RESUMO
Colon cancer results from erroneous renewal of the enteric epithelium. Mutations in stem cells, or their proliferative progenitors, cause accumulation of cells that invade into the stroma and continue to divide rather than migrating on top of the basement membrane prior to entering into apoptosis. Many of these changes in invasive activity appear to be related to the invasion-suppressor role of E-cadherin. We have also investigated Listeria monocytogenes and other enteric bacteria, since these bacteria stimulate invasion through the production of a beta-casein-derived 13-amino acid peptide which is produced by enzymes present in the colon cancer ecosystem. The pro-invasive 13-amino acid peptide signals via small guanosine triphosphatases, which modulate the actin cytoskeleton, and via phosphorylation of the delta opioid receptor. The pro-invasive activity of this peptide is neutralized by the delta opioid receptor antagonist, naloxone. Since the delta opioid receptor belongs to the family of G protein-coupled receptors, implicated in colon cancer cell invasion signalling pathways, it is tempting to speculate that opioids could play a role in mediating this trait of malignant tumours.
Assuntos
Neoplasias do Colo/fisiopatologia , Transdução de Sinais/fisiologia , Apoptose/fisiologia , Neoplasias do Colo/patologia , Humanos , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Listeriose/patologia , Listeriose/fisiopatologia , Modelos Biológicos , Invasividade Neoplásica/patologia , Invasividade Neoplásica/fisiopatologia , Receptores Opioides/fisiologiaRESUMO
PURPOSE: We have previously demonstrated that conditioned medium from bacteria, some of which were isolated from the colon of cancer patients, stimulate cancer cell invasion in vitro through a 13-mer beta-casein-derived peptide. Since invasion signalling pathways are coordinated by the balance between protein kinases and phosphatases, we investigated the effect of conditioned medium from bacteria on the overall cellular tyrosine phosphorylation. METHODS: The tyrosine phosphorylation level of HCT-8/E11 human colon cancer cells treated with the pro-invasive conditioned medium of Listeria, prepared on top of collagen type I gels (CM(Coll) Listeria/TSB), were analysed by means of immunoprecipitation and Western blot, with specific anti-phosphotyrosine antibodies. RESULTS: We demonstrated that CM(Coll) Listeria/TSB increases the tyrosine phosphorylation level of ErbB2 and ErbB3, members of the epidermal growth factor receptor (EGFR) family, and the association between ErbB3 and the phosphatidylinositol 3-kinase (PI3K) regulatory subunit (p85alpha). CM(Coll) Listeria/TSB-stimulated ErbB3 tyrosine phosphorylation and cancer cell invasion were independent from EGFR expression and activity but dependent on ErbB2 activity. CONCLUSIONS: The interaction between Listeria and collagen type I produces, next to the 13-mer peptide, at least another pro-invasive factor that signals via ErbB2/ErbB3 heterodimers.
Assuntos
Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Listeria monocytogenes/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Transdução de Sinais , Tirosina/metabolismo , Western Blotting , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Neoplasias do Colo/microbiologia , Humanos , Imunoprecipitação , Listeriose/metabolismo , Invasividade Neoplásica , FosforilaçãoRESUMO
Transfer of antigens frequently follows adhesion of protozoan parasites to host cells. We were interested in such transfer from the Entamoeba surface to enterocytes following adhesion of trophozoites. Therefore, cocultures of enterocytes in vitro and ex vivo with Entamoeba histolytica (strain HM-1:IMSS) or Entamoeba dispar (strain SAW760) trophozoites were processed for immunocytochemistry. The EH5 monoclonal antibody against amoebic proteophosphoglycans marked a dotted pattern on the apical side of enterocytes in in vitro cocultures with HM-1:IMSS and SAW760 trophozoites. Basolateral staining was present in cocultures following dysfunction of tight junctions, or when trophozoites made direct contact with the basolateral side of enterocytes in in vitro and ex vivo cocultures. Based on the molecular mass in Western blot, the transferred proteophosphoglycan was identified as a lipophosphopeptidoglycan. In conclusion, trophozoites transfer LPPG to the apical side of enterocytes following adhesion and prior to dysfunction of tight junctions.
