RESUMO
Hypoxia and dysfunctional tumor vessels represent a prominent feature of pancreatic cancer, being, at least in part, responsible for chemotherapy resistance and immune suppression in these tumors. We tested whether the increase of oxygen delivery induced in vivo by myo-inositol trispyrophosphate (ITPP) can reverse hypoxia, control tumor growth and improve chemotherapy response. Tumor size, metastatic development (microcomputed tomography scan follow-up) and the survival of rats and nude or NOD.SCID mice, (bearing syngenic rat and MiaPaCa2- or patient-derived pancreatic tumors), were determined on ITPP and/or gemcitabine treatment. Partial oxygen pressure, expression of angiogenic factors and tumor histology were evaluated. Infiltration and oxidative status of immune cells, as well as chemotherapy penetration in tumors, were determined by fluorescence-activated cell sorting, fluorometry, nitric oxide release assays, Western blot and confocal microscopy. Weekly intravenous ITPP application resulted in the inhibition of metastasis development and restricted primary tumor growth, showing a superior effect on the rats' survival compared with gemcitabine. ITPP treatment restored tumor normoxia and caused a reduction in hypoxia inducible factor-1α levels, with subsequent VEGF and Lox downregulation, resulting in improved vessel structure and decreased desmoplasia. The latter effects translated into elevated immune cells influx and improved susceptibility to gemcitabine treatment. Growth of human pancreatic tumor xenografts was strongly inhibited by administration of ITPP. ITPP exploits a two-stage mechanism causing rapid, early and sustainable late stage normoxia. This is due to the angiogenic factor modulation and vascular normalization, leading to enhanced chemotherapy delivery and synergistic life prolongation, on combination with low doses of gemcitabine.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Desoxicitidina/análogos & derivados , Hipóxia/tratamento farmacológico , Fosfatos de Inositol/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/uso terapêutico , Sinergismo Farmacológico , Imunofluorescência , Humanos , Hipóxia/metabolismo , Hipóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neovascularização Patológica , Oxigênio/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , GencitabinaRESUMO
BACKGROUND/AIMS: We aimed to analyze substance P (SP) and neprilysin (NEP), the membrane metallopeptidase that degrades SP, in chronic pancreatitis (CP). METHODS: SP and NEP mRNA levels were analyzed by qRT-PCR in tissue samples from 30 patients with CP and 8 organ donors. In addition, SP serum levels were determined before and after surgery in the same patients, by means of a competitive ELISA assay. Genetic and epigenetic analyses of the NEP gene were also performed. RESULTS: SP mRNA expression levels were higher in CP tissues compared to controls (p = 0.0152), while NEP mRNA showed no significant differences between CP and healthy subjects (p = 0.2102). In CP patients, SP serum levels correlated with those in tissue, and after surgical resection SP serum levels were reduced compared to the preoperative values. Failure of NEP to overexpress in CP tissues was associated with significant miR-128a overexpression (p = 0.02), rather than with mutations in the NEP coding region or the presence of hypermethylation sites in the NEP promoter region. CONCLUSION: Tissue and serum levels of SP were increased in CP, while NEP levels remained unaltered. In an SP/NEP-mediated pathway, it would appear that NEP fails to provide adequate surveillance of SP levels. Failure of NEP to overexpress could be associated with miRNA regulation.
Assuntos
Neprilisina/fisiologia , Pancreatite Crônica/etiologia , Substância P/fisiologia , Adulto , Idoso , Metilação de DNA , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neprilisina/sangue , Neprilisina/genética , Pancreatite Crônica/sangue , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Substância P/sangue , Substância P/genéticaRESUMO
BACKGROUND: The transcription factor HOXC8 regulates many genes involved in tumour progression. This study was to investigate the role of HOXC8 in pancreatic ductal adenocarcinoma (PDAC) growth and metastasis. METHODS: The Hoxc8 expression was determined in 15 PDAC cell lines and human specimens by RT-polymerase chain reaction and/or immunohistochemistry. The effects of HOXC8 silencing by RNA interference were investigated by functional tests. RESULTS: The Hoxc8 mRNA expression in PDAC cell lines was negatively related to their growth in vivo. Except for Suit2-007 cells, only those with low Hoxc8 mRNA expression grew in nude rats. Successful down-regulation of HOXC8 expression caused increased proliferation, migration (P ≤ 0.05) and colony formation (P ≤ 0.05) in Suit2-007, Panc-1 and MIA PaCa-2 PDAC cells, respectively. The Hoxc8 mRNA levels in diseased human pancreas tissues were significantly increased over normal in PDAC and autoimmune chronic pancreatitis specimens (P<0.01, respectively), but negatively related to tumour stage (P=0.09). In primary and metastatic tumour samples, immunohistochemical staining for HOXC8 was stronger in surrounding than in neoplastic tissues. Furthermore, grading of primary carcinomas was negatively associated with HOXC8 staining (P=0.03). Liver metastases showed the lowest HOXC8 expression of all neoplastic lesions. CONCLUSION: These data indicate that HOXC8 expression is inversely related to PDAC progression and metastasis.
