RESUMO
Resistance of cancer stem-like and cancer tumor bulk cells to radiochemotherapy and destructive infiltration of the brain fundamentally influence the treatment efficiency to cure of patients suffering from Glioblastoma (GBM). The interplay of adhesion and stress-related signaling and activation of bypass cascades that counteract therapeutic approaches remain to be identified in GBM cells. We here show that combined inhibition of the adhesion receptor ß1 integrin and the stress-mediator c-Jun N-terminal kinase (JNK) induces radiosensitization and blocks invasion in stem-like and patient-derived GBM cultures as well as in GBM cell lines. In vivo, this treatment approach not only significantly delays tumor growth but also increases median survival of orthotopic, radiochemotherapy-treated GBM mice. Both, in vitro and in vivo, effects seen with ß1 integrin/JNK co-inhibition are superior to the monotherapy. Mechanistically, the in vitro radiosensitization provoked by ß1 integrin/JNK targeting is caused by defective DNA repair associated with chromatin changes, enhanced ATM phosphorylation and prolonged G2/M cell cycle arrest. Our findings identify a ß1 integrin/JNK co-dependent bypass signaling for GBM therapy resistance, which might be therapeutically exploitable.
Assuntos
Adaptação Biológica , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Integrina beta1/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Tolerância a Radiação , Estresse Fisiológico , Animais , Neoplasias Encefálicas/radioterapia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Movimento Celular/efeitos da radiação , Quimiorradioterapia , Montagem e Desmontagem da Cromatina , Reparo do DNA , Modelos Animais de Doenças , Glioma/mortalidade , Glioma/patologia , Glioma/radioterapia , Histona Desacetilases , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Camundongos , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Estresse Fisiológico/efeitos da radiação , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Each year more than 450,000 Germans are expected to be diagnosed with cancer subsequently receiving standard multimodal therapies including surgery, chemotherapy and radiotherapy. On top, molecular-targeted agents are increasingly administered. Owing to intrinsic and acquired resistance to these therapeutic approaches, both the better molecular understanding of tumor biology and the consideration of alternative and complementary therapeutic support are warranted and open up broader and novel possibilities for therapy personalization. Particularly the latter is underpinned by the increasing utilization of non-invasive complementary and alternative medicine by the population. One investigated approach is the application of low-dose electromagnetic fields (EMF) to modulate cellular processes. A particular system is the BEMER therapy as a Physical Vascular Therapy for which a normalization of the microcirculation has been demonstrated by a low-frequency, pulsed EMF pattern. Open remains whether this EMF pattern impacts on cancer cell survival upon treatment with radiotherapy, chemotherapy and the molecular-targeted agent Cetuximab inhibiting the epidermal growth factor receptor. Using more physiological, three-dimensional, matrix-based cell culture models and cancer cell lines originating from lung, head and neck, colorectal and pancreas, we show significant changes in distinct intermediates of the glycolysis and tricarboxylic acid cycle pathways and enhanced cancer cell radiosensitization associated with increased DNA double strand break numbers and higher levels of reactive oxygen species upon BEMER treatment relative to controls. Intriguingly, exposure of cells to the BEMER EMF pattern failed to result in sensitization to chemotherapy and Cetuximab. Further studies are necessary to better understand the mechanisms underlying the cellular alterations induced by the BEMER EMF pattern and to clarify the application areas for human disease.
Assuntos
Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/efeitos da radiação , Radiação Eletromagnética , Magnetoterapia/métodos , Tolerância a Radiação/efeitos da radiação , Linhagem Celular Tumoral , Humanos , Magnetoterapia/instrumentação , Espécies Reativas de Oxigênio/metabolismoRESUMO
Radiation and chemotherapy are the main pillars of the current multimodal treatment concept for cancer patients. However, tumor recurrences and resistances still hamper treatment success regardless of advances in radiation beam application, particle radiotherapy, and optimized chemotherapeutics. To specifically intervene at key recurrence- and resistance-promoting molecular processes, the development of potent and specific molecular-targeted agents is demanded for an efficient, safe, and simultaneous integration into current standard of care regimens. Potential targets for such an approach are integrins conferring structural and biochemical communication between cells and their microenvironment. Integrin binding to extracellular matrix activates intracellular signaling for regulating essential cellular functions such as survival, proliferation, differentiation, adhesion, and cell motility. Tumor-associated characteristics such as invasion, metastasis, and radiochemoresistance also highly depend on integrin function. Owing to their dual functionality and their overexpression in the majority of human malignancies, integrins present ideal and accessible targets for cancer therapy. In the following chapter, the current knowledge on aspects of the tumor microenvironment, the molecular regulation of integrin-dependent radiochemoresistance and current approaches to integrin targeting are summarized.
