The combination of A-966492 and Topotecan for effective radiosensitization on glioblastoma spheroids

Abstract

Radiotherapy is one of the modalities in the treatment of glioblastoma patients, but glioma tumors are resistant to radiation and also chemotherapy drugs. Thus, researchers are investigating drugs which have radiosensitization capabilities in order to improve radiotherapy. PARP enzymes and topoisomerase I enzymes have a critical role in repairing DNA damage in tumor cells. Thus, inhibiting activity of these enzymes helps stop DNA damage repair and increase DSB lethal damages. In the current study, we investigated the combination of TPT as a topoisomerase I inhibitor, and A-966492 as a novel PARP inhibitor for further radiosensitization. U87MG cells (a human glioblastoma cell line) were cultured in Poly-Hema coated flasks to reach 300 μm-diameter spheroids. Treatments were accomplished by using non-toxic concentrations of A-966492 and Topotecan. The surviving fraction of treated cells was determined by clonogenic assay after treatment with drugs and 6 MV X-ray. The γ-H2AX expression was measured by an immunofluorescence staining method to examine the influence of A-966492, TPT and radiation on the induction of double stranded DNA breaks. Treatments using the A-966492 drug were conducted in concentration of 1 μM. Combining A-966492 and TPT with radiation yielded enhanced cell killing, as demonstrated by a sensitizer enhancement ratio at 50% survival (SER₅₀) 1.39 and 1.16 respectively. Radio- and chemo-sensitization was further enhanced when A-966492 was combined with both X-ray and TPT, with SER₅₀ of 1.53. Also γ-H2AX expression was higher in the group treated with a combination of drugs and radiation. A-966492 is an effective PARP inhibitor and has significant radio-sensitivity on U87MG spheroids. By accumulating cells in the S phase and by inhibiting the DNA damage repair, TPT enhanced radio-sensitivity. A-966492 combined with TPT as a topoisomerase I inhibitor had additive radio-sensitizing effects. As a result, applying PARP and topoisomerase I inhibitors can be a suitable strategy for improving radiotherapy in clinics.

Keywords: Glioblastoma; PARP inhibitor; Radiosensitivity; Topoisomerase I inhibitor; Topotecan.