Assuntos
Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Proteínas de Homeodomínio/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Estudos de Casos e Controles , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Nus , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer, fibrotic diseases and arteriosclerosis. The PDGF family currently consists of at least three gene products, PDGF-A, PDGF-B and PDGF-C, which selectively signal through two PDGF receptors (PDGFRs) to regulate diverse cellular functions. After two decades of searching, PDGF-A and B were the only ligands identified for PDGFRs. Recently, however, database mining has resulted in the discovery of a third member of the PDGF family, PDGF-C, a functional analogue of PDGF-A that requires proteolytic activation. PDGF-A and PDGF-C selectively activate PDGFR-alpha, whereas PDGF-B activates both PDGFR-alpha and PDGFR-beta. Here we identify and characterize a new member of the PDGF family, PDGF D, which also requires proteolytic activation. Recombinant, purified PDGF-D induces DNA synthesis and growth in cells expressing PDGFRs. In cells expressing individual PDGFRs, PDGF-D binds to and activates PDGFR-beta but not PDGFR-alpha. However, in cells expressing both PDGFRs, PDGF-D activates both receptors. This indicates that PDGFR-alpha activation may result from PDGFR-alpha/beta heterodimerization.
Assuntos
Linfocinas , Fator de Crescimento Derivado de Plaquetas/química , Fator de Crescimento Derivado de Plaquetas/fisiologia , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Bromodesoxiuridina/metabolismo , DNA Complementar/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Fibroblastos/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Fosforilação , Reação em Cadeia da Polimerase , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Distribuição Tecidual , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
Lymphoproliferation, chronic B cell activation resulting in hypergammaglobulinemia, and profound immunodeficiency are prominent features of a retrovirus-induced syndrome designated murine acquired immunodeficiency syndrome (MAIDS). In vivo treatment of infected mice with recombinant interleukin 12 (IL-12) beginning at the time of infection or up to 9 wk after virus inoculation markedly inhibited the development of splenomegaly and lymphadenopathy, as well as B cell activation and Ig secretion. Treatment with IL-12 also had major effects in preventing induction of several immune defects including impaired production of interferon gamma (IFN-gamma) and IL-2 and depressed proliferative responses to various stimuli. The therapeutic effects of IL-12 on the immune system of mice with MAIDS were also associated with reduced expression of the retrovirus that causes this disease (BM5def), with lesser effects on expression of ecotropic MuLV. IL-12 treatment was not effective in IFN-gamma knockout mice or in infected mice treated simultaneously with IL-12 and anti-IFN-gamma. These results demonstrate that induction and progression of MAIDS are antagonized by IL-12 through high-level expression of IFN-gamma and may provide an experimental basis for developing treatments of retrovirus-induced immune disorders with similar immunopathogenic mechanisms.
