γ-Glutamyl transpeptidase catalyses the extracellular detoxification of cisplatin in a human cell line derived from the proximal convoluted tubule of the kidney

Abstract

Nephrotoxicity is a side-effect and the main factor limiting the clinical use of cisplatin. In vivo, the administration of the cysteine-containing tripeptide glutathione (GSH) has been found to reduce nephrotoxicity, but the biochemical mechanism of this protective action is not fully understood. The present study was designed to gain insights into the mechanism by which GSH prevents cisplatin nephrotoxicity. We also wanted to verify the hypothesis of whether the protective action of GSH is mediated by products of the extracellular breakdown of GSH catalysed by gamma-glutamyl transpeptidase (GGT), an enzyme that is highly expressed in kidney tubular cells. The study was performed in HK-2 cells, derived from the immortalisation of human kidney proximal tubule cells. We investigated the influence of modulators of GGT activity and/or thiols on the antiproliferative activity of cisplatin and on the intracellular GSH content. We determined the antiproliferative activity of cisplatin, platinum cellular accumulation and DNA platination following precomplexing of the drug with thiols. The antiproliferative effect of cisplatin was minimally affected by the addition of GSH. However, when the antiproliferative assay was performed in the presence of glycyl-glycine (GlyGly), to serve as a transpeptidation acceptor and thus to stimulate GGT-mediated GSH catabolism, cisplatin-induced growth inhibition was largely prevented. This effect was not mediated through an increase of intracellular GSH levels, which were not affected by the GlyGly supplementation. The thiol dipeptide cysteinyl-glycine, i.e. the GSH catabolite generated by GGT activity, showed a higher reactivity against cisplatin in vitro than GSH, as was shown by the more rapid oxidation of its -SH groups. The cisplatin/GSH or cisplatin/cysteinyl-glycine adducts did not display an antiproliferative effect. However, 2 h precomplexing with GSH in the presence of GGT, or directly with the GSH catabolite cysteinyl-glycine, decreased the antiproliferative effect of cisplatin and drug-induced DNA platination to a greater extent than precomplexing with GSH alone. The results of the present study show that, in HK-2 cells, extracellular GSH decreases the antiproliferative effects of cisplatin only upon its hydrolysis by GGT, thereby supporting the hypothesis that the extracellular metabolism of GSH by GGT plays a role in modulating cisplatin nephrotoxicity. A primary role in the protection of HK-2 cells appears to be played by cysteinyl-glycine, the proximal product of the GGT-mediated hydrolysis of GSH, which shows a high reactivity against CDDP resulting in the rapid inactivation of the drug.

Controversial role of Gamma-Glutamyl Transferase activity in cisplatin nephrotoxicity

“…Preincubation of cells before cisplatin treatment indeed rescued from cisplatin sensitivity by more than 200-fold in RPteC/SV40 from increased sensitivity. these data would suggest that GGt is a "good" activity involved in detoxifiying cisplatin, and are consistent with previously published proposed mechanisms of GGt-dependent resistance to cisplatin (Daubeuf et al, 2002(Daubeuf et al, , 2003Paolicchi et al, 2003). these proposed mechanisms were all based on findings using 2D cell cultures with cell lines that had varying degrees of similarity with RPteCs down to overexpression of GGT in HeLa cells.…”

“…these results led Hanigan and co-workers to the conclusion that cisplatin is successively metabolized by GGT, dipeptidase and finally cystein-conjugate beta-lyase, finally forming an active thiol that causes nephrotoxicity (townsend, 2003;Zhang and Hanigan, 2003). Paolicchi et al (2003) reinterpreted these findings, as it had already been shown that systemic inhibition of GGt by acivicin lead to increased plasmatic levels of GSH in mice (Griffith and Meister, 1979). This finding was corroborated in GGT knockout mice (lieberman et al, 1996).…”

“…However, even if in vivo models of GGt knockout mice and the use of GGt inhibitors in mice favor a role of GGT in toxification of cisplatin, results from in vitro models do not yet match the in vivo results. Specifically, some reports show toxic effects caused by metabolism of cisplatin through a pathway including GGt (Hanigan et al, 2001), whereas others see GGT as the main detoxification mechanism, where the direct GGt product cysgly or the GGt substrate glutathione (GSH) are covalently attached to cisplatin, rendering it nontoxic (Daubeuf et al, 2002(Daubeuf et al, , 2003Paolicchi et al, 2003).…”

γ-Glutamyl transpeptidase is induced by 4-hydroxynonenal via EpRE/Nrf2 signaling in rat epithelial type II cells

“…γ-Glutamyl transpeptidase (γ-glutamyl transferase, GGT), an enzyme located on the outer surface of the plasma membrane, catalyzes the transfer of the γ-glutamyl moiety from glutathione (GSH), glutathione S-conjugates, or other γ-glutamyl compounds to γ-glutamyl acceptors such as amino acids, dipeptides, or H 2 O. GGT plays key roles in the maintenance of glutathione homeostasis [1][2][3][4][5][6][7] and metabolism of biomolecules such as leukotriene C4 and xenobiotics following their conjugation with GSH [8].…”

Can gamma-glutamyl transferase levels contribute to a better prognosis for patients with hepatocellular carcinoma?

“…A study of U937 lymphoma cells found that γ-GTP may play a role in anti-apoptotic signaling (36). A study has confirmed that cysteinyl-glycine, which is catalyzed by γ-GTP, is able to form complexes with cisplatin and that such adducts are not readily transported through the cell membrane (37). These mechanisms are thought to account for the progression of HCC.…”