Publications by authors named "Masatoshi Tagawa"

123 Publications

Diffuse pleural thickening and thoracic contraction: An indistinguishable case from malignant pleural mesothelioma.

SAGE Open Med Case Rep 2020 14;8:2050313X20948716. Epub 2020 Aug 14.

Department of Pathology, Tokyo Women's Medical University Yachiyo Medical Center, Yachiyo, Japan.

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.
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http://dx.doi.org/10.1177/2050313X20948716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446552PMC
August 2020

Combination of a p53-activating CP-31398 and an MDM2 or a FAK inhibitor produces growth suppressive effects in mesothelioma with wild-type p53 genotype.

Apoptosis 2020 08;25(7-8):535-547

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba, 260-8717, Japan.

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.
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http://dx.doi.org/10.1007/s10495-020-01612-6DOI Listing
August 2020

Distinct roles of BCNP1 in B-cell development and activation.

Int Immunol 2020 01;32(1):17-26

Department of Immunology, School of Basic Medical Sciences.

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.
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http://dx.doi.org/10.1093/intimm/dxz055DOI Listing
January 2020

AMPK activation induced in pemetrexed-treated cells is associated with development of drug resistance independently of target enzyme expression.

Mol Oncol 2019 06 15;13(6):1419-1432. Epub 2019 May 15.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Japan.

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.
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http://dx.doi.org/10.1002/1878-0261.12496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547620PMC
June 2019

A p53-stabilizing agent, CP-31398, induces p21 expression with increased G2/M phase through the YY1 transcription factor in esophageal carcinoma defective of the p53 pathway.

Am J Cancer Res 2019 1;9(1):79-93. Epub 2019 Jan 1.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute 666-2 Nitona, Chuo-ku, Chiba 260-8717, Japan.

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 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 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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356922PMC
January 2019

Dual-vector prodrug activator gene therapy using retroviral replicating vectors.

Cancer Gene Ther 2019 05 22;26(5-6):128-135. Epub 2018 Oct 22.

Department of Cell Biology and Pathology, University of Miami, Miami, USA.

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.
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http://dx.doi.org/10.1038/s41417-018-0051-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760537PMC
May 2019

Role of Membrane Cholesterol Levels in Activation of Lyn upon Cell Detachment.

Int J Mol Sci 2018 06 19;19(6). Epub 2018 Jun 19.

Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.

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.
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http://dx.doi.org/10.3390/ijms19061811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032236PMC
June 2018

Heat shock protein 90 inhibitors augment endogenous wild-type p53 expression but down-regulate the adenovirally-induced expression by inhibiting a proteasome activity.

Oncotarget 2018 May 25;9(40):26130-26143. Epub 2018 May 25.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chuo-ku, Chiba 260-8717, Japan.

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 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 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.
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http://dx.doi.org/10.18632/oncotarget.25452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995238PMC
May 2018

Multi-panel assay of serum autoantibodies in colorectal cancer.

Int J Clin Oncol 2018 Oct 24;23(5):917-923. Epub 2018 Apr 24.

Department of Surgery, School of Medicine, Toho University, 6-11-1 Omori-Nishi, Ota-ku, Tokyo, 143-8541, Japan.

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.
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http://dx.doi.org/10.1007/s10147-018-1278-3DOI Listing
October 2018

Augmented expression of cardiac ankyrin repeat protein is induced by pemetrexed and a possible marker for the pemetrexed resistance in mesothelioma cells.

Cancer Cell Int 2017 11;17:120. Epub 2017 Dec 11.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba, 260-8717 Japan.

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 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.
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http://dx.doi.org/10.1186/s12935-017-0493-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5725641PMC
December 2017

An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced.

Virol J 2017 11 10;14(1):219. Epub 2017 Nov 10.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba, 260-8717, Japan.

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.
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http://dx.doi.org/10.1186/s12985-017-0888-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681831PMC
November 2017

Strong antitumor efficacy of a pancreatic tumor-targeting oncolytic adenovirus for neuroendocrine tumors.