Assuntos
Linfócitos B/imunologia , Interleucina-12/uso terapêutico , Síndrome de Imunodeficiência Adquirida Murina/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Linfócitos B/efeitos dos fármacos , Sequência de Bases , Primers do DNA , Feminino , Citometria de Fluxo , Hipoxantina Fosforribosiltransferase/biossíntese , Interferon gama/imunologia , Interleucina-12/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Síndrome de Imunodeficiência Adquirida Murina/fisiopatologia , Síndrome de Imunodeficiência Adquirida Murina/terapia , Tamanho do Órgão/efeitos dos fármacos , Reação em Cadeia da Polimerase , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Esplenomegalia/prevenção & controle , Fatores de TempoRESUMO
Mice with a null mutation of the gene encoding interferon consensus sequence-binding protein (ICSBP) develop a disease with marked expansion of granulocytes and macrophages that frequently progresses to a fatal blast crisis, thus resembling human chronic myelogenous leukemia (CML). One important feature of CML is decreased responsiveness of myeloid cells to apoptotic stimuli. Here we show that myeloid cells from mice deficient in ICSBP exhibit reduced spontaneous apoptosis and a significant decrease in sensitivity to apoptosis induced by DNA damage. In contrast, apoptosis in thymocytes from ICSBP-deficient mice is unaffected. We also show that overexpression of ICSBP in the human U937 monocytic cell line enhances the rate of spontaneous apoptosis and the sensitivity to apoptosis induced by etoposide, lipopolysaccharide plus ATP, or rapamycin. Programmed cell death induced by etoposide was specifically blocked by peptides inhibitory for the caspase-1 or caspase-3 subfamilies of caspases. Studies of proapoptotic genes showed that cells overexpressing ICSBP have enhanced expression of caspase-3 precursor protein. In addition, analyses of antiapoptotic genes showed that overexpression of ICSBP results in decreased expression of Bcl-X(L). These data suggest that ICSBP modulates survival of myeloid cells by regulating expression of apoptosis-related genes.
Assuntos
Apoptose/imunologia , Sequência Consenso/imunologia , Células-Tronco Hematopoéticas/patologia , Interferons/farmacologia , Proteínas Repressoras/fisiologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/genética , Células da Medula Óssea , Caspases/biossíntese , Caspases/genética , Caspases/metabolismo , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Etoposídeo/antagonistas & inibidores , Etoposídeo/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Fatores Reguladores de Interferon , Linfonodos , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Baço , Células U937 , Proteína bcl-XRESUMO
Mice with a null mutation of the gene encoding interferon consensus sequence-binding protein (ICSBP) develop a chronic myelogenous leukemia-like syndrome and mount impaired responses to certain viral and bacterial infections. To gain a mechanistic understanding of the contributions of ICSBP to humoral and cellular immunity, we characterized the responses of control and ICSBP-/- mice to infection with influenza A (flu) and Leishmania major (L. major). Mice of both genotypes survived infections with flu, but differed markedly in the isotype distribution of antiflu antibodies. In sera of normal mice, immunoglobulin (Ig)G2a antibodies were dominant over IgG1 antibodies, a pattern indicative of a T helper cell type 1 (Th1)-driven response. In sera of ICSBP-/- mice, however, IgG1 antibodies dominated over IgG2a antibodies, a pattern indicative of a Th2-driven response. The dominance of IgG1 and IgE over IgG2a was detected in the sera of uninfected mice as well. A seeming Th2 bias of ICSBP-deficient mice was also uncovered in their inability to control infection with L. major, where resistance is known to be dependent on IL-12 and IFN-gamma as components of a Th1 response. Infected ICSBP-deficient mice developed fulminant, disseminated leishmaniasis as a result of failure to mount a Th1-mediated curative response, although T cells remained capable of secreting IFN-gamma and macrophages of producing nitric oxide. Compromised Th1 differentiation in ICSBP-/- mice could not be attributed to hyporesponsiveness of CD4(+) T cells to interleukin (IL)-12; however, the ability of uninfected and infected ICSBP-deficient mice to produce IL-12 was markedly impaired. This indicates that ICSBP is a deciding factor in Th responses governing humoral and cellular immunity through its role in regulating IL-12 expression.
