Abstract
BackgroundRadiotherapy is routinely used to combat glioblastoma (GBM). However, the treatment efficacy is often limited by the radioresistance of GBM cells.MethodsTwo GBM lines MO59K and MO59J, differing in intrinsic radiosensitivity and mutational status of DNA-PK and ATM, were analyzed regarding their response to DNA-PK/PI3K/mTOR inhibition by PI-103 in combination with radiation. To this end we assessed colony-forming ability, induction and repair of DNA damage by γH2AX and 53BP1, expression of marker proteins, including those belonging to NHEJ and HR repair pathways, degree of apoptosis, autophagy, and cell cycle alterations.ResultsWe found that PI-103 radiosensitized MO59K cells but, surprisingly, it induced radiation resistance in MO59J cells. Treatment of MO59K cells with PI-103 lead to protraction of the DNA damage repair as compared to drug-free irradiated cells. In PI-103-treated and irradiated MO59J cells the foci numbers of both proteins was higher than in the drug-free samples, but a large portion of DNA damage was quickly repaired. Another cell line-specific difference includes diminished expression of p53 in MO59J cells, which was further reduced by PI-103. Additionally, PI-103-treated MO59K cells exhibited an increased expression of the apoptosis marker cleaved PARP and increased subG1 fraction. Moreover, irradiation induced a strong G2 arrest in MO59J cells (~ 80% vs. ~ 50% in MO59K), which was, however, partially reduced in the presence of PI-103. In contrast, treatment with PI-103 increased the G2 fraction in irradiated MO59K cells.ConclusionsThe triple-target inhibitor PI-103 exerted radiosensitization on MO59K cells, but, unexpectedly, caused radioresistance in the MO59J line, lacking DNA-PK. The difference is most likely due to low expression of the DNA-PK substrate p53 in MO59J cells, which was further reduced by PI-103. This led to less apoptosis as compared to drug-free MO59J cells and enhanced survival via partially abolished cell-cycle arrest. The findings suggest that the lack of DNA-PK-dependent NHEJ in MO59J line might be compensated by DNA-PK independent DSB repair via a yet unknown mechanism.
Highlights
IntroductionThe treatment efficacy is often limited by the radioresistance of GBM cells
Radiotherapy is routinely used to combat glioblastoma (GBM)
The findings suggest that the lack of DNA-PK-dependent Non-homologous end-joining (NHEJ) in MO59J line might be compensated by DNA-PK independent Double-strand breaks (DSB) repair via a yet unknown mechanism
Summary
The treatment efficacy is often limited by the radioresistance of GBM cells. Tremendous advances in physical targeting and tumor imaging [1, 2] and optimization of ionizing radiation (IR) treatment protocols have yielded significant advances in patient outcome. Radioresistance of tumor cells remains a major cause of treatment failure, resulting in a lower progression-free survival rate in many types of cancers, including glioblastoma (GBM), pancreatic and lung cancers. The success rate of curing GBM remains very low with only about 10% of patients alive after 5 years following radiochemotherapy treatments [3]. [4,5,6,7] This strategy is based on the rationale that additional artificially induced DNA damage may lower the threshold of the IR mediated cancer cell death. DSBs affect the genomic integrity of cells and, if insufficiently repaired or misrepaired, they may lead to chromosome breaks, gene deletions and translocations [8]
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