Biomedical subjects

Takaki Hiwasa

Publications and source records attributed to Takaki Hiwasa.

At least 19 recordsLinked to original sources

PubMed2007

Serum anti-myomegalin antibodies in patients with esophageal squamous cell carcinoma.

In order to identify new serum markers of esophageal squamous cell carcinoma (SCC), we performed serological identification of antigens by recombinant cDNA expression cloning (SEREX). E. coli was transformed with a lambdaZAPII phage cDNA library prepared from mRNA of an esophageal cancer cell line (T.Tn), and IPTG-induced cDNA products were screened for interaction with antibodies in allogeneic sera of patients with esophageal SCC. We identified myomegalin (MMGL, phosphodiesterase 4D interacting protein/PDE4DIP) as a new SEREX antigen for esophageal SCC. Western blot analysis revealed that serum anti-myomegalin antibodies (s-MMGL-Abs) were present in 43 (47%) of 91 patients, but in only one (2.2%) of 45 healthy controls. Of the 21 patients with stage I disease, 8 (38%) were sero-positive. The positive rate of s-MMGL-Abs was greater than those of other conventional tumor markers. Reverse transcription-PCR analysis suggested that alternative splicing from myomegalin variant 1 to variant 5 may explain, in part, the development of s-MMGL-Abs. Although the presence of s-MMGL-Abs was not related to any clinicopathological features of the patients, multivariate analysis indicated that the presence of s-MMGL-Abs was significantly associated with a favorable prognosis. Consequently, s-MMGL-Abs may be a useful tumor marker to diagnose and establish a prognosis in patients with esophageal SCC.

Aged↗

PubMed2006

Presence of serum tripartite motif-containing 21 antibodies in patients with esophageal squamous cell carcinoma.

SEREX has been applied to esophageal SCC, and the TRIM21 gene was identified as a novel SEREX antigen of esophageal SCC. The presence of s-TRIM21-Abs was confirmed by Western blotting using bacterially expressed TRIM21 gene product and was evaluated for clinicopathological significance in patients with esophageal SCC. s-TRIM21-Abs were detected in 18 (20%) of 91 patients with esophageal SCC but not in 42 healthy donors. The presence of s-TRIM21-Abs was partly associated with tumor size (P = 0.063) and poor survival (P = 0.067). To measure serum antibody levels, ELISA using purified recombinant TRIM21 protein was developed. The levels of s-TRIM21-Abs were significantly higher in patients with esophageal SCC than in healthy donors (P = 0.013). s-TRIM21-Abs may be a useful tumor marker to diagnose and predict disease progression in patients with esophageal SCC.

Adult↗

PubMed2006

Relationship between pancreatic secretory trypsin inhibitor and early recurrence of intrahepatic cholangiocarcinoma following surgical resection.

OBJECTIVES: The extremely unfavorable prognosis of intrahepatic cholangiocarcinoma (ICC), even after surgical resection, is mainly attributed to a high rate of recurrence. The aim of this study was to identify the molecules associated with early recurrence of ICC following resection. METHODS: Between December 1984 and July 2003, 46 patients with ICC underwent surgical resection. The clinical outcome of these patients was evaluated in view of the time of recurrence. Consequently, we categorized ICC patients into subgroups, based on the clinical results, and screened differentially expressed genes by DNA microarray analysis. Furthermore, the obtained results were validated in an independent sample set by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemistry was performed to assess the expressed genes at the protein level. RESULTS: The survival of patients with early recurrence, occurring within a year after surgical resection, was significantly poor after surgery and even after recurrence, as compared to that of patients whose recurrence occurred beyond a year after surgery. By the DNA microarray analysis, 13 differentially expressed genes were picked up, and quantitative RT-PCR reaction identified the pancreatic secretory trypsin inhibitor (PSTI) as a candidate gene associated with early recurrence of ICC after resection. This observation was confirmed through examination of an independent set of samples, in which the patients with higher levels of PSTI mRNA expression had significantly shorter recurrence-free survival. Immunohistochemically, PSTI was expressed in the cytoplasm of cancer cells. CONCLUSIONS: PSTI might be a potential marker for identifying ICC patients with an increased risk of early recurrence after surgical resection.