Assuntos
Proteínas de Transporte/imunologia , Proteínas de Transporte/fisiologia , Interleucina-12/biossíntese , Proteínas Repressoras , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular/imunologia , Sequência Consenso/imunologia , Suscetibilidade a Doenças , Vírus da Influenza A/imunologia , Fatores Reguladores de Interferon , Interferons/fisiologia , Interleucina-12/deficiência , Interleucina-12/metabolismo , Leishmania major/imunologia , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/imunologia , Células Th1/citologia , Células Th2/citologiaRESUMO
PURPOSE: Tumour hypoxia activates hypoxia-inducible factor-1 (HIF-1) and indluences angiogenesis, cell survival and invasion. Prolyl hydroxylase-3 (PHD3) regulates degradation of HIF-1α. The effects of PHD3 in tumour growth are largely unknown. EXPERIMENTAL DESIGN: PHD3 expression was analysed in human pancreatic cancer tissues and cancer cell lines by real-time quantitative PCR and immunohistochemistry. PHD3 overexpression was established by stable transfection and downregulation by short interfering RNA technology. VEGF was quantified by enzyme-linked immunosorbent assay. Matrigel invasion assays were performed to examine tumour cell invasion. Apoptosis was measured by annexin-V staining and caspase-3 assays. The effect of PHD3 on tumour growth in vivo was evaluated in an established orthotopic murine model. RESULTS: PHD3 was upregulated in well-differentiated human tumours and cell lines, and regulated hypoxic VEGF secretion. PHD3 overexpression mediated tumour cell growth and invasion by induction of apoptosis in a nerve growth factor-dependent manner by the activation of caspase-3 and phosphorylation of focal adhesion kinase HIF-1 independently. In vivo, PHD3 inhibited tumour growth by abrogation of tumour angiogenesis. CONCLUSION: Our results indicate essential functions of PHD3 in tumour growth, apoptosis and angiogenesis and through HIF-1-dependent and HIF-1-independent pathways.
Assuntos
Dioxigenases/genética , Neovascularização Patológica/patologia , Neoplasias Pancreáticas/patologia , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Animais , Anexina A5/análise , Apoptose , Carcinoma Ductal Pancreático/enzimologia , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/cirurgia , Caspase 3/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Dioxigenases/fisiologia , Ensaio de Imunoadsorção Enzimática , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Prolina Dioxigenases do Fator Induzível por Hipóxia , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/cirurgia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
Despite spontaneous or vaccination-induced immune responses, pancreatic cancer remains one of the most deadly immunotherapy-resistant malignancies. We sought to comprehend the spectrum of pancreatic tumor-associated antigens (pTAAs) and to assess the clinical relevance of their immunogenicity. An autologous SEREX-based screening of a cDNA library constructed from a pancreatic T3N0M0/GIII specimen belonging to a long-term survivor (36 months) revealed 18 immunogenic pTAA. RT-PCR analysis displayed broad distribution of the identified antigens among normal human tissues. PNLIPRP2 and MIA demonstrated the most distinct pancreatic cancer-specific patterns. ELISA-based screening of sera for corresponding autoantibodies revealed that although significantly increased, the immunogenicity of these molecules was not a common feature in pancreatic cancer. QRT-PCR and immunohistochemistry characterized PNLIPRP2 as a robust acinar cell-specific marker whose decreased expression mirrored the disappearance of parenchyma in the diseased organ, but was not related to the presence of PNLIPRP2 autoantibodies. Analyses of MIA-known to be preferentially expressed in malignant cells-surprisingly revealed an inverse correlation between intratumoral gene expression and the emergence of autoantibodies. MIA(high) patients were autoantibody-negative and had shorter median survival when compared with autoantibody-positive MIA(low) patients (12 vs. 34 months). The observed pTAA spectrum comprised molecules associated with acinar, stromal and malignant structures, thus presenting novel targets for tumor cell-specific therapies as well as for approaches based on the bystander effects. Applying the concept of cancer immunoediting to interpret relationships between gene expression, antitumor immune responses, and clinical outcome might better discriminate between past and ongoing immune responses, consequently enabling prognostic stratification of patients and individual adjustment of immunotherapy.
Assuntos
Adenocarcinoma/diagnóstico , Adenocarcinoma/imunologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/imunologia , Adenocarcinoma/sangue , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Idoso , Antígenos Glicosídicos Associados a Tumores/genética , Antígenos Glicosídicos Associados a Tumores/imunologia , Antígenos Glicosídicos Associados a Tumores/metabolismo , Autoanticorpos/sangue , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Feminino , Biblioteca Gênica , Humanos , Imunoquímica , Lipase/genética , Lipase/imunologia , Lipase/metabolismo , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , PrognósticoRESUMO
The v-sis oncogene encodes a platelet-derived growth factor (PDGF)-related product whose transforming activity is mediated by its functional interaction with the PDGF receptor. PDGF, as well as processed forms of the v-sis gene product, is a disulfide-linked dimer with eight conserved cysteine residues in the minimum region necessary for biologic activity. Site-directed mutagenesis of the v-sis gene revealed that each conserved cysteine residue was required directly or indirectly for disulfide-linked dimer formation. However, substitution of serine for cysteine codons at any of four positions had no detrimental effect on transforming activity of the encoded v-sis protein. These results establish that interchain disulfide bonds are not essential in order for this protein to act as a functional ligand for the PDGF receptor. The remaining four substitutions of serine for cysteine each inactivated transforming function of the molecule. In each case this was associated with loss of a conformation shown to involve intramolecular disulfide bonds. These studies provide insight into the role of individual cysteine residues in determining the structure of the sis/PDGF molecule critical for biological activity.
