RESUMO
BACKGROUND: Biological laboratories and companies involved in antibody development need convenient and versatile methods to detect highly active antibodies. METHODS: To develop a mammalian cell-based ZZ display system for antibody quantification, the eukaryotic ZZ-displayed plasmid was constructed and transfected into CHO cells. After screening by flow cytometric sorting, the stable ZZ display cells were incubated with reference IgG and samples with unknown IgG content for 40 min at 4â, the relative fluorescence intensity of cells was analyzed and the concentration of IgG was calculated. RESULTS: By investigating the effects of different display-associated genetic elements, a eukaryotic ZZ-displaying plasmid with the highest display efficiency were constructed. After transfection and screening, almost 100% of the cells were able to display the ZZ peptide (designated CHO-ZZ cells). These stable CHO-ZZ cells were able to capture a variety of IgG, including human, rabbit, donkey and even mouse and goat. CHO-ZZ cells could be used to quantify human IgG in the range of approximately 12.5-1000 ng/mL, and to identify high-yielding engineered monoclonal cell lines. CONCLUSIONS: We have established a highly efficient CHO-ZZ display system in this study, which enables the quantification of IgG from various species under physiological conditions. This system offers the advantage of eliminating the need for antibody purification and will contribute to antibody development.
Assuntos
Imunoglobulina G , Cricetinae , Camundongos , Coelhos , Animais , Humanos , Cricetulus , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Citometria de Fluxo , PlasmídeosRESUMO
The existing mercury whole-cell biosensors (WCBs, parts per billion range) are not able to meet the real-world requirements due to their lack of sensitivity for the detection of ultratrace mercury in the environment. Ultratrace mercury is a potential threat to human health via the food chain. Here, we developed an ultrasensitive mercury WCB by directed evolution of the mercury-responsive transcriptional activator (MerR) sensing module to detect ultratrace mercury. Subsequently, the mutant WCB (m4-1) responding to mercury in the parts per trillion range after 1 h of induction was obtained. Its detection limit (LOD) was 0.313 ng/L, comparable to those of some analytical instruments. Surprisingly, the m4-1 WCB also responded to methylmercury (LOD = 98 ng/L), which is far more toxic than inorganic mercury. For more convenient detection, we have increased another green fluorescent protein reporter module with an optimized 5' untranslated region (5' UTR) sequence. This yields two visual WCBs with an enhanced fluorescence output. At a concentration of 2.5 ng/L, the fluorescence signals can be directly observed by the naked eye. With the combination of mobile phone imaging and image processing software, the 2GC WCB provided simple, rapid, and reliable quantitative and qualitative analysis of real samples (LOD = 0.307 ng/L). Taken together, these results indicate that the ultrasensitive visual whole-cell biosensors for ultratrace mercury detection are successfully designed using a combination of directed evolution and synthetic biotechnology.
Assuntos
Técnicas Biossensoriais , Mercúrio , Compostos de Metilmercúrio , Humanos , Mercúrio/análise , Regiões 5' não Traduzidas , Técnicas Biossensoriais/métodosRESUMO
Bacterial biosensors have great potential in contaminant detection for sensitivity, specificity, cost-effectiveness, and easy operation. However, the existing cadmium-responsive bacterial biosensors cannot meet the real-world detection requirements due to lack of sensitivity, specificity, and anti-interference capability. This study aimed to develop a bacterial biosensor for detecting the total and extractable cadmium in actual environmental samples. We constructed the cadmium-responsive biosensor with the regulatory element (cadmium resistance transcriptional regulatory, CadR) and the reporting element (GFP) and improved its performance by directed evolution. The mutant libraries of biosensors were generated by error-prone PCR and screened by continuous five-round fluorescence-activated cell sorting (FACS), and a bacteria variant epCadR5 with higher performance was finally isolated. Biosensor fluorescence intensity was measured by a microplate reader, and results showed that the evolved cadmium-responsive bacterial biosensor was of high sensitivity and specificity in detecting trace cadmium, with a detection limit of 0.45 µg/L, which is 6.8 times more specific to cadmium than that of the wild-type. Furthermore, microscopic qualitative analysis results showed that the bacteria could produce fluorescence response in a cadmium-contaminated soil matrix, and quantitative analysis results showed that the values of cadmium from epCadR5 bacteria were close to that from inductively coupled plasma-mass spectrometry. These results suggest that the biosensor may have a broad application prospect in the detection of cadmium-contaminated soil and water.
