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1.
Cell Death Dis ; 12(7): 663, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34230456

RESUMO

A majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.


Assuntos
Adenoviridae/genética , Proteínas E1 de Adenovirus/genética , Antineoplásicos/farmacologia , Imidazóis/farmacologia , Imidazolinas/farmacologia , Mesotelioma/terapia , Neurofibromina 1/metabolismo , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Adenoviridae/crescimento & desenvolvimento , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Regulação Neoplásica da Expressão Gênica , Mesotelioma/genética , Mesotelioma/metabolismo , Mesotelioma/virologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Neurofibromina 1/genética , Vírus Oncolíticos/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Carga Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Replicação Viral , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Anticancer Res ; 41(8): 3731-3740, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34281831

RESUMO

BACKGROUND: The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is thought to have promising clinical potential. However, the off-target effects of Cas9 are a major concern for its application. Therefore, we hypothesized that the adverse effects of off-target gene editing might be minimized if the human codon-optimized Streptococcus pyogenes Cas9 (hCas9) could be specifically expressed in cancer cells. MATERIALS AND METHODS: We constructed a chimeric adenoviral vector, Ad5F35-MKp-hCas9, and infected human bladder cancer cell lines with this vector. The confirmation of hCas9 gene expression was performed in 3-4 days after from infection. RESULTS: hCas9 gene expression was observed in Ad5F35-MKp-hCas9 infected bladder cancer cells but not in non-malignant cells. CONCLUSION: Our study showed that the Ad5F35-MKp-hCas9 vector is capable of expressing the hCas9 gene with high specificity in bladder cancer cells. These findings may help in minimizing the risk of off-target effects of gene editing.


Assuntos
Adenoviridae/genética , Proteína 9 Associada à CRISPR/genética , Vetores Genéticos/genética , Transfecção/métodos , Neoplasias da Bexiga Urinária/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Edição de Genes/métodos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Regiões Promotoras Genéticas , Neoplasias da Bexiga Urinária/patologia
3.
SAGE Open Med Case Rep ; 8: 2050313X20948716, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922794

RESUMO

The differential diagnosis of reactive mesothelial hyperplasia and mesothelioma is difficult. We present a rare case of diffuse pleural thickening with thoracic contraction that was indistinguishable from mesothelioma. A 66-year-old woman with no history of asbestos exposure visited our hospital with a complaint of dyspnea. The clinical findings included circumferential pleural thickening on chest computed tomography image and a high concentration of hyaluronic acid in the pleural fluid. Pleural biopsies obtained by thoracoscopy under local anesthesia were pathologically consistent with mesothelioma, but the patient refused to take any kind of mesothelioma treatments. Four months later, she consented to a surgical pleural biopsy under general anesthesia to obtain larger tissue samples, which included typical proliferating polygonal cells positive for CAM5.2, calretinin, WT-1, D2-40, CK5/6, epithelial membrane antigen, and glucose transporter-1 and negative for carcinoembryonic antigen, BerEP4, and MOC31. The analysis was consistent with diagnosis of epithelioid mesothelioma. Fluorescence in situ hybridization, however, showed the presence of p16 gene, and the expression of BRCA1-associated protein-1 was detected by immunohistochemistry. Our final diagnosis was diffuse pleural thickening unrelated to asbestos exposure. Differential diagnosis of diffuse pleural thickening and malignant mesothelioma is thus difficult and routine immunohistochemical examinations are often insufficient for accurate diagnosis. Multiple diagnostic methods are required for correct diagnosis in a clinically marginal case.

