RESUMEN
BACKGROUND: HER2-targeted therapies have improved the outcomes of HER2-positive gastric cancer (GC), yet resistance remains a challenge. We sought to explore the effects of reversible and irreversible HER2 tyrosine kinase inhibitors (TKIs) alone or in combination with the HER2-targeting antibody drug conjugate trastuzumab deruxtecan (T-Dxd). METHODS: The effects of HER2-TKIs on HER2 and downstream signaling were evaluated via Western blotting. Proteasomal inhibitors and co-immunoprecipitation assays were performed to explore the role of proteasomal degradation in HER2 expression modulation, and immunofluorescence assays were employed to explore mechanisms of HER2 internalization. The synergistic potential of the irreversible HER2-TKI pyrotinib in combination with T-Dxd was validated using growth and viability assays in anti-HER2-positive GC cell cultures and tumor growth and immunohistochemical staining assays in a mouse xenograft model. RESULTS: Our study revealed that reversible HER2-TKIs elevated HER2 protein levels, whereas irreversible HER2-TKIs decreased them. Pyrotinib triggered HER2 degradation within the proteasome by promoting ubiquitination and dissociation from HSP90. Furthermore, pyrotinib substantially induced HER2 internalization, which led to improved cellular uptake of T-Dxd. The increased T-Dxd uptake was accompanied by greater efficacy in suppressing the growth of GC cells and enhanced anti-tumor effects in an animal model. CONCLUSION: In summary, our research reveals the molecular mechanisms of irreversible HER2-TKIs in regulating HER2 protein expression by promoting HER2 internalization. These findings advance our comprehension of targeted therapy for GC and provide a promising therapeutic combination strategy with enhanced efficacy against HER2-positive GC.
Asunto(s)
Acrilamidas , Aminoquinolinas , Camptotecina/análogos & derivados , Inmunoconjugados , Neoplasias Gástricas , Humanos , Animales , Ratones , Neoplasias Gástricas/patología , Receptor ErbB-2/metabolismo , Línea Celular Tumoral , Trastuzumab/uso terapéuticoRESUMEN
Pan-HER TKIs (pyrotinib, lapatinib) are potent HER2 inhibitors, however, their anti-tumor efficacy on esophageal cancer remains to be elucidated. Using two HER2-positive esophageal cancer cell lines, we observed that both pyrotinib and lapatinib could significantly suppress the activation of HER2 and its downstream signaling. However, pyrotinib showed a potent inhibitory effect at 0.1 µM treatment relative to 1 µM of lapatinib. Moreover, treatment with pyrotinibm, but not lapatinib, markedly reduced the protein level of HER2 through enhancing HER2 ubiquitination level and proteasomal degradation. In vitro and in vivo experiments further revealed that pyrotinib effectively suppresses cancer cell invasion and migration, as well as the growth of tumors in nude mice. Overall, our results suggest that pyrotinib is a superior TKI over lapatinib in inhibiting esophageal cancer cell proliferation and tumorigenic potential, and can be chosen as a neo-adjuvant for esophageal cancer treatment.
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Acrilamidas , Aminoquinolinas , Neoplasias Esofágicas , Receptor ErbB-2 , Animales , Ratones , Lapatinib/farmacología , Receptor ErbB-2/metabolismo , Ratones Desnudos , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/metabolismoRESUMEN
Circularly permuted TRAIL (CPT), a novel recombinant TRAIL mutant, is a potent antitumor agent. However, its efficacy in triple-negative breast cancer (TNBC) remains unclear. Treatment with CPT alone and in combination with doxorubicin (Dox) is explored for its effects on the proliferation and apoptosis of MDA-MB-231 (MB231) and MDA-MB-436 (MB436) breast cancer cells in vitro and in vivo. Here, we show that CPT combined with Dox exhibits time- and dose-dependent synergy to inhibit cell viability and enhance apoptosis of MB231 and MB436 cells. Combined treatment substantially increases caspase-8, caspase-3, and PARP cleavage in both cell lines and significantly suppresses tumor growth in nude mice bearing MB231 xenografts. Collectively, our findings demonstrate that treatment with CPT in combination with Dox exerts synergistic antitumor effects through activation of the caspase cascade pathway, a mechanism that is partly dependent on the Dox-induced upregulation of death receptor 4 and death receptor 5. Therefore, CPT combined with Dox may be a feasible therapeutic strategy for the management of TNBC.
