Koichiro Wada

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.

Peroxisome Proliferator-activated Receptor γ-mediated Regulation of Neural Stem Cell Proliferation and Differentiation

Peroxisome proliferator-activated receptor γ (PPARγ) plays an important role in insulin sensitivity, tissue homeostasis, and regulating cellular functions. We found high-level expression of PPARγ in embryo mouse brain and neural stem cells (NSCs), in contrast to extremely low levels in adult mouse brain. Here, we show that PPARγ mediates the proliferation and differentiation of murine NSCs via up-regulation of the epidermal growth factor receptor and activation of the ERK pathway. Cell growth rates of NSCs prepared from heterozygous PPARγ-deficient mouse brains, PPARγ-RNA-silenced NSCs, and PPARγ dominant-negative NSCs were significantly decreased compared with those of wild-type NSCs. Physiological concentrations of PPARγ agonists, rosiglitazone and pioglitazone, stimulated NSC growth, whereas antagonists caused cell death in a concentration-dependent manner via activation of the caspase cascade. The stimulation of cell growth by PPARγ was associated with a rapid activation of the ERK pathway by phosphorylation and up-regulation of epidermal growth factor receptor and cyclin B protein levels. In contrast, activation of PPARγ by agonists inhibited the differentiation of NSCs into neurons. The inhibition of differentiation was associated with an activation of STAT3. These data indicate that PPARγ regulates the development of the central nervous system during early embryogenesis via control of NSC proliferation.

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)

Leukotriene B4 and lipoxin A4 are regulatory signals for neural stem cell proliferation and differentiation

Leukotrienes (LTs) and lipoxins (LXs) are lipid mediators that play a key role in regulating acute inflammatory responses. Their roles in neural stem cell (NSC) functions are of interest. We showed here that LTB4 and LXA4 regulated proliferation and differentiation of murine NSCs that were isolated from embryo brains. Proliferation of NSCs was stimulated by LTB4 (3 to 100 nM) and blocked by receptor antagonist (IC50=2.7 µM). In contrast, LXA4, and its aspirin‐triggered‐15‐epi‐LXA4 stable analog attenuated growth of NSCs at as little as 1 nM. Both lipoxygenase (LOX) inhibitors and LTB4 receptor antagonists caused apoptosis and cell death. Gene chip analysis revealed that growth‐related gene expressions such as epidermal growth factor (EGF) receptor, cyclin E, p27, and caspase 8 were tightly regulated by LTB4; LXA4 gave the opposite gene expressions. In addition to proliferation, LTB4 induced differentiation of NSCs into neurons as monitored by neurite outgrowth and MAP2 expression. These results indicate for the first time that LTB4 and LXA4 directly regulate proliferation and differentiation of NSCs, suggesting these new pathways may be useful in restoring stem cells.—Wada, K., Arita, M., Nakajima, A., Katayama, K., Kudo, C., Kamisaki, Y., Serhan, C. N. Leukotriene B4 and lipoxin A4 are regulatory signals for neural stem cell proliferation and differentiation. FASEB J. 20, 1785–1792 (2006)

Aspirin-triggered lipoxin A4 and lipoxin A4 up-regulate transcriptional corepressor NAB1 in human neutrophils

Aspirin‐triggered 15‐epi‐lipoxin A4 (ATL) is an endogenous lipid mediator that mimics the actions of native lipoxin A4, a putative “stop signal” involved in regulating resolution of inflammation. A metabolically more stable analog of ATL, 15‐epi‐16‐(para‐fluoro)‐phenoxy‐lipoxin A4 analog (ATLa), inhibits neutrophil recruitment in vitro and in vivo and displays potent anti‐inflammatory actions. ATLa binds with high affinity to the lipoxin A4 receptor, a G protein‐coupled receptor on the surface of leukocytes. In this study, we used freshly isolated human neutrophils to examine ATLas potential for initiating rapid nuclear responses. Using differential display reverse transcription polymerase chain reaction, we identified a subset of genes that was selectively up‐regulated upon short exposure of polymorphonuclear leukocytes to ATLa but not to the chemoattractant leukotriene B4 or vehicle alone. We further investigated ATLa regulation of one of the genes, NAB1, a transcriptional corepressor identified previously as a glucocorticoid‐responsive gene in hamster smooth muscle cells. Treatment of human neutrophils with pertussis toxin blocked ATLa up‐regulation of NAB 1. In addition, ATLa stimulated NAB1 gene expression in murine lung vascular smooth muscle in vivo. These findings provide evidence for rapid transcriptional induction of a cassette of genes via an ATLa‐stimulated G protein‐coupled receptor pathway that is potentially protective and overlaps with the anti‐inflammatory glucocorticoid regulatory circuit.

