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BACKGROUND AND AIMS: The dynamic N6-methyladenosine (m6 A) mRNA modification is essential for acute stress response and cancer progression. Sublethal heat stress from insufficient radiofrequency ablation (IRFA) has been confirmed to promote HCC progression; however, whether m6 A machinery is involved in IRFA-induced HCC recurrence remains open for study. APPROACH AND RESULTS: Using an IRFA HCC orthotopic mouse model, we detected a higher level of m6 A reader YTH N6-methyladenosine RNA binding protein 1-3 (YTHDF1) in the sublethal-heat-exposed transitional zone close to the ablation center than that in the farther area. In addition, we validated the increased m6 A modification and elevated YTHDF1 protein level in sublethal-heat-treated HCC cell lines, HCC patient-derived xenograft (PDX) mouse model, and patients' HCC tissues. Functionally, gain-of-function/loss-of-function assays showed that YTHDF1 promotes HCC cell viability and metastasis. Knockdown of YTHDF1 drastically restrains the tumor metastasis evoked by sublethal heat treatment in tail vein injection lung metastasis and orthotopic HCC mouse models. Mechanistically, we found that sublethal heat treatment increases epidermal factor growth receptor (EGFR) m6 A modification in the vicinity of the 5' untranslated region and promotes its binding with YTHDF1, which enhances the translation of EGFR mRNA. The sublethal-heat-induced up-regulation of EGFR level was further confirmed in the IRFA HCC PDX mouse model and patients' tissues. Combination of YTHDF1 silencing and EGFR inhibition suppressed the malignancies of HCC cells synergically. CONCLUSIONS: The m6 A-YTHDF1-EGFR axis promotes HCC progression after IRFA, supporting the rationale for targeting m6 A machinery combined with EGFR inhibitors to suppress HCC metastasis after RFA.
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Carcinoma Hepatocelular/cirugía , Neoplasias Hepáticas/cirugía , Procesamiento Postranscripcional del ARN/efectos de la radiación , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/efectos de la radiación , Ablación por Radiofrecuencia/efectos adversos , Animales , Carcinoma Hepatocelular/genética , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Receptores ErbB/genética , Receptores ErbB/metabolismo , Receptores ErbB/efectos de la radiación , Regulación Neoplásica de la Expresión Génica , Respuesta al Choque Térmico/efectos de la radiación , Humanos , Neoplasias Hepáticas/genética , Metilación/efectos de la radiación , Ratones , Metástasis de la Neoplasia , Trasplante de Neoplasias , Procesamiento Postranscripcional del ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Insuficiencia del TratamientoRESUMEN
Purpose: Although EGFR inhibitor (EGFRi) is used in cancer therapy to suppress tumor growth and resistance to treatment including radiotherapy, EGFRi resistance frequently developed, which significantly reduced treatment outcomes. Therefore, developing alternative approaches for EGFRi is of great importance. Based on our recent observation that ATM inhibitor (ATMi) efficiently inhibited ionizing radiation (IR)-induced EGFR activation in mouse embryo fibroblasts (MEF), the main purpose of this study is to determine whether ATMi could inhibit IR-induced EGFR activation in human tumor cell lines and explore its potential in EGFRi-alternative therapies.Materials and methods: We compared the effects of ATMi, EGFRi individually or in combination on IR-induced EGFR phosphorylation, cell growth and radio-sensitization in nine human tumor cell lines including lung adenocarcinoma (A549 and H358), glioblastoma (LN229), cervical cancer (HeLa), colorectal carcinoma (SW480 and HCT116) and nasopharygeal carcinoma (5-8 F, 6-10B and HK1) cell lines. In addition, we detected the effects of ATMi, EGFRi alone or both on the efficiency of non-homologous end-joining (NHEJ) and homologous recombination (HR) using I-SceI -GFP based NHEJ or HR reporter cell lines.Results: Compared to EGFRi treatment, ATMi treatment decreased IR-induced EGFR phosphorylation, suppressed growth and increased IR sensitization in tested cell lines at a similar or even more efficient level. Combining ATMi and EGFRi did not significantly increased the effects on these phenotypes as ATMi treatment alone. Also, similar to ATMi, EGFRi mainly reduced the efficiency of HR but not NHEJ although combining ATMi and EGFRi further inhibited the HR efficiency.Conclusions: Our study demonstrates that ATMi can function like EGFRi in human tumor cells to inhibit tumor cell growth and sensitize the tumor cells to IR, suggesting that ATMi treatment as an alternative approach may exert anticancer effects on EGFRi-resistant tumor cells and facilitate radiotherapy.
