RESUMEN
EphA2 (EPH receptor A2) (erythropoietinproducing hepatocellular receptor tyrosine kinase subtype A2) plays a crucial role in human cancers, and is a promising target for the development of new anticancer drugs. In this study, we showed that the interaction of Annexin A1 (ANXA1) and EphA2 increased EphA2 stability by inhibiting its proteasome degradation in gastric cancer (GC) and colon cancer (CC) cells, and the amino acid residues 2030 and 2830 of ANXA1 N terminal were responsible for binding and stabilizing EphA2. Based on the amino acid residues of ANXA1 responsible for binding EphA2, we developed ANXA1derived 3 amino acidlong (SKG) and 11 amino acidlong peptides (EYVQTVKSSKG) in fusion to cellpenetrating peptide, named as A1(2830) and A1(2030) respectively, and found that A1(2830) and A1(2030) blocked the binding of ANXA1 with EphA2, targeted EphA2 degradation, and suppressed the growth of GC and CC cells in vitro and in mice. Our data demonstrated that ANXA1 was able to bind and stabilize EphA2 in GC and CC cells, and disruption of ANXA1EphA2 interaction by the two ANXA1derived peptides inhibited the growth of GC and CC cells by targeting EphA2 degradation, presenting a potential strategy for treating GC and CC with these peptides.
Asunto(s)
Anexina A1/farmacología , Neoplasias del Colon/tratamiento farmacológico , Fragmentos de Péptidos/farmacología , Receptor EphA2/metabolismo , Neoplasias Gástricas/tratamiento farmacológico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologíaRESUMEN
EphA2 is an important oncogenic protein and emerging drug target, but the oncogenic role and mechanism of ligand-independent phosphorylation of EphA2 at tyrosine 772 (pY772-EphA2) is unclear. In this study, we established nasopharyngeal carcinoma (NPC) cell lines with stable expression of exogenous EphA2 and EphA2-Y772A (phosphorylation inactivation) using endogenous EphA2-knockdown cells, and observed that pY772A EphA2 was responsible for EphA2-promoting NPC cell proliferation and anchorage-independent and in vivo growth in mice. Mechanistically, EphA2-Y772A mediated EphA2-activating Shp2/Erk-1/2 signaling pathway in the NPC cells, and Gab1 (Grb2-associated binder 1) and Grb2 (growth factor receptor-bound protein 2) were involved in pY772-EphA2 activating this signaling pathway. Our results further showed that Shp2/Erk-1/2 signaling mediated pY772-EphA2-promoting NPC cell proliferation and anchorage-independent growth. Moreover, we observed that EphA2 tyrosine kinase inhibitor ALW-II-41-27 inhibited pY772-EphA2 and EphA2-Y772A decreased the inhibitory effect of ALW-II-41-27 on NPC cell proliferation. Collectively, our results demonstrate that pY772-EphA2 is responsible for EphA2-dependent NPC cell growth in vitro and in vivo by activating Shp2/Erk-1/2 signaling pathway, and is a pharmacologic target of ALW-II-41-27, suggesting that pY772-EphA2 can serve as a therapeutic target in NPC and perhaps in other cancers.
Asunto(s)
Efrina-A2/genética , Carcinoma Nasofaríngeo/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , China , Efrina-A2/metabolismo , Proteína Adaptadora GRB2/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/genética , Masculino , Ratones , Ratones Desnudos , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patología , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Receptor EphA2/genética , Receptor EphA2/metabolismo , Transducción de Señal/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Overexpression of ANXA1 and EphA2 has been linked to various cancers and both proteins have attracted considerable attention for the development of new anticancer drugs. Here we report that ANXA1 competes with Cbl for binding EphA2 and increases its stability by inhibiting Cbl-mediated EphA2 ubiquitination and degradation in nasopharyngeal carcinoma (NPC). Binding of ANXA1 to EphA2 promoted NPC cell growth and metastasis in vitro and in vivo by elevating EphA2 levels and increasing activity of EphA2 oncogenic signaling (pS897-EphA2). Expression of ANXA1 and EphA2 was positively correlated and both were significantly higher in NPC tissues than in the normal nasopharyngeal epithelial tissues. Patients with high expression of both proteins presented poorer disease-free survival and overall survival relative to patients with high expression of one protein alone. Furthermore, amino acid residues 20-30aa and 28-30aa of the ANXA1 N-terminus bound EphA2. An 11 amino acid-long ANXA1-derived peptide (EYVQTVKSSKG) was developed on the basis of this N-terminal region, which disrupted the connection of ANXA1 with EphA2, successfully downregulating EphA2 expression and dramatically suppressing NPC cell oncogenicity in vitro and in mice. These findings suggest that ANXA1 promotes NPC growth and metastasis via binding and stabilization of EphA2 and present a strategy for targeting EphA2 degradation and treating NPC with a peptide. This therapeutic strategy may also be extended to other cancers with high expression of both proteins. SIGNIFICANCE: These findings show that EphA2 is a potential target for NPC therapeutics and an ANXA1-derived peptide suppresses NPC growth and metastasis. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/20/4386/F1.large.jpg.
