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Tumor priming is considered a promising strategy for improving drug distribution in malignant tissues. Multicellular layers (MCLs) of human cancer cells are potentially useful models for evaluating tumor-priming agents. We evaluated the priming effects of paclitaxel (PTX) on doxorubicin (DOX) penetration using MCLs of the human colorectal cancer cell lines including DLD-1, HCT-116, and HT-29. The penetration of DOX treated at 50 µM for 3 h was highly limited in all three MCLs. The penetration of the priming agent PTX into MCLs was determined using rhodamine-labeled PTX and appeared to be cell line-dependent: full penetration was observed in HCT-116 and HT-29 MCLs, whereas only limited penetration occurred in DLD-1 MCLs. PTX pretreatment at 20 µM for 24 or 48 h induced a tumor-priming effect in DOX distribution, with a 3 to 5.6-fold-increase in HCT-116 and HT-29 MCLs but a less than two-fold increase in DLD-1 MCLs. PTX treatment decreased fibronectin expression in HCT-116 and HT-29 MCLs but not in DLD-1, suggesting that the prominent priming effect of PTX in HCT-116 and HT-29 MCLs may be associated with the downregulation of fibronectin expression. Our study demonstrated that MCLs of human cancer cells are a useful model not only for the study of drug penetration into tumor tissues but also for screening and evaluating tumor-priming agents.
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Neoplasias Colorrectales , Paclitaxel , Humanos , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Fibronectinas , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Células HT29 , Neoplasias Colorrectales/patología , Línea Celular TumoralRESUMEN
Activated pancreatic stellate cells (aPSCs) and M2 macrophages modulate tumor progression and therapeutic efficacy in pancreatic ductal adenocarcinoma (PDAC) via epithelial-mesenchymal transition (EMT). Here, our aim was to analyze the anti-invasion effects of anti-cancer agents where EMT-inducing cancer-stroma interaction occurs under three-dimensional (3D) culture conditions. We used microfluidic channel chips to co-culture pancreatic tumor spheroids (TSs) with aPSCs and THP-1-derived M2 macrophages (M2 THP-1 cells) embedded in type I collagen. Under stromal cell co-culture conditions, PANC-1 TSs displayed elevated expression of EMT-related proteins and increased invasion and migration. When PANC-1 TSs were exposed to gemcitabine, 5-fluorouracil, oxaliplatin, or paclitaxel, 30-50% cells were found unaffected, with no significant changes in the dose-response profiles under stromal cell co-culture conditions. This indicated intrinsic resistance to these drugs and no further induction of drug resistance by stromal cells. Paclitaxel had a significant anti-invasion effect; in contrast, oxaliplatin did not show such effect despite its specific cytotoxicity in M2 THP-1 cells. Overall, our findings demonstrate that the TS-stroma co-culture model of PDAC is useful for activity profiling of anti-cancer agents against cancer and stromal cells, and analyzing the relationship between anti-stromal activity and anti-invasion effects.
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Cocaine and heroin cause impairment of neural plasticity in the brain including striatum. This study aimed to identify genes differentially expressed in the striatum of cynomolgus monkeys in response to cocaine and heroin. After chronic administration of cocaine and heroin in the monkeys, we performed large-scale transcriptome profiling in the striatum using RNA-Seq technology and analysed functional annotation. We found that 547 and 1238 transcripts were more than 1.5-fold up- or down-regulated in cocaine- and heroin-treated groups, respectively, compared to the control group, and 3432 transcripts exhibited differential expression between cocaine- and heroin-treated groups. Functional annotation analysis indicated that genes associated with nervous system development (NAGLU, MOBP and TTL7) and stress granule disassembly (KIF5B and KLC1) were differentially expressed in the cocaine-treated group compared to the control group, whereas gene associated with neuron apoptotic process (ERBB3) was differentially expressed in the heroin-treated group. In addition, IPA network analysis indicated that genes (TRAF6 and TRAF3IP2) associated with inflammation were increased by the chronic administration of cocaine and heroin. These results provide insight into the correlated molecular mechanisms as well as the upregulation and down-regulation of genes in the striatum after chronic exposure to cocaine and heroin.
