6 and E7 mRNAs when shRNA against E6 or E7 were delivered by pseudovirions in HPV-positive cells (CaSki and TC1 cells). E6 silencing resulted in the accumulation of cellular p53 and reduced cell viability. More significant cell death was observed when E7 expression was suppressed. Silencing E6 and E7 and the consequences for cancer cell growth were also investigated in vivo in mice using the capacity of murine TC1 cells expressing HPV-16 E6 and E7 oncogenes to induce fast-growing tumors. Treatment with lentiviruses and HPV virus-like particle vectors coding for an E7 shRNA sequence both resulted in dramatic inhibition of tumor growth. These results show the ability of pseudovirion-delivered shRNA to produce specific gene suppression and provide an effective means of reducing HPV-positive tumor growth.
Assuntos
Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/fisiologia , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias do Colo do Útero/patologia , Vírion/fisiologia , Montagem de Vírus/fisiologia , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Cloretos , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus , Proteínas Repressoras/genética , Transdução Genética , Transfecção , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Compostos de ZincoRESUMO
Ionic channel activity is involved in fundamental cellular behaviour and participates in cancerous features such as proliferation, migration and invasion which in turn contribute to the metastatic process. In this study, we investigated the expression and role of voltage-gated sodium channels in non-small-cell lung cancer cell lines. Functional voltage-gated sodium channels expression was investigated in normal and non-small-cell lung cancer cell lines. The measurement, in patch-clamp conditions, of tetrodotoxin-inhibitable sodium currents indicated that the strongly metastatic cancerous cell lines H23, H460 and Calu-1 possess functional sodium channels while normal and weakly metastatic cell lines do not. While all the cell lines expressed mRNA for numerous sodium channel isoforms, only H23, H460 and Calu-1 cells had a 250 kDa protein corresponding to the functional channel. The other cell lines also had another protein of 230 kDa which is not addressed to the membrane and might act as a dominant negative isoform to prevent channel activation. At the membrane potential of these cells, channels are partially open. This leads to a continuous entry of sodium, disrupting sodium homeostasis and down-stream signaling pathways. Inhibition of the channels by tetrodotoxin was responsible for a 40-50% reduction of in vitro invasion. These experiments suggest that the functional expression of voltage-gated sodium channels might be an integral component of the metastatic process in non-small-cell lung cancer cells probably through its involvement in the regulation of intracellular sodium homeostasis. These channels could serve both as novel markers of the metastatic phenotype and as potential new therapeutic targets.
Assuntos
Movimento Celular/fisiologia , Canais de Sódio/fisiologia , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Homeostase/efeitos dos fármacos , Humanos , Líquido Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/fisiopatologia , Potenciais da Membrana/efeitos dos fármacos , Microscopia Confocal , Invasividade Neoplásica , Técnicas de Patch-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sódio/metabolismo , Canais de Sódio/genética , Canais de Sódio/metabolismo , Tetrodotoxina/farmacologiaRESUMO
Matrix metalloproteinases (MMP) including MMP-2 and MMP-9 play a major role in tumour invasion by proteolysing the extracellular matrix. Their activation, particularly that of MMP-9, is partly dependent on plasmin that is inhibited by TFPI-2 (tissue factor pathway inhibitor-2), a serine protease inhibitor whose gene expression is decreased in about one-third of non-small cell lung cancers (NSCLC). In addition, MMP-2 and MMP-9 are essential in the development of NSCLC and can be regulated by functional promoter polymorphisms. In this study, the -1306C/T MMP-2, -735C/T MMP-2 and -1562C/T MMP-9 polymorphisms were analysed in 90 NSCLC patients and 90 controls. In addition, the promoter region of the TFPI-2 gene was screened for sequence variations in both groups by DHPLC. A -167G/A polymorphism was identified in 3% of controls whereas none of the 90 patients exhibited this genetic variation in the TFPI-2 promoter region. Moreover, no difference in -1306C/T MMP-2, -735C/T MMP-2 and -1562C/T MMP-9 genotypes was found between cases and controls. However, the homozygous -1562CC MMP-9 genotype was more frequent in patients with