Asunto(s)
Anexina A1/metabolismo , Efrina-A2/metabolismo , Carcinoma Nasofaríngeo/patología , Neoplasias Nasofaríngeas/patología , Animales , Anexina A1/química , Anexina A1/genética , Sitios de Unión , Unión Competitiva , Línea Celular Tumoral , Efrina-A2/química , Efrina-A2/genética , Humanos , Masculino , Ratones Endogámicos BALB C , Simulación del Acoplamiento Molecular , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/mortalidad , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/mortalidad , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Receptor EphA2 , Ubiquitina/metabolismoRESUMEN
HDAC7 plays a crucial role in cancers, and is the main drug target of several HDAC inhibitors. However, the role and mechanism of HDAC7 in nasopharyngeal carcinoma (NPC) are still unclear. In this study, we observed that HDAC7 was significantly upregulated in the NPC tissues relative to normal nasopharyngeal mucosa (NNM) tissues, HDAC7 expression levels were positively correlated with NPC progression and negatively correlated with patient prognosis, and HDAC7 knockdown dramatically inhibited the in vitro proliferation, migration, and invasion of NPC cells, and the growth of NPC xenografts in mice, indicating the HDAC7 promotes the oncogenicity of NPC. Mechanistically, HDAC7 promoted the in vitro proliferation, migration, and invasion of NPC cells by upregulating EphA2, in which miR-4465 mediated HDAC7-regulating EphA2, a direct target gene of miR-4465. We further showed that miR-4465 was significantly downregulated in the NPC tissues relative to NNM tissues, and inhibited the in vitro proliferation, migration, and invasion of NPC cells by targeting EphA2 expression. Moreover, we observed that the expressions of HDAC7, miR-4465, and EphA2 in NPC tissues were correlated. The results suggest that HDAC7 promotes the oncogenicity of NPC by downregulating miR-4465 and subsequently upregulating EphA2, highlighting HDAC7 as a potential therapeutic target for NPC.
Asunto(s)
Efrina-A2/metabolismo , Histona Desacetilasas/metabolismo , MicroARNs/genética , Animales , Apoptosis/genética , Carcinoma/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Efrina-A2/genética , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasas/genética , Humanos , Ratones , MicroARNs/metabolismo , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/patología , Pronóstico , Receptor EphA2 , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A heterozygous point mutation of p53 gene at codon 280 from AGA to ACA (R280T) frequently occurs in nasopharyngeal carcinoma (NPC) cell lines, and about 10% NPC tissues. However, the role of this mutation in the pathogenesis of NPC remains unclear. In this study, we generated p53 knockout (KO) NPC cell lines from CNE2 cells carrying heterozygous p53 R280T (p53-R280T) mutation and C666-1 cells carrying wild-type p53 by CRISPR-Cas9 gene editing system, and found that KO of heterozygous p53-R280T significantly decreased NPC cell proliferation and increased NPC cell apoptosis, whereas KO of wild-type p53 had opposite effects on NPC cell proliferation and apoptosis. Moreover, KO of heterozygous p53-R280T inhibited the anchorage-independent growth and in vivo tumorigenicity of NPC cells. mRNA sequencing of heterozygous p53-R280T KO and control CNE2 cells revealed that heterozygous p53-R280T mutation activated PI3K-Akt signaling pathway. Moreover, blocking of PI3K-Akt signaling pathway abolished heterozygous p53-R280T mutation-promoting NPC cell proliferation and survival. Our data indicate that p53 with heterozygous R280T mutation functions as an oncogene, and promotes the oncogenicity of NPC cells by activating PI3K-Akt signaling pathway.
