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1.
Proteomics ; 24(15): e2400071, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38700387

RESUMO

Plasma is an abundant source of proteins and potential biomarkers to aid in the detection, diagnosis, and prognosis of human diseases. These proteins are often present at low levels in the blood and difficult to identify and measure due to the large dynamic range of proteins. The goal of this work was to characterize and compare various protein precipitation methods related to how they affect the depth and breadth of plasma proteomic studies. Abundant protein precipitation with perchloric acid (PerCA) can increase protein identifications and depth of plasma proteomic studies. Three acid- and four solvent-based precipitation methods were evaluated. All methods tested provided excellent plasma proteomic coverage (>600 identified protein groups) and detected protein in the low pg/mL range. Functional enrichment analysis revealed subtle differences within and larger changes between the precipitant groups. Methanol-based precipitation outperformed the other methods based on identifications and reproducibility. The methods' performance was verified using eight lung cancer patient samples, where >700 protein groups were measured and proteins with an estimated plasma concentration of ∼10 pg/mL were detected. Various protein precipitation agents are amenable to extending the depth and breadth of plasma proteomes. These data can guide investigators to implement inexpensive, high-throughput methods for their plasma proteomic workflows.


Assuntos
Proteínas Sanguíneas , Precipitação Química , Proteômica , Humanos , Proteômica/métodos , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/química , Proteoma/análise , Neoplasias Pulmonares/sangue , Espectrometria de Massas em Tandem/métodos
2.
Proteomics ; 22(17): e2200125, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35708973

RESUMO

Traditional data-independent acquisition (DIA) workflows employ off-column fractionation with data-dependent acquisition (DDA) to generate spectral libraries for data extraction. Recent advances have led to the establishment of library-independent approaches for DIA analyses. The selection of a DIA workflow may affect the outcome of plasma proteomics studies. Here, we establish a gas-phase fractionation (GPF) workflow to create DIA libraries for DIA with parallel accumulation and serial fragmentation (diaPASEF). This workflow along with three other workflows, fractionated DDA libraries, fractionated DIA libraries, and predicted spectra libraries, were evaluated on 20 plasma samples from nonsmall cell lung cancer patients with low or high levels of IL-6. We sought to optimize protein identification and total experiment time. The novel GPF workflow for diaPASEF outperformed the traditional ddaPASEF workflow in the number of identified and quantified proteins. A library-independent workflow based on predicted spectra identified and quantified the most proteins in our experiment at the cost of computational power. Overall, the choice of DIA library workflow seemed to have a limited effect on the overall outcome of a plasma proteomics experiment, but it can affect the number of proteins identified and the total experiment time.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Proteoma/metabolismo , Proteômica , Fluxo de Trabalho
3.
Proteomics ; 19(3): e1800135, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30585401

RESUMO

The current state of proteomics requires a choice between targeted and global discovery methods. A method, that combines targeted and data-independent acquisition for absolute quantification of all identified plasma proteins, in a single sequential window acquisition of all theoretical fragment ions (SWATH) acquisition run, using a panel of spike-in standards (SIS), is established and optimized. The absolute quantification (AQ) of SWATH and multiple-reaction monitoring-high resolution (MRM-HR) acquisition methods are compared using the 100 protein PlasmaDive SIS panel spiked into non-depleted human plasma. SWATH provides equivalent quantification and differentially abundant protein profiles as MRM-HR. Absolute quantities of the SIS peptides from the SWATH data are used to estimate the absolute quantities (eAQ) for all the proteins in the run. The eAQ values provide similar quantification and differentially abundant protein profiles as AQ and protein area (PA) values. As a proof-of-concept, the eAQ method is applied to 12 plasma samples from six non-small cell lung cancer (NSCLC) patients and the performance of eAQ values versus peak area quantification is evaluated. There is a strong correlation between AQ and peak area ratios producing significant overlap of differentially abundant proteins. This eAQ method can provide quantitative data equivalent to AQ or peak area values.


