RESUMO
By a proteomics-based approach, we identified an overexpression of fascin in colon adenocarcinoma cells (FPCKpP-3) that developed from nontumorigenic human colonic adenoma cells (FPCK-1-1) and were converted to tumorigenic by foreign-body-induced chronic inflammation in nude mice. Fascin overexpression was also observed in the tumors arising from rat intestinal epithelial cells (IEC 6) converted to tumorigenic in chronic inflammation which was induced in the same manner. Upregulation of fascin expression in FPCK-1-1 cells by transfection with sense fascin cDNA converted the cells tumorigenic, whereas antisense fascin-cDNA-transfected FPCKpP-3 cells reduced fascin expression and lost their tumor-forming ability in vivo. The tumorigenic potential by fascin expression was consistent with their ability to survive and grow in the three-dimensional multicellular spheroids. We found that resistance to anoikis (apoptotic cell death as a consequence of insufficient cell-to-substrate interactions), which is represented by the three-dimensional growth of solid tumors in vivo, was regulated by fascin expression through caspase-dependent apoptotic signals. From these, we demonstrate that fascin is a potent suppressor to caspase-associated anoikis and accelerator of the conversion of colonic adenoma cells into adenocarcinoma cells by chronic inflammation.
Assuntos
Anoikis/fisiologia , Proteínas de Transporte/metabolismo , Neoplasias do Colo/metabolismo , Inflamação/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/genética , Ratos , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas/metabolismoRESUMO
It has been suggested that nitric oxide (NO) derived from chronically inflamed tissues is a cause of carcinogenesis. We herein demonstrated that administration of an inducible NO synthase inhibitor, aminoguanidine, significantly suppressed the tumorigenic conversion of human colonic adenoma (FPCK-1-1) cells into adenocarcinoma (FPCK/Inflam) cells accelerated by foreign body-induced chronic inflammation in nude mice. To determine whether NO directly promotes carcinogenesis, we exposed FPCK-1-1 cells continuously to chemically generated NO (FPCK/NO), and periodically examined their tumorigenicity. FPCK/NO cells formed tumors, whereas vehicle-treated cells (FPCK/NaOH) did not. We selected a tumorigenic population from FPCK/NO cells kept it in three-dimensional (3D) culture where in vivo-like multicellular spheroidal growth was expected. FPCK/Inflam cells developed large spheroids whereas FPCK/NO cells formed tiny but growing compact aggregates in 3D culture. Meanwhile, FPCK-1-1 and FPCK/NaOH cells underwent anoikis (apoptotic cell death consequential on insufficient cell-to-substrate interactions) through activation of caspase 3. The survived cells in the 3D culture (FPCK/NO/3D), which were derived from FPCK/NO cells, showed a similar tumor incidence to that of FPCK/Inflam cells. These results showed that NO was one of the causative factors for the acceleration of colon carcinogenesis, especially in the conversion from adenoma to adenocarcinoma in the chronic inflammatory environment.
Assuntos
Adenocarcinoma/patologia , Adenoma/patologia , Neoplasias do Colo , Inflamação , Óxido Nítrico/metabolismo , Adenocarcinoma/fisiopatologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Feminino , Guanidinas/farmacologia , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos NusRESUMO
The cells of a weakly tumorigenic and non-metastatic murine fibrosarcoma (QR-32) are converted into highly malignant tumors (acquiring metastatic potential) once they have grown in vivo after being co-implanted with gelatin sponge which induces inflammation. In the present study, we examined whether nitric oxide (NO) is involved in the inflammation-based tumor progression by administrating a specific inhibitor to inducible nitric oxide synthase, aminoguanidine (AG). First, we co-implanted 1 x 10(5) QR-32 cells with gelatin sponge (10 x 5 x 3 mm piece) into a subcutaneous space in C57BL6 mice. Administration of AG in drinking water (1%) had started 2 days before the tumor implantation and continued until the termination of the experiment. The incidence of tumor formation and the tumor growth did not differ between AG-treated group and -untreated group. On day 28, we excised the arising tumors to establish culture cell lines for evaluation of their acquisition of metastatic phenotype in other normal mice. Metastasis incidence and the number of metastatic colonies were significantly reduced in the tumor cell lines obtained from AG-treated mice compared to those from non-treated mice (p < 0.05). Immunohistochemical analysis demonstrated that inducible nitric oxide synthase and nitrotyrosine in the inflamed lesion were reduced in the AG-administered mice. However, intensity of 8-hydroxy-2-deoxyguanosine was not different between the groups. These results showed that nitric oxide and its reactive nitrogen oxide species cooperatively play a pivotal role in the progression of benign tumor cells in inflamed lesions.