RESUMEN
OBJECTIVE: To construct plasmids with Hre2.Grp78 chimeric promoter regulating fusion gene TK/VP3 and elaborate the effects of overexpressed TK/VP3 on nasopharyngeal carcinoma cells. METHODS: Four plasmids were constructed, including pcDNA3.1-CMV-TK/VP3, pcDNA3.1-Hre2.TK/VP3, pcDNA3.1-Grp78.TK/VP3, and pcDNA3.1-Hre2.Grp78.TK/VP3. The human nasopharyngeal carcinoma cell line HNE1 cells were transfected with the 4 plasmids, respectively. Cell viabilities were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was conducted using flow cytometry analysis. The expression of TK, VP3, Grp78, and hypoxia-inducible factor 1α and apoptosis-related proteins was determined by real-time quantitative polymerase chain reaction and Western blotting. RESULTS: The recombinant plasmids that could steadily overexpress TK and VP3 were successfully constructed. Expression of TK and VP3 in cells transfected with pcDNA3.1-Hre2.TK/VP3 and pcDNA3.1-Grp78.TK/VP3 was significantly higher than pcDNA3.1-CMV-TK/VP3, and expression in cells transfected with pcDNA3.1-Hre2.Grp78.TK/VP3 was the highest. Under glucose deprivation or hypoxia condition, Grp78 or hypoxia-inducible factor 1α was overexpressed so that expression of TK and VP3 was significantly upregulated, which could further inhibit cell proliferation and enhance cell apoptosis. CONCLUSION: We successfully constructed 4 plasmids with Hre2.Grp78 chimeric promoter regulating fusion gene TK/VP3, which could significantly inhibit the proliferation as well as enhance the apoptosis of nasopharyngeal carcinoma cells under glucose deprivation or hypoxia condition.
Asunto(s)
Proteínas de Choque Térmico/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta , Transactivadores/metabolismo , Apoptosis/genética , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Chaperón BiP del Retículo Endoplásmico , Regulación Neoplásica de la Expresión Génica , Humanos , Hipoxia/genética , Hipoxia/metabolismo , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Plásmidos/genética , Proteínas Recombinantes de Fusión/genética , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Transactivadores/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Following the publication of the above article, the authors have realized that one of the data panels featured in Fig. 5D was selected incorrectly. Specifically, the wrong image was selected for the A1 (2830), HCT116 experiment. The authors have revisited their original sources to identify the correct data panel, and can confirm that the error arose unintentionally during the process of compiling the figure. The correct version of Fig. 5, featuring corrected data panel for Fig. 5D, is shown on the next page. The authors confirm that this error did not affect the conclusions reported in this study, and are grateful to the Editor of International Journal of Oncology for allowing them the opportunity to publish this corrigendum. Furthermore, the authors apologize to the readership of the Journal for any inconvenience caused. [the original article was published in International Journal of Oncology 57: 12031213, 2020; DOI: 10.3892/ijo.2020.5119].
RESUMEN
OBJECTIVE: To construct hypoxia/radiation inducible promotor HRE1.Egr-1, and to observe its promotive effect on the expression of yCDglyTK gene in nasopharyngeal cancer HNE-1 cells and the anti-tumor effect of yCDglyTK and to lay an experimental foundation for further exploration of new gene-radio therapy of nasopharyngeal cancer. METHODS: pcDNA3.1(-)HRE1.Egr-1.yCDglyTK was constructed by gene recombination technique. Stable yCDglyTK-expressing HNE-1 cells were generated by transfecting the recombinant plasmid into the target cells with liposome. The expression of yCDglyTK was detected by Western blot in 4 groups: a normoxia group, a radiation group, a hypoxia group, and a hypoxia and radiation group. The killing effect of 5-FC in different circumstances was determined by MTT. RESULTS: The expression of yCDglyTK/5-FC gene in all the groups was significantly different(P<0.01),especially in the hypoxia and radiation group. The killing effect of 5-FC on HNE1 cells varied under different conditions, especially in the hypoxia and radiation group. CONCLUSION: Hypoxia and radiation can induce the activity of fusion promoter HRE1.Egr-1, and obviously promote the anti-tumor effect of yCDglyTK/5-FC system, suggesting that yCDglyTK may be a candidate suicide gene for gene-radio therapy of NPC.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Fusión Génica/fisiología , Terapia Genética/métodos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias Nasofaríngeas/terapia , Flucitosina/farmacología , Humanos , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/radioterapia , Elementos de Respuesta/genética , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Células Tumorales CultivadasAsunto(s)
Proteínas de la Cápside/genética , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/terapia , Animales , Secuencia de Bases , Carcinoma , Genes Supresores de Tumor , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Datos de Secuencia Molecular , Carcinoma NasofaríngeoRESUMEN
