松下 一之

To investigate changes in protein expression by proteomic analysis in the sera of patients with sepsis and to identify new biomarkers of sepsis. A total of 45 consecutive patients with severe sepsis or septic shock (sepsis group), 22 healthy volunteers, and 23 patients undergoing off-pump coronary artery bypass grafting (control group). Serum samples from eight patients of each group underwent proteomic analysis involving removal of 12 major proteins and subsequent reversed-phase high-performance liquid chromatography fractionation and one-dimensional electrophoresis. The intensity of 41 bands (with 12 proteins identified) increased and that of 42 bands (with 22 proteins identified) decreased in the sepsis group. Results of proteomic analysis successfully validated by Western blotting and/or enzyme-linked immunosorbent assay for three proteins (YKL-40, lipocalin 2, and S100A9) increased in the sepsis group as well as two proteins (retinol-binding protein, vitamin D-binding protein) decreased. Serum YKL-40 levels (sYKL-40) on intensive care unit (ICU) admission were assessed by enzyme-linked immunosorbent assay between the two groups; resulting YKL-40 was significantly higher in the sepsis group (P < 0.001). Furthermore, sYKL-40 on ICU admission was significantly higher in patients with positive blood culture (P < 0.005), patients with septic shock (P < 0.05), and patients requiring continuous hemodiafiltration (P < 0.05) or hydrocortisone replacement therapy (P < 0.005) during subsequent treatment. A positive correlation between sYKL-40 and blood IL-6 level on ICU admission was noted in the sepsis group (r = 0.465, P < 0.01). YKL-40 identified by proteomic analysis is considered as a biomarker of sepsis. However, further investigation is needed to clarify its roles and clinical usefulness as a biomarker.

To investigate changes in protein expression by proteomic analysis in the sera of patients with sepsis and to identify new biomarkers of sepsis. A total of 45 consecutive patients with severe sepsis or septic shock (sepsis group), 22 healthy volunteers, and 23 patients undergoing off-pump coronary artery bypass grafting (control group). Serum samples from eight patients of each group underwent proteomic analysis involving removal of 12 major proteins and subsequent reversed-phase high-performance liquid chromatography fractionation and one-dimensional electrophoresis. The intensity of 41 bands (with 12 proteins identified) increased and that of 42 bands (with 22 proteins identified) decreased in the sepsis group. Results of proteomic analysis successfully validated by Western blotting and/or enzyme-linked immunosorbent assay for three proteins (YKL-40, lipocalin 2, and S100A9) increased in the sepsis group as well as two proteins (retinol-binding protein, vitamin D-binding protein) decreased. Serum YKL-40 levels (sYKL-40) on intensive care unit (ICU) admission were assessed by enzyme-linked immunosorbent assay between the two groups; resulting YKL-40 was significantly higher in the sepsis group (P < 0.001). Furthermore, sYKL-40 on ICU admission was significantly higher in patients with positive blood culture (P < 0.005), patients with septic shock (P < 0.05), and patients requiring continuous hemodiafiltration (P < 0.05) or hydrocortisone replacement therapy (P < 0.005) during subsequent treatment. A positive correlation between sYKL-40 and blood IL-6 level on ICU admission was noted in the sepsis group (r = 0.465, P < 0.01). YKL-40 identified by proteomic analysis is considered as a biomarker of sepsis. However, further investigation is needed to clarify its roles and clinical usefulness as a biomarker.

Background Serological identification of antigens by recombinant cDNA expression cloning (SEREX) is an established method for detecting new tumor-specific antigens. Antibodies to SEREX antigens may be useful for the detection of esophageal squamous cell carcinoma (SCC). Methods A phage cDNA library of a human esophageal SCC cell line was screened using sera of patients with esophageal SCC. The presence and levels of serum antibodies to SEREX antigens were established by Western blotting and enzyme-linked immunosorbent assay (ELISA) using purified recombinant antigen proteins, respectively. Results The newly identified esophageal SCC antigen is encoded by a novel gene located on chromosome 1, here designated CUEC-23. SerumCUEC-23-antibodies (s-CUEC-23-Abs) were detected in 14 of 54 patients with esophageal SCC (26%) by Western blot analysis. Esophageal SCCs were positive for s-CUEC-23-Abs together with CEA, SCC-Ag or CYFRA21-1 in 44, 41 and 52% of cases, respectively. There was no detectable association between the presence of s-CUEC-23-Abs and clinicopathological variables. ELISA showed that the levels of s-CUEC-23-Abs were significantly higher in patients with esophageal SCC than in healthy volunteers (17% in the former using the mean + 3 SD of s-CUEC-23- Abs in healthy controls as the cutoff). Conclusion A new tumor antigen, CUEC-23, was identified by SEREX screening. s-CUEC-23-Abs might be a useful serum marker to detect esophageal SCC.

Background/Aims: Treatment strategy for T2/T3 esophageal carcinoma has become controversial because of recent improvements in chemoradiation therapy. Only a few study analyzed the prognostic impact of clinicopathological factors for surgical outcome of patients with esophageal carcinoma focused on T2/T3 tumors. Methodology: Subjects of this study were 187 patients with pathological T2 (n=46) or pathological T3 (n=141) thoracic esophageal squamous cell carcinoma who received surgical treatment without neoadjuvant therapy. The impact of clinicopathological factors on survival was evaluated by univariate and multivariate analysis. Results: Overall 5-year survival rate of all patients was 38%. Dismal 5-year overall survival rates were observed in patients with 5 or more positive nodes (11%). Multivariate analysis indicated that lymph node metastases (hazard ratio; 2.07, 95%IC, 1.20-3.56, p<0.01) and the number of metastatic nodes (hazard ratio; 1.77, 95%IC, 1.112.82, p=0.02) were independent risk factors for poor survival. However, tumor depth itself was not an independent risk factor for survival. Conclusions: Although survival of patients with pathological T2 or T3 was partly dependent on tumor depth, it was mainly dependent on lymph node status. Multiple positive nodes were independent risk factors for poor survival.

