Takako Serizawa

Interleukin-6 mediates epithelial-stromal interactions and promotes gastric tumorigenesis.

Interleukin-6 (IL-6) is a pleiotropic cytokine that affects various functions, including tumor development. Although the importance of IL-6 in gastric cancer has been documented in experimental and clinical studies, the mechanism by which IL-6 promotes gastric cancer remains unclear. In this study, we investigated the role of IL-6 in the epithelial–stromal interaction in gastric tumorigenesis. Immunohistochemical analysis of human gastritis, gastric adenoma, and gastric cancer tissues revealed that IL-6 was frequently detected in the stroma. IL-6–positive cells in the stroma showed positive staining for the fibroblast marker α-smooth muscle actin, suggesting that stromal fibroblasts produce IL-6. We compared IL-6 knockout (IL-6−/−) mice with wild-type (WT) mice in a model of gastric tumorigenesis induced by the chemical carcinogen N-methyl-N-nitrosourea. The stromal fibroblasts expressed IL-6 in tumors from WT mice. Gastric tumorigenesis was attenuated in IL-6−/− mice, compared with WT mice. Impaired tumor development in IL-6−/− mice was correlated with the decreased activation of STAT3, a factor associated with gastric cancer cell proliferation. In vitro, when gastric cancer cell line was co-cultured with primary human gastric fibroblast, STAT3–related genes including COX-2 and iNOS were induced in gastric cancer cells and this response was attenuated with neutralizing anti-IL-6 receptor antibody. IL-6 production from fibroblasts was increased when fibroblasts were cultured in the presence of gastric cancer cell–conditioned media. IL-6 production from fibroblasts was suppressed by an interleukin-1 (IL-1) receptor antagonist and siRNA inhibition of IL-1α in the fibroblasts. IL-1α mRNA and protein were increased in fibroblast lysate, suggesting that cell-associated IL-1α in fibroblasts may be involved. Our results suggest the importance of IL-6 mediated stromal-epithelial cell interaction in gastric tumorigenesis.

Role of Interleukin-32 in Helicobacter pylori-Induced Gastric Inflammation

ABSTRACT Helicobacter pylori infection is associated with gastritis and gastric cancer. An H. pylori virulence factor, the cag pathogenicity island (PAI), is related to host cell cytokine induction and gastric inflammation. Since elucidation of the mechanisms of inflammation is important for therapy, the associations between cytokines and inflammatory diseases have been investigated vigorously. Levels of interleukin-32 (IL-32), a recently described inflammatory cytokine, are increased in various inflammatory diseases, such as rheumatoid arthritis and Crohns disease, and in malignancies, including gastric cancer. In this report, we examined IL-32 expression in human gastric disease. We also investigated the function of IL-32 in activation of the inflammatory cytokines in gastritis. IL-32 expression paralleled human gastric tissue pathology, with low IL-32 expression in H. pylori-uninfected gastric mucosa and higher expression levels in gastritis and gastric cancer tissues. H. pylori infection increased IL-32 expression in human gastric epithelial cell lines. H. pylori-induced IL-32 expression was dependent on the bacterial cagPAI genes and on activation of nuclear factor κB (NF-κB). IL-32 expression induced by H. pylori was not detected in the supernatant of AGS cells but was found in the cytosol. Expression of the H. pylori-induced cytokines CXCL1, CXCL2, and IL-8 was decreased in IL-32-knockdown AGS cell lines compared to a control AGS cell line. We also found that NF-κB activation was decreased in H. pylori-infected IL-32-knockdown cells. These results suggest that IL-32 has important functions in the regulation of cytokine expression in H. pylori-infected gastric mucosa.

Distribution of intestinal metaplasia as a predictor of gastric cancer development

Helicobacter pylori, gastritis, and intestinal metaplasia (IM) are known risk factors for gastric cancer. In the present study, we conducted a cohort study to evaluate the predictive value of the distribution of IM for gastric cancer development.

Gastric Metaplasia Induced by Helicobacter pylori Is Associated with Enhanced SOX9 Expression via Interleukin-1 Signaling

ABSTRACT Histopathological changes of the gastric mucosa after Helicobacter pylori infection, such as atrophy, metaplasia, and dysplasia, are considered to be precursors of gastric cancer, yet the mechanisms of histological progression are unknown. The aim of this study was to analyze the histopathological features of the gastric mucosa in mice infected with H. pylori strain PMSS1 in relation to gastric stem cell marker expression. C57BL/6J mice infected with PMSS1 were examined for histopathological changes, levels of proinflammatory cytokines, and expression of stem cell markers. Histopathological gastritis scores, such as atrophy and metaplasia, and levels of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), were increased after PMSS1 infection. Expression levels of the cell proliferation and stem cell markers CD44 and SOX9 were also significantly increased in PMSS1-infected mice. Importantly, almost all metaplastic cells induced by PMSS1 infection expressed SOX9. When IL-1 receptor (IL-1R) knockout mice were infected with PMSS1, metaplastic changes and expression levels of stem cell markers were significantly decreased compared with those in wild-type (WT) mice. In conclusion, H. pylori infection induced the expression of cytokines and stem cell markers and histopathological metaplasia in the mouse gastric mucosa. SOX9 expression, in particular, was strongly associated with metaplastic changes, and these changes were dependent on IL-1 signaling. The results suggested the importance of SOX9 in gastric carcinogenesis.