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Antineoplásicos , Neoplasias de la Mama , Neoplasias de la Mama Triple Negativas , Animales , Ratones , Humanos , Femenino , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Ratones Desnudos , Línea Celular Tumoral , Doxorrubicina/farmacología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis , Neoplasias de la Mama/tratamiento farmacológicoRESUMEN
Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), including gefitinib and erlotinib, have shown notable effects in lung adenocarcinoma patients harboring EGFR mutations, there are significant differences between individual patients in the degree of benefits provided by EGFR-TKIs. Some evidence supports a role for caveolin-1 (Cav-1) in modulating drug sensitivity. This study aimed to investigate whether Cav-1 plays an important role in sensitivity to EGFR-TKIs in lung adenocarcinoma cells. Downregulation of Cav-1 in PC-9 cells were performed to investigate changes in sensitivity to EGFR-TKIs in vitro and in vivo. Knockdown of Cav-1 dramatically enhanced sensitivity to EGFR-TKIs by down-regulating phosphorylation of EGFR. These results suggest that Cav-1 may be a predictor of the poor efficacy of EGFR-TKIs treatment in lung adenocarcinoma with EGFR mutations.
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Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Apoptosis/efectos de los fármacos , Caveolina 1/metabolismo , Receptores ErbB/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Adenocarcinoma/genética , Adenocarcinoma del Pulmón , Animales , Antineoplásicos/administración & dosificación , Caveolina 1/genética , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Resultado del TratamientoRESUMEN
BACKGROUND Circularly permuted tumor necrosis factor-related apoptosis-inducing ligand, a mutant form of tumor necrosis factor-related apoptosis-inducing ligand, is an effective antitumor cytokine. However, its antitumor effect in colorectal cancer is unclear. This study assessed the antitumor effect of circularly permuted tumor necrosis factor-related apoptosis-inducing ligand alone or with 5-fluorouracil in colorectal cancer cells in vitro and explored the underlying mechanisms. MATERIAL AND METHODS We used the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay to analyze cell proliferation inhibition. The apoptotic effects of circularly permuted tumor necrosis factor-related apoptosis-inducing ligand, 5-fluorouracil, or both in human colorectal cancer cells were evaluated using flow cytometry. Furthermore, the levels of apoptosis-related proteins were examined by Western blotting. RESULTS Compared to either agent alone, cotreatment with 5-fluorouracil and circularly permuted tumor necrosis factor-related apoptosis-inducing ligand showed obvious antitumor effects and induced significant apoptosis of colorectal cancer cells. 5-Fluorouracil enhanced circularly permuted tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by increasing death receptor 4 and 5 levels in HCT116 cells, but only of death receptor 4 in SW480 cells. Moreover, 5-fluorouracil plus circularly permuted tumor necrosis factor-related apoptosis-inducing ligand increased apoptosis-related protein levels such as cleaved caspase-3, caspase-8, and poly-ADP-ribose polymerase and downregulated that of the survival protein B-cell lymphoma-extra-large. Pretreatment with the pan-caspase inhibitor, z-VAD-FMK, attenuated the caspase-dependent apoptosis induced by circularly permuted tumor necrosis factor-related apoptosis-inducing ligand alone or combined with 5-fluorouracil. CONCLUSIONS Cotreatment with 5-fluorouracil and circularly permuted tumor necrosis factor-related apoptosis-inducing ligand showed enhanced antitumor effects on colorectal cancer cells.
