RESUMO
Reduced expression in immortalized cells (REIC)/dickkopf-3 (Dkk-3), a tumor suppressor gene, is downregulated in various cancers. We previously reported the tumor-inhibitory effects of the REIC/Dkk-3 gene, delivered by a conventional adenoviral vector (Ad-CAG-REIC) in pancreatic cancer. Here, we developed an Ad-REIC vector with a novel gene expression system, termed the super gene expression (SGE) system, and assessed its therapeutic effects relative to those of Ad-CAG-REIC in pancreatic cancer cells. Human pancreatic cancer cell lines ASPC1 and MIAPaCa2 were used. REIC/Dkk-3 expression was assessed by western blot analysis. Relative cell viability and apoptotic effects were examined in vitro. The anti-tumor effects of Ad-REIC treatment were assessed in the mouse xenograft model. Compared with Ad-CAG-REIC, Ad-SGE-REIC elicited a significant increase in REIC protein expression in the cells studied. Relative to Ad-CAG-REIC, Ad-SGE-REIC reduced cell viability and induced apoptosis in the ASPC1 and MIAPaCa2 cell lines in vitro, and achieved superior tumor growth inhibition in the mouse xenograft model. Compared with conventional Ad-REIC agents, Ad-SGE-REIC provided enhanced inhibitory effects against tumor growth. Our results indicate that Ad-SGE-REIC is an innovative therapeutic tool for pancreatic cancer.
Assuntos
Adenoviridae/genética , Terapia Genética , Vetores Genéticos/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neoplasias Pancreáticas/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Quimiocinas , Modelos Animais de Doenças , Expressão Gênica , Ordem dos Genes , Vetores Genéticos/administração & dosagem , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Transdução de Sinais , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: We previously reported that expressions of the pro-angiogenic cytokines angiopoietin-2 (Ang-2), follistatin, granulocyte colony-stimulating factor, hepatocyte growth factor, leptin, platelet-derived growth factor-BB, platelet endothelial cell adhesion molecule-1, and vascular endothelial growth factor were associated with the response to sorafenib in patients with advanced hepatocellular carcinoma (HCC). The aim of the present study is to examine the same relationship in a larger cohort. METHODS: In the current retrospective cohort study, we measured serum levels of the eight cytokines in 120 consecutive HCC patients who were treated with sorafenib. We evaluated the effects of increased expression of serum cytokines on progression-free survival (PFS) and overall survival (OS). RESULTS: Elevated expression of Ang-2 correlated both with significantly shorter PFS (hazard ratio (HR), 1.84; 95% confidence interval (CI), 1.21-2.81), and OS (HR, 1.95; 95% CI, 1.21-3.17). Patients with more than three cytokines expressed above the median similarly had significantly shorter PFS (HR, 1.98; 95% CI, 1.30-3.06) and OS (HR, 1.94; 95% CI, 1.19-3.22). Differences in OS were evident in cases with the evidence of macroscopic vascular invasion or extrahepatic metastasis. CONCLUSION: High expression of Ang-2 or more than cytokines in serum is associated with poor PFS and OS in HCC patients treated with sorafenib.
Assuntos
Carcinoma Hepatocelular/sangue , Citocinas/sangue , Neoplasias Hepáticas/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Angiopoietina-2/sangue , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/irrigação sanguínea , Carcinoma Hepatocelular/tratamento farmacológico , Estudos de Coortes , Feminino , Humanos , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/sangue , Niacinamida/análogos & derivados , Niacinamida/uso terapêutico , Compostos de Fenilureia/uso terapêutico , Estudos Retrospectivos , SorafenibeRESUMO
BACKGROUND: The surveillance of hepatocellular carcinoma (HCC) has become prevalent, and the modalities for its treatment have improved. AIM: To understand the changes that occur in the characteristics and prognostic factors of HCC with time. METHODS: Newly diagnosed HCC patients were divided into two groups; patients treated before 31 December 2000 (n = 504), and after 1 January 2001 (n = 746), and their clinical backgrounds and prognostic factors were analysed. RESULTS: The number of patients negative for both Hepatitis B surface antigen (HBsAg) and Hepatitis C virus antibody (HCVAb) increased with time (NBNC-HCC). The size of HCC decreased in patients who were positive for HBsAg (B-HCC) or HCVAb (C-HCC), whereas no difference was observed in NBNC-HCC. The patient survival of C-HCC improved; however, no difference was detected for NBNC-HCC. In multivariate analysis, low albumin, high aspartate aminotransferase (AST), ascites, large tumour size, multiple tumour number and high alpha-fetoprotein were risk factors for survival before 2000, whereas the presence of HBsAg was additionally selected as a good prognostic factor and AST was excluded after 2001. CONCLUSIONS: The prognostic factors as well as clinical background of HCC changed with time, and the presence of HBsAg was found to be an additional good prognostic factor after 2001.