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Trastornos Relacionados con Cocaína/patología , Cocaína/efectos adversos , Cuerpo Estriado/patología , Dependencia de Heroína/patología , Heroína/efectos adversos , Animales , Cocaína/administración & dosificación , Trastornos Relacionados con Cocaína/genética , Cuerpo Estriado/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Heroína/administración & dosificación , Dependencia de Heroína/genética , Humanos , Cinesinas , Macaca fascicularis , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/genética , RNA-Seq , Autoadministración , Transcriptoma/efectos de los fármacosRESUMEN
OBJECTIVE: The aim of this study was to investigate differentially expressed genes and their functions in the hippocampus and striatum after heroin administration in cynomolgus macaques of different ages. METHODS: Cynomolgus monkeys were divided by age as follows: 1 year (A1, n = 2); 3 to 4 years (A2, n = 2); 6 to 8 years (A3, n = 2); and older than 11 years (A4, n = 2). After heroin was injected intramuscularly into the monkeys (0.6 mg/kg), we performed large-scale transcriptome profiling in the hippocampus (H) and striatum (S) using RNA sequencing technology. Some genes were validated with real-time quantitative PCR. RESULTS: In the hippocampus, the gene expression of A1H was similar to that of A4H, while the gene expression of A2H was similar to that of A3H. Genes associated with the mitogen-activated protein kinase signaling pathway (STMN1, FGF14, and MAPT) and γ-aminobutyric acid-ergic synapses (GABBR2 and GAD1) were differentially expressed among control and heroin-treated animals. Differential gene expression between A1S and A4S was the least significant, while differential gene expression between A3S and A2S was the most significant. Genes associated with the neurotrophin signaling pathway (NTRK1 and NGFR), autophagy (ATG5), and dopaminergic synapses (AKT1) in the striatum were differentially expressed among control and heroin-treated animals. CONCLUSION: These results suggest that even a single heroin exposure can cause differential gene expression in the hippocampus and striatum of nonhuman primates at different ages.
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BACKGROUND AND AIM: Anti-tumor necrosis factor (TNF) agents, such as infliximab (IFX), have been increasingly used to induce and maintain disease remission in patients with Crohn's disease (CD). Despite a considerable non-response rate, little is known about the genetic predictors of response to anti-TNF therapy in CD. Our aim in this study was to investigate the genetic factors associated with response to anti-TNF therapy in patients with CD. METHODS: We performed a two-stage genome-wide association study (GWAS) to identify loci influencing the response to IFX among Korean patients with CD, comprising 42 good responders with mucosal healing and 70 non-responders. The achievement of mucosal healing was assessed by endoscopy and imaging. The functional significance of TRAP1 (TNF receptor associated protein 1) was examined using dextran sodium sulfate-induced colitis model in TRAP1 transgenic mice. RESULTS: The GWAS identified rs2158962, an intronic single nucleotide polymorphism (SNP) of TRAP1, significantly associated with mucosal healing (odds ratio = 4.94; Pcombined = 1.35 × 10-7 ). In the dextran sodium sulfate-induced acute colitis, TRAP1 transgenic mice showed a better response to IFX than the wild-type mice. CONCLUSIONS: The TRAP1 gene is associated with mucosal healing in CD patients following IFX therapy. Identifying the genetic predictors of mucosal healing to anti-TNF therapy can prevent patients from exposure to ineffective therapies.