Cancer Med 2017 Oct 21;6(10):2385-2397. Epub 2017 Sep 21.

Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan.

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.
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http://dx.doi.org/10.1002/cam4.1185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633550PMC
October 2017

Cytotoxicity of replication-competent adenoviruses powered by an exogenous regulatory region is not linearly correlated with the viral infectivity/gene expression or with the E1A-activating ability but is associated with the p53 genotypes.

BMC Cancer 2017 Sep 5;17(1):622. Epub 2017 Sep 5.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chiba, Japan.

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.
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http://dx.doi.org/10.1186/s12885-017-3621-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584036PMC
September 2017

A Tumor-targeting Adenovirus with High Gene-transduction Efficiency for Primary Pancreatic Cancer and Ascites Cells.

Anticancer Res 2017 07;37(7):3599-3605

Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan

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.
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http://dx.doi.org/10.21873/anticanres.11730DOI Listing
July 2017

Metformin produces growth inhibitory effects in combination with nutlin-3a on malignant mesothelioma through a cross-talk between mTOR and p53 pathways.

BMC Cancer 2017 05 2;17(1):309. Epub 2017 May 2.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba, 260-8717, Japan.

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.
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http://dx.doi.org/10.1186/s12885-017-3300-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414226PMC
May 2017

Midkine is a potential novel marker for malignant mesothelioma with different prognostic and diagnostic values from mesothelin.

BMC Cancer 2017 03 23;17(1):212. Epub 2017 Mar 23.

Department of Chest Diseases, Medical Faculty, Lung and Pleural Cancers Research and Clinical Center, Eskisehir Osmangazi University, 26 000, Eskisehir, Turkey.

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.
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http://dx.doi.org/10.1186/s12885-017-3209-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5362983PMC
March 2017

Panel of autoantibodies against multiple tumor-associated antigens for detecting gastric cancer.

Cancer Sci 2017 Mar;108(3):308-315

Department of Surgery, School of Medicine, Toho University, Tokyo, Japan.

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.
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http://dx.doi.org/10.1111/cas.13158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5378227PMC
March 2017

Usefulness of p16/CDKN2A fluorescence in situ hybridization and BAP1 immunohistochemistry for the diagnosis of biphasic mesothelioma.

Ann Diagn Pathol 2017 Feb 22;26:31-37. Epub 2016 Oct 22.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Nitona 666-2, Chuo-ku, Chiba, Chiba 260-8717, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.anndiagpath.2016.10.010DOI Listing
February 2017

Combination of a third generation bisphosphonate and replication-competent adenoviruses augments the cytotoxicity on mesothelioma.

BMC Cancer 2016 07 12;16:455. Epub 2016 Jul 12.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba, 260-8717, Japan.

Background: Approximately 80 % of mesothelioma specimens have the wild-type p53 gene, whereas they contain homozygous deletions in the INK4A/ARF locus that encodes p14 (ARF) and the 16 (INK4A) genes. Consequently, the majority of mesothelioma is defective of the p53 pathways. We examined whether zoledronic acid (ZOL), a third generation bisphosphonate, and adenoviruses with a deletion of the E1B-55kD gene (Ad-delE1B55), which augments p53 levels in the infected tumors, could produce combinatory anti-tumor effects on human mesothelioma cells bearing the wild-type p53 gene.

Methods: Cytotoxicity of ZOL and Ad-delE1B55 was assessed with a WST assay. Cell cycle changes were tested with flow cytometry. Expression levels of relevant molecules were examined with western blot analysis to investigate a possible mechanism of cytotoxicity. Furthermore, the expressions of Ad receptors on target cells and infectivity were estimated with flow cytometry. Viral replication was assayed with the tissue culture infection dose method.

