Enhancement of arsenic trioxide-induced apoptosis in renal cell carcinoma cells by L-buthionine sulfoximine.
Enhancement of arsenic trioxide-induced apoptosis in renal cell carcinoma cells by L-buthionine sulfoximine.
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DOI：
10.3892/ijo.24.6.1489
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发表时间：
2004-06
影响因子：
5.2
通讯作者：
X. Wu;O. Ogawa;Y. Kakehi
中科院分区：
文献类型：
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作者：
X. Wu;O. Ogawa;Y. Kakehi
三氧化二砷(As_2O_3)在体外可诱导急性早幼粒细胞白血病患者临床缓解和多种肿瘤细胞的凋亡。为建立以三氧化二砷为基础的联合化疗方案，我们观察了三氧化二砷与化疗药物或谷胱甘肽合成抑制剂L丁硫氨酸亚磺胺联合应用对肾癌的细胞毒作用。采用四甲基偶氮唑盐比色法和等位图法分别检测其细胞毒性和协同作用。采用Hoechst 33342染色、流式细胞仪分析、DNA片段分析等方法检测细胞凋亡率。三氧化二砷联合阿霉素、长春花碱或5-氟尿嘧啶处理ACHN细胞产生拮抗作用。而三氧化二砷和硫代巴比妥钠联合作用具有协同的细胞毒作用。Caki-1、Caki-2、NC65细胞和6例患者新鲜来源的肾癌细胞也有协同作用。As_2O_3和BSO联合作用对ACHN细胞的细胞毒作用明显高于As_2O_3组、BSO组、BSO组和As_2O_3组。我们进一步探讨了这种协同作用的机制，发现As_2O_3和BSO的协同细胞毒性是通过诱导细胞凋亡来实现的。联合应用可显著降低细胞内谷胱甘肽含量和谷胱甘肽S转移酶活性。然而，三氧化二砷与阿霉素、长春花碱或5-氟尿嘧啶合用均不降低细胞内GSH和GST。此外，GSH增强剂N-乙酰半胱氨酸和硫辛酸显著抑制As_2O_3和BSO的联合细胞毒作用。这些结果表明，BSO通过下调细胞内GSH氧化还原系统，使肾癌细胞对As_2O_3诱导的细胞凋亡敏感，提示联合应用As_2O_3和BSO治疗肾癌具有潜在的应用价值。
Arsenic trioxide (As2O3) induces clinical remission in acute promyelocytic leukemic patients and apoptosis in various tumor cells in vitro. To develop As2O3-based combination chemotherapy for renal cell carcinoma (RCC), we investigated the cytotoxic effects of As2O3 in combination with chemotherapeutic agents or L-buthionine sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor. Cytotoxicity and synergy were assessed by the MTT assay and isobolographic analysis, respectively. Apoptosis was monitored by Hoechst 33342 staining, flow cytometrical analysis, and DNA fragmentation assay. Treatment of ACHN cells with As2O3 in combination with adriamycin, vinblastine, or 5-fluorouracil induced an antagonistic effect. However, combination treatment with As2O3 and BSO resulted in a synergistic cytotoxic effect. Synergy was also obtained in Caki-1, Caki-2, NC65 cells and freshly derived RCC cells from 6 patients. Simultaneous treatment of ACHN cells with As2O3 and BSO caused significantly more cytotoxicity than the As2O3 first BSO second or the reverse treatment. We further explored the mechanisms underlying this synergistic effect and found that the synergistic cytotoxicity of As2O3 and BSO was realized by inducing apoptosis. This combination markedly decreased intracellular GSH content and GSH-S-transferase (GST) activity. However, neither the intracellular GSH nor GST was decreased by As2O3 with adriamycin, vinblastine, or 5-fluorouracil. Furthermore, the GSH-increasing agents N-acetylcysteine and lipoic acid significantly inhibited the combined cytotoxicity of As2O3 and BSO. These findings indicate that BSO sensitizes RCC cells to As2O3-induced apoptosis through the down-regulation of the intracellular GSH redox system, suggesting the potential application of a combination of As2O3 and BSO for the treatment of RCC.