Quantification of (-)-epigallocatechin-3-gallate Inhibition of Migration and Invasion of Oral Squamous Cell Carcinoma Cell Lines Using Real-time Cell Analysis

Catechins found in green tea, in particular (−)-epigallocatechin-3-gallate (EGCG), have antitumor activity. The primary antitumor actions of catechins are anti-oxidative, anti-angiogenic, and anti-metastatic effects. Cell migration and invasion contribute to the metastatic potential of tumors. Real-time cell analysis (RTCA) measures cell migration and invasion in vitro. In the present study, using RTCA, we investigated whether the cell migration and invasion of oral squamous cell carcinomas (OSCCs) of the tongue and floor of the mouth were inhibited by EGCG. Studies were performed using the human SCC-4 and SAS cell lines, which are poorly differentiated OSCCs of the tongue, and the HO-1-u-1 cell line, an OSCC of the floor of the mouth. SCC-4 cells exhibited high cell migration and invasion compared with the SAS and HO-1-u-1 cells. EGCG was most effective in inhibiting the migration and invasion of SCC-4 cells, and inhibited OSCC cell invasion more strongly than it inhibited cell migration. EGCG inhibited the expression of matrix metalloproteinase (MMP)-2, MMP-9, and integrin α1 and β1 mRNA in the OSCC cell lines, particularly SCC-4 cells. The findings of the present study suggest that EGCG inhibits OSCC cell migration and invasion by inhibiting MMP-2, MMP-9, and integrin α1and β1 expression. Thus, EGCG may be a suitable agent or lead compound for the inhibition of OSCC metastasis

The Role of a Brain-specific Splice Variant of Ryanodine Receptor Type 1

The ryanodine receptor type 1 (RyR1) is capable of homotetrameric assembly to form a Ca2+ release channel at intracellular Ca2+ storage sites such as endoplasmic reticulum (ER). The mRNA transcript encoding full-length RyR1 is approximately 16kb and is mainly distributed in excitable cells. A 2.4-kb mRNA splice variant from the 3\u27-terminal region of the RyR1 gene coexists specifically in brain together with the full-length form, although the functions of this brain-specific splice variant remain unclear. To investigate the short form of RyR1 in intracellular Ca2+ signaling in brain at the cellular level, we established an experimental system whereby the green fluorescent protein (GFP) -tagged brain-specific variant of RyR1 is coexpressed with the full-length protein in the same cell. Both forms of RyR1 were localized in the ER. Caffeine-induced Ca2+-release activities in cells expressing both the brain-specific and full-length RyR1 were reduced compared to cells expressing only the full-length form of RyR1. These results suggested that coexpression of the brain-specific splice variant of RyR1 with its full-length counterpart modulates intracellular Ca2+ signaling by acting as a dominant-negative subunit of the Ca2+ release channel in a tissue-specific fashion

Association of phosphorylation site of tau protein with neuronal apoptosis in Alzheimer\u27s disease

金沢大学大学院医学系研究科In addition to neuritic changes and amyloid deposits, neuronal and glial cell apoptosis is an important pathological feature of Alzheimer\u27s disease (AD). Several factors have been postulated as causes or triggers of cellular apoptotic change. This study focused on a quantifiable relationship between phosphorylation sites of tau protein in the neurofibrillary tangles (NFT) and neuronal apoptosis. Five monoclonal anti-tau antibodies (AT180, AT8, HT7, Tau2 and Tau5) for NFT labeling and TdT-mediated UTP nick-end labeling (TUNEL) for localizing apoptotic change were employed. TUNEL-stained neuronal nuclei showed significantly high density in the entorhinal cortex, cornu ammonis (CA) and the parietal cortex. In all regions, density of TUNEL-stained neuronal nuclei showed significantly direct correlation with that of AT8-, AT180- and Tau2-positive neurons. Correlation of TUNEL-stained neuronal nuclei with tau-positive neurons differed depending on the cerebral regions. Density of TUNEL-stained neuronal nuclei showed inverse correlation with that of both AT8-positive and Gallyas-stained NFT in the CA and showed significantly direct correlation with AT8- and HT7-positive neurons in the frontal cortex. Density of tau-positive and Gallyas-stained NFT was higher than that of TUNEL-stained nuclei. We conclude that phosphorylation sites of tau, 159-163 and 202-205, are probably associated with neuronal apoptosis and apoptotic change follows abnormal phosphorylation of tau

Investigation of Cell Migration and Invasion Using Real-time Cell Analysis, as well as the Association with Matrix Metalloproteinase-9 in Oral Squamous Cell Carcinomas

The recently developed technology of real-time cell analysis (RTCA) was designed to analyze cell migration and invasion in vitro. In this study, we investigated these cellular factors in oral squamous cell carcinomas (OSCCs) of the tongue and floor of the mouth with RTCA. We also examined the associated matrix metalloproteinases (MMPs) and integrins. We used the cell lines SCC-4 and SAS, which are human poorly differentiated OSCCs from the tongue, and HO-1-u-1, which are human poorly differentiated OSCCs from the floor of the mouth. Using RTCA, cell migration was assessed on fibronectin–coated CIM-Plates, and invasion was assessed on fibronectin- and matrigel-coated CIM-Plates. SCC-4 cells demonstrated a high ability for cell migration and invasion compared with SAS and HO-1-u-1 cells. The SCC-4 cells also expressed high levels of MMP-9 and integrin α1 mRNA compared with SAS and HO-1-u-1 cells. The MMP inhibitor Marimastat blocked migration and invasion of all OSCCs. The findings suggest that MMP-9 is associated with cell migration and invasion in OSCCs, and indicate that RTCA will be useful for analyzing the metastatic capability of OSCCs and developing more effective new drugs for this disease

