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Colorectal cancer is the second leading cause of cancer-related deaths in the U.S. Met, the receptor for hepatocyte growth factor (HGF), is over-expressed in colon tumors and is associated with poor prognosis. Recently, the green tea polyphenol (−)-epigallocatechin gallate (EGCG) was reported to suppress Met activation in breast cancer cells. However, the possible confounding effect of hydrogen peroxide (H2O2), produced when EGCG is added to cell culture media, was not assessed. In the present study, the human colon cancer cell lines HCT116 and HT29 were used to examine the relationships between Met activation, EGCG treatment, and H2O2 generation. At concentrations of 0.5, 1 and 5 μM, EGCG suppressed markedly the activation of Met in the presence of HGF. Concentrations of 10 μM EGCG and below generated low amounts of H2O2 (<1.5 μM), whereas higher H2O2 concentrations (>5 μM) were required to directly increase the phosphorylation of Met. Moreover, suppression of Met activation by EGCG occurred in the presence or absence of catalase, suggesting that such effects were not an ‘artifact’ of H2O2 generated from EGCG in cell culture media. We conclude that EGCG might be a beneficial therapeutic agent in the colon, inhibiting Met signaling and helping to attenuate tumor spread/metastasis, independent of H2O2-related mechanisms.
According to the American Cancer Society, colorectal cancer is the second most common cause of cancer-related deaths in the US. Disease survival is related to tumor stage. For example, Stage I colorectal cancer, in which the carcinoma remains localized in the sub-mucosa of the colon epithelium, has an overall 5-year survival rate of over 90%. In contrast, the 5-year survival rate for metastatic disease (Stage IV) drops to less than 10%. Since metastasis accounts for >90% of colon cancer deaths , therapies that target this process and block disease progression are of major interest. The receptor tyrosine kinase, Met, has been identified as an important protein that is essential for metastasis in colorectal carcinogenesis [2–6]. When Met is activated by its ligand, hepatocyte growth factor (HGF), it induces proliferation, motility, and invasion; all processes that are required for metastasis to occur.
Green tea and its associated polyphenols have been shown to reduce intestinal tumor formation in the ApcMin/+ mouse [7, 8]. EGCG, which is the most abundant polyphenol in green tea, has been shown to interfere with several cancer-related signaling pathways in vitro and in vivo. However, the ability of EGCG to suppress Met signaling in colon cancer cells remains to be studied. After oral ingestion, most EGCG accumulates in the intestine . This makes the use of EGCG an attractive therapeutic approach for the possible prevention of colon tumor metastasis.
EGCG has been shown to produce large amounts of hydrogen peroxide (H2O2) when added to cell culture media . This has been proposed to represent a possible ‘artifact’ when working with tea polyphenols in vitro. Therefore, empirical evidence is needed whenever cell culture studies use EGCG, to verify that effects are due to EGCG rather than H2O2 generated in the experiment. The present study provides evidence that EGCG suppresses Met signaling in human colon cancer cells, and that this effect is independent of H2O2 generated under the conditions used here.
HCT116 and HT29 human colon cancer cells were obtained from American Type Culture Collection (Manassas, VA). They were maintained in McCoy’s 5A media (Invitrogen, Carlsbad, CA) with 10% FBS and 1% penicillin/streptomycin. Cells were grown at 37°C with 5% CO2.
Cells (0.8 × 106) were plated in 60 mm dishes and grown in serum-containing media for 48 h. Cells were then incubated in serum-free media for 4 h. After serum starvation, cells were pretreated for 30 min with various concentrations of EGCG (Sigma-Aldrich, St. Louis, MO) followed by treatment with 30 ng/ml HGF (Calbiochem, San Diego, CA). In some experiments, 30 U/ml catalase (Roche Applied Science, Indianapolis, IN) were added to the media 10 min prior to addition of EGCG.
Cells (0.8 × 106) were plated in 60 mm dishes and grown in serum-containing media for 48 h. Cells were then serum-starved for 4 h. Various concentrations of H2O2 (Sigma-Aldrich, MO) were added and cells were incubated for an additional 30 min prior to immunoblot analyses.
