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1.
PLoS One ; 10(2): e0117254, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25692292

RESUMO

Primary pancreatic carcinoma has an unfavourable prognosis and standard treatment strategies mostly fail in advanced cases. Virotherapy might overcome this resistance to current treatment modalities. However, data from clinical studies with oncolytic viruses, including replicating adenoviral (Ad) vectors, have shown only limited activity against pancreatic cancer and other carcinomas. Since pancreatic carcinomas have a complex tumor architecture and frequently a strong stromal compartment consisting of non-neoplastic cell types (mainly pancreatic stellate cells = hPSCs) and extracellular matrix, it is not surprising that Ad vectors replicating in neoplastic cells will likely fail to eradicate this aggressive tumor type. Because the TGFß receptor (TGFBR) is expressed on both neoplastic cells and hPSCs we inserted the TGFBR targeting peptide CKS17 into the hypervariable region 5 (HVR5) of the capsid protein hexon with the aim to generate a replicating Ad vector with improved activity in complex tumors. We demonstrated increased transduction of both pancreatic cancer cell lines and of hPSCs and enhanced cytotoxicity in co-cultures of both cell types. Surface plasmon resonance analysis demonstrated decreased binding of coagulation factor X to CKS17-modified Ad particles and in vivo biodistribution studies performed in mice indicated decreased transduction of hepatocytes. Thus, to increase activity of replicating Ad vectors we propose to relax tumor cell selectivity by genetic hexon-mediated targeting to the TGFBR (or other receptors present on both neoplastic and non-neoplastic cells within the tumor) to enable replication also in the stromal cell compartment of tumors, while abolishing hepatocyte transduction, and thereby increasing safety.


Assuntos
Adenoviridae/genética , Proteínas do Capsídeo/genética , Terapia Genética , Vetores Genéticos/genética , Vírus Oncolíticos/genética , Neoplasias Pancreáticas/patologia , Engenharia de Proteínas , Adenoviridae/fisiologia , Sequência de Aminoácidos , Animais , Transporte Biológico , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Fator X/metabolismo , Vetores Genéticos/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Imunoglobulina M/metabolismo , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Vírus Oncolíticos/fisiologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/virologia , Células Estreladas do Pâncreas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Células Estromais/virologia , Transdução Genética , Tropismo Viral , Replicação Viral
2.
Mol Cancer Res ; 11(12): 1611-23, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24145173

RESUMO

UNLABELLED: Glioblastoma multiforme, the most common primary brain tumor, is highly refractory to therapy, mainly due to its ability to form micrometastases, which are small clusters or individual cells that rapidly transverse the brain and make full surgical resection impossible. Here, it is demonstrated that the invasive phenotype of glioblastoma multiforme is orchestrated by the transcription factor NF-κB which, via metalloproteinases (MMP), regulates fibronectin processing. Both, cell lines and tumor stem cells from primary glioblastoma multiforme, secrete high levels of fibronectin which when cleaved by MMPs forms an extracellular substrate. Subsequently, forming and interacting with their own microenvironment, glioblastoma multiforme cells are licensed to invade their surroundings. Mechanistic study revealed that NF-κB inhibition, either genetically or pharmacologically, by treatment with Disulfiram, significantly abolished the invasive phenotype in the chick chorioallantoic membrane assay. Furthermore, having delineated the underlying molecular mechanism of glioblastoma multiforme invasion, the potential of a disulfiram-based therapy was revealed in a highly invasive orthotrophic glioblastoma multiforme mouse model. IMPLICATIONS: This study defines a novel therapeutic approach that inhibits micrometastases invasion and reverts lethal glioblastoma into a less aggressive disease.


Assuntos
Dissulfiram/farmacologia , Inibidores Enzimáticos/farmacologia , Fibronectinas/metabolismo , Glioblastoma/patologia , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Metaloproteases/genética , Metaloproteases/metabolismo , Camundongos , NF-kappa B/genética , Invasividade Neoplásica , Microambiente Tumoral
3.
Mol Cancer Ther ; 12(9): 1783-95, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23832120

RESUMO

Glioblastoma is the most frequent brain tumor of glial origin in adults. With the best available standard-of-care, patients with this disease have a life expectancy of only approximately 15 months after diagnosis. Because the EGF receptor (HER1/EGFR) is one of the most commonly dysregulated oncogenes in glioblastoma, HER1/EGFR-targeted agents, such as erlotinib, were expected to provide a therapeutic benefit. However, their application in the clinical setting failed. Seeking an explanation for this finding, we previously identified several candidate genes for resistance of human glioblastoma cell lines toward erlotinib. On the basis of this panel of genes, we aimed at identifying drugs that synergistically enhance the antiproliferative effect of erlotinib on established and primary glioblastoma cell lines. We found that NSC23766, an inhibitor of RAC1, enhanced the antineoplastic effects of erlotinib in U87MG, T98MG, and A172MG glioblastoma cell lines for the most part in a synergistic or at least in an additive manner. In addition, the synergistic antiproliferative effect of erlotinib and NSC23766 was confirmed in primary cultured cells, indicating a common underlying cellular and molecular mechanism in glioblastoma. Therefore, agents that suppress RAC1 activation may be useful therapeutic partners for erlotinib in a combined targeted treatment of glioblastoma.


