松下 一之

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) encompasses a wide spectrum of diseases, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which carries a significant risk of progression to cirrhosis and hepatocellular carcinoma. Since NASH is a progressive but reversible condition, it is desirable to distinguish NASH from simple steatosis, and to treat NASH patients at an early stage. To establish appropriate diagnosis and therapy, the pathological mechanisms of the disease should be elucidated; however, these have not been fully clarified for both NASH and simple steatosis. This study aims to reveal the differences between simple steatosis and NASH. METHODS: This study used fatty liver Shionogi (FLS) mice as a NASH model, for comparison with dd Shionogi (DS) mice as a model of simple steatosis. Genome-wide gene expression analysis was performed using Affymetrix GeneChip Mouse Genome 430 2.0 Array, which contains 45101 probe sets for known and predicted genes. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to investigate gene expression changes and protein localizations. RESULTS: DNA microarray analysis of the liver transcriptomes and qRT-PCR of both types of mice revealed that LCN2, CXCL1 and CXCL9 mRNAs were overexpressed in FLS mouse livers. Immunohistochemistry showed that CXCL1 protein was mainly localized to steatotic hepatocytes. CXCL9 protein-expressing hepatocytes and sinusoidal endothelium were localized in some areas of inflammatory cell infiltration. Most interestingly, hepatocytes expressing LCN2, a kind of adipokine, were localized around almost all inflammatory cell clusters. Furthermore, there was a positive correlation between the number of LCN2-positive hepatocytes in the specimen and the number of inflammatory foci. CONCLUSIONS: Overexpression and distinct localization of LCN2, CXCL1 and CXCL9 in the liver of fatty liver Shionogi mice suggest significant roles of these proteins in the pathogenesis of NASH.

The prognosis of advanced colorectal cancer after surgical resection remains poor if curative resection cannot be achieved. Neoadjuvant chemotherapy( NAC) may increase the curative resection rate and reduce the recurrence rate following resection of marginally resectable advanced colorectal cancer by ensuring adequate surgical margin and controlling micro-metastases. Herein, we report the treatment regimen and outcomes of NAC for advanced colorectal cancer at our institute. Between April 2005 and December 2012, 10 patients with marginally resectable advanced colorectal cancer received NAC before undergoing laparotomy. NAC consisted of 4 to 8 courses of the FOLFIRI-3 regimen combined with molecular targeted agents. Laparotomy was performed 4 to 6 weeks after the last course of NAC, and 12 courses of mFOLFOX6 were recommended as adjuvant chemotherapy after surgery. A partial response (PR) according to Response Evaluation Criteria in Solid Tumors ver. 3 was observed in 5 patients and progressive disease (PD) was not observed in any patient. Curative resection was achieved in 9 patients. All patients are currently alive, and the 2-year relapse-free survival rate was 62.2%. This is a retrospective study of a small number of subjects however, the results suggest that NAC for marginally resectable advanced colorectal cancer increases the curative resection rate and reduces the recurrence rate.

Cancer arises through accumulation of epigenetic and genetic alteration. Aberrant promoter methylation is a common epigenetic mechanism of gene silencing in cancer cells. We here performed genome-wide analysis of DNA methylation of promoter regions by Infinium HumanMethylation27 BeadChip, using 14 clinical papillary thyroid cancer samples and 10 normal thyroid samples. Among the 14 papillary cancer cases, 11 showed frequent aberrant methylation, but the other three cases showed no aberrant methylation at all. Distribution of the hypermethylation among cancer samples was non-random, which implied existence of a subset of preferentially methylated papillary thyroid cancer. Among 25 frequently methylated genes, methylation status of six genes (HIST1H3J, POU4F2, SHOX2, PHKG2, TLX3, HOXA7) was validated quantitatively by pyrosequencing. Epigenetic silencing of these genes in methylated papillary thyroid cancer cell lines was confirmed by gene re-expression following treatment with 5-aza-2'-deoxycytidine and trichostatin A, and detected by real-time RT-PCR. Methylation of these six genes was validated by analysis of additional 20 papillary thyroid cancer and 10 normal samples. Among the 34 cancer samples in total, 26 cancer samples with preferential methylation were significantly associated with mutation of BRAF/RAS oncogene (P = 0.04, Fisher's exact test). Thus, we identified new genes with frequent epigenetic hypermethylation in papillary thyroid cancer, two subsets of either preferentially methylated or hardly methylated papillary thyroid cancer, with a concomitant occurrence of oncogene mutation and gene methylation. These hypermethylated genes may constitute potential biomarkers for papillary thyroid cancer.

Endoscopic intervention is less invasive than percutaneous or surgical approaches and should be considered the primary drainage procedure in most cases with obstructive jaundice. Recently, therapeutic endoscopic retrograde cholangiopancreatography (ERCP) using double-balloon enteroscopy (DBE) has been shown to be feasible and effective, even in patients with surgically altered anatomies. On the other hand, endoscopic partial stent-in-stent (PSIS) placement of self-expandable metallic stents (SEMSs) for malignant hilar biliary obstruction in conventional ERCP has also been shown to be feasible, safe and effective. We performed PSIS placement of SEMSs for malignant hilar biliary obstruction due to liver metastasis using a short DBE in a patient with Roux-en-Y anastomosis and achieved technical and clinical success. This procedure can result in quick relief from obstructive jaundice in a single session and with short-term hospitalization, even in patients with surgically altered anatomies. (C) 2012 Baishideng. All rights reserved.

