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1.
J Cancer ; 10(24): 6025-6036, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31762812

RESUMO

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.

2.
Anal Chem ; 91(5): 3327-3335, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30701963

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/imunologia
3.
Cancer Sci ; 109(11): 3591-3601, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30142229

RESUMO

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 Xenoenxerto
4.
Drug Des Devel Ther ; 11: 3281-3290, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29180852

RESUMO

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/metabolismo
5.
Mol Cancer Ther ; 15(10): 2323-2333, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27512117

RESUMO

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%∼46% 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 Xenoenxerto
6.
Oncotarget ; 7(21): 31122-36, 2016 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-27105500

RESUMO

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 Cima
7.
Oncotarget ; 6(33): 35073-86, 2015 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-26551021

RESUMO

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 , Prognóstico
8.
Oncotarget ; 6(13): 11098-113, 2015 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-25860938

RESUMO

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 Xenoenxerto
9.
Leuk Lymphoma ; 56(9): 2674-82, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25535814

RESUMO

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 Xenoenxerto
10.
Int J Cancer ; 135(4): 809-19, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24477565

RESUMO

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 Sinais
11.
PLoS One ; 8(7): e69354, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23894455

RESUMO

In previous research, we found α-enolase to be inversely correlated with progression-free and overall survival in lung cancer patients and detected α-enolase on the surface of lung cancer cells. Based on these findings, we hypothesized that surface α-enolase has a significant role in cancer metastasis and tested this hypothesis in the current study. We found that α-enolase was co-immunoprecipitated with urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen in lung cancer cells and interacted with these proteins in a cell-free dot blotting assay, which can be interrupted by α-enolase-specific antibody. α-Enolase in lung cancer cells co-localized with these proteins and was present at the site of pericellular degradation of extracellular matrix components. Treatment with antibody against α-enolase in vitro suppressed cell-associated plasminogen and matrix metalloproteinase activation, collagen and gelatin degradation, and cell invasion. Examination of the effect of treatment with shRNA plasmids revealed that down regulation of α-enolase decreases extracellular matrix degradation by and the invasion capacity of lung cancer cells. Adoptive transfer of α-enolase-specific antibody to mice resulted in accumulation of antibody in subcutaneous tumor and inhibited the formation of tumor metastasis in lung and bone. This study demonstrated that surface α-enolase promotes extracellular matrix degradation and invasion of cancer cells and that targeting surface α-enolase is a promising approach to suppress tumor metastasis.


Assuntos
Membrana Celular/metabolismo , Matriz Extracelular/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Fosfopiruvato Hidratase/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Movimento Celular , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Humanos , Hospedeiro Imunocomprometido , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Metástase Neoplásica , Neoplasias/mortalidade , Fosfopiruvato Hidratase/antagonistas & inibidores , Plasminogênio/metabolismo , Ligação Proteica , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
12.
PLoS One ; 8(6): e65762, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23840364

RESUMO

Advanced gastrointestinal stromal tumors (GIST), a KIT oncogene-driven tumor, on imatinib mesylate (IM) treatment may develop secondary KIT mutations to confer IM-resistant phenotype. Second-line sunitinib malate (SU) therapy is largely ineffective for IM-resistant GISTs with secondary exon 17 (activation-loop domain) mutations. We established an in vitro cell-based platform consisting of a series of COS-1 cells expressing KIT cDNA constructs encoding common primary±secondary mutations observed in GISTs, to compare the activity of several commercially available tyrosine kinase inhibitors on inhibiting the phosphorylation of mutant KIT proteins at their clinically achievable plasma steady-state concentration (Css). The inhibitory efficacies on KIT exon 11/17 mutants were further validated by growth inhibition assay on GIST48 cells, and underlying molecular-structure mechanisms were investigated by molecular modeling. Our results showed that SU more effectively inhibited mutant KIT with secondary exon 13 or 14 mutations than those with secondary exon 17 mutations, as clinically indicated. On contrary, at individual Css, nilotinib and sorafenib more profoundly inhibited the phosphorylation of KIT with secondary exon 17 mutations and the growth of GIST48 cells than IM, SU, and dasatinib. Molecular modeling analysis showed fragment deletion of exon 11 and point mutation on exon 17 would lead to a shift of KIT conformational equilibrium toward active form, for which nilotinib and sorafenib bound more stably than IM and SU. In current preclinical study, nilotinib and sorafenib are more active in IM-resistant GISTs with secondary exon 17 mutation than SU that deserve further clinical investigation.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Gastrointestinais/tratamento farmacológico , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Motivos de Aminoácidos , Animais , Benzamidas/farmacologia , Células COS , Chlorocebus aethiops , Dasatinibe , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligação de Hidrogênio , Mesilato de Imatinib , Indóis/farmacologia , Simulação de Acoplamento Molecular , Mutação de Sentido Incorreto , Piperazinas/farmacologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/química , Pirimidinas/farmacologia , Pirróis/farmacologia , Sunitinibe , Tiazóis/farmacologia
13.
Autophagy ; 9(2): 220-33, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23196876

RESUMO

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.


