Apatinib enhances the radiosensitivity of the esophageal cancer cell line KYSE-150 by inducing apoptosis and cell cycle redistribution

Abstract

To determine the radiosensitizing effect of apatinib on esophageal cancer cells, and to preliminarily investigate the underlying mechanism, KYSE-150 cells were treated with apatinib, x-ray or apatinib combined with x-ray, and compared with a blank control. It was observed that apatinib significantly inhibited vascular endothelial growth factor (VEGF) secretion and the proliferation of KYSE-150 cells in a dose-dependent manner. As the concentration of apatinib increased, the radiobiological parameters inactivation dose (D0), quasi domain does (Dq) and survival fraction (SF2) of KYSE-150 cells decreased, while the sensitization enhancement ratio SERD0 increased. The rate of apoptosis in cells treated with apatinib and x-ray was markedly higher compared with those of the blank control, x-ray and apatinib alone groups (P<0.05). The proportion of cells in the G2/M phase was significantly increased in the apatinib, x-ray and combination groups compared with the blank control group (P<0.05). Compared with the control and x-ray groups, combination treatment did not significantly alter the expression level of polyADP-ribose polymerase (PARP), although it significantly increased the expression of cleaved-PARP (P<0.05). Moreover, the expression of cell serine/threonine-protein kinase-2 (CHK2) was downregulated (P<0.05), whilst expression of the phosphorylated form, pCHK2, was significantly increased (P<0.05) in the combination group when compared with the control and x-ray groups. In conclusion, the present study suggested that apatinib increases the radiosensitivity of KYSE-150 esophageal cancer cells by inhibiting VEGF secretion and cell proliferation, and promoting apoptosis and cell cycle redistribution.

Keywords: apatinib; apoptosis; cell cycle; esophageal cancer; radiosensitivity.

Figures

Figure 1.

Apatinib inhibits VEGF secretion in KYSE-150 cells. KYSE-150 cells were irradiated with (A) 0 Gy and (B) 6 Gy x-ray following treatment with different concentrations of apatinib for 48 h. The VEGF concentration was calculated by ELISA, which indicated that the secretion of VEGF was significantly decreased in apatinib-treated KYSE-150 cells. All data are presented as the mean ± standard deviation, from three independent experiments. *P

Figure 2.

Apatinib inhibits the proliferation rate of KYSE-150 cells. KYSE-150 cells were treated with various concentrations of apatinib (0, 5, 10, 20, 30 and 40 µmol/l) for 24, 48 and 72 h. Cell proliferation rate was calculated using a Cell Counting Kit-8 assay. Apatinib significantly inhibited the proliferation rate of KYSE-150 cells in a time- and dose-dependent manner. All data are presented as the mean ± standard deviation from three independent experiments. *P

Figure 3.

Survival fraction of KYSE-150 cells pretreated with apatinib, followed by irradiation. Apatinib remarkably increased the radiosensitivity of KYSE-150 cells, with SERD0 of 1.36 for 20 and 40 µmol/l apatinib. All data are presented as the mean ± standard deviation from three independent experiments.

Figure 4.

Effects of apatinib combined with x-ray iradiation on apoptosis in KYSE-150 cells. KYSE-150 cells were pretreated with 20 µmol/l apatinib for 48 h prior to irradiation (4 Gy). Following irradiation, the cells were further incubated for 24 h. The apoptosis rate of the cells was analyzed by Annexin V/PI staining and flow cytometry. The apoptosis rates of the apatinib, x-ray and apatinib combined with x-ray groups were higher compared with that of the control group. Representative images of flow cytometric analysis are presented as follows: (A) Control group; (B) apatinib group; (C) radiation alone group; (D) combination group. All data are presented as the mean ± standard deviation from three independent experiments. PI, propidium iodide; FITC, fluorescein isothiocyanate; PI, propidium iodide; Q, quadrant.

Figure 5.

Effects of apatinib and x-ray on the expression of PARP and cleaved-PARP in KYSE-150 cells. (A) The protein levels of PARP and cleaved-PARP were determined by western blotting. Lane 1, control group; lane 2, apatinib group; lane 3, x-ray group; and lane 4, combination group. (B) Quantitative analysis of the protein expression of PARP. (C) Quantitative analysis of the protein expression of cleaved-PARP. All data are presented as the mean ± standard deviation from three independent experiments. *P#P<0.05 vs. the same index sample treated with 4 Gy x-ray. PARP, poly (ADP-ribose) polymerase.

Figure 6.

Effect of combination treatment of apatinib and x-ray irradiation on cell cycle distribution in KYSE-150 cells. KYSE-150 cells were pretreated with 20 µmol/l apatinib for 48 h prior to being exposed to 4 Gy radiation. The cells were incubated for another 24 h following irradiation and cell cycle distribution in the cells was analyzed by flow cytometry. The proportions of cells in the G2/M phase in the apatinib, x-ray and apatinib combined with x-ray groups were significantly higher compared with that of the control group. Representative images of flow cytometric analysis were presented as follows: (A) Control group; (B) apatinib group; (C) radiation alone group; (D) combination group. All data are presented as the mean ± standard deviation from three independent experiments. PE, phycoerythrin.

Figure 7.

Effects of apatinib and x-ray on the expression of CHK2 and pCHK2 in KYSE-150 cells. (A) The protein levels of CHK2 and pCHK2 were determined by western blotting. Lane 1, control group; lane 2, apatinib group; lane 3, x-ray group; and lane 4, combination group. (B) Quantitative analysis of the protein expression of CHK2. (C) Quantitative analysis of the protein expression of pCHK2. All data are presented as the mean ± standard deviation from three independent experiments. *P#P<0.05 vs. the same index sample treated with 4 Gy x-ray. CHK2, serine/threonine-protein kinase-2; p, phosphorylated.