RESUMO
Lung cancer is the primary cause of cancer-associated mortality worldwide, which makes the identification of reliable lung cancer biomarkers a pressing need for early diagnosis and prognosis. RGS11, which is a regulator of G-protein signaling and also a lung cancer biomarker, plays an important role in cancer-related metastasis. However, trace levels of RGS11 (in the range of pg/mL) in serum samples make it difficult to quantify using currently available enzyme-linked immunosorbent assay (ELISA) kits and, therefore, this hinders progress in the discovery of new approaches for treating lung cancer. The aim of this study is to develop a rapid, sensitive, and reliable platform for the detection of RGS11 lung cancer biomarker based on a suspension immunoassay coupled with an isothermal exponential amplification strategy. Our study was initiated by the functionalization of magnetic beads with anti-RGS11 antibodies (Ab-MB) by EDC (1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide)/NHS ( N-hydroxysulfosuccinimide) activation. Ab-MB served as a sensing probe for the competitive immunorecognitions between known concentrations of His-tag RGS11 and unknown concentrations of target RGS11 in serum. The reporter anti-His antibodies, which were modified with primers that induced an isothermal exponential amplification reaction, were subsequently introduced to the reaction mixture that resulted in the formation of immunosandwich complexes. The exponentially amplified DNA duplex that was intercalated with SYBR Green was designated as a signal reporter for the assessment of RGS11 in an inversely proportional relationship. The sensing platform was excellent for the determination of RGS11 with an exceptional detection limit of 148 fg/mL and a linear dynamic range of 0.1-10 pg/mL using a minimal sample volume (20 µL) and with a reaction time of 1.5 h. In addition, we challenged the sensing platform with RGS11-spiked samples (in 2× diluted serum), and an acceptable recovery rate (>90%) was observed. Finally, 24 clinical samples acquired from patients with advanced lung cancer (C), inflammation (I), and heart failure (H) were analyzed by this newly developed sensing platform and a commercial ELISA kit for validation. This sensing platform has potential in biomedical applications for clinically diagnosing liquid biopsy samples for patients with lung cancer. Moreover, the universal design of our proposed system is easily adapted to detect any other protein if a His-tag recombinant protein is available.
Assuntos
Biomarcadores Tumorais/sangue , Imunoensaio/métodos , Neoplasias Pulmonares/diagnóstico , Proteínas RGS/sangue , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Reações Antígeno-Anticorpo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Histidina/genética , Histidina/imunologia , Histidina/metabolismo , Humanos , Limite de Detecção , Neoplasias Pulmonares/metabolismo , Magnetismo , Técnicas de Amplificação de Ácido Nucleico , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Oligopeptídeos/metabolismo , Proteínas RGS/genética , Proteínas RGS/imunologiaRESUMO
Gastrointestinal stromal tumor (GIST) is a type of KIT-driven cancer. KIT gene mutations are found in approximately 80% of GISTs, and most of these mutations occur in exon 9 and exon 11. Imatinib has been successfully used as a first-line treatment for advanced GIST, with a significant improvement in progression-free survival (PFS) and overall survival. However, disease progression might develop due to primary or secondary resistance to imatinib. Sunitinib and regorafenib have been approved as second- and third-line treatments for advanced GIST patients, with median PFS values of 6.8 and 4.8 months, respectively. However, these relatively modest improvements in PFS underscore the need for more effective KIT inhibitors. BPR1J373 is a multitargeted kinase inhibitor that has been shown to inhibit the proliferation of KIT-driven acute myeloid leukemia cells in vitro and in vivo. In this study, we found that BPR1J373 inhibited proliferation and induced apoptosis by targeting KIT in GIST cells with KIT gene mutations. BPR1J373 also induced cell cycle arrest and senescent change in KIT-mutant GIST48 cells, probably by targeting aurora kinase A. In the KIT-null COS-1 cell-based system, BPR1J373 effectively inhibited KIT with single or double mutations of KIT developed in GIST. The antiproliferative effect was also consistently evident in GIST430 tumor-grafted mice. The results suggest that BPR1J373 could be a potential anticancer drug for GIST and deserves further investigation for clinical applications.
