Effect of cell cycle and radiosensitivity by CoCl2 induced hypoxia in Eca109 cells

Abstract

Hypoxia in solid tumors is common, which not only a major problem for radiation therapy but also leads to resistance to most anticancer drugs, importantly, appears to accelerate malignant progression and increase metastasis. We observed the change of the cell cycle and apoptosis in order to investigate effect of radiosensitivity induced by CoCl2 in esophageal cancer line Eca109 cells in vitro. Materials and methods Cell cultureEsophageal cancer line Eca109 cells, which was obtained from the Cancer Research Center of Forth Affiliated Hospital, Hebei Medical University, were cultured in RPMI1640 (Sigma, USA) supplemented with 10% FBS and penicillin/streptomycin (100 IU/50 μg/mL) in a humidified atmosphere containing 5% CO2 in air at 37 . Cells in exponential phase of growth were used to test. Hypoxia treatment induced by CoCl 2We studied the effect of increasing concentrations of CoCl2 (0, 50, 100, 150 and 200 μM) on Eca109 cells and observed a dose-dependent morphological change of the cells. Since 200 μM of CoCl2 appeared to be toxic for these cells, we chose the 150 μM concentration as hypoxia group and 0 μM as control group. Subgroup and irradiationAll Eca109 cells were divided into control, hypoxia, hypoxia plus irradiation and only irradiation groups. The cells were irradiated in ordinary temperature using 60 Co γ ray from therapeutic machine of dose rate 110 cGy/min with field area 20 × 20 cm and source-skin distance 80 cm. 0.5 cm tissue compensation was putted on culture flask. Cell cycle and apoptosis analysisCells cultured in control-oxygen, anerobic cultivation induced by CoCl2 for 8, 16, 24 h and irradiation groups were gathered and washed in ice-cold PBS before trypsinization, diluted to 1 × 10 5 /mL. After fixation with 75% ethanol, cells were digested with DNAse-free RNAse in PBS containing 50 mg/mL propidium iodide for DNAstaining. Cell cycle and apoptosis was detected by using Abstract Objective: To investigate the change of the cell cycle, apoptosis and radiosensitivity effect by CoCl2 induced hypoxia in esophageal cancer line Eca109 cells in vitro. Methods: The hypoxia culture model induced by 150 microM CoCl2 was established. The cell cycle and apoptosis were measured with flow cytometry (FCM). The radiosensitivity was analysized with clonogenic assay after irradiation alone or combined with hypoxia in Eca109 cells in vitro. Results: Eca109 cells were treated with 150 microM CoCl2 for 24 h, cell cycle arrest in G0/G1 phase increase and decreasing arrest in S phase with longer of hypoxiac time (0-24 h), the other rate of cell cycle and apoptosis did not change obviously. The G2/M phase block was arrested obviously in radiation alone comparing with the hypoxia plus irradiated group, apoptosis did not occur in Eca109 cell line following irradiation. The D0 value and cell surviving fraction of Eca109 cell was 2.48 Gy, 2.44 Gy and 97.33%, 96.33% in hypoxia and control group, respectively; the Dq value of Eca109 cell was 2.89 Gy, 0.52 Gy, the cell surviving fraction after radiation with 4 Gy was 48.3%...