Effects of riluzole (RZ) and ionizing radiation (IR) in metabotropic glutamate receptor-1 (GRM1) positive human melanoma

Abstract

9083 Background: Melanoma has long been known to be relatively radio-resistant. GRM1 is a metabotropic glutamate receptor that has been detected in human melanoma cell lines and biopsies. Riluzole (RZ), a glutamate release inhibitor, has been shown to arrest GRM1 positive human melanoma cells in G2/M and sub-G1 phases of the cell cycle. The purpose of this study was to determine if RZ enhances the lethal effects of IR in human melanoma. Methods: ATP luminescence assays were performed. Clonogenic assays were performed and cell survival curves generated. Cell cycle analysis was performed utilizing flow cytometry. Western blot analysis was performed utilizing cleaved PARP and caspase-3 antibodies as markers of apoptosis. Results: Luminescence assays revealed 25uM Riluzole to be the necessary concentration for clonogenic assays. At 2Gy, there was a 48% reduction (p≤0.05) in cell survival in RZ-treated cells. At 4 Gy, there was a 19% reduction (p≤0.05) in cell survival in RZ-treated cells. No differences were seen at 6 and 8 Gy. Cell cycle analysis showed that the combination of IR and RZ was superior to IR alone in increasing the number of cells in sub-G1, which represents apoptotic death. Western blot analysis showed that the combination of IR and RZ showed yielded increased cleaved PARP and caspase-3 activity when compared to IR alone. Conclusions: Riluzole is a FDA approved drug that has long been used in ALS. It is relatively non-toxic and crosses the blood brain barrier. Our data shows that Riluzole in combination with radiation eliminates the radio-resistant shoulder of the C8161 survival curve. RZ and IR, as combination therapy are more lethal than IR or RZ alone in human melanoma, as demonstrated by flow cytometry and WB analysis. This data has promising implications for melanoma patients with brain metastases. No significant financial relationships to disclose.