Assuntos
Tumores do Estroma Gastrointestinal/tratamento farmacológico , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-kit/genética , Pirimidinas/administração & dosagem , Animais , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Tumores do Estroma Gastrointestinal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
SOX2 is a transcription factor essential for self-renewal and pluripotency of embryonic stem cells. Recently, SOX2 was found overexpressed in the majority of the lung squamous cell carcinoma (SQC), in which it acts as a lineage-survival oncogene. However, downstream targets/pathways of SOX2 in lung SQC cells remain to be identified. Here, we show that BMP4 is a downstream target of SOX2 in lung SQC. We found that SOX2-silencing-mediated inhibition of cell growth was accompanied by upregulation of BMP4 mRNA and its protein expression. Meta-analysis with 293 samples and qRT-PCR validation with 73 clinical samples revealed an inversely correlated relationship between levels of SOX2 and BMP4 mRNA, and significantly lower mRNA levels in tumor than in adjacent normal tissues. This was corroborated by immunohistochemistry analysis of 35 lung SQC samples showing lower BMP4 protein expression in tumor tissues. Cell-based experiments including siRNA transfection, growth assay and flow cytometry assay, further combined with a xenograft tumor model in mice, revealed that reactivation of BMP4 signaling could partially account for growth inhibition and cell cycle arrest in lung SQC cells upon silencing SOX2. Finally, chromatin immunoprecipitation analysis and luciferase reporter assay revealed that SOX2 could negatively regulate BMP4 promoter activity, possibly through binding to the promoter located in the first intron region of BMP4. Collectively, our findings suggest that BMP4 could act as a tumor suppressor and its downregulation by elevated SOX2 resulting in enhanced growth of lung SQC cells.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Pulmonares/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Bases de Dados Genéticas , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Camundongos , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Transdução de SinaisRESUMO
Lithium has been used or explored to treat psychiatric and neurodegenerative diseases that are frequently associated with an abnormal immune status. It is likely that lithium may work through modulation of immune responses in these patients. Because dendritic cells (DC) play a central role in regulating immune responses, this study investigated the influence of lithium chloride (LiCl) on the development and function of DC. Exposure to LiCl during the differentiation of human monocyte-derived immature DCs (iDC) enhances CD86 and CD83 expression and increases the production of IL-1ß, IL-6, IL-8, IL-10, and TNF-α. However, the presence of LiCl during LPS-induced maturation of iDC has the opposite effect. During iDC differentiation, LiCl suppresses the activity of glycogen synthase kinase (GSK)-3ß, and activates PI3K and MEK. In addition, LiCl activates peroxisome proliferator-activated receptor γ (PPARγ) during iDC differentiation, a pathway not described before. Each of these signaling pathways appears to have distinct impact on the differentiating iDC. The enhanced CD86 expression by LiCl involves the PI3K/AKT and GSK-3ß pathway. LiCl modulates the expression of CD83 in iDC mainly through MEK/ERK, PI3K/AKT, and PPARγ pathways, while the increased production of IL-1ß and TNF-α mainly involves the MEK/ERK pathway. The effect of LiCl on IL-6/IL-8/IL-10 secretion in iDC is mediated through inhibition of GSK-3ß. We have also demonstrated that PPARγ is downstream of GSK-3ß and is responsible for the LiCl-mediated modulation of CD86/83 and CD1 expression, but not IL-6/8/10 secretion. The combined influence of these molecular signaling pathways may account for certain clinical effect of lithium.
Assuntos
Células Dendríticas/efeitos dos fármacos , Células Dendríticas/fisiologia , Cloreto de Lítio/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos CD/imunologia , Antígeno B7-2/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/citologia , Inibidores Enzimáticos/metabolismo , Quinase 3 da Glicogênio Sintase/imunologia , Glicogênio Sintase Quinase 3 beta , Humanos , Imunoglobulinas/imunologia , Interleucinas/imunologia , Glicoproteínas de Membrana/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Monócitos/citologia , PPAR gama/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/imunologia , Antígeno CD83RESUMO
BACKGROUND: α-Enolase (ENO1) is a key glycolytic enzyme implicated in the development of many human cancers including breast cancer. Increased expression of ENO1 has recently been reported in estrogen (ER)-positive human breast cancer patients. The present study examined the expression of ENO1 and assessed its significance in canine mammary carcinoma. RESULTS: Immunohistochemical staining was employed to investigate the expression of ENO1 in 82 cases of canine mammary tumor (32 benign tumors and 50 carcinomas). Quantification of immunohistochemistry was carried out using Quick score and the results showed cytoplasmic ENO1 overexpression in 9 of the 50 carcinomas (18%). Overexpression of ENO1 correlated significantly with shorter cause-specific survival (P = 0.019), but was not associated with ER positivity in canine mammary carcinoma. CONCLUSIONS: Our findings suggest that overexpression of ENO1 may be used as a prognostic marker for poor outcome in canine mammary carcinoma.
