Miyajima A, Nakashima J, Yoshioka K, Tachibana M, Tazaki H, Murai M
Department of Urology, Keio University School of Medicine, Shinjuku, Tokyo, Japan.
Br J Cancer. 1997;76(2):206-10. doi: 10.1038/bjc.1997.363.
This study was undertaken to investigate the intracellular induction of reactive oxygen species (ROS) by cis-dichlorodiammineplatinum (CDDP) and the augmentation of their cytotoxicity in bladder cancer cells (KU7) by enhancement of ROS generation by the glutathione (GSH) depletors buthionine sulphoximine (BSO) and diethylmaleate (DEM). CDDP-induced cytotoxicity in KU7 cells and its modulation by GSH depletors were determined using spectrophotometric measurement with crystal violet staining. The effects of GSH depletors on intracellular GSH levels were confirmed using the GSH reductase-DTNB recycling method. Intracellular ROS generation induced by CDDP with or without GSH depletors was estimated from the amount of intracellular dichlorofluorescein (DCF), an oxidized product of dichlorofluorescein (DCFH), which was measured with an anchored cell analysis and sorting system. The cytotoxic effects of CDDP (IC50 15.0 +/- 2.5 microM) were significantly enhanced by BSO (IC50 9.3 +/- 2.6 microM, P < 0.01) and DEM (IC50 10.3 +/- 0.3 microM, P <0.01). BSO and DEM produced a significant depletion in intracellular GSH levels (9.6 +/- 0.4 nmol 10(-6) cells, 17.9 +/- 1.0 nmol 10(-6) cells) compared with the controls (30.5 +/- 0.6 nmol 10(-6) cells). Intracellular DCF production in KU7 cells treated with CDDP (1.35 +/- 0.33 microM) was significantly enhanced by the addition of BSO (4.43 +/- 0.33 microM) or DEM (3.12 +/- 0.22 microM) at 150 min. These results suggest that ROS may play a substantial role in CDDP-induced cytotoxicity and that GSH depletors augment its cytotoxicity through an enhancement of ROS generation in bladder cancer cells.
本研究旨在探讨顺二氯二氨铂（CDDP）对膀胱癌细胞（KU7）内活性氧（ROS）的诱导作用，以及通过谷胱甘肽（GSH）消耗剂丁硫氨酸亚砜胺（BSO）和马来酸二乙酯（DEM）增强ROS生成来增强其细胞毒性。使用结晶紫染色的分光光度法测定CDDP诱导的KU7细胞毒性及其被GSH消耗剂的调节作用。使用GSH还原酶-DTNB循环法确认GSH消耗剂对细胞内GSH水平的影响。根据细胞内二氯荧光素（DCF）的量估计有无GSH消耗剂时CDDP诱导的细胞内ROS生成，DCF是二氯荧光素（DCFH）的氧化产物，通过锚定细胞分析和分选系统进行测量。BSO（IC50 9.3 +/- 2.6 microM，P < 0.01）和DEM（IC50 10.3 +/- 0.3 microM，P <0.01）显著增强了CDDP的细胞毒性作用（IC50 15.0 +/- 2.5 microM）。与对照组（30.5 +/- 0.6 nmol 10(-6)个细胞）相比，BSO和DEM使细胞内GSH水平显著降低（9.6 +/- 0.4 nmol 10(-6)个细胞，17.9 +/- 1.0 nmol 10(-6)个细胞）。在150分钟时，添加BSO（4.43 +/- 0.33 microM）或DEM（3.12 +/- 0.22 microM）显著增强了用CDDP（1.35 +/- 0.33 microM）处理的KU7细胞内DCF的产生。这些结果表明，ROS可能在CDDP诱导的细胞毒性中起重要作用，并且GSH消耗剂通过增强膀胱癌细胞中的ROS生成来增强其细胞毒性。
