RESUMO
Dickkopf1 (DKK1), a secreted protein involved in embryonic development, is a potent inhibitor of the Wnt signaling pathway and has been postulated to be a tumor suppressor or tumor promoter depending on the tumor type. In this study, we showed that DKK1 was expressed differently among non-small-cell lung cancer cell lines. The DKK1 expression level was much higher in A549 cells than in H460 cells. We revealed that blockage of DKK1 expression by silencing RNA in A549 cells caused up-regulation of intracellular reactive oxygen species (ROS) modulator (ROMO1) protein, followed by partial cell death, cell growth inhibition, and loss of epithelial-mesenchymal transition property caused by ROS, and it also increased γ-radiation sensitivity. DKK1 overexpression in H460 significantly inhibited cell survival with the decrease of ROMO1 level, which induced the decrease of cellular ROS. Thereafter, exogenous N-acetylcysteine, an antioxidant, or hydrogen peroxide, a pro-oxidant, partially rescued cells from death and growth inhibition. In each cell line, both overexpression and blockage of DKK1 not only elevated p-RB activation, which led to cell growth arrest, but also inactivated AKT/NF-kB, which increased radiation sensitivity and inhibited cell growth. This study is the first to demonstrate that strict modulation of DKK1 expression in different cell types partially maintains cell survival via tight regulation of the ROS-producing ROMO1 and radiation resistance.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Tolerância a Radiação , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Transição Epitelial-Mesenquimal/efeitos da radiação , Raios gama , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína do Retinoblastoma/metabolismoRESUMO
Fibulin-3, an extracellular glycoprotein, has been suggested as having functions in tissue regeneration and organogenesis. However, its role in cancer remains unclear. We show here that fibulin-3 was silenced by hypermethylation of the promoter region in the relatively invasive A549 non-small cell lung cancer (NSCLC) cells compared with less invasive H460 NSCLC cells. Enforced expression of fibulin-3 in A549 cells down-regulated cellular MMP-7 and MMP-2, which was followed by inhibition of cell invasiveness. Conversely, suppression of fibulin-3 expression with siRNA in H460 cells showed the opposite effect. These results indicate that fibulin-3 is a negative regulator of invasiveness in NSCLC and further studies are needed for its therapeutic applications in treatment of NSCLC.
Assuntos
Metilação de DNA , Proteínas da Matriz Extracelular/genética , Regulação Neoplásica da Expressão Gênica , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 7 da Matriz/metabolismo , Regiões Promotoras Genéticas , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/farmacologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Invasividade Neoplásica , Transdução de SinaisRESUMO
Smooth muscle protein 22-alpha (SM22α) is known as a transformation- and shape change-sensitive actin cross-linking protein found in smooth muscle tissue and fibroblasts; however, its functional role remains uncertain. We reported previously that SM22α overexpression confers resistance against anti-cancer drugs or radiation via induction of metallothionein (MT) isozymes in HepG2 cells. In this study, we demonstrate that SM22α overexpression leads cells to a growth arrest state and promotes cellular senescence caused by treatment with a subclinical dose of γ-radiation (0.05 and 0.1 Gy) or doxorubicin (0.01 and 0.05 µg/ml), compared to control cells. Senescence growth arrest is known to be controlled by p53 phosphorylation/p21(WAF1/Cip1) induction or p16(INK4a)/retinoblastoma protein (pRB) activation. SM22α overexpression in HepG2 cells elevated p16(INK4a) followed by pRB activation, but did not activate the p53/p21(WAF1/Cip1) pathway. Moreover, MT-1G, which is induced by SM22α overexpression, was involved in the activation of the p16(INK4a)/pRB pathway, which led to a growth arrest state and promoted cellular senescence caused by damaging agents. Our findings provide the first demonstration that SM22α modulates cellular senescence caused by damaging agents via regulation of the p16(INK4a)/pRB pathway in HepG2 cells and that these effects of SM22α are partially mediated by MT-1G.
Assuntos
Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Metalotioneína/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Proteína do Retinoblastoma/metabolismo , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Raios gama , Humanos , Metalotioneína/genética , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genéticaRESUMO
The expression of the Wnt-antagonist Dickkopf gene (DKK) is downregulated in several types of tumors as a consequence of epigenetic DNA modification; four DKK members, DKK1, DKK2, DKK3, and DKK4, have been identified. In this study, we investigated another function of DKK3 in non-small cell lung cancer H460 cells, in which DKK3 was hypermethylated (44%) but still expressed, by interfering with DKK3 expression using DKK3-silencing RNA (SiRNA). We found that knockdown of DKK3 expression by DKK3 SiRNA transfection led to the detachment of H460 cells from the bottom of the culture plate and caused apoptosis. The expression of cyclin-dependent kinases D1 and E were increased by DKK3 knockdown, indicating that cells with blocked DKK3 expression entered the apoptotic pathway. We also found that the intracellular level of reactive oxygen species was higher in cells with blocked DKK3 expression than in normal H460 cells, and levels of p53, p21, and Bax were also increased by the gene knockdown. These results indicate that DKK3 acts as an antiapoptotic molecule by decreasing the intracellular level of reactive oxygen species.