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Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Receptores ErbB/antagonistas & inhibidores , Neoplasias/radioterapia , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Receptores ErbB/fisiología , Receptores ErbB/efectos de la radiación , Recombinación Homóloga/efectos de los fármacos , Humanos , Neoplasias/patología , Fosforilación , Tolerancia a RadiaciónRESUMEN
Background and objectives: The light-curing unit is considered an essential piece of equipment in every dental office. This study was conducted to evaluate the effect of Light-Emitting Diodes (LEDs) by the light cure (LC) device on gingival tissues of albino rats histologically and by regarding the expression of P53 and epidermal growth factor receptor (EGFR). Materials and methods: Gingival tissues of the rats were exposed to LEDs for 30 s with an interval of 30 s for periods of 2 and 5 min and were examined after two and four weeks of light exposure. After the set time, histological sections were studied and the P53 and EGFR expressions were evaluated immunohistochemically and by molecular methods. Results: Mild hyperplasia and mild inflammatory response were detected in higher rates after two weeks of exposure when compared to 4 weeks postexposure. Whereas fibrosis was found at a higher rate after four weeks than that found after two weeks postexposure, parakeratosis was seen only in the group that was exposed for 5 min to LC and when biopsies were taken after 2 weeks. We found that the immunohistochemical expression of P53 was not changed. Similarly, the alteration of EGFR expression was statistically nonsignificant (p > 0.05) when compared to the control group. The data obtained from the qRT-PCR reaction was analyzed using the comparative CT (2-ΔΔCT) method. Statistically, there was no significant difference in the expression of EGER and P53 gene transcripts. Conclusions: LED causes no serious alteration in P53 and EGFR expression, and only trivial histopathological changes occurred, most of which recovered after a 4-week interval.
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Luces de Curación Dental , Encía/efectos de la radiación , Proteína p53 Supresora de Tumor/efectos de la radiación , Animales , Receptores ErbB/genética , Receptores ErbB/efectos de la radiación , Modelos Animales , Ratas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
PURPOSE: Exposure to extremely low frequency electromagnetic fields (ELF-EMFs) could elicit biological effects including carcinogenesis. However, the detailed mechanisms by which these ELF-EMFs interact with biological system are currently unclear. Previously, we found that a 50-Hz magnetic field (MF) exposure could induce epidermal growth factor receptor (EGFR) clustering and phosphorylation on cell membranes. In the present experiment, the possible roles of reactive oxygen species (ROS) in MF-induced EGFR clustering were investigated. MATERIALS AND METHODS: Human amnion epithelial (FL) cells were exposed to a 50-Hz MF with or without N-acetyl-l-cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC). EGFR clustering on cellular membrane surface was analyzed using confocal microscopy after indirect immunofluorescence staining. The intracellular ROS level and acid sphingomyelinase (ASMase) activity were detected using an ROS assay kit and an Amplex® Red Sphingomyelinase Assay Kit, respectively. RESULTS: Results showed that exposure of FL cells to a 50-Hz MF at 0.4 mT for 15 min significantly enhanced the ROS level, induced EGFR clustering and increased ASMase activity. However, pretreatment with NAC or PDTC, the scavenger of ROS, not only counteracted the effects of a 50-Hz MF on ROS level and AMS activity, but also inhibited the EGFR clustering induced by MF exposure. CONCLUSIONS: The present and previous data suggest that ROS mediates the MF-induced EGFR clustering via ASMase activation.
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Campos Magnéticos , Especies Reactivas de Oxígeno/metabolismo , Esfingomielina Fosfodiesterasa/fisiología , Células Cultivadas , Activación Enzimática , Receptores ErbB/química , Receptores ErbB/efectos de la radiación , HumanosRESUMEN
Three γ-heteroatom-substituted N-methylpyrroletetrazole-lysines (mPyTXKs) were synthesized and subsequently incorporated into proteins site-specifically via genetic code expansion. The γ-seleno-substituted derivative, mPyTSeK, showed excellent incorporation efficiency in Escherichia coli and allowed site-selective photo-cross-linking of the GST dimer. Furthermore, the mPyTSeK-cross-linked GST dimer can be cleaved under mild oxidative conditions. The incorporation of mPyTXKs into proteins in mammalian cells was also demonstrated. Lastly, the recombinantly expressed mPyTSeK-encoded Grb2 was shown to covalently capture its interaction partner, EGFR, in mammalian cell lysate, which was subsequently released after treatment with H2O2.