4.
Apoptosis ; 25(7-8): 535-547, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32468177

RESUMO

A majority of mesothelioma had the wild-type p53 genotype but was defective of p53 functions primarily due to a genetic defect in INK4A/ARF region. We examined a growth suppressive activity of CP-31398 which was developed to restore the p53 functions irrespective of the genotype in mesothelioma with wild-type or mutated p53. CP-31398 up-regulated p53 levels in cells with wild-type p53 genotype but induced cell growth suppression in a p53-independent manner. In contrasts, nutlin-3a, an MDM2 inhibitor, increased p53 and p21 levels in mesothelioma with the wild-type p53 genotype and produced growth suppressive effects. We investigated a combinatory effect of CP-31398 and nutlin-2a and found the combination produced synergistic growth inhibition in mesothelioma with the wild-type p53 but not with mutated p53. Western blot analysis showed that the combination increased p53 and the phosphorylation levels greater than treatments with the single agent, augmented cleavages of PARP and caspase-3, and decreased phosphorylated FAK levels. Combination of CP-31398 and defactinib, a FAK inhibitor, also achieved synergistic inhibitory effects and increased p53 with FAK dephosphorylation levels greater than the single treatment. These data indicated that a p53-activating CP-31398 achieved growth inhibitory effects in combination with a MDM2 or a FAK inhibitor and suggested a possible reciprocal pathway between p53 elevation and FAK inactivation.


Assuntos
Antineoplásicos/farmacologia , Quinase 1 de Adesão Focal/genética , Imidazóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Pirimidinas/farmacologia , Proteína Supressora de Tumor p53/genética , Protocolos de Quimioterapia Combinada Antineoplásica , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Sinergismo Farmacológico , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Epitélio/patologia , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Genótipo , Humanos , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/agonistas , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação/efeitos dos fármacos
5.
Int Immunol ; 32(1): 17-26, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31412363

RESUMO

B-cell novel protein 1 (BCNP1) has recently been identified as a new B-cell receptor (BCR) signaling molecule but its physiological function remains unknown. Here, we demonstrate that mice deficient in BCNP1 exhibit impaired B-cell maturation and a reduction of B-1a cells. BCNP1-deficient spleen B cells show enhanced survival, proliferation and Ca2+ influx in response to BCR cross-linking as compared with wild-type spleen B cells. Consistently, mutant B cells show elevated phosphorylation of SYK, B-cell linker protein (BLNK) and PLCγ2 upon BCR cross-linking. In vivo, BCNP1-deficient mice exhibit enhanced humoral immune responses to T-independent and T-dependent antigens. Moreover, aged mutant mice contain elevated levels of serum IgM and IgG3 antibodies and exhibit polyclonal and monoclonal B-cell expansion in lymphoid organs. These results reveal distinct roles for BCNP1 in B-cell development, activation and homeostasis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Linfócitos B/imunologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
6.
Mol Oncol ; 13(6): 1419-1432, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31033201

RESUMO

Pemetrexed (PEM) inhibits DNA and RNA synthesis and is currently one of the first-line agents for mesothelioma. PEM suppresses the activities of several enzymes involved in purine and pyrimidine synthesis, and elevated activity of these enzymes in tumors is often linked with resistance to PEM. The agent also stimulates AMP-activated protein kinase (AMPK) and consequently influences the mammalian target of rapamycin complex 1 (mTORC1) pathways. Nevertheless, it remains unclear whether PEM resistance is linked to the AMPK or mTORC1 pathways. Here, we established two independent PEM-resistant mesothelioma cell lines in which expression of the PEM-target enzymes was not elevated, and found that levels of phosphorylated AMPK and p70S6K and, to a lesser extent, levels of phosphorylated AKT and p53, were increased in these cells as compared with the respective parent cells. PEM stimulation also augmented phosphorylation of AMPK, p70S6K, AKT and p53 in most cases. An AMPK activator increased phosphorylation and PEM resistance in parental cells, and the inhibitor decreased the resistance of PEM-resistant cells. In contrast, inhibitors for p70S6K and AKT did not influence PEM resistance; furthermore, increased levels of endogenous p53 did not affect PEM sensitivity. These data collectively indicate that constitutive activation of AMPK is associated with PEM resistance, and that this is unconnected with elevated DNA and RNA synthesis.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Mesotelioma/metabolismo , Pemetrexede/farmacologia , Western Blotting , Linhagem Celular Tumoral , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo
7.
Am J Cancer Res ; 9(1): 79-93, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30755813