Assuntos
Biomarcadores Tumorais/sangue , Proteínas Sanguíneas/análise , Carcinoma Pulmonar de Células não Pequenas/sangue , Neoplasias Pulmonares/sangue , Humanos , Proteômica
4.
Proteomics ; 15(18): 3267-77, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26058877

RESUMO

Biomarkers to identify subjects at high-risk for developing lung cancer will revolutionize the disease outlook. Most biomarker studies have focused on patients already diagnosed with lung cancer and in most cases the disease is often advanced and incurable. The objective of this study was to use proteomics to identify a plasma biomarker for early detection of lung lesions that may subsequently be the harbinger for cancer. Plasma samples were obtained from subjects without lung cancer grouped as never, current, or ex-smokers. An iTRAQ-based proteomic analysis was performed on these pooled plasma samples. We identified 31 proteins differentially abundant in current smokers or ex-smokers relative to never smokers. Western blot and ELISA analyses confirmed the iTRAQ results that demonstrated an increase of apolipoprotein E (APOE) in current smokers as compared to both never and ex-smokers. There was a strong and significant correlation of the plasma APOE levels with development of premalignant squamous metaplasia. Additionally, we also showed that higher tissue levels of APOE are seen with squamous metaplasia, supporting a direct relationship. Our analysis reveals that elevated plasma APOE is associated with smoking, and APOE is a novel predictive protein biomarker for early morphological changes of squamous metaplasia in the lung.


Assuntos
Apolipoproteínas E/análise , Biomarcadores Tumorais/sangue , Proteínas Sanguíneas/análise , Pulmão/patologia , Metaplasia/sangue , Fumar/sangue , Adulto , Idoso , Feminino , Humanos , Marcação por Isótopo , Masculino , Metaplasia/epidemiologia , Pessoa de Meia-Idade , Proteômica/métodos , Curva ROC , Fumar/epidemiologia , Adulto Jovem
5.
J Biol Chem ; 288(23): 16348-16360, 2013 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-23625920

RESUMO

In lung cancers, TTF-1 displays seemingly paradoxical activities. Although TTF-1 is amplified in primary human lung cancers, it inhibits primary lung tumors from metastasizing in a mouse model system. It was reported that the oncogenic proepithelial mesenchymal transition (EMT) high mobility group AT-hook 2 gene (HMGA2) mediates the antimetastatic function of TTF-1. To gain mechanistic insight into the metastasis-critical signaling axis of TTF-1 to HMGA2, we used both reverse and forward strategies and discovered that microRNA-33a (miR-33a) is under direct positive regulation of TTF-1. By chromatin immunoprecipitation, we determined that TTF-1 binds to the promoter of SREBF2, the host gene of miR-33a. The 3'-untranslated region (UTR) of HMGA2 contains three predicted binding sites of miR-33a. We showed that the first two highly conserved sites are conducive to HMGA2 repression by miR-33a, establishing HMGA2 as a genuine target of miR-33a. Functional studies revealed that enforced expression of miR-33a inhibits the motility of lung cancer cells, and this inhibition can be rescued by overexpression of the form of HMGA2 without the 3'-UTR, suggesting that TTF-1 keeps the prometastasis gene HMGA2 in check via up-regulating miR-33a. This study reports the first miRNAs directly regulated by TTF-1 and clarifies how TTF-1 controls HMGA2 expression. Moreover, the documented importance of SREBF2 and miR-33a in regulating cholesterol metabolism suggests that TTF-1 may be a modulator of cholesterol homeostasis in the lung. Future studies will be dedicated to understanding how miRNAs influence the oncogenic activity of TTF-1 and the role of TTF-1 in cholesterol metabolism.


Assuntos
Regiões 3' não Traduzidas/fisiologia , Proteína HMGA2/biossíntese , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Elementos de Resposta/fisiologia , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Colesterol/genética , Colesterol/metabolismo , Proteína HMGA2/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , MicroRNAs/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Proteínas Nucleares/genética , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/biossíntese , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/genética
6.
Proteomics Clin Appl ; 18(1): e2300006, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37650339

RESUMO

PURPOSE: Plasma is an abundant source of protein biomarkers. Mass spectrometry (MS) is an effective means to measure a large number of proteins in a single run. The recent development of data-independent acquisition with parallel accumulation and serial fragmentation (diaPASEF) on a trapped ion mobility spectrometer (TIMS) affords deep proteomic coverage with short liquid chromatography gradients. In this work, we utilized a process optimization approach, design of experiments (DoE), to maximize precursor identification for a plasma proteomic diaPASEF workflow. EXPERIMENTAL DESIGN: A partial factorial design was used to screen 11 sample preparation factors and six diaPASEF MS acquisition factors. Selected factors were optimized using the response surface method. RESULTS: Three important sample preparation factors and the two important MS acquisition factors were identified in the screening experiments and were selected for separate optimization experiments. The optimal parameters were compared to our standard plasma proteomics workflows using either a 1-h or overnight trypsin digestion. The optimized method outperformed the 1-h digestion, and it was similar in performance to the overnight digestion, however, the optimized method could be completed in a day. CONCLUSION AND CLINICAL RELEVANCE: We have used DoE to report an optimized plasma proteomics workflow for diaPASEF, however, established methods are already highly optimized, and resources may be better spent on running samples than comprehensive optimization.