Assuntos
Apoptose/fisiologia , Proliferação de Células , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Interferência de RNA/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Quimiocinas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Metilação de DNA , Citometria de Fluxo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
The sensitivity or resistance of cancer cells and normal tissues to ionizing radiation plays an important role in the clinical setting of lung cancer treatment. However, to date the exact molecular mechanisms of intrinsic radiosensitivity have not been well explained. In this study, we compared the radiosensitivity or radioresistance in two non-small cell lung cancers (NSCLCs), H460 and A549, and investigated the signaling pathways that confer radioresistance. H460 cells showed a significant G(2)/M arrest after 12 h of irradiation (5 Gy), reaching 60% of G(2)/M phase arrest. A549 cells also showed a significant G(2)/M arrest after 12 h of exposure; however, this arrest completely disappeared after 24 h of exposure. A549 has higher methylated CpG sites in PTEN, which is correlated with tumor radioresistance in some cancer cells, than H460 cells, and the average of the extent of the methylation was approximately 4.3 times higher in A549 cells than in H460 cells. As a result, PTEN expression was lower in A549 than in H460. Conducting Western blot analysis, we found that PTEN acted as a negative regulator for pAkt, and the pAkt acted as a negative regulator for p53 expression. According to the above results, we concluded that the radiosensitivity shown in H460 cells may be due to the higher expression of PTEN through p53 signaling pathway.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Neoplasias Pulmonares/enzimologia , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tolerância a Radiação , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Sequência de Bases , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Morte Celular/efeitos da radiação , Linhagem Celular Tumoral , Ilhas de CpG/genética , Metilação de DNA/genética , Metilação de DNA/efeitos da radiação , Fase G2/efeitos da radiação , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Mitose/efeitos da radiação , PTEN Fosfo-Hidrolase/genética , Tolerância a Radiação/efeitos da radiação , Radiação Ionizante , Análise de Sequência de DNA , Transdução de Sinais/efeitos da radiação , TemperaturaRESUMO
TSPYL5, encoding testis-specific Y-like protein, has been postulated to be a tumor suppressor gene, and its hypermethylation is often associated with human disease, especially cancer. In this study, we report that the TSPYL5 gene was less methylated (30%) in A549 lung adenocarcinoma cells, which are relatively resistant to gamma-radiation, than in H460 lung cancer cells, in which the TSPYL5 gene was hypermethylated (95%); thus, the expression level of TSPYL5 is much higher in A549 cells than in H460 cells. We showed that TSPYL5 suppression with silencing RNA in A549 cells up-regulated cellular PTEN, followed by down-regulation of AKT activation. Therefore, blockage of TSPYL5 sensitized A549 cells to cytotoxic agents such as gamma-radiation. In addition, TSPYL5 suppression also showed an increased level of p21(WAF1/Cip1) and subsequently induced inhibition of cell growth in A549 cells. The overexpression of TSPYL5 in H460 cells showed the opposite effects. This study provides the first demonstration that TSPYL5 modulates cell growth and sensitization of cells to the detrimental effects of damaging agents via regulation of p21(WAF1/Cip1) and PTEN/AKT pathway.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Neoplasias Pulmonares/genética , Proteínas Nucleares/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tolerância a Radiação/genética , Apoptose/genética , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Ilhas de CpG , Inibidor de Quinase Dependente de Ciclina p21/genética , Metilação de DNA , Raios gama , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , PTEN Fosfo-Hidrolase/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
We previously demonstrated that combined treatment with extracts of the medicinal mushroom Ganoderma lucidum and the herb Duchesnea chrysantha (GDE) significantly suppresses cell growth and selectively induces apoptosis in human leukemia HL-60 cells, but not in normal cells. GDE?s mechanism of action and its activity against HL-60 cells suggest that it could be suitable for the combined-modality treatment of hematological malignancies. In the present study, we examined whether treatment with a combination of GDE and ionizing radiation enhances the therapeutic effect. We demonstrated that, when used in combination with radiation at a clinically relevant dose of 2 Gy, GDE further suppressed cell proliferation and induced apoptosis as well as micronuclei formation in HL-60 cells, leading to increased cell death. Furthermore, GDE pretreatment not only reduced radiation-induced G2/M-phase arrest, but also induced G1-phase arrest. These events are associated with the inhibition of cyclin-dependent kinase 1 (CDK1) phosphorylation and the dephosphorylation of retinoblastoma protein (pRB). Collectively, these data show that combined treatment with GDE and radiation enhances radiation-induced apoptosis and overall cell death. These findings may be clinically relevant and suggest a novel therapeutic strategy for increasing the efficacy of radiotherapy.