Assuntos
Doenças do Cão/metabolismo , Neoplasias Mamárias Animais/metabolismo , Fosfopiruvato Hidratase/metabolismo , Animais , Biomarcadores/análise , Doenças do Cão/mortalidade , Cães , Feminino , Regulação Neoplásica da Expressão Gênica , Immunoblotting/veterinária , Glândulas Mamárias Animais/química , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/química , Neoplasias Mamárias Animais/mortalidade , Fosfopiruvato Hidratase/análiseRESUMO
The development of rapid and sensitive methods for the detection of immunogenic tumor-associated antigen is important not only for understanding their roles in cancer immunology but also for the development of clinical diagnostics. Alpha-enolase (ENO1), a p48 molecule, is widely distributed in a variety of tissues, whereas gamma-enolase (ENO2) and beta-enolase (ENO3) are found exclusively in neuron/neuroendocrine and muscle tissues, respectively. Because ENO1 has been correlated with small cell lung cancer, nonsmall cell lung cancer, and head and neck cancer, it can be used as a potential diagnostic marker for lung cancer. In this study, we developed a simple, yet novel and sensitive, electrochemical sandwich immunosensor for the detection of ENO1; it operates through physisorption of anti-ENO1 monoclonal antibody on polyethylene glycol-modified disposable screen-printed electrode as the detection platform, with polyclonal secondary anti-ENO1-tagged, gold nanoparticle (AuNP) congregates as electrochemical signal probes. The immunorecognition of the sample ENO1 by the congregated AuNP@antibody occurred on the surface of the electrodes; the electrochemical signal from the bound AuNP congregates was obtained after oxidizing them in 0.1 M HCl at 1.2 V for 120 s, followed by the reduction of AuCl(4-) in square wave voltammetry (SWV) mode. The resulting sigmoidally shaped dose-response curves possessed a linear dynamic working range from 10(-8) to 10(-12) g/mL. This AuNP congregate-based assay provides an amplification approach for detecting ENO1 at trace levels, leading to a detection limit as low as 11.9 fg (equivalent to 5 microL of a 2.38 pg/mL solution).
Assuntos
Antígenos de Neoplasias/análise , Técnicas Eletroquímicas/métodos , Ouro/química , Neoplasias Pulmonares/diagnóstico , Nanopartículas Metálicas/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/imunologia , Técnicas Biossensoriais/métodos , Eletrodos , Humanos , Fosfopiruvato Hidratase/análise , Fosfopiruvato Hidratase/imunologia , Polietilenoglicóis/químicaRESUMO
Enolase-alpha (ENO-1) is a key glycolytic enzyme that has been used as a diagnostic marker to identify human lung cancers. To investigate the role of ENO-1 in breast cancer diagnosis and therapy, the mRNA levels of ENO-1 in 244 tumor and normal paired tissue samples and 20 laser capture-microdissected cell clusters were examined by quantitative real-time PCR analysis. Increased ENO-1 mRNA expression was preferentially detected in estrogen receptor-positive (ER+) tumors (tumor/normal ratio >90-fold) when compared to ER-negative (tumor/normal ratio >20-fold) tumor tissues. The data presented here demonstrate that those patients whose tumors highly expressed ENO-1 had a poor prognosis with greater tumor size (>2 cm, *P = .017), poor nodal status (N > 3, *P = .018), and a shorter disease-free interval (<==1 year, *P < .009). We also found that higher-expressing ENO-1 tumors confer longer distance relapse (tumor/normal ratio = 82.8-92.4-fold) when compared to locoregional relapse (tumor/normal ratio = 43.4-fold) in postsurgical 4-hydroxy-tamoxifen (4-OHT)-treated ER+ patients (*P = .014). These data imply that changes in tumor ENO-1 levels are related to clinical 4-OHT therapeutic outcome. In vitro studies demonstrated that decreasing ENO-1 expression using small interfering RNA (siRNA) significantly augmented 4-OHT (100 nM)-induced cytotoxicity in tamoxifen-resistant (Tam-R) breast cancer cells. These results suggest that downregulation of ENO-1 could be utilized as a novel pharmacological approach for overcoming 4-OHT resistance in breast cancer therapy.