Assuntos
Carcinoma Hepatocelular/diagnóstico , Antígenos de Superfície da Hepatite B , Anticorpos Anti-Hepatite C , Neoplasias Hepáticas/diagnóstico , Idoso , Biomarcadores Tumorais/imunologia , Carcinoma Hepatocelular/epidemiologia , Carcinoma Hepatocelular/imunologia , DNA Viral/imunologia , Feminino , Humanos , Japão/epidemiologia , Neoplasias Hepáticas/epidemiologia , Neoplasias Hepáticas/imunologia , Masculino , Pessoa de Meia-Idade , Prevalência , Prognóstico , Fatores de RiscoRESUMO
How m-calpain is activated in cells has challenged investigators because in vitro activation requires near-millimolar calcium. Previously, we demonstrated that m-calpain activation by growth factors requires extracellular signal-regulated kinase (ERK); this enables tail deadhesion and allows productive motility. We now show that ERK directly phosphorylates and activates m-calpain both in vitro and in vivo. We identified serine 50 as required for epidermal growth factor (EGF)-induced calpain activation in vitro and in vivo. Replacing the serine with alanine limits activation by EGF and subsequent cell deadhesion and motility. A construct with the serine converted to glutamic acid displays constitutive activity in vivo; expression of an estrogen receptor fusion construct produces a tamoxifen-sensitive enzyme. Interestingly, EGF-induced m-calpain activation occurs in the absence of increased intracellular calcium levels; EGF triggers calpain even in the presence of intracellular calcium chelators and in calcium-free media. These data provide evidence that m-calpain can be activated through the ERK cascade via direct phosphorylation and that this activation may occur in the absence of cytosolic calcium fluxes.
Assuntos
Calpaína/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Substituição de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Cálcio/metabolismo , Calpaína/química , Calpaína/genética , Linhagem Celular , Movimento Celular , DNA Complementar/genética , Ativação Enzimática/efeitos dos fármacos , Humanos , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/químicaRESUMO
Connective tissue growth factor (CTGF) is up-regulated by TGF-beta1 during wound healing. The present study examined the expression of CTGF during regeneration after 70% partial hepatectomy (PH) or d-galactosamine (GalN)-injured liver in rats. CTGF, TGF-beta1, and type I collagen mRNAs were semiquantified by a ribonuclease protection assay. After PH, TGF-beta1 and type I collagen were increased at 2-6 h and at 12-48 h. CTGF increased at 6 h and returned to the control level thereafter. The ribonuclease protection assay of cultured hepatic stellate cells (HSC) and in situ hybridization suggest that the cells express CTGF along sinusoid might be HSCs. After GalN administration, CTGF increased at 2-96 h with a shoulder peak at 6-12 h followed by a main peak at 24 h. TGF-beta1 and type I collagen were up-regulated with kinetics similar to those of CTGF. The different kinetics between PH and GalN regenerations indicate that regulation of CTGF in the two processes is different. Higher TGF-beta1 expression after inflammatory/necrotic process in the GalN regeneration may caused the prolonged CTGF expression.