Background: Diabetic nephropathy (DN) is a significant cause of hemodialysis, and its early detection is extremely important to prevent or delay end-stage renal disease. The significance of the renal function marker serum cystatin C (sCysC) and its relationship with glomerular filtration rate in chronic kidney disease (CKD) and DN in Japanese patients with type 2 diabetes remains uncertain. In this study, we examined the effectiveness of sCysC as a marker of early DN and CKD in Japanese subjects. Methods: A total of 325 Japanese patients with type 2 diabetes and 88 healthy subjects were studied retrospectively. sCysC concentration (mg/L) was determined by a latex turbidmetric immunoassay using a BioMajesty 8040 analyzer. The renal function of the diabetic patients was evaluated using the albumin-creatinine ratio (ACR) and Kidney Disease Outcome Quality Initiative-Kidney Disease Improving Global Outcomes (K/DOQI-KDIGO) classification. Results: There was a significant increase in sCysC but not in serum creatinine (sCr) or serum beta(2)-microglobulin (s beta 2M) in patients with grade 2 DN (ACR 30-300 mg/g) compared to grade 1 patients. Receiver operating characteristic analysis in grade 2 and 3 DN patients showed that sCysC had superior sensitivity and specificity than sCr and s beta 2M for early detection of DN. In addition, sCysC showed particularly high sensitivity and specificity in DN patients with stage 2 CKD. Conclusions: sCysC was effective for detection of grade 2 DN and would be especially useful for screening stage 2 CKD patients (K/DOQI-KDIGO).

Long-term outcomes of the surgically treated unruptured intracranial aneurysms (UIA) are not well known. We conducted this study to clarify the surgical results and the long-term outcomes of the surgically treated UIA. <br> Seventy-three consecutive patients who were surgically treated and were followed over five years were enrolled in this study. Mean (median) age was 58.3 (59) years. The location of aneurysm was as follows: internal carotid in 32%, middle cerebral in 27%, multiple in 23% and so on. In the aneurysmal size, medium (4–11 mm) was most common (55%), followed by large or giant (12 mm or more) one (16%).<br> Results: 1) Permanent surgical morbidity was 5.5% and mortality was 1.4% (one case due to fulminant hepatitis). 2) Surgical treatment of the single aneurysm: craniotomy in 57 patients and intervention (GDC embolization) in four. 3) Mean (median) follow-up period was 8.6 (8) years. 4) Regrowth of the aneurysm was observed in one patient (1.4%), and de novo aneurysms were visualized in three (4.5%). 5) Long-term outcomes: modified Rankin scale 0–1 in 65 patients (89%), and six in five (7%). 6) Factors related to outcomes: cardiovascular events were seen in five patients; cerebrovascular in three patients and cardiac in two.<br> It is concluded that long-term outcomes of UIA were fairly favorable, and cardio- and cerebrovascular events were the main factor related to unfavorable outcome.<br>

Esophageal squamous cell carcinoma (SCC) is frequently associated with a high mortality rate as a result of late diagnosis and/or aggressive behavior. Although multimodal treatment is applied for advanced tumors, many patients suffer from progressive disease and rapid recurrence. Besides various imaging techniques, serum biomarkers should also be useful for early diagnosing and treatment monitoring. A comprehensive review is provided here mainly on advancements of our clinical research in serum biomarkers to diagnose and/or monitor esophageal SCC. First, we focused on conventional secretory type serum markers, SCC-antigen and CYFRA 21-1. Both serum markers are useful to predict high-risk patients to develop recurrent disease. Second, we reviewed the clinicopathological significance of various angiogenic factors, vascular endothelial growth factor, thymidine phosphorylase, fibroblast growth factor, midkine, and hepatocyte growth factor. These growth factors could be useful biomarkers to predict lymph node and/or distant metastases. Finally, we reviewed advancements of clinical research on autoantibodies against tumor-specific antigens, particularly focused on serum p53 antibody. Because serum antibodies frequently respond to a small volume of tumors, they are useful in early tumor detection and prediction of residual cancer cells. Serum biomarkers may be a useful tool in the management of esophageal SCC.

Esophageal squamous cell carcinoma (SCC) is frequently associated with a high mortality rate as a result of late diagnosis and/or aggressive behavior. Although multimodal treatment is applied for advanced tumors, many patients suffer from progressive disease and rapid recurrence. Besides various imaging techniques, serum biomarkers should also be useful for early diagnosing and treatment monitoring. A comprehensive review is provided here mainly on advancements of our clinical research in serum biomarkers to diagnose and/or monitor esophageal SCC. First, we focused on conventional secretory type serum markers, SCC-antigen and CYFRA 21-1. Both serum markers are useful to predict high-risk patients to develop recurrent disease. Second, we reviewed the clinicopathological significance of various angiogenic factors, vascular endothelial growth factor, thymidine phosphorylase, fibroblast growth factor, midkine, and hepatocyte growth factor. These growth factors could be useful biomarkers to predict lymph node and/or distant metastases. Finally, we reviewed advancements of clinical research on autoantibodies against tumor-specific antigens, particularly focused on serum p53 antibody. Because serum antibodies frequently respond to a small volume of tumors, they are useful in early tumor detection and prediction of residual cancer cells. Serum biomarkers may be a useful tool in the management of esophageal SCC.