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Enfermedad de Crohn/tratamiento farmacológico , Proteínas HSP90 de Choque Térmico/fisiología , Infliximab/uso terapéutico , Mucosa Intestinal/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Adolescente , Adulto , Animales , Enfermedad de Crohn/genética , Enfermedad de Crohn/fisiopatología , Femenino , Fármacos Gastrointestinales/uso terapéutico , Regulación de la Expresión Génica/fisiología , Estudio de Asociación del Genoma Completo , Genotipo , Proteínas HSP90 de Choque Térmico/genética , Humanos , Mucosa Intestinal/fisiología , Masculino , Ratones Transgénicos , Fenotipo , Polimorfismo de Nucleótido Simple , ARN Mensajero/genética , Sistema de Registros , Cicatrización de Heridas/genética , Adulto JovenRESUMEN
The induction of senescence in cancer cells has recently been implicated as a mechanism of tumor regression in response to various modes of stress. 14-3-3 proteins are conserved scaffolding molecules that are involved in various cellular functions. Among the seven isoforms, 14-3-3ß is specifically expressed in astrocytoma in correlation with the malignancy grade. We investigated the possible role of 14-3-3ß in the regulation of senescence induction in A172 glioblastoma cells. The knockdown of 14-3-3ß by specific small interfering RNA resulted in a significant change in cellular phenotypes and an increase in cells staining positive for senescence-associated ß-galactosidase. Western blotting of the 14-3-3ß-depleted A172 cells revealed increased p27 expression and decreased SKP2 expression, while the expression of p53 and p21 was not altered. Subsequently, we demonstrated that ERK is a key modulator of SKP2/p27 axis activity in 14-3-3ß-mediated senescence based on the following: (1) 14-3-3ß knockdown decreased p-ERK levels; (2) treatment with U0126, an MEK inhibitor, completely reproduced the senescence morphology as well as the expression profiles of p27 and SKP2; and (3) the senescence phenotypes induced by 14-3-3ß depletion were considerably recovered by constitutively active ERK expression. Our results indicate that 14-3-3ß negatively regulates senescence in glioblastoma cells via the ERK/SKP2/p27 pathway. Furthermore, 14-3-3ß depletion also resulted in senescence phenotypes in U87 glioblastoma cells, suggesting that 14-3-3ß could be targeted to induce premature senescence as a therapeutic strategy against glioblastoma progression.
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Proteínas 14-3-3/metabolismo , Senescencia Celular/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Proteínas 14-3-3/genética , Ciclo Celular/genética , Línea Celular Tumoral , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
PPARγ is a nuclear receptor that regulates differentiation and proliferation and is highly expressed in many cancer cells. Its synthetic ligands, such as rosiglitazone and ciglitazone, and its inhibitor GW9662, were shown to induce cellular differentiation, inhibit proliferation, and lead to apoptosis. Glioblastoma is a common brain tumor with poor survival prospects. Recently, glioblastoma stem cells (GSCs) have been examined as a potential target for anticancer therapy; however, little is known about the combined effect of various agents on GSCs. In this study, we found that cotreatment with PPARγ ligands and GW9662 inhibited stem-like properties in GSC-like spheres, which significantly express SOX2. In addition, this treatment decreased the activation of STAT3 and AKT and decreased the amounts of 14-3-3 gamma and BIS proteins. Moreover, combined administration of small-interfering RNA (siRNA) transfection with PPARγ ligands induced downregulation of SOX2 and MMP2 activity together with inhibition of sphere-forming activity regardless of poly(ADP-ribose) polymerase (PARP) cleavage. Taken together, our findings suggest that a combination therapy using PPARγ ligands and its inhibitor could be a potential therapeutic strategy targeting GSCs.
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Glioblastoma/genética , Metaloproteinasa 2 de la Matriz/genética , Células Madre Neoplásicas/efectos de los fármacos , PPAR gamma/genética , Factores de Transcripción SOXB1/genética , Proteínas 14-3-3 , Proteínas Adaptadoras Transductoras de Señales/genética , Anilidas/administración & dosificación , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Humanos , Ligandos , Células Madre Neoplásicas/patología , Transducción de Señal/efectos de los fármacosRESUMEN
Airway epithelial cells are often infected by respiratory syncytial virus (RSV), one of the most common causes of asthma, bronchiolitis, chronic obstructive pulmonary disease, and pneumonia. During the infection process, excessive mucins instigate airway inflammation. However, the mechanism underlying RSV-induced airway hyper-responsiveness and inflammation is poorly understood. Furthermore, no reliable vaccines or drugs for antiviral therapy are available. In this study, the effect of the natural compound grape seed proanthocyanidin (GSP) on RSV-infected human airway epithelial cells A549 was evaluated. After pretreatment of the cells with or without exposure to RSV with 5-10 µg GSP/mL, the expression of various mucins (MUC1, MUC2, MUC5AC, MUC5B, and MUC8) was evaluated by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting, as well as confocal microscopy. We found that GSP significantly decreased RSV-induced mucin synthesis at the mRNA and protein levels. In addition, GSP suppressed the RSV-induced signaling pathways, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, together with nuclear factor kappa B (NF-κB) and activating protein-1 family members (c-Jun and c-Fos). Concomitantly, GSP inhibited the replication of RSV within A549 cells. Taken together, all our results suggest that GSP could be a potent therapeutic agent to suppress excessive mucus production and viral replication in RSV-induced airway inflammatory disorders.