Assuntos
Substâncias de Crescimento/biossíntese , Substâncias de Crescimento/genética , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular , Fígado/fisiologia , Fígado/cirurgia , Regeneração , Animais , Células Cultivadas , Colágeno/biossíntese , Fator de Crescimento do Tecido Conjuntivo , Matriz Extracelular/metabolismo , Galactosamina/metabolismo , Hibridização In Situ , Cinética , Fígado/efeitos dos fármacos , Fígado/lesões , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-fos/biossíntese , RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Ribonucleases/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta1RESUMO
Wound healing is compromised in aging adults in part due to decreased responsiveness of fibroblasts to extracellular signals. However, the cellular mechanisms underlying this phenomenon are not known. Aged dermal fibroblasts with reduced remaining replicative capacities demonstrated decreased epidermal growth factor (EGF)-induced cell migrative and cell proliferative capacities, as reported previously. Thus, as cells approach senescence, programmed in vivo or in vitro, EGF responsiveness is preferentially lost. To define the rate-limiting signaling event, we found that the activity of two different EGF receptor (EGFR)-signaling pathways to cell migration (phospholipase-C gamma) and/or mitogenesis (extracellular signal/regulated-mitogen-activated kinases) were decreased in near senescent cells despite unchanged levels of effector molecules. Aged cells presented decreased levels of EGFR, although insulin receptor and transferrin receptor levels were relatively unchanged. EGFR mRNA levels and production of new transcripts decreased during aging, suggesting that this preferential loss of EGFR was due to diminished production, which more than counteracts the reduced ligand-induced receptor loss. Since these data suggested that the decrement in EGF was rate-limiting, higher levels of EGFR were established in near senescent cells by electroporation of EGFR cDNA. These cells presented higher levels of EGFR and recovered their EGF-induced migration and proliferation responsiveness. Thus, the defect in EGF responsiveness of aged dermal fibroblasts is secondary to reduced EGFR message transcription. Our experimental model suggests that EGFR gene delivery might be an effective future therapy for compromised wound healing.
Assuntos
Senescência Celular , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Transdução de Sinais , Divisão Celular , Movimento Celular , AMP Cíclico/metabolismo , DNA Complementar , Receptores ErbB/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Regiões Promotoras GenéticasRESUMO
During wound healing, fibroblasts are recruited from the surrounding tissue to accomplish repair. The requisite migration and proliferation of the fibroblasts is promoted by growth factors including those that activate the epidermal growth factor receptor (EGFR). Counterstimulatory factors in wound fluid are postulated to limit this response; among these factors is the ELR-negative CXC chemokine, interferon inducible protein-10 (IP-10). We report here that IP-10 inhibited EGF- and heparin-binding EGF-like growth factor-induced Hs68 human dermal fibroblast motility in a dose-dependent manner (to 52% and 44%, respectively, at 50 ng/ml IP-10), whereas IP-10 had no effect on either basal or EGFR-mediated mitogenesis (96 +/- 15% at 50 ng/ml). These data demonstrate for the first time a counterstimulatory effect of IP-10 on a specific induced fibroblast response, EGFR-mediated motility. To define the molecular basis of this negative transmodulation of EGFR signaling, we found that IP-10 did not adversely impact receptor or immediate postreceptor signaling as determined by tyrosyl phosphorylation of EGFR and two major downstream effectors phospholipase C-gamma and erk mitogen-activated protein kinases. Morphological studies suggested which biophysical steps may be affected by demonstrating that IP-10 treatment resulted in an elongated cell morphology reminiscent of failure to detach the uropod; in support of this, IP-10 pretreatment inhibited EGF-induced cell detachment. These data suggested that calpain activity may be involved. The cell permeant agent, calpain inhibitor I, limited EGF-induced motility and de-adhesion similarly to IP-10. IP-10 also prevented EGF- induced calpain activation (reduced by 71 +/- 7%). That this inhibition of EGF-induced calpain activity was secondary to IP-10 initiating a cAMP-protein kinase A-calpain cascade is supported by the following evidence: (a) the cell permeant analogue 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) prevented EGF-induced calpain activity and motility; (b) other ELR-negative CXC chemokines, monokine induced by IFN-gamma and platelet factor 4 that also generate cAMP, inhibited EGF-induced cell migration and calpain activation; and (c) the protein kinase A inhibitor Rp-8-Br-cAMPS abrogated IP-10 inhibition of cell migration, cell detachment, and calpain activation. Our findings provide a model by which IP-10 suppresses EGF-induced cell motility by inhibiting EGF-induced detachment of the trailing edges of motile cells.