Assuntos
Integrinas/antagonistas & inibidores , Terapia de Alvo Molecular/métodos , Neoplasias/terapia , Radioterapia (Especialidade)/métodos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Anticorpos Monoclonais/uso terapêutico , Quimiorradioterapia , Humanos , Integrinas/metabolismo , Modelos Biológicos , Neoplasias/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos da radiaçãoRESUMO
Cyclin-dependent kinase 9 (CDK9), mainly involved in regulation of transcription, has recently been shown to impact on cell cycling and DNA repair. Despite the fact that CDK9 has been proposed as potential cancer target, it remains largely elusive whether CDK9 targeting alters tumor cell radiosensitivity. Five human head and neck squamous cell carcinoma (HNSCC) cell lines (SAS, FaDu, HSC4, Cal33, UTSCC5) as well as SAS cells stably transfected with CDK9-EGFP-N1 plasmid or empty vector controls were used. Upon either CDK9 small interfering RNA knockdown or treatment with a pan-CDK inhibitor (ZK304709), colony formation, DNA double strand breaks (DSBs), apoptosis, cell cycling, and expression and phosphorylation of major cell cycle and DNA damage repair proteins were examined. While CDK9 overexpression mediated radioprotection, CDK9 depletion clearly enhanced the radiosensitivity of HNSCC cells without an induction of apoptosis. While the cell cycle and cell cycle proteins were significantly modulated by CDK9 depletion, no further alterations in these parameters were observed after combined CDK9 knockdown with irradiation. ZK304709 showed concentration-dependent cytotoxicity but failed to radiosensitize HNSCC cells. Our findings suggest a potential role of CDK9 in the radiation response of HNSCC cells. Additional studies are warranted to clarify the usefulness to target CDK9 in the clinic.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/radioterapia , Ciclo Celular/genética , Quinase 9 Dependente de Ciclina/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/radioterapia , Apoptose/efeitos da radiação , Carcinoma de Células Escamosas/patologia , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Sobrevivência Celular/efeitos da radiação , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/genética , Radiossensibilizantes/administração & dosagem , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
PURPOSE: To evaluate matrix metalloproteinase (MMP) activity and invasion after ionizing radiation (IR) exposure and to determine whether MMP could be epigenetically modulated by histone deacetylase (HDAC) inhibition. MATERIAL AND METHODS: Two human breast cancer cell lines (MDA-MB-231 and MCF-7) were cultured in monolayer (2D) and in laminin-rich extracellular matrix (3D). Invasion capability, collagenolytic and gelatinolytic activity, MMP and TIMP protein and mRNA expression and clonogenic survival were analyzed after IR exposure, with and without a HDAC inhibition treatment [1.5 mM valproic acid (VA) or 1 µM trichostatin-A (TSA)]. RESULTS: IR exposure resulted in cell line-dependent stimulation of invasion capacity. In contrast to MCF-7 cells, irradiated MDA-MB-231 showed significantly enhanced mRNA expression of mmp-1, mmp-3 and mmp-13 and of their regulators timp-1 and timp-2 relative to unirradiated controls. This translated into increased collagenolytic and gelatinolytic activity and could be reduced after valproic acid (VA) treatment. Additionally, VA also mitigated IR-enhanced mmp and timp mRNA expression as well as IR-increased invasion capability. Finally, our data confirm the radiosensitizing effect of VA. CONCLUSION: These results suggest that IR cell line-dependently induces upregulation of MMP mRNA expression, which appears to be mechanistically linked to a higher invasion capability that is modifiable by HDAC inhibition.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Metaloproteinases da Matriz/metabolismo , Ácido Valproico/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Raios Infravermelhos/uso terapêutico , Células MCF-7 , Metaloproteinases da Matriz/genética , Invasividade Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-2/genéticaRESUMO
