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Stratification of patients for multidrug response is a promising strategy for cancer treatment. Genome-based prediction models have great potential for this purpose because the extent of drug sensitivity may be attributed to the heterogeneity of the underlying genetic characteristics of cancer. However, microarray data is difficult to analyze and is not reproducible. Several machine-learning algorithms have therefore been developed in a repeatable manner. Random forests algorithm, which uses an ensemble approach based on classification and regression trees, appears to be superior for predicting multidrug sensitivity. This is because ensemble methods are more effective when there are much more predictors than samples. Here, we review recent advances in the development of classification algorithms using microarray technology for prediction of anticancer sensitivity, discuss the availability of ensemble methods for prediction models, and present data regarding the identification of potential responders to FOLFOX therapy using random forests algorithm.

BACKGROUND: NEDD8 activating enzyme (NAE) regulates NEDD8 conjugation in neddylation pathway, a part of the ubiquitin-proteasome system, and controls cell cycle progression, DNA replication, and signal transduction via activation of cullin-RING ligases (CRLs) and subsequent degradation of their substrates. Through one such pathway, the constitutively active nuclear factor κB (NF-κB) triggers the progression of some types of hematological cancer. Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) remains the least curable form of DLBCL despite recent therapeutic advances. Because either phosphatidylinositol-3 kinase (PI3K)/AKT signaling or NF-κB signaling constitutively activated by B cell receptor signaling and MYD88 are reported to promote cancer cell growth and survival in ABC-DLBCL, the key kinases in these pathways [PI3Kδ, Bruton's tyrosine kinase (BTK), and Interleukin-1 receptor-associated kinases (IRAK)] are considered to be potential targets for ABC-DLBCL therapy. We previously reported an NAE inhibitor, TAS4464, that could inactivate NF-κB by accumulating phosphorylated IκBα (p-IκBα). Here, we report on the possible application of TAS4464 to treat ABC-DLBCL that is insensitive to kinase inhibitors. MATERIALS AND METHODS: Human ABC-DLBCL cell lines, OCI-LY10, SU-DHL-2, and TMD8 were used to evaluate the biological activity of TAS4464. Intracellular ATP levels were measured to assess in vitro cell growth. The effects of TAS4464 on NEDD8 conjugation and levels of CRLs substrates were evaluated by Western blot analysis. The levels of NF-κB-targeted gene transcripts were assessed by qRT-PCR. The antitumor activities of TAS4464 were evaluated in both subcutaneous and systemic xenograft model of TMD8. RESULTS: TAS4464 led to growth arrest and death in ABC-DLBCL cell lines in the nanomolar range through inhibition of cullin neddylation. Similar effects were also observed in a cell line insensitive to the kinase inhibitors that inhibit divergent activator of NF-κB. qRT-PCR analysis revealed TAS4464 inhibited the NF-κB pathway in all ABC-DLBCL cell lines tested through accumulation of p-IκBα, which directly inhibit NF-κB activity. Intravenous administration of TAS4464 showed strong antitumor activity in a TMD8 xenograft model, with inhibited cullin neddylation and activation of caspases 3 and 8 in the tumors. Futhermore, in a TMD8 systemic model, intravenous administration of TAS4464 prolonged mice survival more than two-fold. CONCLUSION: TAS4464 leads to cell growth through inhibition of NF-κB pathway regardless of the activation signaling pathway of NF-κB pathway. Though NF-kB activation signaling is complicated, the downstream inhibition of NF-κB pathway by TAS4464 indicated the potential for treatment of ABC-DLBCL in which NF-kB was dominant for their survival. In addition, TAS4464 demonstrates marked antitumor activity and survival benefit in a human ABC-DLBCL model. Therefore, TAS4464 may be a valuable addition to current options for chemotherapy in ABC-DLBCL. Citation Format: Hiromi Muraoka, Chihoko Yoshimura, Shingo Tsuji, Akihiro Hashimoto, Takashi Mizutani, Shuichi Ohkubo, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS4464, a novel and highly potent inhibitor of NEDD8 activating enzyme, overcomes insensitivity to BTK, PI3Kδ, and IRAK4 inhibitors in activated B-cell like diffuse large B-cell lymphoma via inactivation of the NF-κB pathway. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C177.