Assuntos
Calpaína/metabolismo , Movimento Celular/efeitos dos fármacos , Quimiocinas CXC/farmacologia , Fator de Crescimento Epidérmico/antagonistas & inibidores , Receptores ErbB/antagonistas & inibidores , Sequência de Aminoácidos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Calpaína/antagonistas & inibidores , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Quimiocina CXCL10 , Quimiocinas CXC/química , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Derme/citologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Humanos , Isoenzimas/metabolismo , Fosfolipase C gama , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fosfolipases Tipo C/metabolismoRESUMO
Cell motility is induced by many growth factors acting through cognate receptors with intrinsic tyrosine kinase activity (RPTK). However, most of the links between receptor activation and the biophysical processes of cell motility remain undeciphered. We have focused on the mechanisms by which the EGF receptor (EGFR) actuates fibroblast cell motility in an attempt to define this integrated process in one system. Our working model is that divergent, but interconnected pathways lead to the biophysical processes necessary for cell motility: cytoskeleton reorganization, membrane extension, formation of new adhesions to substratum, cell contraction, and release of adhesions at the rear. We postulate that for any given growth factor some of the pathways/processes will be actively signaled and rate-limiting, while others will be permissive due to background low-level activation. Certain couplings have been defined, such as PLCgamma and actin modifying proteins being involved in cytoskeletal reorganization and lamellipod extension and MEK being implicated in detachment from substratum. Others are suggested by complementary investigations in integrin-mediated motility, including rac in membrane protrusion, rho in new adhesions, myosin II motors in contraction, and calpain in detachment, but have yet to be placed in growth factor-induced motility. Our model postulates that many biochemical pathways will be shared between chemokinetic and haptokinetic motility but that select pathways will be activated only during RPTK-enhanced motility.
Assuntos
Movimento Celular/fisiologia , Receptores ErbB/fisiologia , Fibroblastos/fisiologia , Actinas/metabolismo , Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Adesão Celular/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Modelos Biológicos , Fosfatidilinositol 3-Quinases/fisiologia , Fosfolipases/metabolismo , Proteína Quinase C/fisiologia , Proteínas Tirosina Quinases/metabolismoRESUMO
Two hepatitis B virus (HBV) carriers who had antibodies to HBV surface antigen (anti-HBs) were studied. Case 1 was a 47 year old woman positive for hepatitis B e antigen (HBeAg), and case 2 was a 61 year old man positive for antibody to HBeAg (anti-HBe) and DNA-polymerase (DNA-p). Neither case had received the HBV vaccine. The nucleotide sequences of the HBV-DNA extracted from the patients' sera were determined within the pre-S2 and S genes. Seven out of nine S gene clones from case 1 and six out of nine S gene clones from case 2 had an amino acid replacement from Thr or Ile to Ser at codon 126 in the alpha-determinant of the S gene. Amino acid substitution of codon 145 of the S gene previously reported was not observed. Although two previous reports on HBV escape mutant carriers with both anti-HBs and HBeAg described some deletions in the pre-S2 gene, our cases did not show these deletions. Our analysis indicated that carriers with the HBV escape mutant did not always have pre-S2 gene deletions. We found two HBV escape mutant carriers who had amino acid substitutions at codon 126 in the S gene due to point mutation without any deletions in the pre-S2 gene.