Aged↗

PubMed2006

Identification of a novel SEREX antigen, SLC2A1/GLUT1, in esophageal squamous cell carcinoma.

We have carried out SEREX (serological identification of antigens by recombinant cDNA expression cloning), and identified SLC2A1 (solute carrier family 2/facilitated glucose transporter, member 1) as an antigen recognized by serum IgG antibodies in patients with esophageal squamous cell carcinoma (SCC). The levels of serum anti-SLC2A1 antibodies (s-SLC2A1-Abs), examined by enzyme-linked immunosorbent assay using bacterially expressed glutathione-S-transferase-SLC2A1 fusion protein, were significantly higher in patients with esophageal SCC than in healthy donors. When using a cut-off level as the mean + 2x standard deviations of healthy donors, a total of 12 (21%) out of 57 SCC patients were revealed as positive for s-SLC2A1-Abs. The presence of s-SLC2A1-Abs was not associated with either clinicopathological factors or survival. Because s-SLC2A1-Abs were not associated with the positivity of other conventional serum markers, a combination assay of s-SLC2A1-Abs with these conventional serum markers may be useful for the diagnosis and monitoring of esophageal SCC.

Adult↗

PubMed2005

Cathepsin D is a potential serum marker for poor prognosis in glioma patients.

Cathepsin D is an aspartyl protease involved in protein catabolism and tissue remodeling which can be secreted from cancer cells. To identify a potential serum marker for gliomas, we investigated the gene expression levels of cathepsin D in 87 tissue samples and measured the protein concentrations in sera of glioma patients. The tissue samples consisted of 43 glioblastomas, 13 anaplastic astrocytomas, 22 astrocytomas, and 9 normal brain tissues. The results of real-time quantitative reverse transcription-PCR analysis showed that cathepsin D transcript levels became significantly higher as the glioma grade advanced (P = 0.0466, glioblastoma and anaplastic astrocytoma; P = 0.0008, glioblastoma and astrocytoma; P = 0.0271, glioblastoma and normal brain tissue; unpaired t test). Immunohistochemical analysis with anti-cathepsin D antibody revealed dense and spotty staining in the tumor cells with high transcript levels. The low expression of cathepsin D significantly correlated with long survival of the glioma patients. Furthermore, the glioblastoma patients with high gene expression of cathepsin D lived significantly shorter than those with low expression (P = 0.0104, Cox-Mantel log-rank test) and frequently had leptomeningeal dissemination (P = 0.0016, chi2 test). The multivariate analysis confirmed that the cathepsin D expression level was an independent predictor for short survival (P = 0.0102, Cox proportional hazard regression model). Measurement of the serum cathepsin D concentrations by ELISA showed a significant increase in the patients with high-grade gliomas as compared with the low-grade tumors (P = 0.0081, chi2 test). These results collectively suggest that cathepsin D could be a potential serum marker for the prediction of aggressive nature of human gliomas.

Astrocytoma↗

PubMed2005

Serological identification of tumor antigens of esophageal squamous cell carcinoma.