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Extracto de Semillas de Uva/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mucinas/biosíntesis , Proantocianidinas/farmacología , Infecciones por Virus Sincitial Respiratorio/metabolismo , Virus Sincitiales Respiratorios/efectos de los fármacos , Virus Sincitiales Respiratorios/fisiología , Células A549 , Humanos , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Infecciones por Virus Sincitial Respiratorio/genética , Infecciones por Virus Sincitial Respiratorio/virología , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Replicación Viral , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Heat shock factor 1 (HSF1), a transcription factor activated by various stressors, regulates proliferation and apoptosis by inducing expression of target genes, such as heat shock proteins and Bcl-2 (B-cell lymphoma 2) interacting cell death suppressor (BIS). HSF1 also directly interacts with BIS, although it is still unclear whether this interaction is critical in the regulation of glioblastoma stem cells (GSCs). In this study, we examined whether small interfering RNA-mediated BIS knockdown decreased protein levels of HSF1 and subsequent nuclear localization under GSC-like sphere (SP)-forming conditions. Consistent with BIS depletion, HSF1 knockdown also reduced sex determining region Y (SRY)-box 2 (SOX2) expression, a marker of stemness, accompanying the decrease in SP-forming ability and matrix metalloprotease 2 (MMP2) activity. When HSF1 or BIS knockdown was combined with temozolomide (TMZ) treatment, a standard drug used in glioblastoma therapy, apoptosis increased, as measured by an increase in poly (ADP-ribose) polymerase (PARP) cleavage, whereas cancer stem-like properties, such as colony-forming activity and SOX2 protein expression, decreased. Taken together, our findings suggest that targeting BIS or HSF1 could be a viable therapeutic strategy for GSCs resistant to conventional TMZ treatment.
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Antineoplásicos Alquilantes/farmacología , Proteínas de Unión al ADN/genética , Dacarbazina/análogos & derivados , Resistencia a Antineoplásicos/genética , Glioblastoma/genética , Glioblastoma/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Dacarbazina/farmacología , Expresión Génica , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Glioblastoma/metabolismo , Factores de Transcripción del Choque Térmico , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Temozolomida , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
B-cell lymphoma (BCL)-2-interacting cell death suppressor (BIS) has diverse cellular functions depending on its binding partners. However, little is known about the effects of biochemical modification of BIS on its various activities under oxidative stress conditions. In this study, we showed that H2O2 reduced BIS mobility on SDS-polyacrylamide gels in a time-dependent manner via the activation of extracellular signaling-regulated kinase (ERK). The combined results of mass spectroscopy and computational prediction identified Thr285 and Ser289 in BIS as candidate residues for phosphorylation by ERK under oxidative stress conditions. Deletion of these sites resulted in a partial reduction in the H2O2-induced mobility shift relative to that of the wild-type BIS protein; overexpression of the deletion mutant sensitized A172 cells to H2O2-induced cell death without increasing the level of intracellular reactive oxygen species. Expression of the BIS deletion mutant decreased the level of heat shock protein (HSP) 70 mRNA following H2O2 treatment, which was accompanied by impaired nuclear translocation of heat shock transcription factor (HSF) 1. Co-immunoprecipitation assays revealed that the binding of wild-type BIS to HSF1 was decreased by oxidative stress, while the binding of the BIS deletion mutant to HSF1 was not affected. These results indicate that ERK-dependent phosphorylation of BIS has a role in the regulation of nuclear translocation of HSF1 likely through modulation of its interaction affinity with HSF1, which affects HSP70 expression and sensitivity to oxidative stress.