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Neoplasias Colorrectales/tratamiento farmacológico , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Inhibidores de Caspasas , Caspasas/metabolismo , Línea Celular , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/patología , Neoplasias Colorrectales/metabolismo , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND Although downregulation of caveolin-1 (Cav-1), which is a key constituent of membrane caveolae and a regulator of cellular processes, is associated with colorectal cancer (CRC), its involvement in the disease progression is largely unknown. This study aimed to explore the role of Cav-1 in CRC and the associated mechanisms. MATERIAL AND METHODS Fresh tissues from patients with CRC and human CRC SW480 cells were used to evaluate Cav-1 and Ki-67 expression using immunohistochemistry and Western blotting. The MTS and Transwell assays were performed to determine the effects of Cav-1 overexpression via pcDNA3.1/Cav-1 plasmid on cell proliferation and metastasis. The effect of Cav-1 on the epidermal growth factor receptor (EGFR) activation was evaluated by Western blotting. The correlation of Cav-1 expression with clinicopathological factors was statistically analyzed. RESULTS Overexpression of Cav-1 significantly reduced proliferation, migration, and invasion of SW480 cancer cells in vitro. The EGF-induced phosphorylation of EGFR and activations of the RAF-MEK-ERK and PI3K-AKT pathways were adversely regulated by Cav-1 overexpression in vitro. In 76 cases of CRC patients with EGFR expression, a negative correlation was observed between the level of Cav-1 and tumor-node-metastasis stage, lymph node metastasis, and distant metastasis (All p<0.05). Finally, the expression level of Cav-1 was negatively correlated with that of Ki-67. CONCLUSIONS This report is the first to show that overexpression of Cav-1significantly inhibits the proliferation, migration, and invasion potential of SW480 cells, possibly through reducing EGF-induced EGFR activation. High Cav-1 expression level may be a predictor of positive outcomes in patients with colorectal cancer.
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Caveolina 1/genética , Neoplasias Colorrectales/metabolismo , Receptores ErbB/genética , Caveolina 1/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación hacia Abajo , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Antígeno Ki-67/metabolismo , Metástasis Linfática , Masculino , Fosforilación/genética , Transducción de Señal/genéticaRESUMEN
Filamin A (FLNa) is a ubiquitously expressed cytoplasmic protein, which composes of an N-terminal actin binding domain (ABD) followed by 24 Ig-like repeats. FLNa functions as a cytoskeletal protein that links transmembrane receptors, including integrins, to F-actin and serves as a signaling intermediate. Recent studies have identified FLNa as a scaffold protein that interacts with over 90 proteins and plays vital roles in cellular signaling transduction. Mutations or defects in human FLNa gene have been shown to cause numerous developmental defects. Moreover, aberrant expression of FLNa has been observed in many cancers, such as parathyroid tumor, cervical cancer, and breast cancer. However, its role in lung adenocarcinoma has seldom been discussed. In the present study, our in vitro and in vivo studies demonstrated that silencing FLNa expression in lung cancer cell line A549 cells promoted proliferation, migration, and invasiveness of A549 cells by enhancing the activation of epidermal growth factor receptor and ERK signaling pathway. These results shed light on novel functions of FLNa in lung cancer and uncovered novel mechanisms, these results provided possible targets for the prediction and treatment for lung adenocarcinoma.
