Toshio Fujisawa

Adiponectin suppresses colorectal carcinogenesis under the high-fat diet condition

Background and aims: The effect of adiponectin on colorectal carcinogenesis has been proposed but not fully investigated. We investigated the effect of adiponectin deficiency on the development of colorectal cancer. Methods: We generated three types of gene-deficient mice (adiponectin-deficient, adiponectin receptor 1-deficient, and adiponectin receptor 2-deficient) and investigated chemical-induced colon polyp formation and cell proliferation in colon epithelium. Western blot analysis was performed to elucidate the mechanism which affected colorectal carcinogenesis by adiponectin deficiency. Results: The numbers of colon polyps were significantly increased in adiponectin-deficient mice compared with wild-type mice fed a high-fat diet. However, no difference was observed between wild-type and adiponectin-deficient mice fed a basal diet. A significant increase in cell proliferative activity was also observed in the colonic epithelium of the adiponectin-deficient mice when compared with wild-type mice fed a high-fat diet; however, no difference was observed between wild-type and adiponectin-deficient mice fed a basal diet. Similarly, an increase in epithelial cell proliferation was observed in adiponectin receptor 1-deficient mice, but not in adiponectin receptor 2-deficient mice. Western blot analysis revealed activation of mammalian target of rapamycin, p70 S6 kinase, S6 protein and inactivation of AMP-activated protein kinase in the colon epithelium of adiponectin-deficient mice fed with high-fat diet. Conclusions: Adiponectin suppresses colonic epithelial proliferation via inhibition of the mammalian target of the rapamycin pathway under a high-fat diet, but not under a basal diet. These studies indicate a novel mechanism of suppression of colorectal carcinogenesis induced by a Western-style high-fat diet.

Metformin suppresses intestinal polyp growth in ApcMin/+ mice

Metformin is a biguanide derivative that is widely used in the treatment of diabetes mellitus. One of the pharmacological targets of metformin is adenosine monophosphate‐activated protein kinase (AMPK). We investigated the effect of metformin on the suppression of intestinal polyp formation in ApcMin/+ mice. Administration of metformin (250 mg/kg) did not reduce the total number of intestinal polyp formations, but significantly reduced the number of intestinal polyp formations larger than 2 mm in diameter in ApcMin/+ mice. To examine the indirect effect of metformin, the index of insulin resistance and serum lipid levels in ApcMin/+ mice were assessed. These factors were not significantly attenuated by the treatment with metformin, indicating that the suppression of polyp growth is not due to the indirect drug action. The levels of tumor cell proliferation as determined by 5‐bromodeoxyuridine and proliferating cell nuclear antigen immunohistochemical staining, and apoptosis, via transferase deoxytidyl uridine end labeling staining, in the polyps of metformin‐treated mice were not significantly different in comparison to those of control mice. Gene expression of cyclin D1 and c‐myc in intestinal polyps were also not significantly different between those two groups. In contrast, metformin activated AMPK in the intestinal polyps, resulting in the inhibition of the activation of mammalian target of rapamycin, which play important roles in the protein synthesis machinery. Metformin suppressed the polyp growth in ApcMin/+ mice, suggesting that it may be a novel candidate as a chemopreventive agent for colorectal cancer. (Cancer Sci 2008; 99: 2136–2141)

A Novel Role of Interleukin-13 Receptor α2 in Pancreatic Cancer Invasion and Metastasis

