Combined p14 ARF and Antisense EGFR Potentiate the Efficacy of Adenovirus-Mediated Gene Therapy in Laryngeal Squamous Cell Carcinoma (LSCC)

The tumor suppressor p14^ARF and protooncogene epidermal growth factor receptor (EGFR) play an important role in the development of laryngeal squamous cell carcinoma (LSCC). We explored the inhibition of proliferation and induction of differentiation in human larynx cancer cells (Hep-2) in vitro when p14^ARF couples with antisense complementary DNA of EGFR to transfect into Hep-2 cells via the AdEasy-1 vector system. In vitro studies, using standard isobologram analyses, identified whether Ad-antisense EGFR is synergistic with Ad-14^ARF. To evaluate the cytotoxicity of these agents the gold standard clonogenic survival assay was used. Western blotting analyses, 3′(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and flow cytometer (FCM) analysis was used to detect protein expression, proliferation, and cell cycle distribution of Hep-2 cells, respectively. Meanwhile, empty vector and PBS were set as a control. The activity of proliferation of Hep-2 cells was inhibited markedly by infection of Ad-p14^ARF combined with Ad-antisense EGFR compared with Ad-p14^ARF or Ad-antisense EGFR alone (P = 0.001, P = 0.002, respectively), with Ad-sense EGFR (P = 0.0005), with vector control (Ad-Ctrl) (P = 0.0001), and with PBS (P = 0.0001). FCM revealed that the proportion in the G₀/G₁ phases increased by up to 86.9% when Ad-p14^ARF was associated with Ad-antisense EGFR to transfect Hep-2 cells. A weakened expression of EGFR protein and P14 (ARF) protein overexpression was observed. Our study in vitro indicated that association of Ad-p14^ARF with Ad-antisense EGFR remarkably inhibited activity of proliferation and inducted differentiation of Hep-2 cells. Therefore, not only EGFR, but also p14^ARF, plays a major role in the genesis and in modulating cell growth and differentiation of LSCC, and their synergistic effect was obvious. An effective potential target of gene therapy to prevent LSCC proliferation was provided.