Results: A combinatory use of ZOL and Ad-delE1B55 suppressed cell growth and increased sub-G1 or S-phase populations compared with a single agent, depending on cells tested. The combinatory treatment up-regulated p53 levels and subsequently enhanced the cleavage of caspase-3, 8, 9 and poly (ADP-ribose) polymerase, but expression of molecules involved in autophagy pathways were inconsistent. ZOL-treated cells also increased Ad infectivity with a dose-dependent manner and augmented Ad replication although the expression levels of integrin molecules, one of the Ad receptors, were down-regulated.

Conclusions: These findings indicated that ZOL and Ad-delE1B55 achieved combinatory anti-tumor effects through augmented apoptotic pathways or increased viral replication.
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http://dx.doi.org/10.1186/s12885-016-2483-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4942884PMC
July 2016

Unfavorable neuroblastoma prognostic factor NLRR2 inhibits cell differentiation by transcriptional induction through JNK pathway.

Cancer Sci 2016 Sep 2;107(9):1223-32. Epub 2016 Sep 2.

Saga-Ken Medical Center Koseikan, Saga, Japan.

The novel human gene family encoding neuronal leucine rich repeat (NLRR) proteins were identified as prognostic markers from our previous screening of primary neuroblastoma (NB) cDNA libraries. Of the NLRR gene family members, NLRR1 and NLRR3 are associated with the regulation of cellular proliferation and differentiation, respectively. However, the functional regulation and clinical significance of NLRR2 in NB remain unclear. Here, we evaluated the differential expression of NLRR2, where high expressions of NLRR2 were significantly associated with a poor prognosis of NB (P = 0.0009), in 78 NBs. Enforced expression of NLRR2 in NB cells enhanced cellular proliferation and induced resistance to retinoic acid (RA)-mediated cell growth inhibition. In contrast, knockdown of NLRR2 exhibited growth inhibition effects and enhanced RA-induced cell differentiation in NB cells. After RA treatment, NLRR2 expression was increased and correlated with the upregulation of c-Jun, a member of the activator protein-1 (AP-1) family in NB cells. Moreover, the expressions of NLRR2 and c-Jun were suppressed by treatment with a JNK inhibitor, which ameliorated the promoter activity of the NLRR2 gene while knockdown of c-Jun reduced NLRR2 expression. We then searched AP-1 binding consensus in the NLRR2 promoter region and confirmed c-Jun recruitment at a consensus. Conclusively, NLRR2 must be an inducible gene regulated by the JNK pathway to enhance cell survival and inhibit NB cell differentiation. Therefore, NLRR2 should have an important role in NB aggressiveness and be a potential therapeutic target for the treatment of RA resistant and aggressive NB.
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http://dx.doi.org/10.1111/cas.13003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5021041PMC
September 2016

Cytologic Differential Diagnosis of Malignant Mesothelioma and Reactive Mesothelial Cells With FISH Analysis of p16.

Diagn Cytopathol 2016 Jul 15;44(7):591-8. Epub 2016 Apr 15.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chiba, Japan.

Background: Mesothelioma patients often present with serosal effusions, which are ideal for cytopathological diagnoses. However, the morphological overlap between malignant and benign mesothelial proliferation can make a conclusive cytological diagnosis of mesothelioma elusive because immunohistochemical staining does not discriminate definitively between the two in this setting. p16 is deleted in up to 80% of pleural mesotheliomas. The aim of this study was to establish the correlation between the p16 deletion status of the cell block with that of its corresponding tumor using fluorescence in situ hybridization (FISH) analysis for individual patient tumors.

Methods: Twenty-two biopsies and 24 corresponding cell blocks, containing serosal effusions with atypical mesothelial cells from 22 patients with histologically confirmed pleural mesotheliomas, were analyzed with p16 FISH. Seventeen cell blocks consisting of serosal effusions with reactive mesothelial cells from nonmesothelioma cases were also analyzed. Combined immunofluorescence and FISH were also performed.

Results: Seventeen of the 22 mesothelioma patients (77.3%) showed homozygous deletions of p16 in the tumor tissue and in the atypical mesothelial cells from the cell blocks. p16 FISH followed by immunofluorescence with EMA was helpful towards identifying the mesothelioma cells in the cell blocks.