Glioblastoma multiforme (GBM) is a highly aggressive and extremely lethal cancer and novel molecular therapies are required for optimized multimodal therapy regimes. While focal adhesion kinase (FAK) is regarded as a therapeutic target, its radiosensitizing potential remains to be elucidated in glioblastoma. Thus, FAK was inhibited using the pharmaco-logical inhibitor TAE226 and cytotoxicity and radiosensitization of glioblastoma cells were investigated in vitro. Monolayer and suspension cell cultures of a panel of glioblastoma cell lines (A172, LN229, U87MG, U138MG, U343MG, DD-HT7607, and DD-T4) were treated with increasing TAE226 concentrations (0-10 µM) alone or in combination with X-rays (0-6 Gy). Subsequently, clonogenic cell survival, expression and the phosphorylation of FAK downstream signaling, apoptosis and autophagy were analyzed. Efficient FAK inhibition by TAE226 mediated significant cytotoxicity and reduced sphere formation in a dose- and time-dependent manner. Two out of seven glioblastoma cell lines showed radiosensitization. Apoptotic induction by TAE226 was cell line-dependent. The results demonstrated that pharmacological FAK inhibitor TAE226 efficiently reduced clonogenicity and sphere formation in glioblastoma cells without generally modifying their radiosensitivity. However, future studies are necessary to define the potential of FAK inhibition by TAE226 or other pharmacological inhibitors in combination with radiochemotherapy.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , Glioblastoma/tratamento farmacológico , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Humanos , Morfolinas/farmacologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND AND PURPOSE: Inhibition of histone deacetylases (HDACs) has preclinically and clinically shown promise to overcome radio- and chemoresistance of tumor cells. NDACI054 is a novel HDAC inhibitor, which has been evaluated here for its effects on cell survival and radiosensitization of human tumor cell lines from different origins cultured under more physiological three-dimensional (3D), extracellular matrix (ECM)-based conditions. MATERIAL AND METHODS: A549 lung, DLD-1 colorectal, MiaPaCa2 pancreatic and UT-SCC15 head and neck squamous cell carcinoma cells were treated with increasing NDACI054 concentrations (0-50 nM, 24 h) either alone or in combination with X-rays (single dose, 0-6 Gy). Subsequently, 3D clonogenic cell survival, HDAC activity, histone H3 acetylation, apoptosis, residual DNA damage (γH2AX/p53BP1 foci assay 24h post irradiation) and phosphorylation kinetics of Ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), Caspase-3 and Poly(ADP-ribose)-Polymerase 1 (PARP 1) cleavage were analyzed. RESULTS: NDACI054 potently decreased HDAC activity with concomitant increase in acetyl-histone H3 levels, mediated significant cytotoxicity and radiosensitization. These effects were accompanied by a significant increase of residual γH2AX/p53BP1-positive foci, slightly elevated levels of Caspase-3 and PARP 1 cleavage but no induction of apoptosis. CONCLUSIONS: Our data show potent antisurvival and radiosensitizing effects of the novel HDAC inhibitor NDACI054 encouraging further preclinical examinations on this compound for future clinical use.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Neoplasias/patologia , Radiossensibilizantes/farmacologia , Acetilação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Relação Dose-Resposta a Droga , Histonas/metabolismo , Humanos , FosforilaçãoRESUMO
Inherent and acquired resistance to targeted therapeutics continues to emerge as a major clinical obstacle. For example, resistance to EGF receptor targeting occurs commonly, more so than was expected, on the basis of preclinical work. Given emerging evidence that cancer cell-substrate interactions are important determinants of therapeutic sensitivity, we examined the impact of cell-fibronectin interactions on the efficacy of the EGF receptor antibody cetuximab, which is used widely for lung cancer treatment. Our results revealed the potential for cell-fibronectin interactions to induce radioresistance of human non-small cell lung cancer cells. Cell adhesion to fibronectin enhanced tumor cell radioresistance and attenuated the cytotoxic and radiosensitizing effects of cetuximab. Both in vitro and in vivo, we found that cetuximab treatment led to a remarkable induction of fibronectin biosynthesis. Mechanistic analyses revealed the induction was mediated by a p38-MAPK-ATF2 signaling pathway and that RNAi-mediated inhibition of fibronectin could elevate the cytotoxic and radiosensitizing potential of cetuximab. Taken together, our findings show how cell adhesion blunts cetuximab, which, by inducing fibronectin, generates a self-attenuating mechanism of drug resistance.