Whereas interleukin-13 receptor alpha2 chain (IL-13Ralpha2) is overexpressed in a variety of human solid cancers including pancreatic cancer, we investigated its significance in cancer invasion and metastasis. We used two pancreatic cancer cell lines, IL-13Ralpha2-negative HPAF-II and IL-13Ralpha2-positive HS766T, and generated IL-13Ralpha2 stably transfected HPAF-II as well as IL-13Ralpha2 RNA interference knocked-down HS766T cells. Ability of invasion and signal transduction was compared between IL-13Ralpha2-negative and IL-13Ralpha2-positive cells and tumor metastasis was assessed in murine model for human pancreatic cancer with orthotopic implantation of tumors. IL-13 treatment enhanced cell invasion in IL-13Ralpha2-positive cancer cell lines but not in IL-13Ralpha2-negative cell lines. Furthermore, gene transfer of IL-13Ralpha2 in negative cell lines enhanced invasion, whereas its silencing downmodulated invasion of pancreatic cell lines in a Matrigel invasion assay. In vivo study revealed that IL-13Ralpha2-positive cancer metastasized to lymph nodes, liver, and peritoneum at a significantly higher rate compared with IL-13Ralpha2-negative tumors. The expression of IL-13Ralpha2 in metastatic lesions was found to be increased compared with primary tumors, and mice with IL-13Ralpha2-positive cancer displayed cachexia and poor prognosis. Invasion and metastasis also correlated with increased matrix metalloproteinase protease activity in these cells. Mechanistically, IL-13 activated extracellular signal-regulated kinase 1/2 and activator protein-1 nuclear factors in IL-13Ralpha2-positive pancreatic cancer cell lines but not in IL-13Ralpha2-negative cell lines. Taken together, our results show for the first time that IL-13 can signal through IL-13Ralpha2 in pancreatic cancer cells and IL-13Ralpha2 may serve as a prognostic biomarker of invasion and metastasis in pancreatic cancer.

Predominant T helper type 2-inflammatory responses promote murine colon cancers

Colon cancer is one of the most serious complications of inflammatory bowel diseases, especially ulcerative colitis (UC). Previous studies have shown that characteristic immunological event during inflammation in UC is the expression of T helper‐type 2 (Th2) cell‐derived cytokines. In this study, we investigated the influence of a predominant Th2‐type cytokine response in colitis on carcinogen‐induced colon tumors. Wild type (WT), interferon gamma (IFN‐γ) gene deficient (−/−) [Th2 dominant] or interleukin (IL)‐4−/− [Th1‐dominant] mice of BALB/c background were used in this study. To compare tumor formation, mice were given the carcinogen azoxymethane (AOM) and intrarectal administration of trinitrobenzene sulfonic acid (TNBS), to induce colitis. Thirty‐three weeks after initial treatment, the total colon was examined. When IFN‐γ−/− mice were treated with AOM and TNBS, significantly higher number of tumors were seen (8.4 ± 1.7) than in WT (3.3 ± 2.9) or IL‐4−/− (3.1 ± 3.4) mice, which received identical treatments. A separate set of experiment, using less doses of AOM and TNBS also showed the higher frequency of tumor formation in IFN‐γ−/− mice than in IL‐4−/− mice. Histologically, the tumors were well‐ or moderately‐differentiated adenocarcinomas. No invasion into the submucosal or serosal layers of the intestine was seen. In immunohistological staining, some tumors in IFN‐γ−/− mice showed distinct nuclear expression of β‐catenin, in contrast to the strong membrane staining seen in tumors of IL‐4−/− mice. In conclusion, colonic inflammation associated with Th2‐donimant cytokine responses enhanced the formation of malignant neoplasms.

Involvement of JNK pathway in the promotion of the early stage of colorectal carcinogenesis under high-fat dietary conditions

Background and aims: The molecular mechanisms underlying the promotion of colorectal carcinogenesis by a high-fat diet (HFD) remain unclear. We investigated the role of the insulin-signal pathway and the c-Jun N-terminal kinase (JNK) pathway, which reportedly play crucial roles in insulin resistance, during colorectal carcinogenesis in the presence of hyperinsulinaemia induced by a HFD. Methods: Azoxymethane-induced aberrant crypt foci formation and cell proliferation in the colonic epithelium were compared between mice fed a normal diet (ND) and mice fed a HFD. A western blot analysis was performed to elucidate the mechanism affecting colorectal carcinogenesis by a HFD. Results: The number of aberrant crypt foci and the colonic epithelial cell proliferative activity were significantly higher in the HFD group than in the ND group. While the plasma insulin level was significantly higher in the HFD group than in the ND group, a western blot analysis revealed the inactivation of Akt, which is located downstream of the insulin receptor, in the colonic epithelia of the HFD group. On the other hand, JNK activity was significantly higher in the HFD group than in the ND group. A JNK specific inhibitor significantly suppressed the increase in epithelial cell proliferation only under a HFD, but not under a ND. Conclusions: Colonic cell proliferation was promoted via the JNK pathway in the presence of a HFD but not in the presence of a ND. This novel mechanism may explain the involvement of the JNK pathway in the effect of dietary fat intake on colon carcinogenesis.

