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Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has threatened public health globally, and the emergence of viral variants has exacerbated an already precarious situation. To prevent further spread of the virus and determine government action required for virus control, accurate and rapid immunoassays for SARS-CoV-2 diagnosis are urgently needed. In this study, we generated monoclonal antibodies (mAbs) against the SARS-CoV-2 nucleocapsid protein (NP), compared their reactivity using an enzyme-linked immunosorbent assay (ELISA), and selected four mAbs designated 1G6, 3E10, 3F10, and 5B6 which have higher reactivity to NP and viral lysates of SARS-CoV-2 than other mAbs. Using an epitope mapping assay, we identified that 1G6 detected the C-terminal domain of SARS-CoV-2 NP (residues 248-364), while 3E10 and 3F10 bound to the N-terminal domain (residues 47-174) and 3F10 detected the N-arm region (residues 1-46) of SARS-CoV-2 NP. Based on the epitope study and sandwich ELISA, we selected the 1G6 and 3E10 Abs as an optimal Ab pair and applied them for a microfluidics-based point-of-care (POC) ELISA assay to detect the NPs of SARS-CoV-2 and its variants. The integrated and automatic microfluidic system could operate the serial injection of the sample, the washing solution, the HRP-conjugate antibody, and the TMB substrate solution simply by controlling air purge via a single syringe. The proposed Ab pair-equipped microsystem effectively detected the NPs of SARS-CoV-2 variants as well as in clinical samples. Collectively, our proposed platform provides an advanced protein-based diagnostic tool for detecting SARS-CoV-2.
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Tumor-associated (TA) autoantibodies have been identified at the early tumor stage before developing clinical symptoms, which holds hope for early cancer diagnosis. We identified a TA autoantibody from HBx-transgenic (HBx-tg) hepatocellular carcinoma (HCC) model mouse, characterized its target antigen, and examined its relationship to human HCC. The mimotopes corresponding to the antigenic epitope of TA autoantibody were screened from a random cyclic peptide library and used for the detection of serum TA autoantibody. The target antigen of the TA autoantibody was identified as an oncogenic bi-functional purine biosynthesis protein, ATIC. It was upregulated in liver cancer tissues of HBx-tg mouse as well as human HCC tissues. Over-expressed ATIC was also secreted extracellularly via the cancer-derived exosomes, which might cause auto-immune responses. The cyclic peptide mimotope with a high affinity to anti-ATIC autoantibody, CLPSWFHRC, distinguishes between serum samples from HCC patients and healthy subjects with 70.83% sensitivity, 90.68% specificity (AUC = 0.87). However, the recombinant human ATIC protein showed a low affinity to anti-ATIC autoantibody, which may be incompatible as a capture antigen for serum TA autoantibody. This study indicates that anti-ATIC autoantibody can be a potential HCC-associated serum biomarker and suggests that autoantibody biomarker's efficiency can be improved by using antigenic mimicry to native antigens present in vivo.
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Autoanticorpos/sangue , Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/diagnóstico , Epitopos/imunologia , Hidroximetil e Formil Transferases/imunologia , Neoplasias Hepáticas/diagnóstico , Complexos Multienzimáticos/imunologia , Nucleotídeo Desaminases/imunologia , Peptídeos Cíclicos/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Autoanticorpos/imunologia , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/imunologia , Feminino , Humanos , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Biblioteca de Peptídeos , Prognóstico , Adulto JovemRESUMO
Studies have shown that cancer stem cells (CSCs) are involved in resistance and metastasis of cancer; thus, therapies targeting CSCs have been proposed. Here, we report that heat shock 70-kDa protein 1-like (HSPA1L) is partly involved in enhancing epithelial-mesenchymal transition (EMT) and CSC-like properties in non-small cell lung cancer (NSCLC) cells. Aldehyde dehydrogenase 1 (ALDH1) is considered a CSC marker in some lung cancers. Here, we analyzed transcriptional changes in genes between ALDH1high and ALDH1low cells sorted from A549 NSCLC cells and found that HSPA1L was highly expressed in ALDH1high cells. HSPA1L played two important roles in enhancing CSC-like properties. First, HSPA1L interacts directly with IGF1Rß and integrin αV to form a triple complex that is involved in IGF1Rß activation. HSPA1L/integrin αV complex-associated IGF1Rß activation intensified the EMT-associated cancer stemness and γ-radiation resistance through its downstream AKT/NF-κB or AKT/GSK3ß/ß-catenin activation pathway. Secondly, HSPA1L was also present in the nucleus and could bind directly to the promoter region of ß-catenin to function as a transcription activator of ß-catenin, an important signaling protein characterizing CSCs by regulating ALDH1 expression. HSPA1L may be a novel potential target for cancer treatment because it both enhances IGF1Rß activation and regulates γß-catenin transcription, accumulating CSC-like properties.
