UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
Neurochem Int. 2010 Mar;56(4):611-9. doi: 10.1016/j.neuint.2010.01.004. Epub 2010 Jan 12.
Cancer cells have a high demand for cysteine as precursor of the antioxidant, glutathione, that is required to promote cell growth and division. Uptake of cystine by the x(c)(-) cystine-glutamate exchanger provides the majority of cysteine, but a significant percentage may be derived from methionine, via a transsulfuration pathway. Our aim was to evaluate the relative contribution of the exchanger and the transsulfuration pathway to glutathione synthesis in astrocytoma/glioblastoma cells, using the C6 glioma cell line as a model system. Blockade of the x(c)(-) exchanger with the gliotoxins l-alphaaminoadipate or l-beta-N-oxalylamino-l-alanine (400 microM) caused a loss of cellular cysteine and depletion in glutathione to 51% and 54% of control, respectively, after 24 h. Inhibition of the transsulfuration pathway with propargylglycine (1 mM, 24 h) depleted glutathione to 77% of control. Co-incubation of cells with gliotoxin and propargylglycine reduced glutathione to 39% of control at 24 h and to 20% at 48 h. Expression of cystathionine-gamma-lyase, the rate-limiting enzyme of the transsulfuration pathway, was significantly increased following incubation of the cells with gliotoxins. Incubation of C6 cells with diethylmaleate for 3 h led to a significant reduction in glutathione (63%), whereas expression of cystathionine-gamma-lyase was increased by 1.5-fold. Re-feeding methionine to diethylmaleate-treated cells incubated in the absence of cystine or methionine resulted in a significant recovery in glutathione that was blocked by propargylglycine. Co-incubation of C6 cells with diethylmaleate and the JNK-inhibitor, SP600125, abolished the increase in expression of cystathionine-gamma-lyase that had been observed in the presence of diethylmaleate alone. Similar results were obtained with the p38(MAPK) inhibitor, SB203580. It is concluded that glutathione depletion causes a JNK- and p38(MAPK)-mediated increase in expression of cystathionine-gamma-lyase that promotes flux through the transsulfuration pathway to compensate for loss of glutathione in C6 glioma cells.
癌细胞对半胱氨酸的需求很高，半胱氨酸是抗氧化剂谷胱甘肽的前体，谷胱甘肽是促进细胞生长和分裂所必需的。x(c)(-)胱氨酸-谷氨酸交换器摄取胱氨酸为细胞提供了大部分的半胱氨酸，但仍有相当一部分可能来自蛋氨酸，通过转硫途径。我们的目的是评估交换器和转硫途径对星形细胞瘤/胶质母细胞瘤细胞谷胱甘肽合成的相对贡献，使用 C6 神经胶质瘤细胞系作为模型系统。用神经毒素 l-α-氨基己二酸或 l-β-N-氧代-l-丙氨酸（400μM）阻断 x(c)(-)交换器 24 小时后，细胞内半胱氨酸丧失，谷胱甘肽耗竭至对照的 51%和 54%。用炔丙基甘氨酸（1mM，24 小时）抑制转硫途径使谷胱甘肽耗竭至对照的 77%。细胞与神经毒素和炔丙基甘氨酸共孵育 24 小时可使谷胱甘肽降低至对照的 39%，48 小时时降低至 20%。用神经毒素孵育细胞后，胱硫醚-γ-裂解酶的表达显著增加，胱硫醚-γ-裂解酶是转硫途径的限速酶。用二乙基马来酸酐孵育 C6 细胞 3 小时，谷胱甘肽显著减少（63%），而胱硫醚-γ-裂解酶的表达增加了 1.5 倍。在用二乙基马来酸酐处理的细胞中，在缺乏胱氨酸或蛋氨酸的情况下孵育，补充蛋氨酸可显著恢复谷胱甘肽，但被炔丙基甘氨酸阻断。C6 细胞与二乙基马来酸酐和 JNK 抑制剂 SP600125 共孵育，消除了单独用二乙基马来酸酐时观察到的胱硫醚-γ-裂解酶表达的增加。用 p38（MAPK）抑制剂 SB203580 也得到了类似的结果。研究结果表明，谷胱甘肽耗竭导致 JNK 和 p38（MAPK）介导的胱硫醚-γ-裂解酶表达增加，从而促进转硫途径的通量，以补偿 C6 神经胶质瘤细胞中谷胱甘肽的丧失。
