RESUMO
OBJECTIVE: To investigate the effect of chronic exposure to sodium arsenite at a dose of 1. 0 µmol / L on proliferation of human bronchial epithelial cells( HBE) and human keratinocytes( HaCaT) and discuss the mechanism of arsenic carcinogenesis. METHODS: Malignant transformation model of HBE and HaCaT cells cultured in vitro were used in this study. MTT assay was used to detect the capacity of proliferation. Flow cytometry was used to detect cell cycle. The expression of cell cycle related protein like cyclin E, cyclin D1 and cyclin A protein were inspected by Western blot. RESULTS: The treated cells, including passage 36 and 43 of HBE cells and passage 28 and 35 of HaCaT cells grow faster than the control group( P < 0. 01 and P < 0. 05). The treated cells in the G1 phase were decreased( P < 0. 05), however cells in the S phase were increased( P <0. 05). In addition, the expression of cyclin E displayed a trend of up-regulation( P <0. 05), and it was maintained at a high level in advanced period. CONCLUSION: By increasing the expression of cyclin E in HBE and HaCaT cells, low dose of sodium arsenite made cells escaping from the G1 phase to S phase, accelerating cell cycle progression and proliferation, a way that may lead to malignant transformation.
Assuntos
Arsênio/farmacologia , Arsenitos/toxicidade , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/induzido quimicamente , Células Epiteliais/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Compostos de Sódio/toxicidade , Ciclo Celular , Linhagem Celular , Ciclina D1 , Células Epiteliais/metabolismo , Humanos , Queratinócitos/metabolismo , Regulação para CimaRESUMO
Long-term exposure to arsenite leads to human lung cancer, but the underlying mechanisms of carcinogenesis remain obscure. The transcription factor of nuclear factor-erythroid-2 p45-related factor (Nrf2)-mediated antioxidant response represents a critical cellular defense mechanism and protection against various diseases. Paradoxically, emerging data suggest that the constitutive activation of Nrf2 is associated with cancer development, progression and chemotherapy resistance. However, the role of Nrf2 in the occurrence of cancer induced by long-term arsenite exposure remains to be fully understood. By establishing transformed human bronchial epithelial (HBE) cells via chronic low-dose arsenite treatment, we showed that, in acquiring this malignant phenotype, continuous low level of ROS and sustained enhancement of Nrf2 and its target antioxidant enzyme levels were observed in the later-stage of arsenite-induced cell transformation. The downregulation of Keap1 level may be responsible for the over-activation of Nrf2 and its target enzymes. To validate these observations, Nrf2 was knocked down in arsenite-transformed HBE cells by SiRNA transfection, and the levels of Nrf2 and its target antioxidant enzymes, ROS, cell proliferation, migration, and colony formation were determined following these treatments. Results showed that blocked Nrf2 expression significantly reduced Nrf2 and its target antioxidant enzyme levels, restored ROS levels, and eventually suppressed cell proliferation, migration, and colony formation of the transformed cells. In summary, the results of the study strongly suggested that the continuous activation of Nrf2 and its target antioxidant enzymes led to the over-depletion of intracellular ROS levels, which contributed to arsenite-induced HBE cell transformation.
