Tomohiko Mori

N-Cadherin Expression and Epithelial-Mesenchymal Transition in Pancreatic Carcinoma

Purpose: Loss of intercellular adhesion and increased cell motility promote tumor cell invasion. In the present study, E- and N-cadherin, members of the classical cadherin family, are investigated as inducers of epithelial-to-mesenchymal transition (EMT) that is thought to play a fundamental role during the early steps of invasion and metastasis of carcinomas. Cell growth factors are known to regulate cell adhesion molecules. The purpose of the study presented here was to investigate whether a gain in N-cadherin in pancreatic cancer is involved in the process of metastasis via EMT and whether its expression is affected by growth factors. Experimental Design: We immunohistochemically examined the expression of N- and E-cadherins and vimentin, a mesenchymal marker, in pancreatic primary and metastatic tumors. Correlations among the expressions of N-cadherin, transforming growth factor (TGF)β, and fibroblast growth factor 2 was evaluated in both tumors, and the induction of cadherin and vimentin by growth factors was examined in cultured cell lines. Results: N-cadherin expression was observed in 13 of 30 primary tumors and in 8 of 15 metastatic tumors. N-cadherin expression correlated with neural invasion (P = 0.008), histological type (P = 0.043), fibroblast growth factor expression in primary tumors (P = 0.007), and TGF expression (P = 0.004) and vimentin (P = 0.01) in metastatic tumors. Vimentin, a mesenchymal marker, was observed in a few cancer cells of primary tumor but was substantially expressed in liver metastasis. TGF stimulated N-cadherin and vimentin protein expression and decreased E-cadherin expression of Panc-1 cells with morphological change. Conclusion: This study provided the morphological evidence of EMT in pancreatic carcinoma and revealed that overexpression of N-cadherin is involved in EMT and is affected by growth factors.

In vivo antitumor effect of the mTOR inhibitor CCI‐779 and gemcitabine in xenograft models of human pancreatic cancer

Mammalian target of rapamycin (mTOR) is considered to be a major effector of cell growth and proliferation that controls protein synthesis through a large number of downstream targets. We investigated the expression of the phosphatidylinositol 3′‐kinase (PI3K)/mTOR signaling pathway in human pancreatic cancer cells and tissues, and the in vivo antitumor effects of the mTOR inhibitor CCI‐779 with/without gemcitabine in xenograft models of human pancreatic cancer. We found that the Akt, mTOR and p70 S6 kinase (S6K1) from the PI3K/mTOR signaling pathway were activated in all of the pancreatic cancer cell lines examined. When surgically resected tissue specimens of pancreatic ductal adenocarcinoma were examined, phosphorylation of Akt, mTOR and S6K1 was detected in 50, 55 and 65% of the specimens, respectively. Although CCI‐779 had no additive or synergistic antiproliferative effect when combined with gemcitabine in vitro, it showed significant antitumor activity in the AsPC‐1 subcutaneous xenograft model as both a single agent and in combination with gemictabine. Furthermore, in the Suit‐2 peritoneal dissemination xenograft model, the combination of these 2 drugs achieved significantly better survival when compared with CCI‐779 or gemcitabine alone. These results demonstrate promising activity of the mTOR inhibitor CCI‐779 against human pancreatic cancer, and suggest that the inhibition of mTOR signaling can be exploited as a potentially tumor‐selective therapeutic strategy.

Endogenous decoy receptor 3 blocks the growth inhibition signals mediated by Fas ligand in human pancreatic adenocarcinoma

