15-deoxy-Δ^12,14-prostaglandin J₂ upregulates glutamate cysteine ligase expression through activation of PI3K and Nrf2 signaling in MCF-7 human breast cancer cells: a potential role of ROS

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15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂), a representative J-series cyclopentenone prostaglandin, has been reported to exert cytoprotective effects by inducing various phase 2 detoxifying/antioxidant enzymes. In the present study, we have found that 15d-PGJ₂ induces expression of glutamate cysteine ligase (GCL), the rate-limiting enzyme in the glutathione (GSH) synthesis, in the human breast cancer cell line (MCF-7). While GCL and its product GSH play key roles in elimination of electophilic carcinogens, their elevated levels in tumor cells may confer resistance to anti-cancer drugs. Thus, we investigated the molecular mechanism underlying 15d-PGJ₂-induced GCL expression in MCF-7 cells and its patho-physiologic implacations in mammary carcinogenesis. Treatment of MCF-7 cells with 15d-PGJ₂ (10 μM) caused a time-dependent induction of the GCL catalytic (GCLC) subunit with maximal induction observed at 24 h. Nrf2 is a major transcription factor involved in the transactivation of many genes encoding phase 2 detoxifying and antioxidant enzymes via the antioxidant response element (ARE). 15d-PGJ₂ treatment resulted in nuclear translocation of Nrf2 and its subsequent interaction with ARE. Transfection of MCF-7 cells with dominant negative Nrf2 abrogated 15d-PGJ₂-mediated induction of GCLC. LY294002, the phosphatidylinositol 3-kinase inhibitor, attenuated 15d-PGJ₂-mediated GCLC expression as well as nuclear translocation of Nrf2. 15d-PGJ₂ contains an α,β-unsaturated carbonyl moiety and readily forms adducts with various thiols including GSH. It is likely that the formation of a 15d-PGJ₂-GSH conjugate can result in the initial reduction in the GSH level, which may trigger the generation of reactive oxygen species (ROS). N-Acetyl-L-cysteine attenuated 15d-PGJ₂-mediated induction of GCLC expression, suggesting the possible involvement of ROS in 15d-PGJ₂-induced GCLC expression. 9,10-Dihydro-15d-PGJ₂, which lacks an electrophilic α,β-unsaturated carbonyl group and cannot form a GSH conjugate, failed to induce GCLC expression as well as ROS production. Multidrug resistance-associated protein 1 (MRP1) is a transporter able to efflux GSH conjugate of many electrophiles out of the cell. 15d-PGJ₂-induced GCLC expression was attenuated by the MRP1 antagonist MK571, suggesting that MRP1 contributes to 15d-PGJ2-mediated upregulation of GCLC by pumping out the 15d-PGJ₂-GSH conjugate. We also observed that 15d-PGJ₂ abrogated doxorubicin-induced cytotoxicity, which was blocked by co-treatment with the GCL inhibitor buthionine sulfoximine. These results, taken together, suggest that the induction of GCLC expression and subsequent elevation of GSH levels by 15d-PGJ₂ are mediated by ROS. Such enhancement of the GSH level may confer resistance to doxorubicin-induced MCF-7 cell death.