Inhibition of metabotropic glutamate receptor 5 facilitates hypoxia-induced glioma cell death

Highlights

• Inhibition of mGluR5 under hypoxia promoted cell death.

• Inhibition of mGluR5 induced expression of genes of mitochondrial oxidative function.

• Akt activation reversed mGluR5 inhibition-induced facilitation of glioma cell death.

Abstract

Glioma is a primary brain tumor with high frequency and dismal prognosis. As there is no permanent cure available, identifying new therapy or mediator to augment the effectiveness of existing therapy is urgently needed. In the current study we tested the effect of group I metabotropic glutamate receptors (mGluRs): mGluR1 and mGluR5 on the viability of glioma cell lines. We analyzed cell viability using lactate dehydrogenase (LDH) release assay and evaluated apoptosis by propidium iodide (PI) staining. We used qPCR to evaluate change in mitochondrial gene expression and Western blot to evaluate the phosphorylation of Akt and ERK. Inhibition of mGluR5 by a selective antagonist MPEP under hypoxia promoted cell death, and induced expression of mitochondrial oxidative function related genes, with concurrent lowering of AKT phosphorylation level in glioma cell lines. Akt activation reversed mGluR5 inhibition on hypoxia-induced glioma cell death. These results suggest mGluR5 as a potential therapeutic target for hypoxic tumors such as malignant glioma.

Introduction

Gliomas, as the most frequent primary brain tumor, have rapid progression and dismal prognosis of less than 1-year median survival (Ohgaki et al., 2004). However, there is still no effective treatment available due to the fact that glioma cells have complex composition, diffuse invasiveness and are able to evade therapy as a result of the blood brain barrier (Westphal and Lamszus, 2011).

Current treatments of glioma in some clinics include first line treatment tumor resection and then radio-chemotherapy with the alkylating drug temozolomide (Stupp et al., 2005). However, such treatment couldn’t benefit imminent tumor recurrence which accounts for most glioblastoma mortality. Follow-up treatments are urgently needed. For instance, the vascular endothelial growth factor (VEGF)-targeting antibody bevacizumab is commonly used to reduce micro-vessel density and increase intra-tumor hypoxia (Rapisarda et al., 2009, Weller et al., 2013), since VEGF enhances angiogenesis which is typical in glioblastoma (Rubenstein et al., 2000).

Hypoxia is prevalent in the microenvironment of gliomas with changed metabolism (Darbin et al., 2000). However, it impedes radiotherapy, as oxygen is required to generate free radicals to destroy tumor cell (Steinbach et al., 2003), and chemotherapy, as it reduced drug uptake, stability and penetration into tumors (Reichert et al., 2002). Therefore, hypoxia in the glioma microenvironment needs to be addressed for better treatment.

mGluRs are G-protein-coupled receptors mostly expressed in the central nervous system, which function in neuronal excitability and synaptic plasticity, as well as modulate the feedback inhibition of neurotransmitter release. mGluR family has three groups according to their sequence homology, pharmacology and associated second messenger signaling pathways (Niswender and Conn, 2010). Group I mGluR signaling is mediated by GTP-binding proteins (G-proteins) and downstream second messengers such as cyclic adenosine monophosphate, diacylglycerol and inositol 1,4,5-triphosphate. On the other hand, signaling pathways of group II and group III mGluRs are mostly mediated by Gi/o proteins, which can liberate the Gβγ subunits to modulate activity of adenylyl cyclase, ion channel and other downstream signaling molecules (Hermans and Challiss, 2001, Marinissen and Gutkind, 2001).

Accumulating evidences indicate that, aside from neuro-modulation and maintenance of cellular homeostasis of the central nervous system (Pereira et al., 2017), mGluRs are expressed in other types of tissues and play other functional roles, such as in human malignancies. mGluR5 is found to be expressed in oligodendroglial progenitor cells and oligodendrocytes in rats by immunocytochemical analysis and pharmaceutical function verification (Luyt et al., 2003). Positive correlation between mGluR5 expression and survival rate has been documented in human oral squamous cell carcinoma tissues and cell lines (Park et al., 2007). mGluR5 is also expressed in human laryngeal cancer cell lines, and mGluR5 antagonist leads to inhibition of cell proliferation (Stepulak et al., 2011). Melanocyte-specific mGluR5 transgenic mice develop melanoma by targeting extracellular-signal-regulated kinase (ERK) (Choi et al., 2011). Ectopic mGluR1 expression renders normal melanocytes hyperproliferative in vitro and melanoma tumor prone in vivo (Marin and Chen, 2004). mGluR1 signaling mediates proliferation of endothelial cells and angiogenesis in breast cancer (Speyer et al., 2014). In prostate cancer and lung adenocarcinoma, single nucleotide polymorphisms and mutation are detected in mGluR1 (Ali et al., 2014).

In the current work, we studied the function of group I mGluRs in glioma. Specifically, we tested the effect of mGluR1 and mGluR5 on the viability of glioma cell lines. Inhibition of mGluR5 by a selective antagonist MPEP under hypoxia promoted cell death, and induced expression of genes related to mitochondrial oxidative function with concurrent lowering of AKT phosphorylation level in glioma cell lines. Akt activation reversed mGluR5 inhibition on hypoxia-induced glioma cell death. These results suggested that mGluR5 promoted glioma cell death under hypoxia, raising the possibility that mGluR5 could be used as secondary drug for glioma treatment.