DNA-PKcs inhibition sensitizes cancer cells to carbon-ion irradiation via telomere capping disruption

Abstract

Heavy-ion irradiation induces a higher frequency of DNA double strand breaks (DSBs) which must be properly repaired. Critical shortening of telomeres can trigger DNA damage responses such as DSBs. Telomeres are very sensitive to oxidative stress such as ionizing radiation. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the central component in the non-homologous end joining (NHEJ) repair complex and participates in telomere maintenance. Therefore, it is expected to enhance the cell killing effect of heavy-ion irradiation via DNA-PKcs inhibition. To test this hypothesis, cellular radiosensitivity was measured by the clonal genetic assay. DNA damage repair was relatively quantified by long PCR. Apoptosis was quantified by flow-cytometric analysis of annexin V/PI double staining, and senescence was analyzed by galactosidase activity. Telomere length was semi-quantified by real-time PCR. P53 and p21 expression was determined by western blotting. Our data demonstrated that MCF-7 and HeLa cells with DNA-PKcs inhibition were more susceptible to carbon-ion irradiation than Those without DNA-PKcs inhibition. Even though NHEJ was inhibited by the DNA-PKcs specific inhibitor, NU7026, most DNA damage induced by carbon-ion irradiation was repaired within 24 hours after irradiation in both cell lines. However, potential lethal damage repair (PLDR) could not restore cellular inactivation in DNA-PKcs inhibited cells. MCF-7 cells showed extensive senescence and accelerated telomere length reduction, while HeLa cells underwent significant apoptosis after irradiation with NU7026 incubation. In addition, both cell lines with shorter telomere were more susceptible to carbon-ion radiation. Our current data suggested that DNA-PKcs inhibition could enhance cellular sensitivity to carbon-ion radiation via disturbing its functional role in telomere end protection. The combination of DNA-PKcs inhibition and carbon-ion irradiation may be an efficient method of heavy-ion therapy.

Figure 1. Effect of NU7026 on clonogenic survival of MCF-7 (A) and HeLa cells (B) after ionizing radiation.

Cells were incubated with 10 µM NU7026 for 3 hours before irradiation.

Figure 2. Cellular senescence of MCF-7 cells with/without 10 µM NU7026 treatment 30 days after 1 Gy carbon-ion irradiation.

Figure 3. NU7026 enhanced radiosensitivity independent of its role in DNA damage repair.

A. The survival fraction of irradiated cells after 24 hours recovery; B. The kinetics of DNA damage repair after 1 Gy carbon-ion irradiation with/without NU7026 treatment, as measured by relative long PCR amplification.

Figure 4. Telomere length reduction in MCF-7 cells with/without NU7026 treatment 30 days after carbon-ion irradiation.

MCF-7 cells were incubated with 10 µM NU7026. *: p<0.01 versus control; ^#: p<0.01 versus 1 Gy carbon-ion irradiation.

Figure 5. A. Real-time monitoring of adherent cells by the RT-CES System.

Real-time monitoring of the growth and proliferation of MCF-7 and HeLa cells in the presence of MST312 using the RT-CES platform. B. Relative telomere length in MCF-7 and HeLa cells after prolonged MST312 incubation. C. Senescence of MCF-7 cells 50 days after MST312 incubation; D. Senescence of MCF-7 cells with shorter telomeres 5 days after 1 Gy carbon-ion irradiation.