Assuntos
Entamoeba/fisiologia , Enterócitos/fisiologia , Glicosilfosfatidilinositóis/fisiologia , Peptidoglicano/metabolismo , Proteínas de Protozoários/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/imunologia , Western Blotting/métodos , Células CACO-2 , Adesão Celular/fisiologia , Técnicas de Cocultura , Colo/fisiologia , Entamoeba/imunologia , Entamoeba histolytica/imunologia , Entamoeba histolytica/fisiologia , Glicosilfosfatidilinositóis/imunologia , Humanos , Imuno-Histoquímica/métodos , Lectinas/fisiologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/fisiologia , Peptidoglicano/imunologia , Junções Íntimas/fisiologiaRESUMO
Tight junctions and microvilli constitute an anti-invasive barrier at the luminal side of enteric cell layers. Both subcellular structures are disrupted following adhesion of Entamoeba histolytica trophozoites to enteric cell layers in vitro. It was our aim to analyse the molecular mechanism underlying this disruption. Therefore, we cocultured enteric T84 cell layers established on filter inserts with E. histolytica trophozoites and tested various modulators of enteric molecules, involved in the functional regulation of tight junctions, as well as inhibitors of trophozoite virulence factors on their capacity to maintain the transepithelial electrical resistance. Pretreatment of trophozoites with the proteinase inhibitor N-Tosyl-Phenylalanine chloromethyl ketone or N-Tosyl-l-Lysine chloromethyl ketone prevented the decrease in transepithelial electrical resistance whereas none of the modulators used to pretreat enterocytes were successful. Moreover, zymography and Western blot analysis revealed that both N-Tosyl-Phenylalanine chloromethyl ketone and N-Tosyl-l-Lysine chloromethyl ketone inhibited E. histolytica cysteine proteinases and prevented proteolysis of tight junction molecules ZO-1 and ZO-2 and of villin, the major actin bundling molecule in microvilli. Immunocytochemistry with an antibody against ezrin, an actin-binding molecule in microvilli, and phase contrast microscopy demonstrated that pretreatment of trophozoites with N-Tosyl-Phenylalanine chloromethyl ketone or N-Tosyl-l-Lysine chloromethyl ketone also prevented disturbance of microvilli and destruction of Caco-2 enteric cell layers in cocultures. Taken together, our results indicate that trophozoites use their proteinases to overcome microvilli and tight junction barriers during the invasion of enteric cell layers, that these phenomena could be prevented by pretreatment of trophozoites with N-Tosyl-Phenylalanine chloromethyl ketone or N-Tosyl-l-Lysine chloromethyl ketone, and that such pretreatment disabled trophozoites to destroy enteric cell layers in vitro.
Assuntos
Entamoeba histolytica/fisiologia , Enterócitos/parasitologia , Inibidores da Síntese de Proteínas/farmacologia , Junções Íntimas/parasitologia , Tosilina Clorometil Cetona/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Animais , Células Cultivadas , Cisteína Endopeptidases/metabolismo , Impedância Elétrica , Entamoeba histolytica/efeitos dos fármacos , Entamoeba histolytica/enzimologia , Enterócitos/efeitos dos fármacos , Enterócitos/fisiologia , Proteínas dos Microfilamentos/metabolismo , Microvilosidades/efeitos dos fármacos , Microvilosidades/parasitologia , Microvilosidades/fisiologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/fisiologiaRESUMO
Alkylphospholipids, such as the antitumor ether lipid 1-O-octadecyl-2-O-methylglycero-3-phosphocholine, modulate cancer cell invasion through changes in the adherens junction E-cadherin complex, a major organizer of epithelia. We wanted to know whether alkylphospholipids would also change tight junctions, molecular complexes that seal cell-to-cell contacts in polarised epithelia. Therefore, human colorectal cancer cell layers T84 were established in two-compartment culture chambers and the functional integrity of tight junctions was evaluated through their transepithelial electrical resistance. Incorporation of alkylphospholipids causes a rapid and reversible decrease of transepithelial electrical resistance. This decrease is due to an increased paracellular permeability and is temperature-independent. Unlike methyl-beta-cyclodextrin, alkyl-phospholipids do not specifically displace lipids from raft-like membrane domains. Nevertheless, alkylphospholipids change the detergent-solubility of zonula occludens-protein and occludin. Our data, together with the literature, indicate that non-toxic doses of alkylphospholipids affect more than one cell-cell adhesion complex, probably through their incorporation into the plasma membrane lipid bilayer.