RESUMO

Restoration of p53 functions is one of the therapeutic strategies for esophageal carcinoma which is often defective of the p53 pathway. We examined effects of CP-31398 which potentially increased expression of wild-type p53 or converted mutated p53 to the wild-type. We used 9 kinds of human squamous esophageal carcinoma cells with different p53 genotypes and examined expression of p53 and the related molecules in CP-31398-treated cells. Cisplatin, a DNA damaging agent, induced cleavages of PARP and caspase-3 without increase of p53 levels, indicating that the p53 down-stream pathway was disrupted in these cells. CP-31398 induced growth retardation but the cytotoxic effects were irrelevant to p53 genotype. CP-31398 influenced expression of p53 and the downstream molecules in a cell-dependent manner, but constantly increased p21 expression at the transcriptional level with decreased YY1 expression. Knockdown experiments with siRNA demonstrated that the CP-31398-mediated p21 up-regulation was unrelated with p53 expression but was associated with YY1 expression. We also showed that CP-31398-induced cell cycle changes including increase of G2/M populations was attributable to the up-regulated p21. These data collectively indicated that CP-31398 augmented endogenous p21 levels and induced cell cycle changes through regulation of YY1, and that YY1 was a novel target of CP-31398 in p53 dysfunctional cells.

8.
Cancer Gene Ther ; 26(5-6): 128-135, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30348946

RESUMO

Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefits in a variety of cancer models. In the present study, we evaluated a possible combinatorial effect of prodrug activator genes delivered by two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV) on human hepatocellular carcinoma Hep3B cells. Both RRVs showed efficient replicative spread in culture and can overcame superinfection resistance each other. Notably, the replication and spread of each RRV in culture remained unaffected by pretransduction with the counterpart RRV. We further transduced cells with RRVs which individually possessed the prodrug activator genes yeast cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) alone or in combination, and evaluated the cytotoxic effects of RRV-mediated gene therapy with CD and TK in the presence of the respective prodrugs, 5-fluorocytosine and ganciclovir. All combinations of the two prodrug activator genes produced synergistic cytocidal effects, but the combined effects of the different genes were significantly greater than those of the same genes when delivered by two different vectors. The present findings indicate the potential utility of dual-vector gene therapy using two different RRVs carrying different prodrug activator genes.


Assuntos
Terapia Genética/métodos , Vetores Genéticos/genética , Pró-Fármacos/uso terapêutico , Retroviridae/genética , Linhagem Celular Tumoral , Humanos , Pró-Fármacos/farmacologia
9.
Oncotarget ; 9(40): 26130-26143, 2018 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-29899847

RESUMO

Heat shock protein 90 (HSP90) inhibitors suppressed MDM4 functions which mediated p53 ubiquitination, and blocked a chaperon function which influenced expression of the client proteins. We examined cytotoxic effects of the inhibitors, 17-allylamino-17-demetheoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG), on mesothelioma and investigated combinatory effects of the inhibitors and adenoviruses expressing the wild-type p53 gene (Ad-p53). A majority of mesothelioma lacks p14 and p16 expression, which leads to defective p53 pathway despite bearing the wild-type p53 genotype. The HSP90 inhibitors up-regulated endogenous wild-type p53 expression and induced cell death. Furthermore, the inhibitors increased the endogenous p53 levels that were induced by cisplatin. Nevertheless, the HSP90 inhibitors suppressed Ad-p53-induced exogenous p53 expression primarily at a posttranscriptional level and inhibited the Ad-p53-mediated cell death. HSP90 inhibitors suppressed ubiquitination processes which were involved in p53 degradation, but a proteasome inhibitor, MG-132, prevented the HSP90 inhibitors-induced p53 down-regulation. In contrast, an inhibitor for HSP70 with a chaperon function, pifithrin-µ, did not produce the p53 down-regulation. The HSP90 inhibitors did not suppress expression of Ad receptor molecules but rather increased expression of green fluorescence protein transduced by the same Ad vector. These data collectively indicated that an HSP90 inhibitor possessed a divalent action on p53 expression, as an activator for endogenous wild-type p53 through inhibited ubiquitination and a negative regulator of exogenously over-expressed p53 through the proteasome pathway.

