Mizutani Y, Yoshida O
Department of Urology, Faculty of Medicine, Kyoto University, Japan.
Cancer. 1994 Feb 1;73(3):730-7. doi: 10.1002/1097-0142(19940201)73:3<730::aid-cncr2820730338>3.0.co;2-x.
Previous studies have reported the glutathione plays a central role in a wide range of cellular functions, including protection, detoxification, transport, and metabolism. Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamyl-cysteine synthetase, depletes intracellular glutathione. The study investigates the cytotoxic effect of BSO and tumor necrosis factor-alpha (TNF-alpha) used in combination on TNF-alpha-resistant human renal and ovarian cancer cells.
Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. TNF-alpha mRNA was examined by Northern blot analysis.
Combination treatment of TNF-alpha-resistant R4 and R11 human renal cell carcinoma cells with BSO and TNF-alpha overcame their resistance to TNF-alpha. In addition, the combination of BSO and TNF-alpha resulted in a synergistic cytotoxic effect on TNF-alpha-resistant OVC-8 and C30 human ovarian cancer cells. Treatment of R4, R11, and OVC-8 cells with TNF-alpha in combination with glutathione or N-acetyl-cysteine (NAC) showed an antagonistic cytotoxic effect. A possible mechanism of resistance to TNF-alpha in tumor cells is the expression of TNF-alpha mRNA or protein. R4 cells and OVC-8 cells constitutively expressed mRNA for TNF-alpha. Treatment of R4 cells or OVC-8 cells with BSO down-regulated the expression of TNF-alpha mRNA; however, treatment with TNF-alpha up-regulated the expression of TNF-alpha mRNA. When BSO was used in combination with TNF-alpha, the level of TNF-alpha mRNA enhanced by TNF-alpha was markedly reduced. Incubation of R4 cells with glutathione or NAC also down-regulated the expression of TNF-alpha mRNA. R11 and C30 cells did not constitutively express mRNA for TNF-alpha, and the BSO treatment had no effect on the TNF-alpha mRNA level.
This study demonstrates that the combination of BSO and TNF-alpha can overcome the TNF-alpha resistance of tumor cells and that depletion of intracellular glutathione and down-regulation of TNF-alpha mRNA by BSO may play a role in the enhanced cytotoxicity seen with the combination of BSO and TNF-alpha. There may not be always a correlation between the expression of TNF-alpha mRNA in tumor cells and their resistance to TNF-alpha. The synergistic effect obtained with established renal cell carcinoma cells and ovarian cancer cells suggests that combination treatment with TNF-alpha and BSO could have clinical application in the therapy of TNF-alpha-resistant tumors.
以往研究报道，谷胱甘肽在广泛的细胞功能中发挥核心作用，包括保护、解毒、转运和代谢。丁硫氨酸亚砜胺（BSO）是γ-谷氨酰-半胱氨酸合成酶的特异性抑制剂，可消耗细胞内谷胱甘肽。本研究调查了联合使用BSO和肿瘤坏死因子-α（TNF-α）对TNF-α耐药的人肾癌细胞和卵巢癌细胞的细胞毒性作用。
通过1天的微量培养四氮唑染料法测定细胞毒性。通过Northern印迹分析检测TNF-α mRNA。
联合使用BSO和TNF-α处理TNF-α耐药的R4和R11人肾癌细胞可克服其对TNF-α的耐药性。此外，BSO和TNF-α的联合对TNF-α耐药的OVC-8和C30人卵巢癌细胞产生协同细胞毒性作用。用TNF-α联合谷胱甘肽或N-乙酰半胱氨酸（NAC）处理R4、R11和OVC-8细胞显示出拮抗细胞毒性作用。肿瘤细胞对TNF-α耐药的一种可能机制是TNF-α mRNA或蛋白的表达。R4细胞和OVC-8细胞组成性表达TNF-α的mRNA。用BSO处理R4细胞或OVC-8细胞可下调TNF-α mRNA的表达；然而，用TNF-α处理可上调TNF-α mRNA的表达。当BSO与TNF-α联合使用时，TNF-α上调的TNF-α mRNA水平明显降低。用谷胱甘肽或NAC孵育R4细胞也可下调TNF-α mRNA的表达。R11和C30细胞不组成性表达TNF-α的mRNA，BSO处理对TNF-α mRNA水平无影响。
本研究表明，BSO和TNF-α联合可克服肿瘤细胞对TNF-α的耐药性，BSO消耗细胞内谷胱甘肽并下调TNF-α mRNA可能在BSO和TNF-α联合产生的增强细胞毒性中起作用。肿瘤细胞中TNF-α mRNA的表达与其对TNF-α的耐药性之间可能并不总是存在相关性。在已建立的肾癌细胞和卵巢癌细胞中获得的协同效应表明，TNF-α和BSO联合治疗可能在TNF-α耐药肿瘤的治疗中具有临床应用价值。