Quantification of (–)-Epigallocatechin-3-gallate Inhibition of Anaplastic Thyroid Cancer Cell Line Adhesion and Proliferation Using Real-time Cell Analysis

Anaplastic thyroid cancer (ATC) has a poor prognosis because of immediate metastasis. Several studies in humans and animals have suggested that the ingestion of green tea or its active ingredient (–)-epigallocatechin-3-gallate (EGCG) may decrease the risk of cancer. Using a recently developed real-time cell analysis (RTCA) system, we have shown previously that EGCG inhibits cell migration and the invasion of oral cavity cancers by suppressing matrix metalloproteinases. In the present study we used RTCA to investigate the effects of EGCG on cell adhesion to fibronectin-coated plates using three cancer cell lines: one ATC cell line (TCO-1) and two poorly differentiated oral squamous cell carcinomas (OSCCs) cell lines (SAS and HO-1-u-1; originating from the tongue and floor of the mouth, respectively). EGCG (50µM) inhibited the adhesion of all three cell lines. In addition to its effects on cell adhesion, 50µM EGCG inhibited the cell proliferation of TCO-1 cells. Furthermore, EGCG decreased αV integrin (ITGAV) mRNA levels in all three cell lines, suggesting that EGCG inhibits the cell adhesion and proliferation of OSCC and ATC cells via suppression of integrin expression. Therefore, EGCG represents a useful dietary constituent or a lead compound for counteracting metastasis of oral cavity cancers and thyroid cancers

A Study of the Populations of X-ray Sources in the Small Magellanic Cloud with ASCA

The Advanced Satellite for Cosmology and Astrophysics (ASCA) has made multiple observations of the Small Magellanic Cloud (SMC). X-ray mosaic images in the soft (0.7--2.0 keV) and hard (2.0--7.0 keV) bands are separately constructed, and the latter provides the first hard X-ray view of the SMC. We extract 39 sources from the two-band images with a criterion of S/N>5, and conduct timing and spectral analyses for all of these sources. Coherent pulsations are detected from 12 X-ray sources; five of which are new discoveries. Most of the 12 X-ray pulsars are found to exhibit long-term flux variabilities, hence they are likely to be X-ray binary pulsars (XBPs). On the other hand, we classify four supernova remnants (SNRs) as thermal SNRs, because their spectra exhibit emission lines from highly ionized atoms. We find that XBPs and thermal SNRs in the SMC can be clearly separated by their hardness ratio (the ratio of the count rate between the hard and soft bands). Using this empirical grouping, we find many XBP candidates in the SMC, although no pulsations have yet been detected from these sources. Possible implications on the star-formation history and evolution of the SMC are presented by a comparison of the source populations in the SMC and our Galaxy.Comment: 11 pages, 39 Figures, to be published in ApJ Supplement. Tables (body and figures also) are available at http://www-cr.scphys.kyoto-u.ac.jp/member/jun/job

Quantification of Cell Migration and Invasion, and Their Association with Periostin in Anaplastic Thyroid Cancer, Using a Real-time Cell Analyzer

Anaplastic thyroid cancer (ATC) is known to be a highly malignant cancer of the thyroid with a high mortality rate. In a previous study, we used real-time cell analysis (RTCA) to analyze cell migration and invasion of oral squamous cell carcinomas (OSCCs) of the tongue and floor of the mouth. In the present study, we investigated cell migration and invasion of ATC using RTCA, as well as their association with periostin, matrix metalloproteinases (MMPs), and integrins. Experiments were performed on TCO-1 and HTC/C3 cells, which are human ATC cell lines. OSCC cell lines were used for comparison. Using the cell analysis system, cell migration was assessed on fibronectin-coated CIM-Plates, whereas invasion was assessed on fibronectin- and matrigel-coated CIM-Plates. SCC-4 cells exhibited high cell migration and invasion activity compared with other OSCC cell lines. TCO-1 cells exhibited equivalent cell invasion but stronger migration than SCC-4 cells. Although TCO-1 cells had strong invasive activity, they did not express MMP-9, unlike SCC-4 cells. Conversely, periostin expression was high in TCO-1 cells. Therefore, periostin expression appears to be associated with the cell migration and invasion activity of ATC. The RTCA system will be useful for the analysis of the metastatic characteristics of ATC in head and neck cancer

Ablation of the scaffold protein JLP causes reduced fertility in male mice

金沢大学がん研究所がん分子細胞制御The specific and efficient activation of mitogen-activated protein kinase (MAPK) signaling modules is mediated, at least in part, by scaffold proteins. c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP) was identified as a scaffold protein for JNK and p38 MAPK signaling modules. JLP is expressed nearly ubiquitously and is involved in intracellular signaling pathways, such as the Gα13 and Cdo-mediated pathway, in vitro. To date, however, JLP expression has not been analyzed in detail, nor are its physiological functions well understood. Here we investigated the expression of JLP in the mouse testis during development. Of the tissues examined, JLP was strongest in the testis, with the most intense staining in the elongated spermatids. Since the anti-JLP antibody used in this study can recognize both JLP and sperm-associated antigen 9 (SPAG9), a splice variant of JLP that has been studied extensively in primates, we also examined its expression in macaque testis samples. Our results indicated that in mouse and primate testis, the isoform expressed at the highest level was JLP, not SPAG9. We also investigated the function of JLP by disrupting the Jlp gene in mice, and found that the male homozygotes were subfertile. Taken together, these observations may suggest that JLP plays an important role in testis during development, especially in the production of functionally normal spermatozoa. © 2008 Springer Science+Business Media B.V