Satoshi Ishizone

Inhibition of NADPH oxidase 4 activates apoptosis via the AKT/apoptosis signal-regulating kinase 1 pathway in pancreatic cancer PANC-1 cells.

Pancreatic adenocarcinoma is an aggressive human malignancy and is characterized by resistance to apoptosis. Recently, NADPH oxidase (Nox) 4-mediated generation of intracellular reactive oxygen species (ROS) was proposed to confer antiapoptotic activity and thus a growth advantage to pancreatic cancer cells. The signaling mechanism by which Nox4 transmits cell survival signals remains unclear. Here, we show that both a flavoprotein inhibitor, diphenylene iodonium (DPI), and small interfering RNAs designed to target Nox4 mRNA (siNox4RNAs) inhibited superoxide production in PANC-1 pancreatic cancer cells, and depletion of ROS by DPI or siNox4RNAs induced apoptosis. Parallely, DPI treatment and siNox4RNA transfection blocked activation of the cell survival kinase AKT by attenuating phosphorylation of AKT. Furthermore, AKT phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) on Ser-83 was reduced by DPI and siNox4RNAs. When ASK1Ser83Ala (an AKT phosphorylation-defective ASK1 mutant) was introduced into PANC-1 cells, this mutant alone induced apoptosis. But, addition of DPI or co-transfection of siNox4RNA had no additive effect, indicating that the mutant can substitute for these reagents in apoptosis induction. Taken together, these findings suggest that ROS generated by Nox4, at least in part, transmit cell survival signals through the AKT–ASK1 pathway in pancreatic cancer cells and their depletion leads to apoptosis.

Implantation-Dependent Expression of Trophinin by Maternal Fallopian Tube Epithelia during Tubal Pregnancies: Possible Role of Human Chorionic Gonadotrophin on Ectopic Pregnancy

Trophinin, tastin, and bystin have been identified as molecules potentially involved in human embryo implantation. Both trophoblasts and endometrial epithelial cells express trophinin, which mediates apical cell adhesion through homophilic trophinin-trophinin binding. We hypothesized that trophinins function in embryo implantation is unique to humans and investigated the expression of trophinin, tastin, and bystin in ectopic pregnancy, a condition unique to humans. In tubal pregnancies, high levels of all three were found in both trophoblasts and fallopian tubal epithelia. Trophinin expression in maternal cells was particularly high in the area adjacent to the trophoblasts, whereas trophinin was barely detectable in intact fallopian tubes from women with in utero pregnancies or without pregnancies. When explants of intact fallopian tube were incubated with the human chorionic gonadotrophin (hCG), trophinin expression was enhanced in epithelial cells. Since the trophectoderm of the human blastocyst secretes hCG before and after implantation, these results suggest that hCG from the human embryo induces trophinin expression by maternal cells. As both beta-subunit of hCG and trophinin genes have diverged in mammals, the present study suggests a unique role of hCG and trophinin in human embryo implantation, including the pathogenesis of ectopic pregnancy.

Surgical treatment for anorectal malignant melanoma: report of five cases and review of 79 Japanese cases

IntroductionAnorectal malignant melanoma (AMM) is a relatively rare disease. Because of its poor prognosis, the optimal surgical treatment for AMM is still controversial and difficult to determine. In this paper, we report five cases of AMM that have been treated by surgery and/or other methods at Shinshu University Hospital within the last decade. We also review the present five cases along with 74 other Japanese cases reported between 1997 and 2006 and discuss the role of surgery in the treatment of AMM.Results and discussionAmong our AMM patients, two who underwent radical abdominoperineal resection had long survival, while the other three patients who underwent palliative surgery had a poor outcome. On the total of 79 AMM patients, those who underwent curative surgery had a better outcome than those who underwent palliative surgery (p < 0.0001). Furthermore, the outcome of AMM patients at stages 0 and I was better than that of AMM patients at stages II, III, and IV (p < 0.0001). There was no significant difference in survival between AMM patients with and without adjuvant chemotherapy.ConclusionIn conclusion, AMM patients treated by curative surgery can expect long-term survival, although the usefulness of adjuvant chemotherapy for AMM patients is controversial.

Natural history of gastric mucosal cytokine expression in Helicobacter pylori gastritis in Mongolian gerbils.