Many cancers are resistant to Fas‐mediated apoptosis despite the expression of Fas. To investigate the mechanisms by which Fas signals are attenuated, we focused on decoy receptor 3 (DcR3). DcR3 is a soluble receptor against Fas ligand belonging to the tumor necrosis factor receptor superfamily and overexpresses in some forms of cancers. Exogenous DcR3 inhibits Fas‐mediated apoptosis in Fas‐sensitive Jurkat cells. In our study, we examined the expression and function of DcR3 in pancreatic cancers. TaqMan RT‐PCR showed that DcR3 mRNA was highly expressed in pancreatic cancer cell lines (71%) and tissues (67%). Its expression significantly correlated with cancer invasion to veins. Western blotting showed that the DcR3 protein was produced and secreted in 4 of 6 cell lines. The protein expressions were compatible with the mRNA expression. Five of 7 pancreatic cancer cell lines became sensitive to agonistic anti‐Fas antibody (CH‐11) to various extents, without Fas upregulation, when exposed to CH‐11 for 48 hr after pretreatment with IFNγ. Four of 7 pancreatic cancer cell lines were inhibited from growing, compared to control cells, when cocultured with membrane‐bounded Fas ligand (mFasL) transfected lymphomas for 48 hr after pretreatment with IFNγ. DcR3 reduced this growth inhibition when added exogenously. Regression analysis showed that the DcR3 expression significantly correlated with the sensitivity to mFasL, and not to CH‐11. These results suggest that DcR3 is highly expressed in many pancreatic cancers and endogenous DcR3 blocks the growth inhibition signals mediated by mFasL. DcR3 can be a candidate target molecule for the therapeutic intervention.

Clinical Significance of Focal Adhesion Kinase in Resectable Pancreatic Cancer

Focal adhesion kinase (FAK) is a non-receptor, cytoplasmic protein tyrosine kinase that is involved in the regulation of cellular signaling, migration, apoptosis, and cell cycle progression. Previous reports have shown that FAK is expressed in various kinds of cancer tissues and cancer cell lines; however, no information is available about human pancreatic carcinoma specimens. Tissue such specimens were obtained from 50 patients who underwent pancreatic resection for pancreatic invasive ductal carcinoma at our institute from 1996 to 2002. Immunohistochemical analysis of FAK was performed in the resected specimens. Focal adhesion kinase expression in seven human pancreatic cancer cell lines was analyzed by reverse transcription polymerase chain reaction (PCR) analysis and Western blot analysis. Focal adhesion kinase expression was detected in 24 of 50 cases (48%). There was a statistically significant correlation between FAK expression and tumor size (P = 0.004), although FAK expression did not significantly correlate with other factors such as tumor histological grade, lymph node metastasis, distant metastasis, histological stage, and overall survival. Reverse transcription PCR analysis and Western blot analysis showed that FAK was expressed in all seven pancreatic cancer cell lines. Focal adhesion kinase expression was not directly related to clinicopathological factors except tumor size in pancreatic carcinoma. Focal adhesion kinase expression may not be a prognostic marker for pancreatic cancer patients.

Enhanced susceptibility to transglutaminase reaction of α-lactalbumin in the molten globule state

The susceptibility of alpha-lactalbumin to transglutaminase reactions was studied using an enzyme from Streptoverticillium which can catalyze the reactions irrespective of the presence or absence of Ca2+. Transglutaminase-catalyzed polymerization of alpha-lactalbumin in the native state occurred to a very limited extent. Transformation from the native state to the molten globule state brought about by Ca(2+)-removal from holo-alpha-lactalbumin enhanced the polymerization of the protein catalyzed by transglutaminase. The incorporation of Carbobenzoxy-Gln-Gly into alpha-lactalbumin through the enzyme reaction was investigated to determine the amounts of lysine residues which are present at molecular surface and available to the enzyme. There was no significant difference in the amount of available lysine residues between the native and the molten globule molecule. However, the amount of surface glutamine residues incorporated with monodansylcadaverine by transglutaminase was remarkably higher in the molten globule state than that in the native state. The monodansylcadaverine-incorporated site of alpha-lactalbumin in the molten globule state was identified as Gln-54 by amino-acid sequence analysis of fluorescence-labeled peptides separated from chymotryptic digests of the protein. Possible reason for selective labeling of Gln-54 in molten globule alpha-lactalbumin was proposed.

Characterization of texture and mechanical properties of heat-induced soy protein gels

Heat-set gels were prepared from acid-precipitated soybean proteins at various heating temperatures (80–100°C), protein concentrations (18–20%), and proportions of glycinin. The gels were evaluated for mechanical parameters by means of a compression-decompression test. Gels formed at higher heating temperature and protein concentration were firm, tough and unfracturable. The elasticities of the gels were similar at all protein concentrations and were lower when heated at higher temperature. Heating above 93°C was necessary for formation of rigid gels. The glycinin/β-conglycinin ratio affected the texture of the gels. Three-dimensional representation of the gels through factor analysis of instrumental data and calculation of factor scores was useful to evaluate the texture of the gels.