10.
Int J Mol Sci ; 19(6)2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29921831

RESUMO

Cholesterol, a major component of the plasma membrane, determines the physicalproperties of biological membranes and plays a critical role in the assembly of membranemicrodomains. Enrichment or deprivation of membrane cholesterol affects the activities of manysignaling molecules at the plasma membrane. Cell detachment changes the structure of the plasmamembrane and influences the localizations of lipids, including cholesterol. Recent studies showedthat cell detachment changes the activities of a variety of signaling molecules. We previously reportedthat the localization and the function of the Src-family kinase Lyn are critically regulated by its membrane anchorage through lipid modifications. More recently, we found that the localization andthe activity of Lyn were changed upon cell detachment, although the manners of which vary betweencell types. In this review, we highlight the changes in the localization of Lyn and a role of cholesterolin the regulation of Lyn's activation following cell detachment.


Assuntos
Adesão Celular , Membrana Celular/metabolismo , Colesterol/metabolismo , Quinases da Família src/metabolismo , Animais , Humanos , Transdução de Sinais , Quinases da Família src/genética
11.
Int J Clin Oncol ; 23(5): 917-923, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29691673

RESUMO

BACKGROUND: Although serum p53 autoantibodies (s-p53-Abs) are induced even in the early stages of colorectal cancer, their positive rate is only approximately 20%. Therefore, we assessed the possibility of using other serum autoantibodies to increase the positive rates for detecting colorectal cancer. METHODS: Autoantibodies against 17 tumor antigens (p53, RalA, HSP70, Galectin1, KM-HN-1, NY-ESO-1, p90, Sui1, HSP40, CyclinB1, HCC-22-5, c-myc, PrxVI, VEGF, HCA25a, p62, and Annexin II) were evaluated in 279 patients with colorectal cancer and 74 healthy controls. Cutoff values were fixed at mean + 3 standard deviations of serum titers in healthy controls. RESULTS: Autoantibodies with the highest positive rates were p53 (20%), RalA (14%), HSP70 (12%), and Galectin1 (11%). Combination assays using multiple autoantibodies increased the positive rates based on the number of autoantibodies used. Positive rates of 56, 62, 66, 71, and 73% were obtained with 6, 9, 11, 14, and 17 antibodies, respectively, for the overall disease. Moreover, these autoantibodies showed relatively high positive rates even during stage 0/I disease (55 and 70% with 6 and 17 antibodies, respectively). CONCLUSION: The measurement of set of 17 autoantibodies allowed autoantibody profiling in patients with colorectal cancer. The combination assay of six tumor antigens (p53, RalA, HSP70, Galectin1, KM-HN-1, and NY-ESO-1) achieved a positive rate of 56%. Such high positive rates will be helpful for detecting colorectal cancer regardless of tumor stages.


Assuntos
Antígenos de Neoplasias/sangue , Autoanticorpos/sangue , Biomarcadores Tumorais/sangue , Neoplasias Colorretais/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/imunologia , Autoanticorpos/imunologia , Estudos de Casos e Controles , Neoplasias Colorretais/sangue , Neoplasias Colorretais/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
12.
Cancer Cell Int ; 17: 120, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29238267

RESUMO

Background: Pemetrexed (PEM) is an anti-cancer agent targeting DNA and RNA synthesis, and clinically in use for mesothelioma and non-small cell lung carcinoma. A mechanism of resistance to PEM is associated with elevated activities of several enzymes involved in nucleic acid metabolism. Methods: We established two kinds of PEM-resistant mesothelioma cells which did not show any increase of the relevant enzyme activities. We screened genes enhanced in the PEM-resistant cells with a microarray analysis and confirmed the expression levels with Western blot analysis. A possible involvement of the candidates in the PEM-resistance was examined with a WST assay after knocking down the expression with si-RNA. We also analyzed a mechanism of the up-regulated expression with agents influencing AMP-activated protein kinase (AMPK) and p53. Results: We found that expression of cardiac ankyrin repeat protein (CARP) was elevated in the PEM-resistant cells with a microarray and Western blot analysis. Down-regulation of CARP expression with si-RNA did not however influence the PEM resistance. Parent and PEM-resistant cells treated with PEM increased expression of CARP, AMPK, p53 and histone H2AX. The CARP up-regulation was however irrelevant to the p53 genotypes and not induced by an AMPK activator. Augmented p53 levels with nutlin-3a, an inhibitor for p53 degradation, and DNA damages were not always associated with the enhanced CARP expression. Conclusions: These data collectively suggest that up-regulated CARP expression is a potential marker for development of PEM-resistance in mesothelioma and that the PEM-mediated enhanced expression is not directly linked with immediate cellular responses to PEM.