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Glioblastoma stem cells (GSCs) are a subpopulation of highly tumorigenic and stem-like cells that are responsible for resistance to conventional therapy. Bcl-2-intreacting cell death suppressor (BIS; also known as BAG3) is an anti-apoptotic protein that is highly expressed in human cancers with various origins, including glioblastoma. In the present study, to investigate the role of BIS in GSC subpopulation, we examined the expression profile of BIS in A172 and U87-MG glioblastoma cell lines under specific in vitro culture conditions that enrich GSC-like cells in spheres. Both BIS mRNA and protein levels significantly increased under the sphere-forming condition as compared with standard culture conditions. BIS depletion resulted in notable decreases in sphere-forming activity and was accompanied with decreases in SOX-2 expression. The expression of STAT3, a master regulator of stemness, also decreased following BIS depletion concomitant with decreases in the nuclear levels of active phosphorylated STAT3, while ectopic STAT3 overexpression resulted in recovery of sphere-forming activity in BIS-knockdown glioblastoma cells. Additionally, immunoprecipitation and confocal microscopy revealed that BIS physically interacts with STAT3. Furthermore, BIS depletion increased STAT3 ubiquitination, suggesting that BIS is necessary for STAT3 stabilization in GSC-like cells. BIS depletion also affected epithelial-to-mesenchymal transition-related genes as evidenced by decrease in SNAIL and MMP-2 expression and increase in E-cadherin expression in GSC-like cells. Our findings suggest that high levels of BIS expression might confer stem-cell-like properties on cancer cells through STAT3 stabilization, indicating that BIS is a potential target in cancer therapy.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias Encefálicas/patología , Glioblastoma/patología , Células Madre Neoplásicas/metabolismo , Factor de Transcripción STAT3/metabolismo , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/fisiología , Glioblastoma/metabolismo , Humanos , Células Madre Neoplásicas/patologíaRESUMEN
Tumor necrosis factor receptor-associated protein 1 (TRAP1), a member of the HSP90 family, controls a variety of physiological functions, including cell proliferation, differentiation, and survival. Most studies have been devoted to understanding the anti-apoptotic roles of TRAP1 in cancer and targeting it for tumor control in clinical settings. Additionally, we have identified a new role for TRAP1 in regulation of liver regeneration after partial hepatectomy in TRAP1 transgenic mice and cellular proliferation in TRAP1-overexpressing cells, via mitochondrial alterations. Moreover, recent works have indicated a role for TRAP1 in the regulation of cancer stem cells (CSCs) as well as a metabolic switch between mitochondrial respiration and aerobic glycolysis called as "Warburg effect." This review discusses the implications of TRAP1 action for both metabolism and the regulation of CSCs.
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Proteínas HSP90 de Choque Térmico/metabolismo , Mitocondrias/metabolismo , Células Madre Neoplásicas/metabolismo , Animales , Apoptosis/fisiología , Proliferación Celular , Senescencia Celular/fisiología , Glucólisis/fisiología , Humanos , Regeneración Hepática/fisiología , RatonesRESUMEN
Glioblastoma multiforme (GBM), also known as glioblastoma, is the most common and aggressive brain tumor. GBM has a poor survival rate and high resistance to standard therapy, leading to recurrence and metastasis to adjacent normal regions. Glioblastoma stem cells (GSCs) are regarded as an emerging target for therapy of GBM. Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor that functions in a variety of cancers and in normal adipocyte differentiation. The newly discovered connection between PPARγ ligands and cancer stem cells (CSCs) raises important implications for the potential therapeutic use of synthetic PPARγ ligands, such as thiazolidinediones (TZDs), in glioblastoma. Here, I hypothesize that synthetic PPARγ ligands serve to modulate stemness-related molecules and several signaling pathway in GSCs and I propose potential experimental approaches to investigate the effects of these ligands on GSCs in vitro and in vivo.