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Receptores ErbB/metabolismo , Fibronectinas/metabolismo , Neoplasias Pulmonares/patologia , Radiossensibilizantes/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos da radiação , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/efeitos da radiação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Cetuximab , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Feminino , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Nus , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Raios X , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PURPOSE: Cyclin-dependent kinase 2 (CDK2) is critically involved in cell cycling and has been proposed as a potential cancer target. It remains largely elusive whether CDK2 targeting alters the tumor cell radiosensitivity. MATERIALS AND METHODS: CDK2(-/-) and wild type (WT) mouse embryonic fibroblasts (MEF) as well as six human head and neck squamous cell carcinoma (HNSCC) cell lines (SAS, FaDu, Cal-33, HSC-4, UTSCC-5, UTSCC-8) were used. Upon CDK2 knockdown using small interfering technology, colony formation, DNA double-strand breaks (DSB), cell cycle distribution and expression and phosphorylation of major proteins regulating cell cycle and DNA damage repair were examined. RESULTS: CDK2(-/-) MEF and CDK2 HNSCC knockdown cell cultures were more radiosensitive than the corresponding controls. Repair of DSB was attenuated under CDK2 knockout or knockdown. In contrast to data in MEF, combined CDK2 knockdown with irradiation showed no cell cycling alterations in SAS and FaDu cultures. Importantly, CDK2 knockdown failed to radiosensitize SAS and FaDu when cultured in a more physiological three-dimensional (3D) extracellular matrix environment. CONCLUSIONS: Our findings suggest that targeting of CDK2 radiosensitizes HNSCC cells growing as monolayer. Additional studies performed under more physiological conditions are warranted to clarify the potential of CDK2 as target in radiotherapy.
Assuntos
Apoptose/efeitos da radiação , Quinase 2 Dependente de Ciclina/metabolismo , Técnicas de Silenciamento de Genes , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/fisiopatologia , Tolerância a Radiação , Animais , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/genética , Relação Dose-Resposta à Radiação , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Camundongos , Doses de RadiaçãoRESUMO
PURPOSE: Cell invasion represents one of the major determinants that treatment has failed for patients suffering from glioblastoma. Contrary findings have been reported for cell migration upon exposure to ionizing radiation. Here, the migration and invasion capability of glioblastoma cells on and in collagen type I were evaluated upon irradiation with X-rays or carbon ions. METHODS AND MATERIALS: Migration on and invasion in collagen type I were evaluated in four established human glioblastoma cell lines exposed to either X-rays or carbon ions. Furthermore, clonogenic radiation survival, proliferation (5-bromo-2-deoxyuridine positivity), DNA double-strand breaks (γH2AX/53BP1-positive foci), and expression of invasion-relevant proteins (eg, ß1 integrin, FAK, MMP2, and MMP9) were explored. Migration and invasion assays for primary glioblastoma cells also were carried out with X-ray irradiation. RESULTS: Neither X-ray nor carbon ion irradiation affected glioblastoma cell migration and invasion, a finding similarly observed in primary glioblastoma cells. Intriguingly, irradiated cells migrated unhampered, despite DNA double-strand breaks and reduced proliferation. Clonogenic radiation survival was increased when cells had contact with extracellular matrix. Specific inhibition of the ß1 integrin or proliferation-associated signaling molecules revealed a critical function of JNK, PI3K, and p38 MAPK in glioblastoma cell invasion. CONCLUSIONS: These findings indicate that X-rays and carbon ion irradiation effectively reduce proliferation and clonogenic survival without modifying the migration and invasion ability of glioblastoma cells in a collagen type I environment. Addition of targeted agents against members of the MAPK and PI3K signaling axis to conventional chemoradiation therapy seems potentially useful to optimize glioblastoma therapy.
