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1.
J Thorac Cardiovasc Surg ; 149(4): 1185-91, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25595377

RESUMO

OBJECTIVE: Obesity has been linked to esophageal adenocarcinoma (EAC). We hypothesize that adipokines, which are altered by obesity, could affect EAC growth rates and potentially serve as biomarkers of disease and targets for treatment. We have developed a potential murine model to investigate the effects of obesity-altered adipokines on EAC in vivo. METHODS: Severe combined immune-deficient mice were fed either a high-fat diet (HFD) containing 60% animal fat, or a control diet with 10% animal fat, and monitored for weight gain for 5 weeks. All mice were subcutaneously implanted with EAC cells (OE33), and tumor volume was monitored for an additional 4 weeks by direct measurement and uptake of fluorescently labeled 2-D-deoxyglucose. At sacrifice, serum triglyceride levels and abdominal fat-pad weight were measured to assess obesity state. Adipokine levels were measured within abdominal fat of tumor-bearing mice. RESULTS: Mice fed the HFD displayed increased body weight, visceral fat, and serum leptin and triglycerides. All mice developed tumors; OE33 EAC cells in HFD mice displayed increased growth rates, proliferation, and metabolic activity relative to tumors of EAC in control diet mice. Adipokine expression in the abdominal fat revealed distinct changes associated with the HFD and increased body weight. CONCLUSIONS: Ad libitum feeding of the HFD was correlated with more-proliferative EAC tumors in vivo. This phenotype was associated with alterations to secreted adipokines, representing a potential mechanism for our observations. Further studies are necessary to explore findings, as they have potential to improve treatment of EAC.


Assuntos
Adenocarcinoma/etiologia , Adipocinas/metabolismo , Dieta Hiperlipídica , Neoplasias Esofágicas/etiologia , Gordura Intra-Abdominal/metabolismo , Obesidade/complicações , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adiposidade , Animais , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Humanos , Gordura Intra-Abdominal/fisiopatologia , Camundongos Endogâmicos NOD , Camundongos SCID , Obesidade/metabolismo , Obesidade/fisiopatologia , Fenótipo , Fatores de Tempo , Carga Tumoral , Aumento de Peso
2.
J Vis Exp ; (89)2014 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-25079877

RESUMO

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett's esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.


Assuntos
Esôfago de Barrett/patologia , Imunofluorescência/métodos , Linhagem Celular , Corantes Fluorescentes/química , Humanos , Coloração e Rotulagem/métodos
3.
Cancer Lett ; 352(2): 187-95, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25011082

RESUMO

Five-year survival rates for non-small cell lung cancer (NSCLC) have seen minimal improvement despite aggressive therapy with standard chemotherapeutic agents, indicating a need for new treatment approaches. Studies show inactivating mutations in the LKB1 tumor suppressor are common in NSCLC. Genetic and mechanistic analysis has defined LKB1-deficient NSCLC tumors as a phenotypically distinct subpopulation of NSCLC with potential avenues for therapeutic gain. In expanding on previous work indicating hypersensitivity of LKB1-deficient NSCLC cells to 2-deoxy-D-glucose (2DG), we find that 2DG has in vivo efficacy in LKB1-deficient NSCLC using transgenic murine models of NSCLC. Deciphering of the molecular mechanisms behind this phenotype reveals that loss of LKB1 in NSCLC cells imparts increased sensitivity to pharmacological compounds that aggravate ER stress. In comparison to NSCLC cells with functional LKB1, treatment of NSCLC cells lacking LKB1 with the ER stress activators (ERSA), tunicamycin, brefeldin A or 2DG, resulted in aggravation of ER stress, increased cytotoxicity, and evidence of ER stress-mediated cell death. Based upon these findings, we suggest that ERSAs represent a potential treatment avenue for NSCLC patients whose tumors are deficient in LKB1.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Desoxiglucose/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Serina-Treonina Quinases/deficiência , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP , Animais , Apoptose/efeitos dos fármacos , Brefeldina A/farmacologia , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Imageamento por Ressonância Magnética , Camundongos Knockout , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Espécies Reativas de Oxigênio/metabolismo , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos
4.
J Thorac Cardiovasc Surg ; 145(2): 531-8, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23142123

RESUMO

BACKGROUND: Only local ablation (radiofrequency ablation, cryotherapy) or esophagectomy currently is available to treat high-grade dysplasia in Barrett's esophagus. Alternative treatments, specifically chemopreventive strategies, are lacking. Our understanding of the molecular changes of high-grade dysplasia in Barrett's esophagus offers an opportunity to inhibit neoplastic progression of high-grade dysplasia in Barrett's esophagus. Increased activity of the Src kinase and deregulation of the tumor suppressor p27 are features of malignant cells and high-grade dysplasia in Barrett's esophagus. Src phosphorylates p27, inhibiting its regulatory function and increasing cell growth and proliferation. We hypothesized that a small molecule inhibitor of Src might reduce the growth and reverse Src-mediated deregulation of p27 in Barrett's esophagus cells. METHODS: Immortalized Barrett's esophagus cell lines established from patient biopsies were treated with the Src kinase inhibitor dasatinib and evaluated for p27 localization and protein levels, as well as for effects on the cell cycle and apoptosis using flow cytometry, viability assays, and protein and RNA markers. RESULTS: Dasatinib reduced both Src activation and p27 phosphorylation and increased p27 protein levels and nuclear localization. These effects correlated with decreased proliferation, cell-cycle arrest, and activation of apoptosis. Analysis of biopsies of patients with Barrett's esophagus revealed the presence of phosphorylated p27 in high-grade dysplasia, consistent with in vitro findings. CONCLUSIONS: Dasatinib has considerable antineoplastic effects on Barrett's esophagus cell lines carrying genetic markers associated with dysplasia, which correlates with the reversal of p27 deregulation. These findings suggest that dasatinib has potential as a treatment for patients with high-grade dysplasia and Barrett's esophagus and that p27 holds promise as a biomarker in the clinical use of dasatinib in patients with high-grade dysplasia and Barrett's esophagus.


Assuntos
Apoptose/efeitos dos fármacos , Esôfago de Barrett/enzimologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Esofágicas/enzimologia , Esôfago/efeitos dos fármacos , Lesões Pré-Cancerosas/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia , Quinases da Família src/antagonistas & inibidores , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Biópsia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Dasatinibe , Relação Dose-Resposta a Droga , Ativação Enzimática , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Esôfago/enzimologia , Esôfago/patologia , Citometria de Fluxo , Humanos , Fosforilação , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismo
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