The protein composition of gingival crevicular fluid (GCF) may reflect the pathophysiology of periodontal diseases. A standard GCF proteomic pattern of healthy individuals would serve as a reference to identify biomarkers of periodontal diseases by proteome analyses. However, protein profiles of GCF obtained from apparently healthy individuals have not been well explored. As a step toward detection of proteomic biomarkers for periodontal diseases, we applied both gel-based and gel-free methods to analyze GCF obtained from healthy subjects as compared with supragingival saliva. To ensure optimized protein extraction from GCF, a novel protocol was developed. The proteins in GCF were extracted with high yield by urea buffer combined with ultrafiltration and the intensity of spots with supragingival saliva and GCF was compared using agarose two-dimensional electrophoresis. Eight protein spots were found to be significantly more intense in GCF. They included superoxide dismutase 1 (SOD1), apolipoprotein A-I (ApoA-I), and dermcidin (DCD). Moreover, GCF proteins from healthy subjects were broken down into small peptide fragments and then analyzed directly by LC-MS/MS analysis. A total of 327 proteins including ApoA-I, SOD1, and DCD were identified in GCF. These results may serve as reference for future proteomic studies searching for GCF biomarkers of periodontal diseases.

The protein composition of gingival crevicular fluid (GCF) may reflect the pathophysiology of periodontal diseases. A standard GCF proteomic pattern of healthy individuals would serve as a reference to identify biomarkers of periodontal diseases by proteome analyses. However, protein profiles of GCF obtained from apparently healthy individuals have not been well explored. As a step toward detection of proteomic biomarkers for periodontal diseases, we applied both gel-based and gel-free methods to analyze GCF obtained from healthy subjects as compared with supragingival saliva. To ensure optimized protein extraction from GCF, a novel protocol was developed. The proteins in GCF were extracted with high yield by urea buffer combined with ultrafiltration and the intensity of spots with supragingival saliva and GCF was compared using agarose two-dimensional electrophoresis. Eight protein spots were found to be significantly more intense in GCF. They included superoxide dismutase 1 (SOD1), apolipoprotein A-I (ApoA-I), and dermcidin (DCD). Moreover, GCF proteins from healthy subjects were broken down into small peptide fragments and then analyzed directly by LC-MS/MS analysis. A total of 327 proteins including ApoA-I, SOD1, and DCD were identified in GCF. These results may serve as reference for future proteomic studies searching for GCF biomarkers of periodontal diseases.

The Far UpStream Element (FUSE)-binding protein-interacting repressor (FIR), a c-myc transcriptional suppressor, is alternatively spliced removing the transcriptional repression domain within exon 2 (FIR Delta exon2) in colorectal cancers. SAP155 is a subunit of the essential splicing factor 3b (SF3b) subcomplex in the spliceosome. This study aims to study the significance of the FIR-SAP155 interaction for the coordination of c-myc transcription, pre-mRNA splicing, and c-Myc protein modification, as well as to interrogate FIRDexon2 for other functions relating to altered FIR pre-mRNA splicing. Knockdown of SAP155 or FIR was used to investigate their reciprocal influence on each other and on c-myc transcription, pre-mRNA splicing, and protein expression. Pull down from HeLa cell nuclear extracts revealed the association of FIR, FIR Delta exon2, and SF3b subunits. FIR and FIR Delta exon2 were coimmunoprecipitated with SAP155. FIR and FIR Delta exon2 adenovirus vector (Ad-FIR and Ad-FIR Delta exon2, respectively) were prepared to test for their influence on c-myc expression. FIR, SAP155, SAP130, and c-myc were coordinately upregulated inhuman colorectal cancer. These results reveal that SAP155 and FIR/FIR Delta exon2 forma complex and are mutually upregulating. Ad-FIR Delta exon2 antagonized Ad-FIR transcriptional repression of c-myc in HeLa cells. Because FIRDexon2 still carries RRM1 and RRM2 and binding activity to FUSE, it is able to displace repression competent FIR from FUSE in electrophoretic mobility shift assays, thus thwarting FIR-mediated transcriptional repression by FUSE. Thus aberrant FIR Delta exon2 production in turn sustained c-Myc expression. In conclusion, altered FIR and c-myc pre-mRNA splicing, in addition to c-Myc expression by augmented FIR/FIR Delta exon2-SAP155 complex, potentially contribute to colorectal cancer development. Mol Cancer Res; 10(6); 787-99. (C) 2012 AACR.

Background: Carbon-ion radiotherapy (CIR) has been under development. We report the results of a phase I/II clinical trial of preoperative CIR for esophageal squamous cell carcinoma (ESCC). Methods: Thirty-one thoracic ESCC patients were enrolled. They were first treated with CIR. The radiation dose was escalated from the initial dose of 28.8 GyE up to 36.8. Four to 8 weeks after CIR followed by clinical evaluation of the therapy, surgery was performed. Thereafter, a pathological evaluation was made. Results: Acute toxicity was not seen except in one case (3.2%), and there were no late toxicities. Throughout the study period, there were no cases of withdrawal due to the effects of preoperative CIR. Twelve out of 31 (38.7%) patients achieved a clinical complete response (CR) and 13 patients (41.9%) achieved a partial response. Twelve out of 31 patients (38.7%) achieved a pathological CR. The overall 1-, 3-, and 5-year survival rates in the stage I cases were 91%, 81%, and 61%, and was 100%, 85%, and 77% for the stage II, and 71%, 43%, and 29% for the stage III cases, respectively. Conclusions: CIR showed strong local tumor control and is highly effective as a neoadjuvant therapy without severe adverse events. J. Surg. Oncol. 2012; 105:750-755. (C) 2011 Wiley Periodicals, Inc.