Assuntos
Genes Virais/genética , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Hepatite B/virologia , Mutação Puntual/genética , Precursores de Proteínas/genética , Proteínas do Envelope Viral/genética , DNA Viral/genética , Feminino , Hepatite B/imunologia , Anticorpos Anti-Hepatite B/imunologia , Antígenos E da Hepatite B/imunologia , Vírus da Hepatite B/genética , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Inactivation of the retinoblastoma (Rb) gene is considered to play a fundamental role in the genesis and progression of several human cancers. In retinoblastoma, the inactivation of Rb promoter by mutations or hypermethylation has been reported. Although genetic changes of Rb gene have been described in hepatocellular carcinoma (HCC), an epigenetic change such as hypermethylation of the Rb promoter as reported in retinoblastoma has not been described. MATERIALS AND METHODS: We examined the hypermethylation in the promoter region of Rb gene by restriction fragment length polymorphism in 19 HCCs, as well as the expression of Rb mRNA and protein by RT-PCR and by immunoblotting, respectively. RESULTS: We found no evidence of hypermethylation in the promoter region of the Rb gene in all HCCs analyzed. However, the expression of Rb mRNA and protein was lost in one HCC, and no mutation was detected in the Rb promoter region of this patient. The inactivation of Rb promoter by hypermethylation or by inhibition of binding of transcription factors due to point mutations did not contribute to the loss of mRNA and protein in the patient. CONCLUSIONS: Hypermethylation in the Rb promoter region appeared to have little causal effect on HCC.
Assuntos
Carcinoma Hepatocelular/genética , Genes do Retinoblastoma/genética , Neoplasias Hepáticas/genética , RNA Mensageiro/genética , Idoso , Carcinoma Hepatocelular/patologia , Feminino , Expressão Gênica , Humanos , Immunoblotting , Neoplasias Hepáticas/patologia , Masculino , Metilação , Pessoa de Meia-Idade , Fosforilação , Mutação Puntual , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , DNA Polimerase Dirigida por RNARESUMO
We designed a bioartificial liver support system in which encapsulated multicellular spheroids of rat hepatocytes were utilized as a bioreactor in a hollow fiber cartridge. The spheroids, formed in a positively charged polystyrene dish that contained hormonally defined medium, were encapsulated into microdroplets of agarose that contained about 9 x 10(7) rat hepatocytes. The medium, including 150 mL reservoir volume, was circulated in a closed circuit in which the cartridge was inserted. The pH and levels of dissolved oxygen were monitored and automatically regulated so that they were maintained within a constant range for 72 h. Albumin accumulated in the circuit at the rate of 2.0 mg/L/h in this system. When the bioreactor cells in the system were replaced with Hep G2 cells, a human hepatoblastoma cell line, albumin accumulated at the rate of 0.15 mg/L/h. The spheroids of primary culture hepatocytes had 13 times higher albumin-producing capacity than the aggregates of Hep G2. The serum of a patient with fulminant hepatic failure was circulated in this system with the spheroids of primary culture hepatocytes. The concentration of branched amino acid (BCAA) in the circuit significantly increased during the 48 h circulation, while the concentration of aromatic amino acid (AAA) and methionine decreased. The ratio of BCAA/AAA increased from 0.640 to 0.772, indicating that the hepatocyte spheroids had improved the imbalance of the amino acid profile in the serum. These findings indicate that this system may be a useful model for an artificial liver support. (c) 1996 John Wiley & Sons, Inc.
RESUMO
Adult rat hepatocytes assemble to form multicellular spheroids under non-adherent environments such as immobilized chondroitin sulfate-proteoglycan in primary culture. Previously, we demonstrated that hepatocyte spheroids exhibited various differentiated structures as observed in the liver tissue. It was also shown that hepatocyte growth was highly suppressed and several differentiated functions, including albumin production and gluconeogenesis, were well preserved in spheroids. To investigate the differentiated functions of cultured hepatocytes in relation to cell morphology, we compared the expression of the albumin and transferrin genes in spheroids with those in monolayers by Northern blot analysis. Production of these proteins in the culture medium was simultaneously examined by ELISA. Gene expression and protein production of both albumin and transferrin were better preserved in spheroids. We also examined changes in the expression of liver-specific genes in response to IL-6. Reduced mRNA levels of both albumin and transferrin was only found in spheroids and no change was observed in monolayers. These results suggest that the regulation of tissue-specific gene expression is better preserved in spheroids, in which hepatocytes are in close contact with each other.