BACKGROUND: NEDD8 activating enzyme (NAE) is an essential E1 enzyme in the NEDD8 conjugation (neddylation) pathway, which controls cancer cell growth and survival through activation of cullin-RING ligase complexes (CRLs). Therefore, NAE is considered to be a promising target for cancer therapy. Here we describe the profile of a novel, highly potent, and selective NAE inhibitor, TAS4464, which inactivates CRLs in tumors and has strong antitumor efficacy in a preclinical model. MATERIALS AND METHODS: The selectivity of TAS4464 to E1 enzymes were assessed by various ubiquitin-like protein conjugation assays. The effects of TAS4464 on NEDD8 conjugation and the amounts of CRL substrates were evaluated by Western blot analysis. Cell viability was measured with a Cell Titer-Glo assay. The antitumor activity of TAS4464 was evaluated in an acute lymphoblastic leukemia (CCRF-CEM) xenograft model. RESULTS: We demonstrated that TAS4464 was a mechanism-based NAE inhibitor. TAS4464 formed an NEDD8-TAS4464 adduct that could achieve nanomolar inhibition of NAE. TAS4464 selectively inhibited NAE enzyme activity relative to the other E1s, ubiquitin activating enzyme (UAE) and SUMO activating enzyme (SAE). TAS4464 treatment inhibited cullin neddylation and induced accumulation of key CRL substrate proteins such as CDT1, p27, and phosphorylated IκB in human cancer cell lines. Enzyme inhibitory and cellular NAE inhibitory activities of TAS4464 were higher than those of a known NAE inhibitor, MLN4924. Cytotoxicity profiling of TAS4464 revealed widespread antiproliferative activity against a panel of cancer cell lines. Almost all cell lines derived from hematological malignancies were highly sensitive to TAS4464. Whereas carbonic anhydrase inhibition by MLN4924 resulted in accumulation in red blood cells, the blood-to-plasma ratio of TAS4464 was close to equivalent because of higher selectivity. In addition, TAS4464 did not inhibit major cytochrome p450 enzymes and showed good metabolic stability in isolated hepatocytes in vitro. The pharmacodynamic action of TAS4464 in tumors assessed by measuring the amount of cullin-NEDD8 conjugation in CCRF-CEM xenografts revealed strong cullin neddylation inhibitory activity of TAS4464; the inhibitory activity was sustained longer than that of MLN4924. In the CCRF-CEM xenograft model, weekly administration of TAS4464 at a dose of 100 mg/kg was more efficacious than twice-weekly administration of MLN4924 at a dose of 120 mg/kg: TAS4464 led to complete tumor regression without marked weight loss. CONCLUSION: TAS4464 is the most potent and selective NAE inhibitor reported to date, with good pharmaceutical properties and superior efficacy. Hematological cancer cell lines are highly sensitive to TAS4464. Based on its promising preclinical activity, a phase 1 clinical trial of TAS4464 is being planned. Citation Format: Chihoko Yoshimura, Hiromi Muraoka, Hiroaki Ochiiwa, Shingo Tsuji, Akihiro Hashimoto, Toru Takenaka, Hiromi Kazuno, Keiji Ishida, Takashi Mizutani, Shuichi Ohkubo, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS4464, a novel, highly potent, and selective inhibitor of NEDD8 activating enzyme demonstrates sustained target inhibition and antitumor activity in a preclinical model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C176.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by numerous genetic mutations and chromosomal abnormalities. Current chemotherapies have shown limited efficacy in AML patients, most of whom develop chemoresistance and relapse repeatedly. Because the expression of anti-apoptotic proteins reportedly correlates with poor prognosis in AML, the modulation of apoptosis-related proteins emerges as a promising treatment strategy. Apoptosis occurs mainly via two signaling pathways. In the mitochondria-mediated intrinsic apoptotic pathway, the release of cytochrome c triggers caspase-9 activation, which subsequently activates effector caspases (e.g., caspase-3). In the death receptor-mediated extrinsic apoptotic pathway, the binding of death receptor ligands to their receptors activates caspase-8 and, in turn, effector caspases, as in the intrinsic pathway. NEDD8 activating enzyme (NAE) inhibitors, which act on the NEDD8 conjugation pathway, induce quantitative changes in the substrates of E3 cullin-RING ligases (CRLs) and cause apoptosis in AML. Here, we describe the mechanism by which our highly potent and selective NAE inhibitor TAS4464 promotes apoptosis in AML. MATERIALS AND METHODS: Intracellular ATP levels were measured to assess in vitro cell growth. The effects of TAS4464 on NEDD8 conjugation, the protein levels of CRL substrates, and the activation of various caspases were evaluated by means of Western blotting. The transcription levels of the Noxa and c-FLIP genes were assessed by using qRT-PCR. The antitumor activity and in vivo pharmacodynamic activity of TAS4464 were evaluated in a THP-1 xenograft model. RESULTS: TAS4464 led to cell growth arrest and cell death in AML cell lines with various genetic backgrounds, including the MLL-AF9 fusion gene and FLT3-ITD mutation. TAS4464 activated both caspase-9 (intrinsic apoptotic pathway) and caspase-8 (extrinsic apoptotic pathway); combined treatment with inhibitors of these caspases markedly diminished the TAS4464-associated induction of apoptosis. By analyzing apoptosis-related factors in each apoptotic pathway, we found that TAS4464 increased the pro-apoptotic factor Noxa protein (intrinsic apoptotic pathway) and decreased the anti-apoptotic factor c-FLIP protein (extrinsic apoptotic pathway) with changes evident at the mRNA transcriptional level. In addition, treatment with TAS4464 led to accumulation of the CRL substrate c-Myc, and c-Myc knockdown by siRNA ablated the TAS4464-induced decrease in cFLIP protein. Intravenous administration of TAS4464 on day 1, 3 and 5 for 3 weeks resulted in tumor growth inhibition (TGI) of 100% including complete response in human THP-1 xenograft model, compared with twice-weekly administration of cytarabine that resulted in a TGI of only 6%. Pharmacodynamics analysis revealed that TAS4464 inhibited cullin neddylation and induced activation of caspases in THP-1 xenografted tumor. CONCLUSION: TAS4464 exerts a strong apoptosis-inducing effect in AML cell lines via both intrinsic and extrinsic apoptotic pathway by modulating apoptosis-related proteins. In addition, TAS4464 demonstrates marked antitumor activity in the THP-1 xenograft model. Given its potent preclinical activities, TAS4464 is a promising agent for treating AML. Citation Format: Hiroaki Ochiiwa, Chihoko Yoshimura, Hiromi Muraoka, Keiji Ishida, Tomonori Haruma, Shingo Tsuji, Akihiro Hashimoto, Takashi Mizutani, Shuichi Ohkubo, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS4464, a novel NEDD8 activating enzyme inhibitor, potently induces cell death via both intrinsic and extrinsic apoptotic pathways in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C178.

In this study, an electronic endoscope was applied for observation of gastric submucosal vessels, with infrared illumination, in anesthetized dogs. An in-vivo spectrophotometry showed that infrared light at 620-820 nm penetrates the abdominal and gastric wall. During the endoscopy performed in dogs, the infrared radiation penetrated the abdominal wall and gastric wall from the outside, and was detected by the endoscope's charge-coupled device. A television monitor displayed a network of gastric vasculature, which was identified as veins in the gastric wall by injecting saline or indocyanine green into the vein. Using this system, it was possible to measure venous diameters of more than 0.2 mm by comparison with a reference wire. The diameter obtained by the image analysis correlated lineally to that of a vascular template prepared from the same stomach. Thus, it may become possible to assess gastric submucosal hemodynamics using infrared endoscopy, a new application in electronic endoscopy.

The purpose of this study was to clarify the role of endogenous nitric oxide in ethanol-induced perturbation of microcirculation and hepatic injury in perfused rat liver. Infusion of ethanol into the portal vein at 25 and 100 mmol/L increased portal pressure, which is an indicator of hepatic vasoconstriction, in a concentration-dependent fashion. Portal pressure started to rise immediately after ethanol load, then decreased gradually and remained at higher than basal levels throughout the period of ethanol infusion. Release of lactate dehydrogenase into the effluent perfusate began to increase after 30 min of ethanol infusion and continued to increase during the 60-min period of ethanol infusion. The lactate dehydrogenase level in the effluent perfusate at 60 min was dependent on the ethanol concentration (0 mmol/L, 8 +/- 3 IU/L; 25 mmol/L, 16 +/- 2 IU/L; 100 mmol/L, 52 +/- 6 IU/L). Simultaneous infusion of NG-monomethyl-L-arginine, a nitric oxide synthesis inhibitor, enhanced significantly the ethanol-induced increase in portal pressure by 100% to 400% and increased lactate dehydrogenase release by 40% to 80%. The effect of NG-monomethyl-L-arginine on the ethanol-induced increase in portal pressure was completely reversed by the co-infusion of an excess dose of L-arginine. Change in portal pressure averaged over 60 min of ethanol infusion correlated with levels of lactate dehydrogenase release 60 min after the initiation of ethanol infusion (r = 0.77, p < 0.01). In conclusion, inhibition of the action of endogenous nitric oxide was associated with an increase in hepatic vasoconstriction and hepatocellular damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Background/Aims: Gastric microcirculatory disturbances are involved in the pathogenesis of stress ulcers; however, vasomodulators regulating this process are not fully understood. This study was conducted to investigate the role of endothelin 1 (ET-1) in hemorrhagic shock-induced gastric mucosal damage in rats. Methods: ET-1 contents in plasma and gastric mucosa were measured and gastric mucosal damage was evaluated during a control period, 60 minutes of ischemia, 15 minutes of reperfusion, and 30 minutes of postreperfusion. Next, effects of BQ-123, an endothelin a receptor antagonist, on the gastric mucosal damage and hemodynamics were studied. Results: Both plasma and mucosal ET-1 significantly increased after ischemia and reperfusion compared with the control values, but only mucosal ET-1 continued to increase after reperfusion, leading to the development of gastric mucosal damage. BQ-123, administered just before reperfusion, reduced mucosal damage in the postreperfusion period dose-dependently and improved mean gastric mucosal blood flow and mucosal hemoglobin oxygen saturation during the 30-minute postreperfusion period. Conclusions: These results suggest that endogenous ET-1 plays an important role in the pathogenesis of hemorrhagic shock-induced gastric mucosal damage through impairment of mucosal microcirculation. Further, endothelin a antagonists may have therapeutic benefits for shock-induced gastric mucosal damage.