Assuntos
Técnicas Biossensoriais , Cádmio , Bactérias , Técnicas Biossensoriais/métodos , Solo , ÁguaRESUMO
During radiotherapy procedures, radiation-induced bystander effect (RIBE) can potentially lead to genetic hazards to normal tissues surrounding the targeted regions. Previous studies showed that RIBE intensities in cell cluster models were much higher than those in monolayer cultured cell models. On the other hand, low-concentration carbon monoxide (CO) was previously shown to exert biological functions via binding to the heme domain of proteins and then modulating various signaling pathways. In relation, our previous studies showed that exogenous CO generated by the CO releasing molecule, tricarbonyldichlororuthenium (CORM-2), at a relatively low concentration (20 µM), effectively attenuated the formation of RIBE-induced DNA double-strand breaks (DSB) and micronucleus (MN). In the present work, we further investigated the capability of a low concentration of exogenous CO (CORM-2) of attenuating or inhibiting RIBE in a mixed-cell cluster model. Our results showed that CO (CORM-2) with a low concentration of 30 µM could effectively suppress RIBE-induced DSB (p53 binding protein 1, p53BP1), MN formation and cell proliferation in bystander cells but not irradiated cells via modulating the inducible nitric oxide synthase (iNOS) andcyclooxygenase-2 (COX-2). The results can help mitigate RIBE-induced hazards during radiotherapy procedures.
Assuntos
Efeito Espectador/efeitos dos fármacos , Efeito Espectador/efeitos da radiação , Monóxido de Carbono/farmacologia , Modelos Biológicos , Radiação , Animais , Células CHO , Agregação Celular/efeitos dos fármacos , Agregação Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Cricetinae , Cricetulus , Ciclo-Oxigenase 2/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Raios XRESUMO
BACKGROUND: Recent technological advances in atmospheric plasmas have made the creation of non-thermal atmospheric pressure plasma (NTP) possible for utilization in the medical field. Although accumulated evidence suggests that NTP induces cell death in various cancer cell types thus offering a promising alternative treatment strategy, the mechanism underlying its therapeutic effect is not fully understood. RESULTS: We analyzed relevant signaling cascades associated with the tumor protein p53, in particular the cell cycle arrest, DNA damage as well as the underlying apoptosis pathways. Based on our results, the major effect from plasma exposure was found to be the activation of MAPK and p53 signaling pathways, resulting in changes in gene expression of MEKK, GADD, FOS and JUN. Finally, a significant modulation in expression of genes related to cellular proliferation and differentiation was observed. CONCLUSION: Overall, the presented data of the tumor transcriptome helped identify the key players in modulated gene expression following exposure to plasma at the molecular level, and also helped interpret the downstream processes. The present work laid the foundation for further studies to clarify the roles of multiple pathways in plasma-induced biological processes. Further investigation of these genes in other cell lines may reveal comprehensive mechanisms of plasma induced effects.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Gases em Plasma/farmacologia , Transcriptoma/genética , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Dano ao DNA/genética , Perfilação da Expressão Gênica/métodos , Humanos , Proteínas Quinases Ativadas por Mitógeno , Transdução de Sinais/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
Radiation-induced bystander effect (RIBE) has been proposed to have tight relationship with the irradiation-caused secondary cancers beyond the irradiation-treated area after radiotherapy. Our previous studies demonstrated a protective effect of low concentration carbon monoxide (CO) on the genotoxicity of RIBE after α-particle irradiation. In the present work, a significant inhibitory effect of low-dose exogenous CO, generated by tricarbonyldichlororuthenium (II) dimer [CO-releasing molecule (CORM-2)], on both RIBE-induced proliferation and chromosome aberration was observed. Further studies on the mechanism revealed that the transforming growth factor ß1/nitric oxide (NO) signaling pathway, which mediated RIBE signaling transduction, could be modulated by CO involved in the protective effects. Considering the potential of exogenous CO in clinical applications and its protective effect on RIBE, the present work aims to provide a foundation for potential application of CO in radiotherapy.
