Glutathione depletion sensitizes cisplatin- and temozolomide-resistant glioma cells in vitro and in vivo

L C De Andrade-Lima,C R R Rocha,A Quinet,J E Belizário,D B Vieira,C C M Garcia,C F M Menck,V Munford

doi:10.1038/cddis.2014.465

Abstract

Malignant glioma is a severe type of brain tumor with a poor prognosis and few options for therapy. The main chemotherapy protocol for this type of tumor is based on temozolomide (TMZ), albeit with limited success. Cisplatin is widely used to treat several types of tumor and, in association with TMZ, is also used to treat recurrent glioma. However, several mechanisms of cellular resistance to cisplatin restrict therapy efficiency. In that sense, enhanced DNA repair, high glutathione levels and functional p53 have a critical role on cisplatin resistance. In this work, we explored several mechanisms of cisplatin resistance in human glioma. We showed that cellular survival was independent of the p53 status of those cells. In addition, in a host-cell reactivation assay using cisplatin-treated plasmid, we did not detect any difference in DNA repair capacity. We demonstrated that cisplatin-treated U138MG cells suffered fewer DNA double-strand breaks and DNA platination. Interestingly, the resistant cells carried higher levels of intracellular glutathione. Thus, preincubation with the glutathione inhibitor buthionine sulfoximine (BSO) induced massive cell death, whereas N-acetyl cysteine, a precursor of glutathione synthesis, improved the resistance to cisplatin treatment. In addition, BSO sensitized glioma cells to TMZ alone or in combination with cisplatin. Furthermore, using an in vivo model the combination of BSO, cisplatin and TMZ activated the caspase 3–7 apoptotic pathway. Remarkably, the combined treatment did not lead to severe side effects, while causing a huge impact on tumor progression. In fact, we noted a remarkable threefold increase in survival rate compared with other treatment regimens. Thus, the intracellular glutathione concentration is a potential molecular marker for cisplatin resistance in glioma, and the use of glutathione inhibitors, such as BSO, in association with cisplatin and TMZ seems a promising approach for the therapy of such devastating tumors.

Highlights

Cisplatin is one of the most effective anticancer drugs and is used as a first-line treatment for a wide spectrum of solid tumors, such as ovarian, lung and testicular cancer,[6] and it is used for adjuvant therapy in gliomas.[7]
It has been demonstrated that functional p53 has an opposing effect in glioma treated with TMZ or nimustine (ACNU).[4,5]
It was observed that U138MG (p53mt) cells were less sensitive to cisplatin treatment, whereas the other lineages displayed quite similar survival rates, despite their different p53 status (Figure 1a)

Summary

Introduction

Cisplatin is one of the most effective anticancer drugs and is used as a first-line treatment for a wide spectrum of solid tumors, such as ovarian, lung and testicular cancer,[6] and it is used for adjuvant therapy in gliomas.[7]. The GSH content and glutathione S-transferase (GST) have long been associated with cisplatin resistance in numerous cell lines and tumor tissues.[9,10,11]. Considering these possible pathways, it is not clear, which one determinates cisplatin resistance in glioma cells. The GSH levels within the cell were shown to act as a decisive resistance barrier to cisplatin, reducing the induction of DNA damage in the treated cells Both in an in vitro and in vivo model depletion of GSH by an inhibitor (buthionine sulfoximine, BSO) sensitized the glioma cell lines to cisplatin. Combination with BSO, cisplatin and TMZ turned out to be an extremely powerful approach to improve cytotoxicity in glioma, providing an exciting alternative for glioma treatment

Methods

Results

Conclusion