squamous cell carcinoma than in controls (p=0.018). When genotype distributions were compared to MMP-2 and MMP-9 gene expression in tumours, no relationship was found with the -1306 MMP-2 and -1562 MMP-9 polymorphisms. In contrast, tumour MMP-2 gene expression was lower in homozygous -735CC patients than in those with CT or TT genotypes. In addition, the survival time was longer in patients with the MMP-2 -735T allele than in those with the CC genotype (p=0.02). The relative risk of death was increased 2.6-fold in -735CC patients (p=0.045; 95% CI=1.0-6.7). The results of this study suggest that the -735C/T MMP-2 polymorphism might be an independent prognostic marker in NSCLC, but this should be confirmed in a larger cohort of patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Polimorfismo Genético , Idoso , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Primers do DNA , Feminino , Expressão Gênica , Genótipo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The proinflammatory chemokine CC chemokine ligand 5 (CCL5) is a potent chemoattractant of immature dendritic cells (iDCs). It remains to be elucidated whether CCL5 may also enhance iDC migration through the basement membrane by affecting matrix metalloproteinase (MMP)-9 secretion. In this study, iDCs were differentiated in vitro from human monocytes of healthy donors. Zymographic analysis of cellular membranes of nontreated iDCs revealed a basal secretion of the pro- and active MMP-9, whereas only pro-MMP-9 was detected in conditioned media. Increasing concentrations of CCL5 significantly enhanced MMP-9 secretion by iDCs, peaking at 100 ng/ml, which optimally increased iDC migration through a reconstituted basement membrane (Matrigel) in vitro. The CCL5-enhanced secretion of MMP-9 occurred early (2 h) and was maintained at least for 10 h. A significant increase in MMP-9 mRNA synthesis was detected by reverse transcriptase-polymerase chain reaction, only at 6 h of CCL5 treatment, which suggests that the early effect of CCL5 (0-4 h) on MMP-9 secretion was independent of mRNA synthesis, whereas the more delayed effect (6-10 h) could be mediated through an increase in MMP-9 gene expression. In a Matrigel migration assay, the CCL5-enhanced iDC migration was reduced significantly by specific inhibitors of MMP-9, such as tissue inhibitor of metalloproteinase-1 or an anti-MMP-9 antibody, which indicates that iDC migration through the basement membrane depends on MMP-9. These results suggest that under inflammatory conditions, the chemokine CCL5 may enhance iDC migration through the basement membrane by rapidly increasing their MMP-9 secretion.
Assuntos
Membrana Basal/metabolismo , Movimento Celular/imunologia , Quimiocinas CC/farmacologia , Células Dendríticas/fisiologia , Metaloproteinase 9 da Matriz/metabolismo , Anticorpos/farmacologia , Membrana Basal/imunologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Movimento Celular/efeitos dos fármacos , Quimiocina CCL5 , Quimiocinas CC/antagonistas & inibidores , Quimiocinas CC/imunologia , Colágeno/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Humanos , Técnicas In Vitro , Laminina/imunologia , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Proteoglicanas/imunologia , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , Valores de Referência , Fatores de Tempo , Inibidores Teciduais de Metaloproteinases/farmacologiaRESUMO
Graft endothelium has a key role in organ transplantation because it regulates graft infiltration by allogeneic activated T cells. Overexpression of death molecules that could induce apoptosis of alloreactive T cells might be an alternative to the immunosuppressive treatment currently used in graft transplantation. Several studies have shown that immune-privileged sites express Fas ligand (FasL) and induce apoptosis of activated T-cells. We propose that endothelial cells engineered to express FasL could inhibit alloreactive T cell-proliferation by inducing apoptosis. An expression vector was constructed with human FasL cDNA and used to transfect an endothelial cell line (ECV304 cells). We demonstrated that FasL-transfected ECV304 cells were effective in inducing apoptosis of Jurkat T cell lymphoma as an agonist anti-Fas antibody. Using a mixed lymphocyte-endothelial cell culture model we observed that FasL-transfected ECV304 cells which conserved their two principal costimulatory pathways inhibited alloreactive T cell-proliferation by inducing activated T-cell apoptosis. These results suggest that endothelial cells could be interesting candidates to convey a death signal and induce hyporesponsiveness of alloreactive T cells during organ transplantation.