Conclusion: We confirmed that the p16 FISH results obtained from the cell blocks are as reliable as those from the tissue sections. Cell block analysis is recommended for patients with serosal effusions of unknown origins with the following methods: immunohistochemistry should be performed to validate the mesothelial origin, and p16 FISH should be performed to confirm malignancy. Diagn. Cytopathol. 2016;44:591-598. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dc.23490DOI Listing
July 2016

An intrapleural administration of zoledronic acid for inoperable malignant mesothelioma patients: a phase I clinical study protocol.

Springerplus 2016 27;5:195. Epub 2016 Feb 27.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba, 260-8717 Japan ; Department of Molecular Biology and Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan.

Background: The third generation of bisphosphonates is clinically in use for patients of osteoporosis or malignancy-linked hypercalcemia. The agents can also produce anti-tumor effects on bone metastasis of several types of tumors. We recently found that one of the agents achieved cytotoxicity to mesothelioma in vitro and in an orthotopic animal model. Mesothelioma is resistant to a number of chemotherapeutic agents, and suppression of local tumor growth is beneficial to the patients since metastasis to extra-thoracic organs is relatively infrequent until a late stage.

Methods/design: We demonstrated in an orthotopic mouse model that an intrapleural but not intravenous injection of zoledronic acid, one of the third generation bisphosphonates, at a clinically equivalent dose suppressed the tumor growth. Nevertheless, a high concentration of zoledronic acid administrated in the pleural cavity produced pleural adhesion. We also showed that zoledronic acid produced synergistic cytotoxic effects with cisplatin, the first-line chemotherapeutic agent for mesothelioma. We then planned to conduct a phase I clinical study to investigate any adverse effects and a possible clinical benefits produced by an intrapleural administration of zoledronic acid to mesothelioma patients who became resistant to the first-line chemotherapeutic agents. The clinical trial is a dose escalation study starting with 0.4, 1, 4, 8 and 16 mg per person since safety of administration of zoledronic acid into the pleural cavity remains unknown. Each dose group consists of three persons and the protocol allows to repeat administration of the same dose into the pleural cavity at a 4-weeks interval.

Discussion: We will conduct a possible combinatory study of intrapleural administration of zoledronic acid and systemic administration of the first-line agent to a chemotherapy-naïve patient based on the maximum tolerance dose of zoledronic acid determined by the present clinical trial. We propose that administration of bisphosphonates in a closed cavity is a treatment strategy for tumors developed in the cavity probably through the direct cytotoxic activity.

Trial Registration: UMIN clinical trials registry, Japan. Register ID: UMIN8093.
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http://dx.doi.org/10.1186/s40064-016-1893-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4769234PMC
March 2016

Replication-competent adenoviruses with the type 35-derived fiber-knob region achieve reactive oxygen species-dependent cytotoxicity and produce greater toxicity than those with the type 5-derived region in pancreatic carcinoma.

Apoptosis 2015 Dec;20(12):1587-98

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba, 260-8717, Japan.

Pancreatic carcinoma is relatively resistant to chemotherapy and cell death induced by replication of adenoviruses (Ad) can be one of the therapeutic options. Transduction efficacy of conventional type 5 Ad (Ad5) is however low and the cytotoxic mechanism by replication-competent Ad was not well understood. We constructed replication-competent Ad5 of which the E1A promoter region was replaced with a transcriptional regulatory region of the midkine, the survivin or the cyclooxygenase-2 gene, all of which were expressed at a high level in human tumors. We also prepared replication-competent Ad5 that were activated with the same region but had the type 35 Ad-derived fiber-knob region (AdF35) to convert the major cellular receptor for Ad infection from the coxsackie adenovirus receptor to CD46 molecules. Replication-competent AdF35 that were activated with the exogenous region produced cytotoxic effects on human pancreatic carcinoma cells greater than the corresponding Ad5 bearing with the same regulatory region. Cells infected with the AdF35 showed cytopathic effects and increased sub-G1 fractions. Caspase-9, less significantly caspase-8 and poly (ADP-ribose) polymerase, but not caspase-3 was cleaved and expression of molecules involved in autophagy and caspase-independent cell death pathways remained unchanged. Nevertheless, H2A histone family member X molecules were phosphorylated, and N-acetyl-L-cystein, an inhibitor for reactive oxygen species, suppressed the AdF35-mediated cytotoxicity. These data indicated a novel mechanism of Ad-mediated cell death and suggest a possible clinical application of the fiber-knob modified Ad.
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http://dx.doi.org/10.1007/s10495-015-1171-8DOI Listing
December 2015