Assuntos
Aminoquinolinas/farmacologia , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Glioblastoma/patologia , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Aminoquinolinas/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Humanos , Masculino , Camundongos Endogâmicos NOD , Terapia de Alvo Molecular , Pirimidinas/uso terapêutico , Quinazolinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas rac1 de Ligação ao GTP/metabolismo
4.
Shock ; 38(1): 98-106, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22552016

RESUMO

More than 50% of severely injured patients have chest trauma. Second insults frequently result in acute lung injury (ALI), with sepsis being the main underlying condition. We aimed to develop a standardized, reproducible, and clinically relevant double-hit mouse model of ALI induced by chest trauma and polymicrobial sepsis and to investigate the pathophysiologic role of activated neutrophils. Lung contusion was applied to C57Bl/6 mice via a focused blast wave. Twenty-four hours later, sepsis was induced by cecal ligation and puncture. For polymorphonuclear leukocyte (PMN) depletion, animals received intravenous injections of PMN-depleting antibody. In response to blunt chest trauma followed by sepsis as well as after sepsis alone, a significant local and systemic inflammatory response with increased cytokine/chemokine levels in lung and plasma was observed. In contrast, lung apoptosis was markedly elevated only after a double hit. Intra-alveolar neutrophils and total bronchoalveolar lavage protein concentrations were markedly increased following isolated chest trauma or the combined insult, but not after sepsis alone. Lung myeloperoxidase activity was enhanced only in response to the double hit accompanied by histological disruption of the alveolar architecture, lung congestion, and marked cellular infiltrates. Neutrophil depletion significantly diminished lung interleukin 1ß and interleukin 6 concentrations and reduced the degree of septic ALI. Here we have established a novel and highly reproducible mouse model of chest trauma-induced septic ALI characterizing a clinical relevant double-hit scenario. In particular, the depletion of neutrophils substantially mitigated the extent of lung injury, indicating a pathomechanistic role for neutrophils in chest trauma-induced septic ALI.


Assuntos
Lesão Pulmonar Aguda/etiologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Sepse/complicações , Traumatismos Torácicos/complicações , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Animais , Apoptose/imunologia , Líquido da Lavagem Broncoalveolar/química , Dióxido de Carbono/sangue , Caspases/metabolismo , Quimiocinas/sangue , Citocinas/sangue , Modelos Animais de Doenças , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/sangue , Pressão Parcial , Sepse/imunologia , Síndrome de Resposta Inflamatória Sistêmica/etiologia , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Ferimentos não Penetrantes/complicações , Receptor fas/sangue
5.
Carcinogenesis ; 33(1): 84-93, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22095073

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis, representing one risk factor for PDAC, are characterized by a marked desmoplasia enriched of pancreatic myofibroblasts (PMFs). Thus, PMFs are thought to essentially promote pancreatic tumorigenesis. We recently demonstrated that the adhesion molecule L1CAM is involved in epithelial-mesenchymal transition of PMF-cocultured H6c7 human ductal epithelial cells and that L1CAM is expressed already in ductal structures of chronic pancreatitis with even higher elevation in primary tumors and metastases of PDAC patients. This study aimed at investigating whether PMFs and L1CAM drive malignant transformation of pancreatic ductal epithelial cells by enhancing their tumorigenic potential. Cell culture experiments demonstrated that in the presence of PMFs, H6c7 cells exhibit a profound resistance against death ligand-induced apoptosis. This apoptosis protection was similarly observed in H6c7 cells stably overexpressing L1CAM. Intrapancreatic inoculation of H6c7 cells together with PMFs (H6c7co) resulted in tumor formation in 7/8 and liver metastases in 6/8 severe combined immunodeficiency (SCID) mice, whereas no tumors and metastases were detectable after inoculation of H6c7 cells alone. Likewise, tumor outgrowth and metastases resulted from inoculation of L1CAM-overexpressing H6c7 cells in 5/7 and 3/7 SCID mice, respectively, but not from inoculation of mock-transfected H6c7 cells. Treatment of H6c7co tumor-bearing mice with the L1CAM antibody L1-9.3/2a inhibited tumor formation and liver metastasis in 100 and 50%, respectively, of the treated animals. Overall, these data provide new insights into the mechanisms of how PMFs and L1CAM contribute to malignant transformation of pancreatic ductal epithelial cells in early stages of pancreatic tumorigenesis.


