Purpose: Effective chemopreventive strategies could save millions of lives. We have previously reported that the combination of the EGFR-TKI erlotinib and the green tea constituent epigallocatechin-3-gallate (EGCG) exhibited synergistic chemopreventive effects in head and neck cancers by inducing the expression of Bim, p21, p27, and inhibiting the phosphorylation of ERK and AKT and expression of Bcl-2. In the current study, we further investigated the mechanism of regulation of Bim, Bcl-2, p21 and p27, and their role in apoptosis.

Methods: Well characterized and genetically validated squamous cell carcinoma of the head and neck cell lines were used throughout the study. Annexin V staining was conducted for apoptosis assay. Expression of mRNAs and proteins were measured by RT-PCR and Western blotting, respectively. Gene overexpression and knockdown strategies were used to activate or shut down the expression of specific proteins.

Results: siRNA-mediated silencing of Bim significantly inhibited apoptosis induced by the combination of erlotinib and EGCG (p=). On the other hand, overexpression of Bcl-2 significantly protected cells from apoptosis (p=), whereas overexpression of constitutively AKT had minimal effect on apoptosis (actual values). Analysis of mRNA expression by RT-PCR revealed that neither erlotinib, EGCG nor their combination had any significant effects on the mRNA expression of Bim, p21, p27 or Bcl-2, suggesting post-transcriptional regulation of Bim, p21, p27 and Bcl-2 by the combination of erlotinib and EGCG. Furthermore, we found that erlotinib or the combination of EGCG and erlotinib inhibited the phosphorylation of Bim, and that inhibition of protein translation by cycloheximide pretreatment stabilized Bim, suggesting posttranslational regulation of Bim.

Conclusion: Our results strongly suggest that the combination of erlotinib and EGCG induces apoptosis of SCCHN cells by regulating Bim and Bcl-2 at the post-translational level. Currently, a clinical trial is underway at Winship Cancer Institute of Emory University to inhibit or reverse the progression of oral premalignant lesions using this combination. (This study is supported by R03CA159369, P50CA128613 and Robbins Scholar Award of Winship Cancer Institute).

Citation Format: Abedul Haque, Mohammad A. Rahman, Zhuo G. Chen, Dong M. Shin, A.R.M. Ruhul Amin. Combination of erlotinib and epigallocatechin-3-gallate induces apoptosis of squamous cell carcinoma of the head and neck through posttranslational regulation of Bim and Bcl-2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2269. doi:10.1158/1538-7445.AM2014-2269