RESUMO
Expression of the protease inhibitor elafin is deregulated in several human cancers. However, functions of the protein in cancer are yet to be established. Here, we show that elafin elicits pro-apoptotic effects in melanoma cells but not in normal melanocytes. Elafin triggered the intrinsic apoptotic pathway as evidenced by the increased caspase 9 activity and unaltered caspase 8 activity. Caspase 9-specific siRNA, but not caspase 8-specific siRNA, dramatically abrogated elafin-induced apoptosis. Elevated level of p53 was observed, resulting in increased transcriptional activation and consequent expression of downstream effector molecules (Bax, Puma, Noxa, p21). Moreover, the apoptotic effect of elafin was inhibited by p53-specific siRNA and the p53 inhibitor pifithrin-alpha. Elafin treatment of xenograft mice of melanoma cells led to significantly smaller tumor sizes compared with those of untreated control mice. Immunohistochemical analysis revealed decreased elafin expression in melanoma tissue specimens. Western blot and reverse transcription analyses indicated transcriptional repression of the elafin gene in melanoma cells. Our results collectively indicate that elafin induces apoptosis in melanoma cells through a p53-dependent intrinsic apoptotic pathway, and that repression of elafin expression in melanoma may contribute to disease progression.
Assuntos
Apoptose/efeitos dos fármacos , Elafina/farmacologia , Melanoma/patologia , Inibidores de Proteases/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Humanos , Imuno-Histoquímica , Melanoma/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , RNA Interferente Pequeno , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The identification of novel tumor-specific proteins or antigens is of great importance for diagnostic and therapeutic applications in pancreatic cancer. Using oligonucleotide microarrays, we identified a broad spectrum of differentially expressed pancreatic cancer-related genes. Of these, we selected an overexpressed expressed sequence taq and cloned a 721-bp full-length cDNA with an open reading frame of 196 amino acids. This novel gene was localized on the Homo sapiens 16p13.3 chromosomal locus, and its nucleotide sequence matched the Homo sapiens similar to common salivary protein 1 (LOC124220). We named the gene pancreatic adenocarcinoma up-regulated factor. The pancreatic adenocarcinoma up-regulated factor was secreted into the culture medium of pancreatic adenocarcinoma up-regulated factor-overexpressing Chinese hamster ovary cells, had an apparent molecular mass of approximately 25 kDa, and was N-glycosylated. The induction of pancreatic adenocarcinoma up-regulated factor in Chinese hamster ovary cells increased cell proliferation, migration, and invasion ability in vitro. Subcutaneous injection of mice with Chinese hamster ovary/pancreatic adenocarcinoma up-regulated factor cells resulted in 3.8-fold greater tumor sizes compared to Chinese hamster ovary/mock cells. Reverse transcription-polymerase chain reaction and western blotting with antirecombinant human pancreatic adenocarcinoma up-regulated factor antibodies confirmed that pancreatic adenocarcinoma up-regulated factor was highly expressed in six of eight pancreatic cancer cell lines. Immunohistochemical staining of human pancreatic cancer tissues also showed pancreatic adenocarcinoma up-regulated factor overexpression in the cytoplasm of cancer cells. Transfection with pancreatic adenocarcinoma up-regulated factor-specific small-interfering RNA reduced cancer cell migration and invasion in vitro. Treatment with antirecombinant human pancreatic adenocarcinoma up-regulated factor in vitro and in vivo reduced proliferation, migration, invasion, and tumorigenic ability. Collectively, our results suggest that pancreatic adenocarcinoma up-regulated factor is a novel secretory protein involved in pancreatic cancer progression and might be a potential target for the treatment of pancreatic cancer.
Assuntos
Adenocarcinoma/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Lectinas/metabolismo , Regulação para Cima , Adenocarcinoma/genética , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Carcinoma Ductal Pancreático/genética , Linhagem Celular , Cricetinae , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Lectinas/química , Lectinas/genética , Camundongos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica/genética , Regulação para Cima/genéticaRESUMO
BACKGROUND: We intended to identify proteins that are differentially expressed in human atherosclerotic plaques. METHODS AND RESULTS: Comparative 2-dimensional electrophoretic analysis on carotid atherosclerotic endarterectomy specimens (n = 10) revealed that heat shock protein 27 (Hsp27) expression was significantly increased in the nearby normal-appearing area compared with the plaque core area from the same vessel specimen, which was further confirmed by Western blot analysis. The Hsp27 expression in the adjacent normal-appearing vessel areas was much higher than that in nonatherosclerotic reference arteries. The phosphorylation of Hsp27 showed a gradation in the degree of phosphorylation: greatest in the reference arteries, intermediate in the adjacent normal-appearing area, and lowest in plaque core area. Immunohistochemical analysis showed that the phosphorylation of Hsp27 of smooth muscle cells in the carotid endarterectomy specimens was decreased compared with that in the reference artery specimen. The mean plasma level of Hsp27 was significantly higher in patients with acute coronary syndrome (ACS) (n = 27; 106.1 +/- 74.1 ng/mL) than in the normal reference subjects (n = 29; 45.8 +/- 29.5 ng/mL; P < 0.005). The plasma levels of Hsp27 were significantly correlated with those of heat shock protein 70 (Hsp70) (r = 0.422, P < 0.0005), with adjustment for ACS/reference status. CONCLUSIONS: In the atherosclerotic lesion, Hsp27 expression is increased in the normal-appearing vessel adjacent to atherosclerotic plaque, whereas levels in the plaque itself are significantly decreased. Both plaque and adjacent artery show decreased Hsp27 phosphorylation compared with reference vessel. In ACS, plasma Hsp27 and Hsp70 are increased, and levels of Hsp27 correlate with Hsp70, C-reactive protein, and CD40L levels.
Assuntos
Estenose das Carótidas/cirurgia , Proteínas de Choque Térmico/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Estenose das Carótidas/sangue , Eletroforese em Gel Bidimensional , Endarterectomia das Carótidas , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas de Choque Térmico/sangue , Proteínas de Choque Térmico/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Infarto do Miocárdio/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Reactive oxygen species (ROS), generated by ionizing radiation, has been implicated in its effect on living tissues. We confirmed the changes in the oxidative stress markers upon irradiation. We characterized the changes in the proteome profile in rat liver after administering irradiation, and the affected proteins were identified by MALDI-TOF-MS and ESI-MS/MS. The identified proteins represent diverse sets of proteins participating in the cellular metabolism. Our results demonstrated that proteomics analysis is a useful method for characterization of a global proteome change caused by ionizing radiation to unravel the molecular mechanisms involved in the cellular responses to ionizing radiation.