Radiosensitization of DNA-dependent protein kinase catalytic subunit inhibitor NU7026 on HCT116 colorectal cancer stem cells

Abstract

Objective To evaluate the radiosensitization and mechanism of DNA-dependent protein kinase catalytic subunit inhibitor NU7026 on HCT116 colorectal cancer stem cells. Methods The flow cytometry was used to determine the sub-population of cancer stem cells with the markers CD133/CD44. The cells were divided into four groups: control group, 20 μmol/L NU7926 group, 2 Gy irradiation group, and 20 μmol/L NU7026 combined with 2 Gy irradiation group. Cell proliferation and survival were evaluated by colony-formation experiment. Flow cytometry was used to analyze the cell cycle distribution and apoptosis induction. γ-H2AX foci were detected with immune-fluorescence staining analysis by a laser confocal microscopy for investiating the DNA double-strand break repair. Results The flow cytometric data of CD133+ /CD44+ positive cells indicated that the sub-population of cancer stem cell (CSC) took the ratio of (88.14±0.47)% of the cultured HCT116 cells. The colony-formation efficiency of HCT116 cells was (84.75±1.35)% in serum-free mediums in vitro culture. Compared to 2 Gy irradiation alone group, the NU7026 combined with 2 Gy irradiation group had a lower cell colony-forming ratio (t=7.21, P<0.01) and a lower survival ratio (t=7.22, P<0.01). The proportion of CSCs sub-population increased at 48 h post-2 Gy irradiation, suggesting that HCT116 CSCs was more resistant to ionizing radiation. Importantly, NU7026 largely decreased the proportion of CSCs sub-population in 2 Gy-irradiated cells. The difference of CSC proportion between 2 Gy irradiation alone group and 2 Gy combined with NU7026 treatment group was statistically significant (t=9.55, P<0.01). In addition, the group of NU7026 combined with 2 Gy irradiation had a higher ratio of G2/M arrest 24 h post-irradiation (t=7.67, P< 0.01) and an increased induction of cell early apoptosis (t=8.24, P< 0.05). 48 h post irradiation as compared to 2 Gy irradiation alone group. NU7026 treatment significantly inhibited the cellular capacity of repairing DNA double-strand breaks induced by γ-ray irradiation. The γ-H2AX foci of the combined treatments group were much higher than that of 2 Gy irradiation alone group at 2, 4, 8, 24 h post-irradiation (t=19.58, 11.95, 7.01, 9.45, P<0.01). Conclusions DNA-dependent protein kinase catalytic subunit inhibitor NU7026 can significantly sensitize the cancer stem cells of colorectal carcinoma HCT116 cells to γ-ray irradiation. Multiple mechanisms are involved in the radiosensitization effect of NU7026, including DNA repair inhibition, elongation of G2/M arrest, and increase of radiation-induced apoptosis. Key words: DNA-PKcs; HCT116; Radiosensitization; Cancer stem cells