title: Combined p14ARF and antisense EGFR potentiate the efficacy of adenovirus-mediated gene therapy in laryngeal squamous cell carcinoma (LSCC).
abstract: 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 G(0)/G(1) 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.
authors.affiliation: Department of Otolaryngology, West China Hospital, Sichuan University, Chengdu, People's Republic of China.
authors.forename: Junming
authors.initials: J
authors.lastname: Xian
authors.forename: Yinghe
authors.initials: Y
authors.lastname: Lin
authors.forename: Yafeng
authors.initials: Y
authors.lastname: Liu
authors.forename: Ping
authors.initials: P
authors.lastname: Gong
authors.forename: Shixi
authors.initials: S
authors.lastname: Liu
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D002294:Carcinoma, Squamous Cell / Q000473:pathology* / Q000628:therapy; D002453:Cell Cycle; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D016373:DNA, Antisense / Q000235:genetics*; D066246:ErbB Receptors / Q000096:biosynthesis / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007822:Laryngeal Neoplasms / Q000473:pathology* / Q000628:therapy; D014162:Transfection; D025803:Tumor Suppressor Protein p14ARF / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D016373:DNA, Antisense; D025803:Tumor Suppressor Protein p14ARF; D066246:ErbB Receptors