title: Hypoxia promotes radioresistance of CD133-positive Hep-2 human laryngeal squamous carcinoma cells in vitro.
abstract: Hypoxia promotes the radioresistance of laryngeal carcinomas and CD133 is one of the markers expressed by tumor-initiating, human laryngeal carcinoma cells. In order to investigate whether CD133-positive Hep-2 cells exhibit a radioresistant phenotype and to determine whether hypoxia promotes this phenotype, we performed a series of experiments. Hep-2 cells, and Hep-2 cells stably expressing hypoxia-inducible factor (HIF)-targeted small interfering RNA (siRNA) were cultured under hypoxic and normoxic conditions and were treated with varying doses of γ-rays (0, 5, 10, 15 and 20 Gy). MTT and cell cycle assays were subsequently performed. Using fluorescence-activated cell sorting (FACS), CD133-positive Hep-2 cells and CD133-positive HIF-siRNA Hep-2 cells were isolated. These cells were grown as spheres under hypoxic and normoxic conditions for MTT and soft agar colony formation assays. The expression levels of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), survivin, p53 and ataxia-telangiectasia mutated (ATM) were also assayed using flow cytometry. The data showed that the growth of Hep-2 cells exposed to hypoxic conditions and treated with 10 Gy radiation (group A) was less compared to that of groups B-D (P<0.05). In addition, more cells in group A were arrested in the G1 phase of the cell cycle compared to groups B-D (P<0.05). The percentage of CD133+ cells detected after radiation increased and was the highest for group A (P<0.05). In sphere formation assays, significantly more CD133+ cells grew in spheres than CD133- cells (P<0.001). Moreover, sphere formation was the highest for CD133+ Hep-2 cells grown under hypoxic conditions and exposed to irradiation (group E) (P<0.05). Lastly, expression of DNA-PKcs and survivin for group E was the highest (P<0.05), while ATM and p53 levels remained largely unchanged (P>0.05). In conclusion, CD133-positive Hep-2 cells exhibited a radioresistant phenotype that was enhanced with hypoxia. Furthermore, an increase in DNA-PK activity was associated with this enhancement.
authors: Otolaryngology Department, Fuzhou General Hospital of PLA, Fuzhou, Fujian 350025, P.R. China.
authors: Maoxin
authors: M
authors: Wang
authors: Xiaoming
authors: X
authors: Li
authors: Yongtao
authors: Y
authors: Qu
authors: Ou
authors: O
authors: Xu
authors: Qingjia
authors: Q
authors: Sun
mesh_terms: D000071916:AC133 Antigen; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D017209:Apoptosis / Q000528:radiation effects; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology* / Q000532:radiotherapy; D015687:Cell Hypoxia / Q000528:radiation effects; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D005720:Gamma Rays; D015972:Gene Expression Regulation, Neoplastic / Q000528:radiation effects; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D007822:Laryngeal Neoplasms / Q000378:metabolism / Q000473:pathology* / Q000532:radiotherapy*; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology / Q000528:radiation effects; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D011836:Radiation Tolerance / Q000235:genetics*
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D006023:Glycoproteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C000606023:PROM1 protein, human; D010455:Peptides; D034741:RNA, Small Interfering