Assuntos
Carcinoma Ductal Pancreático/etiologia , Miofibroblastos/fisiologia , Molécula L1 de Adesão de Célula Nervosa/fisiologia , Neoplasias Pancreáticas/etiologia , Animais , Apoptose , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Humanos , Neoplasias Hepáticas/secundário , Camundongos , Camundongos SCID , Neoplasias Pancreáticas/patologia
6.
Shock ; 37(2): 210-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22089186

RESUMO

Polymorphonuclear granulocytes (PMNs) have been attributed a primarily deleterious role in the pathogenesis of acute lung injury (ALI). However, evidence exists that PMNs might also act beneficially in certain types of ALI. In this regard, we investigated the role of activated neutrophils in the pathophysiology of lung contusion-induced ALI. We used the model of blunt chest trauma accompanied by PMN-depletion in male C3H/HeN mice. Animals received 25 µg/g body weight PMN-depleting antibody Gr-1 intravenously 48 h before trauma. Bronchoalveolar lavage (BAL) and lung tissue interleukin 6 (IL-6) were similarly elevated in PMN-depleted and control animals after trauma, whereas macrophage inflammatory protein 2 and monocyte chemoattractant protein 1 in BAL and lungs, IL-10 in BAL, and lung keratinocyte chemoattractant (KC) were even further increased in the absence of PMNs. Plasma IL-6 and KC were also increased in response to the insult and even further in the absence of PMNs. Chest trauma induced an enhanced release of IL-6, tumor necrosis factor α, macrophage inflammatory protein 2, monocyte chemoattractant protein 1, and IL-10 from isolated KU, which was blunted in the absence of PMNs. In the presence of PMNs, BAL protein was further increased at 30 h when compared with the 3-h time point, which was not the case in the absence of PMNs. Taken together, in response to lung trauma, activated neutrophils control inflammation including mediator release from distant immune cells but simultaneously mediate pulmonary tissue damage. Thus, keeping in mind potential inflammatory adverse effects, modulation of neutrophil activation or trafficking might be a reasonable therapeutic approach in chest trauma-induced lung injury.


Assuntos
Granulócitos/citologia , Células de Kupffer/citologia , Lesão Pulmonar/metabolismo , Neutrófilos/citologia , Baço/citologia , Animais , Peso Corporal , Lavagem Broncoalveolar , Queratinócitos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Peroxidase/metabolismo , Fatores de Tempo , Ferimentos não Penetrantes/metabolismo
8.
Gut ; 60(2): 225-37, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20876774

RESUMO

BACKGROUND: Evasion of apoptosis is a hallmark of pancreatic cancer. However, the underlying mechanisms are still only partly understood and may involve antiapoptotic proteins such as c-FLIP. Here, the role of c-FLIP in the regulation of death receptor-mediated apoptosis in pancreatic cancer was investigated. METHODS: Expression of c-FLIP(L) and c-FLIP(S) was analysed in primary pancreatic carcinoma samples, pancreatic carcinoma cell lines and primary tumour cells together with its function as a regulator of death receptor-induced apoptosis by knockdown and overexpression studies and through modulation by chemotherapeutics. RESULTS: c-FLIP is expressed in pancreatic intraepithelial neoplasm (PanIN) lesions and in pancreatic ductal adenocarcinomas, whereas normal pancreatic ducts were consistently negative for c-FLIP. Simultaneous downregulation of c-FLIP(L) and c-FLIP(S) as well as individual knockdown of either isoform by RNA interference significantly enhances TRAIL (tumour necrosis factor-related apoptosis-inducing ligand)- and CD95-induced caspase activation and caspase-dependent apoptosis. Also, pretreatment with chemotherapeutic drugs--that is, 5-fluorouracil (5-FU), cisplatin or gemcitabine--downregulates c-FLIP and renders cells sensitive to death receptor-triggered apoptosis. Similarly, primary cultured pancreatic cancer cells are primed for TRAIL-induced apoptosis by pre-exposure to 5-FU or cisplatin. Mechanistic studies revealed that 5-FU-mediated suppression of c-FLIP results in increased TRAIL-induced recruitment and activation of caspase-8 at the death-inducing signalling complex (DISC), leading to caspase-3 activation and caspase-dependent cell death. Overexpression of c-FLIP(L) rescues cells from 5-FU- or cisplatin-mediated sensitisation for TRAIL-induced apoptosis, indicating that c-FLIP suppression is a key event in this chemotherapy-mediated sensitisation to TRAIL. Further, concomitant neutralisation of c-FLIP and XIAP acts in concert to potentiate TRAIL-induced apoptosis. CONCLUSIONS: Both the long and the short isoform of the antiapoptotic protein c-FLIP are critical regulators of death receptor-induced apoptosis in pancreatic carcinoma cells and are suppressed by chemotherapeutics. Targeting either c-FLIP(L) or c-FLIP(S) is sufficient to promote death receptor-induced apoptosis in pancreatic carcinoma cells. These findings have important implications for the design of TRAIL-based combination protocols in pancreatic cancer.