Assuntos
Efeito Espectador/efeitos da radiação , Monóxido de Carbono/farmacologia , Proliferação de Células , Animais , Células CHO , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Aberrações Cromossômicas , Cricetulus , Óxido Nítrico/fisiologia , Fator de Crescimento Transformador beta1/fisiologiaRESUMO
A whole-cell biosensor (WCB) is a convenient and cost-effective method for detecting contaminants. However, the practical application of the cadmium WCBs has been hampered by performance deficiencies, such as low sensitivity, specificity, and responsive strength. In this study, to improve the performance of cadmium WCBs, the cadmium transcription factor (CadC) and its DNA binding site (CadO), the key sensing module of the biosensor, were successively and separately subjected to a two-step directed evolution: 6-round random mutagenesis for CadC and 2-round saturation mutagenesis for CadO. For practical application, the GFP reporter gene was replaced with the lacZ gene and a facile and rapid smartphone detection platform for actual water samples was established by optimizing the reaction systems with detergents. The results showed that the evolved cadmium fluorescent biosensor CadO66 exhibited a higher specificity and a detection limit of 0.034 µg/L, representing a 19-fold reduction compared to the wild-type cadmium biosensor. The detergent sodium dodecylbenzenesulfonate effectively enhanced the visualization of WCB B0033-lacZ. Using the fluorescent WCB CadO66 and the visual WCB B0033-lacZ to analyze the cadmium contents of the actual water samples, the results were also consistent with a graphite furnace atomic absorption spectrometer. Taken together, this study indicates that the two-step directed evolution of CadC and CadO can efficiently improve the performance of cadmium WCBs, further promoting the utilization of WCB in actual sample detection and presenting a promising and feasible method for rapid sample detection.
Assuntos
Técnicas Biossensoriais , Cádmio , Cádmio/análise , Cádmio/metabolismo , Águas Residuárias , Genes Reporter , Água , Técnicas Biossensoriais/métodosRESUMO
Antibiotic abuse is the main reason for the drug resistance of pathogenic bacteria, posing a potential health risk. Antibiotic surveillance is critical for preventing antibiotic contamination. This study aimed to develop a sensitive and broad-spectrum whole-cell biosensor for tetracycline antibiotics (TCs) detection. Wild-type TCs-responsive biosensor was constructed by introducing a tetracycline operon into a sfGFP reporter plasmid. Using error-prone PCR, mutation libraries containing approximately 107 variants of the tetracycline repressor (TetR) gene were generated. The tigecycline-senstive mutants were isolated using high-throughput flow cytometric sorting. After 2 rounds of directed evolution, a mutant epS2-22 of TerR was isolated and assembled as a TCs biosensor. The epS2-22 biosensor was more sensitive and broad-spectrum than the wild-type biosensors. The detection limits of the epS2-22 biosensor for seven TCs were 4- to 62-fold lower than the wild-type biosensor (no response to tigecycline). Meanwhile, the epS2-22 biosensor had a shorter detection time and a stronger signal output than the wild type. In addition, the evolved epS2-22 biosensor showed excellent performance in detecting low traces of TCs in environmental water. These results suggest that directed evolution is a powerful tool for developing high-performance whole-cell biosensors, and the evolved epS2-22 biosensors have the potential for wider applications in real-world TCs detection.
Assuntos
Antibacterianos , Tetraciclina , Tigeciclina , Tetraciclina/farmacologia , Antibacterianos/farmacologia , Movimento Celular , Contaminação de Medicamentos , Fatores de TranscriçãoRESUMO
Ambient PM2.5 is one of the environmental risk factors and was correlated with senescence-related diseases based on the epidemiologic investigation. However, little is known about senescence induced by PM2.5 as well as the underlying mechanisms. In this study, we demonstrated that PM2.5 exposure aggravated cellular senescence in vivo and in vitro, and disrupted micronuclei (MN) played a vital role in this process. Our results suggested that the nuclear envelope (NE) of PM2.5-induced MN was ruptured. Subsequently, cGAS was found to localize to approximately 80% of the disrupted MN but few for intact MN. Upon examination of cGAMP and SA-ß-Gal, the cGAS-STING pathway was found activated and related to cellular senescence induced by PM2.5. Taken together, we reported a novel finding that PM2.5 exposure causes cellular senescence via DNA damage, MN formation, and cGAS activation. These results revealed the potential toxicity of PM2.5 and its related mechanisms in cellular senescence.