Assuntos
Antineoplásicos Hormonais/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fosfopiruvato Hidratase/metabolismo , Tamoxifeno/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/antagonistas & inibidores , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Células Cultivadas , Proteínas de Ligação a DNA/antagonistas & inibidores , Ensaios de Seleção de Medicamentos Antitumorais , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Fosfopiruvato Hidratase/antagonistas & inibidores , Prognóstico , RNA Mensageiro/análise , RNA Interferente Pequeno , Análise de Sobrevida , Proteínas Supressoras de Tumor/antagonistas & inibidoresRESUMO
OBJECTIVE: Elevation of serum autoantibodies to alpha-enolase (ENO1) is often seen in inflammation diseases. However, it is unclear whether the levels of serum ENO1 autoantibodies could be affected during tumor progression. Hence, we attempted to determine the relative serum ENO1 autoantibody levels in healthy individuals and various stages of patients with lung and breast cancers. METHODS: Sera were obtained from 99 normal individuals, 21 patients with non-cancer-associated diseases and 178 cancer patients, including Stage I, II and IV non-small cell lung cancer, small cell lung cancer and breast cancer. The ENO1 autoantibody levels were determined by enzyme-linked immunosorbent assay. RESULTS: Compared with the healthy individuals, the levels of ENO1 autoantibodies were significantly decreased in Stage IV non-small cell lung cancer, small cell lung cancer and breast cancer patients. Consistently, this phenomenon was also observed in tumor-grafted mice. Using logistic regression analyses, data show that the titer status of ENO1 autoantibody level is highly associated with the late stage of lung and breast cancers when compared with those of healthy controls. In contrast, there were no statistic differences between healthy controls and early stages of non-small cell lung cancer patients, and total amounts of serum immunoglobulin A, immunoglobulin G and immunoglobulin M levels in Stage IV non-small cell lung cancer patients were not significantly distinct from those of the healthy controls. Thus, the decreased ENO1 autoantibody event in malignant stage of cancer patients is not contributed by reduction in total immunoglobulin. CONCLUSIONS: Marked decrease in the basal level of serum ENO1 autoantibodies is a common malignant event of lung and breast cancers, suggesting that ENO1 autoantibody may serve as a prognostic marker to monitor the disease progression of these cancer patients.
Assuntos
Autoanticorpos/sangue , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/imunologia , Proteínas de Ligação a DNA/imunologia , Neoplasias Pulmonares/imunologia , Fosfopiruvato Hidratase/imunologia , Proteínas Supressoras de Tumor/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma de Células Pequenas/imunologia , Regulação para Baixo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Adulto JovemRESUMO
Atherosclerosis and its associated complications represent major causes of morbidity and mortality in the industrialized or Western countries. Monocyte chemoattractant protein-1 (MCP-1) is critical for the initiating and developing of atherosclerotic lesions. Interleukin-8 (IL-8), a CXC chemokine, stimulates neutrophil chemotaxis. Ticlopidine is one of the antiplatelet drugs used to prevent thrombus formation relevant to the pathophysiology of atherothrombosis. In this study, we found that ticlopidine dose-dependently decreased the mRNA and protein levels of TNF-alpha-stimulated MCP-1, IL-8, and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs). Ticlopidine declined U937 cells adhesion and chemotaxis as compared to TNF-alpha stimulated alone. Furthermore, the inhibitory effects were neither due to decreased HUVEC viability, nor through NF-kB inhibition. These results suggest that ticlopidine decreased TNF-alpha induced MCP-1, IL-8, and VCAM-1 levels in HUVECs, and monocyte adhesion. Therefore, the data provide additional therapeutic machinery of ticlopidine in treatment and prevention of atherosclerosis.
Assuntos
Quimiocina CCL2/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Interleucina-8/metabolismo , Ticlopidina/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais/citologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Quimiocina CCL2/genética , Quimiotaxia/efeitos dos fármacos , Células Endoteliais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-8/genética , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição RelA/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Background: Epithelial ovarian cancer (EOC) has a high tumor-associated mortality rate among gynecological cancers. Although CA125 is a well-studied biomarker for ovarian cancer, it is also elevated under numerous conditions, resulting in decreased specificity. Recently, we identified a novel tumor-associated antigen, salt-inducible kinase 3 (SIK3), during tumorigenesis in ovarian cancer. However, the association between SIK3 expression and patient outcomes in ovarian cancer remains unclear. Materials and Methods: We collected EOC samples from 204 patients and examined tumor SIK3 expression by immunohistochemistry (IHC) and CA125 expression in tumors and serum. The expression levels of SIK3 and CA125 were correlated with patient survival. SIK3 expression was silenced with SIK3-specific shRNAs to investigate the possible mechanisms related to chemoresistance in serous-type ovarian cancer cell lines OVCAR4 and SKOV3. Results: In advanced-stage serous ovarian cancer, patients with low SIK3 expression have poorer overall survival (OS) and progression-free survival (PFS) than patients with high SIK3 expression. Ovarian cancer cells with SIK3 knockdown display increased chemoresistance to Taxol plus cisplatin treatment, which is associated with the upregulation of the ABCG2 transporter. In addition, in serous ovarian cancer, SIK3 expression is inversely correlated to ABCG2 expression, and patients with low SIK3 and high ABCG2 expression have worse prognosis than patients with high SIK3 and low ABCG2 expression. Conclusion: Our results demonstrated that serous EOC patients with low SIK3 expression have poor prognosis, which is associated with chemoresistance mediated by ABCG2 upregulation. SIK3 and ABCG2 expression levels may be potential prognostic markers to predict the outcome in serous EOC patients.