Autoantibodies are often detected in the patients with esophageal cancer. We applied serological analysis of recombinant cDNA expression libraries (SEREX) to a case of esophageal squamous cell carcinoma in order to identify tumor antigens. A cDNA library derived from an esophageal cancer cell line was bacterially expressed and screened for interaction with antibodies in five allogeneic sera of patients with esophageal squamous cell carcinoma. To examine the specific immunoreactivity of the antigens, sera from 16 more patients with esophageal squamous cell carcinoma, 16 patients with gastric cancer, 16 patients with colon cancer, 16 patients with breast cancer and 37 healthy volunteers were screened. We identified 11 independent cDNA clones that potentially encoded esophageal cancer tumor antigens. The identified cDNA clones were SURF1, HOOK2, CENP-F, ZIC2, hCLA-iso, Ki-1/57, enigma, HCA25a, SPK and two EST clones named LOC146223 and AGENCOURT_7565913. The sero-positive rates of antibodies against SURF1 (48%), LOC146223 (38%), HOOK2 (14%) and AGENCOURT_7565913 (14%) were significantly higher in esophageal cancer patients than in healthy controls. At least one of these antibodies was detected in 18 (86%) of 21 sera from esophageal cancer patients. A disease-specific humoral immune response against SURF1, LOC146223, HOOK2 or AGENCOURT_7565913 was observed in most patients with esophageal squamous cell carcinoma. Antibodies against these SEREX antigens may represent a pool of candidates for serum tumor markers of esophageal squamous cell carcinoma.

Adult↗

PubMed2005

Proteome-based identification of molecular markers predicting chemosensitivity to each category of anticancer agents in human gliomas.

To identify the protein markers that are clinically useful for predicting efficacy of anticancer agents, we investigated the correlation between the proteome profiling patterns and the in vitro chemosensitivity in human gliomas. The proteome of 93 surgical samples were analyzed with two-dimensional gel electrophoresis (2DE) and mass spectrometry. The in vitro chemosensitivities to 10 different kinds of anticancer agents (cyclophosphamide, nimustine, cisplatin, cytosine arabinoside, mitomycin C, peplomycin, adriamycin, etoposide, vincristine, paclitaxel) were measured by flow cytometric detection of apoptosis. We identified a set of 41 proteins that significantly affected the in vitro chemosensitivity to each category of anticancer agents. Many of the proteins that correlated with chemoresistance were categorized into the signal transduction proteins including the G-proteins. The present study showed that the proteome analysis using 2DE could provide a list of proteins that may be the potential predictive markers for chemosensitivity in human gliomas. They can also be direct and rational targets for anti-glioma therapy and be used for sensitization to the conventional chemotherapeutic regimens.

Antineoplastic Agents↗

PubMed2004

Serological identification of TROP2 by recombinant cDNA expression cloning using sera of patients with esophageal squamous cell carcinoma.

We applied serological analysis of recombinant cDNA expression libraries (SEREX) to cases of esophageal squamous cell carcinoma (SCC) to identify tumor antigens. One of the clones identified was TROP2, which is known as calcium signal transducer. To evaluate the clinical significance of serum anti-TROP2 antibodies (s-TROP2-Abs) in patients with esophageal SCC, the presence of s-TROP2-Abs was analyzed by Western blotting using bacterially expressed TROP2 protein. We found that 23 of 75 (31%) patients were positive for s-TROP2-Abs. Positivity in terms of s-TROP2-Abs showed a significant association with tumor size but not with other clinicopathological features. The protein expression levels of TROP2 were much higher in esophageal SCC cell lines as compared to those in normal esophageal mucosa and its immortalized cells although the mRNA expression levels were not necessarily elevated in malignant cell lines and tissues. Immunohistochemical studies showed that the expression of TROP2 protein in esophageal SCC specimens was noticeably higher than that found in mild hyperplasia of esophageal mucosae. Thus, s-TROP2-Abs seemed useful in the diagnosis of SCC and may be a candidate for serum tumor markers.

Aged↗

PubMed2004

Increased expression of collagen XVIII and its prognostic value in nonsmall cell lung carcinoma.