We performed SEREX (serological identification of antigens by recombinant cDNA expression cloning) using the sera of patients with esophageal squamous cell carcinoma (SCC), and examined whether some of the SEREX antigens can affect chemosensitivity against anticancer drugs. We isolated a novel gene which was designated as AISEC (antigen identified by SEREX for esophageal carcinoma). RT-PCR analysis showed that the mRNA expression levels of AISEC were higher in esophageal SCC tissues than in their normal counterparts. By transfection into activated Ha-ras-transformed NIH3T3 (ras-NIH) mouse fbroblasts, we isolated a clone, FAISEC-3, which stably expressed AISEC. FAISEC-3 cells were more resistant to anticancer drugs, such as mitomycin C, ifosfamide, vincristine, camptothecin and etoposide, than parental ras-NIH cells. Luciferase reporter assay after a transient transfection with AISEC cDNA or the control vector revealed that the transactivity of p53 was suppressed by AISEC in a dose-dependent manner. These results suggested that esophageal SCC tissues produce AISEC in increased amounts, which can reduce the chemosensitivity against anticancer drugs possibly by suppressing the p53 transactivation ability.

Background Although the presence of serum p53 antibody (s-p53-Abs) before treatment has been shown to correlate with poor prognosis and lymph node metastasis in esophageal cancer, there has been little information about postoperative s-p53-Abs titer and perioperative changes of s-p53-Abs titers in patients with esophageal carcinoma. Methods A highly specific enzyme-linked immunosorbent assay was used to analyze s-p53-Abs in 110 patients with esophageal squamous cell carcinoma before and 1 month after surgery. The cutoff level of 1.3 U/ml was used to indicate seropositive patients. Impact of postoperative s-p53-Abs titer and perioperative changes of s-p53-Abs on survival was evaluated. Results Forty (36%) of 110 patients were positive for s-p53-Abs before surgery and 35 patients (32%) were positive after surgery. s-p53-Abs titer generally decreased after surgery. Among sero-positive patients, the patients who remained sero-positive after surgery (n = 28) had a worse prognosis than patients who showed sero-conversion (P = 0.02). Among sero-positive patients, the nondecreased titer group showed significantly unfavorable survival (P < 0.01). Multivariate analysis revealed that postoperative s-p53-Abs was an independent risk factor for worse overall survival (adjusted hazard ratio = 3.05; 95% confidence interval = 1.11-8.33; P = 0.03). Conclusions Perioperative monitoring of s-p53-Abs titers was useful to identify patients with esophageal cancer with a high risk for tumor recurrence and a poor prognosis. Continuous sero-positive patients and/or nondecreased titer group, even after surgery, showed significantly unfavorable survival.

Background Although the presence of serum p53 antibody (s-p53-Abs) before treatment has been shown to correlate with poor prognosis and lymph node metastasis in esophageal cancer, there has been little information about postoperative s-p53-Abs titer and perioperative changes of s-p53-Abs titers in patients with esophageal carcinoma. Methods A highly specific enzyme-linked immunosorbent assay was used to analyze s-p53-Abs in 110 patients with esophageal squamous cell carcinoma before and 1 month after surgery. The cutoff level of 1.3 U/ml was used to indicate seropositive patients. Impact of postoperative s-p53-Abs titer and perioperative changes of s-p53-Abs on survival was evaluated. Results Forty (36%) of 110 patients were positive for s-p53-Abs before surgery and 35 patients (32%) were positive after surgery. s-p53-Abs titer generally decreased after surgery. Among sero-positive patients, the patients who remained sero-positive after surgery (n = 28) had a worse prognosis than patients who showed sero-conversion (P = 0.02). Among sero-positive patients, the nondecreased titer group showed significantly unfavorable survival (P < 0.01). Multivariate analysis revealed that postoperative s-p53-Abs was an independent risk factor for worse overall survival (adjusted hazard ratio = 3.05; 95% confidence interval = 1.11-8.33; P = 0.03). Conclusions Perioperative monitoring of s-p53-Abs titers was useful to identify patients with esophageal cancer with a high risk for tumor recurrence and a poor prognosis. Continuous sero-positive patients and/or nondecreased titer group, even after surgery, showed significantly unfavorable survival.

Based on the genetic background of cancer, we have been trying to develop novel diagnostic and therapeutic strategies against human cancers. c-myc gene activation has been detected in many human cancers, indicating a key role of c-myc in tumor development. Thus targeting c-myc gene suppression is a promising strategy for cancer treatment. Recently, an interaction between FIR (FUSE-Binding Protein-Interacting Repressor) and TFIIH/p89/XPB helicase was found to repress c-myc transcription and so might be important for suppressing tumor formation. Previously, we have shown that the expression of splicing variant of FIR is elevated in colorectal cancer tissues and promotes tumor development by disabling FIR-repression to sustain high levels of c-Myc, opposing apoptosis in cancer cells. In this study, FIR recombinant adenovirus vector induces tumor growth suppression against tumor xenografts in animal model experiment. Together, one clue to the development of cancer diagnosis and therapies directed against c-Myc may go through FIR and its splicing variant.

【背景】 本邦における原発性肝細胞癌の８割はC型肝炎ウイルスによる慢性的な肝障害に起因しており、C型肝炎ウイルスへの推定罹患者数は200万人と言われている。肝細胞癌に特異性の高い腫瘍マーカーとしてAFP・AFP-L3・PIVKA-_II_が有用とされているが、腫瘍マーカー単独での早期癌の検出率は非常に低く、確定診断には超音波検査等の画像診断や、患者への負担の大きい肝生検を必要としているのが現状である。超音波検査による小病変の拾い上げは検者の熟練度に左右されやすく、絶対的なものではない。したがって早期診断、治療方針の検討を行う上で、侵襲性が低く、簡便である血液を用いた検査として、高感度な新規腫瘍マーカーの発見が望まれる。<BR> 【方法】 血清プロテオーム解析の問題点として、「存在量の多いタンパク質の除去」・「キャリアータンパク質に結合しているタンパク質の抽出」等が挙げられる。この問題点を解決した方法として、北里大学で開発されたペプチド抽出法（K法）がある。K法はアセトン沈殿法や限外ろ過膜法に比較して低分子量タンパク質及びペプチドの抽出効率及び再現性に優れ、またアルブミン等のキャリアプロテインに結合している成分の抽出も可能にしている。 C型肝炎ウイルス感染をバックグラウンドに持つ原発性肝細胞癌患者及び肝硬変患者血清と健常者血清各5例に応用した。 血清中のペプチド成分を抽出し、逆相HPLCで分画後、各フラクションをMALDI-TOF MS（AutoFlex II, Bruker Daltonics Inc）で比較解析した。<BR> 【結果・考察】 原発性肝細胞癌患者血清中で有意に増大しているピークを３本検出し、そのうち2ピークの同定に成功した。2ピークは同一のタンパク質の断片であり、肝細胞癌患者血清での報告がなされていないペプチドである。本ペプチドは原発性肝細胞癌の診断の新たな指標になる可能性があり、残り1ピークの同定と平行して、今後多検体での検討を進めていく。

