Yoshiji Miyazaki

Peroxisome Proliferator-activated Receptor γ Induces Growth Arrest and Differentiation Markers of Human Colon Cancer Cells

Peroxisome proliferator‐activated receptor γ (PPARγ), one of the nuclear receptors expressed in adipose tissue, plays an important role in adipocyte differentiation. In this study, we investigated the expression of PPARγ and its role in cellular growth and differentiation in six colon cancer cell lines: HT‐29, CaCo‐2, SW‐480, DLD‐1, LoVo, and T‐84. All six expressed PPARγ mRNA and protein, shown respectively on northern and western blot analyses. Luciferase assay in HT‐29 cells, which strongly express PPARγ showed that troglitazone, a selective ligand for PPAR?, transacti‐vated the transcription of a peroxisome proliferator response element (PPRE)‐driven promoter. Furthermore, troglitazone caused a marked decrease in [3H] thymidine incorporation and G1 cell‐cycle arrest determined by flow cytometry. Finally, troglitazone induced expression of mRNAs for villin and intestinal alkaline phosphatase, markers for enterocyte differentiation. In conclusion, human colon cancer cells express PPARγ, the ligands of which inhibit cell growth and induce differentiation markers.

Gastrin Induces Heparin-Binding Epidermal Growth Factor-like Growth Factor in Rat Gastric Epithelial Cells Transfected With Gastrin Receptor

BACKGROUND & AIMS Parietal cells express heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). However, it is unknown whether HB-EGF mediates the trophic action of gastrin. The purpose of this study was to determine whether gastrin modulates the expression of HB-EGF, which mediates the proliferative effects of gastrin on gastric epithelial cells. METHODS RGM1 cells, a rat gastric epithelial cell line, were transfected with a human gastrin receptor complementary DNA. Gastrin induction of messenger RNAs (mRNAs) for EGF-related polypeptides was assayed by Northern blotting. Processing of cell surface-associated proHB-EGF and secretion of HB-EGF were determined by flow cytometry and Western blotting, respectively. Tyrosine phosphorylation of the EGF receptor was assayed by immunoprecipitation and Western blotting with an antiphosphotyrosine antibody. Cell growth was evaluated by [3H]thymidine incorporation. RESULTS Gastrin induced expression of HB-EGF mRNA, processing of proHB-EGF, release of HB-EGF into the medium, and tyrosine phosphorylation of the EGF receptor. The growth-stimulatory effects of gastrin were partly inhibited by anti-rat HB-EGF serum and completely blocked by AG1478, an EGF receptor-specific tyrphostin. CONCLUSIONS The findings suggest that HB-EGF at least partially mediates the proliferative effects of gastrin on gastric epithelial cells.

Deficiency of KIT-positive cells in the colon of patients with diabetes mellitus.

Diabetes mellitus is a well‐known cause of gastrointestinal dysmotility. The pathogenesis of diabetic gastroenteropathy is mainly considered to be a neuropathy, but the cause of dysmotility remains unknown. Interstitial cells of Cajal (ICC), which express c‐kit receptor tyrosine kinase (KIT), are considered to be pacemaker cells for the gastrointestinal movement. Therefore, we investigated a possible involvement of ICC in the pathogenesis of diabetic gastroenteropathy in humans.

Peroxisome proliferator-activated receptor γ reduces the growth rate of pancreatic cancer cells through the reduction of cyclin D1

Peroxisome proliferator-activated receptor gamma (PPARgamma) forms a heterodimeric DNA-binding complex with the retinoid X receptor (RXR) and regulates the transcription of its target genes. Activation of PPARgamma has been shown to induce G1 arrest and to inhibit cell growth of human pancreatic carcinoma cell lines. The purpose of the present study was to examine the effect of ligand activation of PPARgamma and RXR on cell growth and on the expression of G1 cyclins in a pancreatic cancer cell line PANC-1, which expresses PPARgamma at high levels. Troglitazone, a specific ligand for PPARgamma, was found to cause a reduction in the growth rate and induced G1 cell cycle arrest and this effect was additive with that of 9-cis retinoic acid (9-cis RA), a ligand for RXR. Of the G1 cyclins tested, troglitazone specifically reduced the expression of cyclin D1 mRNA and the corresponding protein and this effect was also additive with 9-cis RA. These results suggest that the activation of PPARgamma together with RXR may be useful for the suppression of pancreatic cancer cell growth through the reduction in cyclin D1 levels.

