mTOR) Inhibitor PI-103 on the Radiation Sensitivity of Glioblastoma Cell Lines MO59K and MO59J Differing in DNA-PK and ATM Status (original) (raw)

2020, Research Square (Research Square)

Background: Radiotherapy is routinely used to combat glioblastoma multiforme (GBM). However, the treatment e cacy is often limited by the radioresistance of GBM cells. Methods: Two isogenic 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, expression of marker proteins, including those belonging to NHEJ and HR repair pathways, degree of apoptosis, autophagy, and cell cycle alterations. Results: We found that PI-103 radiosensitized MO59K cells but, surprisingly, it induced radiation resistance in MO59J cells. In MO59K cells, combined PI-103 and radiation treatment induced much higher γH2AX expression measured by Western blot as compared to MO59J. Another cell line-speci c 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. In contrast, PI-103-treated MO59J cells showed an increased level of LC3BII, indicative of cytoprotective autophagy. Moreover, irradiation induced a strong G2 arrest in MO59J cells (~80% vs. 50% in MO59K), which was, however, partially abolished by PI-103 thus allowing cell-cycle progression of a fraction of cells. In contrast, treatment with PI-103 increased the G2 fraction in irradiated MO59K cells. Conclusions: The 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 ndings 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. Future research on an extended cell panel should focus on nding ways to enhance the radiosensitivity of cell lines with de ciencies in DNA-PK and ATM, the key proteins involved in the DNA damage response. Background Radiation therapy (RT) constitutes an important approach to treating local and regional cancer. About 50-70% of all tumor patients receive RT during treatment. Tremendous advances in physical targeting and tumor imaging [1, 2] and optimization of ionizing radiation (IR) treatment protocols have yielded signi cant advances in patient outcome. Yet, 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 multiforme (GBM), pancreatic and lung cancers. Particularly, the success rate of curing GBM remains very low with only about 10% of patients alive after 5 years following radiochemotherapy treatments [3].