OBJECTIVE: To explore the in vitro, in situ and in vivo killing effects to CNE-2 cells of human nasopharyngeal carcinoma (NPC) by FCU/5-FC system combined with gamma irradiation for predicting the treatment effect on NPC. METHODS: Plasmid pcDNA3.1(-)CMVe.Egr-1. FCU was introduced into CNE-2 cells by electroporation. The transfected cells were selected by G418 (600 microg/ml) for 14 days to yield cells expressing FCU stably. The FCU protein in transfected CNE-2 cells was tested by Western blotting. In vitro response of FCU-expressing CNE-2 cells to 5-FC or gamma irradiation, alone or in combination was detected by MTT assay. Furthermore, A NPC model was employed by inoculating CNE-2 cells in the right flank of nude mice for in situ gene therapy, and after 12 days of inoculation, those rats were randomized to seven groups, then the suppression of NPC growth in model was observed after giving different treatments. Finally, FCU-expressing CNE-2 cells were inoculating in the right flank of nude mice to generate NPC xenografts for in vivo gene therapy, and after 5-day of implantation, those rats were randomized to seven groups, then the delaying of tumour growth was observed in xenografts treated with different conditions. RESULTS: A anticipated relative molecular quality 42,000 protein was obtained from total protein of FCU-expressing CNE-2 cells. The growth of FCU-positive CNE-2 cells were inhibited by 5-FC or gamma irradiation, alone or in combination, but cells treated with both 5-FC and gamma irradiation resulted in enhanced cell killing when compared with cells treated with gamma irradiation or 5-FC alone. In vitro study showed that the relative survival rates of FCU-expressing CNE-2 cells treated with gamma irradiation were 15.85% - 97.88%, while that of gamma irradiation + 5-FC (100 microg/ml) group were 6.58% - 50.00%, and there was a significant difference (P < 0.01). The MTT results also demonstrated that the relative survival rate has a striking different (P < 0.01) between 5-FC group (12.11% - 99.51%) and 5-FC + gamma irradiation (1.0 Gy) group (2.37% - 35.87%). Not only in situ but also in vivo, potent growth inhibition on the explanted NPC tumours was observed in mice treated with 5-FC or gamma irradiation, alone or in combination, among which interference of both 5-FC and gamma irradiation leaded to most distinct suppression of tumour growth. Tumour volumes in groups interfered by 5-FC and or gamma irradiation were extinctly different with the control group and PBS treatment group (P < 0.05). CONCLUSION: CNE-2 cells or nasopharyngeal carcinoma venograph could be killed by FCU/5-FC suicide gene prodrug system or gamma irradiation, and there is a synergistic therapeutic effect on NPC between FCU/5-FC and gamma irradiation.
Asunto(s)
Fluorouracilo/uso terapéutico , Genes Transgénicos Suicidas/genética , Neoplasias Nasofaríngeas/terapia , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Terapia Combinada , Fluorouracilo/administración & dosificación , Terapia Genética/métodos , Humanos , Ratones , Ratones Desnudos , Neoplasias Nasofaríngeas/patología , Profármacos/administración & dosificación , Profármacos/uso terapéutico , Radioterapia/métodos , Factores de Tiempo , TransfecciónRESUMEN
OBJECTIVE: To investigate the killing effects of VP(3) on nasopharyngeal carcinoma cell line CNE-2. METHODS: Plasmid expression vector pcDNA3.1(-) CMV.VP(3)-His was constructed and identified by Kpn I/EcoR I endonuclease analysis, and then sequenced to verify successful insertion in the sense direction of VP(3) gene. pcDNA3.1(-) CMV.VP(3)-His and pcDNA3.1(-)-His expression plasmid was transiently transfected into nasopharyngeal carcinoma cell line CNE-2 . VP(3) protein expression was detected by Western blotting. MTT assay was used to determine the killing effects of VP(3) gene on nasopharyngeal carcinoma cell line CNE-2. RESULTS: Endonuclease analysis and sequencing confirmed the recombinant plasmid contained the complete VP(3) CDS sequence. Western blotting detected a 14.03 kD protein expression from the transfected cells, which was the expecting band of VP(3) gene. The growth of CNE-2 cells that expressed VP(3) gene was inhibited,while the growth of CNE-2 cells that did not express VP(3) gene was not inhibited. CONCLUSION: VP(3) gene can kill nasopharyngeal carcinoma cell CNE-2.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de la Cápside/fisiología , Terapia Genética , Neoplasias Nasofaríngeas/patología , Secuencia de Bases , Proteínas de la Cápside/genética , Línea Celular Tumoral , Humanos , Datos de Secuencia Molecular , TransfecciónRESUMEN
OBJECTIVE: To discuss the etiology, diagnosis, treatment, and prevention of pneumomediastinum or pneumothorax during the removal of bronchial foreign bodies in children. METHODS: We analyzed the clinical data of 10 cases of pneumomediastinum or pneumothorax during the removal of bronchial foreign bodies in children. RESULTS: Two patients died and the other 8 were cured. CONCLUSION: Pneumomediastinum or pneumothorax is mainly caused by the intrapulmonary hyper-pressure and fracture of pulmonary bubbles. The prognosis of pneumomediastinum or pneumothorax is closely related to such factors as correct and punctual diagnosis and quick removal of the airway obstruction.