Assuntos
Endotélio Vascular/citologia , Ativação Linfocitária , Glicoproteínas de Membrana/fisiologia , Linfócitos T/imunologia , Apoptose , Linhagem Celular , Citotoxicidade Imunológica , Endotélio Vascular/fisiologia , Proteína Ligante Fas , Humanos , TransfecçãoRESUMO
Tissue factor pathway inhibitor-2 (TFPI-2) displays structural similarities with TFPI-1, the major inhibitor of tissue factor (TF)/, factor VIIa. It is synthesized mostly by syncytiotrophoblast in the placenta, but its physiological functions are not fully understood. We studied the synthesis of TFPI-2 mRNA and that of TFPI-1 and TF in three human trophoblast cell lines, JAR, BeWo, and JEG-3. We first developed specific competitive reverse transcription-polymerase chain reaction (RT-PCR) assays for each gene studied using human umbilical vein endothelial cells (HUVEC). The three trophoblast cell lines strongly synthesized TF mRNA whereas the synthesis of TFPI-1 mRNA was very low. TFPI-2 mRNA was not detected in unstimulated or stimulated JAR cells. In contrast, JEG-3 and, to a lesser extent, BeWo produced significant amounts of TFPI-2 mRNA, which were significantly increased after stimulation with phorbol 12-myristate 13-acetate (PMA). However, tumor necrosis factor-alpha (TNF-alpha) had no effect on this synthesis. JEG-3 and BeWo are thus two cell lines that could be used to study TFPI-2 gene regulation and to investigate the role of TF, TFPI-1, and TFPI-2 during trophoblast differentiation.
Assuntos
Glicoproteínas/biossíntese , Trofoblastos/metabolismo , Linhagem Celular , Feminino , Glicoproteínas/genética , Humanos , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Tissue factor pathway inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin, a protease which is involved in tumour progression by activating (MMPs). This therefore makes TFPI-2 a potential inhibitor of invasiveness and the development of metastases. In this study, low levels of TFPI-2 expression were found in 65% of patients with small cell lung cancer (SCLC), the most aggressive type of lung cancer. To study the impact of TFPI-2 in tumour progression, TFPI-2 was overexpressed in NCI-H209 SCLC cells which were orthotopically implanted in nude mice. Investigations showed that TFPI-2 inhibited lung tumour growth. Such inhibition could be explained in vitro by a decrease in tumour cell viability, blockade of G1/S phase cell cycle transition and an increase in apoptosis shown in NCI-H209 cells expressing TFPI-2. We also demonstrated that TFPI-2 upregulation in NCI-H209 cells decreased MMP expression, particularly by downregulating MMP-1 and MMP-3. Moreover, TFPI-2 inhibited phosphorylation of the MAPK signalling pathway proteins involved in the induction of MMP transcripts, among which MMP-1 was predominant in SCLC tissues and was inversely expressed with TFPI-2 in 35% of cases. These results suggest that downregulation of TFPI-2 expression could favour the development of SCLC.
RESUMO
Human small cell lung carcinoma (SCLC) is the most aggressive type of lung cancer but no clinically relevant animal model has been developed to date. Such a model would be valuable to study the molecular aspects of tumour progression and to test the effectiveness of new treatment agents. We generated a reproducible and reliable nude mouse orthotopic model of human SCLC with NCI-H209 tumour cells genetically modified to express firefly luciferase. Cells were analysed for long-term stability of bioluminescence and a clone was passaged twice subcutaneously to enhance tumorigenicity. Cells resuspended in Matrigel and/or EDTA RPMI medium containing a (99m)Tc-labelled tin colloid used as tracer were implanted intrabronchially with a catheter inserted into the trachea and positioned in the main bronchus using X-ray-guided imaging. Deposition of cells into the lung was then assessed by scintigraphy. The growth of the primary tumour was sensitively and non-invasively followed by bioluminescence imaging that allowed real-time monitoring of tumour progression in the same animals over a 2-12-week period. Additional 3D bioluminescence imaging and computed tomography scanning were used to document tumour location and measurements that were confirmed by histological analyses. In conclusion, this original nude mouse orthotopic model resembles various stages of human small cell lung cancer, and therefore could be used to evaluate new treatment strategies.