Assuntos
Proteômica , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Fluxo de Trabalho , Proteômica/métodos , Biomarcadores , Proteoma/análise
7.
J Biol Chem ; 287(34): 28790-801, 2012 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-22761434

RESUMO

The thyroid transcription factor 1 gene (TTF-1 or NKX2-1) is essential to lung development; however, it is also a critical factor in lung cancer. TTF-1 is amplified in lung cancers, suggesting that it is a gain-of-function lung oncogene. Conversely, TTF-1 counters epithelial to mesenchymal transition in cell-based studies and inhibits progression of primary lung adenocarcinomas to metastases in an animal model of lung adenocarcinomas. The unifying theory regarding TTF-1 is that it exhibits both pro-oncogenic and anti-metastatic function depending on the cellular context. Occludin is the first discovered constituent of the epithelial tight junction; in recent years, a functional role of occludin as a tumor suppressor has begun to emerge. Here, we demonstrate that TTF-1 transactivated the expression of the epithelial tight junction molecules occludin (OCLN) and claudin-1 (CLDN1). We show that transcriptional activation occurred through a direct interaction of TTF-1 with the OCLN and CLDN1 promoters. Furthermore, in cells that lack TTF-1, exogenous TTF-1 expression dampened the inhibitory effect of TGF-ß on occludin and claudin-1 content. Using cells derived from a genetically engineered mouse model of lung adenocarcinomas, we observed that silenced TTF-1 expression down-regulated occludin, which we supported with additional siRNA experiments. Finally, TTF-1 knockdown conferred human lung cancer cells resistance to anoikis, and expression of occludin restored cellular sensitivity to anoikis. Overexpression of occludin impeded migration and induced anoikis in lung carcinoma cells. Collectively, these data suggest that TTF-1 transcriptionally regulates occludin, which represents another avenue of TTF-1-mediated metastasis suppression.


Assuntos
Adenocarcinoma/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/biossíntese , Neoplasias Experimentais/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/biossíntese , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Anoikis/genética , Linhagem Celular Tumoral , Movimento Celular , Claudina-1 , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Membrana/genética , Camundongos , Metástase Neoplásica , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteínas Nucleares/genética , Ocludina , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/genética , Transcrição Gênica/genética , Proteínas Supressoras de Tumor/genética
8.
9.
PLoS One ; 16(12): e0260988, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34855926

RESUMO

Blood-based biomarkers including systemic inflammation (SI) indicators or circulating factors (cytokines, chemokines, or growth factors) are associated with a poor prognosis for lung cancer patients. Collectively these biomarkers can predict the immune state of a patient. We wanted to define and compare the immune states of small cell and non-small cell lung cancer patients, in the hopes that the information gained could lead to overall improvements in patient care and outcomes. Specimens and data from 235 patients was utilized, 49 surgically resected non-small cell lung cancer (NSCLC) patients with no evidence of disease (DF), 135 advanced non-small cell lung cancer (NSCLC), 51 small cell lung cancer (SCLC). SI markers neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte (PLR), systemic inflammation index (SII), and systemic inflammation response index (SIRI) were determined from blood counts. Forty-seven plasma cytokines were measured using a multiplex bead-based assay. Progression-free survival (PFS) and overall survival (OS) were assessed using Kaplan-Meier and Cox Proportional Hazards models. NSCLC patients had significantly high levels of SI markers than SCLC and DF patients, while NLR, PLR and SII were also higher in SCLC than DF patients. SI optimized marker values to differentiate SI value were; 6.04 (NLR), 320 (PLR), 1615 (SII), and 7.3 (SIRI). Elevated levels NLR (p<0.001), PLR (p<0.001), and SII (p = 0.018) were associated with a worse PFS and OS in NSCLC, while none of the markers were associated with PFS in SCLC patients. NSCLC patients with a poor outcome displayed heterogeneous immune states relative to systemic inflammation and circulating IL-6 markers. These groups could be distinguished based on the cytokines IL-8, TNFα, and IL-27. We identified heterogeneity of immune states in SCLC and NSCLC patients and in NSCLC patients with the poorest prognosis. This heterogeneity could be exploited to improve outcomes for these patients.