Based on the genetic background of cancer, we have been trying to develop novel diagnostic and therapeutic strategies against human cancers. c-myc gene activation has been detected in many human cancers, indicating a key role of c-myc in tumor development. Thus targeting c-myc gene suppression is a promising strategy for cancer treatment. Recently, an interaction between FIR (FUSE-Binding Protein-Interacting Repressor) and TFIIH/p89/XPB helicase was found to repress c-myc transcription and so might be important for suppressing tumor formation. Previously, we have shown that the expression of splicing variant of FIR is elevated in colorectal cancer tissues and promotes tumor development by disabling FIR-repression to sustain high levels of c-Myc, opposing apoptosis in cancer cells. In this study, FIR recombinant adenovirus vector induces tumor growth suppression against tumor xenografts in animal model experiment. Together, one clue to the development of cancer diagnosis and therapies directed against c-Myc may go through FIR and its splicing variant.

Based on the genetic background of cancer, we have been trying to develop novel diagnostic and therapeutic strategies against human cancers. c-myc gene activation has been detected in many human cancers, indicating a key role of c-myc in tumor development. Thus targeting c-myc gene suppression is a promising strategy for cancer treatment. Recently, an interaction between FIR (FUSE-Binding Protein-Interacting Repressor) and TFIIH/p89/XPB helicase was found to repress c-myc transcription and so might be important for suppressing tumor formation. Previously, we have shown that the expression of splicing variant of FIR is elevated in colorectal cancer tissues and promotes tumor development by disabling FIR-repression to sustain high levels of c-Myc, opposing apoptosis in cancer cells. In this study, FIR recombinant adenovirus vector induces tumor growth suppression against tumor xenografts in animal model experiment. Together, one clue to the development of cancer diagnosis and therapies directed against c-Myc may go through FIR and its splicing variant.

Early diagnosis of hepatocellular carcinoma (HCC) greatly improves its prognosis. However, the distinction between benign and malignant tumors is often difficult, and novel immunohistochemical markers are necessary. Using agarose two-dimensional fluorescence difference gel electrophoresis, we analyzed HCC tissues from 10 patients. The fluorescence volumes of 48 spots increased and 79 spots decreased in tumor tissues compared with adjacent nontumor tissue, and 83 proteins were identified by mass spectrometry. Immunoblot confirmed that the expression of clathrin heavy chain (CHC) and Ku86 significantly increased, whereas formiminotransferase cyclodeaminase (FTCD), rhodanese, and vinculin decreased in tumor. The protein expression in tumor and nontumor tissues was further evaluated by immunostaining. Interestingly, CHC and FTCD expression was strikingly different between tumor and nontumor tissues. The sensitivity and specificity of individual markers or a combination for the detection of HCC were 51.8% and 95.6% for CHC, 61.4% and 98.5% for FTCD, and 80.7% and 94.1% for CHC+FTCD, respectively. Strikingly, the sensitivity and specificity increased to 86.7% and 95.6% when glypican-3, another potential biomarker for HCC, was used with FTCD. Moreover, CHC and FTCD were useful to distinguish early HCC from benign tumors such as regenerative nodule or focal nodular hyperplasia, because the sensitivity and specificity of the markers are 41.2% and 77.8% for CHC, 44.4% and 80.0% for FTCD, which is comparable with those of glypican-3 (33.3% and 100%). The sensitivity significantly increased by combination of these markers, 72.2% for CHC+FTCD, and 61.1% for CHC+glypican-3 and FTCD+glypican-3, as 44.4% of glypican-3 negative early HCC were able to be detected by either CHC or FTCD staining. Conclusion: Immunostaining of CHC and FTCD could make substantial contributions to the early diagnosis of HCC.

Background: Recently, the use of histone deacetylase inhibitors (HDACI) with gene therapy has been shown to improve the effect of this therapy. The effectiveness of one of the novel HDACIs, FK228, was examined in adenovirus-mediated p53 gene therapy of esophageal squamous cell carcinoma (ESCC). Materials and Methods: The expression levels of coxsackie and adenovirus receptor (CAR) in ESCC patients were examined immunohistochemically. CAR induction by FK228 in ESCC cells was analyzed by real-time PCR and Western blotting. The efficiencies of adenoviral transduction treated with FK228 were determined using AV1. OCMV-beta gal. The acetylation of p53 protein was detected by Western blotting. Results: CAR expression was reduced in some tumor specimens compared to that in normal specimens. CAR expression was increased by FK228 in both in vitro and in vivo experiments. FK228 improved the efficiency of adenovirus infection. Acetylated p53 protein was increased in a dose- and a time-dependent manner. Conclusion: Our findings suggest that FK228 has a potent ability to augment the effect of adenovirus-mediated p53 gene therapy in ESCC cells.

Extensive lymphadenectomy, including upper mediastinum, for thoracic esophageal carcinoma was introduced at the beginning of 1980s. However, the efficacy has not been analyzed in large series at a single institute. We evaluated factors potentially related to improved surgical results in patients with thoracic esophageal squamous cell carcinoma (SCC). From 1959 to 1998, a total of 792 patients with thoracic esophageal SCC underwent R0 surgery. A variety of clinicopathological factors were compared among patients treated from 1990 to 1998 (recent group, n=164) and 1959 to 1989 (former group, n=628). The recent group showed significantly better survival than the former group (5-year survival rates: 51 versus 17%, P=0.01), partly because earlier stage disease was included in the recent group than in the former group. Multivariable analysis, using the Cox regression analysis, indicated the time period of surgery, age, tumor location, the number of positive nodes (&gt;5), venous invasion, and tumor-node-metastasis stage. Upper mediastinum lymphadenectomy was also an independent factor to improve survival of patients with thoracic esophageal SCC.