Asunto(s)
Proliferación Celular/genética , Receptores ErbB/genética , Filaminas/genética , Regulación Neoplásica de la Expresión Génica , Interferencia de ARN , Células A549 , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Movimiento Celular/genética , Receptores ErbB/metabolismo , Filaminas/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Sistema de Señalización de MAP Quinasas/genética , Metástasis de la NeoplasiaRESUMEN
Pyrrolidine dithiocarbamate (PDTC) can lower the blood glucose level and improve the insulin sensitivity in diabetic rats. However, the mechanisms underlying this effect of PDTC treatment in diabetic rats remained uncertain. In this study, we evaluated the mechanisms by which PDTC conferred protection against oxidative damage to pancreatic islet ß-cells in rats with experimental type 2 diabetes mellitus (DM). DM in the rats was elicited by long-term high-fat diet accompanied with a single intraperitoneal (i.p.) injection of a low dose of streptozotocin. After a 7-day administration of PDTC (50 mg/kg/day i.p.), blood glucose levels were measured and pancreatic tissues were collected for the determination of various biochemical and enzymatic activities using immunohistochemistry, immunofluorescence, and western blot techniques. The percentage of apoptotic pancreatic islet ß-cells was detected by flow cytometry. The results showed that diabetic rats had elevated blood glucose levels and insulin resistance, accompanied with an increase in malondialdehyde content, nitrotyrosine production, and inducible nitric oxide synthase expression. A decrease in superoxide dismutase and glutathione peroxidase activities was also observed in DM rats, culminating with elevated ß-cell apoptosis. PDTC treatment significantly reduced the oxidative damage and the ß-cell apoptosis, and also increased the insulin production through down-regulating FoxO1 acetylation and up-regulating nuclear PDX-1 level. These data suggested that PDTC can protect islet ß-cells from oxidative damage and improve insulin production through regulation of PDX-1 and FoxO1 in a DM rat model.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Factores de Transcripción Forkhead/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Estrés Oxidativo/efectos de los fármacos , Pirrolidinas/farmacología , Tiocarbamatos/farmacología , Animales , Antioxidantes/farmacología , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/patología , Glutatión Peroxidasa/metabolismo , Insulina/biosíntesis , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Masculino , Malondialdehído/metabolismo , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismoRESUMEN
The aim of the present study was to examine the effects of pyrrolidine dithiocarbamate (PDTC) on hepatic glycogen synthesis and FoxO1 transcriptional activity in type 2 diabetic rats and the mechanism underlying these effects. Fasting blood glucose and glycogen deposition, together with expressions of two key genes related to gluconeogenesis, were studied in the liver of rats fed a normal diet (NC), high-fat diet (HFD)-induced insulin-resistant rats made type 2 diabetic by a single intraperitoneal injection of streptozotocin (DM), and a DM with intervention of PDTC (DM + PDTC) for 1 wk. The phosphorylation of Akt, GSK-3ß, and FoxO1 was assessed in liver extracts of fasted rats by Western blot, whereas indirect immunofluorescence staining was performed to determine the cellular distribution of FoxO1. The DM rats exhibited obvious increases in fasting blood glucose as well as decreased hepatic glycogen content compared with the NC group. Activation of the Akt/GSK-3ß pathway and inactivating phosphorylation of FoxO1 were reduced greatly in DM rat livers (P < 0.01). By contrast, PDTC treatment protected DM rats against high fasting blood glucose and hepatic glycogen deposition loss. PDTC also elicited an increase in Akt/GSK-3ß signaling and subsequent inactivation and nuclear export of FoxO1 in DM rat livers, which translated into a significant reduction in the expression of two FoxO1 target genes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. This study suggests that PDTC enhances hepatic glycogen synthesis, whereas it reduces FoxO1 transcriptional activity in DM rats.
Asunto(s)
Antioxidantes/farmacología , Diabetes Mellitus Tipo 2/metabolismo , Factores de Transcripción Forkhead/metabolismo , Glucógeno Hepático/biosíntesis , Hígado/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Pirrolidinas/farmacología , Tiocarbamatos/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Glucemia/efectos de los fármacos , Dieta Alta en Grasa , Ayuno/sangre , Gluconeogénesis/efectos de los fármacos , Glucosa-6-Fosfatasa/biosíntesis , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Resistencia a la Insulina , Hígado/química , Hígado/metabolismo , Masculino , Fosfoenolpiruvato Carboxiquinasa (ATP)/biosíntesis , Fosforilación , Ratas , Ratas WistarRESUMEN
Raf kinase inhibitory protein (RKIP) gene is considered to be a suppressor of metastasis involved in various carcinomas. In the present study, we observed that promoter methylation repressed the expression of RKIP in TE-13 cell line. 5-Aza treatment and stable transfection of RKIP resulted in a significant inhibition of TE-13 cell proliferation. The promoter hypermethylation of RKIP was found to occur in dysplastic tissues and a close correlation was noted between RKIP methylation and the loss of mRNA and protein expression of the gene in ESCC specimens. In summary, RKIP may act as a tumor suppressor gene in esophageal cancer.