Assuntos
Membrana Nuclear , Nucleotidiltransferases , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Membrana Nuclear/metabolismo , Dano ao DNA , Senescência Celular , Material Particulado/toxicidadeRESUMO
PURPOSE: To describe macular thickness measured by optical coherence tomography (OCT) in healthy eyes of adult Chinese persons. DESIGN: Population-based cross-sectional study. PARTICIPANTS: Chinese adults aged 30+ years who were residents of Handan, North China. METHODS: The Handan Eye Study is a population-based study of eye disease in Chinese persons. Eligible residents underwent a comprehensive ophthalmic examination including OCT (Stratus OCT, Carl Zeiss Meditec Inc., Jena, Germany). Fast macular thickness scans were performed over maculae within 6 mm in diameter, divided into 3 regions (central, inner, and outer, with a diameter of 1, 3, and 6 mm, respectively) and 9 quadrants (1 in the central region and 4 each in the inner and outer regions). Retinal thickness (means and standard deviations) was calculated by OCT mapping software, presented for foveal minimum, central macula (within 1 mm diameter), and inner and outer regions divided by 8 quadrants. MAIN OUTCOME MEASURES: Macular thickness measured by OCT. RESULTS: Of the 6830 participants (90.4% response rate) examined, 2230 eyes of healthy subjects with high-quality OCT scans were selected (32.7% of participants; mean age, 46.4+/-9.9 years, 58.4% were women). The mean foveal minimum, central, inner, and outer macular thicknesses were 150.3 (18.1) microm, 176.4 (17.5) microm, 255.3 (14.9) microm, and 237.7 (12.4) microm, respectively (overall differences, P<0.001). The mean foveal volume was 0.139 (0.014) mm(3), and the mean total macular volume was 6.761 (0.516) mm(3). In the inner region, the nasal quadrant was thinner than the superior and inferior quadrants, and in the outer region, the nasal quadrant was the thickest (P<0.001). Age was positively correlated with foveal (beta coefficient = 3.582) and central macular (beta coefficient = 2.422) thicknesses. The foveal minimum, central, inner, and outer macular thicknesses were significantly greater in men than in women. Fasting plasma glucose was negatively correlated with central macular thickness (2.416 mm reduction per millimole/liter increase in glucose), and axial length was positively correlated with central macular thickness (2.138 mm increase per millimeter increase in axial length). CONCLUSIONS: Normal macular thickness measurements using OCT in a large population-based sample of adult Chinese persons aged 30 to 85 years were generally thinner in the foveal and central macular areas than measurements reported in other populations. Age and axial length were positively correlated with macular thickness.
Assuntos
Povo Asiático/estatística & dados numéricos , Macula Lutea/anatomia & histologia , Tomografia de Coerência Óptica , Adulto , Idoso , Idoso de 80 Anos ou mais , China/epidemiologia , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valores de ReferênciaRESUMO
As a potent environmental oxidative stressor, arsenic exposure has been reported to exacerbate cardiovascular diseases and increase vascular endothelial cell monolayer permeability. However, the underlying mechanism of this effect is not well understood. In this paper, we test our hypothesis that reactive oxygen species (ROS)-induced vascular endothelial growth factor (VEGF) expression may play an important role in an arsenic-caused increase of endothelial cell monolayer permeability. The mouse brain vascular endothelial cell bEnd3 monolayer was exposed to arsenite for 1, 3, and 6 days. The monolayer permeability, VEGF protein release, and ROS generation were determined. In addition, VE-cadherin and zonula occludens-1 (ZO-1), two membrane structure proteins, were immunostained to elucidate the effects of arsenite on the cell-cell junction. The roles of ROS and VEGF in arsenite-induced permeability was determined by inhibiting ROS with antioxidants and immuno-depleting VEGF with a VEGF antibody. We observed that arsenite increased bEnd3 monolayer permeability, elevated the production of cellular ROS, and increased VEGF release. VE-cadherin and ZO-1 disruptions were also found in cells treated with arsenite. Furthermore, both antioxidant (N-acetyl cysteine and tempol) and the VEGF antibody treatments significantly lowered the arsenite-induced permeability of the bEnd3 monolayer as well as VEGF expression. VE-cadherin and ZO-1 disruptions were also diminished by N-acetyl cysteine and the VEGF antibody. Our data suggest that the increase in VEGF expression caused by ROS may play an important role in the arsenite-induced increase in endothelial cell permeability.