Background: Recently, the use of histone deacetylase inhibitors (HDACI) with gene therapy has been shown to improve the effect of this therapy. The effectiveness of one of the novel HDACIs, FK228, was examined in adenovirus-mediated p53 gene therapy of esophageal squamous cell carcinoma (ESCC). Materials and Methods: The expression levels of coxsackie and adenovirus receptor (CAR) in ESCC patients were examined immunohistochemically. CAR induction by FK228 in ESCC cells was analyzed by real-time PCR and Western blotting. The efficiencies of adenoviral transduction treated with FK228 were determined using AV1. OCMV-beta gal. The acetylation of p53 protein was detected by Western blotting. Results: CAR expression was reduced in some tumor specimens compared to that in normal specimens. CAR expression was increased by FK228 in both in vitro and in vivo experiments. FK228 improved the efficiency of adenovirus infection. Acetylated p53 protein was increased in a dose- and a time-dependent manner. Conclusion: Our findings suggest that FK228 has a potent ability to augment the effect of adenovirus-mediated p53 gene therapy in ESCC cells.

Background Although thoracic lymph node metastasis in patients with thoracic esophageal squamous cell carcinoma (SCC) has been reported to be a negative risk factor for long-term survival, only a few studies have evaluated the clinicopathologic difference between the impact of metastasis to the paraesophageal lymph nodes and to the nonparaesophageal lymph nodes. The purpose of this study was to evaluate surgical outcome after the clearance of metastatic thoracic lymph nodes. Methods Retrospectively reviewed were 164 consecutive patients with thoracic esophageal SCC who had not had preoperative treatment and underwent surgery from 1980 to 2005 and were found to have thoracic lymph node metastases. Of these patients, 83 underwent surgery from 1980 to 1994 and 81 from 1995 to 2005. Univariate and multivariate analyses were performed to evaluate the impact of nonparaesophageal lymph node metastasis on survival. Results Univariate analysis revealed that T3/T4 tumors and the presence of nonparaesophageal node metastases were associated with only a 20% overall five-year survival rate. The overall five-year survival for the most recent period was significantly better than for the former period (42% vs. 13%, p &lt; 0.01). Based on a multivariate analysis of prognostic impact of each nonparaesophageal node, the presence of metastatic subcarinal and/or posterior mediastinal nodes was an independent risk factor for reduced survival. Conclusion Surgical outcome for patients with thoracic esophageal cancer and metastatic thoracic lymph nodes has improved during the last 25 years. Although postoperative chemotherapy might improve survival, the presence of T3/T4 tumors and/or metastatic nonparaesophageal nodes were unfavorable factors for survival.

In recent years, the development of gene therapy systems as new treatment or prevention strategies for various malignant diseases has been explored. Based on the genetic background of esophageal cancer, several molecular therapies have been developed. In this article, we review p53 genetic alterations and angiogenesis in esophageal cancer. Our recent clinical results from a phase I/II study of adenoviral-mediated p53 gene therapy for patients with un-resectable esophageal cancer are reviewed. Lastly we review the available experimental therapeutic models from the literature and our experimental work over the past few years are reviewed. (Ann Thorac Cardiovasc Surg 2008; 14: 3-8)

Background Although thoracic lymph node metastasis in patients with thoracic esophageal squamous cell carcinoma (SCC) has been reported to be a negative risk factor for long-term survival, only a few studies have evaluated the clinicopathologic difference between the impact of metastasis to the paraesophageal lymph nodes and to the nonparaesophageal lymph nodes. The purpose of this study was to evaluate surgical outcome after the clearance of metastatic thoracic lymph nodes. Methods Retrospectively reviewed were 164 consecutive patients with thoracic esophageal SCC who had not had preoperative treatment and underwent surgery from 1980 to 2005 and were found to have thoracic lymph node metastases. Of these patients, 83 underwent surgery from 1980 to 1994 and 81 from 1995 to 2005. Univariate and multivariate analyses were performed to evaluate the impact of nonparaesophageal lymph node metastasis on survival. Results Univariate analysis revealed that T3/T4 tumors and the presence of nonparaesophageal node metastases were associated with only a 20% overall five-year survival rate. The overall five-year survival for the most recent period was significantly better than for the former period (42% vs. 13%, p &lt; 0.01). Based on a multivariate analysis of prognostic impact of each nonparaesophageal node, the presence of metastatic subcarinal and/or posterior mediastinal nodes was an independent risk factor for reduced survival. Conclusion Surgical outcome for patients with thoracic esophageal cancer and metastatic thoracic lymph nodes has improved during the last 25 years. Although postoperative chemotherapy might improve survival, the presence of T3/T4 tumors and/or metastatic nonparaesophageal nodes were unfavorable factors for survival.

Background: p53 gene therapy has been examined in several clinical trials, however, the results of those trials have mostly been unsatisfactory due to the low efficacy of this therapy. Leptomycin B (LMB) is an antibiotic originally isolated from Streptomyces that has the ability to inhibit the export of proteins containing a nuclear export signal from the nucleus to the cytoplasm. Currently, it has been shown that p53 protein has a nuclear export signal. In this study, we assessed whether LMB augments the transduced p53 gene effect. Methods: Antiproliferative effect of LMB was assessed in human esophageal squamous cancer cell lines. Accumulation of p53 protein into the nucleus by LMB was observed by fluorescence microscopy. The combined effect of p53 and LMB was evaluated in in vitro experiments. Results: LMB induced cell death in a dose-dependent manner and p53 drastically accumulated in the nucleus after LMB treatment. The combinatory treatment of p53 gene and LMB significantly increases the efficiency compared to either agent alone. Conclusions: Our findings suggest that LMB has a potent ability to augment the effect of the tumor suppressor p53 in esophageal squamous cancer cell lines and that it is a promising component in p53 gene therapy. Copyright (C) 2008 S. Karger AG, Basel.