Background: Hereditary colorectal cancer accounts for approximately 4-5% of all colorectal cancers. The causative genes for familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer are large, making comprehensive analyses difficult. Therefore, high-throughput and practical methods are required to make an early diagnosis of hereditary colorectal cancers and identify high-risk individuals. For this purpose, we developed a novel gene scanning method by high-resolution melting (HRM) analysis. Methods: High-resolution melting (HRM) analysis is a promising prescreening method for nucleic acid sequence variants because of its high sensitivity and high-throughput capability. We evaluated HRM for screening APC, MLH1, MSH2, and MSH6 genes for point mutations, small deletions, and insertions. Simultaneously, we evaluated quantitative polymerase chain reaction-HRM (qPCR-HRM) for screening the MSH2 gene for large rearrangements. Results: All 28 point mutations and 1 large rearrangement were successfully detected by qPCR-HRM analysis. Conclusions: A fast and reliable mutation detection strategy with HRM and qPCR-HRM was used to diagnose hereditary colorectal cancers. Because this method is simple and economical, it may be useful in diagnostic laboratories.

Hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, is one of the most common cancers worldwide and the third most common cause of cancer-related death. Imaging studies including ultrasound and computed tomography are recommended for early detection of HCC, but they are operator dependent, costly and involve radiation. Therefore, there is a need for simple and sensitive serum markers for the early detection of hepatocellular carcinoma (HCC). In our recent proteomic studies, a number of proteins overexpressed in HCC tissues were identified. We thought if the serum autoantibodies to these overexpressed proteins were detectable in HCC patients. Of these proteins, we focused on Ku86, a nuclear protein involved in multiple biological processes and aimed to assess the diagnostic value of serum anti-Ku86 in the early detection of HCC. Serum samples were obtained prior to treatment from 58 consecutive patients with early or relatively early hepatitis C virus (HCV)-related HCC and 137 patients with HCV-related liver cirrhosis without evidence of HCC. Enzyme immunoassays were used to measure serum levels of autoantibodies. Serum levels of anti-Ku86 antibodies were significantly elevated in HCC patients compared to those in liver cirrhosis patients (0.41 +/- 0.28 vs. 0.18 +/- 0.08 Abs at 450 nm, P &lt; 0001). Setting the cut-off level to give 90% specificity, anti-Ku86 was positive in 60.7% of stage I solitary tumor &lt;2 cm in diameter, whereas the sensitivities of alpha-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist II (PIVKA-II) were 17.8% and 21.4%, respectively. The results of ROC analyses indicated the better performance of anti-Ku86 for early detection of HCC. Serum anti-Ku86 levels decreased after surgical resection of the tumors in the 12 HCC cases tested, Elevation of anti-Ku86 in solid tumors other than liver was minimal. Serum anti-Ku86 is a potential biomarker for early detection of HCV-related HCC. Further studies in a larger number of HCC patients with various etiologies are needed to further evaluate the diagnostic and pathophysiological roles of elevation of serum anti-Ku86 in early HCC. (C) 2012 Elsevier Inc. All rights reserved.

Hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, is one of the most common cancers worldwide and the third most common cause of cancer-related death. Imaging studies including ultrasound and computed tomography are recommended for early detection of HCC, but they are operator dependent, costly and involve radiation. Therefore, there is a need for simple and sensitive serum markers for the early detection of hepatocellular carcinoma (HCC). In our recent proteomic studies, a number of proteins overexpressed in HCC tissues were identified. We thought if the serum autoantibodies to these overexpressed proteins were detectable in HCC patients. Of these proteins, we focused on Ku86, a nuclear protein involved in multiple biological processes and aimed to assess the diagnostic value of serum anti-Ku86 in the early detection of HCC. Serum samples were obtained prior to treatment from 58 consecutive patients with early or relatively early hepatitis C virus (HCV)-related HCC and 137 patients with HCV-related liver cirrhosis without evidence of HCC. Enzyme immunoassays were used to measure serum levels of autoantibodies. Serum levels of anti-Ku86 antibodies were significantly elevated in HCC patients compared to those in liver cirrhosis patients (0.41 +/- 0.28 vs. 0.18 +/- 0.08 Abs at 450 nm, P &lt; 0001). Setting the cut-off level to give 90% specificity, anti-Ku86 was positive in 60.7% of stage I solitary tumor &lt;2 cm in diameter, whereas the sensitivities of alpha-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist II (PIVKA-II) were 17.8% and 21.4%, respectively. The results of ROC analyses indicated the better performance of anti-Ku86 for early detection of HCC. Serum anti-Ku86 levels decreased after surgical resection of the tumors in the 12 HCC cases tested, Elevation of anti-Ku86 in solid tumors other than liver was minimal. Serum anti-Ku86 is a potential biomarker for early detection of HCV-related HCC. Further studies in a larger number of HCC patients with various etiologies are needed to further evaluate the diagnostic and pathophysiological roles of elevation of serum anti-Ku86 in early HCC. (C) 2012 Elsevier Inc. All rights reserved.

Head and neck squamous cell carcinoma (HNSCC) is usually found at a late stage and distant metastasis occurs at high frequency; therefore, novel prognostic markers are needed. This study was aimed to identify novel tumor markers in HNSCC. We identified 65 proteins which were significantly increased or decreased in the tumors by 2D-DIGE using 12 HNSCC and adjacent non-cancer tissues. Western blotting and immunohistochemical analysis confirmed that the expression of plectin was significantly increased in most cancer tissues as compared with non-cancer tissues. Strikingly, the suppression of endogenous plectin using siRNA inhibited the proliferation, migration and invasion of HNSCC cells and down-regulated Erk 112 kinase. Furthermore, immunohistochemistry using paraffin-embedded tissues from 62 patients showed not only that the frequency of recurrence was correlated with the plectin expression but that the prognosis of patients with a high plectin was extremely poor. Moreover, the survival rate of patients with a high plectin was significantly lower than that of patients with low E-cadherin levels, which is known to correlate With the poor prognosis of HNSCC. Our findings suggest that plectin promotes the migration and invasion of HNSCC cells through activation of Erk 1/2 kinase and is a potential prognostic biomarker of HNSCC. (C) 2011 Elsevier B.V. All rights reserved.

