Riluzole enhances ionizing radiation-induced cytotoxicity in human melanoma cells that ectopically express metabotropic glutamate receptor 1 in vitro and in vivo.
Riluzole enhances ionizing radiation-induced cytotoxicity in human melanoma cells that ectopically express metabotropic glutamate receptor 1 in vitro and in vivo.
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DOI：
10.1158/1078-0432.ccr-10-1276
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发表时间：
2011-04-01
期刊：
影响因子：
--
通讯作者：
Haffty BG
中科院分区：
文献类型：
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作者：
Khan AJ;Wall B;Ahlawat S;Green C;Schiff D;Mehnert JM;Goydos JS;Chen S;Haffty BG
脑转移是转移性黑色素瘤患者常见的终末前事件，需要放射治疗。我们的小组先前已经证明，表达人GRM 1（hGRM 1）的黑色素瘤细胞释放过量的细胞外谷氨酸，并被谷氨酸释放抑制剂利鲁唑抑制生长。阿曲唑处理的细胞在24小时时在细胞周期的G2/M期积累，然后经历凋亡性细胞死亡。我们评估了利鲁唑是否增强了黑色素瘤细胞的放射敏感性。进行克隆形成测定以评价在表达和不表达hGRM 1的黑素瘤细胞中处理后的克隆形成存活。进行Western免疫印迹以确认凋亡细胞死亡。异种移植小鼠模型用于验证体外实验。将从异种移植物收获的肿瘤固定并染色用于细胞凋亡和DNA损伤标记物。在hGRM 1阳性细胞系C8161和UACC 903中，利鲁唑增强了电离辐射的致死作用;在hGRM 1阴性的UACC 930细胞系中没有观察到差异。用利鲁唑加辐射处理的C8161细胞也显示出最高水平的PARP和半胱天冬酶-3的裂解形式;通过免疫组织化学，切除的C8161异种移植物显示出最大数量的凋亡细胞（p<0.001）。在细胞周期分析中，证实了与利鲁唑和辐射的组合在G2/M期的序列依赖性富集。用利鲁唑和每周放射部分处理的异种移植物表现出显著的生长抑制，并显示DNA损伤显著增加。我们已经证明，在体外和体内，利鲁唑和电离辐射的组合导致更大的细胞毒性。这些结果对接受全脑放射治疗的脑转移患者具有临床意义。
Brain metastases are a common pre-terminal event in patients with metastatic melanoma and require radiation therapy. Our group has previously demonstrated 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 G2/M phase of the cell cycle at 24 hours, and then undergo apoptotic cell death. We evaluated whether riluzole enhanced radiosensitivity in melanoma cells. Clonogenic assays were performed to evaluate clonogenic survival after treatment in hGRM1 expressing and non-expressing 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. 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 demonstrated the greatest number of apoptotic cells by immunohistochemistry (p<0.001). On cell cycle analysis, a sequence-dependent enrichment in the G2/M phase was demonstrated with the combination of riluzole and irradiation. Xenografts treated with riluzole and weekly radiation fractions demonstrated significant growth inhibition and revealed markedly increased DNA damage. We have demonstrated, 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.