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Reactivos de Enlaces Cruzados/efectos de la radiación , Glutatión Transferasa/genética , Lisina/análogos & derivados , Lisina/genética , Ingeniería de Proteínas , Tetrazoles/efectos de la radiación , Animales , Reactivos de Enlaces Cruzados/síntesis química , Reactivos de Enlaces Cruzados/toxicidad , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/efectos de la radiación , Escherichia coli , Proteína Adaptadora GRB2/química , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/efectos de la radiación , Glutatión Transferasa/química , Glutatión Transferasa/efectos de la radiación , Células HEK293 , Humanos , Peróxido de Hidrógeno/química , Lisina/efectos de la radiación , Lisina/toxicidad , Schistosoma japonicum , Tetrazoles/síntesis química , Tetrazoles/toxicidad , Rayos UltravioletaRESUMEN
Static magnetic fields (SMFs) can affect cell proliferation in a cell-type and intensity-dependent way but the mechanism remains unclear. At the same time, although the diamagnetic anisotropy of proteins has been proposed decades ago, the behavior of isolated proteins in magnetic fields has not been directly observed. Here we show that SMFs can affect isolated proteins at the single molecular level in an intensity-dependent manner. We found that Epidermal Growth Factor Receptor (EGFR), a protein that is overexpressed and highly activated in multiple cancers, can be directly inhibited by SMFs. Using Liquid-phase Scanning Tunneling Microscopy (STM) to examine pure EGFR kinase domain proteins at the single molecule level in solution, we observed orientation changes of these proteins in response to SMFs. This may interrupt inter-molecular interactions between EGFR monomers, which are critical for their activation. In molecular dynamics (MD) simulations, 1-9T SMFs caused increased probability of EGFR in parallel with the magnetic field direction in an intensity-dependent manner. A superconducting ultrastrong 9T magnet reduced proliferation of CHO-EGFR cells (Chinese Hamster Ovary cells with EGFR overexpression) and EGFR-expressing cancer cell lines by ~35%, but minimally affected CHO cells. We predict that similar effects of magnetic fields can also be applied to some other proteins such as ion channels. Our paper will help clarify some dilemmas in this field and encourage further investigations in order to achieve a better understanding of the biological effects of SMFs.
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Proliferación Celular/efectos de la radiación , Receptores ErbB/química , Receptores ErbB/efectos de la radiación , Campos Magnéticos , Neoplasias/patología , Animales , Células CHO , Proliferación Celular/genética , Cricetinae , Cricetulus , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HCT116 , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Dominios Proteicos/genética , Dominios Proteicos/efectos de la radiación , TransfecciónRESUMEN
BACKGROUND AND PURPOSE: EGFR is translocated into the cell nucleus in response to irradiation, where it is involved in regulation of radio-sensitivity. The aim of this study is to elucidate the functional role of nuclear EGFR. MATERIAL AND METHODS: To identify EGFR-bound nuclear proteins and mRNAs, Maldi-TOF analysis and mRNA gene arrays were used. Complex formation of proteins was shown by confocal microscopy, immunoprecipitation and Western blotting. The effect of EGFR binding to mRNAs was exhibited by quantitative RT-PCR. Cellular endpoints were shown by Western blotting, mitochondrial mass quantification, lactate quantification and clonogenic survival assays. RESULTS: Maldi-TOF analysis of proteins bound to nuclear EGFR in response to irradiation showed colocalization with Lamin A and heterogeneous nuclear ribonucleoproteins. Confocal microscopy and Western blotting confirmed this colocalization. Both Lamin A and heterogeneous nuclear ribonucleoproteins are involved in mRNA processing. To support a role of nEGFR in this context after irradiation, we isolated EGFR-bound mRNA and observed an EGFR kinase-dependent mRNA stabilizing effect. With the help of DNA microarrays, we identified mRNAs associated with the Warburg effect that were bound to nuclear EGFR. In this context, we observed radiation-induced HIF1α expression, which triggers inhibition of pyruvate dehydrogenase and blocks the tricarboxylic acid cycle. Consequently, we detected mitophagy and increased lactate production, which is associated with increased treatment resistance. Reduction of nEGFR decreased radiation-induced expression of Hif1α and lactate production. CONCLUSIONS: We showed that nuclear EGFR selectively binds and stabilizes mRNA involved in the Warburg effect in response to irradiation. As a consequence, cells switch from aerobic to anaerobic glucose metabolism, which can be prevented by HIF1α inhibitor BAY87-2243, Dasatinib, Erlotinib or EGFR siRNA.