Hepatic microcirculatory perturbation is observed after ischemia/reperfusion. Endothelin-1, a potent vasoconstrictive peptide, is known to modulate local circulation. This study was designed to examine whether endothelin-1 participates in the mechanism of microcirculatory disturbance and damage of the liver after ischemia/reperfusion. Ischemia in the median and left lateral lobes of the liver was induced for 60 min; it was followed by reperfusion for 24 hr. In some rats, endothelin-1 antiserum or control serum without endothelin-1–blocking activity was administered intravenously just before reperfusion. Rats were divided into three groups: an ischemia/reperfusion group that was injected with control serum, an endothelin-1 antiserum-treated group and a sham-operated group. Endothelin-1 concentrations in blood collected from the suprahepatic vena cava were measured before and after ischemia/reperfusion by use of a sandwich enzyme immunoassay. Index of blood volume in regional hepatic tissue and index of blood oxygenation in regional hepatic tissue were assessed with an organ reflectance spectrophotometry system before and at 5 min and 1, 2, and 24 hr after reperfusion. The endothelin-1 concentration in the ischemia/reperfusion group started to rise immediately at onset of reperfusion from basal values around 1 pg/ml and reached a value of 5 to 6 pg/ml 5 min after reperfusion; it was maintained at significantly high levels during the reperfusion period compared with the sham-operated group. Hepatic microcirculatory disturbance indicated by lowered index of blood volume in regional hepatic tissue and index of blood oxygenation in regional hepatic tissue levels was observed in the early phase of reperfusion in the ischemia/reperfusion group. Endothelin-1 antiserum injection diminished significantly the microcirculatory disturbance in the liver after ischemia/reperfusion. The serum ALT levels in the endothelin-1 antiserum–treated group 2 hr after reperfusion were decreased by 40 to 50 compared with the nontreated group. Moreover, liver damage assessed histologically was reduced dramatically in the endothelin-1 antiserum–treated group. In conclusion, endothelin-1 appears to be involved in the microcirculatory disturbance and histological damage in the liver after ischemia/reperfusion. Blockade of endothelin-1 action may prevent liver injury after ischemia/reperfusion. (Hepatology 1994;19:675–681).

Background: Helicobacter pylori produces ammonia in the stomach from urea. The present study was undertaken to clarify whether ammonia has an etiological role in H. pylori-associated gastric mucosal atrophy. Methods: Ammonia at 0.01% was administered as drinking water for 8 weeks and mucosal cell migration rate and cell proliferation were investigated in rat stomach. Results: Long-term administration of 0.01% ammonia for 4–8 weeks decreased mucosal thickness in the antrum but not in the body. Acceleration of cell migration preceded the occurrence of mucosal atrophy. Labeling indices in both antral and body mucosa significantly increased in all ammonia-treated groups, compared with those of the control group. In the antrum, the proliferative zone was significantly enlarged as mucosal atrophy developed, whereas, in body mucosa, enlargement of the proliferative zone occurred despite the absence of mucosal atrophy. Conclusion: Ammonia at 0.01% accelerates epithelial migration, especially in the antrum, leading to mucosal atrophy. Acceleration of epithelial proliferation occurs during the development of mucosal atrophy.

BACKGROUND: Mutations in the H3F3A gene, which encodes histone H3.3, were recently reported in cases of pediatric glioblastoma. H3F3A K27M mutations occur in gliomas that arise at midline locations, including the pons, thalamus, and spine; moreover, this particular mutation is found mainly in tumors in children and adolescents. In this study, we aimed to determine the association between H3F3A mutations and adult thalamic glioma. METHODS: Genomic H3F3A was sequenced from 20 separate thalamic gliomas. Additionally, for 14 of the 20 gliomas, 639 genes—including cancer-related genes and chromatin-modifier genes—were sequenced, and the Infinium HumanMethylation450K BeadChip was used to examine DNA methylation across the genome. RESULTS: Of the 20 tumors, 18 were high-grade thalamic gliomas, and of these 18, 11 were from patients under 50 years of age (median age, 38 years; range, 17–46), and seven were from patients more than 50 years of age. Among the 18 high-grade thalamic gliomas, the H3F3A K27M mutation was present in 10 (91%) of the 11 tumors from patients under 50 years of age, and was absent in all seven tumors from patients over 50 years of age. The H3F3A K27M mutation was not detected in two diffuse astrocytomas. By additional sequencing, recurrent mutations were identified in TP53, ATRX, NF1 and EGFR. In addition, a KDM6A mutation was found in one case of diffuse astrocytoma and a CREBBP mutation was identified in one case of glioblastoma with the H3F3A K27M mutation. Gliomas with H3F3A K27M from pediatric or young-adult patients had similar, characteristic DNA methylation profiles. In contrast, thalamic gliomas with wild-type H3F3A had DNA methylation profiles similar to those of hemispheric glioblastomas. The patients with high-grade thalamic gliomas harboring the H3F3A K27M mutation that occur frequently in young adults had a poor prognosis similar to those with wild-type H3F3A tumors that occur mainly in older adults. CONCLUSIONS: We found that high-grade thalamic gliomas from young adults, like those from children and adolescents, frequently had H3F3A K27M. SECONDARY CATEGORY: Neuropathology & Tumor Biomarkers.

The mechanism for Helicobacter pylori -induced gastric mucosal injury remains obscure. H. pylori has high urease activity to produce ammonia from urea in the stomach. In this study, the effects of ammonia on (a) gastric mucosal integrity, (b) gastric mucosal hemodynamics, (c) mucosal cellular viability, (d) mitochondrial respiration, and (e) energy metabolism of gastric mucosa were investigated. Ammonia (pH 10.3) at concentrations of >125 mmol/L caused acute macroscopic gastric mucosal lesions in a dose-dependent manner, whereas glycine-NaOH buffer (pH 10.3) or ammonium chloride (pH 4.5) did not. The decrease in energy charge preceded the occurrence of gastric mucosal lesions, but ammonia caused no change in mucosal hemodynamics. Oxygen consumption of isolated cells and mitochondria of gastric mucosa was inhibited by ammonia dose-dependently. The present results indicate that ammonia impairs mitochondrial and cellular respiration and energy metabolism and that ammonia decreases mucosal cell viability, leading subsequently to mucosal damage.

Background— Heart failure is a typical age-associated disease. Although age-related changes of heart are likely to predispose aged people to heart failure, little is known about the molecular mechanism of cardiac aging. Methods and Results— We analyzed age-associated changes in murine heart and the manner in which suppression of the p110α isoform of phosphoinositide 3-kinase activity modified cardiac aging. Cardiac function declined in old mice associated with the expression of senescence markers. Accumulation of ubiquitinated protein and lipofuscin, as well as comprehensive gene expression profiling, indicated that dysregulation of protein quality control was a characteristic of cardiac aging. Inhibition of phosphoinositide 3-kinase preserved cardiac function and attenuated expression of the senescence markers associated with enhanced autophagy. Suppression of target of rapamycin, a downstream effector of phosphoinositide 3-kinase, also prevented lipofuscin accumulation in the heart. Conclusions— Suppression of phosphoinositide 3-kinase prevented many age-associated changes in the heart and preserved cardiac function of aged mice.

A 47-year-old man who was diagnosed as Crohn's disease at the age of 27 became aware of a mass on the forehead. CT and PET scan revealed many scattered tumors in his body. The CEA level was 4424 ng/ml. Primary lesion was not detected by the surveillance of whole gastro-intestinal tract. Liver tumor biopsy samples were histologicaly analyzed and were diagnosed as adenocarcinoma. Further immunohistochemical analysis revealed that the biopsy material had colonic character determined by positive CK20 and negative CK7 staining. Therefore, we performed 12 cycles of mFOLFOX6 chemotherapy. It was effective with the reduced size of tumors and the decreased level of CEA.