ABSTRACT Data regarding the chronological changes in gastric mucosal cytokines in the different phases of Helicobacter pylori infection are unavailable. We examined Mongolian gerbils for up to 52 weeks after H. pylori (ATCC 43504) inoculation. Levels of mRNAs of mucosal cytokines (interleukin-1β [IL-1β], gamma interferon [IFN-γ], IL-4, IL-6, and IL-10) were assessed using real-time reverse transcription-PCR. Starting 26 weeks after H. pylori inoculation, two clinicohistologic patterns appeared: gastric ulcers in 32% and hyperplastic polyps in 68% of gerbils. High levels of mucosal IL-1β mRNA were observed early in the infection, reaching maximum at 4 weeks and then rapidly declining. Mucosal IFN-γ mRNA also reached maximal levels at 4 weeks but remained high thereafter. Both IL-1β and IFN-γ mRNA levels were consistently higher in the pyloric mucosa than in the fundic mucosa. In contrast, IL-4, IL-6, and IL-10 mRNA levels peaked at 8 to 26 weeks and levels were similar in the pyloric mucosa and the fundic mucosa. IFN-γ mRNA levels were significantly higher in gerbils with ulcers than in those with hyperplastic polyps (median IFN-γ/glyceraldehyde-3-phosphate dehydrogenase ratio × 100,000 = 650 versus 338, respectively [antrum], and 172 versus 40, respectively [corpus]) (P < 0.05). We propose that the different outcomes (e.g., ulcers or hyperplastic polyps) might relate to imbalances among cytokines.

Clinical utility of quantitative RT-PCR targeted to α1,4-N-acetylglucosaminyltransferase mRNA for detection of pancreatic cancer

α1,4‐N‐Acetylglucosaminyltransferase (α4GnT) is a glycosyltransferase responsible for the biosynthesis of α1,4‐GlcNAc‐capped O‐glycans, and is frequently expressed in pancreatic cancer cells but not peripheral blood cells. In the present study, we tested the clinical utility of α4GnT mRNA expressed in the mononuclear cell fraction of peripheral blood as a biomarker of pancreatic cancer. Total RNA isolated from the peripheral blood mononuclear cells from 55 pancreatic cancer patients, 10 chronic pancreatitis patients, and 70 cancer‐free volunteers was analyzed quantitatively by reverse transcription‐polymerase chain reaction with primers specific for α4GnT, and the expression level of α4GnT mRNA relative to that of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was measured. When the ratio of α4GnT to GAPDH transcripts exceeded a defined cut‐off value, patients were considered to have pancreatic cancer. By these standards, 76.4% of the pancreatic cancer patients were detected by this assay. A strong correlation was obtained between positivity in this assay and the expression of α4GnT protein detected immunohistochemically in pancreatic cancer tissues resected subsequently, suggesting that α4GnT mRNA detected in the peripheral blood is derived from circulating pancreatic cancer cells. Although increased levels of α4GnT mRNA was detected in 40.0% of chronic pancreatitis patients and 17.1% of cancer‐free volunteers, the expression levels were significantly lower than those seen in pancreatic cancer patients. These results suggest that quantitative analysis of α4GnT mRNA expressed in the mononuclear cell fraction of peripheral blood will contribute to the detection of pancreatic cancer. (Cancer Sci 2006; 97: 119  – 126)

Efficacy of S-1 for patients with peritoneal metastasis of gastric cancer.

Background: This study was designed to examine the efficacy and compliance of S-1 for the patients with peritoneal metastasis of gastric cancer. Methods: Sixteen consecutive patients with peritoneal metastasis of gastric cancer were treated with S-1. Their survival was compared with that of the historical control group (25 patients). Thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase and orotate phosphoribosyl transferase mRNA expression in the tumor were evaluated. Results: The median survival time of S-1-treated patients was 550 days, which was significantly longer than that of the historical control group (215 days). We elucidated some factors to prolong the survival of the patients treated with S-1 for peritoneal metastasis: peritoneal metastasis without other distant metastases, the combination of S-1 treatment and gastrectomy, and low expression of thymidine phosphorylase mRNA in primary tumors. Conclusions: S-1 showed a surprisingly long-term survival with minimum toxicity in patients with peritoneal metastasis of gastric cancer.