Assuntos
Apoptose/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias Pancreáticas/patologia , Receptores de Morte Celular/fisiologia , Antimetabólitos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Fluoruracila/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Pâncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Células Tumorais Cultivadas , Receptor fas/fisiologia
9.
Eur J Clin Invest ; 41(2): 151-8, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20860757

RESUMO

BACKGROUND: Pancreatic stellate cells (PSC) play a central role in fibrogenesis associated with acute and chronic pancreatitis. Pancreatic stone protein/regenerating protein (PSP/reg) belongs to a family of secretory stress proteins (SSP) that are constitutively synthesized by pancreatic acinar cells and upregulated dramatically during acute and chronic pancreatitis. Assuming a protective role of this stress protein, we investigated its effects on human PSC. MATERIAL AND METHODS: Pancreatic stellate cells were obtained by outgrowth from fibrotic human pancreas tissue. PSP/reg was expressed in the yeast Pichia pastoris and purified from medium supernatants. PSP/reg was added at concentrations of 100 ng/mL to cultured PSC. Cell proliferation was determined by bromodeoxyuridine incorporation. PSC migration was assessed by a wound healing assay. Extracellular matrix (collagen type I and fibronectin), matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) were demonstrated on protein level. RESULTS: Pancreatic stone protein/regenerating protein inhibited PSC proliferation and migration. Soluble collagen I and fibronectin were reduced after the addition of PSP/reg. PSP/reg slightly decreased the synthesis of MMP-1 and MMP-2 and strongly decreased TIMP-1 and TIMP-2 concentrations in PSC supernatants. CONCLUSIONS: Our work describes a novel aspect that in vitro PSP/reg reduces PSC activity (proliferation and migration) and stimulates fibrolysis by increasing MMP/TIMP ratio. The findings suggest that PSP/reg might have a protective function in the repair phase of acute and chronic pancreatitis by promoting resolution of fibrosis. We highlight PSP/reg as an antifibrogenic protein in pancreatic injury.


Assuntos
Litostatina/metabolismo , Metaloproteinases da Matriz/metabolismo , Células Estreladas do Pâncreas/metabolismo , Pancreatite/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , Idoso , Análise de Variância , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo
10.
Cancers (Basel) ; 2(3): 1661-82, 2010 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-24281180

RESUMO

Pancreatic cancer is characterized by its late detection, aggressive growth, intense infiltration into adjacent tissue, early metastasis, resistance to chemo- and radiotherapy and a strong "desmoplastic reaction". The dense stroma surrounding carcinoma cells is composed of fibroblasts, activated stellate cells (myofibroblast-like cells), various inflammatory cells, proliferating vascular structures, collagens and fibronectin. In particular the cellular components of the stroma produce the tumor microenvironment, which plays a critical role in tumor growth, invasion, spreading, metastasis, angiogenesis, inhibition of anoikis, and chemoresistance. Fibroblasts, myofibroblasts and activated stellate cells produce the extracellular matrix components and are thought to interact actively with tumor cells, thereby promoting cancer progression. In this review, we discuss our current understanding of the role of pancreatic stellate cells (PSC) in the desmoplastic response of pancreas cancer and the effects of PSC on tumor progression, metastasis and drug resistance. Finally we present some novel ideas for tumor therapy by interfering with the cancer cell-host interaction.

11.
Am J Physiol Gastrointest Liver Physiol ; 297(6): G1163-71, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19779015

RESUMO

Mechanisms leading to acute pancreatitis after a fat-enriched meal combined with excess alcohol are incompletely understood. We have studied the effects of alcohol and fat (VLDL) on pancreatic acinar cell (PAC) function, oxidative stress, and repair mechanisms by pancreatic stellate cells (PSC) leading to fibrogenesis. To do so, PAC (rat) were isolated and cultured up to 24 h. Ethanol and/or VLDL were added to PAC. We measured PAC function (amylase, lipase), injury (lactic dehydrogenase), apoptosis (TUNEL, Apo2.7, annexin V binding), oxidative stress, and lipid peroxidation (conjugated dienes, malondialdehyde, chemoluminescence); we also measured PSC proliferation (bromodeoxyuridine incorporation), matrix synthesis (immunofluorescence of collagens and fibronectin, fibronectin immunoassay), and fatty acids in PAC supernatants (gas chromatography). Within 6 h, cultured PAC degraded and hydrolyzed VLDL completely. VLDL alone (50 microg/ml) and in combination with alcohol (0.2, 0.5, and 1% vol/vol) induced PAC injury (LDL, amylase, and lipase release) within 2 h through generation of oxidative stress. Depending on the dose of VLDL and alcohol, apoptosis and/or necrosis were induced. Antioxidants (Trolox, Probucol) reduced the cytotoxic effect of alcohol and VLDL. Supernatants of alcohol/VLDL-treated PAC stimulated stellate cell proliferation and extracellular matrix synthesis. We concluded that, in the presence of lipoproteins, alcohol induces acinar cell injury. Our results provide a biochemical pathway for the clinical observation that a fat-enriched meal combined with excess alcohol consumption can induce acinar cell injury (acute pancreatitis) followed by repair mechanisms (proliferation and increased matrix synthesis in PSC).