TGF-β Signaling in Dendritic Cells Governs Colonic Homeostasis by Controlling Epithelial Differentiation and the Luminal Microbiota

Dendritic cells (DCs) mediate host immune responses to gut microbes and play critical roles in inflammatory bowel disease. In this study, we examined the role of TGF-β signaling in DCs in colonic homeostasis. CD11c-cre Tgfbr2fl/fl mice developed spontaneous colitis, and CD11c-cre Tgfbr2fl/+ mice exhibited susceptibility to dextran sulfate sodium–induced colitis. Colitis in these mice was characterized by goblet cell depletion and dysbiosis caused by Enterobacteriaceae enrichment. Wild-type mice gavaged with Enterobacteriaceae from CD11c-cre Tgfbr2fl/fl mice feces showed severe colitis after dextran sulfate sodium treatment, whereas those treated with Notch inhibitor exhibited attenuated colonic injury with increased goblet cell numbers, thickened mucus layer, and fewer fecal Enterobacteriaceae. Wild-type mice transplanted with CD11c-cre Tgfbr2fl/fl bone marrow developed colitis showing increased Jagged1 and Jagged2 in DCs, increased Hes1 levels in epithelium, and goblet cell depletion. These findings suggest that TGF-β signaling in DCs regulates intestinal homeostasis by modulating epithelial cell differentiation and fecal microbiota.

Efficacy of triple therapy with esomeprazole, amoxicillin, and sitafloxacin as a third-line Helicobacter pylori eradication regimen

OBJECTIVE To examine the efficacy of third-line Helicobacter pylori eradication therapy with esomeprazole, amoxicillin, and sitafloxacin for patients with clarithromycin- and metronidazole-based first- and second-line therapy failure. METHODS Thirty patients with first- and second-line H. pylori eradication failure were treated prospectively with esomeprazole 20mg twice daily, amoxicillin 750mg twice daily, and sitafloxacin 100mg twice daily for 7 days. After 8-12 weeks, the outcome of eradication therapy was assessed by 13C-urea breath test or stool antigen test. RESULTS All 30 patients completed the study. Eradication was successful in 25 patients and the eradication rate was 83% in the intention-to-treat and per-protocol analyses. No specific or significant adverse events were recorded in the 30 patients. Patient characteristics such as sex, body mass index, and pepsinogen I/II ratio did not differ between patients who were treated successfully and those who were not treated successfully. CONCLUSIONS Third-line H. pylori eradication therapy with esomeprazole, amoxicillin, and sitafloxacin is as safe and effective as previously reported sitafloxacin-based triple therapy.

Safety of Regular-Dose Imatinib Therapy in Patients with Gastrointestinal Stromal Tumors Undergoing Dialysis

The number of cancer patients undergoing dialysis has been increasing, and the number of these patients on chemotherapy is also increasing. Imatinib is an effective and safe therapy for KIT-positive gastrointestinal stromal tumors (GIST), but the efficacy and safety of imatinib in dialysis patients remain unclear. Because clinical trials have not been conducted in this population, more investigations are required. We report on a 75-year-old Japanese man undergoing dialysis who presented with massive tarry stool from a duodenal GIST. The duodenal GIST was 14 cm in diameter with multiple liver and bone metastases. The patient underwent an urgent pancreaticoduodenectomy to achieve hemostasis. After surgery, he was administered imatinib 400 mg/day. No severe adverse event including myelosuppression, congestive heart failure, liver functional impairment, intestinal pneumonia, or Steven-Johnson syndrome occurred, and the liver metastasis remained stable for 4 months. During chemotherapy, hemodialysis continued three times per week without adverse events. We suggest that regular-dose imatinib is an effective and safe treatment in patients with GIST undergoing dialysis. In addition, we present a literature review of the effectiveness and safety of imatinib treatment in dialysis patients.

Mo1653 CK19-Specific Autophagy Knockout Mice Model to Examine the Colon Cancer Progression