Assuntos
Radiossensibilizantes/administração & dosagem , Reishi , Rosaceae , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteína Quinase CDC2/metabolismo , Caspase 3/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Terapia Combinada , Sinergismo Farmacológico , Medicamentos de Ervas Chinesas/administração & dosagem , Fase G1/efeitos dos fármacos , Fase G1/efeitos da radiação , Raios gama/uso terapêutico , Células HL-60 , Humanos , Testes para Micronúcleos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Fosforilação , Fitoterapia , Extratos Vegetais/administração & dosagem , Proteína do Retinoblastoma/metabolismoRESUMO
The medicinal plant extracts commercially used in Asia were screened for their estrogenic and antiestrogenic activities in a recombinant yeast system featuring both a human estrogen receptor (ER) expression plasmid and a reporter plasmid. Pueraria lobata (flower) had the highest estrogenic relative potency (RP, 7.75×10(-3); RP of 17ß-estradiol=1), followed by Amomum xanthioides (1.25×10(-3)). Next potent were a group consisting of Glycyrrhiza uralensis, Zingiber officinale, Rheum undulatum, Curcuma aromatica, Eriobotrya japonica, Sophora flavescens, Anemarrhena asphodeloides, Polygonum multiflorum, and Pueraria lobata (root) (ranging from 9.5×10(-4) to 1.0×10(-4)). Least potent were Prunus persica, Lycoppus lucidus, and Adenophora stricta (ranging from 9.0×10(-5) to 8.0×10(-5)). The extracts exerting antiestrogenic effects, Cinnamomum cassia and Prunus persica, had relative potencies of 1.14×10(-3) and 7.4×10(-4), respectively (RP of tamoxifen=1). The solvent fractions from selected estrogenic or antiestrogenic herbs had higher estrogenic relative potencies, with their RP ranging from 9.3×10(-1) to 2.7×10(-4) and from 8.2×10(-1) to 9.1×10(-3), respectively. These results support previous reports on the efficacy of Oriental medicinal plants used or not used as phytoestrogens for hormone replacement therapy.
RESUMO
Combined treatment with the medicinal mushroom Ganoderma lucidum and the herb Duchesnea chrysantha extracts (GDE) causes a synergistic induction of mitochondrial damage and apoptosis in HL-60 cells. GDE treatment is selectively toxic to HL-60 leukemia cells whereas no cytotoxic effect is observed in normal peripheral blood mononuclear cells. GDE-induced apoptosis is associated with Bcl-2 down-regulation, Bax translocation, mitochondrial cytochrome c release and caspase-3 activation, suggesting that apoptosis by this combination occurs through the mitochondria-dependent pathway. The present findings suggest that this combination merits further investigation as a potential therapeutic agent for the treatment of cancer.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Basidiomycota/química , Mitocôndrias/efeitos dos fármacos , Rosaceae/química , Clorometilcetonas de Aminoácidos/farmacologia , Antineoplásicos/química , Western Blotting , Caspase 3/metabolismo , Inibidores de Caspase , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Inibidores do Crescimento/química , Inibidores do Crescimento/farmacologia , Células HL-60 , Humanos , Leucemia/metabolismo , Leucemia/patologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Transporte Proteico/efeitos dos fármacos , Fatores de Tempo , Proteína X Associada a bcl-2/metabolismoRESUMO
S-Adenosylmethionine decarboxylase (SAMDC) is a key enzyme for the biosynthesis of spermidine. SAMDC-suppressed HL-60 cells overproduced intracellular reactive oxygen species (ROS), which led to cell growth defect and partial cell death. ROS overproduction was caused by a decrease of the total glutathione (GSH) and the ratio of reduced to oxidized GSH, and by an increase of the intracellular iron uptake. When analyzed by real-time polymerase chain reaction, the transcripts of the genes involved in the GSH synthesis (gamma-glutamyl cysteine synthetase, GSH synthetase), as well as the gene of the GSH-reducing enzyme (NADP+-dependent isocitrate dehydrogenase), were decreased dramatically in these cells. DNA-repairing genes (ATM, PARP, RAD51 and MSH2) also were not activated transcriptionally. In these situations, excessive ROS induced severe DNA damage, which could not be repaired, and ultimately led the cells to a spontaneous cell death or an early senescence state. For such cells, gamma-radiation and cisplatin, which are direct DNA-damaging agents, were very effective for promoting cell death.