Cells were placed in IP lysis buffer, vortexed, and then centrifuged at 10,000 rpm for 5 min. The supernatant was collected and protein concentrations were determined by BCA assay (Pierce, Rockford, IL). Proteins (10–20 mg) were separated by SDS-PAGE on a 4–12% bis-Tris gel (Novex, San Diego, CA) and transferred to nitrocellulose membrane (Invitrogen, Carlsbad, CA). Equal protein loading was confirmed with Amido Black staining and β-actin levels. The membrane was blocked for 1 h with 2% bovine serum albumin, followed by overnight incubation with primary antibody at 4°C, and finally incubated for 1 h with secondary antibody conjugated with horseradish peroxidase (Bio-Rad, Hercules, CA). Antibody dilutions were as follows: Phospho-Met (Tyr1234/1235), 1:1000 (Cell Signaling Technology, Beverly, MA), total Met, 1:1000, (Cell Signaling Technology), and β-actin, 1:5000 (Sigma). Detection was by Western Lightning Chemiluminescence Reagent Plus (PE Life Sciences, Boston, MA) with image analysis on an AlphaInnotech photodocumentation system.
To measure H2O2 production, serum-starved cells were incubated with various concentrations of EGCG in phenol red-free McCoy’s 5A media (Invitrogen) for 30 min. Aliquots of media were analyzed using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes, Eugene, OR), as previously reported .
Cell culture media was pretreated in the presence or absence of 30 U/ml catalase (Roche). EGCG (5 μM) was then added to the media and cells were incubated for 30 min prior to the addition of 30 ng/ml HGF (Calbiochem). Cells were incubated for an additional 15 min and lysed in RIPA buffer containing phosphatase inhibitors (Pierce). The ELISA was performed according to the instruction manual for STAR phospho-Met (Tyr1230/Tyr1234/Tyr1235) ELISA kit (Upstate).
HCT116 cells were incubated with various concentrations of H2O2 for 15 min. The cells were then collected and stained with Guava Viacount® Reagent (Guava Technologies, Hayward, CA) for 5 min. The percentage of viable cells was determined by using the Guava ViaCount Assay on a Guava Personal Cytometer.
Phosphorylation of the Met receptor previously was reported to be inhibited by EGCG in immortalized and tumorigenic breast epithelial cells . To determine if this occurred in human colon cancer cells, HCT116 and HT29 cells were pre-incubated with various concentrations of EGCG for 30 min, followed by the addition of HGF to the media. As expected, in the absence of EGCG pretreatment, HGF alone strongly increased the levels of p-Met in HCT116 cells (Fig. 1A) and HT29 cells (Fig. 1B). However, activation of the Met receptor was suppressed by EGCG concentrations as low as 0.5 μM. This suppression occurred in both cell lines, and was evident after normalizing p-Met to total Met in the immunoblots (Fig. 1C).
The amount of H2O2 produced by EGCG under the current conditions was measured after incubating various concentrations of EGCG in cell culture media for 30 min. With no EGCG present, ~0.1 and 0.25 μM H2O2 was detected in media from HCT116 and HT29 cells, respectively (open bars, Fig. 2A). The concentration of H2O2 increased with EGCG added to the media. However, only at the highest concentration of 10 μM EGCG was H2O2 measured at a level above 1 μM, in each cell line (solid bar, Fig. 2A).
To investigate the direct effects of H2O2 on Met activation, HCT116 cells were incubated with various concentrations of H2O2 for 30 min, in the absence of HGF, and p- Met was analyzed by immunoblotting. Concentrations at or below 2.5 μM H2O2 had no effect on Met phosphorylation, as indicated by the lack of change in p-Met levels compared with the 0 μM H2O2 control (Fig. 2B). When the concentration of H2O2 exceeded 2.5 μM, however, there was increased phosphorylation of Met, most notably at the highest levels of 10 and 20 μM H2O2 (Fig. 2B).