Assuntos
Biomarcadores Tumorais/sangue , Plaquetas/patologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Mediadores da Inflamação/sangue , Linfócitos/patologia , Neutrófilos/patologia , Carcinoma de Pequenas Células do Pulmão/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Terapia Combinada , Citocinas/sangue , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Pneumonectomia/mortalidade , Prognóstico , Carcinoma de Pequenas Células do Pulmão/sangue , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/terapia , Taxa de Sobrevida
10.
Lung Cancer ; 141: 97-100, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31986371

RESUMO

OBJECTIVES: Precision medicine with molecular profiling has revolutionized the management of lung cancer leading to improved outcomes. Patients with actionable mutations receive targeted therapy. As next-generation sequencing (NGS) becomes standard in lung cancer clinics, we sought to use molecular information to identify novel pathways to target in order to improve survival for non-small cell lung cancer (NSCLC) patients. MATERIALS AND METHODS: This retrospective analysis included 183 lung cancer patients who received commercial NGS sequencing as part of their clinical care, as well as the lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) dataset from the Cancer Genome Atlas (TCGA). We grouped mutations using a transcription factor enrichment analysis (TFEA), and the resulting TFEA groups were used to sort patients for survival analyses. RESULTS: Mutations connected to transcription factor 7 like 2/ Transcription Factor 4 (TCF7L2/TCF4) were associated with poor survival in NSCLC patients. Furthermore, Mutations in CCND1, IDH1, SMARC4, and TP53 are the primary contributors to a poor prognosis in these patients. This four gene panel was also found to be associated with a poor prognosis in the LUAD data of TCGA dataset. CONCLUSIONS: We determined that the TCF7L2 pathway is associated with a poor prognosis in patients with lung adenocarcinoma. Therefore, targeting the TCF7L2 pathway may improve outcomes for this group of patients.


Assuntos
Adenocarcinoma de Pulmão/patologia , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/patologia , Neoplasias Pulmonares/patologia , Mutação , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Masculino , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida
11.
J Thorac Oncol ; 15(11): 1727-1737, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32866653

RESUMO

In the summer of 2019, there was a rise in clusters of adolescents and young adults in the United States reporting to emergency departments with acute respiratory distress related to use of e-cigarette (electronic cigarette) or vaping. The number of patients with e-cigarette or vaping-associated lung injury continued to rise through the summer before peaking in September 2019. Through the efforts of state and federal public health agencies, officials were able to define the condition, identify the relationship of the respiratory injury to tetrahydrocannabinol-containing products, and stem the rise in new cases. In this report, we present a comprehensive review of the clinical characteristics and features of patients with e-cigarette or vaping-associated lung injury and guidelines for patient care and management to inform and navigate clinicians who may encounter these patients in their clinical practice.


Assuntos
COVID-19 , Sistemas Eletrônicos de Liberação de Nicotina , Lesão Pulmonar , Neoplasias Pulmonares , Vaping , Adolescente , Feminino , Humanos , Lesão Pulmonar/epidemiologia , Lesão Pulmonar/etiologia , Lesão Pulmonar/terapia , Masculino , Pandemias , SARS-CoV-2 , Estados Unidos/epidemiologia , Vaping/efeitos adversos , Adulto Jovem
12.
J Thorac Oncol ; 15(7): 1119-1136, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32422364

RESUMO

The global coronavirus disease 2019 pandemic continues to escalate at a rapid pace inundating medical facilities and creating substantial challenges globally. The risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in patients with cancer seems to be higher, especially as they are more likely to present with an immunocompromised condition, either from cancer itself or from the treatments they receive. A major consideration in the delivery of cancer care during the pandemic is to balance the risk of patient exposure and infection with the need to provide effective cancer treatment. Many aspects of the SARS-CoV-2 infection currently remain poorly characterized and even less is known about the course of infection in the context of a patient with cancer. As SARS-CoV-2 is highly contagious, the risk of infection directly affects the cancer patient being treated, other cancer patients in close proximity, and health care providers. Infection at any level for patients or providers can cause considerable disruption to even the most effective treatment plans. Lung cancer patients, especially those with reduced lung function and cardiopulmonary comorbidities are more likely to have increased risk and mortality from coronavirus disease 2019 as one of its common manifestations is as an acute respiratory illness. The purpose of this manuscript is to present a practical multidisciplinary and international overview to assist in treatment for lung cancer patients during this pandemic, with the caveat that evidence is lacking in many areas. It is expected that firmer recommendations can be developed as more evidence becomes available.