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Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Receptor IGF Tipo 1/metabolismo , Transcrição Gênica , beta Catenina/biossíntese , Células A549 , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Choque Térmico HSP70/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/patologia , Receptor IGF Tipo 1/genética , beta Catenina/genéticaRESUMO
Microenvironment, such as hypoxia common to cancer, plays a critical role in the epithelial-to-mesenchymal transition (EMT) program, which is a major route of cancer metastasis and confers γ-radiation resistance to cells. Herein, we showed that transgelin 2 (TAGLN2), an actin-binding protein, is significantly induced in hypoxic lung cancer cells and that Snail1 is simultaneously increased, which induces EMT by downregulating E-cadherin expression. Forced TAGLN2 expression induced severe cell death; however, a small population of cells surviving after forced TAGLN2 overexpression showed γ-radiation resistance, which might promote tumor relapse and recurrence. These surviving cells showed high metastatic activity with an increase of EMT markers including Snail1. In these cells, TAGLN2 activated the insulin-like growth factor 1 receptor ß (IGF1Rß)/PI3K/AKT pathway by recruitment of focal adhesion kinase to the IGF1R signaling complex. Activation of the IGF1Rß/PI3K/AKT pathway also induced inactivation of glycogen synthase kinase 3ß (GSK3ß), which is involved in Snail1 stabilization. Therefore, both the IGF1Rß inhibitor (AG1024) and the PI3K inhibitor (LY294002) or AKT inactivation with MK2206 lower the cellular level of Snail1. Involvement of GSK3ß was also confirmed by treatment with lithium chloride, the inducer of GSK3ß phosphorylation, or MG132, the 26S proteasomal inhibitor, which also stabilized Snail1. In conclusion, the present study provides important evidence that hypoxia-inducible TAGLN2 is involved in the selection of cancer cells with enhanced EMT properties to overcome the detrimental environment of cancer cells.
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Carcinoma Pulmonar de Células não Pequenas/metabolismo , Transição Epitelial-Mesenquimal , Adesões Focais/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Tolerância a Radiação , Receptores de Somatomedina/metabolismo , Células A549 , Hipóxia Celular , Linhagem Celular Tumoral , Raios gama , Regulação Neoplásica da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Receptor IGF Tipo 1 , Transdução de Sinais , Regulação para CimaRESUMO
BACKGROUND: Tumor-associated (TA) autoantibodies, which are generated by the immune system upon the recognition of abnormal TA antigens, are promising biomarkers for the early detection of tumors. In order to detect autoantibody biomarkers effectively, antibody-specific epitopes in the diagnostic test should maintain the specific conformations that are as close as possible to those presenting in the body. However, when using patients' serum as a source of TA autoantibodies the characterization of the autoantibody-specific epitope is not easy due to the limited amount of patient-derived serum. METHODS: To overcome these limits, we constructed a B cell hybridoma pool derived from a hepatocellular carcinoma (HCC) model HBx-transgenic mouse and characterized autoantibodies derived from them as tumor biomarkers. Their target antigens were identified by mass spectrometry and the correlations with HCC were examined. With the assumption that TA autoantibodies generated in the tumor mouse model are induced in human cancer patients, the enzyme-linked immunosorbent assays (ELISA) based on the characteristics of mouse TA autoantibodies were developed for the detection of autoantibody biomarkers in human serum. To mimic natural antigenic structures, the specific epitopes against autoantibodies were screened from the phage display cyclic random heptapeptide library, and the streptavidin antigens fused with the specific epitopes were used as coating antigens. RESULTS: In this study, one of HCC-associated autoantibodies derived from HBx-transgenic mouse, XC24, was characterized. Its target antigen was identified as splicing factor 3b subunit 1 (SF3B1) and the high expression of SF3B1 was confirmed in HCC tissues. The specific peptide epitopes against XC24 were selected and, among them, XC24p11 cyclic peptide (-CDATPPRLC-) was used as an epitope of anti-SF3B1 autoantibody ELISA. With this epitope, we could effectively distinguish between serum samples from HCC patients (n = 102) and healthy subjects (n = 85) with 73.53% sensitivity and 91.76% specificity (AUC = 0.8731). Moreover, the simultaneous detection of anti-XC24p11 epitope autoantibody and AFP enhanced the efficiency of HCC diagnosis with 87.25% sensitivity and 90.59% specificity (AUC = 0.9081). CONCLUSIONS: ELISA using XC24p11 peptide epitope that reacts against anti-SF3B1 autoantibody can be used as a novel test to enhance the diagnostic efficiency of HCC.