The direct effects of H2O2 were further examined in the presence and absence of HGF (Figs. 2C,D). In the absence of HGF, p-Met/total Met levels were increased by concentrations of H2O2 in the range 5–100 μM H2O2 (Fig. 2D, open bars). A similar trend was seen in cells treated with H2O2 plus HGF (Fig. 2D, gray bars). However, compared to –HGF controls, receptor activation was enhanced at concentrations below 20 μM H2O2 (compare, for example, white and gray bars at 5 μM H2O2 in Fig. 2D). At concentrations of 20, 50, and 100 μM H2O2, phosphorylation of the Met receptor was decreased slightly when HGF was present, as compared with the corresponding –HGF controls. We also sought to test the upper limit for Met activation in response to H2O2; concentrations of 500 and 1000 μM H2O2 strongly reduced p-Met and total Met in the immunoblots, as well as expression of the loading control β-actin (Fig. 2C).
The viability of HCT116 cells treated with these concentrations of H2O2 was assessed. There was little or no effect on viability when cells were exposed to H2O2 at concentrations up to 100 μM (Fig. 2E). However, at the highest concentrations of 500 and 1000 μM H2O2 the percentage of viable cells was reduced to less than 45% and 8% viability, respectively (Fig. 2E).
To further assess the role of H2O2 and to differentiate its effects from those of EGCG, subsequent experiments included catalase. Immunoblotting revealed that, in the presence or absence of catalase, HGF produced a similar level of Met phosphorylation, and EGCG maintained its inhibitory effects (Fig. 3A). This was confirmed for the same cell lysates using an ELISA kit to detect p-MET, revealing that Met activation was virtually identical in the presence or absence of catalase (Fig. 3B). Thus, inhibition of the Met receptor by EGCG can occur in the absence of H2O2.
Previous studies have shown that tea polyphenols are capable of suppressing receptor tyrosine kinase activity [13–15]. In particular, the Met receptor was shown to be inhibited in breast cancer cells treated with EGCG, the major polyphenol in green tea . Here, EGCG effectively suppressed the activation of the Met receptor in human colon cancer cells, and at a concentration as low as 0.5 μM EGCG, which is likely to be within the physiological range [16, 17].
EGCG rapidly produces H2O2 when added to cell culture media . At a concentration of 25 μM EGCG, for example, 10–12 μM H2O2 was detected in serum-free MEM media . Here, we investigated the H2O2 generated following the addition of EGCG to McCoy’s 5A media, used in the current study. As suggested by Long et al. , excess H2O2 generated in cell culture media from the oxidation of polyphenolic compounds could represent an artifact in mechanistic studies of tea catechins. Thus, it is important to distinguish the effects of EGCG from those of H2O2. Under the present conditions, 10 μM EGCG produced less than 1.5 μM H2O2 in both HCT116 and HT29 cell lines. This concentration of H2O2 did not alter Met phosphorylation levels following direct addition of H2O2 to the experiment. However, the Met receptor was activated when concentrations exceeded 2.5 μM H2O2 in the assay. This might be due to the ability of H2O2 to inhibit protein tyrosine phosphatases , although this was not tested in the present study.
Others have reported that exposing A549 human lung cancer cells to 500 μM H2O2 in the presence of HGF results in the suppression, rather than the activation, of the Met receptor . In the present study, concentrations of 500 and 1000 μM EGCG clearly had a deleterious effect on the viability of human colon cancer cells; thus, the apparent loss of Met phosphorylation must be viewed in this context, along with the reduced levels of total Met and β-actin. Importantly, catalase had no effect in these experiments, either in blocking the HGF-mediated increase in p-MET levels, or diminishing the effectiveness of EGCG to suppress Met activation.
We conclude that, under the present conditions, EGCG can effectively suppress Met signaling in colon cancer cells, at physiologically relevant concentrations. Thus, EGCG may be useful as a therapeutic agent for prevention of metastatic spread of colon tumors. The concern over H2O2 as a possible artifact was ruled out through the use of catalase, and by experiments showing that the concentrations of H2O2 generated by 1–10 μM EGCG were too low to influence Met phosphorylation. Further studies are warranted to determine the mechanism by which EGCG suppresses activation of the Met receptor.
This work was supported in part by grant CA090890 from the US National Cancer Institute.