BACKGROUND: Angiogenesis plays a crucial role in tumor growth and metastasis. Recently, some studies have focused on the angiogenesis inhibitor endostatin. However, the biologic role of the precursor of endostatin, collagen XVIII, in human malignancy is unknown. The purpose of the current study was to evaluate whether the expression of collagen XVIII has additional prognostic value for survival in patients with nonsmall cell lung carcinoma (NSCLC). METHODS: The authors investigated the expression of collagen XVIII in 221 patients using immunohistochemical methods. To confirm the specificity of the collagen XVIII polyclonal antibody used in the current study and to test the expression of collagen XVIII in human lung carcinoma, Western blot analysis was performed on a panel of human lung carcinoma cell lines. RESULTS: Collagen XVIII expression was detected in 162 of 221 patients with NSCLC (73%), primarily in the tumor cell cytoplasm. Low collagen XVIII expression levels were found in 75 tumor specimens, while high collagen XVIII expression levels were noted in 87 tumor specimens. The prevalence of positive collagen XVIII expression was greater in T2-4 tumors than in T1 tumors (P = 0.0235). The prognosis for patients with strongly collagen XVIII-positive NSCLC was significantly worse than the prognosis for patients with collagen XVIII-positive or collagen XVIII-negative NSCLC (P = 0.0010). Multivariate analysis indicated that T status, lymph node status, and the overexpression of collagen XVIII were independent prognostic factors. CONCLUSIONS: The results of the current study indicated that the overexpression of collagen XVIII was associated with NSCLC progression and poor outcome. Thus, collagen XVIII expression may serve as a useful prognostic marker in patients with NSCLC.

Adult↗

PubMed2004

Molecular classification and survival prediction in human gliomas based on proteome analysis.

The biological features of gliomas, which are characterized by highly heterogeneous biological aggressiveness even in the same histological category, would be precisely described by global gene expression data at the protein level. We investigated whether proteome analysis based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry can identify differences in protein expression between high- and low-grade glioma tissues. Proteome profiling patterns were compared in 85 tissue samples: 52 glioblastoma multiforme, 13 anaplastic astrocytomas, 10 atrocytomas, and 10 normal brain tissues. We could completely distinguish the normal brain tissues from glioma tissues by cluster analysis based on the proteome profiling patterns. Proteome-based clustering significantly correlated with the patient survival, and we could identify a biologically distinct subset of astrocytomas with aggressive nature. Discriminant analysis extracted a set of 37 proteins differentially expressed based on histological grading. Among them, many of the proteins that were increased in high-grade gliomas were categorized as signal transduction proteins, including small G-proteins. Immunohistochemical analysis confirmed the expression of identified proteins in glioma tissues. The present study shows that proteome analysis is useful to develop a novel system for the prediction of biological aggressiveness of gliomas. The proteins identified here could be novel biomarkers for survival prediction and rational targets for antiglioma therapy.

Amino Acid Sequence↗

PubMed2004

Growth suppression of esophageal squamous cell carcinoma induced by heavy carbon-ion beams combined with p53 gene transfer.

Heavy carbon-ion beam therapy has revealed several potential advantages over X-rays. Heavy-ion therapy has been applied for various solid tumors including esophageal squamous cell carcinoma (SCC). Although the local control rate in carbon ion radiotherapy for esophageal cancer has revealed better rates than conventional radiotherapy, severe mucosal damage was observed in adjacent normal mucosa. A suitable treatment strategy is required to reduce irradiation dose by introducing combined local therapy. Recently, we initiated clinical p53 gene therapy for esophageal SCC. We herein evaluate the cytotoxic effects of heavy carbon-ion beams combined with p53 gene transfer on human esophageal SCC. We assessed the induction of apoptosis and growth suppression with the use of recombinant adenoviral vector Ad.p53 or heavy carbon-ion beam irradiation or both. Growth suppression was significantly potentiated by combined treatment with heavy carbon-ion beams and Ad.p53 as compared to that treated with either of them alone. Western blot analysis confirmed the expression of both exogenous p53 and p21 proteins after irradiation of Ad.p53 infected cells. Enhanced apoptotic cell death was observed with a terminal deoxynucleotidyl transferase-mediated nick end-labeling assay. These data suggest that heavy carbon-ion beam irradiation combined with Ad.p53 may be a potentially effective therapeutic strategy for locally advanced esophageal SCC.

