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Old 02-02-2006, 04:11 AM   #1
Lani
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more on green tea

1: Oncogene. 2006 Jan 30; [Epub ahead of print] Links

The green tea catechins, (-)-Epigallocatechin-3-gallate (EGCG) and (-)-Epicatechin-3-gallate (ECG), inhibit HGF/Met signaling in immortalized and tumorigenic breast epithelial cells.

Bigelow RL, Cardelli JA.

1Department of Microbiology and Immunology, Feist-Weiller Cancer Center, Louisiana State University-Health Sciences Center, Shreveport, LA, USA.

The hepatocyte growth factor (HGF) receptor, Met, is a strong prognostic indicator of breast cancer patient outcome and survival, suggesting that therapies targeting Met may have beneficial outcomes in the clinic. (-)-Epigallocatechin-3-gallate (EGCG), a catechin found in green tea, has been recognized as a potential therapeutic agent. We assessed the ability of EGCG to inhibit HGF signaling in the immortalized, nontumorigenic breast cell line, MCF10A, and the invasive breast carcinoma cell line, MDA-MB-231. HGF treatment in both cell lines induced rapid, sustained activation of Met, ERK and AKT. Pretreatment of cells with concentrations of EGCG as low as 0.3 muM inhibited HGF-induced Met phosphorylation and downstream activation of AKT and ERK. Treatment with 5.0 muM EGCG blocked the ability of HGF to induce cell motility and invasion. We assessed the ability of alternative green tea catechins to inhibit HGF-induced signaling and motility. (-)-Epicatechin-3-gallate (ECG) functioned similar to EGCG by completely blocking HGF-induced signaling as low as 0.6 muM and motility at 5 muM in MCF10A cells; whereas, (-)-epicatechin (EC) was unable to inhibit HGF-induced events at any concentration tested. (-)-Epigallocatechin (EGC), however, completely repressed HGF-induced AKT and ERK phosphorylation at concentrations of 10 and 20 muM, but was incapable of blocking Met activation. Despite these observations, EGC did inhibit HGF-induced motility in MCF10A cells at 10 muM. These observations suggest that the R1 galloyl and the R2 hydroxyl groups are important in mediating the green tea catechins' inhibitory effect towards HGF/Met signaling. These combined in vitro studies reveal the possible benefits of green tea polyphenols as cancer therapeutic agents to inhibit Met signaling and potentially block invasive cancer growth.Oncogene advance online publication, 30 January 2006; doi:10.1038/sj.onc.1209227.

PMID: 16449979 [PubMed - as supplied by publisher]
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Old 02-04-2006, 11:23 PM   #2
Gina
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Join Date: Oct 2005
Location: Alexandria, VA
Posts: 197
Interesting connection with C-met and her-2 neu

Helicobacter pylori CagA protein targets the c-Met receptor and enhances the motogenic response

Yuri Churin1, Laila Al-Ghoul2, Oliver Kepp1, Thomas F. Meyer1, Walter Birchmeier3 and Michael Naumann2


Infection with the human microbial pathogen Helicobacter pylori is assumed to lead to invasive gastric cancer. We find that H. pylori activates the hepatocyte growth factor/scatter factor receptor c-Met, which is involved in invasive growth of tumor cells. The H. pylori effector protein CagA intracellularly targets the c-Met receptor and promotes cellular processes leading to a forceful motogenic response. CagA could represent a bacterial adaptor protein that associates with phospholipase C but not Grb2-associated binder 1 or growth factor receptorbound protein 2. The H. pyloriinduced motogenic response is suppressed and blocked by the inhibition of PLC and of MAPK, respectively. Thus, upon translocation, CagA modulates cellular functions by deregulating c-Met receptor signaling. The activation of the motogenic response in H. pyloriinfected epithelial cells suggests that CagA could be involved in tumor progression.


The gram-negative bacterium Helicobacter pylori colonizes the stomach of at least half of the world's population and could induce peptic ulcers, mucosa-associated lymphoid tissue lymphoma of the stomach, and gastric atrophy as well as distal gastric adenocarcinoma (Peek and Blaser, 2002). The presence of a pathogenicity island (PAI)* in H. pylori is connected with an increased risk of developing the aforementioned diseases. Several PAI genes are homologous to genes that encode type IV secretion system proteins (Covacci et al., 1999). After H. pylori adherence to epithelial cells, the bacterial PAI-encoded CagA protein is translocated into the host cell (Segal et al., 1999; Asahi et al., 2000; Backert et al., 2000; Odenbreit et al., 2000; Stein et al., 2000), where it undergoes tyrosine phosphorylation at different sites (Higashi et al., 2002b). H. pylori infection also triggers morphological changes and motility in host cells similar to those induced by hepatocyte growth factor (HGF; Segal et al., 1999; Churin et al., 2001). Cell motility is a critical rate-limiting step in the invasive growth program under physiological and pathophysiological conditions. Little is known about the mechanisms that underlie the process of H. pylori–induced cell motility and its putative role in tumor progression.

