β-catenin contributes to cordycepin-induced MGMT inhibition and reduction of temozolomide resistance in glioma cells by increasing intracellular reactive oxygen species

Highlights

• Cordycepin inhibited the viability of glioma cells in vitro and in vivo.

• Cordycepin reduced the expression of MGMT and augmented TMZ-mediated chemotherapy in vitro and in vivo.

• Cordycepin-induced reduction of TMZ resistance in glioma cells was MGMT-dependent.

• β-catenin regulated cordycepin-induced MGMT inhibition and reduction of TMZ resistance in glioma cells.

• ROS is required for cordycepin-induced MGMT inhibition and reduction of TMZ resistance in glioma cells.

Abstract

Glioblastoma multiforme (GBM) is one of the most aggressive human tumors, and it has a poor prognosis. Temozolomide (TMZ) is the primary alkylating agent used to treat GBM. Nevertheless, a number of GBM patients are resistant to TMZ. Therefore, there is an urgent need for more effective therapeutic options. Cordycepin (COR) is a natural chemical with anti-tumor effects, although its mechanism of action is poorly understood. Several lines of evidence suggest that O⁶-methylguanine DNA methyltransferase (MGMT) repairs damaged DNA and contributes to drug resistance to TMZ in gliomas. The Wnt/β-catenin pathway regulates MGMT gene expression. However, whether cordycepin inhibits MGMT expression by downregulating the β catenin pathway and augmenting chemosensitivity to TMZ in glioma cells remains unclear. In the present study, we found that cordycepin inhibited the viability of glioma cells and induced apoptosis, cell cycle arrest, overproduction of reactive oxygen species (ROS) and reduction of glutathione (GSH) in vitro. Moreover, cordycepin significantly reduced tumor volume and prolonged median survival of tumor-bearing rats in vivo. We also found that cordycepin inhibited MGMT expression and augmented chemosensitivity to TMZ in glioma cells in vitro and in vivo, accompanied by downregulation of p-GSK-3β and β-catenin. Moreover, overexpression of MGMT reversed the synergistic effect of cordycepin and TMZ. Pharmacological inhibition of GSK-3β with CHIR-99021 or overexpression of β-catenin reversed cordycepin-induced reduction of cell viability, downregulation of β-catenin and MGMT, increase of apoptosis and reduction of TMZ resistance. Furthermore, we found that β-catenin regulated cordycepin-induced overproduction of ROS by decreasing GSH. Inhibition of ROS production with N-acetyl-l-cysteine (NAC) not only rescued the reduction of cell viability but also eliminated β-catenin and MGMT inhibition, prevented glioma cells apoptosis and reversed the synergistic effect of cordycepin and TMZ. Taken together, we demonstrated that β-catenin contributed to cordycepin-induced MGMT inhibition and reduction of TMZ resistance in glioma cells via increasing intracellular ROS. These results indicate that cordycepin may be a novel agent to improve GBM treatment, especially in TMZ-resistant GBM with high MGMT expression.

Introduction

Glioblastoma multiforme (GBM) is the most common and aggressive type of intrinsic brain tumor, with a very poor prognosis and high recurrence and morbidity [1]. The current standard care for newly diagnosed GBM generally consists of surgical resection followed by radiotherapy in conjunction with the alkylating agent temozolomide (TMZ) [2]. Nevertheless, malignant gliomas are resistant to many kinds of treatments, including radiation, chemotherapy and other adjuvant therapies. Therefore, an alternative therapeutic strategy is needed to effectively treat GBM patients.

Cordycepin (Fig. 1A), isolated from cultured Cordyceps militaris, possesses a variety of clinical effects such as regulation of immunity [3], inflammation [4], protein synthesis [5], platelet aggregation [6] and steroidogenesis [7]. Moreover, cordycepin has been demonstrated to induce apoptosis not only in glioma cells [8,9] but also in other types of cancer cells such as breast cancer cells [10], leukemia cells [11] and gallbladder cancer cells [12]. Whether cordycepin can enhance the sensitivity to TMZ remains unclear.

TMZ is the primary alkylating agent used to treat GBM, however, overall responses are poor, amounting to approximately 26.5% [2]. TMZ induces DNA methylation of guanine at the O⁶ position, leading to double-strand breakage in the genome, resulting in cell cycle arrest and induction of apoptosis. O⁶-metG is quickly repaired by the DNA repair protein O⁶-methylguanine DNA methyltransferase (MGMT) [13]. MGMT expression is considered to be a major determinant of TMZ resistance in GBM, and this is supported by large amounts of data from both clinical and basic studies [[14], [15], [16]]. Several lines of evidence indicate that the prognosis of patients with high MGMT expression is much poorer than of those with low expression [17]. Therefore, other therapeutic agents that suppress MGMT expression and increase sensitivity to TMZ are highly desired.

β-catenin, the primary downstream effector of the canonical Wnt pathway, participates in many cellular processes, including cell proliferation and differentiation, cell cycle, migration and invasion [18,19]. β-catenin stability is controlled by a protein complex composed of several proteins including: Axin, CK, APC and GSK-3β. Complex-associated β-catenin is phosphorylated by GSK-3β and is consequently degraded via the proteasome-dependent pathway [20]. Previous studies have shown that Wnt/β-catenin pathway regulates MGMT gene expression in cancer [21]. Cordycepin inhibited the Wnt/β-catenin pathway in leukemia, and this suppressive effect was mediated by GSK-3β regulation [22]. We speculated that cordycepin may inhibit MGMT expression by downregulating the β-catenin pathway and augmenting chemosensitivity to TMZ in glioma cells. Therefore, we used glioma cell lines and xenograft glioma models to examine whether cordycepin enhanced the efficacy of TMZ and to investigate the underlying mechanisms.