A clinical protocol to inhibit the HGF/c-Met pathway for malignant mesothelioma with an intrapleural injection of adenoviruses expressing the NK4 gene.

Springerplus 2015 16;4:358. Epub 2015 Jul 16.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba, 260-8717 Japan ; Department of Molecular Biology and Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan.

Background: The hepatocyte growth factor (HGF)/c-Met signal pathway is up-regulated in human mesothelioma and suppression of the HGF/c-Met signaling with a competitive inhibitor, NK4 homologous to HGF in the structure, produced anti-tumor effects to mesothelioma in a preclinical study. Mesothelioma is highly resistant to a number of chemotherapeutic agents but distant metastasis to extra-thoracic organs is relatively infrequent until the late stage.

Methods/design: We planned to conduct a clinical study of gene therapy with adenoviruses expressing the NK4 gene (Ad-NK4) to control the local tumor growth. The study is designed to inject Ad-NK4 into the intrapleural cavity with a dose escalation manner from 10(10) to 10(12) virus particles per patient and to examine safety and possible clinical benefits. The clinical investigation is a first-in-human trial to use the NK4 gene and to block the HGF/c-Met pathway with gene medicine. We conducted in vivo animal experiments to examine the safety level as one of the preclinical studies, and showed that Ad DNA administered in the pleural cavity was detected in many parenchymal organs. Biochemical and pathological analyses showed that liver damages were the major adverse effects with little toxicity to other organs. These studies firstly demonstrated biodistribution and transgene expression after an intrapleural injection of Ad vectors in an animal study, which contrasts with an intravenous injection showing relatively rapid clearance of Ad-NK4.

Discussion: The clinical study can also provide information regarding production of NK4 protein and antibody against NK4, and inhibition levels of the HGF/c-Met pathway by detecting dephosphorylation of c-Met in mesothelioma cells. These data will be crucial to judge whether local production of NK4 molecules can be an anti-cancer strategy.

Trial Registration: UMIN clinical trials registry, Japan. Register ID: UMIN15771.
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http://dx.doi.org/10.1186/s40064-015-1123-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4503710PMC
July 2015

Expression of activation-induced cytidine deaminase is associated with a poor prognosis of diffuse large B cell lymphoma patients treated with CHOP-based chemotherapy.

J Cancer Res Clin Oncol 2016 Jan 16;142(1):27-36. Epub 2015 Jun 16.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba, 260-8717, Japan.

Purpose: Activation-induced cytidine deaminase (AID) is involved in somatic hypermutation and class switch recombination processes in the antibody formation. The AID activity induces gene mutations and could be associated with transformation processes of B cells. Nevertheless, the relation between AID expression and the prognosis of B cell lymphoma patients remains uncharacterized.

Methods: We examined expression levels of the AID gene in 89 lymph node specimens from lymphoma and non-lymphoma patients with Northern blot analysis and investigated an association with their survival.

Results: The AID gene was preferentially expressed in B cell lymphoma in particular in diffuse large B cell lymphoma and follicular lymphoma. We confirmed AID protein expression in the mRNA-positive but not in the negative specimens with Western blot analysis and immunohistochemical staining. Survival of the patients treated with cyclophosphamide-/doxorubicin-/vincristine-/prednisone-based chemotherapy demonstrated that the prognosis of diffuse large B cell patients was unfavorable in the mRNA-positive group compared with the negative group, and that AID expression levels were correlated with the poor prognosis. In contrast, AID expression was not linked with the prognosis of follicular lymphoma patients.

