Tatsuro Watanabe

Nucleolin as cell surface receptor for tumor necrosis factor-α inducing protein: a carcinogenic factor of Helicobacter pylori

PurposeTumor necrosis factor-α inducing protein (Tipα) is a unique carcinogenic factor released from Helicobacter pylori (H. pylori). Tipα specifically binds to cells and is incorporated into cytosol and nucleus, where it strongly induces expression of TNF-α and chemokine genes mediated through NF-κB activation, resulting in tumor development. To elucidate mechanism of action of Tipα, we studied a binding protein of Tipα in gastric epithelial cells.MethodsTipα binding protein was found in cell lysates of mouse gastric cancer cell line MGT-40 by FLAG-pull down assay and identified to be cell surface nucleolin by flow cytometry using anti-nucleolin antibody. Incorporation of Tipα into the cells was determined by Western blotting and expression of TNF-α gene was quantified by RT-PCR.ResultsNucleolin was co-precipitated with Tipα-FLAG, but not with del-Tipα-FLAG (an inactive mutant). After treatment with Tipα-FLAG, incorporated Tipα was co-immunoprecipitated with endogenous nucleolin using anti-nucleolin antibody. The direct binding of Tipα to recombinant His-tagged nucleolin fragment (284–710) was also confirmed. Although nucleolin is an abundant non-ribosomal protein of the nucleolus, we found that nucleolin is present on the cell surface of MGT-40 cells. Pretreatment with anti-nucleolin antibody enhanced Tipα-incorporation into the cells through nucleolin internalization. In addition, pretreatment with tunicamycin, an inhibitor of N-glycosylation, decreased the amounts of cell surface nucleolin and inhibited both internalization of Tipα and expression of TNF-α gene.ConclusionsAll the results indicate that nucleolin acts as a receptor for Tipα and shuttles Tipα from cell surface to cytosol and nuclei. These findings provide a new mechanistic insight into gastric cancer development with Tipα.

Epithelial-mesenchymal transition in human gastric cancer cell lines induced by TNF-α-inducing protein of Helicobacter pylori.

Helicobacter pylori strains produce tumor necrosis factor‐α (TNF‐α)‐inducing protein, Tipα as a carcinogenic factor in the gastric epithelium. Tipα acts as a homodimer with 38‐kDa protein, whereas del‐Tipα is an inactive monomer. H. pylori isolated from gastric cancer patients secreted large amounts of Tipα, which are incorporated into gastric cancer cells by directly binding to nucleolin on the cell surface, which is a receptor of Tipα. The binding complex induces expression of TNF‐α and chemokine genes, and activates NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells). To understand the mechanisms of Tipα in tumor progression, we looked at numerous effects of Tipα on human gastric cancer cell lines. Induction of cell migration and elongation was found to be mediated through the binding to surface nucleolin, which was inhibited by the nucleolin‐targeted siRNAs. Tipα induced formation of filopodia in MKN‐1 cells, suggesting invasive morphological changes. Tipα enhanced the phosphorylation of 11 cancer‐related proteins in serine, threonine and tyrosine, indicating activation of MEK‐ERK signal cascade. Although the downregulation of E‐cadherin was not shown in MKN‐1 cells, Tipα induced the expression of vimentin, a significant marker of the epithelial–mesenchymal transition (EMT). It is of great importance to note that Tipα reduced the Youngs modulus of MKN‐1 cells determined by atomic force microscopy: This shows lower cell stiffness and increased cell motility. The morphological changes induced in human gastric cancer cells by Tipα are significant phenotypes of EMT. This is the first report that Tipα is a new inducer of EMT, probably associated with tumor progression in human gastric carcinogenesis.