Usefulness of the Real-Time Reverse Transcription-Polymerase Chain Reaction Assay Targeted to α1,4-N-Acetylglucosaminyltransferase for the Detection of Gastric Cancer

α1,4-N-acetylglucosaminyltransferase (α4GnT) is a glycosyltransferase that forms a unique glycan, GlcNAcα1 → 4Galβ → R, specifically present in gastric gland mucous cell-type mucin. Recently, we molecularly cloned human α4GnT and showed that α4GnT is expressed in the mucous cells that secrete this particular mucin. In the present study, we first demonstrated that α4GnT was frequently expressed in gastric cancer cells but not in peripheral blood cells using immunohistochemistry. To detect gastric cancer cells circulating in the peripheral blood of gastric cancer patients, we quantitatively analyzed the expression level of α4GnT mRNA in the mononuclear cell fraction of peripheral blood using real-time reverse transcription polymerase chain reaction. The transcripts of α4GnT were detected in the mononuclear cell fraction isolated from 62.2% of 37 gastric cancer patients but not from any of 23 healthy individuals. Significant correlation was found in the expression levels of α4GnT mRNA in peripheral blood and α4GnT protein in gastric cancer cells. Surprisingly, α4GnT mRNA was detectable in 80% of five patients with an early stage of gastric cancer when the cancer cells were limited to the gastric mucosa, and the expression levels of α4GnT mRNA were increased in association with tumor progression. In three patients with gastric cancer, during postsurgical follow-up, the expression levels of α4GnT mRNA were decreased after surgical removal of gastric cancer. However, significant amounts of the α4GnT transcripts were again detected in two patients, who eventually developed to the recurrence of gastric cancer. Although α4GnT was detected in 33.3% of nine patients with Helicobacter pylori–infected chronic active gastritis as well as all of four patients with peptic ulcer, the mean expression level of α4GnT mRNA in these benign disorders was lower than that in gastric cancer. These results altogether indicate that the quantitative analysis of α4GnT mRNA expressed in the peripheral blood is useful for the detection and, possibly, monitoring of gastric cancer.

Eradication of Helicobacter pylori decreases mucosal alterations linked to gastric carcinogenesis in Mongolian gerbils

To the Editor: Helicobacter pylori (H. pylori) infection increases the risk of gastric carcinogenesis, and Mongolian gerbils have served as useful models for investigation of H. pylori-induced gastric disorders, including gastric cancer.1–3 It was shown recently that H. pylori eradication decreased N-methyl-N-nitrosourea (MNU)-induced gastric carcinogenesis in gerbils.4 However, no pathological investigation on the relationship between H. pylori eradication and gastric carcinogenicity has been conducted in this model. Therefore, we evaluated the effect of H. pylori eradication on gastric mucosal changes in relation to carcinogenicity. Twelve Mongolian gerbils (specific pathogen-free, 7 weeks old, male; Seac Yoshitomi, Fukuoka, Japan) were inoculated with 8 108 colony-forming-units of H. pylori (ATCC43504; American Type Culture Collection, Rockville, MD, USA). Half were treated with a course of amoxycillin (1mg/kg) plus omeprazole (10mg/kg) 24 weeks after the inoculation; the drugs were suspended in 0.5% hydroxypropylmethylcellulose and administered i.g. twice daily for 10 days. Six gerbils not inoculated with H. pylori served as controls. Forty weeks after the inoculation, all the gerbils were killed. Thirty minutes before being killed, the gerbils were given 5 bromo-2 -deoxyuridine (BrdU) intraperitoneally (200mg/kg), and blood samples were obtained to measure the titer of anti-H. pylori IgG antibody using an enzyme-linked immunosorbent assay. Tissue sections of the excised stomachs were stained with hematoxylin and eosin (H&E) or Alcian blue (pH 2.5)–periodic acid-Schiff. The degree of inflammation was graded according to the Updated Sydney System, as described previously.2 One gerbil from the untreated group died at 32 weeks after H. pylori inoculation. All the gerbils in the H. pylori-inoculated groups were seropositive for H. pylori, and the antibody titer in the treated group was significantly lower than that in the untreated group at the time of death (Table 1). The development of gastric polyps (defined as 2 mm or more elevated mucosal lesion) and the grades of inflammatory cell infiltration in the gastric mucosa were significantly lower in the treated than the untreated group (Fig. 1, Table 1). There were fewer intestinal metaplastic foveolae in the treated than the untreated group, but this finding was not significant. None of the control gerbils showed any pathological changes of the gastric mucosa. BrdU labeling was visualized using mouse monoclonal antiBrdU antibody (1 :50; Dako, Glostrup, Denmark).1,2 The BrdU labeling index was represented by the number of BrdU-positive cells expressed as a percentage of the total epithelial cell number in ten arbitrarily selected areas in the lesser curvature of the midantrum. The labeling index was significantly lower in the treated than the untreated group (see Table 1). This study using the Mongolian gerbil model confirmed that eradication of H. pylori resulted in a significant decrease of polyp formation, inflammatory cell infiltration, and cellular proliferation in the gastric mucosa. In our previous study, gastric inflammation induced by H. pylori was closely related to gastric carcinogenesis.2 Thus, H. Pylori eradication could diminish mucosal alterations linked to gastric carcinogenesis.