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Receptores ErbB/fisiología , Ácido Láctico/biosíntesis , ARN Mensajero/metabolismo , Western Blotting , Neoplasias de los Bronquios/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Dasatinib/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/efectos de la radiación , Clorhidrato de Erlotinib/farmacología , Expresión Génica , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas Nucleares/metabolismo , Oxadiazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Tolerancia a Radiación/efectos de los fármacosRESUMEN
Head and neck squamous cell carcinoma (HNSCC) is strongly associated with alcohol and tobacco consumption. Lately, the incidence of human papillomavirus (HPV)-related tumors has shown a significant increase, and HPV-related tumors show distinctive features if compared with the HPV-negative counterpart. Locally advanced HNSCC can be treated with concomitant chemoradiotherapy, but early recurrences sometimes occur. Relapses are often related to an intrinsic radioresistance of the tumors. Alterations in intracellular pathways, primarily involved in cell proliferation, apoptosis, and DNA repair, can lead to radioresistance. Preclinical and clinical evidence highlighted that 3 main pathways, including the epidermal growth factor receptor (EGFR), the phosphotidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), and the p53 signaling cascades, play a crucial role in radioresistance development. A future approach may consist in the association of radiotherapy (RT) and selective inhibition of the key pathways involved in radioresistance. Phase I, II, and III clinical trials are currently testing these novel treatment strategies.
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Carcinoma de Células Escamosas/radioterapia , Quimioradioterapia/métodos , Neoplasias de Cabeza y Cuello/radioterapia , Terapia Molecular Dirigida/métodos , Tolerancia a Radiación/efectos de la radiación , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Receptores ErbB/efectos de los fármacos , Receptores ErbB/efectos de la radiación , Femenino , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Pronóstico , Tolerancia a Radiación/fisiología , Dosificación Radioterapéutica , Medición de Riesgo , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello , Serina-Treonina Quinasas TOR/efectos de los fármacos , Serina-Treonina Quinasas TOR/efectos de la radiación , Resultado del TratamientoRESUMEN
Recent drug discovery developments in the field of small molecule targeted agents have led to much interest in combining these with radiotherapy. There are good preclinical data to suggest this approach worthy of investigation and in this review we discuss how this has translated into recent clinical trials. The outcome of clinical trials investigating radiotherapy/targeted drug combinations published in the last 5 years is discussed, as are trials in progress. The perceived future opportunities and challenges in the development of this exciting area are considered.
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Antineoplásicos/uso terapéutico , Quimioradioterapia/métodos , Terapia Molecular Dirigida/métodos , Neoplasias/terapia , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Celecoxib , Ensayos Clínicos como Asunto , Inhibidores de la Ciclooxigenasa 2/farmacología , Receptores ErbB/efectos de los fármacos , Receptores ErbB/efectos de la radiación , Humanos , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/radioterapia , Pirazoles/farmacología , Sulfonamidas/farmacología , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/efectos de la radiaciónRESUMEN
Methyl-2-cyano-3,12 dioxoolean-1,9 diene-28-oate (CDDO-Me) is an antioxidative, anti-inflammatory modulator, which activates the nuclear factor-erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. While CDDO-Me has radioprotective activity through Nrf2 activation in vitro and in vivo, its ability to mitigate radiation-induced damage when provided after irradiation has not been studied. Here we investigated whether CDDO-Me mitigates ionizing radiation (IR)-induced DNA damage in immortalized normal human colonic epithelial cells (HCECs) and bronchial epithelial cells (HBECs). DNA damage and clonogenic survival were assessed after treatment with CDDO-Me postirradiation. We observed that treatment with CDDO-Me within 30 min after irradiation improved both DNA damage repair and clonogenic survival independently of Nrf2. CDDO-Me activates the epidermal growth factor receptor (EGFR) related DNA repair responses. In the presence of CDDO-Me, EGFR is phosphorylated and translocates into the nucleus where it interacts with DNA-PKcs. CDDO-Me-mediated mitigation activity can be abrogated through depletion of EGFR, ectopic overexpression of mutant EGFR or inhibition of DNA-PKcs. While post-treatment of CDDO-Me protected noncancerous HCECs and HBECs against IR, cancer cells (HCT116 and MCF7) were not protected by CDDO-Me. These results suggest that targeting EGFR using CDDO-Me is a promising radiation mitigator with potential utility for first responders to nuclear accidents.