The mucosal blood volume in 20 to 24 different regions of stomach was estimated by reflectance spectrophotometry during endoscopy, and an image showing mucosal blood distribution was made by two-dimensional computer color graphics with the aid of a personal computer. In 55 normal controls, the estimated mucosal blood volume (EMBV) was greater in the corpus mucosa than in the antral mucosa, and less in the lesser curvature than in the greater curvature. The volume in the anterior and posterior walls was almost the same. In 15 patients with active gastric ulcers in the angular region, the EMBV was decreased in all regions in the stomach. In 37 patients with healing ulcers, the EMBV increased, resuming the same levels as in normal controls. However, the EMBV around the ulcer increased remarkably at this stage. In 35 patients with ulcers in the scarring stage, the distribution of the EMBV was similar to that in the normal controls. These hemodynamic changes were shown clearly in a color display with the aid of a personal computer. This method could offer new possibilities in endoscopic research.

Early hepatocellular carcinoma (eHCC) originates from the hepatocytes of chronic liver disease and develops into classical hepatocellular carcinoma (HCC). To identify sequential genetic changes in multistep hepatocarcinogenesis, we analyzed molecular karyotypes using oligonucleotide genotyping 50K arrays. First, 1q21.3-44 gain and loss of heterozygosity (LOH) on 1p36.21-36.32 and 17p13.1-13.3 were frequently observed in eHCC, but not in chronic liver diseases, suggesting that such chromosomal aberrations are early, possibly causative events in liver cancer. Next, we detected 25 chromosomal loci associated with liver cancer progression in five HCCs with nodule-in-nodule appearance, in which the inner nodule develops within eHCC lesion. Using these chromosomal regions as independent variables, decision tree analysis was applied on 14 early and 25 overt HCCs, and extracted combination of chromosomal gains on 5q11.1-35.3 and 8q11.1-24.3 and LOH on 4q11-34.3 and 8p11.21-23.3 as distinctive attributes, which can classify early and overt HCCs recursively. In these four altered regions identified as late events of hepatocarcinogenesis, two tumors in 32 overt HCCs analyzed in the present study and one in a set of independent samples of 36 overt HCCs in our previous study harbored a homozygous deletion near the CSMD1 locus on 8p23.2. CSMD1 messenger RNA expression was decreased in HCC without 8p23.2 deletion, possibly due to hypermethylation of the CpG islands in its promoter region. Conclusion: 1q gain and 1p and 17p LOH are early molecular events, whereas gains in 5q and 8q and LOH on 4q and 8p only occur in advanced HCC, and inactivation of the putative suppressor gene, CSMD1, may be the key event in progression of liver cancer. (HEPATOLOGY 2009.)

Abrogating tolerance against unidentified antigens is a critical step in the pathogenesis of ulcerative colitis (UC). T cell anergy, one of the main mechanisms of tolerance, has been shown to be induced by E3 ubiquitin ligases, such as gene related to anergy in lymphocytes (GRAIL), Itch, and c-Cbl in mice. However, it is not well known whether these E3 ligases play roles in human diseases. The pathophysiological role of the E3 ligases in patients with UC was investigated. At first, the expression of GRAIL, Itch, and c-Cbl in human anergic T cells was analyzed by quantitative RT-PCR and Western immunoblotting. Next, the mRNA expression of the E3 ligases was analyzed in peripheral CD4+T cells of 20 patients with UC and 10 healthy volunteers (HV). mRNA expression was analyzed in patients with active UC before and after treatment with prednisolone and leukocytapheresis. Anergic human CD4+T cells expressed significantly higher levels of GRAIL, Itch, and c-Cbl than nonanergic cells. GRAIL expression was significantly higher in patients with UC in remission than in patients with active disease and in HV ( P < 0.01). The level of GRAIL expression was also significantly increased in patients with active disease whose clinical activity index scores improved after treatment ( P < 0.05). There were no significant differences in Itch and c-Cbl expression among patients with active UC, patients with UC in remission, and HV. These data suggest that GRAIL plays an important role in maintaining remission in patients with UC.

Mesenchymal stem cells (MSCs) are attractive cell sources in regenerative medicine. We examined the effects of topical MSCs implantation on an experimental model of inflammatory bowel disease. Putative MSCs, isolated from bone marrow aspirates of male rats by dish adherence and expanded in vitro, were characterized by flow cytometry, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and differentiation assays. Experimental colitis was induced by intraluminal instillation of 2,4,6-trinitrobonzene sulfonic acid (TNBS) in the colons of male rats. The putative MSCs and unselected fresh bone marrow cells were injected into the colonic submucosa surrounding the area exposed to TNBS. The healing process of the injury was examined macroscopically and immunohistologically. Multipotent MSCs positive for CD29 and CD90, and negative for CD31 and CD34, were implanted into colon tissue surrounding the lesion; a majority of the engrafted cells were positive for vimentin. The implantation significantly accelerated healing of the damaged mucosa compared with vehicle-injected controls. The MSCs expressed vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-beta1 in vitro and after the implantation. In conclusion, we found that MSCs were successfully topically implanted in the colon and that they were associated with accelerated healing of TNBS-induced colitis. The beneficial effects of the MSCs might be mediated, at least in part, by their ability to differentiate into colonic interstitial cells and by their ability to provide VEGF and TGF-beta1 to the injured area.

Toll-like receptor 4 (TLR4), which requires a helper molecule, MD-2, is a main receptor for lipopolysaccharide (LPS) from gram-negative bacteria. The expression of TLR4 in H. pylori infection in human gastric mucosa, however, is unclear. The aim of this study was to determine the effect of H. pylori infection on the TLR4 and MD-2 expression in human gastric mucosa.Biopsy samples from the antrum and corpus were obtained from 45 patients (25 patients without H. pylori infection including 5 patients with successful eradication of H. pylori, and 20 patients with H. pylori infection). These samples were used for TLR4, MD-2 mRNA expression study and immunohistochemical study.In patients without H. pylori infection, the expressions of TLR4 and MD-2 were bigger in the corpus mucosa than in the antral mucosa. In patients with H. pylori infection, the expressions of TLR4 and MD-2 significantly increased in the antral and corpus mucosa. Immunohistochemical study revealed similar results as the TLR4 mRNA expression. After the eradication of H. pylori, the expressions of TLR4 and MD-2 were the same levels in both sites as those in patients without H. pylori infection.The results indicated that H. pylori infection significantly increased TLR4 and MD-2 expressions in the antral and corpus mucosa.