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Carcinoma de Células Escamosas/genética , Metilación de ADN/genética , Neoplasias Esofágicas/genética , Genes Supresores de Tumor/fisiología , Proteínas de Unión a Fosfatidiletanolamina/genética , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Humanos , Inmunohistoquímica , Proteínas de Unión a Fosfatidiletanolamina/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Following the publication of this article, an interested reader drew to the authors' attention that various panels in the scratch-wound assays shown in Fig. 1C appeared to contain overlapping sections, such that the data may have been derived from a more limited selection of original sources where the data were intended to show the data from discrete experiments. Furthermore, the results shown in the Kaplan-Meier overall survival analysis plots in Fig. 4C appeared to be inconsistent with the date of publication of this article. Independently, the Editorial Office also investigated the issues of concern in this article, and although the authors attenpted to explain these issues and requested the publication of a corrigendum, the Editor of Molecular Medicine Reports has declined this request and determined that this article should be retracted from the journal on the basis of an overall lack of confidence in the presented data. Upon receiving this decision from the Editor, the authors were not in agreement that the article should be retracted. The Editor apologizes to the readership of the Journal for any inconvenience caused. [Molecular Medicine Reports 20: 3671-3678, 2019; DOI: 10.3892/mmr.2019.10622].
RESUMEN
The metastasis and recurrence rate, and the overall prognosis of colorectal cancer (CRC) remain unsatisfactory. Filamin A (FLNa), as an actinbinding protein, can interact with various signaling molecules and membrane receptors to affect cell signal transduction and function. However, whether FLNa is involved in the progression of CRC remains to be elucidated. The aim of the present study was to explore the role of FLNa in CRC cell proliferation and migration, as well as in the regulation of epidermal growth factor receptor (EGFR) signaling. Following transfection with a FLNatargeting short hairpin RNA plasmid to knockdown expression of FLNa in the EGFtreated SW480 cell line, it was found that decreased expression of FLNa promoted cell proliferation and migration. Additionally, there was a negative correlation between FLNa levels and the activation of EGFR and Akt signaling pathways. Similarly, the expression of FLNa was significantly lower in human CRC tissues compared with adjacent normal tissues and FLNa expression was negatively correlated with the expression of Ki67 in human CRC tissues. Although there was no significant difference in the KaplanMeier estimate of CRC between high expression and low expression of FLNa, there were significant negative associations between FLNa expression and TNM stage. The results suggested that FLNa may participate in EGFinduced cell proliferation and migration in CRC cells. Hence, interventions in the FLNamediated signaling pathway could provide attractive therapeutic targets for CRC.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Receptores ErbB/metabolismo , Filaminas/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Adulto , Anciano , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales/patología , Femenino , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana EdadRESUMEN
Our previous study showed that aspirin induced apoptosis of esophageal cancer cells in vitro by inhibiting the pathway of NF-kappaB downstream regulation of cyclooxygenase-2. The purpose of this study was to determine if similar changes occurred in vivo in the tumors of patients with SCC of the esophagus who were given a preferential COX-2 inhibitor, meloxicam. Fifty-three patients who had an esophagectomy for SCC were allocated randomly to either a Treatment group (n = 25) or a control group (n = 28). Patients in the Treatment group were given 7.5 mg/day of meloxicam, for between 10 and 14 days before surgery. Patients in the control group did not take any type of NSAID during this time interval. Samples of the tumor taken from the resected specimens were collected. Proliferation and apoptosis were measured by flow cytometry. The concentration of 6-keto-prostaglandin F(1)alpha in cancer tissue was determined by radio-immuno-assay. Expression of COX-2 mRNA was measured with RT-PCR and COX-2 protein levels with Western blot analysis. Nuclear NF-kappaB and cytoplasmic I kappaB protein levels were determined by electrophoretic mobility shift assay and Western blot, respectively. There were significantly more apoptotic cells in the tumors of patients who were using meloxicam. It also decreased the levels of COX-2 mRNA, COX-2 protein and nuclear NF-kappaB protein and increased the cytoplasmic I kappaB protein in the cancer. We conclude that meloxicam induces apoptosis in SCC of the esophagus in vivo by inhibiting the pathway of NF-kappaB downstream regulation of COX-2.