Mechanism-based inhibition of cancer metastasis with (−)-epigallocatechin gallate

Cell motility and cell stiffness are closely related to metastatic activity of cancer cells. (-)-Epigallocatechin gallate (EGCG) has been shown to inhibit spontaneous metastasis of melanoma cell line into the lungs of mice, so we studied the effects of EGCG on cell motility, cell stiffness, and expression of vimentin and Slug, which are molecular phenotypes of epithelial-mesenchymal transition (EMT). Treatments of human non-small cell lung cancer cell lines H1299 and Lu99 with 50 and 100 μM EGCG reduced cell motility to 67.5% and 43.7% in H1299, and 71.7% and 31.5% in Lu99, respectively in in vitro wound healing assay. Studies on cell stiffness using atomic force microscope (AFM) revealed that treatment with 50 μM EGCG increased Youngs modulus of H1299 from 1.24 to 2.25 kPa and that of Lu99 from 1.29 to 2.28 kPa, showing a 2-fold increase in cell stiffness, i.e. rigid elasticity of cell membrane. Furthermore, treatment with 50 μM EGCG inhibited high expression of vimentin and Slug in the cells at a leading edge of scratch. Methyl-β-cyclodextrin, a reagent to deplete cholesterol in plasma membrane, showed inhibition of EMT phenotypes similar that by EGCG, suggesting that EGCG induces inhibition of EMT phenotypes by alteration of membrane organization.

Cell-surface nucleolin acts as a central mediator for carcinogenic, anti-carcinogenic, and disease-related ligands.

AbstractPurpose Cell-surface nucleolin in human gastric cancer cell lines is a receptor for TNF-α-inducing protein (Tipα) of Helicobacter pylori. The binding complex of nucleolin and Tipα is internalized into the cells and then induces tumor progression of human gastric cancer. Surface nucleolin is also a receptor of human immunodeficiency virus-1, and the anti-HIV pseudopeptide (HB-19) showed anti-carcinogenic activity in vivo. Surface nucleolin has dual functions depending on the ligands: In order to understand the mechanisms of surface nucleolin, it is necessary to review surface nucleolin and its relation to carcinogenic ligands and anti-carcinogenic ligands. Other ligands can be grouped among disease-related ligands, which is an important new topic for the prevention of various ailments. Results and discussionThis paper mainly deals with two ligands of surface nucleolin, Tipα and pseudopeptide HB-19. The binding complex of nucleolin and Tipα induces expression of TNF-α and chemokine genes and activates NF-κB in gastric cancer cells of humans and mice. However, when human gastric cancer cell line MKN-1 was transfected with nucleolin-targeted siRNA, the result was inhibition of cell migration and elongation induced by Tipα. The amount of surface nucleolin was reduced in membrane fraction of the nucleolin knockdown MKN-1 cells, but the amount of nucleolin in the cytosol or nuclear fractions of the cells did not change. The results indicate that surface nucleolin acts as a carcinogenic mediator for Tipα of H. pylori. In contrast, both the viral external envelop glycoprotein gp120 of HIV and the anti-HIV pseudopeptide HB-19 bind to surface nucleolin. Through this binding, treatment with HB-19 inhibited tumor development in human breast cancer cell line MDA-MB-231 and rhabdoid tumor cell line derived from Wilms’s tumor in xenograft nude mouse models. The results show that surface nucleolin acts as an anti-carcinogenic mediator for HB-19.ConclusionBased on these discrete functions of surface nucleolin, the binding complex of carcinogenic ligands and surface nucleolin seems to be competing with that of anti-carcinogenic ligands and surface nucleolin. Moreover, carcinogenic ligands derived from endogenous sources play a significant role in human cancer development, and the interaction of surface nucleolin with disease-related ligands will be a new research subject for the prevention and treatment of various ailments.

Biophysical Approach to Mechanisms of Cancer Prevention and Treatment with Green Tea Catechins

Green tea catechin and green tea extract are now recognized as non-toxic cancer preventives for humans. We first review our brief historical development of green tea cancer prevention. Based on exciting evidence that green tea catechin, (−)-epigallocatechin gallate (EGCG) in drinking water inhibited lung metastasis of B16 melanoma cells, we and other researchers have studied the inhibitory mechanisms of metastasis with green tea catechins using biomechanical tools, atomic force microscopy (AFM) and microfluidic optical stretcher. Specifically, determination of biophysical properties of cancer cells, low cell stiffness, and high deformability in relation to migration, along with biophysical effects, were studied by treatment with green tea catechins. The study with AFM revealed that low average values of Young’s moduli, indicating low cell stiffness, are closely associated with strong potential of cell migration and metastasis for various cancer cells. It is important to note that treatments with EGCG and green tea extract elevated the average values of Young’s moduli resulting in increased stiffness (large elasticity) of melanomas and various cancer cells. We discuss here the biophysical basis of multifunctions of green tea catechins and green tea extract leading to beneficial effects for cancer prevention and treatment.