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Autoanticorpos/sangue , Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/diagnóstico , Fosfoproteínas/imunologia , Fatores de Processamento de RNA/imunologia , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Epitopos/metabolismo , Humanos , Camundongos Transgênicos , Peptídeos/química , Fosfoproteínas/sangue , Fatores de Processamento de RNA/sangue , Estreptavidina/metabolismo , Transativadores/metabolismo , Proteínas Virais Reguladoras e Acessórias , alfa-Fetoproteínas/metabolismoRESUMO
Amyloid ß precursor protein binding family B member 1(APBB1) was first identified as a binding partner of amyloid precursor protein during brain development, but its function in the context of cancer remain unclear. Here we show for the first time that APBB1 is partly associated with intensifying cancer stem cell(CSC) and epithelial-to-mesenchymal transition (EMT) and enhancing radiation-resistant properties of lung cancer cells. We found that APBB1 was highly expressed in ALDH1high CSC-like cells sorted from A549 lung cancer cells. In APBB1-deficient H460 cells with forced overexpression of APBB1, the protein directly interacted with IGF1Rß, enhanced phosphorylation of IGF1Rß/PI3K/AKT pathway(activation) and subsequently induced the phosphorylation of GSK3ß(inactivation). This phosphorylation stabilized Snail1, a negative regulator of E-cadherin expression, and regulated ß-catenin-mediated ALDH1 expression, which are representative markers for EMT and CSCs, respectively. In contrast, suppression of APBB1 expression with siRNA yielded the opposite effects in APBB1-rich A549 cells. We concluded that APBB1 partly regulates the expression of ALDH1. We also found that APBB1 regulates activation of nuclear factor-κB, which is involved in reducing various stresses including oxidative stress, which suggests that APBB1 is associated with γ-radiation sensitivity. Our findings imply that APBB1 plays an important role in the maintenance of EMT-associated CSC-like properties and γ-radiation resistance via activation of IGF1Rß/AKT/GSK3ß pathway in lung cancer cells, highlighting APBB1 as a potential target for therapeutic cancer treatment.
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Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Transição Epitelial-Mesenquimal , Células-Tronco Neoplásicas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Somatomedina/metabolismo , Células A549 , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Células-Tronco Neoplásicas/efeitos da radiação , Tolerância a Radiação , Dosagem Radioterapêutica , Receptor IGF Tipo 1 , Transdução de Sinais/efeitos da radiaçãoRESUMO
Fibulin-3 (FBLN-3) has been postulated to be either a tumor suppressor or promoter depending on the cell type, and hypermethylation of the FBLN-3 promoter is often associated with human disease, especially cancer. We report that the promoter region of the FBLN-3 was significantly methylated (>95%) in some pancreatic cancer cell lines and thus FBLN-3 was poorly expressed in pancreatic cancer cell lines such as AsPC-1 and MiaPaCa-2. FBLN-3 overexpression significantly down-regulated the cellular level of c-MET and inhibited hepatocyte growth factor-induced c-MET activation, which were closely associated with γ-radiation resistance of cancer cells. Moreover, we also showed that c-MET suppression or inactivation decreased the cellular level of ALDH1 isozymes (ALDH1A1 or ALDH1A3), which serve as cancer stem cell markers, and subsequently induced inhibition of cell growth in pancreatic cancer cells. Therefore, forced overexpression of FBLN-3 sensitized cells to cytotoxic agents such as γ-radiation and strongly inhibited the stemness and epithelial to mesenchymal transition (EMT) property of pancreatic cancer cells. On the other hand, if FBLN3 was suppressed in FBLN-3-expressing BxPC3 cells, the results were opposite. This study provides the first demonstration that the FBLN-3/c-MET/ALDH1 axis in pancreatic cancer cells partially modulates stemness and EMT as well as sensitization of cells to the detrimental effects of γ-radiation.