RESUMO
BACKGROUND: Tumors expressing a transforming growth factor-beta type I receptor (T beta RI) mutant with sequence deletions in a nine-alanine (9A) stretch of the signal peptide are reported to be highly associated with disease progression. Expression of this mutant could interfere with endogenous TGFbeta signaling in the cell. However, little is known about the importance of the remaining part of the signal peptide on the cellular function of T beta RI. RESULTS: We cloned and identified four new in-frame deletion variants of T beta RI, designated DM1 to DM4, in pleural effusion-derived tumor cells. Intriguingly, DM1 and DM2, with a small region truncated in the putative signal peptide of T beta RI, had a serious defect in their protein expression compared with that of the wild-type receptor. Using serial deletion mutagenesis, we characterized a region encoded by nucleotides 16-51 as a key element controlling T beta RI protein expression. Consistently, both DM1 and DM2 have this peptide deleted. Experiments using cycloheximde and MG132 further confirmed its indispensable role for the protein stability of T beta RI. In contrast, truncation of the 9A-stretch itself or a region downstream to the stretch barely affected T beta RI expression. However, variants lacking a region C-terminal to the stretch completely lost their capability to conduct TGFbeta-induced transcriptional activation. Intriguingly, expression of DM3 in a cell sensitive to TGFbeta made it significantly refractory to TGFbeta-mediated growth inhibition. The effect of DM3 was to ablate the apoptotic event induced by TGFbeta. CONCLUSION: We identified four new transcript variants of T beta RI in malignant effusion tumor cells and characterized two key elements controlling its protein stability and transcriptional activation. Expression of one of variants bestowed cancer cells with a growth advantage in the presence of TGFbeta. These results highlight the potential roles of some naturally occurring T beta RI variants on the promotion of tumor malignancy.
Assuntos
Derrame Pleural Maligno/metabolismo , Isoformas de Proteínas , Proteínas Serina-Treonina Quinases , Receptores de Fatores de Crescimento Transformadores beta , Sequência de Aminoácidos , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Humanos , Dados de Sequência Molecular , Derrame Pleural Maligno/genética , Derrame Pleural Maligno/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sinais Direcionadores de Proteínas , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismoRESUMO
The major concern for severe acute respiratory syndrome (SARS), caused by the SARS-associated coronavirus (SARS-CoV), is the lack of diagnostic and therapeutic agents. Using a phage display technology in a chicken system, high-affinity monoclonal antibody fragments against the SARS-CoV spike protein were characterized. Ten truncated spike protein gene fragments were expressed in Escherichia coli cells. Following the immunization of chickens with these recombinant spike proteins, two single-chain variable fragment (scFv) antibody libraries were established with short or long linkers to contain 5x10(7) and 9x10(6) transformants, respectively. After four rounds of panning selection, the scFv antibodies of randomly chosen clones were demonstrated by Coomassie blue staining, and verified by western blot analysis. In a comparison of nucleotide sequences with the chicken germline gene, we found that all clones varied in the complementarity-determining regions, that two scFv antibodies reacted significantly with SARS-CoV-infected Vero cells, and that those two specific scFv antibodies recognized the same region of the spike protein spanning amino acid residues 750-1000. In conclusion, the results suggest that the chicken scFv phage display system can be a potential model for mass production of high-affinity antibodies against the SARS-CoV spike protein.