Conclusions: AID expression is a predictive marker for an unfavorable outcome in DLBCL patients treated with the chemotherapy.
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http://dx.doi.org/10.1007/s00432-015-2001-7DOI Listing
January 2016

Cytotoxic effects of replication-competent adenoviruses on human esophageal carcinoma are enhanced by forced p53 expression.

BMC Cancer 2015 Jun 10;15:464. Epub 2015 Jun 10.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, 260-8717, Chiba, Japan.

Background: Improvement of transduction and augmentation of cytotoxicity are crucial for adenoviruses (Ad)-mediated gene therapy for cancer. Down-regulated expression of type 5 Ad (Ad5) receptors on human tumors hampered Ad-mediated transduction. Furthermore, a role of the p53 pathways in cytotoxicity mediated by replication-competent Ad remained uncharacterized.

Methods: We constructed replication-competent Ad5 of which the E1 region genes were activated by a transcriptional regulatory region of the midkine or the survivin gene, which is expressed preferentially in human tumors. We also prepared replication-competent Ad5 which were regulated by the same region but had a fiber-knob region derived from serotype 35 (AdF35). We examined the cytotoxicity of these Ad and a possible combinatory use of the replication-competent AdF35 and Ad5 expressing the wild-type p53 gene (Ad5/p53) in esophageal carcinoma cells. Expression levels of molecules involved in cell death, anti-tumor effects in vivo and production of viral progenies were also investigated.

Results: Replication-competent AdF35 in general achieved greater cytotoxic effects to esophageal carcinoma cells than the corresponding replication-competent Ad5. Infection with the AdF35 induced cleavages of caspases and increased sub-G1 fractions, but did not activate the autophagy pathway. Transduction with Ad5/p53 in combination with the replication-competent AdF35 further enhanced the cytotoxicity in a synergistic manner. We also demonstrated the combinatory effects in an animal model. Transduction with Ad5/p53 however suppressed production of replication-competent AdF35 progenies, but the combination augmented Ad5/p53-mediated p53 expression levels and the downstream pathways.

Conclusions: Combination of replication-competent AdF35 and Ad5/p53 achieved synergistic cytotoxicity due to enhanced p53-mediated apoptotic pathways.
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http://dx.doi.org/10.1186/s12885-015-1482-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4460641PMC
June 2015

A combinatory use of adenoviruses expressing melanoma differentiation-associated gene-7 and replication-competent adenoviruses produces synergistic effects on pancreatic carcinoma cells.

Tumour Biol 2015 Sep 20;36(10):8137-45. Epub 2015 May 20.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba, 260-8717, Japan.

Type 5 adenoviruses expressing mda-7 gene (Ad-mda-7) induced cell death in various kinds of human tumors, but pancreatic carcinoma cells were relatively resistant to Ad-mda-7-mediated cytotoxicity. We then examined whether infection of Ad-mda-7 together with replication-competent Ad produced combinatory cytotoxic effects. We prepared replication-competent Ad, defective of the E1B55kDa gene or activated by a transcriptional regulatory region of the midkine or the survivin gene of which the expression was up-regulated in human tumors. Type 5 Ad bearing the exogenous regulatory region were further modified by replacing the fiber-knob region with that of type 35 Ad. Pancreatic carcinoma cells were infected with replication-incompetent Ad-mda-7 and the replication-competent Ad. Combinatory effects were examined with the CalcuSyn software and cell cycle analyses. Ad-mda-7 and the replication-competent Ad achieved cytotoxicity to pancreatic carcinoma. A combinatory use of Ad-mda-7 and either Ad defective of the E1B55kDa gene or Ad activated by the regulatory region produced synergistic cytotoxic effects. Cell cycle analyses demonstrated that the combination increased sub-G1 populations. These data collectively suggest that expression of MDA-7 augments cytotoxicity of replication-competent Ad and achieves adjuvant effects on Ad-mediated cell death.
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http://dx.doi.org/10.1007/s13277-015-3555-3DOI Listing
September 2015

NY-ESO-1 autoantibody as a tumor-specific biomarker for esophageal cancer: screening in 1969 patients with various cancers.