Assuntos
Arsenitos/toxicidade , Permeabilidade da Membrana Celular/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Anticorpos/farmacologia , Antioxidantes/farmacologia , Arsenitos/química , Caderinas/metabolismo , Células Endoteliais/metabolismo , Poluentes Ambientais/química , Proteínas de Membrana/metabolismo , Camundongos , Fosfoproteínas/metabolismo , Fatores de Tempo , Fatores de Crescimento do Endotélio Vascular/imunologia , Proteína da Zônula de Oclusão-1RESUMO
Genomic instability can be observed in bystander cells. However, the underlying mechanism(s) is still relatively unclear. In a previous study, we found that irradiated cells released mitochondria-dependent intracellular factor(s) which could lead to bystander gamma-H2AX induction. In this paper, we used normal (rho(+)) and mtDNA-depleted (rho(0)) human-hamster hybrid cells to investigate mitochondrial effects on the genotoxicity in bystander effect through medium transfer experiments. Through the detection of DNA double-strand breaks with gamma-H2AX, we found that the fraction of gamma-H2AX positive cells changed with time when irradiation conditioned cell medium (ICCM) were harvested. ICCM harvested from irradiated rho(+) cells at 10 min post-irradiation (rho(+) ICCM(10 min)) caused larger increases of bystander gamma-H2AX induction comparing to rho(0) ICCM(10 min), which only caused a slight increase of bystander gamma-H2AX induction. The rho(+) ICCM(10 min) could also result in the up-regulation of ROS production (increased by 35% at 10 min), while there was no significant increase in cells treated with rho(0) ICCM(10 min). We treated cells with dimethyl sulfoxide (DMSO), the scavenger of ROS, and quenched gamma-H2AX induction by rho(+) ICCM. Furthermore, after the medium had been transferred and the cells were continuously cultured for 7 days, we found significantly increased CD59(-) gene loci mutation (increased by 45.9%) and delayed cell death in the progeny of rho(+) ICCM-treated bystander cells. In conclusion, the work presented here suggested that up-regulation of the mitochondria-dependent ROS might be very important in mediating genotoxicity of bystander effects.
Assuntos
Efeito Espectador/efeitos da radiação , Dano ao DNA , DNA Mitocondrial/metabolismo , Histonas/metabolismo , Espécies Reativas de Oxigênio/efeitos da radiação , Regulação para Cima , Animais , Células CHO , Morte Celular , Cricetinae , Cricetulus , Humanos , Células Híbridas/efeitos da radiação , Transdução de SinaisRESUMO
Radiation-induced bystander effects (RIBE) have been demonstrated to occur widely in various cell lines. However, very little data is available on the genotoxic effects of RIBE combined with other factor(s). We reported previously that with a low dose of alpha-particle irradiation, the fraction of gamma-H2AX foci-positive cells in non-irradiated bystander cells was significantly increased under elevated NaCl culture conditions. In this study, we further investigated the functional role of NaCl in the enhancement of RIBE using a specially designed co-culture system and micronucleus (MN) test. It was shown that the MN frequency was not increased significantly by elevated NaCl (9.0 g/L) alone or by medium exposure. However, with 1.0 cGy alpha-particle irradiation, the induced MN frequency increased significantly in both irradiated and non-irradiated bystander regions. Additional studies showed that elevated NaCl made the non-irradiated bystander cells more vulnerable to bystander factors. Furthermore, it was found that the induced MN frequency in cells both in irradiated and non-irradiated bystander regions was weakened when the hypertonic medium was changed to normotonic medium for 2h before irradiation. Such observations were quite similar to the co-effect of NaCl and hydrogen peroxide (H(2)O(2)), indicating that elevated NaCl might sensitize non-irradiated cells to bystander factors-induced oxidative stress.
Assuntos
Efeito Espectador/efeitos dos fármacos , Efeito Espectador/efeitos da radiação , Cloreto de Sódio/farmacologia , Partículas alfa/efeitos adversos , Efeito Espectador/genética , Linhagem Celular , Técnicas de Cocultura , Humanos , Peróxido de Hidrogênio/toxicidade , Soluções Isotônicas , Testes para Micronúcleos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Solução Salina HipertônicaRESUMO
Diesel exhaust particle (DEP) has been identified as a class 2A human carcinogen and closely related to the increased incidence of respiratory allergy, cardiopulmonary morbidity and mortality, and risk of lung cancer. However, the molecular mechanisms of DEP mutagenicity/carcinogenicity are still largely unknown. In the present study, we focused on the mutagenicity of DEPs in human-hamster hybrid (A(L)) cells and evaluated the role of cell-particle interaction in mediating mutagenic process. We found that DEPs formed micron-sized aggregates in the medium and located mainly in large cytoplasmic vacuoles of cells by 24h treatment. The cellular granularity was increased by DEP treatment in a dose-dependent manner. DEPs resulted in a dose-dependent increase of mutation yield at CD59 locus in A(L) cells, while inflicting minimal cytotoxicity. There was a more than two-fold increase of mutation yield at CD59 locus in A(L) cells exposed to DEPs at a dose of 50mug/ml. Such induction was significantly reduced by concurrent treatment with phagocytosis inhibitors, cytochalasin B and ammonium chloride (p<0.05). These results provided direct evidence that DEPs was mutagenic in mammalian cells and that cell-particle interaction played an essential role in the process.
