Inhibition of DNA repair for sensitizing resistant glioma cells to temozolomide

Abstract

Object. Temozolomide (TMZ) is a DNA alkylating agent currently used as adjuvant treatment for anaplastic astrocytomas. Its use in managing glioblastoma multiforme has been halted because of the lack of therapeutic effects due to cell resistance. Note that O⁶-alkylguanine—DNA alkyltranferase (AGT) is a DNA repair enzyme that limits the efficacy of TMZ. In this study the authors investigated the ability of O⁶-benzylguanine (BG), an AGT inhibitor, to sensitize a glioblastoma cell line resistant to TMZ. Methods. The effects of TMZ alone (100 µg) and after exposure to BG (50 µg) were assessed in two glioblastoma cell lines, U373-MG and T98G, respectively, sensitive and resistant to TMZ. Cell viability was assessed using trypan blue; cell cycle analysis by fluorescence-activated cell sorter; and apoptosis and autophagy by terminal deoxynucleotidyl transferase—mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and acridine orange staining, respectively. Furthermore, the involvement of an autophagy marker, microtubule-associated light chain 3 (LC3), was assessed. Temozolomide suppressed the growth of and caused cell cycle arrest in the G₂—M phase of U373-MG cells but not T98G cells. Exposure to BG prior to TMZ resulted in a significant decrease in cell viability as well as cell cycle arrest in the G₂—M phase in T98G cells (p < 0.05). Although apoptosis was not detected on TUNEL staining, programmed cell death Type II (autophagy) was detected after exposure to BG and TMZ in T98G cells. Conclusions. These results indicate that inhibition of AGT by BG can render previously resistant glioma cells sensitive to TMZ treatment. The mechanism of cell demise following BG-TMZ treatment seems to be autophagy and not apoptosis. Combination therapy involving TMZ and an AGT inhibitor may be an effective strategy to treat resistant gliomas.