Elsevier

Biochemical Pharmacology

Volume 57, Issue 12, 15 June 1999, Pages 1383-1390
Biochemical Pharmacology

Chemotherapy and Metabolic Inhibitors
Interactions of prostaglandin A2 with the glutathione-mediated biotransformation system

https://doi.org/10.1016/S0006-2952(99)00048-9Get rights and content

Abstract

The cyclopentenone prostaglandin A2 (PGA2) is known to inhibit cell proliferation, and metabolism of this compound thus might be important in controlling its ultimate function. The glutathione-related metabolism of PGA2 was therefore investigated both with purified glutathione S-transferase P1-1 (GSTP1-1) and with IGR-39 human melanoma cells. Firstly, the irreversible inhibition of human GSTP1-1 and its mutants C47S, C101S, and C47S/C101S was studied. PGA2 appeared to inhibit GSTP1-1 mainly by binding to the cysteine 47 moiety of the enzyme. This binding was reversed by a molar excess of GSH, indicating that retro-Michael cleavage occurs. Secondly, after exposing IGR-39 human melanoma cells to PGA2, both diastereoisomers of the PGA2–glutathione conjugate are excreted into the medium, although with a clear excess of the S-form, due to its preferential formation by the GSTP1-1 present in the cells. Thirdly, the effect of PGA2 on intracellular GST activity was determined by quantification of the excreted glutathione conjugate S-(2,4-dinitrophenyl)glutathione (DNPSG) after exposure to 1-chloro-2,4-dinitrobenzene. DNPSG excretion was inhibited after incubation with 10 or 20 μM PGA2 for 1 or 4 hr, as a result of glutathione depletion, reversible GST inhibition, and covalent modification of intracellular GST. Furthermore, PGA2 also inhibited transport of DNPSG by the multidrug resistance-associated protein, an effect that was reversible and competitive. In conclusion, PGA2 modulates all three aspects of the glutathione-mediated biotransformation system, i.e. GSH levels, GSTP1-1 activity, and transport of GSH conjugates. A role for GSTP1-1 as a specific transport protein inside the cell is indicated.

Section snippets

Materials

GSH and NADH were obtained from Boehringer, Mannheim. Prostaglandins A2 and J2 were purchased from Sigma. CDNB was obtained from Aldrich Chemie. HPLC-grade trifluoroacetic acid was obtained from Baker. HPLC-grade methanol was from Labscan. DNPSG was synthesised analogously to Sokolovsky et al. [26].

[3H]Glycine was purchased from Amersham. GSTP1-1 was purified as previously described [27]. The three mutants of GSTP1-1, C47S, C101S, and C47S/C101S, were a generous gift from Dr. M. LoBello (Dept.

Inhibition studies with purified GSTP1-1

Time-dependent inhibition of GSTP1-1 and the mutants C47S, C101S, and C47S/C101S by prostaglandin A2 was studied. In Fig. 1A, the percentage of remaining GST activity after incubating 10, 25, or 250 μM of the prostaglandin A2 with GSTP1-1 is presented. Remaining activity after 3 hr was 74%, 56%, and 34%, respectively. Incubating these concentrations with the C47S mutant still resulted in a decrease in GST activity, albeit less evident than with the parent enzyme and only at the highest

Discussion

Prostaglandin A2 is known to inhibit cell proliferation 1, 2, 3, 4, 5, and numerous studies have been done on the metabolic fate of this prostaglandin. Recent findings concerning the formation and transport of the diastereoisomeric glutathione conjugates shed further light on this metabolism 20, 25. The present study aimed to obtain a more complete picture of the glutathione-related metabolism of PGA2 by investigating interactions with purified enzymes as well as in a cellular system using the

Acknowledgements

We thank Dr. M. LoBello for providing the GSTP1-1 mutants C47S, C101S, and C47S/C101S.

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