Effectiveness of antiplatelet drugs against experimental non-alcoholic fatty liver disease

Objective: No effective drugs have been developed to date to prevent or treat non-alcoholic fatty liver disease (NAFLD), although diet modification and exercise to improve obesity have been attempted. Therefore, development of a novel drug/strategy to treat NAFLD is urgently needed. In the present study, a novel concept is proposed for the treatment of NAFLD. Methods: Fisher 344 male rats were given a choline-deficient, l-amino acid-defined (CDAA) diet or a high-fat high-calorie (HF/HC) diet with or without the antiplatelet agents, aspirin, ticlopidine or cilostazol for 16 weeks. Liver steatosis, inflammation and fibrosis, and the possible mechanisms involved were investigated. Results: All three antiplatelet drugs, namely aspirin, ticlopidine and cilostazol, significantly attenuated liver steatosis, inflammation and fibrosis in the CDAA diet group. Of the three agents, cilostazol was the most effective, and the drug also suppressed HF/HC diet-induced liver steatosis. Cilostazol appeared to exert its beneficial effect against NAFLD by suppressing mitogen-activated protein kinase activation induced by oxidative stress and platelet-derived growth factor via intercepting signal transduction from Akt to c-Raf. Conclusion: Antiplatelet agents, especially cilostazol, offer the promise of becoming key agents for the treatment of NAFLD.

Telmisartan, An Angiotensin II Type 1 Receptor Blocker, Controls Progress of Nonalcoholic Steatohepatitis in Rats

The term nonalcoholic steatohepatitis (NASH) has recently been proposed to identify a fatty liver disease accompanied by diffuse fatty infiltration and inflammation. However, no drug therapy has been established for NASH as yet. In the present study, we demonstrate the effect of the angiotensin II type 1 receptor antagonist telmisartan on the development of NASH in a rat model. Telmisartan, but not the angiotensin receptor antagonist valsartan, markedly attenuated hepatic steatosis, inflammation, and fibrosis in these rats. The quantitative parameters of steatosis, inflammation, and fibrosis were also ameliorated by treatment with telmisartan. Compared with telmisartan, the peroxisome proliferator-activated receptor-γ agonist pioglitazone attenuated hepatic steatosis and fibrosis of the liver to a similar degree. However, telmisartan, but not pioglitazone, dramatically decreased both subcutaneous and visceral fat. In conclusion, these results indicated that telmisartan should be the drug of first choice for the treatment of patients with NASH.

Association between PPARGC1A polymorphisms and the occurrence of nonalcoholic fatty liver disease (NAFLD)

BackgroundGenetic factors as well as environmental factors are important in the development of NAFLD and in this study we investigated associations between polymorphisms of peroxisome proliferators-activated receptor γ coactivator 1α polymorphism (PPARGC1A) and NAFLD.AimsWe recruited 115 patients with biopsy-proven NAFLD, 65 with NASH and 50 with simple steatosis, and 441 healthy control subjects and investigated 15 SNPs of PPARGC1A.ResultsSNP rs2290602 had the lowest p value in the dominant mode (p = 0.00095), and the odds ratio for NAFLD (95% CI) was 2.73 (1.48 – 5.06). rs2290602 was significantly associated with NAFLD even when the most conservative Bonferronis correction was applied (p = 0.0143). The frequency of the T allele of rs2290602 was significantly higher in the NASH patients than in the control subjects (p = 0.00093, allele frequency mode), and its frequency in the NASH patients tended to be higher than in the simple steatosis patients (p = 0.09). The results of the real-time RT-PCR study showed that intrahepatic mRNA expression of PPARGC1A was lower in the TT group than in the GG or GT group at SNP rs2290602 (p = 0.0454).ConclusionThis is the first study to demonstrate a significant association between genetic variations in PPARGC1A and NAFLD. This finding suggested that PPARGC1A polymorphism and lower expression of PPARGC1A mRNA in the liver are an important genetic contribution to etiology of NAFLD.

Critical role of estrogen receptor on anoikis and invasion of squamous cell carcinoma

Estrogen receptor (ER) plays an important role in various physiological functions. We examined whether ERα and ERβ are expressed in squamous cell carcinoma (SCC), and whether ER is a potential target for antitumor therapy. High‐level expression of ERβ, but not ERα, was observed in tumor cells of human primary SCC tissues and various SCC cultured cell lines. Treatment with ER antagonist (tamoxifen), but not agonist (estradiol), caused apoptotic cell death of SCC cells in a concentration‐ and time‐dependent manner. Adhesion of SCC was inhibited by the treatment with tamoxifen, but not with estradiol. Tamoxifen reduced the phosphorylation of focal adhesion kinase (FAK), resulting in decreases in phosphorylation of extracellular signal‐related kinase (Erk) and mitogen‐activated protein kinase. Inhibition of FAK phosphorylation is accompanied by disorder of the cytoskeletal component actin. The cell death caused by tamoxifen is therefore the result of direct interference in cell adhesion, which is called ‘anoikis’, involving a decrease in intracellular FAK signaling. Expression of epidermal growth factor receptor was also inhibited by treatment with a high concentration of tamoxifen. Knockdown of ERβ by small interfering RNA inhibited the proliferation of SCC. In addition, tamoxifen strongly inhibited invasion of SCC. These results imply a potentially important role for ER, whose inhibition may be effective for the treatment of SCC and the prevention of invasion and metastasis. (Cancer Sci 2007; 98: 636–643)

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.