Head and neck squamous cell carcinoma (HNSCC) is usually found at a late stage and distant metastasis occurs at high frequency; therefore, novel prognostic markers are needed. This study was aimed to identify novel tumor markers in HNSCC. We identified 65 proteins which were significantly increased or decreased in the tumors by 2D-DIGE using 12 HNSCC and adjacent non-cancer tissues. Western blotting and immunohistochemical analysis confirmed that the expression of plectin was significantly increased in most cancer tissues as compared with non-cancer tissues. Strikingly, the suppression of endogenous plectin using siRNA inhibited the proliferation, migration and invasion of HNSCC cells and down-regulated Erk 112 kinase. Furthermore, immunohistochemistry using paraffin-embedded tissues from 62 patients showed not only that the frequency of recurrence was correlated with the plectin expression but that the prognosis of patients with a high plectin was extremely poor. Moreover, the survival rate of patients with a high plectin was significantly lower than that of patients with low E-cadherin levels, which is known to correlate With the poor prognosis of HNSCC. Our findings suggest that plectin promotes the migration and invasion of HNSCC cells through activation of Erk 1/2 kinase and is a potential prognostic biomarker of HNSCC. (C) 2011 Elsevier B.V. All rights reserved.

Early diagnosis of biliary tract cancer (BTC) is important for curative surgical resection. Current tumor markers of BTC are unsatisfactory in terms of sensitivity and specificity. In a search for novel biomarkers for BTC, serum samples obtained from 62 patients with BTC were compared with those from patients with benign biliary diseases and from healthy controls, using the MALDI-TOF/TOF ClinProt system. Initial screening and further validation identified a peak at 4204 Da with significantly greater intensity in the BTC samples. The 4204 Da peak was partially purified and identified as a fragment of prothrombin by amino acid sequencing. The sensitivity of the 4204 Da peptide for detection of stage I BTC cancer was greater than those for CEA and CA19-9. Also, serum levels of the 4204 Da peptide were above the cut-off level in 15 (79%) of 19 cases in which the CEA and CA19-9 levels were both within their cut-off values. Receiver operating characteristic analysis showed that the combination of the 4204 Da peptide and CA19-9 was significantly more sensitive for detection of stage I BTC cancer compared to CEA and CA19-9. These results suggest that this protein fragment may be a promising biomarker for biliary tract cancer.

Background: We previously reported that periplakin (PPL) is downregulated in human esophageal cancer tissues compared to the adjacent non-cancer epithelium. Thus PPL could be a useful marker for detection of early esophageal cancer and evaluation of tumor progression, but largely remains unknown in this field. To investigate PPL involvement in carcinogenesis, tumor progression, cellular movement or attachment activity, siRNAs against PPL were transfected into pharyngeal squamous cancer cell lines and their effects on cellular behaviours were examined. Results: PPL knockdown appeared to decrease tumor cell growth together with G2/M phase accumulation in cells attached to a culture dish. However, the extent of cell growth suppression, evaluated by the number of cells attached to the culture dish, was too distinctive to be explained only by cell cycle delay. Importantly, PPL knockdown suppressed cellular movement and attachment to the culture dish accompanied by decreased pAktSer473 phosphorylation. Additionally, LY294002, a PI3K inhibitor that dephosphorylates pAktSer473, significantly suppressed D562 cell migration. Thus PPL potentially engages in cellular movement al least partly via the PI3K/Akt axis. Conclusions: PPL knockdown is related to reduced cellular movement and attachment activity in association with PI3K/Akt axis suppression, rather than malignant progression in pharyngeal cancer cells.

Human malignant pleural mesothelioma (HMPM) is highly resistant to conventional therapy, and therefore novel therapies are required. We previously reported that overexpression of the FUSE-binding protein-interacting repressor (FIR), a c-myc transcriptional repressor, induces apoptosis via c-Myc suppression, and is thus a suitable cancer therapy. In the current preclinical trial, a fusion gene deleted non-transmissible Sendai virus vector encoding FIR (SeV/Delta F/FIR) was prepared and its cytotoxic activity against an orthotopic xenograft model of HMPM, in combination with cisplatin, was assessed. SeV/Delta F/FIR and a fusion gene deleted non-transmissible Sendai virus vector encoding green fluorescent protein (SeV/Delta F/GFP) were prepared. The transduction efficiency of these agents in terms of dose-dependent cytotoxicity and/or apoptosis induction was then assessed in a few HMPM cells. Combination therapy with SeV/Delta F/FIR plus cisplatin was evaluated in vitro and in a mouse model. SeV/Delta F/FIR significantly reduced cell viability in three HMPM cell lines but was less effective in non-tumor immortalized mesothelial cells. SeV/Delta F/FIR cytotoxicity was partly due to apoptosis induction via c-Myc suppression. In addition, SeV/Delta F/FIR showed synergistic antitumor effects in combination with cisplatin, as was revealed by isobologram analysis in MSTO-211H. Moreover, combination therapy with SeV/Delta F/FIR plus cisplatin demonstrated significant tumor reduction and improvement in survival rate in an animal model. Combination therapy with SeV/Delta F/FIR plus cisplatin has therapeutic potential against HMPM. SeV/Delta F/FIR plus cisplatin will be an attractive modality against HMPM in the future. (Cancer Sci 2011; 102: 1366-1373)