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa 2/uso terapéutico , Ciclooxigenasa 2/metabolismo , Neoplasias Esofágicas/tratamiento farmacológico , Tiazinas/uso terapéutico , Tiazoles/uso terapéutico , 6-Cetoprostaglandina F1 alfa/metabolismo , Adulto , Anciano , Apoptosis/efectos de los fármacos , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/cirugía , Proliferación Celular/efectos de los fármacos , Ensayo de Cambio de Movilidad Electroforética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/cirugía , Esofagectomía , Femenino , Citometría de Flujo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas I-kappa B/metabolismo , Masculino , Meloxicam , Persona de Mediana Edad , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Radioinmunoensayo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
The DNA binding protein nuclear factor kappaB (NF-kappaB) and the cellular signaling pathways in which it participates are the central coordinators of many biological processes, including innate and adaptive immune responses, oxidative stress response, and aging. NF-kappaB also plays a key role in diseases, for example, cancer A simple, convenient, and high-throughput detection of NF-kappaB activation is therefore important for systematically studying signaling pathways and for screening therapeutic drug targets. We describe a method based on fluorescence resonance energy transfer (FRET) to directly measure the amount of activated NF-kappaB. More specifically, a double-stranded DNA (dsDNA) probe was designed to contain a pair of FRET fluorophores at the same end of the probe and an endonuclease binding site within the NF-kappaB consensus sequence. The activated NF-kappaB was detected by FRET following the restriction enzyme digestion. Using three different analyte materials--(i) purified recombinant NF- kappaB p50, (ii) nuclear extracts, and (iii) whole cell lysates--we demonstrated that this assay is as sensitive as the traditional, widely used electrophoretic mobility shift assay (EMSA), but much less labor-intensive for measuring NF-kappaB DNA binding activities. In addition, this FRET-based assay can be easily adapted for high-throughput screening of NF-kappaB activation.
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ADN/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , FN-kappa B/metabolismo , Extractos Celulares , Ensayo de Cambio de Movilidad Electroforética , Endonucleasas/metabolismo , Células HeLa , Humanos , FN-kappa B/análisis , Unión Proteica , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND AND AIMS: Caveolin-1 (CAV1) is a multifunctional scaffolding protein and plays an important role in tumorigenesis. However, the epigenetic changes of CAV1 in gastric cardia adenocarcinoma (GCA) have not been investigated so far. The purpose of this study was to clarify the contribution of critical CpG sites in CAV1 to progression/prognosis of GCA and to further elucidate the effect of critical CpG sites on the ectopic expression of ß-catenin in GCA. METHODS: Methylation-specific polymerase chain reaction (MSP) and bisulfite genomic sequencing (BGS) methods were, respectively, applied to examine the methylation status of CAV1. RT-PCR and immunohistochemistry methods were used to determine the mRNA and protein expression of CAV1 and ß-catenin. RESULTS: Decreased mRNA and protein expression of CAV1 were observed in GCA tumor tissues and were associated with hypermethylation of CpG island shore and transcription start site (TSS) regions in CAV1. Hypermethylation of the other two regions within CpG islands in CAV1 was observed both in tumor and corresponding adjacent tissues but was not related to the transcriptional inhibition of CAV1. The methylation status of CpG island shore region in CAV1 was associated with the ectopic expression of ß-catenin and was independently associated with survival in GCA patients. CONCLUSIONS: Hypermethylation of CpG island shore and TSS regions is cancer specific and is closely associated with reduced expression of CAV1. The CpG island shore methylation of CAV1 may play an important role in progression of GCA and may serve as a prognostic methylation biomarker for GCA patients.