Assuntos
Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Movimento Celular/efeitos da radiação , Glioblastoma/patologia , Glioblastoma/radioterapia , Invasividade Neoplásica , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Bromodesoxiuridina/análise , Carbono , Ensaios de Migração Celular/métodos , Proliferação de Células/efeitos da radiação , Colágeno Tipo I , Quebras de DNA de Cadeia Dupla , Glioblastoma/genética , Glioblastoma/metabolismo , Histonas/análise , Humanos , Integrina beta1/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/análise , MAP Quinase Quinase 4/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Cancer resistance to therapy presents an ongoing and unsolved obstacle, which has clear impact on patient's survival. In order to address this problem, novel in vitro models have been established and are currently developed that enable data generation in a more physiological context. For example, extracellular-matrix- (ECM-) based scaffolds lead to the identification of integrins and integrin-associated signaling molecules as key promoters of cancer cell resistance to radio- and chemotherapy as well as modern molecular agents. In this paper, we discuss the dynamic nature of the interplay between ECM, integrins, cytoskeleton, nuclear matrix, and chromatin organization and how this affects the response of tumor cells to various kinds of cytotoxic anticancer agents.
RESUMO
PURPOSE: Assessment of phosphorylated histone H2AX (γH2AX) foci as a measure for double-strand breaks (DSB) is a common technique. Since visual interpretation is time-consuming and influenced by subjective factors, we adapted the pattern recognition algorithms of autoantibodies to automated reading of γH2AX foci. MATERIALS AND METHODS: DSB formation was assessed by detection of γH2AX foci after exposition of thyreocyte rat cell line to (188)Re. We used pattern recognition algorithms of the automated fluorescence interpretation system AKLIDES(®) for evaluation of γH2AX foci. Manual investigation was performed by three laboratories involving five observers. The results were compared by determining correlation and inter-laboratory variability. RESULTS: The study confirmed the adaptation of automated interpretation system AKLIDES® to automated assessment of γH2AX foci in irradiated cells. Both manual and automated quantification resulted in increasing focus numbers depending on dose. Comparison of automated reading with visual assessment for five manual observers resulted in a determination coefficient of R(2) = 0.889. The inter-laboratory variability for five manual investigators of three laboratories was 38.4 %. CONCLUSION: The interpretation system AKLIDES(®) demonstrated a high correlation with visually observed results. High inter-laboratory variability found for manual investigations revealed the usefulness for a standardized technique for evaluation of γH2AX foci.
Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , Histonas/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Reconhecimento Automatizado de Padrão/métodos , Animais , Automação , Partículas beta/efeitos adversos , Linhagem Celular , Ratos , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo , Glândula Tireoide/efeitos da radiaçãoRESUMO
Cell shape and architecture are determined by cell-extracellular matrix interactions and have profound effects on cellular behavior, chromatin condensation, and tumor cell resistance to radiotherapy and chemotherapy. To evaluate the role of chromatin condensation for radiation cell survival, tumor cells grown in three-dimensional (3D) cell cultures as xenografts and monolayer cell cultures were compared. Here, we show that increased levels of heterochromatin in 3D cell cultures characterized by histone H3 deacetylation and induced heterochromatin protein 1alpha expression result in increased radiation survival and reduced numbers of DNA double strand breaks (DSB) and lethal chromosome aberrations. Intriguingly, euchromatin to heterochromatin-associated DSBs were equally distributed in irradiated 3D cell cultures and xenograft tumors, whereas irradiated monolayer cultures showed a 2:1 euchromatin to heterochromatin DSB distribution. Depletion of histone deacetylase (HDAC) 1/2/4 or application of the class I/II pharmacologic HDAC inhibitor LBH589 induced moderate or strong chromatin decondensation, respectively, which was translated into cell line-dependent radiosensitization and, in case of LBH589, into an increased number of DSBs. Neither growth conditions nor HDAC modifications significantly affected the radiation-induced phosphorylation of the important DNA repair protein ataxia telangiectasia mutated. Our data show an interrelation between cell morphology and cellular radiosensitivity essentially based on chromatin organization. Understanding the molecular mechanisms by which chromatin structure influences the processing of radiation-induced DNA lesions is of high relevance for normal tissue protection and optimization of cancer therapy.