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Proteínas da Matriz Extracelular/genética , Isoenzimas/genética , Pâncreas/efeitos da radiação , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/radioterapia , Proteínas Proto-Oncogênicas c-met/genética , Retinal Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Ilhas de CpG/efeitos da radiação , Metilação de DNA/efeitos da radiação , Transição Epitelial-Mesenquimal/efeitos da radiação , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Raios gama , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Isoenzimas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos da radiação , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Regiões Promotoras Genéticas/efeitos da radiação , Proteínas Proto-Oncogênicas c-met/metabolismo , Retinal Desidrogenase/metabolismoRESUMO
Dickkopf1 (DKK1), a secreted protein involved in embryonic development, is a potent inhibitor of the Wnt signaling pathway and has been postulated to be a tumor suppressor or tumor promoter depending on the tumor type. In this study, we showed that DKK1 was expressed differently among non-small-cell lung cancer cell lines. The DKK1 expression level was much higher in A549 cells than in H460 cells. We revealed that blockage of DKK1 expression by silencing RNA in A549 cells caused up-regulation of intracellular reactive oxygen species (ROS) modulator (ROMO1) protein, followed by partial cell death, cell growth inhibition, and loss of epithelial-mesenchymal transition property caused by ROS, and it also increased γ-radiation sensitivity. DKK1 overexpression in H460 significantly inhibited cell survival with the decrease of ROMO1 level, which induced the decrease of cellular ROS. Thereafter, exogenous N-acetylcysteine, an antioxidant, or hydrogen peroxide, a pro-oxidant, partially rescued cells from death and growth inhibition. In each cell line, both overexpression and blockage of DKK1 not only elevated p-RB activation, which led to cell growth arrest, but also inactivated AKT/NF-kB, which increased radiation sensitivity and inhibited cell growth. This study is the first to demonstrate that strict modulation of DKK1 expression in different cell types partially maintains cell survival via tight regulation of the ROS-producing ROMO1 and radiation resistance.
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Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Tolerância a Radiação , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Transição Epitelial-Mesenquimal/efeitos da radiação , Raios gama , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína do Retinoblastoma/metabolismoRESUMO
Approved COVID-19 vaccines primarily induce neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein. However, the emergence of variants of concern with RBD mutations poses challenges to vaccine efficacy. This study aimed to design a next-generation vaccine that provides broader protection against diverse coronaviruses, focusing on glycan-free S2 peptides as vaccine candidates to overcome the low immunogenicity of the S2 domain due to the N-linked glycans on the S antigen stalk, which can mask S2 antibody responses. Glycan-free S2 peptides were synthesized and attached to SARS-CoV-2 virus-like particles (VLPs) lacking the S antigen. Humoral and cellular immune responses were analyzed after the second booster immunization in BALB/c mice. Enzyme-linked immunosorbent assay revealed the reactivity of sera against SARS-CoV-2 variants, and pseudovirus neutralization assay confirmed neutralizing activities. Among the S2 peptide-conjugated VLPs, the S2.3 (N1135-K1157) and S2.5 (A1174-L1193) peptide-VLP conjugates effectively induced S2-specific serum immunoglobulins. These antisera showed high reactivity against SARS-CoV-2 variant S proteins and effectively inhibited pseudoviral infections. S2 peptide-conjugated VLPs activated SARS-CoV-2 VLP-specific T-cells. The SARS-CoV-2 vaccine incorporating conserved S2 peptides and CoV-2 VLPs shows promise as a universal vaccine capable of generating neutralizing antibodies and T-cell responses against SARS-CoV-2 variants.