Assuntos
Antígenos Virais de Tumores/fisiologia , Genes Virais , Proteínas Oncogênicas Virais/fisiologia , Oncogenes , Retroviridae/genética , Vírus do Sarcoma do Macaco-Barrigudo/genética , Antígenos Transformantes de Poliomavirus , Antígenos Virais de Tumores/análise , Transformação Celular Viral , Reações Cruzadas , Cisteína , Mutação , Proteínas Oncogênicas Virais/análise , Fator de Crescimento Derivado de Plaquetas/análise , Fator de Crescimento Derivado de Plaquetas/fisiologia , Conformação Proteica , Processamento de Proteína Pós-Traducional , Receptores de Superfície Celular/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas , Serina , TransfecçãoRESUMO
Human platelet-derived growth factor (PDGF) is a connective tissue cell mitogen comprised of two related chains encoded by distinct genes. The B chain is the homolog of the v-sis oncogene product. Properties that distinguish these ligands include greater transforming potency of the B chain and more efficient secretion of the A chain. By a strategy involving the generation of PDGF A and B chimeras, these properties were mapped to distinct domains of the respective molecules. Increased transforming efficiency segregated with the ability to activate both alpha and beta PDGF receptors. These findings genetically map PDGF B residues 105 to 144 as responsible for conformational alterations critical to beta PDGF receptor interaction and provide a mechanistic basis for the greater transforming potency of the PDGF B chain.
Assuntos
Transformação Celular Neoplásica , Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Proto-Oncogênicas/genética , Linhagem Celular Transformada , Quimera , Vetores Genéticos , Humanos , Ligantes , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/fisiologia , Testes de Precipitina , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-sis , Receptores de Superfície Celular/genética , Receptores do Fator de Crescimento Derivado de Plaquetas , TransfecçãoRESUMO
Efficacy of chemotherapy for pancreatic cancer may be improved by tailoring it to individual chemosensitivity profiles. Identification of nonresponders before initiation of treatment may help to avoid side effects. In this study, primary pancreatic cancer cells were isolated from 18 patients undergoing pancreaticoduodenectomy for pancreatic cancer. Eight commonly used pancreatic cancer cell lines were used as controls. Ex vivo chemosensitivity for gemcitabine, 5-fluorouracil, mitomycin-C, cisplatinum, oxaliplatinum, paclitaxel and a combination of gemcitabine with oxaliplatinum or mitomycin-C was determined using a cellular ATP-based tumour chemosensitivity assay (ATP-TCA). Quantitative real-time-polymerase chain reaction was performed to determine RNA expression levels of genes implicated in chemoresistance. Chemosensitivity towards cytotoxic agents was highly variable in primary pancreatic cancer cells and pancreatic cancer cell lines. ATP-TCA results for gemcitabine correlated to the tissue expression of human equilibrative nucleoside transporter-1 (hENT1). Time to relapse in patients with gemcitabine-sensitive tumours was significantly higher than in patients with chemoresistant pancreatic cancers (P=0.01; 71 vs 269 days). Furthermore, time to relapse in gemcitabine-treated patients was related to hENT1 expression (P=0.0067). Thus, chemosensitivity testing using ATP-TCA in pancreatic cancer is feasible and correlated with time to relapse in gemcitabine-treated patients. This suggests that ATP-TCA testing could be used as a decision-making tool in the adjuvant treatment of pancreatic cancer.