Assuntos
Antineoplásicos/farmacologia , Éteres Fosfolipídicos/farmacologia , Fosforilcolina/análogos & derivados , Junções Íntimas/efeitos dos fármacos , beta-Ciclodextrinas , Western Blotting , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colesterol/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Ciclodextrinas/farmacologia , Detergentes/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Manitol/farmacocinética , Microscopia Confocal , Fosforilcolina/farmacologia , Testes de Precipitina , Trítio , Células Tumorais CultivadasRESUMO
BACKGROUND: Opioids and opioid receptors are an integral part of the tumour microenvironment and hence may influence tumour progression. Studies on direct effects of opioids on invasion-associated cellular activities are equivocal. We wanted to clarify these differences. MATERIALS AND METHODS: The direct effects of the delta opioid receptor (DOR) agonists [D-Pen(2), D-Pen(5)]-enkephalin (DPDPE), leu-enkephalin and [D-Ala(2), D-Leu(5)]-enkephalin (DADLE) on invasion-associated activities of HCT-8 myc-DOR and HCT-8 FLAG-DOR colon cancer cells stably overexpressing DOR were studied. RESULTS: The opioids showed a trend to stimulate invasion of single cells in collagen in one clone, while they did not influence invasion of the other clone. In other invasion assays, no effects were observed. They did not affect cell growth and homotypical cell-cell adhesion. DPDPE at 0.1 muM inhibited directional migration; the other opioids and concentrations tested were inefficient. CONCLUSION: Opioids differently influence invasion-associated cellular activities, depending on the expression level of DOR, experimental set-up, type and concentration of opioid.
Assuntos
Neoplasias do Colo/patologia , Encefalinas/farmacologia , Receptores Opioides delta/agonistas , Animais , Adesão Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Neoplasias do Colo/enzimologia , Relação Dose-Resposta a Droga , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica , Fosforilação/efeitos dos fármacos , Esferoides CelularesRESUMO
Helicobacter pylori interacts with gastric epithelial cells, activating signaling pathways important for carcinogenesis. In this study we examined the role of H. pylori on cell invasion and the molecular mechanisms underlying this process. The relevance of H. pylori cag pathogenicity island-encoded type IV secretion system (T4SS), CagA, and VacA for cell invasion was also investigated. We found that H. pylori induces AGS cell invasion in collagen type I and in Matrigel invasion assays. H. pylori-induced cell invasion requires the direct contact between bacteria and cancer cells. H. pylori-mediated cell invasion was dependent on the activation of the c-Met receptor and on increased MMP-2 and MMP-9 activity. The abrogation of the c-Met receptor using the specific NK4 inhibitor or the silencing of c-Met expression with small interference RNA suppressed both cell invasion and MMP activity. Studies with different H. pylori strains revealed that cell invasion, c-Met tyrosine phosphorylation, and increased MMP-2 and MMP-9 activity were all dependent on the presence of a functional bacterial T4SS, but not on VacA cytotoxicity. Our findings demonstrate that H. pylori strains with a functional T4SS stimulate gastric epithelial cell invasion through a c-Met-dependent signaling pathway that comprises an increase in MMP-2 and MMP-9 activity.