Assuntos
Células Híbridas/efeitos dos fármacos , Mutagênicos/toxicidade , Emissões de Veículos/toxicidade , Cloreto de Amônio/farmacologia , Animais , Antígenos CD59/genética , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Tamanho Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Citocalasina B/farmacologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Células Híbridas/metabolismo , Células Híbridas/ultraestrutura , Corpos de Inclusão , Luz , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Testes de Mutagenicidade/métodos , Mutagênicos/análise , Tamanho da Partícula , Partículas Submitocôndricas/efeitos dos fármacos , Partículas Submitocôndricas/ultraestrutura , Fatores de Tempo , Emissões de Veículos/análise , Emissões de Veículos/prevenção & controleRESUMO
Radioadaptive response (RAR) is an important phenomenon induced by low dose radiation. However, the molecular mechanism of RAR is obscure. In this study, we focused on the possible role of heme oxygenase 1 (HO-1) in RAR. Consistent with previous studies, priming dose of X-ray radiation (1-10cGy) induced significant RAR in normal human skin fibroblasts (AG 1522 cells). Transcription and translation of HO-1 was up-regulated more than two fold by a priming dose of radiation (5cGy). Zinc protoporphyrin â ¨, a specific competitive inhibitor of HO-1, efficiently inhibited RAR whereas hemin, an inducer of HO-1, could mimic priming dose of X-rays to induce RAR. Knocking down of HO-1 by transfection of HO-1 siRNA significantly attenuated RAR. Furthermore, the expression of HO-1 gene was modulated by the nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which translocated from cytoplasm to nucleus after priming dose radiation and enhance the antioxidant level of cells.
Assuntos
Antioxidantes/metabolismo , Fibroblastos/metabolismo , Heme Oxigenase-1/genética , Fator 2 Relacionado a NF-E2/genética , Linhagem Celular , Nucléolo Celular/metabolismo , Nucléolo Celular/efeitos da radiação , Citoplasma/efeitos da radiação , Relação Dose-Resposta à Radiação , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Heme Oxigenase-1/antagonistas & inibidores , Hemina/genética , Hemina/metabolismo , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , Protoporfirinas/genética , Protoporfirinas/metabolismo , RNA Interferente Pequeno/genética , Raios XRESUMO
Non-thermal plasma (NTP) has been proposed as a novel therapeutic method for anticancer treatment. However, the mechanism underlying its biological effects remains unclear. In this study, we investigated the inhibitory effect of NTP on the invasion of HeLa cells, and explored the possible mechanism. Our results showed that NTP exposure for 20 or 40 s significantly suppressed the migration and invasion of HeLa cells on the basis of matrigel invasion assay and wound healing assay, respectively. Moreover, NTP reduced the activity and protein expression of the matrix metalloproteinase (MMP)-9 enzyme. Western blot analysis indicated that NTP exposure effectively decreased phosphorylation level of both ERK1/2 and JNK, but not p38 MAPK. Furthermore, treatment with MAPK signal pathway inhibitors or NTP all exhibited significant depression of HeLa cells migration and MMP-9 expression. The result showed that NTP synergistically suppressed migration and MMP-9 expression in the presence of ERK1/2 inhibitor and JNK inhibitor, but not p38 MAPK inhibitor. Taken together, these findings suggested that NTP exposure inhibited the migration and invasion of HeLa cells via down-regulating MMP-9 expression in ERK1/2 and JNK signaling pathways dependent manner. These findings provide hints to the potential clinical research and therapy of NTP on cervical cancer metastasis.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Metaloproteinase 9 da Matriz/biossíntese , Proteínas de Neoplasias/biossíntese , Gases em Plasma , Movimento Celular , Células HeLa , Humanos , Invasividade NeoplásicaRESUMO
Arsenic is a ubiquitous environmental factor that has been identified as a risk factor for a wide range of human diseases. Alcohol is clearly a toxic substance when consumed in excess. Alcohol abuse results in a variety of pathological effects, including damages to liver, heart, and brain, as well as other organs, and is associated with an increased risk of certain types of cancers. In history, arsenic-contaminated beers caused severe diseases. There are populations who are exposed to relatively high levels of arsenic in their drinking water and consume alcohol at the same time. In this focused review, we aim to discuss important molecular mechanisms responsible for arsenic toxicity and potential combined toxic effects of alcohol and arsenic.