Assuntos
Anticorpos Antivirais/imunologia , Região Variável de Imunoglobulina/imunologia , Glicoproteínas de Membrana/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Western Blotting , Galinhas , Chlorocebus aethiops , Regiões Determinantes de Complementaridade , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Região Variável de Imunoglobulina/química , Dados de Sequência Molecular , Biblioteca de Peptídeos , Proteínas Recombinantes/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/isolamento & purificação , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus , Células VeroRESUMO
PURPOSE: Although existence of humoral immunity has been previously shown in malignant pleural effusions, only a limited number of immunogenic tumor-associated antigens (TAA) have been identified and associated with lung cancer. In this study, we intended to identify more TAAs in pleural effusion-derived tumor cells. EXPERIMENTAL DESIGN: Using morphologically normal lung tissues as a control lysate in Western blotting analyses, 54 tumor samples were screened with autologous effusion antibodies. Biochemical purification and mass spectrometric identification of TAAs were done using established effusion tumor cell lines as antigen sources. We identified a p48 antigen as alpha-enolase (ENO1). Semiquantitative immunohistochemistry was used to evaluate expression status of ENO1 in the tissue samples of 80 patients with non-small cell lung cancer (NSCLC) and then correlated with clinical variables. RESULTS: Using ENO1-specifc antiserum, up-regulation of ENO1 expression in effusion tumor cells from 11 of 17 patients was clearly observed compared with human normal lung primary epithelial and non-cancer-associated effusion cells. Immunohistochemical studies consistently showed high level of ENO1 expression in all the tumors we have examined thus far. Log-rank and Cox's analyses of ENO1 expression status revealed that its expression level in primary tumors was a key factor contributing to overall- and progression-free survivals of patients (P < 0.05). The same result was also obtained in the early stage of NSCLC patients, showing that tumors expressing relatively higher ENO1 level were tightly correlated with poorer survival outcomes. CONCLUSIONS: Our data strongly support a prognostic role of ENO1 in determining tumor malignancy of patients with NSCLC.
Assuntos
Autoantígenos/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Neoplasias Pulmonares/enzimologia , Fosfopiruvato Hidratase/metabolismo , Derrame Pleural Maligno/enzimologia , Adenocarcinoma/enzimologia , Idoso , Carcinoma de Células Grandes/enzimologia , Carcinoma de Células Escamosas/enzimologia , Feminino , Humanos , Masculino , PrognósticoRESUMO
Unlimited growth of cancer cells requires an extensive nutrient supply. To meet this demand, cancer cells drastically upregulate glucose uptake and metabolism compared to normal cells. This difference has made the blocking of glycolysis a fascinating strategy to treat this malignant disease. α-enolase is not only one of the most upregulated glycolytic enzymes in cancer cells, but also associates with many cellular processes or conditions important to cancer cell survival, such as cell migration, invasion, and hypoxia. Targeting α-enolase could simultaneously disturb cancer cells in multiple ways and, therefore, is a good target for anticancer drug development. In the current study, more than 22 million chemical structures meeting the criteria of Lipinski's rule of five from the ZINC database were docked to α-enolase by virtual screening. Twenty-four chemical structures with docking scores better than that of the enolase substrate, 2-phosphoglycerate, were further screened by the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties prediction. Four of them were classified as non-mutagenic, non-carcinogenic, and capable of oral administration where they showed steady interactions to α-enolase that were comparable, even superior, to the currently available inhibitors in molecular dynamics (MD) simulation. These compounds may be considered promising leads for further development of the α-enolase inhibitors and could help fight cancer metabolically.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fosfopiruvato Hidratase/antagonistas & inibidores , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Fosfopiruvato Hidratase/metabolismoRESUMO