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Adenocarcinoma/metabolismo , Cardias/patología , Caveolina 1/metabolismo , Islas de CpG , Neoplasias Gástricas/metabolismo , Adenocarcinoma/diagnóstico , Adenocarcinoma/patología , Caveolina 1/genética , Metilación de ADN , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , ARN Mensajero/metabolismo , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/patología , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
T-cell immunoglobulin and mucin domain-con-taining protein-3 (TIM-3), a negative regulator of antitumor immune response, has been demonstrated to be involved in the onset and progression of several types of malignancies. The present study aimed to determine whether and how TIM3 plays such a role in esophageal squamous cell carcinoma (ESCC). TIM-3 expression was analyzed by immunohistochemistry and realtime fluorescence quantitative PCR (qRTPCR) in ESCC and matched adjacent normal tissues. Functional experiments in vitro were performed to elucidate the effect of TIM3 knockdown on the proliferation, apoptosis, migration, invasion and epithelial-mesenchymal transition (EMT) in Eca109 and TE1 cell lines. Our data revealed that TIM3 expression was significantly elevated at both the mRNA and protein levels in ESCC tissues compared with the levels in the matched adjacent normal tissues (both P<0.001). TIM3 expression was significantly associated with lymph node metastasis (P=0.008), tumornodemetastasis (TNM) stage (P=0.042) and depth of tumor invasion (P=0.042). In addition, we observed a strong correlation between high TIM3 expression and a worse overall survival of ESCC patients (P=0.001). Functional study demonstrated that TIM3 knockdown markedly inhibited proliferation, migration and invasion of ESCC cell lines without affecting apoptosis. In addition, TIM3 depletion was associated with downregulation of matrix metalloproteinase (MMP)-9 and upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, and with reversion of EMT, as reflected by higher levels of the epithelial marker Ecadherin and lower levels of the mesenchymal markers Ncadherin and vimentin. Further study found that TIM3 depletion suppressed the signaling pathway involving pAkt, pGSK3ß and Snail. Taken together, these results suggest that TIM3 is a novel therapeutic target and prognostic biomarker for ESCC and promotes metastasis of ESCC by inducing EMT via, at least partially, the Akt/GSK-3ß/Snail signaling pathway.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/patología , Transición Epitelial-Mesenquimal/fisiología , Neoplasias Esofágicas/patología , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Transducción de Señal/fisiología , Adulto , Anciano , Apoptosis/fisiología , Western Blotting , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción de la Familia Snail/metabolismoRESUMEN
Elevation of the DNA-unwinding helicase RECQL4, which participates in various DNA repair pathways, has been suggested to contribute to the pathogenicity of various human cancers, including gastric cancer. In this study, we addressed the prognostic and chemotherapeutic significance of RECQL4 in human gastric cancer, which has yet to be determined. We observed significant increases in RECQL4 mRNA or protein in >70% of three independent sets of human gastric cancer specimens examined, relative to normal gastric tissues. Strikingly, high RECQL4 expression in primary tumors correlated well with poor survival and gastric cancer lines with high RECQL4 expression displayed increased resistance to cisplatin treatment. Mechanistic investigations revealed a novel role for RECQL4 in transcriptional regulation of the multidrug resistance gene MDR1, through a physical interaction with the transcription factor YB1. Notably, ectopic expression of RECQL4 in cisplatin-sensitive gastric cancer cells with low endogenous RECQL4 was sufficient to render them resistant to cisplatin, in a manner associated with YB1 elevation and MDR1 activation. Conversely, RECQL4 silencing in cisplatin-resistant gastric cancer cells with high endogenous RECQL4 suppressed YB1 phosphorylation, reduced MDR1 expression, and resensitized cells to cisplatin. In establishing RECQL4 as a critical mediator of cisplatin resistance in gastric cancer cells, our findings provide a therapeutic rationale to target RECQL4 or the downstream AKT-YB1-MDR1 axis to improve gastric cancer treatment. Cancer Res; 76(10); 3057-66. ©2016 AACR.