Assuntos
Fibrossarcoma/patologia , Inflamação/complicações , Camundongos , Metástase Neoplásica , Óxido Nítrico Sintase Tipo II/fisiologia , Espécies Reativas de Nitrogênio/fisiologia , Tirosina/análogos & derivados , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Feminino , Fibrossarcoma/metabolismo , Esponja de Gelatina Absorvível , Guanidina/análogos & derivados , Guanina/administração & dosagem , Guanina/análogos & derivados , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Oxirredução , Espécies Reativas de Nitrogênio/metabolismo , Tirosina/fisiologiaRESUMO
We examined the role of phagocyte-derived oxygen radicals in tumor cell acquisition of metastatic phenotype by comparing gp91(phox-/-) mice and C57BL/6J wild-type (WT) mice. The gp91(phox-/-) mouse is deficient in the gp91(phox) gene, an essential subunit of the phagocyte nicotinamide adenine dinucleotide phosphate oxidase that generates superoxide anion. QR-32 fibrosarcoma cells are nonmetastatic but are converted into metastatic tumors once in contact with foreign body (gelatin sponge)-induced phagocytes in vivo. Compared to QR-32 cells co-implanted with the foreign body in WT mice, those in gp91(phox-/-) mice exhibited reduced metastasis. There was no difference in the incidence of primary tumors after injection of B16BL6 melanoma cells in WT and gp91(phox-/-) mice. However, after resection of the primary tumors, metastases were reduced in gp91(phox-/-) mice. Thymosin beta4 gene expression and cell motility/invasion were seen in the tumors from WT mice but not in those from gp91(phox-/-) mice. Adoptive transfer of phagocytes from WT mice, but not those from gp91(phox-/-) mice, restored the metastatic ability of tumors grown in gp91(phox-/-) mice. These findings show that tumor metastatic behavior can primarily be endowed by phagocyte-derived superoxide anion and its oxidative metabolites, which are generated through activation of nicotinamide adenine dinucleotide phosphate oxidase.
Assuntos
NADPH Oxidases/metabolismo , Invasividade Neoplásica , Fagócitos/metabolismo , Superóxidos/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Corpos Estranhos/imunologia , Esponja de Gelatina Absorvível , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , NADPH Oxidase 2 , NADPH Oxidases/deficiência , NADPH Oxidases/genética , Neoplasias Experimentais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
QR-32 tumor cells, a clone derived from a murine fibrosarcoma, are poorly tumorigenic and nonmetastatic when injected into syngeneic C57BL/6 mice. However, they are converted to highly malignant ones once they have grown in vivo after being co-implanted in a subcutaneous site with a foreign body, a gelatin sponge. Early phase of inflammation induced by the gelatin sponge participates in the conversion and histological analysis shows predominant infiltration of neutrophils. The objective of this study was to determine whether the depletion of the infiltrating neutrophils has any effect on the tumor progression. Intraperitoneal administration of a monoclonal anti-granulocyte antibody, RB6-8C5 (RB6), depleted neutrophils from both the peripheral blood circulation and the local inflamed site in mice with co-implantation of QR-32 tumor cells and gelatin sponge. The RB6 administration did not inhibit either tumor development or growth of QR-32 tumor cells. In contrast, tumor cell lines established from RB6-administered mice showed a significant decrease in metastatic incidence as compared with the tumor cell lines obtained from the mice with administration of control rat IgG or saline. Metastatic ability was significantly suppressed when RB6 had been administered in the early phase (from day -2 to day 6 after implantation); however, the administration in the middle (from day 6 to day 14) or late (from day 14 to day 22) phase did not affect the metastatic ability. We confirmed the phenomena by using integrin beta(2) knockout mice that had impaired neutrophil infiltration into inflamed sites. In the knockout mice, neutrophils hardly infiltrated into the gelatin sponge and the tumors showed dramatically suppressed metastatic phenotype as compared with those in wild-type mice or nude mice. Immunohistochemical analysis demonstrated that expressions of 8-hydroxy-2'-deoxyguanosine and nitrotyrosine were parallel to those in the presence of neutrophils. These results suggested that inflammation, especially when neutrophils infiltrate into tumor tissue, is primarily important for benign tumor cells to acquire metastatic phenotype.
Assuntos
Desoxiguanosina/análogos & derivados , Fibrossarcoma/genética , Fibrossarcoma/secundário , Infiltração de Neutrófilos , Tirosina/análogos & derivados , 8-Hidroxi-2'-Desoxiguanosina , Animais , Anticorpos Monoclonais/administração & dosagem , Células Sanguíneas/efeitos dos fármacos , Antígenos CD18/genética , Antígenos CD18/fisiologia , Linhagem Celular Tumoral , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica , Desoxiguanosina/metabolismo , Progressão da Doença , Esquema de Medicação , Fibrossarcoma/complicações , Fibrossarcoma/patologia , Granulócitos/imunologia , Imuno-Histoquímica , Inflamação/etiologia , Inflamação/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout/genética , Camundongos Nus , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fenótipo , Fatores de Tempo , Tirosina/metabolismoRESUMO
We identified a thymosin-beta4 gene overexpression in malignant mouse fibrosarcoma cells (QRsP-30) that were derived from clonal weakly tumorigenic and nonmetastatic QR-32 cells by using a differential display method. Thymosin-beta4 is known as a 4.9-kd polypeptide that interacts with G-actin and functions as a major actin-sequestering protein in cells. All of the six malignant fibrosarcoma cell lines that have been independently converted from QR-32 cells expressed high levels of thymosin-beta4 mRNA and its expression in tumor cells was correlated with tumorigenicity and metastatic potential. Up-regulation of thymosin-beta4 in QR-32 cells (32-S) transfected with sense thymosin-beta4 cDNA converted the cells to develop tumors and formed numerous lung metastases in syngeneic C57BL/6 mice. In contrast, antisense thymosin-beta4 cDNA-transfected QRsP-30 (30-AS) cells reduced thymosin-beta4 expression, and significantly lost tumor formation and metastases to distant organs. Vector-alone transfected cells (32-V or 30-V cells) behaved like their parental cells. We observed that tumor cell motility, cell shape, and F-actin organization is regulated in proportion to the level of thymosin-beta4 expression. These findings indicate that thymosin-beta4 molecule regulates fibrosarcoma cell tumorigenicity and metastasis through actin-based cytoskeletal organization.