Met/HGF receptor modulates bcl-w expression and inhibits apoptosis in human colorectal cancers

The met proto-oncogene is the tyrosine kinase growth factor receptor for hepatocyte growth factor. In the present study, we investigated the role of met expression on the modulation of apoptosis in colorectal tumours. The gene expressions of c- met and the anti-apoptotic bcl -2 family, including bcl -2, bcl -x L and bcl-w, were analysed in human colorectal adenomas and adenocarcinomas by using a quantitative polymerase chain-reaction combined with reverse transcription. In seven of 12 adenomas and seven of 11 carcinomas, the c- met gene was overexpressed. The bcl -w, bcl -2 and bcl -x L genes were over-expressed in nine, five and six of 12 adenomas and in five, two and seven of 11 carcinomas, respectively. The c- met mRNA level in human colorectal adenomas and carcinomas was correlated with bcl -w but not with bcl -2 or with bcl -x L mRNA level. The administration of c- met -antisense oligonucleotides decreased Met protein levels in the LoVo human colon cancer cell line. In the case of c- met -antisense-treated cells, apoptotic cell death induced by serum deprivation was more prominent, compared to control or c- met -nonsense-treated cells. Treatment with c- met -antisense oligonucleotides inhibits the gene expression of bcl -w in LoVo cells. On the other hand, the gene expression of bcl -2 or bcl -x L was not affected by treatment with c- met -antisense oligonucleotides. These findings suggest that Met expression modulates apoptosis through bcl -w expression in colorectal tumours. © 2000 Cancer Research Campaig

Syndecans Reside in Sphingomyelin-Enriched Low-Density Fractions of the Plasma Membrane Isolated from a Parathyroid Cell Line

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) are one of the basic constituents of plasma membranes. Specific molecular interactions between HSPGs and a number of extracellular ligands have been reported. Mechanisms involved in controlling the localization and abundance of HSPG on specific domains on the cell surface, such as membrane rafts, could play important regulatory roles in signal transduction. METHODOLOGY/PRINCIPAL FINDINGS: Using metabolic radiolabeling and sucrose-density gradient ultracentrifugation techniques, we identified [(35)S]sulfate-labeled macromolecules associated with detergent-resistant membranes (DRMs) isolated from a rat parathyroid cell line. DRM fractions showed high specific radioactivity ([(35)S]sulfate/mg protein), implying the specific recruitment of HSPGs to the membrane rafts. Identity of DRM-associated [(35)S]sulfate-labeled molecules as HSPGs was confirmed by Western blotting with antibodies that recognize heparan sulfate (HS)-derived epitope. Analyses of core proteins by SDS-PAGE revealed bands with an apparent MW of syndecan-4 (30-33 kDa) and syndecan-1 (70 kDa) suggesting the presence of rafts with various HSPG species. DRM fractions enriched with HSPGs were characterized by high sphingomyelin content and found to only partially overlap with the fractions enriched in ganglioside GM1. HSPGs could be also detected in DRMs even after prior treatment of cells with heparitinase. CONCLUSIONS/SIGNIFICANCE: Both syndecan-1 and syndecan-4 have been found to specifically associate with membrane rafts and their association seemed independent of intact HS chains. Membrane rafts in which HSPGs reside were also enriched with sphingomyelin, suggesting their possible involvement in FGF signaling. Further studies, involving proteomic characterization of membrane domains containing HSPGs might improve our knowledge on the nature of HSPG-ligand interactions and their role in different signaling platforms

Oxidative Stress Impairs the Heat Stress Response and Delays Unfolded Protein Recovery

Background: Environmental changes, air pollution and ozone depletion are increasing oxidative stress, and global warming threatens health by heat stress. We now face a high risk of simultaneous exposure to heat and oxidative stress. However, there have been few studies investigating their combined adverse effects on cell viability. Principal Findings: Pretreatment of hydrogen peroxide (H2O2) specifically and highly sensitized cells to heat stress, and enhanced loss of mitochondrial membrane potential. H 2O 2 exposure impaired the HSP40/HSP70 induction as heat shock response (HSR) and the unfolded protein recovery, and enhanced eIF2a phosphorylation and/or XBP1 splicing, land marks of ER stress. These H2O2-mediated effects mimicked enhanced heat sensitivity in HSF1 knockdown or knockout cells. Importantly, thermal preconditioning blocked H 2O 2–mediated inhibitory effects on refolding activity and rescued HSF1 +/+ MEFs, but neither blocked the effects nor rescued HSF1-/- MEFs. These data strongly suggest that inhibition of HSR and refolding activity is crucial for H2O2–mediated enhanced heat sensitivity. Conclusions: H2O2 blocks HSR and refolding activity under heat stress, thereby leading to insufficient quality control and enhancing ER stress. These uncontrolled stress responses may enhance cell death. Our data thus highlight oxidative stres

A Multidisciplinary survey on controversies in the use of EUS-guided FNA: assessing perspectives of surgeons, oncologists and gastroenterologists

