Elsevier

Neuroscience

Volume 346, 27 March 2017, Pages 298-308
Neuroscience

The PI3K inhibitor GDC-0941 enhances radiosensitization and reduces chemoresistance to temozolomide in GBM cell lines

https://doi.org/10.1016/j.neuroscience.2017.01.032Get rights and content

Highlights

  • GDC-0941 monotherapy induced cytotoxicity and showed pro-apoptotic effects in glioblastoma multiforme (GBM) cell lines.

  • GDC-0941 enhanced the anti-viability, pro-apoptotic and pro-autophagy effects of standard GBM therapy (TMZ and IR).

  • GDC-0941 reduced the migratory and invasive capacity in GBM cell lines.

  • Treatment with TMZ+GDC-0941+IR induced higher expression of p53 and GSK3β than other groups in three GBM cell line.

  • Combination of TMZ and GDC-0941 with or without IR reduced the levels of p-AKT.

Abstract

Glioblastoma multiforme (GBM) is among the most lethal of all human tumors. It is the most frequently occurring malignant primary brain tumor in adults. The current standard of care (SOC) for GBM is initial surgical resection followed by treatment with a combination of temozolomide (TMZ) and ionizing radiation (IR). However, GBM has a dismal prognosis, and survivors have compromised quality of life owing to the adverse effects of radiation. GBM is characterized by overt activity of the phosphoinositide 3-kinase (PI3K) signaling pathway. GDC-0941 is a highly specific PI3K inhibitor with promising anti-tumor activity in human solid tumors. It is being evaluated in Phase II clinical trials for the treatment of breast and non-squamous cell lung cancer.

We hypothesized that GDC-0941 may act as an antitumor agent and potentiate the effects of TMZ and IR. In this study, GDC-0941 alone induced cytotoxicity and pro-apoptotic effects. Moreover, combined with the standard GBM therapy (TMZ and IR), it suppressed cell viability, showed enhanced pro-apoptotic effects, augmented autophagy response, and attenuated migratory/invasive capacity in three glioma cell lines. Protein microarray analyses showed that treatment with TMZ+GDC-0941+IR induced higher levels of p53 and glycogen synthase kinase 3-beta (GSK3-β) expression in SHG44GBM cells than those induced by other treatments. This was verified in all cell lines by western blot analysis. Furthermore, the combination of TMZ and GDC-0941 with or without IR reduced the levels of p-AKT and O6-methylguanine DNA methyltransferase (MGMT) in T98G cells. The results of this study suggest that the combination of TMZ, IR, and GDC-0941 is a promising choice for future treatments of GBM.

Introduction

Glioblastoma multiforme (GBM) in adults is the most frequent and most lethal primary brain tumor. It is a malignant brain tumor with a dismal prognosis and usually leads to death, with a mean survival of 8–12 months (Network, 2013). Currently, the standard of care (SOC) for malignant glioma is initial surgical resection and treatment with adjuvant temozolomide (TMZ) followed by radiation therapy (NCCN Guidelines, 2015). TMZ shows antitumor effects against a wide range of malignancies, including high-grade glioma. The introduction of TMZ has improved chemotherapy for GBM. TMZ as a monotherapy or in combination with radiotherapy results in better outcomes for GBM patients. However, the current SOC only prolongs post-diagnosis survival by 2–4 months (Stupp et al., 2009). Furthermore, primary or acquired resistance to TMZ and IR is a major therapeutic problem. Therefore, a combination therapy that enhances the efficacy of TMZ and IR is required.

Activity of the phosphoinositide 3-kinase (PI3K)-AKT signaling pathway is correlated with enhanced cellular motility, survival, and resistance to chemotherapeutics. The PI3K signaling pathway plays a critical role in cellular processes involved in regulating tumor metabolism, proliferation, survival, chemoresistance, and motility. Activation of AKT, also known as protein kinase B, is related to increased tumorigenicity and invasiveness (Huang et al., 2012, Li et al., 2015, Wang et al., 2015). Furthermore, overexpression of phosphorylated AKT increases glioma cell resistance to TMZ (Yu et al., 2015). Inhibition of signaling pathways is a promising target for GBM therapy because most human gliomas show activation of a pro-growth or pro-survival signaling pathway (Nonnenmacher et al., 2015). There are many pan-PI3K inhibitors such as LY294002 that have not been used clinically owing to high toxicity (Gharbi et al., 2007, Zhu et al., 2015). Similarly, rapamycin has been shown to impede the survival and recovery in some postsurgical patients (Cloughesy et al., 2008, Workman et al., 2010). To overcome the lack of pharmaceutical inhibitors, we aimed to take advantage of the novel and highly specific pan-PI3K inhibitor GDC-0941 (Folkes et al., 2008). GDC-0941 has exhibited promising anti-neoplastic activity and was well tolerated in Phase I/II trials for the treatment of glioma (Moreno Garcia et al., 2011, Von Hoff et al., 2011, Sarker et al., 2015). Some inhibitors of the PI3K signaling pathway have been shown to sensitize glioma cells and xenografts and enhance the effects of combination TMZ and radiation therapy (Choi et al., 2014).

