Inhibition of Erlotinib-induced checkpoint but not apoptotic targets by EGCG: Implication for their anti-tumor synergism in squamous cell carcinoma of the head and neck

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Previous studies from our laboratory suggested that tyrosine kinase inhibitor, Erlotinib and green tea constituent, EGCG had synergistic anti-tumor activity in cell culture and nude mouse xenograft model of squamous cell carcinoma of the head and neck (SCCHN) (AACR Proceedings, Vol. 47, Abstract # 4892, 2006). However, the mechanism of their anti-tumor synergism is not fully understood. The current study was designed to study the mechanism of anti-tumor synergism by the combined treatment with Erlotinib and EGCG. Propidium iodide and Annexin V-PE staining was performed to study the cell cycle distribution and apoptosis respectively. Expression patterns of cell cycle and apoptosis regulatory proteins were studied by Western blotting. Lenti-virus based expression system using short hairpin RNA (shRNA) was employed to ablate the expression of specific protein. Treatment of head and neck cancer cell lines with Erlotinib time-dependently increased (3-15 folds) the expression of cell cycle regulatory proteins, p21 and p27 and apoptosis regulatory protein Bim. Erlotinib also induced Puma in some cell lines. Cells underwent G1 arrest with very little apoptosis (~10-12%). In contrast, EGCG alone at a concentration of 30 μM had very little or no effect on the expressions of these proteins varying among the cell lines and induced little apoptosis (~8-10%). However, simultaneous use of EGCG with Erlotinib synergistically increased apoptosis (~40-45%, single treatment for 72 h) and strongly inhibited (50-80% reduction) the expression of p21 and p27 without affecting the induction of Bim and Puma. In addition, Erlotinib induced both Foxo1a and p53 transcription factors. In some cell lines, EGCG also induced these proteins to some extent. Ablation of p53 by shRNA suggested that p53 is dispensable for the expression of Bim, p21 or p27 and for the synergistic apoptotic effect. Taken together, our results suggest that Erlotinib induced both checkpoint proteins p21 and p27 and apoptosis regulatory proteins Bim and Puma probably by activating Foxo1a transcription factor. EGCG synergistically increased the anti-tumor activity of Erlotinib by inhibiting the checkpoint proteins p21 and p27 without affecting the expression of Bim and Puma. (Grant Support: U01CA101244; RO1CA112643 and P50CA128613 to DMS).