Mesenchymal stem cells (MSCs), a subpopulation of adult somatic stem cells, are an attractive stem cell source in regenerative medicine because of their multipotentiality. We examined the effects of MSC transplantation on gastric ulcer healing. Putative MSCs, isolated from bone marrow aspirates of male rats by dish adherence and expanded in culture, were characterized by flow cytometry and reverse transcription-polymerase chain reaction. Gastric ulcers were induced by serosal application of acetic acid on the anterior wall of the stomach in female rats. Either MSCs (labeled with PKH67; 1×107 cells) or vehicle was injected into the gastric wall surrounding the ulcer. The healing process of the ulcer and the influence of anti-vascular endothelial growth factor (VEGF) antibody were examined. CD29-positive, CD90-positive, CD34-negative, and CD45-negative MSCs expressed mRNAs for VEGF and hepatocyte growth factor (HGF). The MSCs were transplantable to the gastric tissue surrounding the ulcer, where a majority of the engrafted cells were positive for vimentin. The transplantation significantly accelerated gastric ulcer healing compared with controls. The engrafted MSCs also expressed VEGF and HGF. Administration of anti-VEGF neutralizing antibody dose dependently reduced the MSC-induced promotion of ulcer healing. In conclusion, MSC transplantation accelerated gastric ulcer healing, possibly through the induction of angiogenesis in the gastric mucosa via the secretion of VEGF. The beneficial effects of MSCs might be mediated not only by their differentiation into gastric interstitial cells, but also by their ability to supply angiogenic factors.

Patients with inflammatory bowel disease (IBD) share several immunologic similarities with rheumatoid arthritis (RA). Patients with RA have significantly increased levels of serum agalactosyl immunoglobulin G (IgG). Our aim was to investigate the clinical significance of analyzing the oligosaccharide structure of serum IgG in patients with IBD.Serum IgG oligosaccharide structures were analyzed using high-performance liquid chromatography in 60 patients with Crohn's disease (CD), 58 patients with ulcerative colitis (UC), 27 healthy volunteers (HV), and 15 disease controls (DC). The activity and mRNA level of beta-1,4-galactosyltransferase (Beta4GalT) in antibody-secreting cells were investigated in these subjects.The agalactosyl fraction of the fucosylated IgG oligosaccharides (G0F/G2F) in CD and UC was significantly greater than that in HV and DC (P0.001). The percentage of subjects with a high G0F/G2F in CD, UC, HV, and DC was 72%, 33%, 0%, and 0%, respectively. G0F/G2F, which is significantly correlated with disease severity in both CD and UC, had higher sensitivity to diagnose IBD compared with anti-Saccharomyces cerevisiae antibody. Moreover, G0F/G2F was significantly correlated with the prognosis of UC patients: patients with a high G0F/G2F did not maintain long-term remission. The activity and mRNA level of Beta4GalT were significantly elevated in UC but not in CD.G0F/G2F is a potentially effective diagnostic marker of disease activity in both CD and UC, and of the clinical course in UC. A pathophysiologic difference between CD and UC was also demonstrated.

The therapeutic potential of bone marrow (BM)-derived cells in ulcers is not known. This study aimed to clarify (1) cell types that are derived from the BM which infiltrate ulcers; (2) whether BM-derived cells or gastric myofibroblasts can be used for cell transplantation to treat ulcers; and (3) the phenotypes of such transplantable cells.(1) Wild-type mice were transplanted with BM cells of green fluorescent protein (GFP)-transgenic mice. Acetic acid-induced gastric ulcers were produced in mice after BM transplantation. (2) BM cells and gastric myofibroblasts were isolated from GFP-transgenic mice. Bone marrow cells attached to plastic dishes were selected for expansion. Gastric ulcers were induced, and BM-derived cells, myofibroblasts, or phosphate-buffered saline were injected around ulcers. The ulcer healing process was examined macroscopically and histologically. (3) Expression of growth factors and cytokines in transplantable cells was examined by reverse transcriptase-polymerase chain reaction.(1) GFP-positive cells with interstitial phenotypes were observed at the ulcerated area. (2) Ulcer healing was significantly promoted by the injection of BM-derived cells compared to controls on day 7, but not on day 3. The BM-derived cells were observed in the tissue surrounding the ulcer. However, myofibroblasts were not found. (3) The BM-derived cells expressed hepatocyte growth factor, transforming growth factor-beta(1), and other stromal factors before transplantation, and had mesenchymal phenotypes after transplantation.BM-derived cells are involved in the ulcer healing. BM-derived cells, but not myofibroblasts, are locally implantable to ulcers. Thus, BM-derived cells can be transplanted to accelerate ulcer healing.

Helicobacter pylori (H. pylori)-induced chronic atrophic gastritis is a high-risk factor for gastric cancer. Immune responses to H. pylori are involved in gastric mucosal inflammation, and might affect clinical outcome, including the development of gastric cancer. The present study examines the significance of gene polymorphisms of various cytokines in the development of gastric cancer following H. pylori infection.One hundred Japanese non-cardia gastric cancer patients and 93 dyspeptic patients as controls were enrolled in the study (age range 50-75 years). All patients were positive for H. pylori. Genomic DNA was extracted from peripheral whole blood leukocytes, and we comprehensively analyzed 207 single nucleotide polymorphisms (SNP) in 11 cytokine genes; interleukin (IL)-1alpha, IL-1beta, IL-1 receptor antagonist (RN), IL-4, IL-4R, IL-8, IL-10, IL-12, TNF-alpha, TNF-beta, and IFN-gamma, using either invader assay (163 SNP), direct sequencing (22 SNP), or PCR-restriction fragment length polymorphism (22 SNP).Among the 207 SNP examined, the IL-4 gene diplotypes (984 and 2983 AA/GA) had a significant negative association with gastric cancer development (odds ratio =0.3, 95% confidence interval =0.1-0.9). When we adopted the dyspeptic patients over 66 years of age as the controls, the IL-1RN gene diplotypes (-1102 and 6110 CG/GA) also had a significant negative association (odds ratio =0.2, 95% confidence interval =0.1-0.7).A comprehensive analysis of 207 SNP of 11 cytokine genes revealed that variations in IL-4 and IL-1RN genes are negatively associated with the risk of developing gastric cancer following H. pylori infection. Distinct host cytokine responses in the gastric mucosa might have a role in H. pylori-induced carcinogenesis.

Background and Aim: Cytotoxin-associated gene A (CagA) protein from H. pylori was reported to be injected into host gastric epithelial cells via a bacterial type IV secretion system, thereby modifying signal transduction. It is classified into two major subtypes, Western and East Asian. The present study aimed to compare the effects of East Asian-type and Western-type CagA on host cell growth. Methods: A tetracycline (tet)-off system and cagA genes from Western and East Asian-type H. pylori (NCTC 11637 and F32) were transfected into untransformed rat gastric mucosal (RGM1) cells to establish RGM1-CagA cell lines in which CagA expression could be controlled by tetracycline. These cell lines were used to investigate the effect of CagA protein expression on cell growth with BrdU and water-soluble tetrazolium reagent (WST-8) assays. CagA expression, phosphorylation and extracellular signal-regulated kinase (ERK) activation were examined with immunoprecipitation and Western blotting analysis. Results: 5-Bromo-2′deoxyuridine (BrdU) and WST-8 assays demonstrated significant increases in DNA replication and RGM1 cell growth after CagA protein expression. ERK phosphorylation was enhanced when CagA protein was expressed in RGM1-CagA cells. Moreover, the East Asian-type CagA showed a significantly greater effect on ERK activation and host cell growth than the Western type. PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor, suppressed ERK phosphorylation and the CagA protein-induced increase in RGM1-CagA cell growth. Conclusions: CagA protein expression induces an increase in RGM1-CagA cell proliferation via the mitogen-activated protein kinase (MAPK) signal pathway. The East Asian-type CagA showed a significantly greater effect on cell growth than the Western type, suggesting that the East Asian CagA-positive strain may have an important role in pathogenesis.