Modified Irinotecan/5FU/Leucovorin therapy in advanced colorectal cancer and predicting therapeutic efficacy by expression of tumor-related enzymes

Objective. To evaluate the efficacy and safety of a regimen using Irinotecan, 5FU and Leucovorin for patients with advanced or recurrent colorectal cancer. Material and methods. Irinotecan (75 mg/m2) was administered biweekly, while 5FU (600 mg/m2) and Leucovorin (250 mg/m2) were administered weekly, for 6 weeks. Results. The 21 consecutive patients subjected to this regimen showed a good response rate (43%) with minimal toxicity (incidence of grade 3/4: leukopenia and neutropenia, 5%, respectively, and vomiting, 10%). The mean survival time of all 21 patients was 15.7 months. This regimen could be a valid option for patients with advanced colorectal cancer, especially those seeking a good QoL (quality of life) for the remainder of their lives. We evaluated the expression of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT) mRNAs, and sialyl Lewis X on formalin-fixed, paraffin-embedded colorectal tumor samples. Expression of TS mRNA or sialyl Lewis X was negatively correlated with the response from chemotherapy. Patients with low DPD mRNA expression in the tumor showed a significant longer survival than those with high expression. In patients with high TP mRNA expression, there was a tendency towards a high incidence of leukopenia. Conclusions. Some predictive factors elucidated in this study could contribute to the progress of the tumor-biology based, individualized chemotherapy for colorectal cancer patients.

Roles of virD4 and cagG genes in the cag pathogenicity island of Helicobacter pylori using a Mongolian gerbil model

Background and Aims: The roles of the virD4 and the cagG genes in the cag pathogenicity island of Helicobacter pylori for gastroduodenal pathogenesis are unclear and their roles in vivo have not been examined. Methods: Seven week old male Mongolian gerbils were inoculated with the wild type H pylori TN2GF4, its isogenic virD4, or cagG mutants. Animals were sacrificed at 4, 12, and 24 weeks after inoculation. Gastric inflammation and H pylori density were evaluated by histology, inflammatory response (as measured by interleukin (IL)-1β mRNA levels), proliferative activity (as assessed by 5′-bromo-2′deoxyuridine labelling indices), and host systemic reaction (as measured by anti-H pylori IgG antibody). Results: Degree of gastric inflammation, proliferative activity, and mucosal IL-1β mRNA levels remained low throughout the first 12 weeks in gerbils infected with the virD4 mutants. Degree of gastric inflammation and proliferative activity increased at 24 weeks with the virD4 mutants reaching levels comparative with those seen at four weeks with the wild-type strains. Mucosal IL-1β mRNA levels were also increased at 24 weeks with the virD4 mutants and levels at 24 weeks were similar between the wild-type and virD4 mutants. In contrast, gerbils infected with the cagG mutants had reduced ability to colonise gerbils, and no or little gastric inflammation or proliferative activity was observed. Conclusions: Loss of the virD4 gene temporally retarded but did not abrogate gastric inflammation. Loss of the cagG gene abolished gastric inflammation partially via reduced ability to colonise gerbils. Unknown factors related to the type IV secretion system other than CagA may influence gastric inflammation.