Apoptosis↗

PubMed2004

Increase in metalloproteinase activity in the plasma of smoke-extract-injected rats.

Cigarette smoking is known to be one of the risk factors of subarachnoid hemorrhage (SAH), yet the precise mechanism remains to be proved. Based on the hypothesis that smoke components might enhance protease activity in the arterial wall, we examined the proteinase activities of plasma from smoke-extract-injected rats. Rats were injected intraperitoneally with smoke extract and the protease activity in the plasma was examined by zymography in the presence or the absence of proteinase inhibitors. Metalloproteinase activity was measured using a synthetic substrate. Proteolytic activities of 80- and 100-kDa against gelatin and collagen type I-V were induced 6 h after the injection. Inhibition of the activity by a metalloproteinase inhibitor but not by serine, cysteine or aspartic protease inhibitors suggested that the proteinases can be attributed to metalloproteinases. Cleavage activity in plasma toward a synthetic metalloproteinase substrate also increased within 24 h after the injection of the smoke extract. The results indicate that the induction of plasma metalloproteinase activity by smoke extract might account for the causal role of smoking in the development of SAH.

Animals↗

PubMed2003

Stat6-protease but not Stat5-protease is inhibited by an elastase inhibitor ONO-5046.

A short isoform of Stat6 (65-kDa Stat6), a product of proteolytic processing by an undefined protease (Stat6-protease) in the nucleus, downregulates Stat6-mediated signaling in mast cells. Similarly, Stat5-mediated signaling is downregulated by Stat5-protease in myeloid progenitors. These proteases share a number of characteristics, including their nuclear localization and susceptibility to protease inhibitors. Here, we further investigated these Stat proteases. Interestingly, the activity of Stat6-protease but not of Stat5-protease was inhibited by ONO-5046, an elastase inhibitor that inhibits the activity of neutrophil elastase (NE) and NE-related protease proteinase 3 (PR3). Although both NE and PR3 were able to cleave Stat6 in vitro, the cleavage sites of Stat6 by NE or PR3 differed from that by Stat6-protease in mast cells. In addition, both NE and PR3 could also cleave Stat5, but they differed from Stat5-protease in myeloid progenitors. These results suggest that Stat6-protease may belong to the elastase family but differs from NE or PR3.

Animals↗

PubMed2003

Drug-sensitivity pattern analysis for study of functional relationship between gene products.

We have developed a method that we call 'drug-sensitivity pattern analysis', or DSPA, for analysis of protein function. Cells are transfected with cDNA of the test molecule, followed by analysis of the sensitivity of the transfected cells to multiple growth-inhibitory drugs. If two cDNA products have similar functions, their transfected cells should show similar drug-sensitivity patterns. The cDNAs of some signaling molecules were transfected into NIH3T3 or Ha-ras-transformed NIH3T3 (ras-NIH) cells and stable transfectants, which expressed high amounts of the gene product, were isolated. Chemosensitivity of the transfected clone was compared with the parental cells by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method using more than 40 drugs. The chemosensitivity changes caused by the transfected gene were calculated and expressed numerically as 'drug chemosensitivity index' (DCI). When the DCI values were analyzed by regression analysis, a significant positive relationship between IkappaBalpha superrepressor and dominant-negative IKKbeta and an inverse relationship between p53 and Mdm2 were consistent with previous reports. Thus, the DSPA method is useful for identifying functional similarities between gene products.

3T3 Cells↗

PubMed2003

Acquisition of drug resistance by constitutive suppression of NF-kappaB in ras-transformed NIH3T3 mouse fibroblasts.