Assuntos
Adenosilmetionina Descarboxilase/metabolismo , Antineoplásicos/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Senescência Celular/efeitos dos fármacos , Senescência Celular/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Relação Dose-Resposta à Radiação , Raios gama , Células HL-60 , Humanos , Doses de Radiação , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/efeitos da radiaçãoRESUMO
Despite receiving much attention as a biodegradable substitute for conventional non-biodegradable plastics, the commercial use of polyhydroxybutyrate (PHB) remains limited because of its high production cost. In order to reduce the recovery/purification cost, which forms over half of the total production cost, we have developed a new cultivation method which enables spontaneous liberation of PHB by modulation of the initial inoculum size and the medium composition in recombinant Escherichia coli harboring Alcaligenes eutrophus phbCAB genes. In flask cultivation using a low cell inoculum and 2x LB medium containing 21% glucose, autolysis of 80.2% as well as yields of 85.2 g/l of PHB and a PHB content of 99.0% (w/w) were obtained. The glucose conversion rate was 0.43. The strategies developed in this study can minimize complex and unfavorable efforts required for efficient recovery/purification processes, thereby enabling biodegradable plastic to be produced by the recombinant E. coli so as to compete with conventional non-biodegradable plastic.
Assuntos
Escherichia coli/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Alcaligenes/genética , Meios de Cultura/química , Escherichia coli/genética , Escherichia coli/ultraestrutura , Fermentação , Glucose/metabolismoRESUMO
An increment of thioredoxin-1 (TRX) is observed in many human primary cancers and appears to contribute to an increase of cell growth and a resistance to chemotherapy. On the contrary, when TRX was overexpressed in the HT-1080 fibrosarcoma cells, the cell growth was retarded and chromosomal polyploidy and cellular senescence were induced. TRX-overexpression made HT-1080 cells resistant to an oxidative stress caused by H2O2 or paraquat. But these cells were significantly sensitive to ionizing radiation, showing an abrogation of the G2 checkpoint. Their DNA contents were twice of the controls and they expressed typical senescence markers. Their expression levels of p53 and cyclin-dependent kinase inhibitors (CDKI) were about 2-3-fold higher than the control. Nevertheless, cyclin D1 and D3, which are negatively regulated by CDKIs, were also increased. Overall, in HT-1080 cells the TRX-overexpression created a state of cellular senescence caused by a simultaneous stimulation of the mitogen-activated pathways and an inhibition of the cyclin-dependent kinases, which is known as a hypermitogenic arrest.
Assuntos
Senescência Celular , Raios gama , Tiorredoxinas/metabolismo , Western Blotting , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Oxidantes/farmacologia , PoliploidiaRESUMO
Reactive oxygen species including hydrogen peroxide (H(2)O(2)) and hydroxyl radical (OH) can be generated by ionizing radiation and has the potential to induce diseases. We provide the evidence that NAD-dependent malate dehydrogenase (MDH) is involved in the antioxidant role in preventing H(2)O(2) or γ-radiation-induced damage in Escherichia coli through the action of oxaloacetate. The E. colimdh mutant strain defective in MDH activity was more sensitive to H(2)O(2) or γ-radiation than was the wild type strain, when challenged in the exponential growth phase. The mdh mutant cells pretreated with oxaloacetate (2.5 mM), a product of NAD-dependent MDH activity, prior to H(2)O(2) treatment or γ-irradiation are resistant to H(2)O(2) or γ-radiation-induced damage, so cell survivability is restored to similar levels with the wild type. The SOS induction of umu'-'lacZ fusion gene by H(2)O(2) is significantly repressed by pretreatment of oxaloacetate in a dose-dependent way. These results indicate that oxaloacetate effectively protects E. coli cells against damage caused by oxidative stress. Oxaloacetate strongly prevented the DNA strand breaks by OH in a metal-catalyzed oxidation (MCO) system that generated H(2)O(2) as a mediator. By contrast, the prevention of DNA damage by oxaloacetate in an γ-irradiation system that directly generates OH from H(2)O in vitro was far less than that in an MCO system. Our results demonstrated that oxaloacetate, metabolite of NAD-dependent MDH action, plays a role as an antioxidant, possibly by scavenging H(2)O(2).