Assuntos
Antineoplásicos/uso terapêutico , Desoxicitidina/análogos & derivados , Perfilação da Expressão Gênica , Neoplasias Pancreáticas/tratamento farmacológico , Adulto , Idoso , Linhagem Celular Tumoral , Desoxicitidina/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Reação em Cadeia da Polimerase , GencitabinaRESUMO
Pancreatic cancer has an abysmal prognosis. Targets for early detection, prevention and therapy are desperately needed. Inflammatory pathways have an important impact on tumour growth and progression. Expression of BLT2 (the second leukotriene B(4) receptor) was investigated by real-time RT-PCR and immunohistochemistry. Cell proliferation was studied after stable transfection with BLT2, after treatment with siRNA and Compound A as an agonist. BLT2 is expressed in all pancreatic cancer cell lines. Results from real-time RT-PCR revealed significant overexpression of BLT2 in malignant intraductal papillary mucinous neoplasias (IPMNs) and pancreatic adenocarcinoma. Intense staining was evident in IPMNs, infiltrating tumour cells and advanced pancreatic intraepithelial neoplasias (PanINs) but not in normal ductal cells. Overexpression of BLT2 as well as stimulation of Colo357, Panc-1 and AsPC1 cells with Compound A caused a significant increase in tumour cell proliferation, an effect reversed after siRNA treatment. This study demonstrates for the first time the expression of BLT2 in the pancreas and overexpression in pancreatic cancers and malignant IPMNs in particular. Upregulation of BLT2 is already evident in precursor lesions (PanINs, IPMNs). Overexpression of this receptor leads to significant growth stimulation. Therefore, we suggest BLT2 as a new target for chemoprevention and therapy for pancreatic cancer.
Assuntos
Proliferação de Células , Neoplasias Pancreáticas/metabolismo , Receptores do Leucotrieno B4/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Humanos , Imuno-Histoquímica , Leucotrieno B4/metabolismo , Ligantes , Neoplasias Pancreáticas/patologia , Pancreatite/metabolismo , RNA Interferente Pequeno , Receptores do Leucotrieno B4/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Little is known about the factors that enable the mobilisation of human mesenchymal stem cells (MSC) from the bone marrow into the blood stream and their recruitment to and retention in the tumour. We found specific migration of MSC towards growth factors present in pancreatic tumours, such as PDGF, EGF, VEGF and specific inhibitors Glivec, Erbitux and Avastin interfered with migration. Within a few hours, MSC migrated into spheroids consisting of pancreatic cancer cells, fibroblasts and endothelial cells as measured by time-lapse microscopy. Supernatant from subconfluent MSC increased sprouting of HUVEC due to VEGF production by MSC itself as demonstrated by RT-PCR and ELISA. Only few MSCs were differentiated into endothelial cells in vitro, whereas in vivo differentiation was not observed. Lentiviral GFP-marked MSCs, injected in nude mice xenografted with orthotopic pancreatic tumours, preferentially migrated into the tumours as observed by FACS analysis of green fluorescent cells. By immunofluorescence and intravital microscopic studies, we found the interaction of MSC with the endothelium of blood vessels. Mesenchymal stem cells supported tumour angiogenesis in vivo, that is CD31(+) vessel density was increased after the transfer of MSC compared with siVEGF-MSC. Our data demonstrate the migration of MSC toward tumour vessels and suggest a supportive role in angiogenesis.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Neovascularização Patológica/metabolismo , Neoplasias Pancreáticas/irrigação sanguínea , Fator A de Crescimento do Endotélio Vascular/metabolismo , Actinas/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Benzamidas , Bevacizumab , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Cetuximab , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Mesilato de Imatinib , Lentivirus/genética , Masculino , Transplante de Células-Tronco Mesenquimais , Camundongos , Camundongos Nus , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Piperazinas/farmacologia , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Fator de Crescimento Derivado de Plaquetas/metabolismo , Pirimidinas/farmacologia , Esferoides Celulares/patologia , Transplante Heterólogo , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
The chemokine receptor CXCR6 has been described on lymphoid cells and is thought to participate in the homing of activated T-cells to non-lymphoid tissue. We now provide evidence that the chemokine receptor CXCR6 is also expressed by activated polymorphonuclear neutrophils (PMN) in vivo: Examination of biopsies derived from patients with pancreatic carcinoma by confocal laser scan microscopy revealed a massive infiltration of PMN that expressed CXCR6, while PMN of the peripheral blood of these patients did not. To answer the question whether CXCR6 expression is a property of infiltrated and activated PMN, leucocytes were collected from patients with localized soft tissue infections in the course of the wound debridement. By cytofluorometry, the majority of these cells were identified as PMN. Up to 50% of these PMN were also positive for CXCR6. Again, PMN from the peripheral blood of these patients were nearly negative for CXCR6, as were PMN of healthy donors. In a series of in vitro experiments, up-regulation of CXCR6 on PMN of healthy donors by a variety of cytokines was tested. So far, a minor, although reproducible, effect of tumour necrosis factor (TNFalpha) was seen: brief exposure with low-dose TNFalpha induced expression of CXCR6 on the surface of PMN. Furthermore, we could show an increased migration of PMN induced by the axis CXCL16 and CXCR6. In summary, our data provide evidence that CXCR6 is not constitutively expressed on PMN, but is up-regulated under inflammatory conditions and mediates migration of CXCR6-positive PMN.