Peroxiredoxins (Prdxs) are a family of antioxidant enzymes that are also known as scavengers of peroxide in mammalian cells. Some reports have shown that the overexpression of Prdx1, which is one of the peroxiredoxins that is a ubiquitously expressed protein, was related to a poor prognosis in several types of human cancers. In this study, we investigated the expression levels of Prdx1 in esophageal squamous cell carcinoma by immunohistochemistry, and the correlation between the Prdx1 expression and the clinical status was elucidated. Immunohistochemical staining was performed in 114 samples which were collected from surgical esophageal cancer specimens. Cytoplasmic staining of Prdx1 was evaluated based on the following scoring criteria: Grade I, negative or weak staining; Grade II, moderate staining; and Grade III, strong staining. The percentage of patients with a Grade I expression of Prx1 was 20% (23 of 114), 44% had Grade II (50 of 114), and 36% had Grade 111 (41 of 114). The Prdx1 immunoreactivity showed an inverse significant correlation with T-category (P &lt; 0.0001), lymph node metastasis (P=0.048), and stage (P=0.001). In addition, the patients with tumors exhibiting a reduced Prdx1 expression had shorter overall survival (P=0.022) in comparison to the patients with tumors which had a higher Prdx1 expression. Currently, Prdx1 has been shown to act as a tumor suppressor. Our results provide strong evidence that the reduced Prdx1 expression is an important factor in esophageal squamous cancer progression and could serve as a useful prognostic marker.

Peroxiredoxins (Prdxs) are a family of antioxidant enzymes that are also known as scavengers of peroxide in mammalian cells. Some reports have shown that the overexpression of Prdx1, which is one of the peroxiredoxins that is a ubiquitously expressed protein, was related to a poor prognosis in several types of human cancers. In this study, we investigated the expression levels of Prdx1 in esophageal squamous cell carcinoma by immunohistochemistry, and the correlation between the Prdx1 expression and the clinical status was elucidated. Immunohistochemical staining was performed in 114 samples which were collected from surgical esophageal cancer specimens. Cytoplasmic staining of Prdx1 was evaluated based on the following scoring criteria: Grade I, negative or weak staining; Grade II, moderate staining; and Grade III, strong staining. The percentage of patients with a Grade I expression of Prx1 was 20% (23 of 114), 44% had Grade II (50 of 114), and 36% had Grade 111 (41 of 114). The Prdx1 immunoreactivity showed an inverse significant correlation with T-category (P &lt; 0.0001), lymph node metastasis (P=0.048), and stage (P=0.001). In addition, the patients with tumors exhibiting a reduced Prdx1 expression had shorter overall survival (P=0.022) in comparison to the patients with tumors which had a higher Prdx1 expression. Currently, Prdx1 has been shown to act as a tumor suppressor. Our results provide strong evidence that the reduced Prdx1 expression is an important factor in esophageal squamous cancer progression and could serve as a useful prognostic marker.

Histone deacetylase inhibitors (HDACIs) are promising therapeutic agents with the potential for regulating cell cycle, differentiation and apoptosis in cancer cells. HDACI activity is associated with selective transcriptional regulation and altering gene expression. However, the exact mechanisms leading to the antitumor effect of HDACIs are not fully understood. FK228, one of the powerful HDACIs, strongly inhibited cell growth of T.Tn and TE2 cells and induced apoptosis. Therefore, comprehensive analysis of the changes in gene expression in human esophageal cancer cell lines by the HDACI FK228 was carried out by microarray analysis. This analysis was used to clarify the expression profiles of genes after exposure to FK228. Of the 4,608 genes analyzed, 93 genes in T.Tn and 65 genes in TE2 were up- or down-reaulated 2-fold or more at least at one time point during FK228 exposure and they were classified into four clusters based on their expression patterns. Among them, 15 genes were contained in both cell lines and their expression patterns were similar. Except p21, Prdx1 (reported by us) and IGFBP3, the behaviour/expression of 12 highly responsive genes has still not been reported in esophageal cancer cells. These observations of the expression patterns of functionally classified genes provided insights into the mechanism of the antitumor effect of FK228 in esophageal squamous carcinoma.

Histone deacetylase inhibitors (HDACIs) are promising therapeutic agents with the potential for regulating cell cycle, differentiation and apoptosis in cancer cells. HDACI activity is associated with selective transcriptional regulation and altering gene expression. However, the exact mechanisms leading to the antitumor effect of HDACIs are not fully understood. FK228, one of the powerful HDACIs, strongly inhibited cell growth of T.Tn and TE2 cells and induced apoptosis. Therefore, comprehensive analysis of the changes in gene expression in human esophageal cancer cell lines by the HDACI FK228 was carried out by microarray analysis. This analysis was used to clarify the expression profiles of genes after exposure to FK228. Of the 4,608 genes analyzed, 93 genes in T.Tn and 65 genes in TE2 were up- or down-reaulated 2-fold or more at least at one time point during FK228 exposure and they were classified into four clusters based on their expression patterns. Among them, 15 genes were contained in both cell lines and their expression patterns were similar. Except p21, Prdx1 (reported by us) and IGFBP3, the behaviour/expression of 12 highly responsive genes has still not been reported in esophageal cancer cells. These observations of the expression patterns of functionally classified genes provided insights into the mechanism of the antitumor effect of FK228 in esophageal squamous carcinoma.

Background: This study aimed to evaluate the surgical outcome of esophagectomy in patients 75 years of age and older with thoracic esophageal carcinoma. Patients and Methods: Between 1980 and 2002, 55 (46%) of 120 patients 75 years of age and older with thoracic esophageal carcinoma underwent an esophagectomy. The risk factors that resulted in decreased survival were analyzed by both univariate and multivariate analyses. Differences in surgical outcome and long-term survival between the earlier time period (1980-1989) and later time period (1990-2002) were analyzed separately. Results: Overall resection rate in elderly patients in both periods was similar (44%, earlier period; 46%, later period). Postoperative complications significantly reduced long-term survival (adjusted hazard ratio for death, 4.05; 95% confidence interval (CI), 1.70-9.62; P &lt; 0.01). Surgical blood loss greater than 1,000 ml was less frequently observed in the later period than in the earlier period (19% vs. 54%, P = 0.01). The postoperative morbidity rate was lower in the later than in the earlier period (29% vs. 63%, P = 0.02). Overall 5-year survival rate was significantly higher in the later period than in the earlier period (57% vs. 18%, P &lt; 0.01). Conclusions: Elderly patients who underwent an esophagectomy in the later period appeared to manifest less neoadjuvant treatment, less surgical stress, fewer postoperative complications, and a better long-term survival than those treated in the earlier period.