Assuntos
Autofagia/efeitos dos fármacos , Tumores do Estroma Gastrointestinal/patologia , Isoxazóis/farmacologia , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Resorcinóis/farmacologia , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Benzoquinonas/farmacologia , Morte Celular/efeitos dos fármacos , Extratos Celulares , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Lactamas Macrocíclicas/farmacologia , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinas/farmacologia
14.
BMC Vet Res ; 7: 62, 2011 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-22014164

RESUMO

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/análise
15.
J Cell Physiol ; 226(2): 424-33, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20672290

RESUMO

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 CD83
16.
FEBS J ; 277(20): 4308-21, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20849415

RESUMO

The c-myc promoter-binding protein-1 (MBP-1) is a transcriptional suppressor of tumorigenesis and thought to be the product of alternative translation initiation of the α-enolase (ENO1) transcript. In the present study, we cloned a 2552-bp novel cDNA with a putative coding sequence of MBP-1 and functionally examined its ability to encode the MBP-1 protein. Similarly to ENO1, the obtained MBP-1 was widely and differentially expressed in a variety of normal tissues and cancer cells. Experiments using MBP-1 promoter-driven luciferase reporter assays, biochemical cell fractionation followed by RT-PCR detection of the cytoplasmic mRNA, and transcription/translation-coupled reactions, consistently demonstrated that this novel transcript was alternatively transcribed from intron III of the ENO1 gene and was feasible for MBP-1 production. Hypoxia treatments significantly increased the transcriptional activation of the MBP-1 gene. Blocking the proteasomal degradation by MG132 stabilized the MBP-1 protein in cells. Compared with the translation efficiency for production of the MBP-1 protein, the MBP-1 transcript was 17.8 times more efficient than the ENO1 transcript. Thus, we suggest that this newly discovered transcript is a genuine template for the protein synthesis of MBP-1 in cells, and optimal expression of this gene in tumors may lead to effective clinical therapies for cancers.


Assuntos
Biomarcadores Tumorais/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Fosfopiruvato Hidratase/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Supressoras de Tumor/genética , Brônquios/citologia , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Humanos , Hipóxia , Íntrons , Proteínas/metabolismo , RNA Mensageiro
17.
Vet Immunol Immunopathol ; 137(3-4): 251-60, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20655599

RESUMO

It was previously reported that up-regulation of alpha-enolase protein was detected in 65% of patients with non-small cell lung cancers (NSCLC). Moreover, a high titer of anti-alpha-enolase antibodies was developed in a smaller proportion (7.4%) of these patients than in non-tumor-associated patients and healthy subjects. In the present study, we characterized polyclonal and single-chain variable fragment (scFv) anti-alpha-enolase antibodies from immunized chickens. The E. coli-derived recombinant alpha-enolase protein was purified to its high homogenicity as verified by SDS-PAGE. After the 4th immunization, a high titer of specific polyclonal anti-alpha-enolase antibodies was elicited in immunized chickens and specifically recognized the purified human alpha-enolase antigen as determined by Western blot and ELISA. The expressed heavy and light chain variable genes (VH and VL) were isolated from spleen B cells and amplified to construct phage antibody libraries containing scFv molecules. After four rounds of panning selection, the scFv antibodies of randomly chosen clones were expressed and their binding specificity to alpha-enolase protein was verified using competitive ELISA, flow cytometry and immunofluorescence staining. Nucleotide sequence analysis from 10 alpha-enolase binding clones showed that 3 (30%) clones used identical heavy and light genes for scFv antibody expression, as represented by EnL5. Notably, amino acid changes in complementarity-determining regions (CDRs) were more frequently observed than those in framework regions (FRs) in all clones, indicating a strong affinity selection through mutations. All together, it is believed that these polyclonal and scFv IgY antibodies may be helpful in the development of molecular diagnostic and therapeutic agents for lung cancers in the future.