5-Aminosalicylic Acid Given in the Remission Stage of Colitis Suppresses Colitis-Associated Cancer in a Mouse Colitis Model

Purpose: The risk of colorectal cancer is increased in patients with inflammatory bowel diseases, especially those with ulcerative colitis (UC). Although 5-aminosalicylic acid (5-ASA) is widely used in the treatment of UC to suppress the colitic inflammation, no studies have been conducted to examine the chemopreventive effect of 5-ASA, given in the remission phase of colitis, against colitis-associated cancer using animal models. We therefore investigated the possible inhibition by peroxisome proliferator-activated receptor-γ (PPARγ) ligands and 5-ASA of colitis-associated colon carcinogenesis in a mouse model. Experimental Design: A dextran sodium sulfate/azoxymethane–induced mouse colon cancer model was used, and the chemopreventive effects of 5-ASA and PPARγ ligands, given in the remission phase of colitis, against colitis-related colon carcinogenesis, were evaluated. Results: The number of neoplasms in the mice treated with 5-ASA was significantly lower than that in the control mice. In addition, the size of the neoplasms in treated mice was also significantly smaller than that in the control mice. In contrast, no significant suppression in the number or size of the tumors was observed in the mice treated with PPARγ ligands. The proliferating cell nuclear antigen–labeling index in the tumor cells of the 5-ASA–treated mice was significantly smaller than that in the control, indicating that 5-ASA reduced tumor cell proliferation. Conclusion: Our results revealed that 5-ASA given in the remission phase of colitis significantly suppressed the development of colitis-associated cancer in a mouse model, which indicates the clinical importance of adopting chemopreventive strategies even in UC patients in remission.

Clinical significance of swollen duodenal papilla in autoimmune pancreatitis.

Objectives: To evaluate the clinical significance of a swollen main duodenal papilla and the associated immunohistopathologic findings in patients with autoimmune pancreatitis (AIP). Methods: Seventeen consecutive patients with AIP registered between April 2001 and October 2005 who underwent both endoscopic retrograde cholangiopancreatography and endoscopic biopsy were enrolled in this study. The endoscopic features, stromal inflammatory cell infiltrate (SICI), and results of immunohistochemical examination of the duodenal papilla using IgG4, CD3, and CD79a antibodies were retrospectively reviewed. These findings in the AIP patients were compared with those in 12 patients with chronic alcoholic tumor-forming pancreatitis (CAP). The numbers of cells in the SICI and of IgG4-positive plasma cells per high-power field were counted in all the histopathologic specimens. Results: A swollen main duodenal papilla was observed in 11 (11 [64.7%]/17) patients with AIP and 4 (4 [33.3%]/12) patients with CAP (P < 0.05). Resolution of the swollen main duodenal papilla was observed in all of these 11 patients with AIP (11 [100%]/11) in response to treatment with corticosteroids. On the other hand, the 6 patients without elevated serum IgG4 or a swollen duodenal papilla, but with a swollen pancreas, improved even without corticosteroid treatment. The number of cells in the SICI in the AIP patients was significantly higher than that in the CAP patients. Although in 13 of 17 AIP patients, infiltration by IgG4-positive plasma cells was detected in the duodenal papilla, no such significant infiltration of the duodenal papilla by IgG4-positive plasma cells was observed in the patients with CAP (P < 0.05). More predominant T-cell infiltration of the duodenal papilla was recognized in the AIP patients than in the CAP patients (P < 0.05). Conclusions: These results suggest that a swollen main duodenal papilla with IgG4-positive plasma cell and T-cell-dominant infiltration and an abundant stromal cell infiltrate are characteristic findings in AIP. We suggest that these findings may be valuable adjuncts to the diagnosis of AIP as well as for selecting suitable candidates for corticosteroid therapy.

