EGFR tyrosine kinase inhibitor AG1478 inhibits cell proliferation and arrests cell cycle in nasopharyngeal carcinoma cells

Abstract

Nasopharyngeal carcinoma (NPC), which occurs with a high incidence in southern China and southeast Asia, is of epithelial origin with overexpression of EGF receptor. To study the effect of inhibition of EGFR signaling on nasopharyngeal carcinoma cell proliferation and cell cycle distribution, EGFR tyrosine kinase inhibitor AG1478 was employed to treat Nasopharyngeal Carcinoma CNE2 cells. The results showed that AG1478 inhibited proliferation of CNE2 cells. Immunoblot showed that AG1478 inhibited EGFR phosphorylation in CNE2 cells without reduced expression of EGFR protein. The activation of Akt and MAPK which are downstream molecules of EGFR signaling pathway, were also inhibited by AG1478. AG1478 induced cell cycle arrest in G1 phase, and the levels of protein p27 were significantly up-regulated. We concluded that inhibition of the EGFR signaling induced cell cycle arrest in G1 phase in CNE2 cells and p27 up-regulation was involved in this process. The EGFR kinase specific inhibitor is of potential to be developed into drugs for NPC treatment.

Introduction

EGFR is a 170 KD protein composed of an extracellular ligand binding domain, a short transmembrane domain, and an intracellular domain, which has tyrosine kinase activity [1]. It has been reported that overexpression of EGF receptor plays a key role in pathological processes such as tumorigenesis and progression, especially epidermal carcinomas. The EGFR is activated by ligand binding to the extracellular domain with subsequent conformational alteration of the extracellular domain leading to receptor hetero- and homodimerization, resulting in intracellular autophosphorylation of tyrosine residues [2]. Overexpression of EGFR has also been shown to correlate with tumor grade, tumor size, lymph node metastasis, survival and resistance to chemotherapy in cancer patients [3]. Binding of EGF to EGF receptor on the surface of tumor cells facilitates its heterodimerization and homodimerization with itself or other members of EGFR family, resulting in activation and autophosphorylation of the PTK domain, which is essential for activation of numerous downstream effectors, including phospholipase Cγ, PI3K/AKT and MAPK, with the ultimate cellular response being DNA synthesis and cell proliferation [4]. After its autophosphorylation of EGFR, it binds the adaptor protein Grb2 through Grb2 SH-2 domain, and activates downstream effectors including Ras, Raf-1, MAPK, etc, in turn. EGFR is also critical to activation of PI3K/AKT pathway [5]. The known oncogenic potential mediated by EGFR and its high level expression in tumor tissue compared with normal cells make this oncoprotein an ideal target for antitumor therapeutic approaches.

Nasopharyngeal carcinoma (NPC) occurs with a high incidence in southern China and southeast Asia. This malignancy is of epithelial origin with overexpression of EGF receptor [6]. Radiotherapy is the predominant treatment modality for NPC, although chemotherapy has drawn much attention in recent years [7]. To exploit that therapeutic potential of EGFR signaling inhibition on NPC, NPC cells were treated with EGFR inhibitor AG1478. We observed that inhibition of NPC cell proliferation and cell cycle arrest in G1 phase and up-regulation of p27 protein following AG1478 treatment.