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Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), had a major impact on both the global health and economy. Numerous virus-neutralizing antibodies were developed against the S1 subunit of SARS-CoV-2 spike (S) protein to block viral binding to host cells and were authorized for control of the COVID-19 pandemic. However, frequent mutations in the S1 subunit of SARS-CoV-2 enabled the emergence of immune evasive variants. To address these challenges, broadly neutralizing antibodies targeting the relatively conserved S2 subunit and its epitopes have been investigated as antibody therapeutics and universal vaccines. Methods: We initiated this study by immunizing BALB/c mice with ß-propiolactone-inactivated SARS-CoV-2 (IAV) to generate B-cell hybridomas. These hybridomas were subsequently screened using HEK293T cells expressing the S2-ECD domain. Hybridomas that produced anti-S2 antibodies were selected, and we conducted a comprehensive evaluation of the potential of these anti-S2 antibodies as antiviral agents and versatile tools for research and diagnostics. Results: In this study, we present a novel S2-specific antibody, 4A5, isolated from BALB/c mice immunized with inactivated SARS-CoV-2. 4A5 exhibited specific affinity to SARS-CoV-2 S2 subunits compared with those of other ß-CoVs. 4A5 bound to epitope segment F1109-V1133 between the heptad-repeat1 (HR1) and the stem-helix (SH) region. The 4A5 epitope is highly conserved in SARS-CoV-2 variants, with a significant conformational feature in both pre- and postfusion S proteins. Notably, 4A5 exhibited broad neutralizing activity against variants and triggered Fc-enhanced antibody-dependent cellular phagocytosis. Discussion: These findings offer a promising avenue for novel antibody therapeutics and insights for next-generation vaccine design. The identification of 4A5, with its unique binding properties and broad neutralizing capacity, offers a potential solution to the challenge posed by SARS-CoV-2 variants and highlights the importance of targeting the conserved S2 subunit in combating the COVID-19.
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COVID-19 , SARS-CoV-2 , Animais , Camundongos , Humanos , SARS-CoV-2/genética , Anticorpos Antivirais , Pandemias , Células HEK293 , EpitoposRESUMO
Tumorassociated (TA) autoantibodies are considered to be promising biomarkers for the early detection of cancer, prior to the development of clinical symptoms. In the present study, a novel TA autoantibody was detected, which may prove to be useful as a diagnostic marker of human HCC using an HBxtransgenic (HBxtg) hepatocellular carcinoma (HCC) mouse model. Its target antigen was identified as the bromodomaincontaining protein 2 (BRD2), a transcriptional regulator that plays a pivotal role in the transcriptional control of diverse genes. BRD2 was upregulated in HCC tissues of the Hras12Vtg mouse and human subjects, as demonstrated using western blotting or immunohistochemical analysis, with the BRD2 autoantibody. In addition, the truncated BRD2 reactive to the BRD2 autoantibody was detected in tumor cellderived exosomes, which possibly activated TA immune responses and the generation of autoantibodies. For the detection of the serum BRD2 autoantibody, epitope mimicries of autoantigenic BRD2 were screened from a random cyclic peptide CX7C library with the BRD2 autoantibody. A mimotope with the sequence of CTSVFLPHC, which was cyclized by one pair of cysteine residues, exhibited high affinity to the BRD2 autoantibody and competitively inhibited the binding of the autoantibody to the cellular BRD2 antigen. The use of this cyclic peptide as a capture antigen in human serum enzymelinked immunosorbent assay allowed the distinction of patients with HCC from healthy subjects with 64.41% sensitivity and 82.42% specificity (area under the ROC curve, 0.7761), which is superior to serum alphafetoprotein (AFP; 35.83% sensitivity; 100% specificity; area under the ROC curve, 0.5337) for the diagnosis of HCC. In addition, the detection of the BRD2 autoantibody combined with other autoantibody biomarkers or AFP has increased the accuracy of HCC diagnosis, suggesting that the combinational detection of cancer biomarkers, including the BRD2 autoantibody, is a promising assay for HCC diagnosis.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Camundongos , Animais , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , alfa-Fetoproteínas , Autoanticorpos , Biomarcadores Tumorais , Peptídeos , Camundongos Transgênicos , Curva ROC , Peptídeos Cíclicos , Fatores de TranscriçãoRESUMO
Phage libraries displaying cDNA or random peptides have been used for profiling autoantibodies in cancer. The detection of autoantibodies in human sera using phages displaying specific epitopes is usually performed by phage-immobilized ELISAs which can detect specific antibodies without identification of whole antigens. However, these ELISAs can give feeble detection signals that are indistinguishable from background signals which are caused by human sera. To improve the usefulness of phage ELISA for human sera, the conditions for each step in phage ELISA were optimized. The antigenicity of phage antigens was maximal when using coating buffer of neutral pH. By using protein-free blocking buffer and pre-adsorbing human sera with phage host cell ER2738 extracts significantly decreased non-specific signals. Finally, when these conditions were applied to phage ELISA using K10P1, the values of the negative controls were concentrated near cutoff values, which made the assay more reliable. The optimized phage ELISA conditions described here would increase the efficacy of detection specific autoantibodies in human sera.