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Bronquios/efectos de la radiación , Colon/efectos de la radiación , Receptores ErbB/efectos de la radiación , Bronquios/citología , Bronquios/metabolismo , Línea Celular Tumoral , Células Cultivadas , Colon/citología , Colon/metabolismo , Daño del ADN , Reparación del ADN , Células Epiteliales/efectos de la radiación , Receptores ErbB/metabolismo , HumanosRESUMEN
Many microRNAs (miRNAs) play crucial roles in regulating expression of oncogenes or tumor suppressor genes. The epidermal growth factor receptor (EGFR) is frequently overexpressed in a wide range of solid tumors and is an important therapeutic target; however, the therapeutic outcome of currently available anti-EGFR agents is often limited due to diverse molecular resistance mechanisms. In this study, we evaluated the potential of targeting miRNA-7 for overcoming radio-resistance of cancer cells with activated EGFR-associated signaling. A panel of human cancer cell lines with increased EGFR-PI3K-Akt signaling was transfected with pre-miR-7 or control miRNA. Ectopic overexpression of miR-7 attenuated EGFR and Akt expression and radiosensitized SQ20B squamous cell carcinoma of the larynx, MDA-MB-468 breast cancer cells, A549 lung carcinoma cells, and U251 and U87 malignant glioma cells. In contrast, antisense-mediated inhibition of mature miR-7 expression led up-regulation of EGFR and its downstream effectors, and increased radio-resistance of U251 glioma cells. Overexpression of miR-7 prolonged radiation-induced γH2AX foci formation and downregulation of DNA-dependent protein kinases (DNA-PKcs). miR-7 may be a useful therapeutic target for overcoming the radio-resistance of human cancers with activated EGFR-PI3K-AKT signaling.
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Receptores ErbB/metabolismo , MicroARNs/genética , Tolerancia a Radiación/genética , Western Blotting , Regulación hacia Abajo , Receptores ErbB/efectos de la radiación , Humanos , Inmunohistoquímica , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/genética , Transducción de Señal/efectos de la radiación , Transfección , Células Tumorales Cultivadas/patología , Células Tumorales Cultivadas/efectos de la radiación , Regulación hacia ArribaRESUMEN
BACKGROUND/PURPOSE: The epidermal growth factor receptor (EGFR) pathway appears to be essential in many cutaneous disorders. It is well established that ultraviolet (UV) irradiation activates the EGFR in the animal and human skin; however, the molecular mechanisms involved in such activation remain unclear. Our aim is to review and analyse them. METHODS: Computerized search and selection of original papers in the MEDLINE database (PubMed) from 1988 to 2009 were performed. Systematic analysis and breakdown of the information selected were carried out. RESULTS: Full manuscripts were retrieved for 32 citations. It was proven that UV light acts directly and indirectly on EGFR (ErbB1/ErbB2) and on numerous intermediaries of extracellular and intracellular signalling. The most closely observed changes imply concentrations and/or molecular activity of the reactive oxygen species group, hydrogen peroxide, matrix metalloproteinases, p38MAPKinase, p21WAF1, p53, signal transducers and activators of transcription 3 and telomerase. CONCLUSION: Our results help to clarify the working and importance of the UV-EGFR system in the human skin.
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Receptores ErbB/efectos de la radiación , Transducción de Señal/efectos de la radiación , Piel/efectos de la radiación , Rayos Ultravioleta , HumanosRESUMEN
Emerging evidence suggests the existence of a new mode of epidermal growth factor receptor (EGFR) signaling in which activated EGFR undergoes nuclear translocation following treatment with ionizing radiation. The authors provide evidence that the nuclear EGFR transport is a stress-specific cellular reaction, which is linked to src-dependent EGFR internalization into caveolae. These flask-shaped pits can fuse with endoplasmic reticulum and the EGFR is sorted into a perinuclear localization. This compartment may serve as a reservoir for nuclear EGFR transport which is regulated by PKCepsilon (protein kinase Cepsilon). Nuclear EGFR is able to induce transcription of genes essential for cell proliferation and cell-cycle regulation. Moreover, nuclear EGFR has physical contact with compounds of the DNA repair machinery and is involved in removal of DNA damage. Anti-EGFR strategies target radiation-associated EGFR nuclear translocation in different manners. EGFR-inhibitory antibodies, i.e., cetuximab (Erbitux((R))), can block nuclear translocation by EGFR immobilization within the cytosol in responder cell lines, whereas tyrosine kinase inhibitors rather target nuclear kinase activity of EGFR linked with cytosolic or nuclear functions. However, both strategies can inhibit DNA repair following irradiation.