Interleukin 13 Mediates Signal Transduction through Interleukin 13 Receptor α2 in Pancreatic Ductal Adenocarcinoma: Role of IL-13 Pseudomonas Exotoxin in Pancreatic Cancer Therapy

Purpose: Interleukin-13 receptor α2 (IL-13Rα2) is a tumor antigen that is overexpressed in certain human tumors. However, its significance and expression in pancreatic cancer is not known. It is also not known whether IL-13 can signal through IL-13Rα2 in cancer. Experimental Design: The expression of IL-13Rα2 was assessed in pancreatic cancer samples by immunohistochemistry and in cell lines by flow cytometry and reverse transcription-PCR. The role of IL-13Rα2 was examined by IL-13–induced signaling in pancreatic cancer cell lines. IL-13Rα2–positive tumors were targeted by IL-13PE cytotoxin in vitro and in vivo in an orthotopic murine model of human pancreatic cancer. Results: Of the pancreatic tumor samples 71% overexpressed moderate to high-density IL-13Rα2 chain compared with normal pancreatic samples. IL-13 induced transforming growth factor-β1 promoter activity in IL-13Rα2–positive tumor cells and in cells engineered to express IL-13Rα2 but not in IL-13Rα2–negative or RNA interference knockdown cells. c-Jun and c-Fos of the AP-1 family of nuclear factors were activated by IL-13 only in IL-13Rα2–positive cells. In the orthotopic mouse model, IL13-PE significantly decreased tumor growth when assessed by whole-body imaging and prolonged the mean survival time. Similar results were observed in mice xenografted with a surgically resected human pancreatic tumor sample. Conclusions: These results indicate that IL-13Rα2 is a functional receptor as IL-13 mediates signaling in human pancreatic cancer cell lines. IL-13 causes transforming growth factor-β activation via AP-1 pathway, which may cause tumor induced immunosuppression in the host. In addition, IL13-PE cytotoxin may be an effective therapeutic agent for the treatment of pancreatic cancer. Clin Cancer Res; 16(2); 577–86

Inhibition of peroxisome proliferator-activated receptor gamma activity in esophageal carcinoma cells results in a drastic decrease of invasive properties.

Esophageal cancer is difficult to treat because of its rapid progression, and more effective therapeutic approaches are needed. The PPARγ is a nuclear receptor superfamily member that is expressed in many cancers. PPARγ expression is a feature of esophageal cancer cell lines, and in the present investigation, the PPARγ antagonists T0070907 and GW9662 could induce loss of invasion but could not induce growth reduction or apoptosis at low concentrations (<10 mM). A high concentration of antagonists (50 µM) inhibited cell growth and induced apoptosis, but these effects did not explain our result at the low concentration. Morphological change, decreased expression of the cell signaling pathway and inhibition of cancer cell invasion were observed in the low concentration. This suggested that PPARγ antagonists inhibited esophageal cancer cell invasion as well as cell adherence, most likely due to alteration in the FAK–MAPK pathway, and this was independent of apoptosis. These results suggested that PPARγ plays an important role in cancer cell invasion and that it might be a novel target for therapy of esophageal cancer. (Cancer Sci 2006; 97: 854–860)

Characterization of K-ras gene mutations in association with mucinous hypersecretion in intraductal papillary-mucinous neoplasms

BACKGROUND/PURPOSE Intraductal papillary-mucinous neoplasms (IPMNs) of the pancreas have a favorable prognosis. However, invasive ductal carcinomas of the pancreas show a rapid progression. The aim of this study was to investigate gene mutations in pure pancreatic juice from IPMN patients and to define these genetic mutations in relation to the histopathological and clinical features of IPMNs. METHODS Twenty-two patients with IPMN, 21 patients with ductal carcinoma, and 20 patients with normal pancreas or chronic pancreatitis were recruited for this study. We measured the main pancreatic ducts largest diameter and the maximum size of a dilated branch was assessed by ultrasonography or endoscopic ultrasonography. Pure pancreatic juice was collected and was investigated for K-ras, p16, and p53 mutations. RESULTS Mutant K-ras gene was detected in 13 of the 22 patients (59.1%) with IPMNs. Different kinds of mutations were detected in the same patient in 4 cases. In the 13 patients with mutant K-ras gene, the diameter of the most dilated part of the main pancreatic duct was 2-8 mm (average, 4.5 mm) and in 7 patients with wild-type K-ras gene, the diameter was 2-5 mm (average, 2.7 mm). There was a significant difference in the diameter of the main pancreatic duct between patients with and without the mutant K-ras gene (P = 0.0323). CONCLUSIONS The incidence of K-ras mutation may be associated with the hypersecretion of mucin.