Assuntos
Infecções por Coronavirus , Controle de Infecções/organização & administração , Neoplasias Pulmonares/terapia , Pandemias , Administração dos Cuidados ao Paciente , Pneumonia Viral , Betacoronavirus/isolamento & purificação , COVID-19 , Comorbidade , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/terapia , Humanos , Comunicação Interdisciplinar , Cooperação Internacional , Neoplasias Pulmonares/epidemiologia , Neoplasias Pulmonares/patologia , Estadiamento de Neoplasias , Pandemias/prevenção & controle , Administração dos Cuidados ao Paciente/métodos , Administração dos Cuidados ao Paciente/organização & administração , Administração dos Cuidados ao Paciente/tendências , Pneumonia Viral/epidemiologia , Pneumonia Viral/prevenção & controle , Pneumonia Viral/terapia , Prognóstico , SARS-CoV-2
13.
Lung Cancer ; 131: 58-61, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31027698

RESUMO

OBJECTIVES: High levels of circulating interleukin-6 (IL-6) are associated with a poor prognosis in many types of cancer including non-small cell lung cancer (NSCLC). While the inflammatory cytokine can stimulate the immune system and promote tumor growth, it remains unclear how circulating IL-6 can potentiate a poor prognosis. We hypothesized that a mechanism for IL-6-associated poor prognosis is that these patients would have altered T-cell populations and impaired T-cell signaling. MATERIALS AND METHODS: Plasma levels of IL-6 were measured using a Cytometric Bead Array. T-cell populations from Non-small cell lung cancer patients were characterized using surface markers by flow cytometry, and signaling in the T-cell populations were measured by PhosFlow cytometry. RESULTS: We determine that patients with high circulating IL-6 levels had distinct T cell characteristics relative to those with low levels. Patients with high levels of IL-6 had significantly more Treg cells and elevated Programmed cell death protein-1 (PD-1) expression on CD4+, CD8+, Treg, and Th17 cells. These patients also showed impaired signal transducer and activator of transcription-1 (STAT1) signaling upon stimulation with IL-6 and phorbol 12-myristate 13-acetate (PMA), and T-Cells from a healthy donor that were treated for four days with IL-6 displayed a similar muting of STAT signaling, which verified the effect seen in patient samples. CONCLUSIONS: This work directly links circulating IL-6 with other poor prognostic indicators, STAT1 and PD-1, and highlights the effects of circulating IL-6 on the immune system. Our data suggest that alteration in T cell populations and function may be a mechanism underlying the poor prognosis seen in NSCLC patients with high IL-6 levels.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Interleucina-6/sangue , Neoplasias Pulmonares/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Circulação Sanguínea , Células Cultivadas , Citometria de Fluxo , Humanos , Imunofenotipagem , Estadiamento de Neoplasias , Receptor de Morte Celular Programada 1/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Regulação para Cima
14.
PLoS One ; 14(5): e0217657, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31150457

RESUMO

Lung cancer is among the common and deadly cancers. Although the treatment options for late-stage cancer patients have continued to increase in numbers, the overall survival rates for these patients have not shown significant improvement. This highlights the need for new targets and drugs to more effectively treat lung cancer patients. In this study, we characterize the MCL-1 inhibitor maritoclax alone or in combination with a BCL-2/xL inhibitor in a panel of lung cancer cell lines. BCL-2 family proteins, phosphorylated proteins, and apoptosis were monitored following the treatments. We found that maritoclax was effective at inhibiting growth in these lung cancer cells. We also establish that cell lines with EGFR mutations were most sensitive to the combined inhibition of MCL-1 and BCL-2/xL. In addition, a high level of phosphorylated AKT (S473) was identified as a marker for sensitivity to the combination treatment. This work has defined EGFR mutations and AKT phosphorylation as markers for sensitivity to combined MCL-1 and BCL-2/xL targeted therapy and establishes a rationale to explore multiple BCL-2 family members in patients who are refractory to EGFR inhibitor treatment. Our data support the design of a clinical trial that aims to employ inhibitors of the BCL-2 family of proteins in lung cancer patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Citometria de Fluxo , Humanos , Mutação/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína Oncogênica v-akt/genética , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Pirróis/farmacologia , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/genética
15.
Histol Histopathol ; 34(5): 479-490, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30302745