Assuntos
Progressão da Doença , Neoplasias Pulmonares/patologia , Carcinoma de Pequenas Células do Pulmão/patologia , Microtomografia por Raio-X , Animais , Linhagem Celular Tumoral , Humanos , Imageamento Tridimensional , Luciferases de Vaga-Lume/biossíntese , Medições Luminescentes , Pulmão/patologia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Proteínas Recombinantes/biossíntese , Carcinoma de Pequenas Células do Pulmão/diagnóstico por imagem , Carcinoma de Pequenas Células do Pulmão/metabolismoRESUMO
Tissue Factor Pathway Inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin which activates metalloproteinases (MMPs) involved in extracellular matrix (ECM) degradation. Its secretion in ECM makes TFPI-2 a potential inhibitor to regulate tumour invasion and metastasis. Moreover, TFPI-2 is frequently downregulated, particularly in aggressive cancers. In this study, we silenced TFPI-2 in the NCI-H460 non-small cell lung cancer cell line and evaluated the role of TFPI-2 in cell invasion and its impact on MMPs expression. As the effects of siRNA are transient, the consequences of both gene silencing and restoration to normal expression could be studied kinetically in the same cells. We showed that TFPI-2 expression by NCI-H460 cells was effectively downregulated using specific small interfering RNA and this silencing was associated with an increase in the invasive potential of tumour cells while migration was not affected. We also showed that mRNA levels and protein expression of MMP-2, -3, -9, -14 were not influenced by TFPI-2 silencing. Moreover, the gelatinase activity of MMP-2 and MMP-9 was unmodified. In contrast, MMP-1 mRNA levels and protein were significantly and similarly increased in cells transfected with TFPI-2 siRNA. In conclusion, this study confirms that TFPI-2 downregulation can contribute to tumour invasion of lung cancer cells.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Glicoproteínas/metabolismo , Neoplasias Pulmonares/metabolismo , RNA/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular , Regulação para Baixo , Matriz Extracelular/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Invasividade Neoplásica , RNA Interferente Pequeno/metabolismoRESUMO
INTRODUCTION: Tissue factor (TF) is the physiological trigger of blood coagulation, but it could also have an important role in cancer by regulating VEGF expression and angiogenesis. METHODS: TF expression was studied by real-time PCR in lung tumors of 64 patients with non-small-cell lung cancer (NSCLC) and by immunohistochemical analysis. The gene expression of two VEGF isoforms, VEGF165 and VEGF189, was also evaluated. Microvascular density (MVD) was studied by measuring Von Willebrand Factor (VWF) mRNA levels and by immunohistochemistry using an anti-CD34 antibody. RESULTS: TF mRNA levels were significantly lower than in corresponding non-affected lung tissues. However, TF expression was higher in T3-T4 tumors and this result was confirmed by immunohistochemistry. VEGF189 mRNA levels were ten times higher than those of VEGF165 and well correlated with TF mRNA levels. MVD was lower in the inner part of tumors than in the adjacent non-affected lung without being related to TF expression. Finally, codon 12 K-ras mutation was found in 8 lung carcinomas, and higher TF and VEGF189 mRNA levels were measured in mutated tissues (p < 0.001). CONCLUSION: These results suggest that high TF expression in lung tumors may result from K-ras mutation and contribute to NSCLC progression, probably via mechanisms other than angiogenesis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Genes ras/genética , Neoplasias Pulmonares/metabolismo , Mutação , Neovascularização Patológica , Tromboplastina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tromboplastina/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator de von Willebrand/metabolismoRESUMO