Inhibitory effects of peptide-bound polysaccharides on lipid oxidation in emulsions

Abstract The inhibitory effects of polysaccharides on lipid oxidation in emulsions were investigated. Methyl linoleate was emulsified by β-casein or surfactants to prepare emulsions, and the oxidation was induced by an azo-compound or FeSO4 addition. The initial oxidation was followed by measuring the oxygen consumption in the emulsions for 30 min, and the extent of the lipid oxidation over a long time (∼48 h) was evaluated by the determination of the unoxidized lipid by gas chromatography and the thiobarbituric acid test. Peptide-bound polysaccharides, such as gum arabic and soluble soybean polysaccharides (SSPS), showed an ability to inhibit lipid oxidation, whereas maltodextrin and pullulan exhibited no inhibitory effects. Especially, SSPS almost perfectly suppressed the lipid oxidation from the initial to the late stages, irrespective of the emulsifiers and oxidation-inducing reagents. The mechanism whereby the peptide-bound polysaccharides inhibited the lipid oxidation in the emulsions is considered in terms of the chemical composition and other properties of the polysaccharides.

Filler effects of oil droplets on the viscoelastic properties of emulsion gels

Abstract Filler effects of oil droplets on the viscoelastic properties of emulsion gels were investigated by small deformation mechanical measurements. The emulsions were made with soya oil and soy 11S globulin (15 wt% oil, 1.5–7% protein concentrations). The emulsion and soy 11S globulin solutions were gelled using Ca ++ -independent transglutaminase from a microorganism. The shear storage modulus (G′) and loss modulus (G″) of the proteinous gels and emulsion gels depended on the protein concentration. The concentration dependence of the modulus was approximated by an exponential function of the form: G ∝ C n , n = 4 and n = 2 being found in the cases of the proteinous gels and the emulsion gels, respectively. Shear moduli of the emulsion gels were much higher than those of the proteinous gels. Gels made from a fine emulsion (containing smaller oil droplets) exhibited higher G′ and G″ values than corresponding gels from a coarse emulsion. Addition of Tween 20 was found to reduce G′ and G″ of emulsion gels even when the protein matrix was not significantly displaced from the oil—water interface by the surfactant.

Heterogeneity of soybean glycinin

Abstract Glycinin from soybean var. Tsuru-no-ko was fractionated on a DEAE-Sephadex A-50 column and the molecular species were investigated by electrophoretic methods. The results obtained indicated the heterogeneity of glycinin molecular species. Four molecular species of apparent MWs 340000, 345000, 360000 and 375000 were detected by gradient gel electrophoresis. A similar heterogeneity was observed in the half-molecule of glycinin, that is, three molecular species of apparent MWs 199000, 211000 and 222000 were detected. Other cultivars which have different subunit compositions from that of Tsuru-no-ko also exhibit a similar heterogeneity of glycinin molecular species. These results together with the evidence obtained in our laboratory on broad bean legumin and by others on some seed globulins suggest that heterogeneity is an inherent property of the major storage proteins of legume seeds.

Interaction of Gum Arabic, Maltodextrin and Pullulan with Lipids in Emulsions

The interaction of gum arabic, maltodextrin and pullulan with lipids in emulsion systems was investigated. Interfacial tension and interfacial viscosity measurements revealed that only gum arabic could adsorb and form a viscoelastic film at the oil-water interface. Good emulsifying activity was demonstrated for gum arabic, whereas fine emulsions could not be produced from the other polysaccharide solutions and oil. Frequency-dependent increases in the storage and loss moduli were observed for all the polysaccharide solutions. Such rheological behavior did not substantially change when maltodextrin and pullulan were mixed with oil to form emulsions. However, the frequency-dependence of the dynamic moduli disappeared in the gum arabic-stabilized emulsion, suggesting the formation of a network structure in which oil droplets could form junctions with gum arabic chains. The results on the inhibition of lipid oxidation by polysaccharides suggest that gum arabic protected lipids from the attack of lipoxygenase and free radicals by adsorbing at the oil droplet surface.