Assuntos
Infecções Bacterianas/imunologia , Proteínas de Neoplasias/metabolismo , Neutrófilos/imunologia , Neoplasias Pancreáticas/imunologia , Receptores de Quimiocinas/metabolismo , Receptores Virais/metabolismo , Doença Aguda , Quimiotaxia de Leucócito/imunologia , Humanos , Ligantes , Microscopia Confocal/métodos , Ativação de Neutrófilo/imunologia , Osteomielite/imunologia , Infecções Relacionadas à Prótese/imunologia , Receptores CXCR6 , Infecções dos Tecidos Moles/imunologia , Células Tumorais Cultivadas , Regulação para Cima/imunologiaRESUMO
Deletion scanning mutagenesis within the transforming region of the v-sis oncogene was used to dissect structure-function relationships. Mutations affecting codons within a domain encoding amino acids 136 through 148 had no effect upon homodimer formation or recognition by antisera which detect determinants dependent upon native intrachain disulfide linkages, yet the same mutations completely abolished transforming activity. A platelet-derived growth factor B (PDGF B) monoclonal antibody that prevents its interaction with PDGF receptors recognized v-sis, delta 142 (deletion of codon 142), and delta 148 but not delta 136, delta 137, or delta 139 mutants. These findings mapped the epitope recognized by this monoclonal antibody to include amino acid residues 136 to 139. Furthermore, mutations in the codon 136 to 148 domain caused markedly impaired ability to induce PDGF receptor tyrosine phosphorylation. Thus, subtle conformational alterations in this small domain critically affect PDGF receptor recognition and/or functional activation.
Assuntos
Fator de Crescimento Derivado de Plaquetas/fisiologia , Receptores de Superfície Celular/fisiologia , Proteínas Oncogênicas de Retroviridae/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Transformação Celular Neoplásica , Códon , Análise Mutacional de DNA , Epitopos , Camundongos , Proteínas Oncogênicas v-sis , Fosfotirosina , Receptores do Fator de Crescimento Derivado de Plaquetas , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
BACKGROUND: Recurrent inflammation in chronic pancreatitis (CP) is not well understood. AIMS: To investigate whether decorin, an extracellular matrix (ECM) proteoglycan with macrophage modulating activity, is a pathogenic factor allowing diseased pancreatic stroma to sustain inflammation by affecting the cytokine profile of accumulating inflammatory cells. METHODS: Decorin was examined in 18 donors and 32 patients with CP by quantitative reverse transcription polymerase chain reaction (QRT-PCR), western blotting, and immunohistochemistry of pancreatic specimens. QRT-PCR was used to assess cytokine expression in donor peripheral blood mononuclear cells (PBMC), exposed or not to decorin in vitro, and to compare it with the cytokine profile of circulating and resident mononuclear cells (MNC) of patients with CP. RESULTS: In CP, desmoplasia is associated with overexpression of decorin in the growing ECM and enlarged pancreatic nerves. In culture, exposure of MNC to decorin stimulated expression of the MNC recruiting chemokine MCP-1. In biopsies, MNC infiltrates in decorin rich CP tissue showed a 300-fold upregulation of MCP-1 compared with decorin free peripheral blood, whereas no difference was found in basal MCP-1 expression in PBMC of patients versus donors. This effect was specific for MCP1-other inflammatory cytokines, such as interleukin 1beta and tumour necrosis factor alpha, were not affected. CONCLUSION: Decorin is a molecular marker of desmoplasia in CP, and excessive decorin may allow fibrotic masses to nourish and protract inflammation by deregulating the process of MNC accumulation and activation. These data provide a molecular basis for surgical resection of diseased tissue as a treatment option in CP.
