Effects of Epigallocatechin-3-gallate on Growth, Epidermal Growth Factor Receptor Signaling Pathways, Gene Expression, and Chemosensitivity in Human Head and Neck Squamous Cell Carcinoma Cell Lines

Effects of EGCG on cell cycle progression and apoptosis. Exponentially growing cells were grown with the indicated concentrations of EGCG and then analyzed by DNA flow cytometry at the indicated time points (A). The distribution of cells in the sub-G₁ fraction (apoptosis) and in the G₁, S, and G₂-M phases of the cell cycle were calculated and plotted (B). An additional separate experiment gave similar results.

Western blot analyses for the p21^Cip1, p27^Kip1, cyclin D1, pRB, Bcl-2, Bcl-X_L, and Bax proteins (A) and for activated caspase 9 protein (B) in extracts of YCU-H891 and YCU-N861 cells. Cells were treated with 10 μg/ml (A) or 50 μg/ml (B) of EGCG for the indicated times, and extracts were prepared. An antibody for actin was used as a loading control (A). The M_r 46,000 and M_r 36,000 caspase 9 protein band represents the inactivated and activated forms of caspase 9, respectively (B). pp-RB, hyperphosphorylated pRB.

Western blot analysis for the EGFR, phosphorylated EGFR (p-EGFR), ERK, phosphorylated ERK (p-ERK), and Stat3 and phosphorylated Stat 3 (p-Stat 3) proteins. Cells were cultured in serum-free medium for 24 h, incubated with or without 10 μg/ml of EGCG for an additional 24 h, and then harvested. For TGF-α stimulation, cells were treated with 50 ng/ml of TGF-α for 6 h (YCU-H891) or for 18 h (YCU-N861) before harvesting. An antibody for actin was used as a loading control.

Reporter assays for c-fos (A) and cyclin D1 (B) in YCU-H891 (left) and YCU-N861 (right) cells. Cells were transfected in opti-MEM I medium with the c-fos promoter-luciferase or cyclin D1 promoter-luciferase reporter plasmids for 16 h and were then cultured in serum-free medium with the indicated concentrations of EGCG for 24 h. For the growth factor stimulation assays, TGF-α was added 30 min after EGCG. Luciferase activities were normalized to parallel assays for β-gal activities; bars, SD.

Cytotoxic effects of the combination of EGCG and 5-FU (A) or of EGCG alone (B) on YCU-H861 and YCU-N861 cells, using cell proliferation assays. Cells were treated with the indicated concentrations of drugs for 48 h, and the extent of inhibition of cell proliferation was determined by proliferation assays (See “Materials and Methods”). A value of 1.0 corresponds to the vehicle-treated control cells; bars, SD.

Cytotoxic effects of EGCG plus 5-FU using colony formation assays with YCU-N861 and YCU-H861 cells. One thousand cells were seeded and cultured for 24 h in 100-mm dishes. The cells were then treated with DMSO alone, with 0.1 μg/ml of EGCG alone, with 5-FU alone, or with 5-FU plus 0.1 μg/ml of EGCG. With YCU-N861 cells, the concentration of 5-FU was 0.1 μg/ml, and with YCU-H891 cells, the concentration of 5-FU was 0.5 μg/ml. The drugs were washed out 48 h later, and the cells were cultured for an additional 7 days. The colonies were then stained with Giemsa solution (A is a photograph of the results obtained with YCU-N861 cells), and the numbers of colonies were counted and plotted as a fraction of the vehicle-treated cultures (B). Each column indicates the average of triplicate dishes; bars, SD. A relative survival of 1.0 corresponds to the control cells; bars, SD.