Background: NEDD8 activating enzyme E1 (NAE) regulates NEDD8 conjugation in the ubiquitin-proteasome system. NAE activates cullin-RING ligases (CRL) and controls the degradation of their substrates, which play key roles in cell cycle progression, DNA replication and signal transduction via various pathways. Through one such pathway, the constitutively active nuclear factor κB (NF-κB) triggers the progression of multiple myeloma (MM). MM is highly treatable but rarely curable, and patients with refractory MM would benefit from additional therapeutic options. NF-κB is activated through two signaling cascades, canonical and non-canonical pathways, which are regulated by the CRL substrates, phosphorylated IκBα (p-IκBα) and phosphorylated p100 (p-p100), respectively. Here, we report on the possible use of an NAE inhibitor that we discovered, TAS4464, to treat MM. Materials and methods: TAS4464 was designed and synthesized at Taiho Pharmaceutical Co., Ltd. Human MM cell lines, KMS-11, KMS-12-BM, KMS-26, KMS-34, and MM.1S were used to evaluate the biological activities of TAS4464. Intracellular ATP levels were measured to assess in vitro cell growth. Effects on the cell cycle were analyzed by using fluorescence-activated cell sorting. The effects of TAS4464 on NEDD8 conjugation and the levels of CRL substrates were evaluated by means of Western blotting. The transcription levels of NF-κB-targeted genes were assessed by using qRT-PCR. Activated p65 and RelB (subunits of NF-κB) were quantified by using commercial DNA-binding ELISA kits. The antitumor activity of TAS4464 was evaluated in an MM.1S xenograft model. Results: TAS4464 inhibited cullin neddylation, leading to cell-growth arrest and apoptosis in MM cell lines in the nanomolar range. These effects were not attenuated after stimulation with interleukin 6 or insulin-like growth factor 1 or in co-culture with stroma cells. TAS4464 led to the accumulation of p-IκBα and p-p100 with subsequent suppression both p65 and RelB activity, whereas bortezomib suppressed only RelB activity. Once-weekly intravenous administration of TAS4464 showed much stronger antitumor activity than that of bortezomib in an MM.1S xenograft model, with inhibited cullin neddylation and induced the cleaved forms of caspases 3 and 8 in the tumors.Conclusion: TAS4464 demonstrated marked antitumor activity in a human MM xenograft model. In addition, TAS4464 inhibited canonical as well as non-canonical pathways of NF-κB, whereas bortezomib inhibited only non-canonical pathway of NF-κB. Therefore, TAS4464 may be a valuable addition to current options for the chemotherapy of MM. Citation Format: Hiromi Muraoka, Chihoko Yoshimura, Shingo Tsuji, Takamasa Suzuki, Akihiro Hashimoto, Takashi Mizutani, Shuichi Ohkubo, Kenichi Matsuo, Yoshikazu Iwasawa, Teruhiro Utsugi. TAS4464, a novel highly potent NEDD8 activating enzyme inhibitor, demonstrates antitumor activity in multiple myeloma through the inactivation of NF-κB pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1730. doi:10.1158/1538-7445.AM2015-1730

e14574 Background: For metastatic colorectal cancer (mCRC) treatment, anti-epidermal growth factor receptor (EGFR) treatment is one of the major options. However, there are few reliable biomarkers ...

High-throughput biological technologies enabled to identify cancer related key molecules and subclasses. However, single experiment could only elucidate one feature of the tumor pathway. In order to identify the clusters and pathways significant for cancer progression, we performed integrated analysis using gene expression and DNA methylation profiles of colorectal cancer samples. Extracting core CpG sites from CpG island with our newly developed method, iClusterPlus, one of integrated analyses methodologies, was applied to expression array and methylation profiling data and 116 colorectal cancer samples were divided into various cluster size (2≤k≤11). We examined clusters (k=3 and k=6), and found that each cluster (k=3) clearly corresponds to three groups consisting of the combination of two clusters in cluster (k=6) with different prognosis. Expression profiles of two clusters in each group were significantly different, which suggests that aberrant DNA methylation is accumulated during the early stage of cancer, while alteration of gene expression profile is the late event of cancer progression. Furthermore, network-based analysis using expression profiles demonstrated that modules including hnRNP family was down-regulated in one of the cluster (k=6) which frequently harbored liver metastasis and poor prognosis. Integrated clustering analysis applied to multidimensional methods is available to identify disease characteristics such as survival rate and distant metastasis, which might be valuable for improving cancer therapy. Citation Format: Shingo Tsuji, Yutaka Midorikawa, Motoaki Seki, Tadatoshi Takayama, Yasuyuki Sugiyama, Hiroyuki Aburatani. Integrative data analysis of colorectal cancer reveals the frequently metastatic sample cluster. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A53.

Geranylgeranylacetone (GGA) effectively protects the gastric mucosa against noxious agents. The precise mechanisms underlying the gastroprotective actions of GGA are not known. To elucidate the precise mechanism of GGA, the effect of GGA treatment on COX-2 expression in rat gastric epithelial (RGM1) cells was investigated. We used a prostaglandin E2 (PGE2) enzyme-linked immunoassay kit and Western blot analysis to measure PGE2 production and COX-2 induction by GGA treatment in serum-starved RGM1 cells. Gel-shift assay, Western blot analysis, and a reporter assay were performed to determine which COX-2 promoter was involved in GGA-induced COX-2 expression. GGA treatment dose dependently increased COX-2 expression and PGE2 production. The nuclear factor (NF)-kappaB sites of the COX-2 gene promoter were critical for GGA-mediated COX-2 expression. GGA induces COX-2 expression and increases PGE2 production in serum-starved RGM1 cells via activation of the NF-kappaB sites of COX-2 gene promoters.

OBJECTIVE: The aim of this study was to evaluate whether oral glucose tolerance test (OGTT) was useful in evaluating the prognosis of patients with liver cirrhosis. METHODS: Fifty-six patients with liver cirrhosis were enrolled in a prospective cohort study. In all cases, glucose tolerance was diagnosed by a 75-g OGTT according to World Health Organization (WHO) criteria. The relationship of clinical variables to the cirrhosis-related prognosis was investigated using univariate and multivariate regression models. RESULTS: Diabetes mellitus (DM) was diagnosed in 21 subjects (38%), impaired glucose tolerance (IGT) in 13 subjects (23%), and normal glucose tolerance (NGT) in 22 subjects (39%) using OGTT. The cumulative survival rates of patients with liver cirrhosis and NGT were 94.7% at 5 yr; liver cirrhosis and IGT, 68.8% at 5 yr; liver cirrhosis and DM, 56.6% at 5 yr. The survival rates of patients with liver cirrhosis and DM significantly differed from those with NGT. Univariate analysis demonstrated that serum albumin, total bilirubin, prothrombin activity, Child-Pugh scores, and glucose intolerance were highly significant prognostic factors. Multiple regression analysis yielded albumin and DM as the most powerful independent negative predictors of survival. CONCLUSIONS: OGTT appears to be useful for evaluating the prognosis of cirrhotic patients. (Am J Gastroenterol 2006;101:70‐75)