Assuntos
Imunoglobulinas/imunologia , Fosfopiruvato Hidratase/imunologia , Anticorpos de Cadeia Única/imunologia , Sequência de Aminoácidos , Animais , Galinhas , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Feminino , Citometria de Fluxo , Imunofluorescência , Imunização , Imunoglobulinas/biossíntese , Microscopia Confocal , Dados de Sequência Molecular , Biblioteca de Peptídeos , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética
18.
Microb Drug Resist ; 16(4): 317-25, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20524901

RESUMO

An increasing incidence of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections has been reported worldwide. The aim of this study was to investigate the mechanism underlying carbapenem resistance and its relationship to antibiotic exposure. Sixteen isolates with various carbapenem susceptibilities recovered from five patients between 2003 and 2006 were subjected to molecular study. The medical records of the patients were also reviewed. All of the patients were admitted for complicated respiratory illness, had a prolonged hospital stay, and were exposed to antibiotics. Carbapenems were prescribed before the emergence of the CRKP. Various combinations of extended-spectrum cephalosporinase genes belonging to the SHV, CTX-M, and AmpC groups were found among the isolates. Other carbapenem resistance-associated genes, such as bla(IMP), bla(VIM), bla(OXA), and bla(KPC), were not found. OmpK35 was not expressed in any of the isolates, and additional loss of OmpK36 was observed in all CRKP isolates. Two insertion elements, ISPa13 or IS5, were found inserted into OmpK36 in the isolates derived from three patients. These IS elements were also identified in their parental carbapenem-susceptible isolates, suggesting that an internal transposition into OmpK36 resulted in resistance. OmpK36 loss represents the major mechanism for the development of CRKP in extended-spectrum cephalosporinase-producing isolates. A prolonged hospital stay and recent carbapenem exposure may predispose patients to CRKP, impacting the clinical outcome.


Assuntos
Antibacterianos , Carbapenêmicos , Farmacorresistência Bacteriana , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/efeitos dos fármacos , Tempo de Internação , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Carbapenêmicos/farmacologia , Carbapenêmicos/uso terapêutico , Farmacorresistência Bacteriana/genética , Feminino , Hospitalização , Humanos , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Taiwan/epidemiologia
19.
Anal Chem ; 82(14): 5944-50, 2010 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-20557064

RESUMO

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/química
20.
Eur J Cancer ; 46(9): 1712-23, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20435467

RESUMO

The success of using glycolytic inhibitors for cancer treatment depends on studying the individual role of frequently deregulated glycolytic genes in cancer. This report aims to study the prognostic implication, and determine the cellular role and action mechanism of glycolytic ENO1 overexpression in head and neck cancer. The relationship of ENO1 mRNA expression in 44-pair clinical specimens with patient clinicopathologic characteristics was analysed by semi-quantitative RT-PCR, Kaplan-Meier survival curve and Cox model analyses. Following ectopic ENO1 expression or knockdown, we studied the proliferative, migratory, invasive, colony-forming and tumourigenic abilities of ENO1-genetically altered cells. DNA microarray analysis was used to identify downstream targets responsible for the ENO1 action in the cells. The expression of ENO1 mRNA was increased in 68% of tumour (T) specimens when compared to their normal (N) counterparts, and positively associated with clinical progression (p<0.05). High ENO1 expression (T/N2) was frequently observed in the patients with large primary tumours, late clinical stages or advanced neck metastasis. Moreover, high ENO1 patients had significantly poorer clinical outcomes than low expressers (T/N<2). Ectopic ENO1 expression stimulated cell transformation, invasion and tongue tumour formation. ENO1 knockdown abrogated the stimulation. Suppression of ENO1-induced proinflammatory CCL20 chemokine expression significantly attenuated its stimulatory effects on cell transformation and invasion. A concordant expression of ENO1 and CCL20 was validated both in ENO1-expressing cells and in clinical specimens. Together, we demonstrate a prognostic role of ENO1 overexpression in head and neck cancer and ENO1-mediated promotion of cell transformation and invasion partly via induced CCL20 expression.


Assuntos
Biomarcadores Tumorais/fisiologia , Transformação Celular Neoplásica/metabolismo , Quimiocina CCL20/metabolismo , Proteínas de Ligação a DNA/fisiologia , Neoplasias Bucais/enzimologia , Fosfopiruvato Hidratase/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Invasividade Neoplásica , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Prognóstico , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Língua/enzimologia , Neoplasias da Língua/patologia , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima
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