<p>Abstract</p> <p>Background</p> <p>EUS-guided FNA can help diagnose and differentiate between various pancreatic and other lesions.</p> <p>The aim of this study was to compare approaches among involved/relevant physicians to the controversies surrounding the use of FNA in EUS.</p> <p>Methods</p> <p>A five-case survey was developed, piloted, and validated. It was collected from a total of 101 physicians, who were all either gastroenterologists (GIs), surgeons or oncologists. The survey compared the management strategies chosen by members of these relevant disciplines regarding EUS-guided FNA.</p> <p>Results</p> <p>For CT operable T2NOM0 pancreatic tumors the research demonstrated variance as to whether to undertake EUS-guided FNA, at p < 0.05. For inoperable pancreatic tumors 66.7% of oncologists, 62.2% of surgeons and 79.1% of GIs opted for FNA (p < 0.05). For cystic pancreatic lesions, oncologists were more likely to send patients to surgery without FNA. For stable simple pancreatic cysts (23 mm), most physicians (66.67%) did not recommend FNA. For a submucosal gastric 19 mm lesion, 63.2% of surgeons recommended FNA, vs. 90.0% of oncologists (p < 0.05).</p> <p>Conclusions</p> <p>Controversies as to ideal application of EUS-FNA persist. Optimal guidelines should reflect the needs and concerns of the multidisciplinary team who treat patients who need EUS-FNA. Multi-specialty meetings assembled to manage patients with these disorders may be enlightening and may help develop consensus.</p

MicroRNA-196b is an independent prognostic biomarker in patients with pancreatic cancer

microRNA-196bは膵癌において異常高発現しており，多変量解析で不良な予後に相関した．その阻害剤は膵癌細胞株において抗腫瘍効果を示すことから，microRNA-196bは診断バイオマーカーおよび治療標的となることが示唆された

Versican G3 Promotes Mouse Mammary Tumor Cell Growth, Migration, and Metastasis by Influencing EGF Receptor Signaling

Increased versican expression in breast tumors is predictive of relapse and has negative impact on survival rates. The C-terminal G3 domain of versican influences local and systemic tumor invasiveness in pre-clinical murine models. However, the mechanism(s) by which G3 influences breast tumor growth and metastasis is not well characterized. Here we evaluated the expression of versican in mouse mammary tumor cell lines observing that 4T1 cells expressed highest levels while 66c14 cells expressed low levels. We exogenously expressed a G3 construct in 66c14 cells and analyzed its effects on cell proliferation, migration, cell cycle progression, and EGFR signaling. Experiments in a syngeneic orthotopic animal model demonstrated that G3 promoted tumor growth and systemic metastasis in vivo. Activation of pERK correlated with high levels of G3 expression. In vitro, G3 enhanced breast cancer cell proliferation and migration by up-regulating EGFR signaling, and enhanced cell motility through chemotactic mechanisms to bone stromal cells, which was prevented by inhibitor AG 1478. G3 expressing cells demonstrated increased CDK2 and GSK-3β (S9P) expression, which were related to cell growth. The activity of G3 on mouse mammary tumor cell growth, migration and its effect on spontaneous metastasis to bone in an orthotopic model was modulated by up-regulating the EGFR-mediated signaling pathway. Taken together, EGFR-signaling appears to be an important pathway in versican G3-mediated breast cancer tumor invasiveness and metastasis

Epigenetic inactivation of TCF2 in ovarian cancer and various cancer cell lines

Transcription factor 2 gene (TCF2) encodes hepatocyte nuclear factor 1β (HNF1β), a transcription factor associated with development and metabolism. Mutation of TCF2 has been observed in renal cell cancer, and by screening aberrantly methylated genes, we have now identified TCF2 as a target for epigenetic inactivation in ovarian cancer. TCF2 was methylated in 53% of ovarian cancer cell lines and 26% of primary ovarian cancers, resulting in loss of the gene's expression. TCF2 expression was restored by treating cells with a methyltransferase inhibitor, 5-aza-2′deoxycitidine (5-aza-dC). In addition, chromatin immunoprecipitation showed deacetylation of histone H3 in methylated cells and, when combined with 5-aza-dC, the histone deacetylase inhibitor trichostatin A synergistically induced TCF2 expression. Epigenetic inactivation of TCF2 was also seen in colorectal, gastric and pancreatic cell lines, suggesting general involvement of epigenetic inactivation of TCF2 in tumorigenesis. Restoration of TCF2 expression induced expression of HNF4α, a transcriptional target of HNF1β, indicating that epigenetic silencing of TCF2 leads to alteration of the hepatocyte nuclear factor network in tumours. These results suggest that TCF2 is involved in the development of ovarian cancers and may represent a useful target for their detection and treatment

Frequent epigenetic inactivation of Wnt antagonist genes in breast cancer

Although mutation of APC or CTNNB1 (β-catenin) is rare in breast cancer, activation of Wnt signalling is nonetheless thought to play an important role in breast tumorigenesis, and epigenetic silencing of Wnt antagonist genes, including the secreted frizzled-related protein (SFRP) and Dickkopf (DKK) families, has been observed in various tumours. In breast cancer, frequent methylation and silencing of SFRP1 was recently documented; however, altered expression of other Wnt antagonist genes is largely unknown. In the present study, we found frequent methylation of SFRP family genes in breast cancer cell lines (SFRP1, 7 out of 11, 64%; SFRP2, 11 out of 11, 100%; SFRP5, 10 out of 11, 91%) and primary breast tumours (SFRP1, 31 out of 78, 40%; SFRP2, 60 out of 78, 77%; SFRP5, 55 out of 78, 71%). We also observed methylation of DKK1, although less frequently, in cell lines (3 out of 11, 27%) and primary tumours (15 out of 78, 19%). Breast cancer cell lines express various Wnt ligands, and overexpression of SFRPs inhibited cancer cell growth. In addition, overexpression of a β-catenin mutant and depletion of SFRP1 using small interfering RNA synergistically upregulated transcriptional activity of T-cell factor/lymphocyte enhancer factor. Our results confirm the frequent methylation and silencing of Wnt antagonist genes in breast cancer, and suggest that their loss of function contributes to activation of Wnt signalling in breast carcinogenesis