Assuntos
Proliferação de Células/efeitos dos fármacos , Etanol/toxicidade , Matriz Extracelular/metabolismo , Lipoproteínas VLDL/toxicidade , Pâncreas Exócrino/efeitos dos fármacos , Pancreatite/etiologia , Doença Aguda , Amilases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fibrose , L-Lactato Desidrogenase/metabolismo , Lipase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lipoproteínas VLDL/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pâncreas Exócrino/metabolismo , Pâncreas Exócrino/patologia , Pancreatite/metabolismo , Pancreatite/patologia , Ratos , Ratos Wistar , Fatores de Tempo
12.
Cancer Res ; 69(10): 4517-26, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19435915

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is thought to originate from ductal structures, exhibiting strong desmoplastic reaction with stromal pancreatic myofibroblasts (PMF), which are supposed to drive PDAC tumorigenesis. Previously, we observed high expression of the adhesion molecule L1CAM (CD171) in PDAC cells accounting for chemoresistance. Thus, this study aimed to investigate whether PMFs are involved in the induction of tumoral L1CAM and whether this contributes to malignant transformation of pancreatic ductal cells and PDAC tumorigenesis. Immunohistochemistry of tissues from chronic pancreatitis specimens revealed considerable L1CAM expression in ductal structures surrounded by dense fibrotic tissue, whereas no L1CAM staining was seen in normal pancreatic tissues. Using the human pancreatic duct cell line H6c7, we show that coculture with PMFs led to a transforming growth factor-beta1 (TGF-beta1)-dependent up-regulation of L1CAM expression. Similarly, L1CAM expression increased in monocultured H6c7 cells after administration of exogenous TGF-beta1. Both TGF-beta1- and PMF-induced L1CAM expression were independent of Smad proteins but required c-Jun NH(2)-terminal kinase activation leading to the induction of the transcription factor Slug. Moreover, Slug interacted with the L1CAM promoter, and its knockdown abrogated the TGF-beta1- and PMF-induced L1CAM expression. As a result of L1CAM expression, H6c7 cells acquired a chemoresistant and migratory phenotype. This mechanism of TGF-beta1-induced L1CAM expression and the resulting phenotype could be verified in the TGF-beta1-responsive PDAC cell lines Colo357 and Panc1. Our data provide new insights into the mechanisms of tumoral L1CAM induction and how PMFs contribute to malignant transformation of pancreatic duct cells early in PDAC tumorigenesis.


Assuntos
Carcinoma Ductal Pancreático/patologia , Molécula L1 de Adesão de Célula Nervosa/genética , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/patologia , Fatores de Transcrição/fisiologia , Fator de Crescimento Transformador beta1/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Carcinoma Ductal Pancreático/genética , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Transformação Celular Neoplásica , Técnicas de Cocultura , Humanos , Camundongos , Ductos Pancreáticos/fisiopatologia , Neoplasias Pancreáticas/genética , Pancreatite/patologia , Pancreatite/cirurgia , RNA Interferente Pequeno/genética , Fatores de Transcrição da Família Snail , Transfecção , Regulação para Cima
13.
Thromb Haemost ; 101(4): 682-6, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19350111

RESUMO

The Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147, basigin) is an immunoglobulin-like receptor expressed in various cell types. During cellular interactions homotypic EMMPRIN-EMMPRIN interactions are known to induce the synthesis of matrix metalloproteinases. Recently, we have identified EMMPRIN as a novel receptor on platelets. To our knowledge EMMPRIN has not been shown to serve as adhesion receptor, yet. Here we characterise platelet glycoprotein VI (GPVI) as a novel adhesion receptor for EMMPRIN. Human platelets were prestimulated with ADP and perfused over immobilised recombinant EMMPRIN-Fc or Fc-fragments under arterial shear conditions. ADP-stimulated platelets showed significantly enhanced rolling (but not enhanced firm adhesion) on immobilised EMMPRIN-Fc compared to Fc. Pretreatment of platelets with blocking mAbs anti-EMMPRIN or anti-GPVI leads to a significant reduction of rolling platelets on immobilised EMMPRIN-Fc, whereas pretreatment with blocking mAbs anti-p-selectin, anti-alpha4-integrin or anti-GPIIb/IIIa complex (20 microg/ml each) had no effect. Consistently, chinese hamster ovary (CHO) cells stably transfected with GPVI showed enhanced rolling (but not adhesion) on immobilised EMMPRIN-Fc in comparison to non-transfected CHO cells. Similarly, CHO cells stably transfected with EMMPRIN showed enhanced rolling on immobilised GPVI-Fc (or EMMPRIN-Fc) compared to non transfected CHO-cells. Finally, specific binding of EMMPRIN to GPVI was demonstrated by a modified ELISA and surface plasmon resonance technology with a dissociation constant of 88 nM. Platelet GPVI is a novel receptor for EMMPRIN and can mediate platelet rolling via GPVI-EMMPRIN interaction.