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Autoanticorpos/sangue , Bacteriófagos/genética , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Imobilizadas , Humanos , Biblioteca de Peptídeos , Sensibilidade e EspecificidadeRESUMO
Cancer stem cells (CSCs) are regarded as essential targets to overcome tumor progression and therapeutic resistance; however, practical targeting approaches are limited. Here, we identify testis-specific Y-like protein 5 (TSPYL5) as an upstream regulator of CSC-associated genes in non-small cell lung cancer cells, and suggest as a therapeutic target for CSC elimination. TSPYL5 elevation is driven by AKT-dependent TSPYL5 phosphorylation at threonine-120 and stabilization via inhibiting its ubiquitination. TSPYL5-pT120 also induces nuclear translocation and functions as a transcriptional activator of CSC-associated genes, ALDH1 and CD44. Also, nuclear TSPYL5 suppresses the transcription of PTEN, a negative regulator of PI3K signaling. TSPYL5-pT120 maintains persistent CSC-like characteristics via transcriptional activation of CSC-associated genes and a positive feedback loop consisting of AKT/TSPYL5/PTEN signaling pathway. Accordingly, elimination of TSPYL5 by inhibiting TSPYL5-pT120 can block aberrant AKT/TSPYL5/PTEN cyclic signaling and TSPYL5-mediated cancer stemness regulation. Our study suggests TSPYL5 be an effective target for therapy-resistant cancer.
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Neoplasias Pulmonares/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Nucleares/antagonistas & inibidores , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Gefitinibe/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular , Proteínas Nucleares/fisiologia , PTEN Fosfo-Hidrolase/fisiologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
TSPYL5, encoding testis-specific Y-like protein, has been postulated to be a tumor suppressor gene, and its hypermethylation is often associated with human disease, especially cancer. In this study, we report that the TSPYL5 gene was less methylated (30%) in A549 lung adenocarcinoma cells, which are relatively resistant to gamma-radiation, than in H460 lung cancer cells, in which the TSPYL5 gene was hypermethylated (95%); thus, the expression level of TSPYL5 is much higher in A549 cells than in H460 cells. We showed that TSPYL5 suppression with silencing RNA in A549 cells up-regulated cellular PTEN, followed by down-regulation of AKT activation. Therefore, blockage of TSPYL5 sensitized A549 cells to cytotoxic agents such as gamma-radiation. In addition, TSPYL5 suppression also showed an increased level of p21(WAF1/Cip1) and subsequently induced inhibition of cell growth in A549 cells. The overexpression of TSPYL5 in H460 cells showed the opposite effects. This study provides the first demonstration that TSPYL5 modulates cell growth and sensitization of cells to the detrimental effects of damaging agents via regulation of p21(WAF1/Cip1) and PTEN/AKT pathway.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Neoplasias Pulmonares/genética , Proteínas Nucleares/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tolerância a Radiação/genética , Apoptose/genética , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Ilhas de CpG , Inibidor de Quinase Dependente de Ciclina p21/genética , Metilação de DNA , Raios gama , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , PTEN Fosfo-Hidrolase/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
Smooth muscle protein 22-alpha (SM22α) is known as a transformation- and shape change-sensitive actin cross-linking protein found in smooth muscle tissue and fibroblasts; however, its functional role remains uncertain. We reported previously that SM22α overexpression confers resistance against anti-cancer drugs or radiation via induction of metallothionein (MT) isozymes in HepG2 cells. In this study, we demonstrate that SM22α overexpression leads cells to a growth arrest state and promotes cellular senescence caused by treatment with a subclinical dose of γ-radiation (0.05 and 0.1 Gy) or doxorubicin (0.01 and 0.05 µg/ml), compared to control cells. Senescence growth arrest is known to be controlled by p53 phosphorylation/p21(WAF1/Cip1) induction or p16(INK4a)/retinoblastoma protein (pRB) activation. SM22α overexpression in HepG2 cells elevated p16(INK4a) followed by pRB activation, but did not activate the p53/p21(WAF1/Cip1) pathway. Moreover, MT-1G, which is induced by SM22α overexpression, was involved in the activation of the p16(INK4a)/pRB pathway, which led to a growth arrest state and promoted cellular senescence caused by damaging agents. Our findings provide the first demonstration that SM22α modulates cellular senescence caused by damaging agents via regulation of the p16(INK4a)/pRB pathway in HepG2 cells and that these effects of SM22α are partially mediated by MT-1G.