Structural basis for the Helicobacter pylori-carcinogenic TNF-α-inducing protein

Stomach cancer is strongly associated with infection by Helicobacter pylori. In 2005, we identified a new H. pylori gene encoding a TNF-alpha inducing protein (Tipalpha) that acts as a carcinogenic factor. Tipalpha is secreted from H. pylori as a homodimer whose subunits are linked by disulfide bonds. We also characterized a Tipalpha deletion mutant (del-Tipalpha) that lacks the N-terminal six amino acid residues (LQACTC), including two cysteines (C5 and C7) that form disulfide bonds, but nonetheless shows a weak ability to induce TNF-alpha expression. Here we report that del-Tipalpha has a novel elongated structure containing a 40-A-long alpha helix, and forms a heart-shaped homodimer via non-covalent bonds. Moreover, their circular dichroism spectra strongly suggest that the structures of the del-Tipalpha and Tipalpha homodimers are very similar. del-Tipalphas unique mode of dimer formation provides important insight into protein-protein interactions and into the mechanism underlying the carcinogenicity of H. pylori infection.

Green tea aroma fraction reduces β-amyloid peptide-induced toxicity in Caenorhabditis elegans transfected with human β-amyloid minigene

Green tea is a popular world-wide beverage with health benefits that include preventive effects on cancer as well as cardiovascular, liver and Alzheimer’s diseases (AD). This study will examine the preventive effects on AD of a unique aroma of Japanese green tea. First, a transgenic Caenorhabditis elegans (C. elegans) CL4176 expressing human β-amyloid peptide (Aβ) was used as a model of AD. A hexane extract of processed green tea was further fractionated into volatile and non-volatile fractions, named roasty aroma and green tea aroma fractions depending on their aroma, by microscale distillation. Both hexane extract and green tea aroma fraction were found to inhibit Aβ-induced paralysis, while only green tea aroma fraction extended lifespan in CL4176. We also found that green tea aroma fraction has antioxidant activity. This paper indicates that the green tea aroma fraction is an additional component for prevention of AD. Graphical Abstract Green tea aroma fraction prevented human β-amyloid peptide-induced paralysis in a transgenic Caenorhabditis elegans CL4176, a model of Alzheimer’s disease.

High Contrast Visualization of Cell-Hydrogel Contact by Advanced Interferometric Optical Microscopy.

Hydrogels with tunable elasticity has been widely used as micromechanical environment models for cells. However, the imaging of physical contacts between cells and hydrogels with a nanometer resolution along the optical axis remain challenging because of low reflectivity at hydrogel-liquid interface. In this work, we have developed an advanced interferometric optical microscopy for the high contrast visualization of cell-hydrogel contact. Here, reflection interference contrast microscopy (RICM) was modified with a confocal unit, high throughput optics and coherent monochromatic light sources to enhance interferometric signals from cell-hydrogel contact zones. The advanced interferomety clearly visualized physical contacts between cells and hydrogels, and thus enabled the quantitative evaluation of the area of cell-hydrogel adhesion.

Inhibition of TNFα-interacting protein α (Tipα)-associated gastric carcinogenesis by BTG2 /TIS21 via downregulating cytoplasmic nucleolin expression