Assuntos
Antioxidantes/metabolismo , Arsenitos/toxicidade , Brônquios/efeitos dos fármacos , Carcinógenos/toxicidade , Transformação Celular Neoplásica/induzido quimicamente , Células Epiteliais/efeitos dos fármacos , Neoplasias Pulmonares/induzido quimicamente , Fator 2 Relacionado a NF-E2/agonistas , Compostos de Sódio/toxicidade , Brônquios/enzimologia , Brônquios/patologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Ativação Enzimática , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Heme Oxigenase-1/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Malondialdeído/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Invasividade Neoplásica , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Arsenic is widely present in nature and is a common environmental poison that seriously damages human health. Chronic exposure to arsenic is a major environmental poisoning factor that promotes cell proliferation and leads to malignant transformation. However, its molecular mechanism remains unclear. In this study, we found that arsenite can promote the transformation of immortalized human keratinocyte cells (HaCaT) from the G0/G1 phase to S phase and demonstrated malignant phenotypes. This phenomenon is accompanied by obviously elevated levels of NRF2, NQO1, Cyclin E, and Cyclin-dependent kinase 2 (CDK2). Silencing the NRF2 expression with small interfering RNA (siRNA) in arsenite-transformed (T-HaCaT) cells was shown to reverse the malignant phenotype. Furthermore, the siRNA silencing of NQO1 significantly decreased the levels of the cyclin E-CDK2 complex, inhibiting the G0/G1 to S phase cell cycle progression and transformation to the T-HaCaT phenotypes. Thus, we hypothesized that the NRF2/NQO1 pathway played a key role in the arsenite-induced malignancy of HaCaT cells. By increasing the expression of Cyclin E-CDK2, the NRF2/NQO1 pathway can affect cell cycle progression and cell proliferation. A new common health effect mechanism of arsenic carcinogenesis has been identified; thus, it would contribute to the development of novel treatments to prevent and treat skin cancer caused by arsenic.
Assuntos
Arsênio , Arsenitos , Arsênio/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Ciclina E/farmacologia , Humanos , Queratinócitos , NAD(P)H Desidrogenase (Quinona)/metabolismo , NAD(P)H Desidrogenase (Quinona)/farmacologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
In arsenic toxicity, activation of the erythroid 2-related factor 2 (NRF2) pathway is regarded as a driver of cancer development and progression; however, the mechanisms by which NRF2 gene expression regulates cell cycle progression and mediates pathways of cellular proliferation and apoptosis in arsenic-induced lung carcinogenesis are poorly understood. In this study, we explored the regulatory functions of NRF2 expression and its target genes in immortalized human bronchial epithelial (HBE) cells continuously exposed to 1.0 µM sodium arsenite over approximately 43 passages (22 weeks). The experimental treatment induced malignant transformation in HBE cells, characterized by increased cellular proliferation and soft agar clone formation, as well as cell migration, and accelerated cell cycle progression from G0/G1 to S phase with increased levels of cyclin E-CDK2 complex,decreased cellular apoptosis rate. Moreover, we observed a sustained increase in NRF2 protein levels and those of its target gene products (NQO1, BCL-2) with concurrently decreased expression of apoptosis-related proteins (BAX, Cleaved-caspase-3/Caspase-3 and CHOP) and increased expression of the anti-apoptotic protein MCL-1. Silencing NRF2 expression with small interfering RNA (siRNA) in arsenite-transformed (T-HBE) cells was shown to reverse the malignant phenotype. Further, siRNA silencing of NQO1 significantly decreased levels of the cyclin E-CDK2 complex, inhibiting G0/G1 to S phase cell cycle progression and transformation to the T-HBE phenotypes. This study demonstrated a novel role for the NRF2/NQO1 signaling pathway in mediating arsenite-induced cell transformation by increasing the expression of cyclin E-CDK2, and accelerating the cell cycle and cell proliferation. Arsenite promotes activation of the NRF2/BCL-2 signaling pathway inhibited CHOP increasing cellular resistance to apoptosis and further promoting malignant transformation.
Assuntos
Arsenitos , Fator 2 Relacionado a NF-E2 , Apoptose , Arsenitos/toxicidade , Proliferação de Células , Transformação Celular Neoplásica/genética , Células Epiteliais , Humanos , Fator 2 Relacionado a NF-E2/genéticaRESUMO
NF-E2-related factor 2 (Nrf2) plays an important role in tumour proliferation and chemoresistance. Nrf2 expression levels may be associated with prognosis of lung cancer, but previous results have been inconsistent. Pooled hazard ratios (HRs) and odds ratios were calculated to assess the prognostic value of the Nrf2 expression in this meta-analysis. Nine studies with 940 patients were included. A high Nrf2 expression level was significantly related to decreased overall survival (OS) (HR = 1.948, 95% CI = 1.564-2.427), lower response rate (HR = 2.675, 95% CI = 1.553-4.610), and poor progression-free survival (HR = 3.078, 95% CI = 1.791-5.293). Subgroup analysis demonstrated that high-Nrf2-expression was significantly correlated with worse OS of patients possessing epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) or undergoing chemotherapeutic treatments (HR = 2.500, 95% CI = 1.556-4.018). Conversely, this high expression was not significantly related to the OS of patients with surgical resection (HR = 1.750, 95% CI = 0.995-3.080, and p=.052). High Nrf2 expression was significantly correlated with worse OS of patients in advanced stage (HR = 2.500, 95% CI = 1.556-4.018), compared with early cancer stage (HR = 1.609, 95% CI = 0.675-3.835, and p=.283). This meta-analysis suggests that high Nrf2 expression may be a predictive factor of poor outcomes in lung cancer. Therefore, Nrf2 likely plays an important role in prognostic evaluation and may be a therapeutic target for EGFR-TKIs therapy and chemotherapy.