Tissue factor pathway inhibitor 2 (TFPI-2), a Kunitz-type proteinase inhibitor, might play an important role during placenta growth by regulating trophoblast invasion and differentiation. Many TFPI-2 transcripts have been detected in syncytiotrophoblast cells, but conflicting results have been reported concerning TFPI-2 synthesis by the cytotrophoblast. To address this issue, we developed a method to isolate pure preparations of human villous cytotrophoblast cells from normal term placentas, and the synthesis of tissue factor, TFPI-1, and TFPI-2 mRNAs was then evaluated. Cells were isolated by trypsin-DNase-EDTA digestion, followed by Percoll gradient separation and immunodepletion of human leukocyte antigen-positive cells. The quality of villous cytotrophoblast cells was verified by electron microscopy. Purity of cell preparations was assessed by labeling cells with GB25, a monoclonal antibody specific to villous trophoblast cells, and by checking the absence of contaminating cells using anti-CD9 antibody. The lack of hCG, CD32 mRNA, and tissue factor mRNA also indicated the absence of contaminating cells. Using competitive reverse transcription polymerase chain reaction, we showed that freshly isolated villous cytotrophoblast cells synthesized significant levels of TFPI-1 mRNA and larger amounts of TFPI-2 mRNA. TFPI-1 and TFPI-2 mRNA synthesis remained unchanged when cytotrophoblast cells were cultured in complete medium and evolved as a multinucleated syncytiotrophoblast. These results indicate that the villous cytotrophoblast and syncytiotrophoblast are both important sites of TFPI-2 synthesis in the human placenta. This study also indicates that tissue factor detection should be used systematically to check the purity of cytotrophoblast cell preparations because it allows detection of contamination by monocytes/macrophages and by syncytial fragments.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glicoproteínas/biossíntese , Glicoproteínas/genética , Placenta/metabolismo , RNA Mensageiro/biossíntese , Trofoblastos/metabolismo , Adulto , Separação Celular , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Gigantes/metabolismo , Humanos , Técnicas In Vitro , Mesoderma/citologia , Mesoderma/metabolismo , Microscopia Eletrônica , Placenta/citologia , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tromboplastina/biossínteseRESUMO
Tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type serine proteinase inhibitor associated with the extracellular matrix, has been shown to reduce tumor invasion. In the present study we identified the presence of a complete CpG island region spanning exon 1 and the three transcription initiation sites. We demonstrate that DNA demethylation by 5'-aza-2'-deoxycytidine restores TFPI-2 transcription in JAR choriocarcinoma cells. The effect of in vitro DNA methylation on TFPI-2 promoter function was also confirmed with TFPI-2/luciferase promoter constructs. Finally, we determined the precise methylation status of CpG sites of the TFPI-2 promoter in normal and tumor trophoblast cells using the bisulfite genomic sequencing method. We conclude that hypermethylation of the TFPI-2 gene is correlated with transcriptional silencing and that the TFPI-2 gene may be a candidate tumor suppressor gene.
Assuntos
Metilação de DNA , Inibidores Enzimáticos/farmacologia , Inativação Gênica , Glicoproteínas/genética , Regiões Promotoras Genéticas , Linhagem Celular Tumoral , Coriocarcinoma , Ilhas de CpG , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Glicoproteínas/metabolismo , Humanos , Luciferases/genética , Luciferases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
The trophoblast, the outermost layer of the human placenta, lacks expression of the classical human leukocyte antigen (HLA) class I molecules. This prevents allorecognition by T cells but raises the question of what protects the trophoblast from natural killer (NK) cells. In a previous study, we have shown that choriocarcinoma cell (CC) resistance to NK lysis was mainly independent of HLA class I molecules. In the present study, we postulated that CC may prevent activation of NK cells by failing to stimulate their triggering receptors (TR). To test this hypothesis, we evaluated the lysis of JAR and JEG-3 CC after effective cross-linking and activation of NK cells by means of lectins or antibodies. Our results show that NK-resistant CC were sensitive to lysis by unstimulated peripheral blood lymphocytes in the presence of phytohemagglutin (PHA), to antibody-dependent cell cytotoxicity in presence of anti-Tja antibodies, and to monoclonal antibody redirected killing using anti-TR antibodies anti-CD16 and anti-CD244/2B4. Finally, CC fail to express CD48, the ligand for CD244/2B4. These results indicate that the resistance of CC to lysis results primarily from defective NK cell activation, at least partially due to the lack of expression of ligands, such as CD48, involved in the triggering of NK cells.