Glutathione depletion causes a JNK and p38MAPK-mediated increase in expression of cystathionine-gamma-lyase and upregulation of the transsulfuration pathway in C6 glioma cells.
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.
Kandil
Sarah
S
UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
Brennan
Lorraine
L
McBean
Gethin J
GJ
D015074:2-Aminoadipic Acid / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000502:physiology; D003542:Cystathionine gamma-Lyase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D003545:Cysteine / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005470:Fluorometry; D005978:Glutathione / Q000502:physiology*; D006801:Humans; D007202:Indicators and Reagents; D048670:MAP Kinase Kinase 4 / Q000037:antagonists & inhibitors / Q000502:physiology*; D051058:Oncogene Protein v-akt / Q000235:genetics / Q000502:physiology; D010766:Phosphorylation; D013455:Sulfur / Q000378:metabolism*; D015854:Up-Regulation / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*
D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D015074:2-Aminoadipic Acid; D013455:Sulfur; D051058:Oncogene Protein v-akt; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D003542:Cystathionine gamma-Lyase; D005978:Glutathione; D003545:Cysteine