Serum biomarkers currently available for gastric cancers are not sufficiently sensitive and specific. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) to generate comparative peptide profiles of serum samples obtained from gastric cancer patients (n = 81) and age- and sex-matched healthy controls (n = 66). Because of initial screening and further validation, we found that the intensities of a 2209 m/z MS peak were increased in the preoperative sera obtained from gastric cancer patients, and we identified this peak, a 2209 Da peptide, as a high molecular weight (HMW) kininogen fragment. Receiver operating characteristic analyses showed that the area under the curve (AUC) for the 2209 Da peptide (AUC = 0.715) was greater than those for conventional tumor markers (carcinoembryonic antigen AUC = 0.593, carbohydrate antigen 19-9 AUC = 0.527) used for the detection of stage I gastric cancers. Inverse correlations were observed between the levels of intact HMW kininogen and the 2209 Da peptide, suggesting that the upregulation of some protease activities is responsible for the overproduction of a kininogen fragment in gastric cancer patients. Serum levels of the 2209 Da peptide identified in this study have a greater diagnostic ability than those of conventional tumor markers used for the early detection of gastric cancer.

Serum biomarkers currently available for gastric cancers are not sufficiently sensitive and specific. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) to generate comparative peptide profiles of serum samples obtained from gastric cancer patients (n = 81) and age- and sex-matched healthy controls (n = 66). Because of initial screening and further validation, we found that the intensities of a 2209 m/z MS peak were increased in the preoperative sera obtained from gastric cancer patients, and we identified this peak, a 2209 Da peptide, as a high molecular weight (HMW) kininogen fragment. Receiver operating characteristic analyses showed that the area under the curve (AUC) for the 2209 Da peptide (AUC = 0.715) was greater than those for conventional tumor markers (carcinoembryonic antigen AUC = 0.593, carbohydrate antigen 19-9 AUC = 0.527) used for the detection of stage I gastric cancers. Inverse correlations were observed between the levels of intact HMW kininogen and the 2209 Da peptide, suggesting that the upregulation of some protease activities is responsible for the overproduction of a kininogen fragment in gastric cancer patients. Serum levels of the 2209 Da peptide identified in this study have a greater diagnostic ability than those of conventional tumor markers used for the early detection of gastric cancer.

Deregulation of protein synthesis plays a critical role in cell transformation. Several translation initiation factors (elFs) have been implicated in malignant transformation; thus, suppression of elFs could be a potential cancer therapy if cancer cells are selectively killed without damaging healthy cells. One of the potential molecular targets is a cancer-specific splicing variant. We have previously shown that one of the splicing variants of elF4H (isoform 1) was overexpressed in primary human colorectal cancer. Our study aimed to explore whether elF4H isoform 1 contributes to carcinogenesis and could be an efficient molecular target for human cancer therapy. We found that its overexpression in immortalized mouse fibroblasts, NIH3T3 cells, generated tumors in nude mice. Conversely, suppression of elF4H isoform 1 expression using specific siRNA inhibited the proliferation of colon cancer cells in vitro and subcutaneously implanted tumor in vivo. Strikingly, elF4H isoform 1 specific siRNA showed no effect on the growth of immortalized human fibroblasts. More interestingly, ectopic expression of elF4H isoform 1 greatly increased the cyclin D1 level. On the other hand, cyclin D1 decreased by shRNA-mediated suppression of elF4H isoform 1. Moreover, cotransfection of elF4H isoform 1 siRNA and cyclin D1 expression plasmid was able to reverse the growth suppression effect of elF4H isoform 1 knockdown. These results suggest that elF4H isoform 1 plays an important role in carcinogenesis through the activation of oncogenic signaling and could be a promising molecular target for cancer therapy.

Background/Aims: Peritoneal immune response as well as systemic response was objectively evaluated in laparoscopy-assisted distal gastrectomy (LADG) compared to open distal gastrectomy (ODG). Methodology: A total of 42 patients with gastric cancer were enrolled, with 23 undergoing LADG and 19 ODG. We evaluated the levels of IL-6 in peritoneal drain fluid, serum C-reactive protein (CRP), white blood cells (WBC), and the postoperative presence of systemic inflammatory response syndrome (SIRS). Results: The serum CRP level was significantly higher in the ODG group than in the LADG group (p=0.007) on postoperative day (POD) 1. WBC counts showed no statistically significant difference between the two groups (p=0.105). The rate of cases exhibiting SIRS was significantly higher in the ODG group than in the LADG group (p&lt;0.001). The IL-6 level of drain fluid was significantly higher in the ODG group than the LADG group (p&lt;0.01) on POD1. Although weak correlation between IL-6 on POD1 and blood loss (R=0.38, p=0.0154) was observed, no significant correlation between IL-6 and operation time was noted. Conclusions: LADG seems to be a lesser traumatic approach for the treatment of gastric cancer.

Deregulation of protein synthesis plays a critical role in cell transformation. Several translation initiation factors (elFs) have been implicated in malignant transformation; thus, suppression of elFs could be a potential cancer therapy if cancer cells are selectively killed without damaging healthy cells. One of the potential molecular targets is a cancer-specific splicing variant. We have previously shown that one of the splicing variants of elF4H (isoform 1) was overexpressed in primary human colorectal cancer. Our study aimed to explore whether elF4H isoform 1 contributes to carcinogenesis and could be an efficient molecular target for human cancer therapy. We found that its overexpression in immortalized mouse fibroblasts, NIH3T3 cells, generated tumors in nude mice. Conversely, suppression of elF4H isoform 1 expression using specific siRNA inhibited the proliferation of colon cancer cells in vitro and subcutaneously implanted tumor in vivo. Strikingly, elF4H isoform 1 specific siRNA showed no effect on the growth of immortalized human fibroblasts. More interestingly, ectopic expression of elF4H isoform 1 greatly increased the cyclin D1 level. On the other hand, cyclin D1 decreased by shRNA-mediated suppression of elF4H isoform 1. Moreover, cotransfection of elF4H isoform 1 siRNA and cyclin D1 expression plasmid was able to reverse the growth suppression effect of elF4H isoform 1 knockdown. These results suggest that elF4H isoform 1 plays an important role in carcinogenesis through the activation of oncogenic signaling and could be a promising molecular target for cancer therapy.

