Hypoxia promotes radioresistance of CD133-positive Hep-2 human laryngeal squamous carcinoma cells in vitro.

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.