Assuntos
Aberrações Cromossômicas , Eucromatina/fisiologia , Neoplasias de Cabeça e Pescoço/patologia , Heterocromatina/fisiologia , Neoplasias Pulmonares/patologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Técnicas de Cultura de Células , Proteínas de Ciclo Celular/metabolismo , Forma Celular , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Feminino , Imunofluorescência , Raios gama , Neoplasias de Cabeça e Pescoço/metabolismo , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Nus , Proteínas Serina-Treonina Quinases/metabolismo , Tolerância a Radiação , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND AND PURPOSE: Resistance of pancreatic ductal adenocarcinoma (PDAC) to chemo- and radiotherapy is a major obstacle. The integral membrane protein Caveolin-1 (Cav-1) has been suggested as a potent target in human pancreatic carcinoma cells. MATERIALS AND METHODS: Human pancreatic tumor cells were examined in a three-dimensional (3D) cell culture model with regard to clonogenic survival, apoptosis, radiogenic DNA-double strand breaks and protein expression and phosphorylation under siRNA-mediated knockdown of Cav-1 without and in combination with irradiation (X-rays, 0-6Gy). Immunohistochemistry was used to assess Cav-1 expression in biopsies from patients with PDAC. RESULTS: Tumor cells in PDAC showed significantly higher Cav-1 expression relative to tumor stroma. Cav-1 knockdown significantly reduced beta1 integrin expression and Akt phosphorylation, induced Caspase 3- and Caspase 8-dependent apoptosis and enhanced the radiosensitivity of 3D cell cultures. While cell cycling and Cav-1 promoter activity remained stable, Cav-1 knockdown-induced radiosensitization correlated with elevated numbers of residual DNA-double strand breaks. CONCLUSIONS: Our data strongly support the concept of Cav-1 as a potent target in pancreatic carcinoma cells due to radiosensitization and Cav-1 overexpression in tumor cells of PDAC. 3D cell cultures are powerful and useful tools for the testing of novel targeting strategies to optimize conventional radio- and chemotherapy regimes for PDAC.
Assuntos
Caveolina 1/metabolismo , Ciclo Celular/efeitos da radiação , Integrina beta1/metabolismo , RNA Interferente Pequeno/metabolismo , Tolerância a Radiação , Apoptose/genética , Apoptose/fisiologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/radioterapia , Caveolina 1/genética , Ciclo Celular/genética , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Integrina beta1/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/radioterapia , Fosforilação/genética , Fosforilação/efeitos da radiação , Probabilidade , RNA Interferente Pequeno/genética , Radiação Ionizante , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Células-Tronco/efeitos dos fármacos , Células-Tronco/efeitos da radiação , TransfecçãoRESUMO
BACKGROUND: The constant increase of cancer cell resistance to radio- and chemotherapy hampers improvement of patient survival and requires novel targeting approaches. Integrin-Linked Kinase (ILK) has been postulated as potent druggable cancer target. On the basis of our previous findings clearly showing that ILK transduces antisurvival signals in cells exposed to ionizing radiation, this study evaluated the impact of the small molecule inhibitor QLT0267, reported as putative ILK inhibitor, on the cellular radiation survival response of human head and neck squamous cell carcinoma cells (hHNSCC). METHODOLOGY/PRINCIPAL FINDINGS: Parental FaDu cells and FaDu cells stably transfected with a constitutively active ILK mutant (FaDu-IH) or empty vectors, UTSCC45 cells, ILK(floxed/floxed(fl/fl)) and ILK(-/-) mouse fibroblasts were used. Cells grew either two-dimensionally (2D) on or three-dimensionally (3D) in laminin-rich extracellular matrix. Cells were treated with QLT0267 alone or in combination with irradiation (X-rays, 0-6 Gy single dose). ILK knockdown was achieved by small interfering RNA transfection. ILK kinase activity, clonogenic survival, number of residual DNA double strand breaks (rDSB; gammaH2AX/53BP1 foci assay), cell cycle distribution, protein expression and phosphorylation (e.g. Akt, p44/42 mitogen-activated protein kinase (MAPK)) were measured. Data on ILK kinase activity and phosphorylation of Akt and p44/42 MAPK revealed a broad inhibitory spectrum of QLT0267 without specificity for ILK. QLT0267 significantly reduced basal cell survival and enhanced the radiosensitivity of FaDu and UTSCC45 cells in a time- and concentration-dependent manner. QLT0267 exerted differential, cell culture model-dependent effects with regard to radiogenic rDSB and accumulation of cells in the G2 cell cycle phase. Relative to corresponding controls, FaDu-IH and ILK(fl/fl) fibroblasts showed enhanced radiosensitivity, which failed to be antagonized by QLT0267. A knockdown of ILK revealed no change in clonogenic survival of the tested cell lines as compared to controls. CONCLUSIONS/SIGNIFICANCE: Our data clearly show that the small molecule inhibitor QLT0267 has potent cytotoxic and radiosensitizing capability in hHNSCC cells. However, QLT0267 is not specific for ILK. Further in vitro and in vivo studies are necessary to clarify the potential of QLT0267 as a targeted therapeutic in the clinic.
Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Radiossensibilizantes/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Dano ao DNA , Fase G2 , Humanos , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
PURPOSE: Integrin-linked kinase (ILK) mediates signals from beta integrins and links integrins to epidermal growth factor receptor (EGFR). Previous studies have identified an antisurvival effect of ILK in irradiated cells. The aim of this study was to evaluate the role of EGFR tyrosine kinase (tk) activity for ILK-mediated radiosensitization. MATERIALS AND METHODS: Human FaDu squamous cell carcinoma (SCC) cells stably transfected with hyperactive ILK (ILK-hk) and ILK(fl/fl) and ILK(-/-) mouse fibroblasts were treated with the pharmacological EGFR-tk inhibitor BIBX1382BS without or in combination with single doses of X-rays. Clonogenic radiation survival, protein expression and phosphorylation (EGFR, v-akt murine thymoma viral oncogene homolog 1 (Akt), p42/44 mitogen-activated protein kinase), DNA-double strand break (DSB) repair measured by gammaH2AX foci, cell morphology and cell cycle distribution were examined. RESULTS: Expression of ILK-hk or ILK(fl/fl) status resulted in significant radiosensitization relative to vector controls or ILK(-/-). Following BIBX1382BS, clonogenic survival of normal fibroblasts and vector controls remained unaffected while ILK-hk-related radiosensitization was significantly diminished. In contrast to BIBX1382BS, which did not affect DNA-DSB repair, ILK-hk-mediated radiosensitization was associated with reduced DNA-DSB repair. At 10 days after BIBX1382BS treatment, FaDu transfectants, in contrast to fibroblasts, showed reduced cell size, accumulation of G1 phase cells and reduced Akt-serine(S)473 phosphorylation. CONCLUSIONS: Our findings confirm ILK as a cell type-independent antisurvival factor in irradiated cells, which actions in terms of radiosensitization critically depend on proper EGFR-tk activity.
Assuntos
Receptores ErbB/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Tolerância a Radiação/fisiologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Compostos Orgânicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Tolerância a Radiação/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
In mammals, the anti-Müllerian hormone (Amh) is responsible for the regression of the Müllerian ducts; therefore, Amh is an important factor of male sex differentiation. The amh gene has been cloned in various vertebrates, as well as in several teleost species. To date, all described species show a sexually dimorphic expression of amh during sex differentiation or at least in differentiated juvenile gonads. We have identified the medaka amh ortholog and examined its expression pattern. Medaka amh shows no sexually dimorphic expression pattern. It is expressed in both developing XY male and XX female gonads. In adult testes, amh is expressed in the Sertoli cells and in adult ovaries in granulosa cells surrounding the oocytes, like in mammals. To better understand the function of amh, we cloned the anti-Müllerian hormone receptor type II (amhrII) ortholog and compared its expression pattern with amh, aromatase (cyp19a1), and scp3. During gonad development, amhrII is coexpressed with medaka amh in somatic cells of the gonads and shows no sexually dimorphic expression. Only the expression level of the Amh type II receptor gene was decreased noticeably in adult female gonads. These results suggest that medaka Amh and AmhrII are involved in gonad formation and maintenance in both sexes.