Apoptotic cell death is frequently suppressed by NF-kappaB transcription factor. We examined the effects of NF-kappaB on the sensitivity to anti-cancer drugs by using two NF-kappaB-inhibitory molecules, IkappaBalpha-super-repressor (IkappaBalpha-SR) and dominant negative IKKbeta (IKKbeta-DN). Ha-ras-transformed NIH3T3 (ras-NIH3T3) mouse fibroblasts were stably transfected with these cDNAs, and suppression of NF-kappaB activity was confirmed by electrophoretic mobility shift assays. Cell viability analysis revealed that IkappaBalpha-SR- or IKKbeta-DN-transfected cells were more resistant to peplomycin, mitomycin C, and camptothecin. Flow cytometric analysis indicated partial G1 arrest of these transfected cells. Consistently, elevated expression of IkappaBalpha-SR and IKKbeta-DN was accompanied by increased levels of p21 but not of Bax. Transfection of p21 into ras-NIH3T3 cells caused similar reduced chemosensitivity to the anti-cancer drugs. These results collectively indicate that constitutive suppression of NF-kappaB activity reduced the chemosensitivity to anti-cancer drugs via enhanced expression of p21.

Animals↗

PubMed2002

CD44 signaling through focal adhesion kinase and its anti-apoptotic effect.

Adhesion molecules can initiate intracellular signaling. Engagement of CD44 either by its natural ligand hyaluronan or a specific antibody on a cell line induced tyrosine phosphorylation and activation of focal adhesion kinase (FAK), which then associated with phosphatidylinositol 3-kinase (PI3K) and activated mitogen-activated protein kinase at its downstream. However, the introduction of dominant negative Rho into the cells inhibited the CD44-stimulated FAK phosphorylation. Cells expressing CD44 were significantly resistant to etoposide-induced apoptosis. This anti-apoptotic effect was cancelled by the inhibition of either Rho, FAK or PI3K. These results may indicate a signaling pathway from CD44 to mediate the resistance against drug-induced apoptosis in cancer cells.

Apoptosis↗

PubMed2002

Proteolytic processing of Stat6 signaling in mast cells as a negative regulatory mechanism.

Accumulating evidence has shown the importance of Stat6-mediated signaling in allergic diseases. In this study, we show a novel regulatory mechanism of Stat6-mediated signaling in mast cells. When Stat6 is activated by interleukin (IL)-4 and translocated to the nucleus, Stat6 is cleaved by a nucleus-associated protease in mast cells. The cleaved 65-kD Stat6 lacks the COOH-terminal transactivation domain and functions as a dominant-negative molecule to Stat6-mediated transcription. The retrovirus-mediated expression of cleavage-resistant Stat6 mutants prolongs the nuclear accumulation of Stat6 upon IL-4 stimulation and enhances IL-4-induced gene expression and growth inhibition in mast cells. These results indicate that the proteolytic processing of Stat6 functions as a lineage-specific negative regulator of Stat6-dependent signaling in mast cells, and thus suggest that it may account for the limited role of Stat6 in IL-4 signaling in mast cells.

Animals↗

PubMed2002

Facilitation of adenoviral wild-type p53-induced apoptotic cell death by overexpression of p33(ING1) in T.Tn human esophageal carcinoma cells.

To investigate the effect of p33(ING1) on wild-type p53 gene therapy, T.Tn human esophageal carcinoma cells were stably transfected with p33(ING1) cDNA. Infection with Ad-p53 (recombinant adenovirus containing wild-type p53) into p33-transfected cells reduced cell viability, while infection with empty vector had little effect. This reduced viability was shown to be due to apoptotic cell death by the TUNEL (terminal deoxynucleotidyl transferase-mediated nick end-labeling) assay. Following infection with Ad-p53, levels of p53 were similar in p33-expressing cells and in the parental line. However, levels of p21 and Mdm2 were elevated in p33-transfected cells. Nonetheless, this enhanced expression of Mdm2 appeared to be ineffective in downregulating p53. Transient transfection with mutant Mdm2 prior to Ad-p53 infection provided a significant protection as compared with cells transfected with wild-type Mdm2. These results imply a synergistic effect between p33 and p53 in the induction of apoptosis of human esophageal carcinoma cells. A role for Mdm2 in this synergism is suggested.

Adenoviridae↗