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Núcleo Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Receptores ErbB/efectos de la radiación , Transducción de Señal/efectos de la radiación , Translocación Genética/efectos de la radiación , Células Tumorales Cultivadas/efectos de la radiación , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Antineoplásicos/farmacología , Caveolas/efectos de la radiación , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , División Celular/genética , División Celular/efectos de la radiación , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Cetuximab , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Reparación del ADN/efectos de la radiación , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Genes src/efectos de la radiación , Humanos , Proteína Quinasa C-epsilon/fisiología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transcripción Genética/genética , Transcripción Genética/efectos de la radiación , Translocación Genética/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Pim-1 is an oncogenic serine/threonine kinase with poorly defined function in epithelial cancers. In this study, we determined 1) associations of Pim-1 expression with clinicopathological parameters including responsiveness to irradiation in squamocellular cancers of head and neck and 2) how Pim-1 expression is controlled subsequent to irradiation. Moderate to high expression of Pim-1 correlated to poor response to radiation therapy (P = .003). It is also associated to the expression of epidermal growth factor receptor (EGFR, P < .0001), which has been shown to be activated by irradiation. In radioresistant tumors, irradiation promoted nuclear translocation of Pim-1 (P < .005). When directly testing EGFR dependence of Pim-1 expression, up-regulation and nuclear translocation of Pim-1 could be induced through stimulation of EGFR with its ligands EGF or transforming growth factor alpha. Both ligand- and irradiation-induced changes in Pim-1 expression and localization could be inhibited by the monoclonal anti-EGFR antibody cetuximab and by the tyrosine kinase inhibitor gefitinib also targeting EGFR. These results suggest that irradiation-induced activation of EGFR upregulates Pim-1, and Pim-1 may be used as a novel predictive marker of radiation response in patients with squamocellular cancers of head and neck.
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Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/metabolismo , Receptores ErbB/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/biosíntesis , Tolerancia a Radiación/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Receptores ErbB/efectos de la radiación , Técnica del Anticuerpo Fluorescente , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Persona de Mediana EdadRESUMEN
Radiotherapy plays a crucial role in the treatment of many malignancies; however, locoregional disease progression remains a critical problem. This has stimulated laboratory research into understanding the basis for tumor cell resistance to radiation and the development of strategies for overcoming such resistance. We know that some cell signaling pathways that respond to normal growth factors are abnormally activated in human cancer and that these pathways also invoke cell survival mechanisms that lead to resistance to radiation. For example, abnormal activation of the epidermal growth factor receptor (EGFR) promotes unregulated growth and is believed to contribute to clinical radiation resistance. Molecular blockade of EGFR signaling is an attractive strategy for enhancing the cytotoxic effects of radiotherapy and, as shown in numerous reports, the radiosensitizing effects of EGFR antagonists correlate with a suppression of the ability of the cells to repair radiation-induced DNA double strand breaks (DSBs). The molecular connection between the EGFR and its governance of DNA repair capacity appears to be mediated by one or more signaling pathways downstream of this receptor. The purpose of this review is to highlight what is currently known regarding EGFR signaling and the processes responsible for repairing radiation-induced DNA lesions that would explain the radiosensitizing effects of EGFR antagonists.
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Reparación del ADN/fisiología , Receptores ErbB/efectos de la radiación , Radiobiología , Transducción de Señal/efectos de la radiación , Animales , Línea Celular Tumoral/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de la radiación , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Receptores ErbB/metabolismo , Humanos , Neoplasias/genética , Neoplasias/radioterapia , Traumatismos por Radiación/prevención & control , Tolerancia a RadiaciónRESUMEN
PURPOSE: Elucidation of the molecular mechanism of radiation-induced activation of src kinase, which initiates EGFR internalization and nuclear transport. MATERIAL AND METHODS: Radiation-induced src activation was investigated in the bronchial carcinoma cell line A549. Proteins were Western blotted and quantified by the help of specific antibodies. Residual DNA-damage was quantified with gammaH(2)AX-foci analysis. Radiation-induced lipid peroxidation was prevented by acetyl-cysteine. RESULTS: The radiation-induced src activation and EGFR stabilization could be mimicked by addition of hydroxy-nonenal (HNE), one of the major lipid peroxidation products. Radiation-generated HNE is bound to EGFR and src and correlated with complex formation between both following radiation. Treatment with HNE activated src and stimulated radiation-associated EGFR and caveolin 1 phosphorylations resulting in increased nuclear transport of EGFR. Consequently, radiation-induced phosphorylation and activation of DNA-PK were increased. This phosphorylation was associated with improved removal of residual damage 24h after irradiation. Inhibition of radiation-induced HNE generation by acetyl-cysteine blocked radiation-induced src activation and EGFR phosphorylation. CONCLUSIONS: HNE generated in response to radiation exposure activates src kinase and is involved in regulation of radiation-stimulated DNA-repair processes.