Background: The location and clinical impact of solitary lymph node metastasis from thoracic esophageal carcinoma have not been evaluated sufficiently. Methods: A consecutive series of 91 patients with a solitary positive lymph node who underwent curative surgery for thoracic esophageal carcinoma was investigated. The prognostic impact was evaluated by univariate analysis and multivariate analysis using Cox's proportional hazards model. Results: A total of 52 (57%) of the 91 patients showed a solitary positive node beyond the thorax. While 29% of the patients with an upper thoracic tumor showed a cervical node, 13% of the patients with a middle tumor and none of the patients with a lower tumor showed a cervical node. Tumor depth and venous invasion were found to be independent risk factors for poor survival. Conclusions: The solitary positive lymph nodes were broadly distributed depending on the tumor location and tumor depth. Tumor depth and venous invasion were risk factors for poor survival in these patients. (c) 2006 Excerpta Medica Inc. All rights reserved.

Purpose. Clinical trials of carbon ion therapy have been performed due to the advantages of high-dose energy delivery with precise localization control to targeted organs and strong cell-killing activities to cancers. Perforated intestines as a late morbidity after carbon ion radiotherapy for uterine cancers were examined to reveal the biological characteristics of carbon ion for future applications for the treatment of gastrointestinal cancers. Methods. Between June 1995 and December 2004, 94 patients with carcinoma of the uterine cervix or corpus were treated with carbon ion therapy. Among them, 9 patients (9.6%) developed major late gastrointestinal (GI) complications. Four out of 9 patients had intestinal perforations excised operatively at our institute. The postoperative clinical courses and histopathological findings of the excised intestine were investigated. Results. Carbon ion irradiation severely damaged smooth muscle layers by coagulation necrosis as well as atrophy of the intestinal epithelium and middle-sized arterial thromboses of the intestines. After evaluating late complications, the dose constraints on the GI tracts were set under 60 GyE to prevent major complications. Thereafter, the incidence of major GI complications markedly decreased. Conclusion. Our findings demonstrated the characteristic histopathological effects of carbon ion radiotherapy and thus are expected to facilitate future additional applications of carbon ion radiotherapy for the treatment of gastrointestinal cancers.

We investigated the feasibility, safety, biological activity and therapeutic efficacy of adenovirus-mediated p53 gene transfer in patients with chemoradiation resistant advanced esophageal carcinoma. Eligible patients were not surgical candidates and had measurable, advanced squamous cell carcinoma of the esophagus that was resistant to chemoradiation therapy. On a 28-day cycle, intratumoral injections of Ad5CMV-p53 (INGN 201; ADVEXIN (R)) were administered on days 1 and 3 at four dose levels (10 x 10(11) particles to 25 x 10(11) particles) and treated for up to five cycles. Ten patients received a total of 26 cycles with no dose-limiting toxicity. Administration of multiple courses was feasible and well-tolerated. Local tumor responses revealed stable disease in nine cases and progressive disease in one case. The overall responses were stable in six and progressive in four cases. Using polymerase chain reaction (PCR) analyses, gene transfer and p53 specific transgene expression were detected in tumor biopsy tissue from all patients. mRNA levels of p53, p21 and MDM2 increased in all but one case. Three patients showed absence of disease upon repeat biopsies. Substantial improvement in swallowing was observed in one patient with stenotic lesions. Intratumoral injection of Ad5CMV-p53 is safe, feasible and biologically active when administered in multiple doses to patients with esophageal cancer. Observations from this study indicate that this treatment results in local antitumor effects in chemoradiation resistant esophageal squamous cell carcinoma.

Recent advances in two-dimensional electrophoresis; (2-DE) such as fluorescent 2-D differential gel electrophoresis (2-D DIGE) has made it possible to detect and quantitate the critical changes involved in disease pathogenesis. We have previously identified novel proteins with altered expression in primary colorectal cancer using agarose 2-DE that has a higher loading capacity than immobilized PH gradient gel. The aim of this study is to identify novel proteins with altered expression in primary esophageal cancer using the powerful method of agarose 2-DE and agarose 2-D DIGE. Excised tissues from 12 patients of primary esophageal cancer were obtained. Proteins with altered expression between cancer and adjacent non-cancer tissues were analyzed by agarose 2-D DIGE and identified by mass spectrometry. Thirty-three proteins out of 74 spots with altered expression in tumors were identified. Among them, a 195-kDa protein, periplakin, was significantly downregulated in esophageal cancer, which was confirmed by immunoblotting. Immunohistochemistry showed that periplakin was mainly localized at cell-cell boundaries in normal epithelium and dysplastic lesions, while it disappeared from cell boundaries, shifted to cytoplasm, in early cancers and scarcely expressed in advanced cancers. These results suggest that periplakin could be a useful marker for detection of early esophageal cancer and evaluation of tumor progression.

Elevated expression of c-myc has been detected in a broad range of human cancers, indicating a key role for this oncogene in tumor development. Recently, an interaction between FUSE-binding protein-interacting repressor (FIR) and TFIIH/p89/XPB helicase was found to repress c-myc transcription and might be important for suppressing tumor formation. In this study, we showed that enforced expression of FIR induced apoptosis. Deletion of the NH2-terminal repression domain of FIR rescued the cells from apoptosis as did coexpression of c-Myc with FIR; thus, repression of Myc mediates FIR-driven apoptosis. Surprisingly, a splicing variant of FIR unable to repress c-myc or to drive apoptosis was frequently discovered in human primary colorectal cancers but not in the adjacent normal tissues. Coexpression of this splicing variant with repressor-competent FIR, either in HeLa cells or in the colon cancer cell line SW480, not only abrogated c-Myc suppression but also inhibited apoptosis. These results strongly suggest the expression of this splicing variant promotes tumor development by disabling FIR repression and sustaining high levels of c-Myc and opposing apoptosis in colorectal cancer.

Strong HLA-DR antigen expression on cancer cells relates to better prognosis of colorectal cancer patients, although the precise mechanism is controversial. From an immunological point of view, HLA-DR antigen, induced by interferon (IFN)-gamma, is required for tumor-associated antigen recognition by CD4(+) T cells. For instance, as reported previously, the expression of HLA-DR antigen in normal colorectal epithelium immediately adjacent to cancer coincided significantly with the existence of IFN-gamma mRNA in the tissue. From another aspect, IFN-gamma has been revealed to suppress c-myc expression in vivo through a stat1-dependent mechanism, which is important for cell growth, cell cycle and chromosome instability. In the present study, strong HLA-DR-positive expression on cancer cells was significantly related to better prognosis for colorectal cancer patients. High IFN-gamma mRNA expression in situ indicated significantly less activation of c-myc mRNA expression. Further, HLA-DR antigen expression in cancer cells, as well as Dukes stages, was an independent factor for better long-term survival by multivariate analysis. Taken together, IFN-gamma, which induces HLA-DR antigens on the cell surface, also suppresses c-myc expression in situ, and is a possible non-immunological mechanism involved in the better long-term survival of colorectal cancer patients.