Endothelin (ET)-1 is a potent inducer of peptic ulcers. The roles of ET-1 in ulcer healing, however, have remained unclear, and these were investigated in mice. Gastric ulcers were induced in mice by serosal application of acetic acid. Three days later, mice were given a neutralizing ET-1 antibody or nonimmunized serum. The ulcer size, amount of fibrosis and myofibroblasts, and localization of ET-1 and ETA/Breceptors were analyzed. To elucidate the mechanisms underlying the effects of ET-1, we examined the proliferation, migration, and release of growth and angiogenic factors in gastric myofibroblasts with or without ET-1. The expression of prepro-ET-1 (an ET-1 precursor) and ET-converting enzyme-1 was examined in gastric myofibroblasts using RT-PCR. Immunoneutralization of ET-1 delayed gastric ulcer healing. The areas of fibrosis and myofibroblasts were smaller in the anti-ET-1 antibody group than in the control. ET-1 was expressed in the gastric epithelium, myofibroblasts, and other cell types. ETAreceptors, but not ETBreceptors, were present in myofibroblasts. ET-1 increased proliferation and migration of gastric myofibroblasts. ET-1 stimulated the release of hepatocyte growth factor, VEGF, PGE2, and IL-6 from gastric myofibroblasts. mRNA for prepro-ET-1 and ET-converting enzyme-1 was also expressed. ET-1 promotes the accumulation of gastric myofibroblasts and collagen fibrils at gastric ulcers. ET-1 also stimulates migration and proliferation of gastric myofibroblasts and enhances the release of growth factors, angiogenic factors, and PGE2. Thus ET-1 has important roles not only in ulcer formation but also in ulcer healing via mobilizing myofibroblasts and inducing production of stroma-derived factors.

Little is known about the clinical efficacy of co-therapy of enprostil, a prostaglandin E2 analogue, with a histamine H2-receptor antagonist. We aimed to assess the additive benefit of enprostil in combination with cimetidine for treating gastric ulcer in a prospective multicenter randomized controlled trial.In 43 hospitals 171 intention-to-treat (ITT) patients, diagnosed as having gastric ulcer by endoscopy, were randomly allocated to receive either enprostil 25microg b.i.d. and cimetidine 400mg b.i.d. (Group E=85), or cimetidine 400mg b.i.d. alone (Group C=86) for 8 weeks. Healing was examined by endoscopy at 4 and 8 weeks.Per protocol (PP) analysis comprised 166 patients (E=82, C=84). Despite no significant advantage at 4 weeks (E=55.3%, C=42.2%), the combination yielded higher healing rates at 8 weeks by ITT (E=89.4%, C=68.6%; p0.001) and PP analysis (E=92.7%, C=70.2%; p0.001). Symptom relief rates [E, C] at 2, 4, and 8 weeks were [80.2%, 68.3%] (not significant), [97.4%, 88.3%] (p0.05), and [95.6%, 87.0%] (p0.05), respectively. Significant advantage was observed in the patients aged 40 or older, with solitary ulcer (5mm in diameter), and without smoking or drinking habits. No adverse effects were critical.Enprostil safely and significantly augmented gastric ulcer healing and symptom relief by cimetidine.

Plaunotol, [(2E,6Z,10E)-7-hydroxymethyl-3,11,15-trimethyl-2,6,10,14-hexadecatetraen-1-ol], a gastroprotective agent, increases the prostaglandin production in the gastric mucosa and accelerates ulcer healing. The precise mechanisms underlying the gastroprotective actions by plaunotol are not known. On the other hand, cyclooxygenase (COX)-2 is a key enzyme in PGE(2) production and its induction is thought to have an important role in ulcer healing. We investigated the mechanism of plaunotol-mediated COX-2 induction in rat gastric epithelial (RGM1) cells. We used a PGE(2) enzyme-linked immunoassay kit and Western blot analysis to measure PGE(2) production and COX-2 induction with plaunotol treatment in serum-starved RGM1 cells. In addition, gel-shift assay, Western blot analysis and a reporter assay were performed to observe which Cox-2 promoter was involved in plaunotol-induced Cox-2 expression. The findings indicated that plaunotol treatment dose-dependently increased COX-2 expression and PGE(2) production. The nuclear factor kappaB (NF-kappaB) and cyclic AMP response element (CRE) sites of the COX-2 gene promoter were critical to plaunotol-mediated COX-2 expression. In conclusion, plaunotol induced COX-2 expression and increased PGE(2) production in serum-starved RGM1 cells via activation of the NF-kappaB and CRE sites of Cox-2 gene promoters.

Genomic alternations, including dosage and allelic imbalance, constitute a major basis of neoplastic and other genetic disorders. Using oligonucleotide genotyping microarrays, here we report the development and usage of an algorithm, called genome imbalance map (GIM) algorithm, for allelic as well as total gene dosage analysis. Using the GIM algorithm, global genome imbalance status at over 100,000 loci was simultaneously analyzed with unprecedented accuracy and allelic discrimination.

Cyclooxyngease-2 (COX-2) is a key enzyme in prostaglandin (PG) synthesis, and COX-2 induction plays an important role in the healing of gastric ulceration. Rebamipide is a gastro-protective agent and attenuates the activity of neutrophils. A number of reports have shown that rebamipide treatment increases PG production in the gastric mucosa {in vivo}. Although its clinical significance in ulcer healing has been demonstrated, {in vitro} evidence remains to be accumulated. Non-transformed rat gastric mucosal cells (RGM1 cells) were stimulated with rebamipide. RT-PCR and Western blot analysis revealed time and dose-dependent transcriptional and translational stimulation of COX-2. PGE(2) was also produced dose-dependently. However, marked COX-2 induction by rebamipide was transient and lasted less than 24 hr. COX-1 expression was unaltered by rebamipide. Reporter assay results confirmed the stimulation of Cox-2 promoter activity by rebamipide. In conclusion, this study provides {in vitro} evidence that rebamipide transcriptionally induces COX-2 and supports the rationale for its clinical use.

Bone marrow is reported to contain hematopoietic stem cells and other adult somatic stem cells that have phenotypes of cells composing tissues other than bone marrow. To explore the implication of bone marrow-derived cells in the treatment of inflammatory bowel diseases, experimental colitis was induced in wild-type rats after transplantation of bone marrow from transgenic rats expressing green fluorescence protein (GFP). Chronic colitis was induced 21 days later using 30 mg 2,4,6-trinitrobenzenesulfonic acid (TNBS). Control rats received saline. At 28, 56, and 224 days after TNBS administration, rats were euthanized, and tissues were removed and processed for paraffin-embedded sections. Cells derived from bone marrow were identified by immunohistochemistry using anti-GFP antibody. To identify the phenotypes of the cells expressing GFP, we conducted serial-section analysis and double-staining analysis using antibodies against cytokeratin (epithelial cells) or vimentin (interstitial cells). In the present study, GFP-positive, bone marrow-derived cells occupied 37.6% and 4.25% of the colonic epithelium at 28 days and 56 days after the induction of TNBS-colitis, respectively. Also, significant amounts of mucosal and submucosal interstitial cells were derived from the bone marrow. These findings showed that a large amount of bone marrow-derived cells were involved in regeneration of the colon after experimental colitis in rats.

Concanavalin A (ConA), directly injected into mice, induces T cell-mediated liver injury. However, it remains unclear whether ConA injection can activate innate immune cells, including natural killer (NK) cells and natural killer T (NKT) cells, both of which exist abundantly in the liver. Here we report that ConA injection stimulated interferon (IFN)-gamma production from liver NKT cells as early as 2 hours after injection and augmented YAC-1 cytotoxicity of liver NK cells. ConA-induced NK cell activation required other types of immune cells and critically depended on IFN-gamma. Because a nonhepatotoxic low dose of ConA was capable of fully activating both NKT cells and NK cells, we next addressed the possibility of ConA injection displaying an antitumor effect in the liver without liver injury. A nonhepatotoxic low-dose ConA injection augmented the cytotoxicity of liver NK cells against Colon-26 colon cancer cells and suppressed hepatic metastasis of Colon-26 cells in a NK cell- and IFN-gamma-dependent manner. In conclusion, a nonhepatotoxic low dose of ConA might serve as an immunomodulator that can preferentially activate the innate immune cells to induce an antitumor effect against metastatic liver tumor.