Assuntos
Pancreatite Crônica/metabolismo , Proteoglicanas/fisiologia , Adolescente , Adulto , Idoso , Western Blotting , Células Cultivadas , Quimiocina CCL2/sangue , Decorina , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Pâncreas/inervação , Proteoglicanas/metabolismo , Proteoglicanas/farmacologia , RNA Mensageiro/genética , Recidiva , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Regulação para CimaRESUMO
The effect of all-trans-retinoic acid (RA) on cellular transformation and on tumorigenicity of retrovirally transformed cells was investigated. RA treatment of NRK and NIH/3T3 cells transformed by BALB/c murine sarcoma virus (MuSV), Kirsten murine sarcoma virus (K-MuSV), and simian sarcoma virus resulted in a significant reduction in anchorage-dependent growth of only K-MuSV-transformed NRK cells. A 62% reduction in cell number was observed at 10(-5) M RA. In contrast, anchorage-independent growth induced by each of the viruses tested was suppressed by RA. Balb/cMSV3T3 cells showed the greatest level of sensitivity with a significant reduction in anchorage-independent growth occurring at 10(-9) M RA. The level of cytoplasmic retinoic acid-binding protein (CRABP) was determined in both parent and transformed cell lines. CRABP was present at a high level in all 3T3 cell types but was absent in all NRK cell lines. For testing the antineoplastic activity of RA in vivo, Balb/cMSV3T3 cells were injected intradermally into nude mice. Subsequent treatment of the tumor sites of these animals by topical application of RA resulted in a significant reduction in both tumor incidence and tumor size, confirming the in vitro results. Analysis of the level of v-onc mRNA revealed that inhibition of retroviral transformation by RA was not due to a decrease in transcription of the v-onc genes.
Assuntos
Transformação Celular Viral/efeitos dos fármacos , Neoplasias Cutâneas/prevenção & controle , Tretinoína/farmacologia , Infecções Tumorais por Vírus/prevenção & controle , Animais , Proteínas de Transporte/análise , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Camundongos , Camundongos Nus , Oncogenes/efeitos dos fármacos , RNA Mensageiro/análise , RNA Viral/análise , Receptores do Ácido Retinoico , Vírus do Sarcoma do Macaco-Barrigudo/genética , Vírus do Sarcoma Murino/genética , Transcrição Gênica/efeitos dos fármacos , Tretinoína/uso terapêuticoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with an overall 5-year survival rate of less than 5%. Invasive tumor growth and early metastasis are two important reasons for this dismal prognosis. Osteopontin (OPN) is a secretory protein with a variety of functions, for example in cell adhesion and migration, inflammatory reaction and apoptosis. In this study the functional role of OPN in human pancreatic cancer and its potential use as a disease marker were analyzed. By real time quantitative PCR, there was a 2.2-fold and 1.6-fold increase of OPN mRNA in pancreatic cancers (n = 23) and chronic pancreatitis samples (n = 22), respectively, compared to normal pancreatic tissues (n = 20). Immunohistochemical analysis demonstrated OPN staining in 60% of the primary pancreatic tumors and in 72% of the lymph node and liver metastases. ELISA analysis of serum samples obtained from pancreatic cancer patients (n = 70), chronic pancreatitis patients (n = 12), and healthy donors (n = 20) showed a 1.6-fold increase in OPN serum levels in patients with tumors and a 1.9-fold increase in patients with chronic pancreatitis. Recombinant human OPN significantly increased the invasiveness of pancreatic cancer cells, without having any impact on cell proliferation. In addition, down regulation of OPN by specific siRNA molecules decreased pancreatic cancer cell invasion. In conclusion, OPN serum levels in pancreatic cancer and chronic pancreatitis patients are not significantly different, thereby restricting its role as a prognostic or follow-up marker. Our results do suggest, however, that blockade of OPN might be useful as a therapeutic approach to inhibit invasion and metastasis of pancreatic cancer cells.