Regulator of G protein signaling 11 (RGS11), a member of the R7 subfamily of RGS proteins, is a well-characterized GTPase-accelerating protein that is involved in the heterotrimeric G protein regulation of the amplitude and kinetics of receptor-promoted signaling in retinal bipolar and nerve cells. However, the role of RGS11 in cancer is completely unclear. Using subtractive hybridization analysis, we found that RGS11 was highly expressed in the lymph-node metastatic tissues and bone-metastatic tumors obtained from patients with lung adenocarcinoma. Characterization of the clinicopathological features of 91 patients showed that around 57.1% of the tumor samples displayed RGS11 overexpression that was associated with primary tumor status, nodal metastasis and increased disease stages. Its high expression was an independent predictive factor for poor prognosis of these patients. Cotransfection of guanine nucleotide-binding protein beta-5 (GNB5) markedly increased RGS11 expression. Enhancement or attenuation of RGS11 expression pinpointed its specific role in cell migration, but not in cell invasion and proliferation. Signaling events initiated by the RGS11-GNB5 coexpression activated the c-Raf/ERK/FAK-mediated pathway through upregulation of the Rac1 activity. Consistently, increasing the cell invasiveness of the transfectants by additional cotransfection of the exogenous urokinase-plasminogen activator gene caused a significant promotion in cell invasion in vitro and in vivo, confirming that RGS11 functions in cell migration, but requires additional proteolytic activity for cell and tissue invasion. Collectively, overexpression of RGS11 promotes cell migration, participates in tumor metastasis, and correlates the clinicopathological conditions of patients with lung adenocarcinoma.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Movimento Celular/fisiologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas RGS/biossíntese , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Animais , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Inclusão em Parafina , Proteínas RGS/genética , Análise de Sobrevida , Transfecção , Regulação para CimaRESUMO
Acute myelogenous leukemia (AML) carrying t(8;21)(q22;q22) or inv(16)/t(16;16)(p13;q22) is classified as core binding factor (CBF)-AML and accounts for approximately 15% of AML. c-KIT mutation can be detected in 17%â¼46% of CBF-AML and is associated with poor prognosis. c-KIT mutation is a crucial hit and cooperates with AML1-ETO resulting from t(8;21)(q22;q22) to cause overt AML. Tyrosine kinase inhibitors (TKI) targeting c-KIT, such as imatinib, has been used successfully to treat c-KIT driven gastrointestinal stromal tumors. However, the effect of TKI on c-KIT-driven leukemia, including CBF-AML and systemic mastocytosis (SM), has not been satisfactory. BPR1J373 is a 5-phenylthiazol-2-ylamine-pyriminide derivative targeting multiple tyrosine kinases. It was shown to inhibit cell proliferation and induce apoptosis in AML cells with constitutively activated c-KIT via inhibiting c-KIT phosphorylation and its downstream signals. The compound induced apoptosis by the mitochondrial intrinsic pathway through upregulation of proapoptotic proteins Bax and Bak and caspase 8 and 9 activation in c-KIT mutant Kasumi-1 cells. Furthermore, it induced cell-cycle arrest via targeting aurora kinase B in c-KIT wild-type KG-1 cells. The antitumor response of BPR1J373 was also shown in subcutaneously grafted SCID mice. BPR1J373 was shown to effectively suppress c-KIT phosphorylation of D816V mutation by treating c-KIT-null COS-1 cells transfected with c-KIT D816V mutant plasmid. In conclusion, BPR1J373 inhibits cell proliferation of c-KIT-driven AML cells via induction of apoptosis and cell-cycle arrest. It is also effective for multiple drug-resistant c-KIT D816V mutation. BPR1J373 deserves further development for clinical use in c-KIT-driven myeloid leukemia. Mol Cancer Ther; 15(10); 2323-33. ©2016 AACR.
Assuntos
Leucemia Mieloide Aguda/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutação , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional , Translocação Genética , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recurrent genetic alterations that are frequently observed in some low-grade lymphomas, such as activated B cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL) and mucosa-associated lymphoid tissue type lymphoma (MALT lymphoma) are usually associated with nuclear factor-κB (NF-κB) activation and confer resistance to therapy. In this study, we investigated the therapeutic efficacy and molecular mechanisms of AUY922, a novel Hsp90 inhibitor, in representative cell lines OCI-Ly3 (ABC-DLBCL) and MA-1 (a low-grade lymphoma cell line with t(14;18)/IgH-MALT1translocation) to explore its potential use in the treatment of refractory B-cell lymphoma. Our results showed that AUY922 effectively induced growth inhibition and apoptosis of OCI-Ly3 and MA-1 cells, which were accompanied by down-regulation of the expression levels of NF-κB and Bcl-2 family proteins, as well as molecules of multiple signaling pathways involving cell proliferation, growth and survival. The growth inhibitory effect of AUY922 was further confirmed in a mouse xenograft model. These findings indicate the potential use of AUY922 in B cell lymphomas.