RESUMO

INTRODUCTION: Port wine stain (PWS) is characterized as a progressive dilatation of immature venule-like vasculatures which result from differentiation-impaired endothelial cells. In this study, we aimed to identify the major biological pathways accounting for the pathogenesis of PWS. METHODS: Sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) was used to identify differentially expressed proteins in PWS lesions, followed by confirmative studies with immunohistochemistry, immunoblot and transmission electron microscopy (TEM). RESULTS: 107 out of 299 identified proteins showed differential expressions in PWS lesions as compared to normal skin, mainly involving the functions of biosynthesis, membrane trafficking, cytoskeleton and cell adhesion/migration. The confirmative studies showed that expressions of membrane trafficking/exocytosis related proteins such as VAT1, IQGAP1, HSC70, clathrin, perlecan, spectrin α1 and GDIR1 were significantly increased in PWS blood vessels as compared to normal ones; while collagen subtypes 6A1 and 6A3 were decreased in PWS skin. Furthermore, TEM studies showed there is a significant upregulation of extracellular vesicle exocytosis from PWS blood vessels as compared to control. CONCLUSIONS: The biological process of membrane trafficking and exocytosis is enhanced in PWS blood vessels. Our results imply that the extracellular vesicles released by lesional endothelial cells may act as potential intercellular signaling mediators to contribute to the pathogenesis of PWS.


Assuntos
Exocitose/fisiologia , Mancha Vinho do Porto/metabolismo , Transporte Proteico/fisiologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Mancha Vinho do Porto/patologia , Regulação para Cima
16.
J Plant Physiol ; 164(7): 815-23, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17240476

RESUMO

The influx of Ca(2+) into the cytosol has long been suggested to serve as a signaling intermediate in the acquisition of tolerance to hyperosmotic and/or salinity stresses. Here we use aequorin-transformed suspension-cultured tobacco cells to directly assess the role of cytosolic calcium (Ca(2+)(cyt)) signaling in salinity tolerance acquisition. Aequorin luminescence recordings and (45)Ca influx measurements using inhibitors of Ca(2+) influx (Gd(3+) and the Ca(2+)-selective chelator EGTA), and modulators of organellar Ca(2+) release (phospholipase C inhibitors U73122 or neomycin) demonstrate that hyperosmolarity, whether imposed by NaCl or by a non-ionic molecule sorbitol, induces a rapid (returning to baseline levels of Ca(2+) within 10 min) and complex Ca(2+)(cyt) pulse in tobacco cells, deriving both from Gd(3+)-sensitive externally derived Ca(2+) influx and from U73122- and neomycin-sensitive Ca(2+) release from an organelle. To determine whether each of the two components of this brief Ca(2+) signal regulate adaptation to hyperosmotic shock, the Ca(2+) pulse was modified by the addition of Gd(3+), U73122, neomycin, or excess Ca(2+), and then cells were treated with salt or sorbitol. After 10 min the cell culture medias were diluted with additional hyperosmotic media to reduce the toxic affects of the modulators, and the growth of cells was measured after 1 week. Gd(3+) treatment reduced growth in salt relative to control cells but not in sorbitol, and exposure to excess Ca(2+) increased growth in salt but not in sorbitol. In contrast, exposure to inhibitors of IP(3) formation had no effect on growth in salt or sorbitol. Therefore, although hyperosmotic treatment stimulates both Ca(2+) influx and Ca(2+) release from an internal Ca(2+) depot, only Ca(2+) influx has a measurable impact on ionic stress tolerance acquisition in tobacco cell suspensions. In contrast, osmoadaptation in these cells appears to occur independent of Ca(2+) signaling.