Assuntos
Neoplasias Pulmonares/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estudos de Coortes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Fator 2 Relacionado a NF-E2/genética , Prognóstico , Intervalo Livre de ProgressãoRESUMO
Dimethylformamide (DMF) is widely used as a solvent in the production of synthetic leather. Previous studies have focused on workers exposed to DMF in leather factories; however, little attention has been paid to the general population. This study was conducted to examine the effects of DMF exposure on elderly residents living near synthetic leather factories. A total of 962 subjects over 60 years of age in proximity to these factories (monitoring points) were enrolled as the exposure group, and 1924 permanent residents living distant from the factories were enrolled as the control group. The exposure group was divided into 3 groups according to their distance from the monitoring points. Physical examination, routine blood tests, and liver and renal function data were collected, and the DMF concentration in the air was analyzed by gas chromatography-mass spectroscopy. The prevalence of abnormal heart rhythm, electrocardiogram and B-mode ultrasound results in the exposure group was significantly greater than in the control group. Aspartate transaminase (AST), alanine transaminase (ALT), and blood urea nitrogen (BUN) levels in the exposure group also were higher than those in the control group (Pâ<â.01). There was an effect of distance from leather factories on liver and kidney dysfunction in the 3 exposure groups. Compared with the exposure group at >3âkm distance from the source, the prevalence of increased AST, ALT, and BUN in the exposure group at <1âkm was significantly greater (Pâ<â.001). It was concluded that DMF exposure was related to an increased risk of a cardiac injury and liver and kidney dysfunction.
Assuntos
Dimetilformamida/efeitos adversos , Exposição Ambiental/efeitos adversos , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Alanina Transaminase/sangue , Aspartato Aminotransferases/sangue , Nitrogênio da Ureia Sanguínea , Estudos de Casos e Controles , Estudos Transversais , Feminino , Humanos , Rim/fisiologia , Fígado/fisiologia , Masculino , Pessoa de Meia-Idade , CurtumeRESUMO
It is well known that long-term exposure to arsenite leads to human skin cancer, but the underlying mechanisms of carcinogenesis remain obscure. The transcription factor Nrf2-mediated antioxidant response represents a critical cellular defense mechanism; however, emerging data suggest that constitutive activation of Nrf2 is associated with cancer development and chemotherapy resistance. The reasons Nrf2 continuously accumulates in cancer cells remain to be fully understood. By establishing transformed human keratinocyte cells via chronic arsenite treatment, we observed a continuous reduction in reactive oxygen species levels and enhanced levels of Nrf2 and its target antioxidant enzymes in the later stage of arsenite-induced cell transformation. We also revealed that hypermethylation of the Keap1 gene promoter region induced by DNA methyltransferase-3 leading to inactivation of its function was responsible for constitutive activation of Nrf2 and its target enzymes. To validate these observations, the expression of Keap1 protein was restored in arsenite-transformed cells by treatment with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-Aza-dC), and protein levels of Nrf2 and colony formation were then determined after these treatments. Results showed that enhancement of Keap1 expression by 5-Aza-dC significantly reduced Nrf2 and its target antioxidant enzyme levels, and that in turn suppressed cell proliferation and colony formation of the transformed cells. Taken together, the present study strongly suggests that loss of Keap1 function by hypermethylation of its promoter region leading to Nrf2 nuclear accumulation appears to play a role in arsenite-induced human keratinocyte transformation.