13.
Virol J ; 14(1): 219, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-29126418

RESUMO

BACKGROUND: Genetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity. METHODS: We used type 5 Ad in which the expression of E1A gene was activated by 5'-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses. RESULTS: The replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level. CONCLUSIONS: Image cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection.


Assuntos
Vetores Genéticos/fisiologia , Citometria por Imagem , Neoplasias Pulmonares/virologia , Mesotelioma/virologia , Terapia Viral Oncolítica/métodos , Adenoviridae/genética , Proteínas E1A de Adenovirus/metabolismo , Proteínas do Capsídeo/metabolismo , Caspase 3/metabolismo , Morte Celular , Linhagem Celular Tumoral , Vetores Genéticos/genética , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Mesotelioma/metabolismo , Mesotelioma/patologia , Mesotelioma Maligno , Análise de Célula Única , Replicação Viral
14.
BMC Cancer ; 17(1): 622, 2017 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-28874135

RESUMO

BACKGROUND: Replication-competent adenoviruses (Ad) produced cytotoxic effects on infected tumors and have been examined for the clinical applicability. A biomarkers to predict the cytotoxicity is valuable in a clinical setting. METHODS: We constructed type 5 Ad (Ad5) of which the expression of E1A gene was activated by a 5' regulatory sequences of survivin, midkine or cyclooxygenase-2, which were highly expressed in human tumors. We also produced the same replication-competent Ad of which the fiber-knob region was replaced by that of Ad35 (AdF35). The cytotoxicity was examined by a colorimetric assay with human tumor cell lines, 4 kinds of pancreatic, 9 esophageal carcinoma and 5 mesothelioma. Ad infectivity and Ad-mediated gene expression were examined with replication-incompetent Ad5 and AdF35 which expressed the green fluorescence protein gene. Expression of cellular receptors for Ad5 and AdF35 was also examined with flow cytometry. A transcriptional activity of the regulatory sequences was investigated with a luciferase assay in the tumor cells. We then investigated a possible correlation between Ad-mediated cytotoxicity and the infectivity/gene expression, the transcriptional activity or the p53 genotype. RESULTS: We found that the cytotoxicity was greater with AdF35 than with Ad5 vectors, but was not correlated with the Ad infectivity/gene expression irrespective of the fiber-knob region or the E1A-activating transcriptional activity. In contrast, replication-competent Ad produced greater cytotoxicity in p53 mutated than in wild-type esophageal carcinoma cells, suggesting a possible association between the cytotoxicity and the p53 genotype. CONCLUSIONS: Sensitivity to Ad-mediated cytotoxic activity was linked with the p53 genotype but was not lineally correlated with the infectivity/gene expression or the E1A expression.


Assuntos
Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Sequências Reguladoras de Ácido Nucleico , Proteína Supressora de Tumor p53/genética , Replicação Viral , Linhagem Celular Tumoral , Efeito Citopatogênico Viral , Genes Reporter , Genótipo , Humanos , Ligação Proteica , Receptores Virais/genética , Receptores Virais/metabolismo , Ativação Transcricional , Transdução Genética , Transgenes , Proteína Supressora de Tumor p53/metabolismo
15.
Cancer Med ; 6(10): 2385-2397, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28941156

