title: Pharmacological genome demethylation increases radiosensitivity of head             and neck squamous carcinoma cells.
abstract: Aberrant inactivation of tumor suppressor genes by promoter hypermethylation has been recognized as a crucial step of tumor development and is related to aggressiveness and therapy resistance. To identify potential novel treatment strategies, we evaluated pharmacological genome demethylation for the increase of irradiation treatment effectiveness in head and neck squamous cell carcinoma (HNSCC) in this in vitro study. HNSCC cells were cultured with 2 different concentrations of 5-azacytidine (5-Aza) for 72 h, followed by a single fraction irradiation with 4 or 50 Gy, respectively. To show successful genome demethylation, the methylation status of the tumor suppressor gene hic1 (hypermethylated in cancer) promoter was analyzed by methylation specific PCR (MSP) as well as hic1 transcription by quantitative RT-PCR. Survival, apoptosis, viability, and migration of the tumor cells were analyzed as functional parameters of combined treatment response. After 5-Aza treatment the hic1 promoter was demethylated and gene transcription restored demonstrating genome demethylation. 5-Aza treated cells tended to be less viable and showed decreased survival indicated by lower colony numbers. Apoptosis and migration were not affected. The combined application of irradiation and 5-Aza significantly reduced survival compared to the single treatments. Accordingly, apoptosis was strongly increased after combined 4 Gy/5-Aza treatment. Viability was not additionally affected by combined treatment. Migration was affected weakly by combined high dosage irradiation/5‑Aza treatment. Our data show that the combined application of 5-Aza and irradiation is effective in vitro. A demethylating concept prior to irradiation should be further evaluated for its potential to reduce irradiation resistance.
authors: Department of Otorhino-laryngology, Head and Neck Surgery, University Medical Center of the Johannes Gutenberg University, Mainz Building 102, 2nd floor, Part D Langenbeckstrasse 1, D-55101 Mainz, Germany. juergen.brieger@unimedizin-mainz.de
authors: Juergen
authors: J
authors: Brieger
authors: Sylvia A
authors: SA
authors: Mann
authors: Warut
authors: W
authors: Pongsapich
authors: Dimitrios
authors: D
authors: Koutsimpelas
authors: Kai
authors: K
authors: Fruth
authors: Wolf J
authors: WJ
authors: Mann
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001374:Azacitidine / Q000494:pharmacology*; D002294:Carcinoma, Squamous Cell / Q000235:genetics*; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006258:Head and Neck Neoplasms / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011836:Radiation Tolerance / Q000187:drug effects*; D000077195:Squamous Cell Carcinoma of Head and Neck; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: D000964:Antimetabolites, Antineoplastic; D001374:Azacitidine