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Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Tiazolidinedionas/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ligandos , PPAR gamma/agonistas , PPAR gamma/metabolismo , Transducción de Señal , Células Tumorales CultivadasRESUMEN
More efficient isolation and identification of cancer stem cells (CSCs) would help in determining their fundamental roles in tumor biology. The classical tool for this purpose is anchorage-independent tumorsphere culture. We compared the effects of differently textured culture plates and serum deprivation on the acquisition of CSC properties of A172 glioblastoma cells. Cells were cultured on standard polystyrene-treated plates, ultra-low attachment, poly (2-hydroxyethyl methacrylate)-coated plates, and 1% agar-coated plates with 10% serum or in serum-free glioblastoma sphere medium (GBM). Based on mitochondrial reductase activity and subG1 proportions, non-adherent conditions had a greater impact on A172 cell viability than serum deprivation. Among the stemness-related genes, SOX-2 expression was significantly upregulated by serum deprivation under non-adherent conditions, while several epithelial-to-mesenchymal transition (EMT)-related genes were less dependent on serum. In addition, reactive oxygen species (ROS) accumulation in A172 cells was significantly increased in GBM under non-adherent conditions. Despite the correlation between SOX-2 induction and ROS accumulation, treatment with the ROS scavenger N-acetyl-l-cysteine did not prevent SOX-2 expression, suggesting that ROS accumulation is not an essential requirement for induction of SOX-2. Our results suggested that cultivation of cancer cells under conditions of serum deprivation in an anchorage-independent manner may enrich SOX-2-expressing CSC-like cells in vitro.
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Técnicas de Cultivo de Célula/instrumentación , Glioblastoma/patología , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción SOXB1/genética , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Supervivencia Celular , Medio de Cultivo Libre de Suero/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Células Madre Neoplásicas/patologíaRESUMEN
The Bis protein is known to be involved in a variety of cellular processes including apoptosis, migration, autophagy as well as protein quality control. Bis expression is induced in response to a number of types of stress, such as heat shock or a proteasome inhibitor via the activation of heat shock factor (HSF)1. We report herein that Bis expression is increased at the transcriptional level in HK-2 kidney tubular cells and A172 glioma cells by exposure to oxidative stress such as H2O2 treatment and oxygen-glucose deprivation, respectively. The pretreatment of HK-2 cells with N-acetyl cysteine, suppressed Bis induction. Furthermore, HSF1 silencing attenuated Bis expression that was induced by H2O2, accompaniedby increase in reactive oxygen species (ROS) accumulation. Using a series of deletion constructs of the bis gene promoter, two putative heat shock elements located in the proximal region of the bis gene promoter were found to be essential for the constitutive expression is as well as the inducible expression of Bis. Taken together, our results indicate that oxidative stress induces Bis expression at the transcriptional levels via activation of HSF1, which might confer an expansion of antioxidant capacity against pro-oxidant milieu. However, the possible role of the other cis-element in the induction of Bis remains to be determined.
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Cancer cells undergo uncontrolled proliferation, and aberrant mitochondrial alterations. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial heat shock protein. TRAP1 mRNA is highly expressed in some cancer cell lines and tumor tissues. However, the effects of its overexpression on mitochondria are unclear. In this study, we assessed mitochondrial changes accompanying TRAP1 overexpression, in a mouse cell line, NIH/3T3. We found that overexpression of TRAP1 leads to a series of mitochondrial aberrations, including increase in basal ROS levels, and decrease in mitochondrial biogenesis, together with a decrease in peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) mRNA levels. We also observed increased extracellular signal-regulated kinase (ERK) phosphorylation, and enhanced proliferation of TRAP1 overexpressing cells. This study suggests that overexpression of TRAP1 might be a critical link between mitochondrial disturbances and carcinogenesis.