RESUMO

Although oncolytic adenoviruses are promising cancer therapy agents, for effective oncolytic activity, viruses need to specifically infect and effectively replicate in cancer cells but not in normal cells. We have previously identified a pancreatic cancer-targeting ligand, SYENFSA (SYE), by screening an adenovirus library displaying random peptides against human pancreatic cancer cells and reported that a survivin promoter-regulated adenovirus, displaying the SYE ligand (AdSur-SYE), provided effective oncolysis of pancreatic ductal adenocarcinoma (PDAC) in a preclinical study. As we examined the infectivity of AdSur-SYE in human surgical specimens of various pancreatic tumors, we unexpectedly found that AdSur-SYE showed high gene transduction efficiency for pancreatic neuroendocrine tumors (PNETs) as well as for PDAC, 9.1- and 6.2-fold, respectively, compared to that of the nontargeting virus (AdSur). The infectivity of both vectors was almost the same in other cancers and organs such as the pancreas. Immunostaining indicated that the cells infected with AdSur-SYE were PNET cells but not stromal cells. AdSur-SYE showed a significantly higher oncolytic potency than that of AdSur in human PNET cell lines, and intratumoral infection with AdSur-SYE completely diminished subcutaneous tumors in a murine model, in which AdSur-SYE effectively proliferated and spread. AdSur-SYE exerted a stronger oncolytic effect in primary PNET cells cocultured with mouse embryonic fibroblasts than AdSur did. Thus, AdSur-SYE shows promise as a next-generation therapy for PNET.


Assuntos
Adenoviridae/genética , Vetores Genéticos/genética , Ligantes , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/patologia , Terapia Viral Oncolítica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Peptídeos/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Genes Reporter , Terapia Genética , Vetores Genéticos/administração & dosagem , Humanos , Imuno-Histoquímica , Proteínas Inibidoras de Apoptose/genética , Camundongos , Tumores Neuroendócrinos/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Neoplasias Pancreáticas/terapia , Regiões Promotoras Genéticas , Survivina , Transdução Genética , Transgenes , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Anticancer Res ; 37(7): 3599-3605, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28668851

RESUMO

BACKGROUND: Optimizing targeting strategies for vectors in order to enhance antitumor activity and secure patient safety is important for cancer gene therapy. We previously identified two pancreatic cancer-targeting ligands (PFWSGAV: PFW and SYENFSA: SYE) by screening an adenovirus library in vivo and in vitro, respectively. MATERIALS AND METHODS: To examine clinical usefulness, we assessed gene-transduction efficiency using surgically-resected pancreatic cancer specimens and ascites cells. RESULTS: For surgical specimens, vectors displaying PFW and SYE improved transduction efficiency by 4.4- and 4.3-fold, respectively. The SYE-displaying vector was >2-fold more efficient for all seven cases, whereas the PFW-displaying vector increased efficiency in two out of four cases. For ascites samples, both vectors increased gene-transduction efficiency of epithelial cell adhesion molecule (EpCAM)-positive ascites cells by >2-fold in two out of five cases. CONCLUSION: Both vectors enhanced adenovirus infectivity of pancreatic cancer cells and have potential for gene therapy of pancreatic cancer; therefore they should be further evaluated in clinical studies.


Assuntos
Adenoviridae/genética , Ascite/genética , Ascite/virologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/virologia , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial/genética , Terapia Genética/métodos , Humanos , Pâncreas/virologia , Transdução Genética/métodos
17.
BMC Cancer ; 17(1): 309, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28464864