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Caveolina 1/metabolismo , Reparación del ADN/fisiología , Receptores ErbB/metabolismo , Peroxidación de Lípido/efectos de la radiación , Familia-src Quinasas/metabolismo , Transporte Activo de Núcleo Celular , Western Blotting , Carcinoma Broncogénico/patología , Carcinoma Broncogénico/radioterapia , Línea Celular Tumoral/metabolismo , Línea Celular Tumoral/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de la radiación , Receptores ErbB/efectos de la radiación , Humanos , Fosforilación/efectos de la radiación , Radiación Ionizante , Sensibilidad y Especificidad , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/efectos de la radiación , Familia-src Quinasas/efectos de la radiaciónRESUMEN
BACKGROUND AND OBJECTIVES: The mechanisms of photodynamic therapy (PDT) have been studied on the cellular and tissue levels. However, the cellular behaviors of cancer cells survived from PDT are still not clear. This study attempted to investigate the influence of 5-aminolevulinic acid (ALA)-based PDT on the invasion ability as well as molecular changes in surviving cancer cells and their progeny. MATERIALS AND METHODS: The systematic effects of ALA-PDT were evaluated using human invasive carcinoma cells (lung adenocarcinoma CL1-5 cells, melanoma A375 cells and breast carcinoma MDA-MB-231 cells). To study the cellular behaviors of surviving cancer cells, PDT-derived variants were established as stable cell lines after consecutive treatment with ALA-PDT. Scratch wound assay and invasion assay were performed to evaluate the migration and invasion ability in the surviving cancer cells and the established PDT-derived variants. RT-PCR and immunoblot analysis were performed to examine the expression levels of epidermal growth factor receptor (EGFR). RESULTS: Though ALA-PDT caused differential phototoxicity among these invasive carcinoma cells, reduced migration was found in all the surviving cancer cells Compared to parental cancer cells, the established PDT-derived variants exerted significant phenotypic changes of cellular morphology, reduced mitochondrial function and a suppressed cellular invasiveness. Furthermore, correlated with the reduced invasion ability, expression of EGFR was downregulated in these established PDT-derived variants. CONCLUSIONS: Except for direct cell killing, ALA-PDT could reduce EGFR expression and invasion ability of the surviving cancer cells and these effects could further pass to the progeny. The results from this study provide insights into a new mechanism by which PDT might affect cellular behaviors and tumor metastasis.
Asunto(s)
Ácido Aminolevulínico/farmacología , Invasividad Neoplásica/genética , Fotoquimioterapia , Células Tumorales Cultivadas/patología , Células Tumorales Cultivadas/efectos de la radiación , Adenocarcinoma/patología , Adenocarcinoma/radioterapia , Neoplasias de la Mama/patología , Neoplasias de la Mama/radioterapia , Línea Celular Tumoral , Movimiento Celular/efectos de la radiación , Regulación hacia Abajo , Receptores ErbB/metabolismo , Receptores ErbB/efectos de la radiación , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/radioterapia , Melanoma/patología , Melanoma/radioterapia , Mitocondrias/efectos de la radiación , Fenotipo , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/radioterapiaRESUMEN
The authors review the current literature on the major biological advances in the molecular testing of brain tumors. The incorporation of several new aspects required for proper disease management into traditional pathology service is the focus of this review. One of the important achievements of the last years in neuro-oncology is the observation that the promoter methylation status of the MGMT (O6-methylguanine DNA methyltransferase) gene determines the treatment efficacy of temozolomide (Temodal) in glioblastomas. This can best be evaluated by methylation-specific PCR (MSP) using tumor tissue obtained for histological evaluation. Furthermore, up-regulation of EGFR signaling through gene amplification has been recognized and targeted by anti-EGFR approaches in high-grade gliomas. The EGFRvIII mutant receptor is practically unique to glioma cells hence analysis of EGFR seems to be justifiably demanded either by oncologists or patients. Immunohistochemistry (IHC) can easily be included in routine laboratory workflow. In addition to this FISH analysis can be performed for the assessment of EGFR gene copy numbers at cellular level. Studying the EGFR status at a genetic and simultaneously at the protein expression level seems to be a valid approach for making treatment decision. Similarly complex and even less clear biological background characterizes the behavior of tumors with oligodendroglial differentiation. The deletion of the chromosomal regions 1p and 19q was found to be associated with favorable outcome and good response to the PCV treatment protocol. Therapeutic decisions are therefore also enabled on the basis of the 1p/19q status. Concurrent temozolomide/radiation therapy is often indicated on the basis of 1p/19q testing. The 1p/19q status can be assessed by FISH or, less frequently, by aCGH or LOH assay. Based on the in-depth overview of the literature the authors highly recommend the adaptation of molecular glioma testing that most efficiently could be done in centralized neuropathology laboratories. This approach would comply with the increasing need for personalized ("tailored") therapy while best satisfying cost/benefit issues.