Tumor cell responses to a novel glutathione S-transferase-activated nitric oxide-releasing prodrug

Abstract

We have used structure-based design techniques to introduce the drug O(2)-[2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino) phenyl] 1-N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), which is efficiently metabolized to potentially cytolytic nitric oxide by the pi isoform of glutathione S-transferase, an enzyme expressed at high levels in many tumors. We have used mouse embryo fibroblasts (MEFs) null for GSTpi (GSTpi(-/-)) to show that the absence of GSTpi results in a decreased sensitivity to PABA/NO. Cytotoxicity of PABA/NO was also examined in a mouse skin fibroblast (NIH3T3) cell line that was stably transfected with GSTpi and/or various combinations of gamma-glutamyl cysteine synthetase and the ATP-binding cassette transporter MRP1. Overexpression of MRP1 conferred the most significant degree of resistance, and in vitro transport studies confirmed that a GSTpi-activated metabolite of PABA/NO was effluxed by MRP1 in a GSH-dependent manner. Additional studies showed that in the absence of MRP1, PABA/NO activated the extracellular-regulated and stress-activated protein kinases ERK, c-Jun NH(2)-terminal kinase (JNK), and p38. Selective inhibition studies showed that the activation of JNK and p38 were critical to the cytotoxic effects of PABA/NO. Finally, PABA/NO produced antitumor effects in a human ovarian cancer model grown in SCID mice.

Fig. 1.

Proposed mechanism by which JS-K and PABA/NO generate NO on activation by GSH. B, structure of non-NO-releasing by-product of PABA/NO, JS-39-94.

Fig. 2.

Molecular surface representation of GSTπ and GSTα with ball- and-stick models of the Meisenheimer complexes formed by GSH and NO-releasing prodrugs. A, GSTπ in complex with the Meisenheimer complex of JS-K (Shami et al., 2003). B, GSTα in complex with the Meisenheimer complex of JS-K (Shami et al., 2003). C, GSTπ in complex with the Meisenheimer complex of PABA/NO (this study). D, GSTα in complex with the Meisenheimer complex of PABA/NO (this study). In C and D, the N-methyl-N-(p-carboxyphenyl)amino group is highlighted with black sticks (bonds).

Fig. 3.

GSTπ^−/− MEF cells are less sensitive to PABA/NO than the parental GSTπ^+/+. Cell survival of GSTπ^−/− (■) and GSTπ^+/+ (●) was assessed 48 h after drug exposure, as detailed under Materials and Methods. IC₅₀ values of 26 ± 13 μM for GSTπ^+/+ and 39 ± 15 μM for GSTπ^−/− were calculated. Data are represented as means ± S.D. of three independent experiments.

Fig. 4.

PABA/NO causes cells to undergo apoptosis. Annexin V staining of WT cells was assessed 2 h after drug exposure as detailed under Materials and Methods.

Fig. 5.

PABA/NO causes protein nitration in WT but not MRP1 overexpressing cells in a dose-dependent manner. Cells were treated for 30 min with the indicated concentrations of PABA/NO.

Fig. 6.

Activation of the ERK, p38, and JNK mitogen-activated protein kinases by treatment with PABA/NO. A, cells were treated with 30 μM PABA/NO for the indicated times. B, cells were treated with the indicated concentrations of PABA/NO for 60 min. 20 μg of protein was loaded in each lane. The protein expression levels of ERK, p38, and JNK were unchanged with the addition of PABA/NO (bottom). Even loading of protein was confirmed by probing for actin (data not shown).

Fig. 7.

Transport of [³H]LTC₄ in the presence of ATP. Uptake of [³H]LTC₄ into MRP1 ‘inside-out’ vesicles was measured in the presence of [³H]LTC₄ alone (□), 25 μM GSH (■), 25 μM PABA/NO (●), or 25 μM GSH and 25 μM PABA/NO (엯) (A) or 500 nM GSH and 1 μg GSTπ (■), 50 μM PABA/NO and 1 μg GSTπ (●), 50 μM PABA/NO and 500 nM GSH (□), or 500 nM GSH, 50 μM PABA/NO, and 1 μg GSTπ (엯) (B), as detailed under Materials and Methods. Data are represented as means ± S.D. of three independent experiments.

Fig. 8.

Treatment with PABA/NO delays growth of A2780 human ovarian tumors in SCID mice. The average tumor volumes ± S.E. in control (●), PABA/NO (엯), and cisplatin (■) treated mice are reported. Mice were treated with 0.8% DMSO (control), 3.36 mg/kg PABA/NO and 2.5 mg/kg cisplatin, respectively.