O6-methylguanine DNA methyltransferase (MGMT) is an independent prognostic factor and a target for personalized therapy (McLendon et al., 2008). Chemotherapeutic resistance in tumors attributable to MGMT protein expression has emerged as a clinical problem (Beharry et al., 2016). Furthermore, MGMT expression is inversely correlated with p53 levels in many tumors (Matsukura et al., 2001, Osanai et al., 2005). Glycogen synthase kinase 3-beta (GSK-3β) regulates cell proliferation and improves survival of bladder cancer patients (Naito et al., 2010). Moreover, GSK-3β is a potential therapeutic target in ovarian, breast, and bladder cancers (Cao et al., 2006). Therefore, it may be a useful indicator of potential therapeutic treatments for glioma. However, the role of GSK-3β in glioma is yet to be elucidated.

Irradiation of cancer cells induces cell death by different mechanisms. Mitotic catastrophe is the leading event to cell death in the form of apoptosis, necrosis, or autophagy (Wouters, 2009). Radioresistance is associated with the activation level of AKT (Jiang et al., 2007). Radiation can induce DNA damage such as double stranded-breaks (DSBs). If this damage is not repaired, it results in G2/M arrest and ultimately leads to cell death.

In our study, we provide evidence that the PI3K inhibitor GDC-0941 reduces migration, inhibits proliferation, induces apoptosis and autophagy, enhances the cytotoxic effect, and acts in combination with TMZ and IR in glioma cell lines. Microarray analysis (AKT/PKB Phospho Antibody Array) was used to confirm these changes at the protein level in SHG44 cells. Furthermore, differences in protein expression were verified by western blot analysis in three GBM cell lines. We found that GDC-0941 reduces p-AKT and MGMT levels and increases p53 and GSK-3β expression. These findings suggest that the combination of GDC-0941 and TMZ inhibits glioma cell growth by down-regulating the PI3K/AKT signal pathway. These results collectively suggest that TMZ+IR+GDC-0941 therapy is a potential therapeutic option for GBM treatment.

Section snippets

Cell culture

The human GBM cell lines, T98G and A172, were obtained from American Type Culture Collection. The SHG44 cell line was purchased from the Chinese Academy of Sciences, Shanghai, China. A172 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 1% penicillin/streptomycin (Invitrogen) and 10% fetal bovine serum (FBS, Gibco). T98G cells were maintained in MEM containing 1% penicillin/streptomycin, 1% non-essential amino acids (NEAA), and 10% FBS. SHG44 glioma

GDC-0941 enhances the suppression of glioblastoma cell viability by TMZ ± ionizing radiation (IR)

The potential of GDC-0941 to suppress cell proliferation was assessed in T98G, SHG44, and A172 GBM cell lines. TMZ (50 μM) had only a minimal effect on viability of these GBM cells, as determined by the CCK8 assay. However, co-treatment with GDC-0941 in the presence or absence of IR resulted in a significant reduction in cell viability. Dimethyl sulfoxide (DMSO) was used as a vehicle control. The GDC-0941 dose was chosen from the range reported in Phase I/II studies in vitro and in vivo (

Discussion

The SOC for GBM is initial surgical resection followed by treatment with TMZ combined with radiation therapy. Despite the aggressive treatment options adopted, GBM still needs therapeutic strategies that are more effective. GDC-0941, an orally administered potent inhibitor of pan-PI3K, shows good anti-neoplastic efficacy with favorable pharmaceutical properties (Ehrhardt et al., 2015, Sarker et al., 2015). A concern for the treatment with GDC-0941 is whether it can cross the blood–brain barrier

Conclusions

GDC-0941 may prove to be a useful addition to the current standard GBM therapy. Taken together, data obtained from this study suggest that targeting the PI3K pathway by GDC-0941 or other PI3K inhibitors combined with standard therapy is a potential strategy for improving the dismal outcome of GBM.

Acknowledgments

This work was supported by the Nature Science Foundation of Jilin Province (201015231) and Research Project of Jilin Provincial Health Department (2011S003), China.

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