RESUMO

BACKGROUND: Mesothelioma is resistant to conventional treatments and is often defective in p53 pathways. We then examined anti-tumor effects of metformin, an agent for type 2 diabetes, and combinatory effects of metformin and nutlin-3a, an inhibitor for ubiquitin-mediated p53 degradation, on human mesothelioma. METHODS: We examined the effects with a colorimetric assay and cell cycle analyses, and investigated molecular events in cells treated with metformin and/or nutlin-3a with Western blot analyses. An involvement of p53 was tested with siRNA for p53. RESULTS: Metformin suppressed cell growth of 9 kinds of mesothelioma including immortalized cells of mesothelium origin irrespective of the p53 functional status, whereas susceptibility to nutlin-3a was partly dependent on the p53 genotype. We investigated combinatory effects of metformin and nutlin-3a on, nutlin-3a sensitive MSTO-211H and NCI-H28 cells and insensitive EHMES-10 cells, all of which had the wild-type p53 gene. Knockdown of p53 expression with the siRNA demonstrated that susceptibility of MSTO-211H and NCI-H28 cells to nutlin-3a was p53-dependent, whereas that of EHMES-10 cells was not. Nevertheless, all the cells treated with both agents produced additive or synergistic growth inhibitory effects. Cell cycle analyses also showed that the combination increased sub-G1 fractions greater than metformin or nutlin-3a alone in MSTO-211H and EHMES-10 cells. Western blot analyses showed that metformin inhibited downstream pathways of the mammalian target of rapamycin (mTOR) but did not activate the p53 pathways, whereas nutlin-3a phosphorylated p53 and suppressed mTOR pathways. Cleaved caspase-3 and conversion of LC3A/B were also detected but it was dependent on cells and treatments. The combination of both agents in MSTO-211H cells rather suppressed the p53 pathways that were activated by nutrin-3a treatments, whereas the combination rather augmented the p53 actions in NCI-H28 and EHMES-10 cells. CONCLUSION: These data collectively indicated a possible interactions between mTOR and p53 pathways, and the combinatory effects were attributable to differential mechanisms induced by a cross-talk between the pathways.


Assuntos
Antineoplásicos/farmacologia , Imidazóis/farmacologia , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Metformina/farmacologia , Piperazinas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Mesotelioma Maligno , Transdução de Sinais/efeitos dos fármacos
18.
BMC Cancer ; 17(1): 212, 2017 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-28335760

RESUMO

BACKGROUND: We evaluated possible diagnostic and prognostic values of serum midkine in malignant pleural mesothelioma in comparison with those of serum mesothelin, a well-established diagnostic biomarker. METHODS: Serum mesothelin and midkine levels were determined with an enzyme-linked immunosorbent assay. We examined specimens from 95 Turkish cases with malignant pleural mesothelioma, 56 metastatic cancers to pleura, 27 other types of benign pleural diseases and 20 benign asbestos pleurisy. The cut-off values were 1.5 nmol/L for mesothelin and 421 pg/mL for midkine. RESULTS: Sensitivity and specificity of mesothelin were 51.6 and 71.4%, 51.6 and 85.2%, and 51.6 and 85% for differentiating mesothelioma from metastatic cancers to pleura, other benign pleural diseases and benign asbestos pleurisy, respectively. Sensitivity and specificity of midkine were 61.1 and 41.1%, 61.1 and 48.1%, and 61.1 and 75% to distinguish mesothelioma from metastatic cancers to pleura, other benign pleural diseases and benign asbestos pleurisy, respectively. Combination of both biomarkers did not improve the differential diagnostic efficacy. Mesothelin levels were elevated in the epitheloid type and in the advanced cases, but were not related to the prognosis. In contrast, elevated baseline levels of midkine were independently associated with a poor prognosis of mesothelioma patients after adjusting for the stage, the histological subtypes and treatment schedules (HR = 1.84; 95% CI: 1.09-3.09) (p = 0.022). CONCLUSIONS: Serum mesothelin showed moderate sensitivity and high specificity to differentiate malignant pleural mesothelioma from metastatic malignancy to pleura and from benign pleural diseases. In contrast, midkine was a useful marker for predicting prognosis of mesothelioma patients.


Assuntos
Biomarcadores Tumorais/sangue , Citocinas/sangue , Proteínas Ligadas por GPI/sangue , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/epidemiologia , Mesotelioma/sangue , Mesotelioma/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Neoplasias Pulmonares/diagnóstico , Masculino , Mesotelioma/diagnóstico , Mesotelioma Maligno , Pessoa de Meia-Idade , Midkina , Prognóstico , Curva ROC , Turquia/epidemiologia
19.
Ann Diagn Pathol ; 26: 31-37, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28038708