Assuntos
Arsênio/toxicidade , Etanol/toxicidade , Animais , Sinergismo Farmacológico , Humanos , Modelos Teóricos , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Diesel exhaust [diesel exhaust particles (DEPs) and their extracts (DPE)] and ultraviolet A radiation (UVA) are two ubiquitous environmental factors that have been identified as essential risk factors for various benign or malignant human diseases, either alone or in combination with other agents. OBJECTIVES: We aimed to investigate the synergistic effects of DPE and UVA at low-dose exposures in human-hamster hybrid (AL) cells and their underlying mechanisms. METHODS: We exposed exponentially growing AL cells to DPE and/or UVA radiation with or without reactive oxygen species (ROS) quenchers and then assayed the cells for survival, mutation induction, apoptosis, and micronucleus generation. In addition, using a singlet oxygen (1O2) trapping probe, 2,2,6,6-tetramethyl-4-piperidone, coupled with electron paramagnetic resonance spectroscopy, we determined the production of 1O2. RESULTS: Treatment of AL cells with DPE+UVA induced significant cytotoxic and genotoxic damage. In contrast, we found no significant damage in cells treated with either UVA or DPE alone at the same doses. Mutation spectra of CD59- mutants showed that treatment with DPE+UVA easily induces multilocus deletions. Sodium azide significantly inhibited both cellular and DNA damage induced by DPE+UVA treatment, whereas other ROS inhibitors had little protecting effect. Furthermore, we found a significant increase of 1O2 in the cells that received DPE+UVA treatment. CONCLUSION: These findings suggest that UVA activated the genotoxicity and cytotoxicity of DPE in mammalian cells and that 1O2 played an important role in these processes.
Assuntos
Material Particulado/efeitos da radiação , Material Particulado/toxicidade , Oxigênio Singlete/química , Raios Ultravioleta , Emissões de Veículos/toxicidade , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Humanos , Células Híbridas , Testes para Micronúcleos , Testes de Mutagenicidade , Triacetonamina-N-Oxil/análogos & derivadosRESUMO
Asbestos fibers are carcinogenic to both humans and experimental animals. The continued discoveries of exposure routes whereby the general public is exposed to asbestos suggest a long-term, low-dose exposure for a large number of people. However, the mechanisms by which asbestos induces malignancy are not entirely understood. In previous studies, we have shown that asbestos is an effective gene and chromosomal mutagen when assayed using the highly sensitive AL mutation assay and that the mutagenicity is mediated by reactive oxygen species. The objective of the present study is to determine the origin of these radical species, particularly reactive nitrogen species, in fiber mutagenesis. Using the radical probe 5',6'-chloromethyl-2',7'-dihydroxyphenoxazine diacetate to trap reactive radical species, we showed that crocidolite increased the levels of oxyradicals in cytoplasts, in the absence of the nucleus, in a dose-dependent manner, which was reduced significantly by cotreatment with the radical scavenger dimethyl sulfoxide. Treatment of enucleated cells with crocidolite asbestos followed by rescue fusion using karyoplasts from control cells resulted in significant mutant induction, indicating that the nuclear-cytoplasmic interaction is necessary for fiber mutagenesis. Using the fluorescent probe 2,3-diaminonaphthotriazole, crocidolite fibers were shown to induce a dose-dependent increase of nitric oxide production, which was suppressed significantly by concurrent treatment with the nitric oxide synthase inhibitor, NG-methyl-L-arginine (L-NMMA). Similarly, there was a dose-dependent decrease in the mutation yield induced by crocidolites in the presence of graded doses of L-NMMA. These data showed that extranuclear targets play an essential role in the initiation of oxidative damage that mediates fiber mutagenesis in mammalian cells.