Purpose: The histone deacetylase inhibitor FK228 shows strong activity as a potent antitumor drug but its precise mechanism is still obscure. The purpose of this study is to reveal the effect of FK228 on gene expression in the cell and to determine the mechanism of the antitumor activity of FK228 for further clinical applications. Experimental Design and Results: Microarray analysis was applied to verify the gene expression profiles of 4,608 genes after FK228 treatment using human esophageal squamous cell cancer cell lines T.Tn and TE2. Among them, peroxiredoxin 1 (Prdx1), a member of the peroxiredoxin family of antioxidant enzymes having cell growth suppression activity, as well WAR as p21(WAF1), were significantly activated by FK288. In addition, FK228 strongly inhibited the cell growth of T.Tn and TE2 by the induction of apoptosis. Further, chromatin immunoprecipitation analysis revealed that FK228 induced the accumulation of acetylated histones H3 and H4 in Prdx1 promoter, including the Sp1-binding site. In mouse xenograft models of T.Tn and TE2 cells, FK228 injection resulted in significant tumor regression as well as activated Prdx1 expression in tumor tissues. Prdx1 suppression by RNA interference hindered the antitumor effect of FK228. Conclusion: Our results indicate that the antitumor effect of FK228 in esophageal cancer cells is shown at least in part through Prdx1 activation by modulating acetylation of histories in the promoter, resulting in tumor growth inhibition with apoptosis induction.

Proteases have received attention as important cellular components responsible for stress response in human cells. However, little is known about the role of proteases in the early steps of cell response after X-ray irradiation. In the present study, we first searched for proteases whose activity levels are changed soon after X-ray irradiation in human RSa cells with a high sensitivity to X-ray cell-killing. RSa cells showed an increased level of fibrinolytic protease activity within 10 min after irradiation with X-ray (up to 3 Gy). The induced protease activity was proved to be inhibited by leupeptin. We next examined whether this protease inducibility is related to the X-ray susceptibility of cells. Treatment of RSa cells with leupeptin prior to X-ray irradiation resulted in lowered colony survival and an increased ratio of G(2)/M-arrested cells and apoptotic cells. These results suggest that leupeptin-sensitive proteases are involved in the resistance of human RSa cells to X-ray cell-killing. (c) 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Chromosomal instability (CIN) has been recognized as a hallmark of human cancer and is caused by continuous chromosome missegregation during mitosis. Proper chromosome segregation requires a physical connection between spindle microtubules and centromeric DNA and this attachment occurs at proteinaceous structures called kinetochore. Several centromere proteins such as CENP-A and CENP-H are the fundamental components of the human active kinetochore, and inappropriate expression of the centromere proteins could be a major cause of CIN. We have previously shown that CENP-A was overexpressed in primary human colorectal cancer. In this study, we show that CENP-H was also up-regulated in all of 15 primary human colorectal cancer tissues as well as in CIN tumor cell lines. Surprisingly, transient transfection of CENP-H expression plasmid into the diploid cell line HCT116 remarkably induced aneupoidy. Moreover, CENP-H stable transfectant of mouse embryonic fibroblast/3T3 cell lines showed aberrant interphase micronuclei, characteristic of chromosome missegregation. In these CENP-H overexpressed cells, CENP-H completely disappeared from the centromere of mitotic chromosomes, which might be the cause of the chromosome segregation defect. These results suggest that the aberrant expression and localization of a kinetochore protein CENP-H plays an important role in the aneuploidy frequently observed in colorectal cancers.

Background/Aims: Electrochemotherapy, which uses two parallel electrodes for delivery of electric pulses, may be useful for treatment of tumor nodules. However, in clinical fields, tumors that are larger than the distance between the two electrodes are frequently observed, and it would be difficult in such cases to deliver electric pulses to the tumor. This study was done to define how host antitumor immunity is generated by repeated electrochemotherapy treatments, and whether it is associated with regression of even large tumor nodules. Methodology: Balb/c mice and Balb/c nude mice were inoculated subcutaneously with colon 26. Electrochemotherapy using bleomycin and electroporation (CUY21) was administered as a treatment for tumor nodules that were larger than the distance between the electrodes. Results: In Balb/c mice, growth of large tumors at the start of treatment is not inhibited by a single electrochemotherapy treatment. However, complete tumor regression was obtained through repeated electrochemotherapy treatments. No tumor cure was observed among Balb/c nude mice under the same therapeutic conditions. Inflammatory cells were accumulated in the tumor tissue seven days after the third electrochemotherapy treatment. Conclusions: Repeated electrochemotherapy is a promising treatment, even for large tumors, such as are usually encountered in clinical practice, by generating T-cell dependent, antitumor immunity.

There has been little research evaluating changes related to tumor cell proliferation between primary and metastatic tumors of gastrointestinal tumors in the same case. We herein report the case of a 50-year-old woman with a gastric gastrointestinal stromal tumor (GIST), who developed metastatic liver tumors three times in the 7 years after proximal gastrectomy for GIST. The primary and all the metastatic liver tumors, except the second, showed fascicular/storiform architecture and the short spindle cell type. The diffuse epithelioid cell proliferation was observed in the second metastatic liver tumor. Although the immunostaining pattern with respect to GIST differentiation markers had been preserved in the primary tumor as well as in all of the metastatic tumors, the latter showed weaker positivity of both Ki-67 and p53 than the primary GIST. The primary tumor showed diffuse positive p53, and the highest value of Ki-67 labeling index (LI) among them. The metastatic liver tumors showed focal, negative or sporadic positive appearances of p53, however, M-67 LI were scattering among them. Immunohistochemical assessment of Ki-67 LI and p53 might be useful for evaluating changes related to tumor cell proliferation between primary and metastatic tumors of GISTs.