Assuntos
Basigina/metabolismo , Plaquetas/metabolismo , Antígenos CD36/metabolismo , Movimento Celular , Ativação Plaquetária , Difosfato de Adenosina/metabolismo , Animais , Anticorpos Monoclonais , Basigina/genética , Basigina/imunologia , Antígenos CD36/genética , Antígenos CD36/imunologia , Células CHO , Adesão Celular , Cricetinae , Cricetulus , Humanos , Camundongos , Selectina-P/metabolismo , Adesividade Plaquetária , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Transfecção
14.
Pancreatology ; 9(1-2): 99-110, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19077460

RESUMO

BACKGROUND/AIMS: Pancreatic stellate cells (PSCs) play a key role in fibrogenesis associated with acute and chronic pancreatitis. Pancreatitis-associated protein (PAP), an acute-phase protein, is dramatically upregulated during acute and chronic pancreatitis. Assuming a protective role of PAP, we investigated its effects on human PSCs. METHODS: PSCs were obtained by outgrowth from fibrotic human pancreas tissue. PAP was expressed in the yeast Pichia pastoris. PAP was added at 10 ng/ml to cultured PSCs. Cell proliferation was determined by bromodeoxyuridine incorporation. PSC migration was assessed by a wound healing assay. Collagen types I and III, fibronectin, matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs) and reversion-inducing cysteine-rich protein with Kazal motifs (RECK) were demonstrated on protein and mRNA level. RESULTS: PAP had no significant effect on PSC proliferation and migration. Cell-associated fibrillar collagen types I and III and fibronectin increased after addition of PAP to PSCs. PAP diminished the expression of MMP-1 and -2 and TIMP-1 and -2 and their concentrations in PSC supernatants. RECK was detected on the surface of PSCs and its expression was reduced after PAP application. CONCLUSIONS: Our data offer new insights into the biological functions of PAP, which may play an important role in wound healing response and cell-matrix interactions.


Assuntos
Antígenos de Neoplasias/fisiologia , Biomarcadores Tumorais/fisiologia , Lectinas Tipo C/fisiologia , Inibidores de Metaloproteinases de Matriz , Glicoproteínas de Membrana/biossíntese , Pâncreas/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/antagonistas & inibidores , Inibidor Tecidual de Metaloproteinase-2/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteínas Ligadas por GPI , Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Pâncreas/citologia , Proteínas Associadas a Pancreatite , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo
15.
Shock ; 30(5): 537-44, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18317405

RESUMO

Alveolar type 2 (AT-2) cell apoptosis is an important mechanism during lung inflammation, lung injury, and regeneration. Blunt chest trauma has been shown to activate inflammatory cells such as alveolar macrophages (AMs) or neutrophils (polymorphonuclear granulocytes [PMNs]), resulting in an inflammatory response. The present study was performed to determine the capacity of different components/cells of the alveolar compartment (AMs, PMNs, or bronchoalveolar lavage [BAL] fluids) to induce apoptosis in AT-2 cells following blunt chest trauma. To study this, male Sprague-Dawley rats were subjected to either sham procedure or blunt chest trauma induced by a single blast wave. Various time points after injury (6 h to 7 d), the lungs were analyzed by immunohistochemistry, for AT-2 cells, or with antibodies directed against caspase 3, caspase 8, Fas, Fas ligand (FasL), BAX, and BCL-2. Bronchoalveolar lavage concentrations of TNF-alpha, IL-1beta, and soluble FasL were determined by enzyme-linked immunosorbent assay. Furthermore, cultures of AT-2 cells isolated from healthy rats were incubated with supernatants of AMs, PMNs, or BAL fluids obtained from either trauma or sham-operated animals in the presence or absence of oxidative stress. Annexin V staining or TUNEL (terminal deoxynucleotidyl transferase) assay was used to detect apoptotic AT-2 cells. Histological evaluation revealed that the total number of AT-2 cells was significantly reduced at 48 h following trauma. Fas, FasL, active caspase 8, and active caspase 3 were markedly up-regulated in AT-2 cells after chest trauma. BAX and BCL-2 did not show any significant changes between sham and trauma. IL-1beta, but not TNF-alpha, levels were markedly increased at 24 h after the injury, and soluble FasL concentrations were significantly enhanced at 6, 12, 24, and 48 h after the insult. Apoptosis of AT-2 cells incubated with supernatants from cultured AMs, isolated at 48 h following chest trauma was markedly increased when compared with shams. In contrast, no apoptosis was induced in AT-2 cells incubated with supernatants of activated PMNs or BAL fluids of traumatized animals. In summary, blunt chest trauma induced apoptosis in AT-2 cells, possibly involving the extrinsic death receptor pathway. Furthermore, mediators released by AMs appeared to be involved in the induction of AT-2 cell apoptosis.