Assuntos
Células Epiteliais/microbiologia , Helicobacter pylori/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Ilhas Genômicas/fisiologia , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Estômago/citologiaRESUMO
Invasion causes cancer malignancy. We review recent data about cellular and molecular mechanisms of invasion, focusing on cross-talk between the invaders and the host. Cancer disturbs these cellular activities that maintain multicellular organisms, namely, growth, differentiation, apoptosis, and tissue integrity. Multiple alterations in the genome of cancer cells underlie tumor development. These genetic alterations occur in varying orders; many of them concomitantly influence invasion as well as the other cancer-related cellular activities. Examples discussed are genes encoding elements of the cadherin/catenin complex, the nonreceptor tyrosine kinase Src, the receptor tyrosine kinases c-Met and FGFR, the small GTPase Ras, and the dual phosphatase PTEN. In microorganisms, invasion genes belong to the class of virulence genes. There are numerous clinical and experimental observations showing that invasion results from the cross-talk between cancer cells and host cells, comprising myofibroblasts, endothelial cells, and leukocytes, all of which are themselves invasive. In bone metastases, host osteoclasts serve as targets for therapy. The molecular analysis of invasion-associated cellular activities, namely, homotypic and heterotypic cell-cell adhesion, cell-matrix interactions and ectopic survival, migration, and proteolysis, reveal branching signal transduction pathways with extensive networks between individual pathways. Cellular responses to invasion-stimulatory molecules such as scatter factor, chemokines, leptin, trefoil factors, and bile acids or inhibitory factors such as platelet activating factor and thrombin depend on activation of trimeric G proteins, phosphoinositide 3-kinase, and the Rac and Rho family of small GTPases. The role of proteolysis in invasion is not limited to breakdown of extracellular matrix but also causes cleavage of proinvasive fragments from cell surface glycoproteins.
Assuntos
Invasividade Neoplásica/patologia , Invasividade Neoplásica/fisiopatologia , Neoplasias/patologia , Neoplasias/fisiopatologia , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Neoplasias/genéticaRESUMO
Invasive microorganisms efface enteric microvilli to establish intimate contact with the apical surface of enterocytes. To understand the molecular basis of this effacement in amebic colitis, we seeded Entamoeba histolytica trophozoites on top of differentiated human Caco-2 cell layers. Western blots of detergent lysates from such cocultures showed proteolysis of the actin-bundling protein villin within 1 min of direct contact of living trophozoites with enterocytes. Mixtures of separately prepared lysates excluded detergent colysis as the cause of villin proteolysis. Caspases were not responsible as evidenced by the lack of degradation of specific substrates and the failure of a specific caspase inhibitor to prevent villin proteolysis. A crucial role for amebic cysteine proteinases was shown by prevention of villin proteolysis and associated microvillar alterations through the treatment of trophozoites before coculture with synthetic inhibitors that completely blocked amebic cysteine proteinase activity on zymograms. Moreover, trophozoites of amebic strains pSA8 and SAW760 with strongly reduced cysteine proteinase activity showed a reduced proteolysis of villin in coculture with enteric cells. Salmonella typhimurium and enteropathogenic Escherichia coli disturb microvilli without villin proteolysis, indicating that the latter is not a consequence of the disturbance of microvilli. In conclusion, villin proteolysis is an early event in the molecular cross-talk between enterocytes and amebic trophozoites, causing a disturbance of microvilli.
Assuntos
Proteínas de Transporte/metabolismo , Cisteína Endopeptidases/metabolismo , Entamoeba histolytica/enzimologia , Mucosa Intestinal/parasitologia , Proteínas dos Microfilamentos/metabolismo , Microvilosidades/parasitologia , Animais , Adesão Celular , Técnicas de Cocultura , Neoplasias Colorretais , Entamoeba histolytica/fisiologia , Humanos , Células Tumorais Cultivadas , Ureia/metabolismoRESUMO
In colon cancer, enteric bacteria and dietary factors are major determinants of the microenvironment but their effect on cellular invasion is not known. We therefore incubated human HCT-8/E11 colon cancer cells with bacteria or bacterial conditioned medium on top of collagen type I gels. Listeria monocytogenes stimulate cellular invasion through the formation of a soluble motility-promoting factor, identified as a 13mer beta-casein-derived peptide (HKEMPFPKYPVEP). The peptide is formed through the combined action of Mpl, a Listeria thermolysin-like metalloprotease, and a collagen-associated trypsin-like serine protease. The 13mer peptide was also formed by tumour biopsies isolated from colon cancer patients and incubated with a beta-casein source. The pro- invasive 13mer peptide-signalling pathway implicates activation of Cdc42 and inactivation of RhoA, linked to each other through the serine/threonine p21- activated kinase 1. Since both changes are necessary but not sufficient, another pathway might branch upstream of Cdc42 at phosphatidylinositol 3-kinase. Delta opioid receptor (deltaOR) is a candidate receptor for the 13mer peptide since naloxone, an deltaOR antagonist, blocks both deltaOR serine phosphorylation and 13mer peptide-mediated invasion.