Assuntos
Antineoplásicos/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Aberrações Cromossômicas , Isoxazóis/farmacologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , NF-kappa B/metabolismo , Resorcinóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 9/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 18 , Modelos Animais de Doenças , Regulação para Baixo , Ativação Enzimática/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Gradação de Tumores , Transdução de Sinais/genética , Translocação Genética , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pancreatic Ductal Adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness and resistance to treatment. We have previously demonstrated that most PDAC patients have circulating antibodies against the glycolytic enzyme alpha-enolase (ENO1), which correlates with a better response to therapy and survival. ENO1 is a metabolic enzyme, also expressed on the cell surface where it acts as a plasminogen receptor. ENO1 play a crucial role in cell invasion and metastasis by promoting plasminogen activation into plasmin, a serine-protease involved in extracellular matrix degradation. The aim of this study was to investigate the role of ENO1 in PDAC cell invasion. We observed that ENO1 was expressed on the cell surface of most PDAC cell lines. Mouse anti-human ENO1 monoclonal antibodies inhibited plasminogen-dependent invasion of human PDAC cells, and their metastatic spreading in immunosuppressed mice was inhibited. Notably, a single administration of Adeno-Associated Virus (AAV)-expressing cDNA coding for 72/1 anti-ENO1 mAb reduced the number of lung metastases in immunosuppressed mice injected with PDAC cells. Overall, these data indicate that ENO1 is involved in PDAC cell invasion, and that administration of an anti-ENO1 mAb can be exploited as a novel therapeutic option to increase the survival of metastatic PDAC patients.
Assuntos
Anticorpos Monoclonais/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Carcinoma Ductal Pancreático/prevenção & controle , Proteínas de Ligação a DNA/antagonistas & inibidores , Neoplasias Hepáticas/prevenção & controle , Neoplasias Pancreáticas/prevenção & controle , Fosfopiruvato Hidratase/antagonistas & inibidores , Proteínas Supressoras de Tumor/antagonistas & inibidores , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Our previous studies suggest that antibodies against ENO1 (anti-ENO1 Ab) have a protective role in patients with non-small cell lung carcinoma. In this study, we evaluated the prognostic value of anti-ENO1 Ab levels in non-small cell lung carcinoma patients undergoing surgery. Circulating levels of anti-ENO1 Ab were assessed in 85 non-small cell lung carcinoma patients before and after surgery, and were correlated with clinical outcome. After surgery, patients with a higher increase of anti-ENO1 Ab had a lower hazard ratio and a better progression-free survival. Using animal models, we demonstrated that tumor cells reduce the circulating levels of anti-ENO1 Ab through physical absorption and neutralization of anti-ENO1 Ab with surface-expressed and secreted ENO1, respectively. Mice transplanted with ENO1-overexpressing tumors generated ENO1-specific regulatory T cells to suppress the production of anti-ENO1 Ab. Our results suggest that the increase of anti-ENO1 Ab may reflect anti-tumor immune responses and serve as a prognostic marker in postoperative lung cancer patients.
Assuntos
Anticorpos Antineoplásicos/sangue , Biomarcadores Tumorais/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Proteínas de Ligação a DNA/imunologia , Neoplasias Pulmonares/imunologia , Fosfopiruvato Hidratase/imunologia , Proteínas Supressoras de Tumor/imunologia , Idoso , Animais , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Intervalo Livre de Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/mortalidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , PrognósticoRESUMO
Gastrointestinal stromal tumor (GIST) is a prototype of mutant KIT oncogene-driven tumor. Prolonged tyrosine kinase inhibitor (TKI) treatment may result in a resistant phenotype through acquired secondary KIT mutation. Heat shock protein 90 (HSP90AA1) is a chaperone protein responsible for protein maturation and stability, and KIT is a known client protein of HSP90AA1. Inhibition of HSP90AA1 has been shown to destabilize KIT protein by enhancing its degradation via the proteasome-dependent pathway. In this study, we demonstrated that NVP-AUY922 (AUY922), a new class of HSP90AA1 inhibitor, is effective in inhibiting the growth of GIST cells expressing mutant KIT protein, the imatinib-sensitive GIST882 and imatinib-resistant GIST48 cells. The growth inhibition was accompanied with a sustained reduction of both total and phosphorylated KIT proteins and the induction of apoptosis in both cell lines. Surprisingly, AUY922-induced KIT reduction could be partially reversed by pharmacological inhibition of either autophagy or proteasome degradation pathway. The blockade of autophagy alone led to the accumulation of the KIT protein, highlighting the role of autophagy in endogenous KIT turnover. The involvement of autophagy in endogenous and AUY922-induced KIT protein turnover was further confirmed by the colocalization of KIT with MAP1LC3B-, acridine orange- or SQSTM1-labeled autophagosome, and by the accumulation of KIT in GIST cells by silencing either BECN1 or ATG5 to disrupt autophagosome activity. Therefore, the results not only highlight the potential application of AUY922 for the treatment of KIT-expressing GISTs, but also provide the first evidence for the involvement of autophagy in endogenous and HSP90AA1 inhibitor-induced KIT degradation.