Assuntos
Adaptação Fisiológica , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Nicotiana/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Citosol/química , Pressão Osmótica , Cloreto de Sódio/farmacologia , Sorbitol/farmacologia , Nicotiana/efeitos dos fármacos , Nicotiana/fisiologia , Fosfolipases Tipo C/metabolismo
17.
Artigo em Inglês | MEDLINE | ID: mdl-28966872

RESUMO

Extracellular vesicles (EVs) can mediate intercellular communication by transferring cargo proteins and nucleic acids between cells. The pathophysiological roles and clinical value of EVs are under intense investigation, yet most studies are limited by technical challenges in the isolation of nanoscale EVs (nEVs). Here, we report a lipid nanoprobe that enables spontaneous labelling and magnetic enrichment of nEVs in 15 minutes, with isolation efficiency and cargo composition similar to what can be achieved by the much slower and bulkier method of ultracentrifugation. We also show that the lipid nanoprobes, which allow for downstream analyses of nucleic acids and proteins, enabled the identification of EGFR and KRAS mutations following nEV isolation from blood plasma from non-small-cell lung-cancer patients. The efficiency and versatility of the lipid nanoprobe opens up opportunities in point-of-care cancer diagnostics.

18.
PM R ; 8(10): 990-996, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27060645

RESUMO

Lung cancer remains a challenging disease with high morbidity and mortality despite targeted therapy. Symptom burden related to cancer impairs quality of life and functional status in patients with lung cancer and in survivors. Pulmonary rehabilitation has been recognized as an effective, noninvasive intervention for patients with chronic respiratory disease. It is well established that pulmonary rehabilitation benefits patients with chronic obstruction pulmonary disease through improved exercise capacity and symptoms. Evidence is increasing that the benefit of pulmonary rehabilitation can be applied to patients with lung cancer. Comprehensive pulmonary rehabilitation has made its way as a cornerstone of integrated care for patients with lung cancer. LEVEL OF EVIDENCE: V.


Assuntos
Neoplasias Pulmonares , Exercício Físico , Terapia por Exercício , Tolerância ao Exercício , Humanos , Qualidade de Vida
19.
Curr Mol Pharmacol ; 9(3): 183-195, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26177648

RESUMO

Lung cancer is among the most prevalent and deadly cancers. Although the development of targeted drugs, erlotinib and crizotinib, has improved lung cancer management, survival rates of lung cancer patients have not shown significant improvement over the past decade. Better therapeutic options are required to treat lung cancer patients. Immunotherapy is a maturing and rapidly growing field, which has recently contributed many novel strategies for addressing cancer treatment. Here, we discuss the current state of cancer vaccines, immune checkpoint blockers, and adoptive cellular therapies, as novel clinical treatment strategies for non-small cell lung cancer. The durability of clinical activity in a subset of patients has led to a great deal of excitement and optimism.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Imunoterapia/tendências , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Vacinas Anticâncer/imunologia , Humanos , Fatores Imunológicos/farmacologia
20.
Oncotarget ; 7(35): 56233-56240, 2016 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-27191652

RESUMO

Recent success of using agents inhibiting the major immune check point, programmed cell death-1 (PD-1) pathway, offers a great promise for effective cancer therapy. Two blocking antibodies for PD-1, nivolumab and pembrolizumab have recently been approved for treating advanced recurrent non-small cell lung cancer (NSCLC). Activation of PD-1 on T cells and PD-L1 on tumor cells or antigen presenting cells leads to T cell exhaustion and ultimately tumor growth. In this study, we performed flow cytometry analysis of peripheral blood samples collected from patients with advanced NSCLC at initial diagnosis. We report that surface expression of PD-1 on CD4+ T cells has a prognostic value in NSCLC patients, as high expression of PD-1 is associated with a shorter progression-free survival and overall survival. Importantly, we also found that high PD-1 expression on peripheral CD4+ T cells is associated with inferior clinical response in a subset of patients who received anti-PD-L1 treatment, indicating a potential predictive value of this marker. This work highlights the potential of a non-invasive and effective method to determine prognostic and predictive biomarkers for inhibiting the PD-1 pathway in NSCLC patients.


Assuntos
Antígeno B7-H1/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Receptor de Morte Celular Programada 1/metabolismo , Idoso , Idoso de 80 Anos ou mais , Células Apresentadoras de Antígenos/metabolismo , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Linfócitos T CD4-Positivos/ultraestrutura , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Membrana Celular/metabolismo , Intervalo Livre de Doença , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Receptor de Morte Celular Programada 1/sangue , Resultado do Tratamento
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