Cd9-mediated activation of the p46 Shc isoform leads to apoptosis in cancer cells

CD9, a member of the tetraspanin family, has been shown to be involved in a range of cellular activities, including migration, proliferation and adhesion, but the molecular mechanisms by which it mediates such events is unclear. Here, we found that anti-CD9 monoclonal antibody ALB6 inhibited cell proliferation, reduced cell viability and induced not only morphological changes specific to apoptosis but also molecular changes, as evidenced by TUNEL and annexin-V staining. For the possible mechanism of ALB6-induced apoptosis, ALB6 activated the c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 mitogen-activated-protein kinase (MAPK) within 5-15 minutes, as well as caspase-3 within 24-48 hours. It is noteworthy that ALB6 induced tyrosine phosphorylation of the p46 Shc isoform specifically and that the overexpression of its dominant-negative form completely suppressed the ALB6-induced activation of JNK/SAPK, p38 MAPK and caspase-3, resulting in the inhibition of apoptotic cell death. These results suggest that CD9 might regulate apoptosis through the specialized signals in human cancer cell lines.

Helicobacter pylori -induced enlarged-fold gastritis is associated with increased mutagenicity of gastric juice, increased oxidative DNA damage, and an increased risk of gastric carcinoma

Background and Aim:  The severe inflammation, increased cell proliferation and marked acid inhibition observed in subjects with Helicobacter pylori‐associated enlarged‐fold gastritis suggest that enlarged‐fold gastritis may be a risk factor for gastric carcinoma. The purpose of the present study was to determine whether a relationship exists between enlarged‐fold gastritis and gastric carcinoma.

PPARγ agonists inhibit cell growth and suppress the expression of cyclin D1 and EGF‐like growth factors in ras‐transformed rat intestinal epithelial cells

Peroxisome proliferator‐activated receptor γ (PPARγ) inhibits the growth of several types of cancer cells. However, the mechanisms by which this occurs are poorly understood. The goal of the present study was to investigate the effects of PPARγ on mutated ras‐induced cell growth, activation of transcription factors and expression of genes associated with cellular transformation in rat intestinal epithelial cells. A human PPARγ cDNA was introduced to the activated H‐ras‐transfected IEC‐6 cells (IECras) and 1 clone (IECrasPR82) that stably expresses both activated ras and PPARγ was obtained. Thiazolidinedione derivatives such as troglitazone and rosiglitazone, selective ligands for PPARγ, inhibited the cellular growth of IECrasPR82 cells in a time‐dependent manner and induced G1 cell cycle arrest. Treatment with troglitazone (20 μM) decreased the expression of cyclin D1, heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) and amphiregulin and suppressed the promoter activities of cyclin D1 and HB‐EGF. Furthermore, a luciferase assay and an electrophoretic mobility shift assay showed that thiazolidinedione derivatives suppressed the transcriptional activities of AP‐1 and Ets, both of which play crucial roles in the expression of cyclin D1 and HB‐EGF. These findings suggest that reduction of EGF‐like growth factors and cyclin D1 through the suppression of AP‐1 and Ets may be 1 mechanism whereby PPARγ inhibits their growth.

Significance of the association between heparin-binding epidermal growth factor-like growth factor and CD9 in human gastric cancer.

Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) is a member of the EGF family. Juxtacrine activity of proHB‐EGF (the membrane‐anchored form of HB‐EGF) has been shown to be significantly potentiated when it is coexpressed with CD9 in vitro. The purpose of our study was to investigate the issue of whether proHB‐EGF and CD9 are coexpressed in gastric cancer. HB‐EGF gene expression and protein production in human gastric cancers was investigated, and EGF receptor and CD9 expressions were also evaluated. HB‐EGF mRNA levels in gastric cancers were elevated, compared with normal gastric tissues, especially in the intestinal type. ProHB‐EGF immunoreactivity was detected primarily in the cytoplasm and plasma membrane of gastric cancer cells. Of 66 patients, 40 (60.6%) exhibited proHB‐EGF immunoreactivity and the level of its expression was significantly associated with tumor status (p < 0.01) and histological differentiation (p < 0.001). In addition, proHB‐EGF mRNA was detected at high levels in the intestinal type by in situ hybridization. CD9 immunoreactivity was found to be preserved in 26 of 36 patients (72.2%) and CD9 protein expression was inversely associated with lymph node status (p < 0.05). A significant correlation between its expression and histological differentiation (p < 0.01) was found, and the association of CD9 with proHB‐EGF was increased in the intestinal type, as evidenced by an immunoprecipitation method. These results indicate that the coexpression of proHB‐EGF and CD9 may be involved in the tumorigenesis and/or proliferation of gastric cancers in a juxtacrine manner.

An increased number of CD40-high monocytes in patients with Crohn’s disease

OBJECTIVE:CD40-CD40 ligand (CD40L) interaction is essential for the T-lymphocyte–dependent immune response. This interaction may be operational in the pathogenesis of inflammatory bowel diseases (IBD). The present study examined the expression of CD40 in peripheral blood mononuclear cells (PBMNCs) and tissue specimens, and CD40-stimulated interleukin (IL)-12 release from PBMNCs in IBD.METHODS:The expression of CD40 in PBMNCs and tissue inflammatory cells was examined by flowcytometry and immunohistochemistry, respectively. IL-12 release was measured in cultured media of PBMNCs by an enzyme-linked immunosorbent assay.RESULTS:Most peripheral blood B-lymphocytes expressed CD40 in all subjects. However, in ulcerative colitis (UC) patients, a significantly increased mean fluorescence intensity (MFI) of CD40 on B-lymphocytes was detected, compared with control subjects and patients with Crohns disease (CD). In contrast, both the percentage positivity and MFI of CD40 on monocytes of active CD subjects were significantly increased, compared with the other groups. In active CD patients, a high level of IL-12 release from PBMNCs was observed by CD40 stimulation, compared with those of the other groups. When primed with IFN-γ, PBMNCs from inactive CD patients released a significantly high level of IL-12, probably via stimulation by the CD40 monoclonal antibody. In the affected mucosa of CD, numerous CD40-positive cells were demonstrated, and they were also CD68-positive, suggesting these double CD40/CD68-positive cells are tissue macrophages.CONCLUSIONS:These results suggest that the examination of CD40 expression in PBMNCs might enable the differentiation of CD from UC. CD40-high monocytes in CD patients may play a role in the pathogenesis of CD.

Effect of cholecystokinin receptor antagonists, MK-329 and L-365,260, on cholecystokinin-induced acid secretion and histidine decarboxylase activity in the rat

To elucidate the regulatory mechanism of acid secretion by cholecystokinin (CCK) in vivo, we compared the effects of CCK and gastrin on acid secretion and histidine decarboxylase (HDC) activity. We also examined the effects of MK-329, a specific antagonist for pancreatic-type CCK receptor, and L-365,260, a specific antagonist for gastrin-type CCK receptor, on the action of CCK. Graded doses of CCK or gastrin were intravenously infused into conscious rats with gastric fistula. Gastrin-17 I infusion up to 10 nmol/kg/h resulted in dose-related increases in acid secretion. CCK-8 infusion also caused an increase in acid secretion. However, it reached a peak with 0.3 nmol/kg/h CCK-8 and attenuated with higher concentrations of CCK-8. This attenuating effect of a higher dose of CCK was reversed by MK-329, but not by L-365,260. Both CCK and gastrin were potent in increasing fundic HDC activity, and the effect of CCK on HDC activity was significantly inhibited by L-365,260, but not by MK-329. Taken together, the present study suggests that CCK and gastrin stimulate histamine formation via a gastrin-type CCK receptor, and the attenuating action of CCK with higher concentrations on acid secretion in vivo is mediated by a pancreatic-type CCK receptor.