Asunto(s)
Cisplatino/farmacología , Resistencia a Antineoplásicos , Proteínas Proto-Oncogénicas c-akt/metabolismo , RecQ Helicasas/metabolismo , Neoplasias Gástricas/patología , Proteína 1 de Unión a la Caja Y/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Técnicas para Inmunoenzimas , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/metabolismo , Células Tumorales CultivadasRESUMEN
BACKGROUND: Aspirin has potential in the prevention or treatment of oesophageal cancer, the seventh most common cancer in the world, but its mechanism of action is still not certain. METHODS: The oesophageal squamous cell carcinoma cell line TE-13 was cultured with aspirin at different concentrations or for different times. Proliferation and apoptosis were measured by MTT reduction and flow cytometry. Expression of COX-2 mRNA was measured by RT-PCR and COX-2 protein levels with Western blot analysis. Nuclear NF-kappaB and cytoplasmic IkappaB protein levels were determined by electrophoretic mobility shift assay and Western blot, respectively. RESULTS: Aspirin significantly inhibited cell proliferation and induced apoptosis at concentrations of 1, 4, 8 mmol/L. Aspirin dose-dependently decreased the levels of COX-2 mRNA, COX-2 protein and nuclear NF-kappaB protein and increased the cytoplasmic IkappaB protein. CONCLUSION: We conclude that aspirin inhibits the proliferation of, and induced apoptosis in, the cultured TE-13 SCC cell line. These changes correlate with a reduction in COX-2 mRNA and protein expression, prostaglandin synthesis, an inhibition of NF-kappaB nuclear translocation, and an increase in cytoplasmic IkappaB. These results support the further investigation of the cyclooxygenase pathway in investigating the potential of aspirin and similar drugs in cancer prevention and therapy.
Asunto(s)
Apoptosis/efectos de los fármacos , Aspirina/farmacología , Carcinoma de Células Escamosas/patología , Ciclooxigenasa 2/metabolismo , Neoplasias Esofágicas/patología , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , División Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/química , Ciclooxigenasa 2/análisis , Citoplasma/química , Humanos , Proteínas de la Membrana/análisis , FN-kappa B/análisis , Prostaglandinas/biosíntesis , ARN Mensajero/análisisRESUMEN
To explore the underlying mechanism(s) of pulmonary arterial hypertension in endotoxic shock, the roles of N-acetylcysteine (NAC), nitric oxide (NO) and carbon monoxide (CO) were investigated. Pulmonary arterial rings (3-mm width) were prepared from 24 rabbits. Lipopolysaccharide (LPS), after 7-hour incubation, decreased the endothelium-dependent relaxation response of the arterial ring (pre-contracted with phenylephrine) to acetylcholine (1 mumol/L), but did not affect the endothelium-independent relaxation response to sodium nitroprusside. The LPS effects were reduced by a concomitant incubation with the free radical scavenger (NAC), NO donor (L-arginine), and CO donor (hemin), respectively. On the other hand, the LPS effects were enhanced by applying heme oxygenase-1 (HO-1) inhibitor (zinc protoporphyrin) to block CO production. The response to acetylcholine changed from relaxation to contraction, however, the contractile response to phenylephrine increased significantly after pre-incubation with nitric oxide synthase (NOS) inhibitor (L-NAME) to block NO production, confirming the importance of CO and NO. These results show that LPS impairs endothelium-dependent relaxation of the pulmonary artery, which can be greatly reduced by the antioxidant, or by supplying with NO and CO. Thus, multiple factors are involved in this model of endotoxin-induced pulmonary hypertension.