To understand the regulation of Helicobacter pylori (H. pylori)-associated gastric carcinogenesis, we examined the effect of B-cell translocation gene 2 (BTG2) expression on the biological activity of Tipα, an oncoprotein secreted from H. pylori. BTG2, the human ortholog of mouse TIS21 (BTG2/TIS21), has been reported to be a primary response gene that is transiently expressed in response to various stimulations. Here, we report that BTG2 is constitutively expressed in the mucous epithelium and parietal cells of the gastric gland in the stomach. Expression was increased in the mucous epithelium following H. pylori infection in contrast to its loss in human gastric adenocarcinoma. Indeed, adenoviral transduction of BTG2/TIS21 significantly inhibited Tipα activity in MKN-1 and MGT-40, human and mouse gastric cancer cells, respectively, thereby downregulating tumor necrosis factor-α (TNFα) expression and Erk1/2 phosphorylation by reducing expression of nucleolin, a Tipα receptor. Chromatin immunoprecipitation proved that BTG2/TIS21 inhibited Sp1 expression and its binding to the promoter of the nucleolin gene. In addition, BTG2/TIS21 expression significantly reduced membrane-localized nucleolin expression in cancer cells, and the loss of BTG2/TIS21 expression induced cytoplasmic nucleolin availability in gastric cancer tissues, as evidenced by immunoblotting and immunohistochemistry. Higher expression of BTG2 and lower expression of nucleolin were accompanied with better overall survival of poorly differentiated gastric cancer patients. This is the first report showing that BTG2/TIS21 inhibits nucleolin expression via Sp1 binding, which might be associated with the inhibition of H. pylori-induced carcinogenesis. We suggest that BTG2/TIS21 is a potential inhibitor of nucleolin in the cytoplasm, leading to inhibition of carcinogenesis after H. pylori infection.

Abstract 2640A: Cell stiffness as a new indicator of diagnosis for human lung cancer cells and their metastasis.

Our previous study with atomic force microscopy (AFM) revealed that highly metastatic B16-F10 cells have lower cell stiffness than low metastatic B16-F1 cells, and that treatment of the cells with a green tea polyphenol, EGCG, increased cell stiffness, associated with inhibition of metastasis. Since cell stiffness measured by Young9s modulus (Pa) using AFM is attracted the attention as a new indicator of cancer diagnosis, we expanded the study to human lung cancer cell lines, comparing with primary cultured normal bronchial epithelial cells. We found that cell stiffness of lung cancer cell lines was significantly lower than that of human immortalized peripheral lung epithelial cells (HPL1D): Young9s moduli for two adenocarcinoma cell lines (A549 and H322) were 2.4 kPa and 2.2 kPa; that of squamous cell carcinoma cell line (H1703): 0.6 kPa; a large cell carcinoma cell line (H1299): 0.8 kPa; and HPL1D: 4.9 kPa. Furthermore, primary cultured normal bronchial epithelial cells (NBEC) established from ten lung cancer patients showed high Young9s moduli from 3.0 kPa to 7.4 kPa. These data indicate that soft cell stiffness is a new indicator of diagnosis for lung cancer. Furthermore, it is important to note that H1703 and H1299 cells with low cell stiffness had high motile activity, compared with A549 and H322 cells with high cell stiffness. This indicates that low cell stiffness of lung cancer cell lines was associated with high motility, suggesting high metastatic ability. Treatments with EGF significantly induced transformation of HPL1D cells with morphological change, resulting in significant reduction of cell stiffness from 3.4 kPa to 1.5 kPa. These data suggest that low cell stiffness is induced in carcinogenic process. A histogram of Young9s moduli indicates that a cancer cell line is a conglomerate of various cells with varying cell stiffness. In order to study the change of cell stiffness during cell-cycle, we established H1299/Fucci cells by introducing fluorescent-cell-cycle-indicator (Fucci) gene into H1299 cells. Young9s moduli for each cell-cycle phase of H1299/Fucci cells were 1.71 kPa for G1, 1.64 kPa for G1→S, 1.55 kPa for S/G2 and 1.64 kPa for M→G1. Thus, changes in cell stiffness were observed depending on cell-cycle regulation, but the difference was small. Based on all data, we think cell stiffness is a dynamic feature of cancer cells, associated with motility and metastasis of lung cancer. Citation Format: Masami Suganuma, Atsushi Takahashi, Tatsuro Watanabe, Hirohiko Akiyama, Yuki Nakajima, Anupom Mondal, Hirota Fujiki. Cell stiffness as a new indicator of diagnosis for human lung cancer cells and their metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2640A. doi:10.1158/1538-7445.AM2013-2640A