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Dacarbazina/análogos & derivados , Receptores ErbB/metabolismo , Glioma/metabolismo , Glioma/patología , O(6)-Metilguanina-ADN Metiltransferasa/genética , Antineoplásicos Alquilantes/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/radioterapia , Quimioterapia Adyuvante , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 19 , Metilación de ADN/efectos de los fármacos , Dacarbazina/farmacología , Dacarbazina/uso terapéutico , Receptores ErbB/efectos de los fármacos , Receptores ErbB/genética , Receptores ErbB/efectos de la radiación , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Glioma/tratamiento farmacológico , Glioma/radioterapia , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Reacción en Cadena de la Polimerasa , Valor Predictivo de las Pruebas , Pronóstico , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Radioterapia Adyuvante , Transducción de Señal , Temozolomida , Regulación hacia ArribaRESUMEN
Recent studies have implicated the lipid mediator platelet-activating factor (PAF) in UVB-mediated systemic immunosuppression known to be a major cause for skin cancers. Previously, our group has demonstrated that UVB irradiation triggers the production of PAF and oxidized glycerophosphocholines that act as PAF-receptor (PAF-R) agonists. The present studies explored the mechanisms by which UVB generates PAF-R agonists. UVB irradiation of human epidermal KB cells resulted in both increased levels of reactive oxygen species (ROS) and PAF-R agonistic activity. Pretreatment of KB cells with antioxidants vitamin C and N-acetylcysteine or the pharmacological inhibitor PD168393 specific for the epidermal growth factor receptor all inhibited UVB-induced ROS as well as PAF-R agonists, yet had no effect on fMLP-mediated PAF-R agonist production. In addition, in vivo production of PAF-R agonists from UVB-irradiated mouse skin was blocked by both systemic vitamin C administration and topical PD168393 application. Moreover, both vitamin C and PD168393 abolished UVB-mediated but not the PAF-R agonist 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine-mediated immunosuppression as measured by the inhibition of delayed type contact hypersensitivity to the chemical dinitrofluorobenzene. These studies suggest that UVB-induced systemic immunosuppression is due to epidermal growth factor receptor-mediated ROS which results in PAF-R agonist formation.
Asunto(s)
Receptores ErbB/fisiología , Factor de Activación Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/agonistas , Glicoproteínas de Membrana Plaquetaria/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/efectos de la radiación , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/efectos de la radiación , Rayos Ultravioleta , Animales , Dermatitis por Contacto/etiología , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/metabolismo , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Receptores ErbB/efectos de la radiación , Humanos , Terapia de Inmunosupresión , Células KB , Ratones , Ratones Pelados , Ratones Endogámicos C57BL , Factor de Activación Plaquetaria/efectos de la radiación , Glicoproteínas de Membrana Plaquetaria/biosíntesis , Especies Reactivas de Oxígeno/farmacología , Receptores Acoplados a Proteínas G/biosíntesis , Piel/inmunología , Piel/metabolismo , Piel/efectos de la radiaciónRESUMEN
Endogenous direct-current electric fields (dcEFs) occur in vivo in the form of epithelial transcellular potentials or neuronal field potentials. A variety of cells respond to dcEFs by migrating directionally, and this is termed galvanotaxis. The mechanism by which dcEFs direct cell movement, however, is not yet understood, and the effects on lung cancer cells are entirely unknown. We demonstrated that cultured human lung adenocarcinoma A549 cells migrate toward the cathode in applied dcEFs at 3 V/cm. Fluorescence microscopy showed that both epidermal growth factor receptors (EGFRs) and F-actin are polarized to the cathode. EGFR inhibitors, cetuximab and AG1478, reduced the migration rate and directed motility in dcEFs. Western blots showed that ERK and AKT signaling pathways were prominently promoted by dcEFs. EGFR inhibitors could reduce this promotion but not completely. These data suggest that polarization of EGFRs and the activation of their downstream signals play an important role in the galvanotaxis of A549 cells in dcEFs.