Assuntos
Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Metalotioneína/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Proteína do Retinoblastoma/metabolismo , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Raios gama , Humanos , Metalotioneína/genética , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genéticaRESUMO
Tumor-associated autoantibodies are promising diagnostic biomarkers for early detection of tumors. We have screened a novel tumor-associated autoantibody in hepatocellular carcinoma (HCC) model mice. Its target antigen was identified as eukaryotic translation initiation factor 3 subunit A (EIF3A) by proteomic analysis, and the elevated expression of EIF3A in HCC tissues of tumor model mice as well as human patients was shown. Also, its existence in tumor-derived exosomes was revealed, which seem to be the cause of tumor-associated autoantibody production. To use serum anti-EIF3A autoantibody as biomarker, ELISA detecting anti-EIF3A autoantibody in human serum was performed using autoantibody-specific epitope. For the sensitive detection of serum autoantibodies its specific conformational epitopes were screened from the random cyclic peptide library, and a streptavidin antigen displaying anti-EIF3A autoantibody-specific epitope, XC90p2(-CPVRSGFPC-), was used as capture antigen. It distinguished patients with HCC (n = 102) from healthy controls (n = 0285) with a sensitivity of 79.4% and specificity of 83.5% (AUC = 0.87). Also, by simultaneously detecting with other HCC biomarkers, including alpha-fetoprotein, HCC diagnostic sensitivity improved from 79.4% to 85%. Collectively, we suggest that serum anti-EIF3A autoantibody is a useful biomarker for the diagnosis of HCC and the combinational detection of related biomarkers can enhance the accuracy of the cancer diagnosis.
Assuntos
Autoanticorpos/sangue , Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/diagnóstico , Fator de Iniciação 3 em Eucariotos/imunologia , Neoplasias Hepáticas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Feminino , Humanos , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-IdadeRESUMO
The mammalian antizyme (AZ) promotes ubiqutin-independent degradation of ornithine decarboxylase, a key enzyme in polyamine biosynthesis. This study shows that AZ suppression in human lung carcinoma A549 cells caused growth defects and death, but made the cells resistant to DNA damaging agents such as gamma-radiation and cisplatin. In these cells, the cellular redox potential (glutathione/glutathione disulfide [GSH/GSSG] ratio) was increased and thus intracellular reactive oxygen species were severely diminished, which might cause growth defects and cell death. The increase of cellular redox potential was mainly caused by dramatic increase of the cytoplasmic nicotinamide adenine dinucleotide phosphate (NADP)(+)-dependent isocitrate dehydrogenase, which generates the reducing equivalents NADPH. In the AZ-suppressed cells, the hypoxia inducible factor 1alpha (HIF-1alpha) was also increased. As in other cases which showed an increment of HIF-1alpha and the cellular redox potential, the AZ-suppressed cells showed resistance to gamma-radiation and anticancer drugs. Therefore, these facts might be considered as important for the use of radio- and chemotherapy on tumor cells which show an unbalance in their polyamine levels.
Assuntos
Regulação Enzimológica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isocitrato Desidrogenase/metabolismo , Oxirredução , Linhagem Celular Tumoral , Sobrevivência Celular , Citoplasma/enzimologia , Citoplasma/metabolismo , Citometria de Fluxo , Raios gama , Humanos , Modelos Biológicos , NADP/química , Poliaminas/química , Interferência de RNA , Espécies Reativas de OxigênioRESUMO
Tescalcin (TESC; also known as calcineurin B homologous protein 3, CHP3) has recently reported as a regulator of cancer progression. Here, we showed that the elevation of TESC in non-small cell lung cancer (NSCLC) intensifies epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties, consequently enhancing the cellular resistance to γ-radiation. TESC expression and the phosphorylation (consequent activation) of signal transducer and activator of transcription 3 (STAT3) were upregulated in CSC-like ALDH1high cells than in ALDH1low cells sorted from A549 NSCLC cells. Knockdown of TESC suppressed CSC-like properties as well as STAT3 activation through inhibition of insulin-like growth factor 1 receptor (IGF1R), a major signaling pathway of lung cancer stem cells. TESC activated IGF1R by the direct recruitment of proto-oncogene tyrosine kinase c-Src (c-Src) to IGF1Rß complex. Treatment of IGF1R inhibitor, AG1024, also suppressed c-Src activation, implicating that TESC mediates the mutual activation of c-Src and IGF1R. STAT3 activation by TESC/c-Src/IGF1R signaling pathway subsequently upregulated ALDH1 expression, which enhanced EMT-associated CSC-like properties. Chromatin immunoprecipitation and luciferase assay demonstrated that STAT3 is a potential transcription activator of ALDH1 isozymes. Ultimately, targeting TESC can be a potential strategy to overcome therapeutic resistance in NSCLC caused by augmented EMT and self-renewal capacity.