Here, we demonstrate that H. pylori activates the HGF/scatter factor receptor c-Met in host cells. H. pylori protein CagA binds c-Met and could represent an adaptor protein, which associates with phospholipase C (PLC ). Thus, upon translocation, CagA modulates cellular functions by deregulating c-Met receptor signaling.


In vitro, HGF promotes epithelial cell growth and survival, as well as epithelial–mesenchymal transition, where it stimulates the dissociation and dispersal of colonies of epithelial cells and the acquisition of a fibroblastic morphology. This results in increased cellular motility and invasiveness (Thiery, 2002). Hence, we tested whether epithelial cell clusters become migratory after infection with H. pylori. Comparison of the same AGS cell colony before and 4 h after H. pylori infection demonstrated the strong stimulation of AGS cell motility (Fig. 1 A) but HGF does not induce motility in AGS cells (not depicted). H. pylori could also stimulate the motility of MDCK cells, which was similar in HGF-treated cells (Fig. 1 A).



Figure 1. H. pylori activates c-Met receptor tyrosine kinase and induces the motogenic response. (A) H. pylori infection induces motility of AGS and MDCK cells. AGS and MDCK cells were infected with H. pylori, or MDCK cells were treated with 50 U/ml HGF. Phase-contrast microscopy was performed at the indicated time points. (B) H. pylori activates the c-Met receptor in AGS cells. AGS cells were infected with H. pylori or treated with HGF. c-Met was immunoprecipitated from lysates prepared at the indicated time points. Immunoprecipitates (IP) were subjected to SDS-PAGE and immunoblot (IB) analysis with antiphosphotyrosine (top) or anti–c-Met (bottom) antibodies. (C) H. pylori infection activates HER2/Neu. AGS cells were pretreated with or without AG1478 and AG825, and either infected with H. pylori for 90 min or treated with 10 ng/ml EGF for 5 min. Cell lysates were prepared, and HER2/Neu was immunoprecipitated and subjected to Western blot analysis using antiphosphotyrosine antibody. (D) AG1478 and AG825 have no effect on c-Met activation. AGS cells were pretreated with or without AG1478 and AG825 and infected with H. pylori for 180 min, and c-Met was immunoprecipitated and subjected to Western blot analysis using antiphosphotyrosine antibody. (E) The inhibitors of EGFR and HER2/Neu had no effect on the motility of AGS cells. AGS cells were treated with the inhibitors of EGFR (AG1478) and HER2/Neu (AG825) and infected with H. pylori. Phase-contrast microscopy was performed 4 h after infection.


Activation of signal transduction pathways in response to HGF stimulation is mediated by autophosphorylation of specific tyrosine residues within the intracellular region of c-Met that form multisubstrate docking sites (
Naldini et al., 1991; Furge et al., 2000). Therefore, we next examined whether H. pylori infection could activate c-Met in AGS cells. Host cells were infected with H. pylori and c-Met was immunoprecipitated from AGS cell lysates prepared at different time points after infection. Western blot analysis of the immunoprecipitated proteins using the phosphotyrosine-specific antibody PY99 demonstrated the stimulation of c-Met tyrosine phosphorylation 30 min immediately after infection (Fig. 1 B).

The activation of EGF receptor (EGFR) in epithelial cells by H. pylori was observed recently (Keates et al., 2001; Wallasch et al., 2002). One of the biological responses to EGFR activation is the stimulation of cell motility (Xie et al., 1998). Therefore, we used inhibitors of EGFR (AG1478) and of the closely related HER2/Neu receptor (AG825) to investigate the role of these receptors in stimulation of AGS cell motility. HER2/Neu was immunoprecipitated from AGS cell lysates infected with H. pylori or treated with EGF. Western blot analysis of the immunoprecipitates using anti-PY antibody revealed that HER2/Neu was activated by H. pylori infection and EGF treatment in AGS cells. This activation was strongly reduced after treatment with the inhibitors (Fig. 1 C), whereas both inhibitors had no effect on the activation of c-Met by H. pylori (Fig. 1 D). In spite of the presence of inhibitors, AGS cells became migratory after infection (Fig. 1 E). These observations indicated that H. pylori induced the sustained activation of c-Met in AGS cells that could lead to the stimulation of host cell motogenic response.

To test whether c-Met is directly involved in the stimulation of host cell motogenic response by H. pylori infection, we used small interfering RNA (siRNA) to silence the expression of the c-Met receptor by RNA interference in epithelial cells. An siRNA to c-Met efficiently and specifically silenced c-Met receptor expression, whereas EGFR expression was not affected. Furthermore, the silencing of c-Met receptor expression had no effect on CagA tyrosine phosphorylation (Fig. 2 A). Epithelial cells transfected with siRNA to c-Met did not express c-Met and were resistant to the induction of motility by H. pylori (Fig. 2, B and C). This effect could not be attributed to manipulations required to introduce siRNA into cells because the inhibition of EGFP expression by siRNA had no effect on H. pylori–induced cell motility (Fig. 2 C). Transfection of siRNA, which blocks c-Met expression, also inhibits H. pylori–induced scattering in AGS cells. Experimental data are shown for HeLa cells because these cells were transfectable with high efficiency. We conclude that c-Met expression is necessary for H. pylori–induced motility in epithelial cells.