Assuntos
Apoptose/imunologia , Contusões/fisiopatologia , Células Epiteliais/citologia , Macrófagos Alveolares/fisiologia , Neutrófilos/fisiologia , Animais , Caspases/metabolismo , Contusões/imunologia , Citocinas/metabolismo , Células Epiteliais/imunologia , Proteína Ligante Fas/metabolismo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Interleucina-1/metabolismo , Lesão Pulmonar/imunologia , Macrófagos Alveolares/imunologia , Masculino , Neutrófilos/imunologia , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo
16.
Cell Signal ; 20(4): 695-704, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18207700

RESUMO

Phagocytosis is an essential event in the complex process of tissue repair. Here we examined the effect of low intensity pulsed ultrasound (US), which promotes fracture and wound healing, on phagocytosis by mouse macrophage cell line J774A.1 and human monocyte-derived macrophages. First, 10 to 40 min low intensity pulsed US increased uptake of serum opsonized E. coli by J774A.1 cells during a 50 min phagocytosis period. In addition, when the E. coli exposure time was varied between 35 to 80 min, the maximum increase in phagocytosis was observed in the first 35 min upon US exposure. In parallel, US induced robust actin polymerization in a time dependent manner in J774A.1 cells, showing the peak effect 30 min after stimulation. Interestingly, a low concentration of cytochalasin D (0.25-0.5 microM) prevented US-induced phagocytosis of E. coli. Furthermore, we demonstrated US enhanced activation of RhoA. Blocking its downstream effector Rho associated kinase (ROCK) with Y27632 abrogated US-induced phagocytosis. We also show that US induced activation of ERK and p38 MAPK. Pretreatment of the cells with the corresponding inhibitors PD98059 and SB203580 reduced US-induced phagocytosis. In addition, activity of tyrosine kinase Src was required for US-induced phagocytosis. Here Src represents an upstream activator of ERK and p38 MAPK. Depolymerization of actin by cytochalasin D prevented US-induced Src, ERK, and p38 activation. Our data provide a new insight into the cellular and molecular mechanisms by which low intensity pulsed US promotes tissue repair.


Assuntos
Actinas/metabolismo , Macrófagos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fagocitose , Transdução de Sinais , Ultrassom , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Amidas/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Ativação do Complemento , Citocalasina D/farmacologia , Escherichia coli/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Imidazóis/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/imunologia , Camundongos , Fagocitose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
17.
Langenbecks Arch Surg ; 393(6): 891-900, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18204855

RESUMO

BACKGROUND: Adenocarcinomas of the pancreas are characterized by a rapid progression, an early metastasis, a limited response to chemo- and radiotherapy, and an intense fibrotic reaction known as tumor desmoplasia. Carcinoma cells are surrounded by a dense stroma consisting of myofibroblast-like cells, collagens, and fibronectin. MATERIALS AND METHODS: This review describes the interaction of activated pancreatic stellate cells (myofibroblast-like cells) with tumor cells in pancreas adenocarcinomas. Our data were obtained in cell culture experiments and in in vivo investigations. RESULTS: Carcinoma cells produce soluble mediators and stimulate motility, proliferation, matrix-, and MMP synthesis of stellate cells. Vice versa-activated stellate cells release mitogens, stimulating proliferation of cancer cells. Cancer cell proliferation and resistance to apoptosis might further be induced by the microenvironment (extracellular matrix), which is primarily provided by stellate cells. A very important aspect in the interaction of stellate cells with cancer cells is the expression of EMMPRIN (extracellular matrix metalloproteinase inducer) by cancer cells, the shedding of the extracellular part of EMMPRIN by matrix metalloproteinases (MMPs), and the induction of MMPs in stellate cells by soluble EMMPRIN. In particular, the stellate cells in close proximity to tumor cells therefore express MMPs and degrade connective tissue. CONCLUSION: Through complex interactions between stellate cells and carcinoma cells, tumor progression and cancer cell invasion are accelerated. As we gain better understanding of these mechanisms, adequate therapies to reduce tumor cell invasion and cancer progression might be developed.


Assuntos
Adenocarcinoma/fisiopatologia , Transformação Celular Neoplásica/patologia , Matriz Extracelular/fisiologia , Pâncreas/fisiopatologia , Neoplasias Pancreáticas/fisiopatologia , Adenocarcinoma/patologia , Animais , Basigina/metabolismo , Comunicação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Colágeno/metabolismo , Matriz Extracelular/patologia , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Pâncreas/patologia , Neoplasias Pancreáticas/patologia , Lesões Pré-Cancerosas/patologia , Lesões Pré-Cancerosas/fisiopatologia
18.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 15(6): 1253-6, 2007 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-18088478

