Altered glutathione metabolism in oxaliplatin resistant ovarian carcinoma cells

Elevation of glutathione (GSH) is commonly observed in cellular resistance to a number of anticancer agents. Most frequently reported change in GSH metabolism that is associated with the elevated GSH levels is increased mRNA expression and activity of γ-glutamyl cysteine synthetase (γGCS), the first enzyme of the GSH biosynthetic pathway. We have isolated sublines of the A2780 ovarian carcinoma cell line (C10 and C25) that are 8- and 12-fold resistant to oxaliplatin by repeatedly exposing the cells to increasing concentrations of the platinum agent. The GSH levels in C10 and C25 cell sublines are 3.1-and 3.8-fold higher than the parent A2780 cell line. The mRNA levels and activities for γGCS and that for γ-glutamyl transpeptidase (γGT), the GSH salvage pathway enzyme, were measured in these cells. The mRNA for γGT and γGCS were measured by RT-PCR, with quantitation of the PCR product by HPLC; mRNA levels are expressed as ratios to β-actin mRNA, used as an endogenous standard. GSH and γGCS activity were measured by HPLC assays and γGT activity by a colorimetric assay. The increase in GSH in C10 and C25 was associated with an elevation in γGT mRNA (2.5- and 8-fold) and γGT activity (2.7- and 2.8-fold). No changes were observed in γGCS mRNA levels or activity. The data indicate that alterations in GSH metabolism leading to elevations in cellular GSH in A2780 ovarian carcinoma cells selected for low levels of resistance to oxaliplatin are mediated by γGT, the ‘salvage’ pathway, rather than an increase in GSH biosynthesis.