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Péptidos y Proteínas de Señalización Intracelular/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Células 3T3 NIH , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
PURPOSE: To investigate possible radiosensitizing activities of the well-known peroxisome proliferator-activated receptor (PPAR)γ ligand ciglitazone and novel PPARγ ligands CAY10415 and CAY10506 in non-small cell lung cancer (NSCLC) cells. METHODS AND MATERIALS: Radiosensitivity was assessed using a clonogenic cell survival assay. To investigate the mechanism underlying PPARγ ligand-induced radiosensitization, the subdiploid cellular DNA fraction was analyzed by flow cytometry. Activation of the caspase pathway by combined PPARγ ligands and γ-radiation treatment was detected by immunoblot analysis. Reactive oxygen species (ROS) were measured using 2,7-dichlorodihydrofluorescein diacetate and flow cytometry. RESULTS: The 3 PPARγ ligands induced cell death and ROS generation in a PPARγ-independent manner, enhanced γ-radiation-induced apoptosis and caspase-3-mediated poly (ADP-ribose) polymerase (PARP) cleavage in vitro. The combined PPARγ ligand/γ-radiation treatment triggered caspase-8 activation, and this initiator caspase played an important role in the combination-induced apoptosis. Peroxisome proliferator-activated receptor-γ ligands may enhance the γ-radiation-induced DNA damage response, possibly by increasing γ-H2AX expression. Moreover, the combination treatment significantly increased ROS generation, and the ROS scavenger N-acetylcysteine inhibited the combined treatment-induced ROS generation and apoptotic cell death. CONCLUSIONS: Taken together, these results indicated that the combined treatment of PPARγ ligands and γ-radiation synergistically induced DNA damage and apoptosis, which was regulated by ROS.
Asunto(s)
Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/terapia , Daño del ADN , Neoplasias Pulmonares/terapia , PPAR gamma/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Tiazolidinedionas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Fluoresceínas , Rayos gamma/uso terapéutico , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Receptores Activados del Proliferador del Peroxisoma , Poli(ADP-Ribosa) Polimerasas/metabolismo , Tolerancia a Radiación/fisiología , Ensayo de Tumor de Célula Madre/métodosRESUMEN
A radioresistant cell line was established by fractionated ionizing radiation (IR) and assessed by a clonogenic assay, flow cytometry, and Western blot analysis, as well as zymography and a wound healing assay. Microarray was performed to profile global expression and to search for differentially expressed genes (DEGs) in response to IR. H460R cells demonstrated increased cell scattering and acidic vesicular organelles compared with parental cells. Concomitantly, H460R cells showed characteristics of increased migration and matrix metalloproteinase activity. In addition, H460R cells were resistant to IR, exhibiting reduced expression levels of ionizing responsive proteins (p-p53 and γ-H2AX); apoptosis-related molecules, such as cleaved poly(ADP ribose) polymerase; and endoplasmic reticulum stress-related molecules, such as glucose-regulated protein (GRP78) and C/EBP-homologous protein compared with parental cells, whereas the expression of anti-apoptotic X-linked inhibitor of apoptosis protein was increased. Among DEGs, syntrophin beta 2 (SNTB2) significantly increased in H460R cells in response to IR. Knockdown of SNTB2 by siRNA was more sensitive than the control after IR exposure in H460, H460R, and H1299 cells. Our study suggests that H460R cells have differential properties, including cell morphology, potential for metastasis, and resistance to IR, compared with parental cells. In addition, SNTB2 may play an important role in radioresistance. H460R cells could be helpful in in vitro systems for elucidating the molecular mechanisms of and discovering drugs to overcome radioresistance in lung cancer therapy.
Asunto(s)
Células Madre Embrionarias/citología , Colorantes Fluorescentes/análisis , Células Madre Pluripotentes Inducidas/citología , Animales , Línea Celular , Línea Celular Tumoral , Fibroblastos/citología , Proteínas Fluorescentes Verdes/genética , Humanos , Ratones , Ratones Transgénicos , Factor 3 de Transcripción de Unión a Octámeros/genéticaRESUMEN
Novel sucrose-based G7 molecular transporters show different patterns of intracellular localization depending on the nature of the linker chains as well as the fluorescent dyes.