J Gastroenterol 2016 Jan 24;51(1):30-4. Epub 2015 Apr 24.

Department of Surgery, School of Medicine, Toho University, Tokyo, Japan.

Background: Although serum NY-ESO-1 antibodies (s-NY-ESO-1-Abs) have been reported in patients with esophageal carcinoma, this assay system has not been used to study a large series of patients with various other cancers.

Patients And Methods: Serum samples of 1969 cancer patients [esophageal cancer (n = 172), lung cancer (n = 269), hepatocellular carcinoma (n = 91), prostate cancer (n = 358), gastric cancer (n = 313), colorectal cancer (n = 262), breast cancer (n = 365)] and 74 healthy individuals were analyzed using an originally developed enzyme-linked immunosorbent assay system for s-NY-ESO-1-Abs. The optical density cut-off value, determined as the mean plus three standard deviations for serum samples from the healthy controls, was fixed at 0.165. Conventional tumor markers were also evaluated in patients with esophageal carcinoma.

Results: The positive rate of s-NY-ESO-1-Abs in patients with esophageal cancer (31 %) was significantly higher than that in the other groups: patients with lung cancer (13 %), patients with hepatocellular carcinoma (11 %), patients with prostate cancer (10 %), patients with gastric cancer (10 %), patients with colorectal cancer (8 %), patients with breast cancer (7 %), and healthy controls (0 %). The positive rate of s-NY-ESO-1-Abs was comparable to that of serum p53 antibodies (33 %), squamous cell carcinoma antigen (36 %), carcinoembryonic antigen (26 %), and CYFRA 21-1 (18 %) and gradually increased with the tumor stage.

Conclusions: The positive rate of s-NY-ESO-1-Abs was significantly higher in patients with esophageal cancer than in patients with the other types of cancers. On the basis of its high specificity and sensitivity, even in patients with stage I tumors, s-NY-ESO-1-Abs may be one of the first choices for esophageal cancer.
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http://dx.doi.org/10.1007/s00535-015-1078-8DOI Listing
January 2016

Fas ligand DNA enhances a vaccination effect by coadministered DNA encoding a tumor antigen through augmenting production of antibody against the tumor antigen.

J Immunol Res 2015 18;2015:743828. Epub 2015 Feb 18.

Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chiba 260-8717, Japan ; Department of Molecular Biology and Oncology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan.

Interaction of Fas and Fas ligand (FasL) plays an important role in the regulation of immune responses by inducing apoptosis of activated cells; however, a possible role of FasL in DNA vaccination has not been well understood. We examined whether administration of DNA encoding FasL gene enhanced antitumor effects in mice that were vaccinated with DNA expressing a putative tumor antigen gene, β-galactosidase (β-gal). Growth of β-gal-positive Colon 26 tumors was retarded in the syngeneic mice immunized with β-gal and FasL DNA compared with those vaccinated with β-gal or FasL DNA. We did not detect increased numbers of β-gal-specific CD8(+) T cells in lymph node of mice that received combination of β-gal and FasL DNA, but amounts of anti-β-gal antibody increased with the combination but not with β-gal or FasL DNA injection alone. Subtype analysis of anti-β-gal antibody produced by the combination of β-gal and FasL DNA or β-gal DNA injection showed that IgG2a amounts were greater in mice injected with both DNA than those with β-gal DNA alone, but IgG2b amounts were lower in both DNA-injected than β-gal DNA-injected mice. These data suggest that FasL is involved in boosting humoral immunity against a gene product encoded by coinjected DNA and enhances the vaccination effects.
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http://dx.doi.org/10.1155/2015/743828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352480PMC
December 2015