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, 2 (6), 538-45

Synergistic Growth Inhibition of Squamous Cell Carcinoma of the Head and Neck by Erlotinib and epigallocatechin-3-gallate: The Role of p53-dependent Inhibition of Nuclear factor-kappaB

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Synergistic Growth Inhibition of Squamous Cell Carcinoma of the Head and Neck by Erlotinib and epigallocatechin-3-gallate: The Role of p53-dependent Inhibition of Nuclear factor-kappaB

A R M Ruhul Amin et al. Cancer Prev Res (Phila).

Abstract

We have previously reported that the green tea polyphenol epigallocatechin-3-gallate (EGCG) and the epidermal growth factor receptor-tyrosine kinase inhibitor erlotinib had synergistic growth-inhibitory effects in cell culture and a nude mouse xenograft model of squamous cell carcinoma of the head and neck. However, the mechanism of their antitumor synergism is not fully understood. In the current study, we investigate the mechanism of their synergistic growth-inhibitory effects. The treatment of squamous cell carcinoma of the head and neck cell lines with erlotinib time-dependently increased the expression of cell cycle regulatory proteins p21 and p27 and apoptosis regulatory protein Bim. EGCG alone had very little or no effect on the expression of these proteins among the cell lines. However, simultaneous treatment with EGCG and erlotinib strongly inhibited erlotinib-induced expression of p21 and p27 without affecting the expression of Bim. Moreover, erlotinib increased the expression of p53 protein, the ablation of which by short hairpin RNA strongly inhibited EGCG- and erlotinib-mediated growth inhibition and the expression of p21, p27, and Bim. In addition, combined treatment with erlotinib and EGCG inhibited the protein level of p65 subunit of nuclear factor-kappaB and its transcriptional target Bcl-2, but failed to do so in cells with ablated p53. Taken together, our results, for the first time, suggest that erlotinib treatment activates p53, which plays a critical role in synergistic growth inhibition by erlotinib and EGCG via inhibiting nuclear factor-kappaB signaling pathway. Characterizing the underlying mechanisms of EGCG and erlotinib synergism will provide an important rationale for chemoprevention or treatment trials using this combination.

Figures

Figure 1
Figure 1
Inhibition of erlotinib-induced cell cycle regulatory protein p21 and p27 by EGCG. (A) Tu686 (B) 1986LN (C) 886LN and (D) SQCCY1 cells were treated with 30 μM EGCG (E), 0.5 μM erlotinib (ER) or a combination of 30 μM EGCG and 0.5 μM erlotinib (C) for the indicated times. Total cell lysates were immunoblotted with anti-p27 (Santa Cruz) and anti-p21 (Santa Cruz). NT is untreated cells. Reproducibility of the data were confirmed with at least three independent experiments for each cell lines.
Figure 2
Figure 2
Expression of Bim following erlotinib and EGCG treatment. (A) Tu686 (B) 886LN (C) Tu212 and (D) SCC38 cells were treated as in Figure 1. Total cell lysates were immunoblotted with anti-Bim (abcam). Reproducibility of the data were confirmed with at least three independent experiments for each cell lines.
Figure 3
Figure 3
Expression of p53 following erlotinib and EGCG treatment. (A) SCCHN cells were treated as in Figure 1 and total cell lysates were immunoblotted with anti-p53 (Santa Cruz). (B) Tu686 cells were treated with 1 μM erlotinib, or 30 μM EGCG or a combination of 1 μM erlotinib and 30 μM EGCG. Total cell lysates were immunoblotted with anti-phospho p53 (Ser 15). Reproducibility of the data were confirmed with three independent experiments.
Figure 4
Figure 4
p53 is required for the growth inhibitory effects of erlotinib, EGCG and their combination. (A) Tu686 and M4e cells were treated with 2 μM erlotinib, 30 μM EGCG and a combination of 2 μM of erlotinib and 30 μM EGCG for 120 h, stained with annexin V-PE and the apoptotic population was determined by flow cytometry. (B) The expression of p53 was knocked down in Tu686 cells by a lentivirus-based shRNA specific for p53. shGFP was used as control. Expression of p53 was determined in total cell lysates. (C) Tu686 cells transduced with shGFP and shp53 were treated with 2 μM erlotinib, 30 μM EGCG and a combination of 2 μM of erlotinib and 30 μM EGCG for 72 h. Expression of p27, p21 and Bim in the total cell lysates was measured by Western blotting. (D) Upper panel: shGFP- and shp53-transduced cells were treated with 2 μM erlotinib, 30 μM EGCG and a combination of 2 μM of erlotinib and 30 μM EGCG for 96 h. Cell growth was measured by SRB assay. Experiments were done in triplicate for each agent and reproducibility was confirmed by multiple independent experiments. Lower panel: cells were treated as in upper panel and the plates were stained with methylene blue. (E) 686LN and M4e cells were treated with 0.5 μM erlotinib, 30 μM EGCG and a combination of 0.5 μM of erlotinib and 30 μM EGCG for 72 h and expression of p21 and p27 were determined by Western blotting.
Figure 5
Figure 5
p53-dependent inhibition of NF-κB. (A) Tu686 cells were treated with 2 μM erlotinib, 30 μM EGCG and a combination of 2 μM of erlotinib and 30 μM EGCG for the indicated times. Expression of p65 and Bcl-2 was measured by immunoblotting. (B) shGFP- and shp53-transduced Tu686 cells were treated with 2 μM erlotinib, 30 μM EGCG and a combination of 2 μM of erlotinib and 30 μM EGCG for 72 h. Total cell lysates were immunoblotted with anti-p65. Lanes 1, 3, 5, 7 are shGFP and lanes 2, 4, 6, 8 are shp53. (C) Cells were treated as in Figure 5B and total cell lysates were used for the expression of Bcl-2. (D) M4e cells were treated with 0.5 μM erlotinib, 30 μM EGCG and a combination of 0.5 μM of erlotinib and 30 μM EGCG for 48 h and total cell lysates were immunoblotted with anti-p65 and anti-Bcl-2. Reproducibility of all results were confirmed by three independent experiments.

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