Article Figures & Data
Figures
- Fig. 1.
Inhibition of cell growth by EGCG (A) or Poly E (B) in human colon cancer cell lines and in FHC fetal colon cells. Results are expressed as percentage of growth with 100% representing control cells treated with DMSO alone. Bars, SD of triplicate assays.
- Fig. 2.
The expression levels of EGFR, p-EGFR, HER2, and p-HER2 proteins in colon cancer cell lines and the FHC fetal colon cell line. Total protein extracts were prepared from 70% confluent cell cultures of the indicated cell lines and equivalent amounts of protein (60 μg/lane) were examined by Western blot analysis for EGFR and p-EGFR (A) or HER2 and p-HER2 (B), using the appropriate antibodies, as described in Materials and Methods. The results were quantitated by densitometry and these values are displayed in the lower panels, respectively. Repeat Western blots gave similar results. C, effects of TGF-α on the growth of HT29 colon cancer and FHC fetal colon cells. The cells were grown in the presence of the indicated concentration of TGF-α in serum-free DMEM medium and growth measured by MTT assays. Growth stimulation ratio is expressed as fold increase versus the TGF-α–untreated cells. Bars, SD of triplicate assays. D, effects of TGF-α on the level of the phosphorylated forms of EGFR and HER2 proteins in FHC cells. The FHC cells were stimulated with 50 ng/mL TGF-α for 24 hours and cell extracts were then examined by Western blot analysis using the respective antibodies. An antibody to actin served as a loading control. Similar results were obtained in a repeat experiment.
- Fig. 3.
Effects of EGCG and Poly E on cell cycle progression, induction of apoptosis, and caspase activities in HT29 cells. The cells were treated with 20 μg/mL EGCG, 20 μg/mL Poly E, or 0.1% DMSO for the indicated times, and then analyzed by DNA flow cytometry (A); cell extracts were examined for DNA fragmentation (C) and caspase-3 and -9 activity (D). A, histograms of the cell cycle analysis (left); distribution of cells in the sub-G1, G0/G1, S, and G2-M phases of the cell cycle are calculated and plotted (right). C and D, data on DNA fragmentation and caspase activity, respectively. Each asterisk indicates a significant difference (P < 0.05) between the control untreated cells and the EGCG- or Poly E-treated cells. Bars, SD of triplicate assays. B, levels of cyclin D1 and Bcl-xL proteins in HT29 cells. The HT29 cells were treated with 20 μg/mL EGCG or Poly E for 24 hours, and cell extracts were then examined by Western blot analysis using the respective antibodies. An antibody to actin served as a loading control. Similar results were obtained in a repeat experiment.
- Fig. 4.
Effects of EGCG and Poly E on activation of EGFR and HER2 and on related downstream signaling pathways. A, HT29 cells were treated with 20 μg/mL EGCG or Poly E for the indicated times (0, 3, 6, 12, 24, and 48 hours), and cell extracts were then examined by Western blot analysis using the respective antibodies. B, as indicated, HT29 cells were treated with 20 μg/mL EGCG or Poly E for 24 hours in the presence or absence of 50 ng/mL TGF-α. Cell extracts were then examined by Western blot analysis using the respective antibodies. An antibody to actin served as a loading control. Similar results were obtained in a repeat experiment.
- Fig. 5.
Effects of EGCG and Poly E on transcriptional activity of the AP-1 (A), c-fos (B), NF-κB (C), and cyclin D1 (D) promoters in HT29 cells. Transient transfection reporter assays were done with the indicated reporter in the presence of the indicated concentrations of DMSO, EGCG, or Poly E in the absence or presence of 50 ng/mL TGF-α, as indicated. Relative luciferase activity was then determined after 24 hours. Asterisks indicate a significant difference (P < 0.05) between control untreated cells and EGCG- or Poly E -treated cells. Bars, SD of triplicate assays. For additional details, see Materials and Methods.
- Fig. 6.
Inhibition of growth and induction of apoptosis in HT29 cells by EC alone, EGCG alone, and various combinations of these agents. A, HT29 cells were treated with the indicated concentrations of EC alone, EGCG alone, and the indicated combinations of these agents for 48 hours, and cell viability assays were then done using the MTT assay system. Bars, SD of triplicate assays. B, combination indices (CI) of EGCG plus EC in HT29 cells. The combination indices were determined as previously described (22, 23). The results are summarized in Table 2. C, induction of apoptosis in HT29 cells by EC alone, EGCG alone, and the combination of these agents. The cells were treated with DMSO or 10 μg/mL EGCG in combination with five different doses of EC (0, 1.0, 10, 50, and 100 μg/mL) for 48 hours, and cell extracts were then examined for DNA fragmentation, as described in Materials and Methods. Each asterisk indicates a significant difference (P < 0.05) between the control untreated cells (blank column) and the catechin-treated cells. Bars, SD of triplicate assays.
- Fig. 7.
Effects of low doses of EGCG and Poly E on inhibition of cell growth, induction of apoptosis, and inhibition of EGFR and HER2 activities in HT29 cells. A, cells were treated with 1 μg/mL EGCG, 1 μg/mL Poly E, 20 μg/mL EGCG, 20 μg/mL Poly E, or 0.1% DMSO for 96 hours, and the numbers of cells were then counted at the indicated times using a Coulter Counter. Points, means of triplicate assays. B and C, cells were treated with 1 μg/mL EGCG, 1 μg/mL Poly E, or 0.1% DMSO for 96 hours. B, cell extracts were examined for DNA fragmentation, as described in Materials and Methods. C, cell extracts were examined by Western blot analysis using the respective antibodies. Each asterisk indicates a significant difference (P < 0.05) between the control untreated cells and the EGCG- or Poly E-treated cells. Bars, SD of triplicate assays. An antibody to actin served as a loading control. Similar results were obtained in a repeat experiment.
Tables
- Table 1.
Effects of EGCG and Poly E on the growth of human colon cancer cells (Caco2, HCT116, HT29, SW480, and SW837) and normal human fetal colon cells (FHC)
IC50* EGCG Poly E Cell lines μg/mL μmol/L μg/mL μmol/L† Caco2 19.2 42.2 22.3 29.4 HCT116 21.7 47.7 20.6 27.2 HT29 22.8 50.2 20.1 26.5 SW480 36.4 80.1 37.0 48.8 SW837 19.6 43.1 19.6 25.9 FHC 48.8 107.4 49.4 65.2 - Table 2.
Combined effects of EGCG and EC on HT29 cells
EGCG concentration (μg/mL) EC concentration (μg/mL) 0.1 1 10 20 1 − − + ++ 10 − ± + ++ 50 ± ± ++ +++ 100 ± ± ++ +++ NOTE: Summary of symbols: −, (CI) 1.1-1.3 (moderate antagonism); ±, CI 0.9-1.1 (additive effect); +, CI 0.8-0.9 (slight synergism); ++, CI 0.6-0.8 (moderate synergism); +++, CI 0.4-0.6 (synergism).
Volume 11, Issue 7
