Riluzole Enhances Ionizing Radiation–Induced Cytotoxicity in Human Melanoma Cells that Ectopically Express Metabotropic Glutamate Receptor 1 In Vitro and In Vivo

Abstract

Purpose: Brain metastases are a common preterminal event in patients with metastatic melanoma and require radiation therapy. Our group has previously shown that human GRM1 (hGRM1) expressing melanoma cells release excess extracellular glutamate and are growth inhibited by riluzole, an inhibitor of glutamate release. Riluzole-treated cells accumulate in G 2 /M phase of the cell cycle at 24 hours, and then undergo apoptotic cell death. We evaluated whether riluzole enhanced radiosensitivity in melanoma cells.Experimental Design: Clonogenic assays were performed to evaluate clonogenic survival after treatment in hGRM1 expressing and nonexpressing melanoma cells. Western immunoblots were performed to confirm apoptotic cell death. A xenograft mouse model was used to validate the in vitro experiments. Tumors harvested from the xenografts were fixed and stained for apoptosis and DNA damage markers.Results: In the hGRM1-positive cell lines C8161 and UACC903, riluzole enhanced the lethal effects of ionizing radiation; no difference was seen in the hGRM1-negative UACC930 cell line. C8161 cells treated with riluzole plus irradiation also showed the highest levels of the cleaved forms of PARP and caspase-3; excised C8161 xenografts showed the greatest number of apoptotic cells by immunohistochemistry (P < 0.001). On cell cycle analysis, a sequence-dependent enrichment in the G 2 /M phase was shown with the combination of riluzole and irradiation. Xenografts treated with riluzole and weekly radiation fractions showed significant growth inhibition and revealed markedly increased DNA damage.Conclusions: We have shown, in vitro and in vivo, that the combination of riluzole and ionizing radiation leads to greater cytotoxicity. These results have clinical implications for patients with brain metastases receiving whole brain radiation therapy.

Metabotropic glutamate receptor 1 disrupts mammary acinar architecture and initiates malignant transformation of mammary epithelial cells

“…In accordance with an important role for Grm1 in melanomagenesis [3], we had previously demonstrated that inhibition of glutamate release by riluzole, an FDA-approved drug for the treatment of amyotrophic lateral sclerosis (ALS), suppressed human melanoma cell growth in vitro and xenograft tumor progression in vivo [21, 26, 27]. In a Phase 0 clinical trial using single agent riluzole for 14 days in patients with resectable stage III/IV melanoma, 34 % of them showed significant decrease in p-AKT and/or p-ERK expression in post-treatment tumor samples, in association with significant decrease in melanoma metabolic activity by FDG-PET [28].…”

Metabotropic glutamate receptors in cancer

“…In particular, mGluRs have been shown empirically to be the predominant mediators of glutamatergic signaling in many cancers (Khan et al, 2011; Koochekpour, 2013; Martino et al, 2013; Speyer et al, 2014; Teh and Chen, 2012; Zhang et al, 2015). The mechanisms by which mGluRs modulate peripheral cell transformation and tumor growth are postulated to be either ectopic expression of wild type mGluRs, increased proliferative signals arising from receptor overexpression, mutations, or expression of polymorphic variants (Ali et al, 2014; Brocke et al, 2010; Mehta et al, 2013; Namkoong et al, 2007; Wall et al, 2014; Zhang et al, 2015).…”

“…Although radiosensitizers have previously not been shown to provide survival benefit in human melanoma, Khan and colleagues (Khan et al, 2011) hypothesized that riluzole may be different, as cells in the G2/M phase rendered by riluzole treatment would be extremely sensitive to radiation (Ballo and Ang, 2004; Ballo et al, 2006; Eichler et al, 2007; Habermalz and Fischer, 1976). In vitro studies showed that mGluR1-positive human melanoma cells were significantly less likely to survive following ionizing radiation treatment at the 2 and 4Gy dose levels when pre-treated with riluzole at 25μM concentration, while there was negligible effect in mGluR1- negative human melanoma cells even at 100μM of riluzole treatment.…”

“…These findings reveal that the mechanism of riluzole is more complex than merely diminish available glutamate. In vivo, mGluR1- positive human melanoma xenografts treated with riluzole plus irradiation resulted in smaller tumor volume and significant increase in apoptotic tumor cells that stained positive for cleaved caspase-3, a marker of apoptosis, and γ-H2AX foci, a marker of DNA-double stranded breaks (Khan et al, 2011). These results suggest that riluzole may be used as a radiosensitizer in a subset of mGluR1 expressing human melanomas.…”

The Akt signaling pathway

“…Riluzole is a noncompetitive inhibitor of metabotropic glutamate receptor-1 (GRM1) that has been shown to slow the progression of amyotropic lateral sclerosis. 176,177 GRM1 is expressed in 60% of human melanomas, and ectopic expression of GRM1 in melanocytes in a murine model resulted in development of melanocytic lesions that were indistinguishable from melanoma. 178 Activation of GRM1 results in activation of the MAPK pathway in a BRAF and N-RAS independent manner.…”