Assuntos
Aldeído Desidrogenase/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/patologia , Fator de Transcrição STAT3/metabolismo , Células A549 , Família Aldeído Desidrogenase 1 , Animais , Proteína Tirosina Quinase CSK , Proteínas de Ligação ao Cálcio/genética , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos da radiação , Feminino , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/radioterapia , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/efeitos da radiação , Proto-Oncogene Mas , RNA Interferente Pequeno/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/administração & dosagem , Receptor IGF Tipo 1 , Receptores de Somatomedina/antagonistas & inibidores , Receptores de Somatomedina/metabolismo , Retinal Desidrogenase , Tirfostinas/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/metabolismoRESUMO
Influenza, which is a highly contagious disease caused by the influenza A virus, continues to be a major health concern worldwide. Although the accurate and early diagnosis of influenza virus infection is important for controlling the spread of this disease and rapidly initiating antiviral therapy, the current influenza diagnostic kits are limited by their low sensitivity. In this study, we developed several new influenza nucleoprotein (NP)-specific monoclonal antibodies (mAbs) and compared their sensitivity and specificity of those with commercially available anti-NP mAbs. Three mAbs, designated M24.11, M34.3, and M34.33, exhibited higher reactivities to recombinant NPs and A/Puerto Rico/8/1934 (H1N1) viral lysates compared with the commercial mAbs, as assessed using enzyme-linked immunosorbent assays. M34.3 and M34.33 showed higher reactivities with A/California/04/09 (pandemic H1N1) and A/Philippines/2/82 (H3N2) viral lysates than the commercial mAbs. In contrast, M24.11 had marked reactivity with H3N2 but not with pandemic H1N1. Immunofluorescent confocal microscopy showed that the three mAbs effectively detected the presence of influenza virus in lung tissues of mice infected with A/Puerto Rico/8/1934. These results indicate that the newly developed M34.3 and M34.33 mAbs could be useful for the development of influenza diagnostics.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/diagnóstico , Nucleoproteínas/imunologia , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/análise , Anticorpos Antivirais/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nucleoproteínas/análise , Nucleoproteínas/metabolismo , Infecções por Orthomyxoviridae/diagnóstico , Proteínas Recombinantes/análise , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Sensibilidade e EspecificidadeRESUMO
Gangliosides abundant in the nervous system have been implicated in a broad range of biological functions, including the regulation of cell proliferation and death. Glutamate-induced cell death, which is accompanied by an accumulation of reactive oxygen species (ROS), is a major contributor to pathological cell death within the nervous system. However, the mechanism underlying this neuronal cell death has not been fully elucidated. In this study, we report that ganglioside GM3 is involved in neuronal cell death. GM3 was up-regulated in the mouse hippocampal cell line HT22 death caused by glutamate. Increment in GM3 levels by both the exogenous addition of GM3 and the overexpression of the GM3 synthase gene induced neuronal cell death. Overexpression of GM3 synthase by microinjecting mRNA into zebrafish embryos resulted in neuronal cell death in the central nervous system (CNS). Conversely, RNA interference-mediated silencing of GM3 synthase rescued glutamate-induced neuronal death, as evidenced by the inhibition of massive ROS production and intracellular calcium ion influx. 12-lipoxygenase (12-lipoxygenase) (12-LOX) was recruited to glycosphingolipid-enriched microdomains (GEM) in a GM3-dependent manner during oxidative glutamate toxicity. Our findings suggest that GM3 acts as not only a mediator of oxidative HT22 death by glutamate but also a modulator of in vivo neuronal cell death.