RESUMO

Malignant mesothelioma is a highly aggressive neoplasm, and the histologic subtype is one of the most reliable prognostic factors. Some biphasic mesotheliomas are difficult to distinguish from epithelioid mesotheliomas with atypical fibrous stroma. The aim of this study was to analyze p16/CDKN2A deletions in mesotheliomas by fluorescence in situ hybridization (FISH) and BAP1 immunohistochemistry to evaluate their potential role in the diagnosis of biphasic mesothelioma. We collected 38 cases of pleural mesotheliomas. The results of this study clearly distinguished 29 cases of biphasic mesothelioma from 9 cases of epithelioid mesothelioma. The proportion of biphasic mesotheliomas with homozygous deletions of p16/CDKN2A in total was 96.6% (28/29). Homozygous deletion of p16/CDKN2A was observed in 18 (94.7%) of 19 biphasic mesotheliomas with 100% concordance of the p16/CDKN2A deletion status between the epithelioid and sarcomatoid components in each case. Homozygous deletion of the p16/CDKN2A was observed in 7 (77.8%) of 9 epithelioid mesotheliomas but not in fibrous stroma. BAP1 loss was observed in 5 (38.5%) of 13 biphasic mesotheliomas and in both epithelioid and sarcomatoid components. BAP1 loss was observed in 5 (62.5%) of 8 epithelioid mesotheliomas but not in fibrous stroma. Homozygous deletion of p16/CDKN2A is common in biphasic mesotheliomas, and the analysis of only one component of mesothelioma is sufficient to show that the tumor is malignant. However, compared with histology alone, FISH analysis of the p16/CDKN2A status and BAP1 immunohistochemistry in the spindled mesothelium provide a more objective means to differentiate between biphasic mesothelioma and epithelioid mesothelioma with atypical stromal cells.


Assuntos
Inibidor de Quinase Dependente de Ciclina p18/genética , Mesotelioma/diagnóstico , Mesotelioma/genética , Neoplasias Pleurais/diagnóstico , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Idoso , Biomarcadores Tumorais/análise , Inibidor p16 de Quinase Dependente de Ciclina , Diagnóstico Diferencial , Feminino , Humanos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Masculino , Pessoa de Meia-Idade , Neoplasias Pleurais/genética
20.
Cancer Sci ; 108(3): 308-315, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28064445

RESUMO

Gastric cancer is the second leading cause of cancer death in the world, and effective diagnosis is extremely important for good outcome. We assessed the diagnostic potential of an autoantibody panel that may provide a novel tool for the early detection of gastric cancer. We analyzed data from patients with gastric cancer and normal controls in test and validation cohorts. Autoantibody levels were measured against a panel of six tumor-associated antigens (TAAs) by ELISA: p53, heat shock protein 70, HCC-22-5, peroxiredoxin VI, KM-HN-1, and p90 TAA. We assessed serum autoantibodies in 100 participants in the test cohort. The validation cohort comprised 248 participants. Autoantibodies to at least one of the six antigens showed a sensitivity/specificity of 49.0% (95% confidence interval [CI], 39.2-58.8%)/92.4% (95% CI, 87.2-97.6%), and 52.0% (95% CI, 42.2-61.8%)/90.5% (95% CI, 84.8-96.3%) in the test and validation cohorts, respectively. In the validation cohort, no significant differences were seen when patients were subdivided based on age, sex, depth of tumor invasion, lymph node metastasis, distant metastasis, peritoneal dissemination, or TNM stage. Patients who were positive for more than two antibodies in the panel tended to have a worse prognosis than those who were positive for one or no antibody. Measurement of autoantibody response to multiple TAAs in an optimized panel assay to discriminate patients with early stage gastric cancer from normal controls may aid in the early detection of gastric cancer.


Assuntos
Antígenos de Neoplasias/imunologia , Autoanticorpos/imunologia , Biomarcadores Tumorais/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias Gástricas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Autoantígenos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas de Choque Térmico HSP70/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas de Membrana/imunologia , Pessoa de Meia-Idade , Proteínas Nucleares/imunologia , Peroxirredoxina VI/imunologia , Prognóstico , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/mortalidade , Proteína Supressora de Tumor p53/imunologia
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