Figure 2. c-Met receptor expression is essential for H. pylori– induced motogenic response in epithelial cells. HeLa cells were transfected with siRNA to c-Met or with siRNA to EGFP (as a control for the effect of transfection). After culturing for 72 h, cells were infected with the H. pylori strain P1 for 6 h. The siRNA to c-Met efficiently silenced c-Met receptor expression analyzed in a Western blot (A, top). Silencing of c-Met expression had no effect on EGFR expression (second panel) and phosphorylation (third panel) of translocated CagA protein (bottom). (B) Cells were transfected with c-Met siRNA and infected with the wild-type H. pylori strain P1. Cells are shown by phase-contrast microscopy (top two panels) or stained with immunofluorescence using c-Met antibody. Actin filaments were visualized with rhodamine-conjugated phalloidin. (C) HeLa cells transfected with c-Met siRNA are resistant to the induction of the motogenic response by H. pylori. Phase-contrast of cells transfected with siRNA to c-Met or siRNA to EGFP.



Figure 3. CagA interacts with the c-Met tyrosine kinase receptor and enhances the motogenic response of AGS cells to H. pylori infection. (A and B) AGS cells were infected with the wild-type H. pylori strain or isogenic mutant strains cagA and virB11. Phase-contrast microscopy was performed at the indicated time points. (B) Different H. pylori strains activate c-Met. Cells were harvested at the indicated time points after infection. c-Met was immunoprecipitated (IP) with anti–c-Met antibody and analyzed by immunoblotting (IB) using antiphosphotyrosine antibody. (C and D) CagA interacts with the c-Met receptor. AGS cells were infected with H. pylori. Cell lysates were prepared at the indicated time points. c-Met (C) or CagA (D) were immunoprecipitated with the corresponding specific antibodies and subjected to immunoblot analysis using anti-CagA (C) and anti–c-Met (D) antibodies. (E) CagA–c-Met interaction depends on c-Met tyrosine phosphorylation. AGS cells were transiently transfected with plasmids expressing either HA-tagged wild-type CagA (CagA) or HA-tagged phosphorylation-resistant CagA (CagA P) and were treated with HGF for 5 min. CagA was precipitated with anti-HA antibody, and immunoprecipitates were analyzed by Western blot analysis using anti–c-Met (top), antiphosphotyrosine (middle) antibodies, and anti–c-Met (bottom) antibodies.
...

We found that CagA was coimmunoprecipitated with c-Met in AGS cells during H. pylori infection (Fig. 3 C, top). The level of CagA phosphorylation increased during infection (Fig. 3 C, bottom). Interaction of CagA and c-Met was confirmed by coimmunoprecipitation using anti-CagA antibody (Fig. 3 D). Next, we investigated whether CagA–c-Met interaction depended on tyrosine phosphorylation of the interactive partners. AGS cells were transfected with HA-tagged wild-type CagA or the HA-tagged phosphorylation-resistant CagA. To induce the c-Met phosphorylation, the cells were treated with HGF or infected with the H. pylori cagA mutant strain before lysis. Western blot analysis of the HA immunoprecipitates using anti–c-Met and antiphosphotyrosine antibodies revealed that CagA only interacted with phosphorylated c-Metl and this interaction was independent of CagA phosphorylation (Fig. 3 E). Furthermore, CagA tyrosine phosphorylation was not affected in epithelial cells, which were silenced of c-Met receptor expression using siRNA to c-Met, indicating that the c-Met receptor is not required for CagA tyrosine phosphorylation (Fig. 2 A).

...
The induction of the motogenic response by H. pylori in epithelial cells represents an example of how human microbial pathogens could activate growth factor receptor tyrosine kinases, and modify signal transduction in the cell using translocated bacterial proteins. H. pylori effector protein CagA targets intracellularly the c-Met receptor and enhances the motogenic response, which suggests that dysregulation of growth factor receptor signaling could play a role in mobility and invasiveness of cells. Numerous experimental and clinical data indicate a particular role of HGF and the proto-oncogene c-Met in tumor invasive growth. The main challenge is to unravel how bacterial effectors interfere with cellular components and direct alterations in growth factor receptor signaling. Our results suggest that H. pylori modulates c-Met receptor signal transduction pathways, which could be responsible for cancer onset and tumor progression. Moreover, this work suggests that H. pylori colonization could not only be associated with stomach cancer development, but could also promote tumor invasion through stimulation of the motogenic response in infected cells.




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Gina
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