Introduction: Autophagy, a basic cell survival mechanism by recycling cellular amino acids, play an important role in cancer and it is vigorously investigated. Though the role of autophagy may be different between cancer initiation and progression, the genetic knockout mice model that could distinguish the influence of autophagy on colon cancer initiation and progression are sparse. To clarify the role of autophagy especially in established colorectal cancer in vivo, we used CK19CreERT mice treated by AOM/DSS (azoxymethane/dextran sodium sulphate) Materials and Methods: To delete ATG5, an essential autophagy gene, in CK19 expressing cells by TAM (tamoxifen) injection, CK19CreERT mice were crossed with ATG5 fl/fl mice. Cre-negative mice were used as controls. The animals, on day 1, were injected intraperitoneally (i.p.) with 12.5 mg/kg AOM. After five days, mice received water with 2.5% DSS for five days. After this, mice were maintained on regular water for 14 days and subjected to two more DSS treatment cycles. On day 60, mice were injected i.p with 10 mg/kg TAM and sacrificed 28 days later. Macroscopic colon tumors were counted and measured with a caliper. Then, the formalin fixed and paraffin embedded colon segments were used for Hematoxylin and eosin staining and immunohistochemical staining examination. The expressions of CK19, ATG5, Ki67, and PCNA in cancerous part and non-cancerous part were compared between autophagy knockout mice and control mice. Result: We generated mutant mice lacking ATG5 caused by TAM injection specifically in intestinal epithelial CK19-positeive cell. In these ATG5 fl/fl CK19CreERT mice, 4 weeks after the TAM injection, about 10% of CK19 positive-cell in the colon were lacking ATG5 by immunohistochemistry. The colon length and body weight, which are commonly used to measure the severity of inflammation, of AOM/DSS treated mice were not significantly different between control mice group and ATG5 fl/fl CK19CreERT mice group. Though the number of colon tumors between control and ATG5 fl/fl CK19CreERT mice group was not significantly different, maximum size of colon tumors were significantly smaller in ATG5 fl/fl CK19CreERT mice group than in control mice group (number of tumors; control (n=5) 9.4 vs ATG5 fl/fl CK19CreERT (n=7) 8.3 , maximum tumor size; control (n=5) 4.26mm vs ATG5 fl/fl CK19CreERT (n=7) 3.36mm (p<0.05) ) . In immunohistochemistry assay, colon tumor of the control group mice was globally strong-positive for CK19, ATG5, Ki67, and PCNA. To the contrast, colon tumor of the ATG5 fl/fl CK19CreERT mice was partly negative for ATG5, Ki67, and PCNA. Conclusion: This animal model, using ATG5 fl/fl CK19Cre ERT mice and AOM/DSS is suitable for examining the role of autophagy in established colon cancer. Blocking autophagy has the potential to treat colon cancer.

598 Transforming Growth Factor-Beta Signaling on Dendritic Cells Regulates Bacterial Communities and Gut Homeostasis

Background: Transcription factor Zinc-finger Binding Protein-89 (ZBP-89, ZNF148) expression is regulated by butyrate and forms protein-protein complexes with tumor suppressor factors, e.g. p53, p300, ataxia-telangiectasia mutated (ATM). To study the role of ZBP-89 in vivo, we generated a conditional knockout in the intestine and colon (ZBP-89ΔInt). Mice exhibited increased morbidity and mortality when challenged with Salmonella typhimurium, in part due to reduced tryptophan hydroxylase 1(Tph1) expression and reduced colonic antimicrobial peptide secretion (defensins). We found that β-catenin cooperates with ZBP89 to induce tryptophan hydroxylase 1(Tph1) expression in enterochromaffin cells. Aim: To determinewhether ZBP-89 interacts directly with β-catenin to prevent colonic transformation. Methods: ZBP-89ΔInt and WT littermates were treated with 7.4mg/kg azoxymethane (AOM) and water containing 2% dextran sulfate sodium (DSS). After 3 cycles, mice were sacrificed 5 weeks later for tumor evaluation, mRNA and histological analysis. Expression of β-cateninTCF gene targets was determined by rt-qPCR on colonic lysates. The ZBP-89 expression vector was co-transfected with or without β-catenin into HEK293 or SW480, a human colorectal cell line with the TCF reporter plasmid TOPFLASH to assess direct regulation of Wnt-β-catenin-TCF transcriptional activity. To demonstrate the association of ZBP-89 with β-catenin, cell lysates of SW480 were used to perform co-immunoprecipitation. Results: We observed 100% increase in tumor incidence and size in the distal colon and rectum of ZBP-89ΔInt compared to WT mice after the administration of AOM/DSS, N=16 mice/group (P<0.05). mRNA for cyclinD1, c-myc and Axin2 was significantly increased 3-fold in the ZBP89ΔInt mice after AOM/DSS treatment. Ectopic expression of ZBP-89 mitigated induction of the TOPFLASH reporter by β-catenin by 60% in both HEK293 and SW480 cell lines. Coimmunoprecipitation/western blotting showed that ZBP-89 and β-catenin formed a complex in cytoplasmic and nuclear extracts. Confocal microscopy demonstrated that ZBP-89 colocalized with β-catenin in the nucleus. In addition to protein-protein interactions, we found that knockdown of ZBP-89 by siRNA reduced β-catenin protein levels, suggesting that ZBP89 regulates β-catenin expression. Indeed, in silico analysis revealed a putative ZBP-89 binding site in the human and mouse β-catenin promoter at -83 to -78. Conclusion: Collectively, the data suggests that ZBP-98 modulates β-catenin activity and subsequently the induction of Wnt-β-catenin-TCF pathway in vivo and in vitro. Pull-down assays demonstrated that ZBP-89 modulates β-catenin activity through both protein-protein interactions and possibly transcriptional regulation of β-catenin expression, providing a possible mechanism by which this transcription factor inhibits colonic tumor formation.