Cyclooxygenase-2 (COX-2) upregulation is recognized to confer advantage in progression in a wide variety of cancers, with colorectal cancer most intensively investigated. Epidemiologically, chemopreventive effects of COX-2 inhibitors have been proven on numerous cancers, but not on hepatocellular carcinoma (HCC). Although the antiapoptotic feature of COX-2 generally supports cancer cell growth, previous reports have shown that COX-2 expression, upregulated in early HCC, is downregulated in advanced HCC. Therefore, COX-2 downregulation may be somehow advantageous and specific for HCC development. However, its mechanism remains unclear. Since promoter hypermethylation often silences the gene expression, we hypothesized that the epigenetic mechanism might regulate COX-2 expression in HCC. We examined the methylation status of the Cox-2 promoter in six human HCC cell lines (Hep3B, HepG2, SK-Hep1, HuH7, PLC, and FLC-7 cells) using methylation-specific PCR. The promoter was remarkably hypermethylated in Hep3B and FLC-7 cells and moderately in HepG2 and SK-Hep1 cells, but not in HuH7 and PLC cells. In Hep3B cells, coincubation with 5-aza-2'-deoxycytidine, a demethylator, demethylated the promoter and upregulated COX-2 expression as well as prostaglandin E2 production dose dependently. On the other hand, no such effects were observed in HuH7 cells. Additionally, the methylator suppressed growth of Hep3B cells dose dependently, accompanied by cyclin D1 downregulation, and the growth suppression was abrogated by potent COX-2 inhibition with a COX-2 selective inhibitor celecoxib, but these responses were not found in HuH7 cells. These results indicated that cell growth was largely retarded by Cox-2 upregulation via promoter demethylation, rather than the potentially reactivated genes concurrently demethylated by 5-aza-2'-deoxycytidine. In conclusion, promoter hypermethylation transcriptionally silences Cox-2 in HCC cells. Epigenetic alteration of Cox-2, at least in part, modulates the growth of HCC cells.

Non-steroidal anti-inflammatory drugs(NSAIDs) have a potential to cause mucosal injury in the gastrointestinal tract. Inhibitions of cyclooxygenase, one of the targets of NSAIDs and direct cytotoxic effects of NSAIDs, are reported to be involved in NSAIDs-related mucosal damage. It is estimated that 15-30% of patients taking NSAIDs develop gastroduodenal ulcers, 2% of patients have life-threatening complications. Normal gastroduodenal mucosa expresses only COX-1, and it is reported that NSAIDs are more strongly inhibiting COX-1 are more harmful to gastroduodenal mucosa. Therefore, selective COX-2 inhibitors have been developed as safer NSAIDs than non-selective NSAIDs. Recent reports have, however, shown that COX-2 is expressing in gastroduodenal ulcers and H. pylori infection, suggesting that COX-2 plays an important role in mucosal healing. In this article, we discuss whether COX-2 selective inhibitors are able to prevent NSAIDs-associated ulceration.

One hundred seventy-one patients with chronic hepatitis C were included in this study (genotype1 and high viral loads (1H), [Formula: see text]; non-1H, [Formula: see text]; N.D., [Formula: see text] ). The combination therapy of interferon and ribavirin for 24 weeks with an additional 24 weeks of interferon monotherapy (48-week treatment) was undergone by 42 1H patients and 5 non-1H patients. The combination therapy of interferon and ribavirin was administered for 24 weeks in 67 1H patients and 22 non-1H patients. Among the 1H patients, the HCV relapse rate was significantly higher in those receiving 24-week combination treatment than in those receiving 48-week treatment (78% versus 42%, [Formula: see text] ). Among the non-1H patients, no significant difference was found between them. Sustained virological response (SVR) rates were observed to decrease as the timing of HCV RNA disappearance was delayed. In spite of the small rate (16%), SVR was obtained from the patients who became negative for HCV RNA by week 24 (beyond week 12) only in those receiving 48-week treatment. In 1H patients, 24-week combination treatment followed by interferon monotherapy for 24 weeks was concluded to be the treatment offering the most hope among those that the medical insurance can be applied in Japan.

Prostaglandin (PG) E(2), a cyclooxygenase (COX) product, and angiotensin II are endogenous and have physiological roles in the body. On the other hand, an inducible isoform of COX (COX-2), insulin-like growth factor (IGF) II, and IGF-I receptor (IGF-IR) are up-regulated in colon carcinoma and might have crucial roles in tumor growth and invasion. The aim of the present study was to investigate the effects of COX-2 inhibitor and drugs blocking the biological activities of angiotensin II [angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs)] on IGF-IR expression and tumor growth in vivo. We also investigated the effects of PGE(2), a major COX-2 product, in cancer cells and the effects of angiotensin II on IGF-IR expression and the underlying mechanism of action. In in vivo studies, tumor growth and IGF-IR expression were investigated in Colon 26 cells inoculated into BALB/c mice. In in vitro studies, the effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on IGF-IR expression were analyzed in three colon cancer cell lines (Colon 26, HCA-7, and LS174T). IGF-II-induced cell growth and invasion were analyzed in Colon 26 cells in the presence and absence of NSAIDs (indomethacin and celecoxib) and angiotensin II. Celecoxib at the lowest effective dose for suppression of PG production (3 mg/kg) or an ACE inhibitor/ARB alone did not have a significant effect as compared with controls, although a high dose of celecoxib (20 mg/kg) suppressed tumor growth. On the other hand, combination therapy with these two categories of drugs significantly reduced tumor growth in vivo. Treatment with both celecoxib and an ACE inhibitor/ARB decreased IGF-IR expression levels in inoculated tumor cells. In in vitro studies, NSAIDs reduced IGF-IR expression in a dose-dependent manner in all three cell lines. NSAIDs also inhibited IGF-II-stimulated growth and invasion in a dose-dependent manner. PGE(2) or angiotensin II treatment reversed the NSAID-induced down-regulation of IGF-IR expression, growth, and invasion. PGE(2) and angiotensin II induced Akt phosphorylation, and LY294002 or wortmannin inhibited PGE(2)- or angiotensin II-induced IGF-IR expression, indicating that PGE(2) and angiotensin II both regulate IGF-IR expression by the same Akt/phosphatidylinositol-3 pathway. Thus, combination therapy with NSAIDs and ACE inhibitors targeting IGF-IR might be a novel and potentially promising strategy for the chemoprevention of colon cancer.

Administration of (1R)-1-benzo[b]thiophen-5-yl-2-[2-(diethylamino)ethoxy]ethan-1-ol hydrochloride (T-588), a cognitive enhancer, like the acetylcholine esterase inhibitors physostigmine and tacrine, stimulated phosphorylation of extracellular signal-regulated kinases (ERK) in the mouse hippocampus. The effect of T-588 on ERK phosphorylation was persistent from 2 to 6 h after injection. Immunohistochemical study showed that T-588 stimulated neuronal ERK phosphorylation in rat hippocampal CA1 pyramidal subfield. These findings suggest that systemic T-588 stimulates the ERK kinase pathway in the hippocampal neurons.