Background: The liver secretes very-low-density lipoproteins (VLDLs) and plays a key role in lipid metabolism. Plasma total triglyceride (TG) level variations have been studied in patients with hepatitis C virus (HCV)-related chronic hepatitis (CH-C). However, the results of these studies are variable. A homogenous assay protocol was recently proposed to directly measure the TG content in VLDL (VLDL-TG) and VLDL remnants. Methodology/Principal Findings: Using the assay protocol, we determined serum VLDL-TG levels in 69 fasting patients with biopsy-proven HCV-related chronic liver disease and 50 healthy subjects. Patients were classified into stages F0-F4 using the 5-point Desmet scale. Serum total TG levels in patients with non-cirrhotic (F1-F3) CH-C did not demonstrate significant differences compared with healthy subjects, but serum VLDL-TG levels did demonstrate significant differences. Mean serum VLDL-TG levels tended to decrease with disease progression from F1 to F4 (cirrhosis). Compared with healthy subjects, serum non-VLDL-TG levels significantly increased in patients with stages F2 and F3 CH-C; however, we observed no significant difference in patients with liver cirrhosis. Furthermore, the serum VLDL-TG/non-VLDL-TG ratio, when taken, demonstrated a significant decrease in patients with CH-C from the mildest stage F1 onward. Conclusions/Significance: The decrease in serum VLDL-TG levels was attenuated by increase in non-VLDL-TG levels in patients with non-cirrhotic CH-C, resulting in comparable total TG levels. Results of previous studies though variable, were confirmed to have a logical basis. The decrease in the serum VLDL-TG/non-VLDL-TG ratio as early as stage F1 demonstrated TG metabolic alterations in early stages of CH-C for the first time. The involvement of TG metabolism in CH-C pathogenesis has been established in experimental animals, while conventional TG measurements are generally considered as poor indicators of CH-C progression in clinical practice. The serum VLDL-TG/non-VLDL-TG ratio, which focuses on TG metabolic alterations, may be an early indicator of CH-C.

Background: The liver secretes very-low-density lipoproteins (VLDLs) and plays a key role in lipid metabolism. Plasma total triglyceride (TG) level variations have been studied in patients with hepatitis C virus (HCV)-related chronic hepatitis (CH-C). However, the results of these studies are variable. A homogenous assay protocol was recently proposed to directly measure the TG content in VLDL (VLDL-TG) and VLDL remnants. Methodology/Principal Findings: Using the assay protocol, we determined serum VLDL-TG levels in 69 fasting patients with biopsy-proven HCV-related chronic liver disease and 50 healthy subjects. Patients were classified into stages F0-F4 using the 5-point Desmet scale. Serum total TG levels in patients with non-cirrhotic (F1-F3) CH-C did not demonstrate significant differences compared with healthy subjects, but serum VLDL-TG levels did demonstrate significant differences. Mean serum VLDL-TG levels tended to decrease with disease progression from F1 to F4 (cirrhosis). Compared with healthy subjects, serum non-VLDL-TG levels significantly increased in patients with stages F2 and F3 CH-C; however, we observed no significant difference in patients with liver cirrhosis. Furthermore, the serum VLDL-TG/non-VLDL-TG ratio, when taken, demonstrated a significant decrease in patients with CH-C from the mildest stage F1 onward. Conclusions/Significance: The decrease in serum VLDL-TG levels was attenuated by increase in non-VLDL-TG levels in patients with non-cirrhotic CH-C, resulting in comparable total TG levels. Results of previous studies though variable, were confirmed to have a logical basis. The decrease in the serum VLDL-TG/non-VLDL-TG ratio as early as stage F1 demonstrated TG metabolic alterations in early stages of CH-C for the first time. The involvement of TG metabolism in CH-C pathogenesis has been established in experimental animals, while conventional TG measurements are generally considered as poor indicators of CH-C progression in clinical practice. The serum VLDL-TG/non-VLDL-TG ratio, which focuses on TG metabolic alterations, may be an early indicator of CH-C.

We searched for novel tumor markers of pancreatic cancer by three-step serum proteome analysis. Twelve serum abundant proteins were depleted using immunoaffinity columns followed by fractionation by reverse-phase high-performance liquid chromatography. Proteins in each fraction were separated by two-dimensional gel electrophoresis. Then the gel was stained by Coomassie Brilliant Blue. Protein spots in which the expression levels were significantly different between cancer and normal control were identified by LC-MS/MS. One hundred and two spots were upregulated, and 84 spots were downregulated in serum samples obtained from patients with pancreatic cancers, and 58 proteins were identified by mass spectrometry. These candidate proteins were validated using western blot analysis and enzyme-linked immunosorbent assay (ELISA). As a result of these validation process, we could confirm that the serum levels of apolipoprotein A-IV, vitamin D-binding protein, plasma retinol-binding protein 4, and tetranectin were significantly decreased in patients with pancreatic cancer.