RESUMO

The aim of this study was to explore the molecular mechanisms of the effect of low intensity pulsed ultrasound (LIPUS) on human primary macrophage functions. Macrophage phagocytosis was analyzed using fluorescein isothiocyanate (FITC)-labelled Escherichia coli (E.Coli); focal complex and extracellular matrix metalloproteinase inducer (EMMPRIN) were observed by fluorescence microscopy; the secretion of metalloproteinases (MMPs) was examined by gelatin zymography, and the expressions of EMMPRIN and extracellular signal-regulated kinases (ERKs) were detected by Western blot. The results indicated that LIPUS accelerated macrophages to phagocytose E.Coli (29.81+/-0.36 vs 18.00+/-0.78), promoted the protein expressions of EMMPRIN and MMPs, increased the level of protein tyrosine phosphorylation, and induced the phosphorylation of ERKs. Furthermore, the above functions were only found in adherent macrophages, and were inhibited or decreased by mitogen activated protein kinase kinase (MAPK kinase, MEK) inhibitor PD98059 and RGD (Arg-Gly-Asp peptide), one of main integrin recognition sequences. It is concluded that the effect of LIPUS on macrophages depends on cell adhesion, and relates to integrin-MEK-ERK pathway.


Assuntos
Macrófagos/imunologia , Macrófagos/efeitos da radiação , Metaloproteinases da Matriz/metabolismo , Fagocitose/efeitos da radiação , Ultrassom , Basigina/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Macrófagos/citologia , Fosforilação
19.
J Cell Sci ; 120(Pt 3): 512-9, 2007 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17227797

RESUMO

The effect of the characteristic desmoplastic reaction of pancreatic cancer on tumor progression is largely unknown. We investigated whether pancreatic stellate cells, which are responsible for the desmoplastic reaction, support tumor progression. Immunohistology revealed that matrix metalloproteinase-2 (MMP-2), which is suggested to promote pancreatic cancer progression, is present in stellate cells adjacent to cancer cells. In vitro, stellate cells exhibited a much higher basal expression of MMP-2 compared with cancer cells. Panc1-, MiaPaCa2- and SW850-conditioned media stimulated MMP-2 release of stellate cells as detected by zymography. Cancer cells expressed and released basigin [BSG, extracellular matrix metalloproteinase inducer (EMMPRIN), CD147], a glycoprotein that is known to stimulate MMP-2 in mesenchymal cells, as detected by immunostaining, western blot and reverse transcription-polymerase chain reaction. Tumor cell-conditioned medium and BSG purified by affinity chromatography from supernatants of cancer cells, but not supernatants depleted from BSG, stimulated expression of MMP-1 and MMP-2 of stellate cells as demonstrated by western blot and zymography. Moreover, the interaction of stellate cells and cancer cells promoted the invasiveness of Panc-1 cells in the chorioallantoic membrane assay and increased the weight of tumors induced by all carcinoma cell lines in nude mice by 2.1-3.7-fold. Our findings support the assumption that the interaction of stellate cells and cancer cells promotes progression of pancreatic cancer.


Assuntos
Membrana Corioalantoide/citologia , Metaloproteinase 2 da Matriz/metabolismo , Pâncreas/citologia , Neoplasias Pancreáticas/enzimologia , Ensaios Antitumorais Modelo de Xenoenxerto , Adenocarcinoma/patologia , Animais , Basigina , Bioensaio , Linhagem Celular Tumoral , Embrião de Galinha , Meios de Cultivo Condicionados/farmacologia , Progressão da Doença , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/patologia
20.
J Gastroenterol Hepatol ; 21 Suppl 3: S92-6, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16958683

RESUMO

Pancreas fibrosis is the result of a dynamic cascade of mechanisms beginning with acinar cell (AC) injury and necrosis and followed by inflammation, activation of macrophages, aggregation of platelets, release of growth factors and reactive oxygen species (ROS), activation of pancreatic stellate cells (PSC), stimulated synthesis of extracellular matrix and reduced matrix degradation. The result is a net matrix accumulation. Numerous in vivo and in vitro studies have provided strong evidence of a central role for PSC in fibrogenesis associated with acute and chronic pancreatitis. The PSC share homologies with hepatic stellate cells (HSC). In normal pancreas, the fat-storing phenotype of PSC is found in low numbers (approx. 4% of the cells) in the periacinar and interlobular space. Similar to the stellate cell-activating mechanisms in the liver, in pancreas injury PSC change their phenotype from the fat-storing to a highly active matrix-producing cell type (activated PSC). The induction of the activated phenotype of PSC has been shown to involve a number of diverse extra- and intracellular effector molecules, including inflammatory cytokines, growth factors, ethanol, acetaldehyde, and oxidative stress.


Assuntos
Matriz Extracelular/patologia , Pâncreas/citologia , Pancreatite/patologia , Doença Aguda , Adipócitos/metabolismo , Adipócitos/patologia , Animais , Doença Crônica , Colágeno/biossíntese , Humanos , Pâncreas/patologia , Pancreatite/metabolismo
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