Purpose: Although numerous proteome studies have been performed recently to identify cancer-related changes in protein expression, only a limited display of relatively abundant proteins has been identified. The aim of this study is to identify novel proteins as potential tumor markers in primary colorectal cancer tissues using a high-resolution two-dimensional gel electrophoresis (2-DE). Experimental Design: 2-DE using an agarose gel for isoelectric focusing was used to compare protein profiling of 10 colorectal cancer tissues and adjacent normal mucosa. Altered expression and post-translational modification of several proteins were examined using Western blot analysis and inummohistochemistry. Results: Ninety-seven proteins of 107 spots (90.7%) that were differentially expressed between matched normal and tumor tissues were identified by mass spectrometry. Among them, 42 unique proteins (49 spots) significantly increased or decreased in the tumors. They include eukaryotic translation initiation factor 411, inorganic pyrophosphatase, anterior gradient 2 homologue, aldolase A, and chloride intracellular channel 1, whose elevated expression in tumor tissues was confirmed by Western blot analysis and immunohistochemistry. Interestingly, only isoform 1 of two transcript variants of eukaryotic translation initiation factor 4H was greatly up-regulated in most of the tumor tissues. Moreover, post-translational modifications of the prolyl-4-hydroxylase beta subunit and annexin A2 also were identified. Conclusions: We identified several novel proteins with altered expression in primary colorectal cancer using agarose 2-DE. This method is a powerful technique with which to search for not only quantitative but also qualitative changes in a biological process of interest and may contribute to the deeper understanding of underlying mechanisms of human cancer.

Purpose: Although numerous proteome studies have been performed recently to identify cancer-related changes in protein expression, only a limited display of relatively abundant proteins has been identified. The aim of this study is to identify novel proteins as potential tumor markers in primary colorectal cancer tissues using a high-resolution two-dimensional gel electrophoresis (2-DE). Experimental Design: 2-DE using an agarose gel for isoelectric focusing was used to compare protein profiling of 10 colorectal cancer tissues and adjacent normal mucosa. Altered expression and post-translational modification of several proteins were examined using Western blot analysis and inummohistochemistry. Results: Ninety-seven proteins of 107 spots (90.7%) that were differentially expressed between matched normal and tumor tissues were identified by mass spectrometry. Among them, 42 unique proteins (49 spots) significantly increased or decreased in the tumors. They include eukaryotic translation initiation factor 411, inorganic pyrophosphatase, anterior gradient 2 homologue, aldolase A, and chloride intracellular channel 1, whose elevated expression in tumor tissues was confirmed by Western blot analysis and immunohistochemistry. Interestingly, only isoform 1 of two transcript variants of eukaryotic translation initiation factor 4H was greatly up-regulated in most of the tumor tissues. Moreover, post-translational modifications of the prolyl-4-hydroxylase beta subunit and annexin A2 also were identified. Conclusions: We identified several novel proteins with altered expression in primary colorectal cancer using agarose 2-DE. This method is a powerful technique with which to search for not only quantitative but also qualitative changes in a biological process of interest and may contribute to the deeper understanding of underlying mechanisms of human cancer.

Aneuploidy is the hallmark of many human cancers. Recent work has strongly suggested that chromosome missegregation during mitosis is the main cause of aneuploidy and contributes to oncogenesis. Centromere protein (CENP) -A is the centromere-specific histone-H3-like variant essential for centromere structure and function. It plays a central role in the assembly of the protein complex, termed kinetochore, which is indispensable for equal chromosome segregation. In this study, we demonstrate that the kinetochore protein CENP-A was overexpressed in all of 11 primary human colorectal cancer tissues. CENP-A mRNA was also up-regulated, indicating that overexpression of CENP-A occurred at the transcriptional level. Immunostaining with anti-CENP-A antibodies showed increased CENP-A signals in the tumor cells. Moreover, coimmunostaining of CENP-B, a centromere-associated DNA binding protein, with CENP-A showed mistargeting of CENP-A to noncentromeric chromatin in the tumor cells. These results suggest that overexpression of CENP-A could play an important role for aneuploidy in colorectal cancers.

Aneuploidy is the hallmark of many human cancers. Recent work has strongly suggested that chromosome missegregation during mitosis is the main cause of aneuploidy and contributes to oncogenesis. Centromere protein (CENP) -A is the centromere-specific histone-H3-like variant essential for centromere structure and function. It plays a central role in the assembly of the protein complex, termed kinetochore, which is indispensable for equal chromosome segregation. In this study, we demonstrate that the kinetochore protein CENP-A was overexpressed in all of 11 primary human colorectal cancer tissues. CENP-A mRNA was also up-regulated, indicating that overexpression of CENP-A occurred at the transcriptional level. Immunostaining with anti-CENP-A antibodies showed increased CENP-A signals in the tumor cells. Moreover, coimmunostaining of CENP-B, a centromere-associated DNA binding protein, with CENP-A showed mistargeting of CENP-A to noncentromeric chromatin in the tumor cells. These results suggest that overexpression of CENP-A could play an important role for aneuploidy in colorectal cancers.

Background. Increased preoperative serum squamous cell carcinoma antigen (SCC-Ag) concentrations have been found to be associated with advanced stage and poor prognosis in lung and cervical cancers. Because little was known about the significance of SCC-Ag concentration in patients with esophageal cancer, the aim of this study was to analyze the clinicopathologic significance of SCC-Ag in patients with esophageal SCC. Patients and methods. Preoperative SCC-Ak concentration was measured with enzyme-linked immunosorbent assay in 309 patients with primary esophageal SCC. All patients underwent curative radical surgery without any preoperative therapy. In 215 of 309 patients, carcinoembryonic antigen (CEA) was also measured to compare clinical significance of CEA with that of SCC-Ag. The prognostic significance for survival of SCC-Ag concentrations was studied with multivariate analysis with Cox proportional hazards model. Results. The SCC-Ag concentration and the positivity rate of SCC-Ag were significantly elevated in patients associated with tumor progression. Statistically significant differences in SCC-Ag concentrations and SCG-Ag positivity rates were observed depending on tumor size, tumor depth, lymph node status, and distant metastasis. Although CFA was not a prognosticfactor (P = .21), a high SCC-Ag concentration was a significant Prognostic factor (P &lt; . 01). Multivariate analyses indicated that T factor had the best predictive power, but SCC-Ag concentration contained additional, independent prognostic information. Conclusion. Our findings suggest that preoperative serum SCC-Ag concentrations might provide a predictive information for tumor progression and survival in patients with esophageal SCC.