A 45-year-old man suffering intermittent melena for several years was referred to our hospital for detailed examinations. A capsule endoscopy and a double-balloon enteroscopy revealed a tumor with a depression on the smooth surface, at approximately 150 cm proximal to the ileocecal valve. We made a diagnosis of ileal carcinoma by biopsy, and a partial ileal resection with lymph node dissection was performed. The resected specimen showed a yellowish tumor measuring 1.2 × 0.8 cm in size with a depression on the smooth surface. Based on the histological examination and immunohistochemical staining we diagnosed ileal carcinoid tumor. The patient is doing well 2 years after the surgery without any signs of recurrence. © 2011 The Japanese Society of Gastroenterological Surgery.

Angiogenesis plays an essential role in the growth and metastasis of esophageal carcinoma. Vascular endothelial growth factor, thymidine phosphorylase, fibroblast growth factor, midkine, and hepatocyte growth factor have been reported to be vital molecules for tumor angiogenesis. Polymorphisms in gene encoding angiogenic factors or their receptors may alter protein expression and/or activity. Increased angiogenic-factor expression and increased serum levels of these molecules were found to be associated with poor treatment response and poor prognosis. We reviewed the clinicopathological significance of angiogenesis-related molecules in patients with esophageal carcinoma. Antiangiogenic molecular-treatment strategies are also discussed. (Ann Thorac Cardiovasc Surg 2010; 16: 389-393)

Background: Serum levels of novel hydroxy polyunsaturated ultra long-chain fatty acids (hPULCFAs) have been previously shown to be reduced in pre-treatment CRC patients compared to disease-free subjects, independent of disease stage. However, whether reduced levels of hPULCFAs result from the presence of cancer is currently unknown, as is the distribution of hPULCFAs in the general population. The following studies were carried out to assess whether conventional therapy would result in restoration of systemic hPULCFAs in CRC patients, and to investigate the relationship between hPULCFA levels and age. Methods: Tandem mass spectrometry was used to determine serum levels of the 28 carbon-containing hPULCFA C28H46O4 (CRC-446) in the following cohorts: two independent Japanese CRC populations following surgical tumor removal (n = 86), a North American Caucasian CRC cohort (n = 150) following post-surgery combination chemo/radiation therapy, 990 randomly selected anonymized serum samples from subjects ranging between 11 and 99 years of age, as well as longitudinally collected serum samples from healthy normals (n = 8, up to 90 weeks) and stage IV CRC subjects on combination therapy (n = 12, up to 63 weeks). Results: Serum CRC-446 levels in CRC subjects were significantly lower than controls (mean of 0.297 +/- 0.07 ug/ml in controls versus 0.092 +/- 0.03 in CRCs, p &lt; 0.001), and were unaffected by surgical tumor removal or by chemo/radiation treatment (p &gt; 0.05 between pre vs post surgery). CRC-446 levels showed a strong inverse association with age (p &lt; E-11) across the randomly-selected cohort of 990 subjects, with no correlation observed in the CRC-positive subjects. Longitudinal intra-subject results, however, showed relatively stable CRC-446 levels over the short term of up to 90 weeks in both disease-free subjects and late-stage CRC patients. Conclusions: Our findings show that CRC-446 levels are not affected by conventional CRC treatment and inversely correlate with age, which suggest that reduced serum CRC-446 levels likely exist prior to the development of CRC. Extrapolation of the results to a simple screening scenario showed that, compared to fecal blood testing, pre-colonoscopy screening using serum CRC-446 levels would require 80% fewer colonoscopies, would identify risk in subjects under the age of 50, and would result in increased numbers of early cases detected. The precise role these serum metabolites play in the aetiology of cancer development remains to be determined.

Background: Serum levels of novel hydroxy polyunsaturated ultra long-chain fatty acids (hPULCFAs) have been previously shown to be reduced in pre-treatment CRC patients compared to disease-free subjects, independent of disease stage. However, whether reduced levels of hPULCFAs result from the presence of cancer is currently unknown, as is the distribution of hPULCFAs in the general population. The following studies were carried out to assess whether conventional therapy would result in restoration of systemic hPULCFAs in CRC patients, and to investigate the relationship between hPULCFA levels and age. Methods: Tandem mass spectrometry was used to determine serum levels of the 28 carbon-containing hPULCFA C28H46O4 (CRC-446) in the following cohorts: two independent Japanese CRC populations following surgical tumor removal (n = 86), a North American Caucasian CRC cohort (n = 150) following post-surgery combination chemo/radiation therapy, 990 randomly selected anonymized serum samples from subjects ranging between 11 and 99 years of age, as well as longitudinally collected serum samples from healthy normals (n = 8, up to 90 weeks) and stage IV CRC subjects on combination therapy (n = 12, up to 63 weeks). Results: Serum CRC-446 levels in CRC subjects were significantly lower than controls (mean of 0.297 +/- 0.07 ug/ml in controls versus 0.092 +/- 0.03 in CRCs, p &lt; 0.001), and were unaffected by surgical tumor removal or by chemo/radiation treatment (p &gt; 0.05 between pre vs post surgery). CRC-446 levels showed a strong inverse association with age (p &lt; E-11) across the randomly-selected cohort of 990 subjects, with no correlation observed in the CRC-positive subjects. Longitudinal intra-subject results, however, showed relatively stable CRC-446 levels over the short term of up to 90 weeks in both disease-free subjects and late-stage CRC patients. Conclusions: Our findings show that CRC-446 levels are not affected by conventional CRC treatment and inversely correlate with age, which suggest that reduced serum CRC-446 levels likely exist prior to the development of CRC. Extrapolation of the results to a simple screening scenario showed that, compared to fecal blood testing, pre-colonoscopy screening using serum CRC-446 levels would require 80% fewer colonoscopies, would identify risk in subjects under the age of 50, and would result in increased numbers of early cases detected. The precise role these serum metabolites play in the aetiology of cancer development remains to be determined.