Nitroalkene fatty acids mediate activation of Nrf2/ARE-dependent and PPARγ-dependent transcription by distinct signaling pathways and with significantly different potencies.

Naturally occurring nitroalkene fatty acids (NAs) derived from oleic (NO(2)-OA) and linoleic (NO(2)-LA) acids mediate a variety of cellular responses. We examined the signaling pathways involved in NA activation of Nrf2/ARE-dependent versus PPARγ/PPRE-dependent transcription in human MCF7 breast cancer cells. Additionally, we compared the relative potencies of NO(2)-OA and NO(2)-LA in activating these two transcriptional programs. Here it is demonstrated that, in addition to the direct adduct formation of NA with the Nrf2 inhibitory protein, Keap1, shown by others, NA activation of Nrf2/ARE-mediated transcription results from increased nuclear Nrf2 levels and depends upon activation of the PI3K/AKT and PKC, but not ERK and JNK MAPK, signaling pathways. Examination of the relationship between NA stimulation of the Nrf2/ARE versus PPARγ/PPRE transcriptional programs revealed concentration-dependent activation of distinct signaling pathways that were readily distinguished by selective attenuation of Nrf2/ARE-dependent, but not PPARγ-dependent, transcription by inhibitors of PI3K and PKC. Moreover, measurable, statistically significant activation of PPARγ/PPRE-dependent transcription occurred at nanomolar concentrations of NAs-the 12-NO(2) isomer of NO(2)-LA showing the most potent activity-whereas significant activation of Nrf2/ARE-dependent transcription occurred at much higher NA concentrations (≥3 µM) with the NO(2)-OA isomers the most potent. These findings have implications for the physiological roles of NAs, suggesting that, at concentrations likely to be encountered in vivo, their direct activation of PPARγ transcription will dominate over their electrophilic activation of Nrf2 antioxidant/protective responses.

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) by nitroalkene fatty acids: importance of nitration position and degree of unsaturation.

Nitroalkene fatty acids are potent endogenous ligand activators of PPARgamma-dependent transcription. Previous studies with the naturally occurring regioisomers of nitrolinoleic acid revealed that the isomers are not equivalent with respect to PPARgamma activation. To gain further insight into the structure-activity relationships between nitroalkenes and PPARgamma, we examined additional naturally occurring nitroalkenes derived from oleic acid, 9-nitrooleic acid (E-9-NO2-18:1 [1]) and 10-nitrooleic acid (E-10-NO2-18:1 [2]), and several synthetic nitrated enoic fatty acids of variable carbon chain length, double bonds, and nitration site. At submicromolar concentrations, E-12-NO2 derivatives were considerably more potent than isomers nitrated at carbons 5, 6, 9, 10, and 13, and chain length (16 versus 18) or number of double bonds (1 versus 2) was of little consequence for PPARgamma activation. Interestingly, at higher concentrations (>2 microM) the nitrated enoic fatty acids (E-9-NO2-18:1 [1], E-9-NO2-16:1 [3], E-10-NO2-18:1 [2], and E-12-NO2-18:1 [7]) deviated significantly from the saturable pattern of PPARgamma activation observed for nitrated 1,4-dienoic fatty acids (E-9-NO2-18:2, E-10-NO2-18:2, E-12-NO2-18:2, and E-13-NO2-18:2).

Differential potencies of naturally occurring regioisomers of nitrolinoleic acid in PPARgamma activation.

Previous studies demonstrated that the naturally occurring electrophile and PPARgamma ligand, nitrolinoleic acid (NO(2)-LA), exists as a mixture of four regioisomers [Alexander, R. L., et al. (2006) Biochemistry 45, 7889-7896]. We hypothesized that these alternative isomers have distinct bioactivities; therefore, to determine if the regioisomers are quantitatively or qualitatively different with respect to PPARgamma activation, NO(2)-LA was separated into three fractions which were identified by NMR (13-NO(2)-LA, 12-NO(2)-LA, and a mixture of 9- and 10-NO(2)-LA) and characterized for PPARgamma interactions. A competition radioligand binding assay showed that all three NO(2)-LA fractions had similar binding affinities for PPARgamma (IC(50) = 0.41-0.60 microM) that were comparable to that of the pharmaceutical ligand, rosiglitazone (IC(50) = 0.25 microM). However, when PPARgamma-dependent transcription activation was examined, there were significant differences observed among the NO(2)-LA fractions. Each isomer behaved as a partial agonist in this reporter gene assay; however, the 12-NO(2) derivative was the most potent with respect to maximum activation of PPARgamma and an EC(50) of 0.045 microM (compare with the rosiglitazone EC(50) of 0.067 microM), while the 13-NO(2) and 9- and 10-NO(2) derivatives were considerably less effective with EC(50) values of 0.41-0.62 microM. We conclude that the regioisomers of NO(2)-LA are not functionally equivalent. The 12-NO(2) derivative appears to be the most potent in PPARgamma-dependent transcription activation, whereas the weaker PPARgamma agonists, 13-NO(2) and 9- and 10-NO(2), may be relatively more important in signaling via other, PPARgamma-independent pathways in which this family of nitrolipid electrophiles is implicated.

Role of glutathione S-transferase P1-1 in the cellular detoxification of cisplatin.

Cells expressing elevated levels of allelic variants of human glutathione S-transferase P1 (GSTP1) and/or efflux transporters, MRP1 or MRP2, were used to evaluate the role of GSTP1-1 in cisplatin resistance. These studies revealed that GSTP1-1 confers low-level resistance (1.4- to 1.7-fold) to cisplatin-induced cytotoxicity in MCF7 cells. However, expression of MRP1 (MCF7 cells) or MRP2 (HepG2 cells) failed to augment or potentiate GSTP1-1-mediated resistance in either cell line. To understand the mechanism by which variants of GSTP1-1 confer resistance to cisplatin, their relative abilities to catalyze conjugation of cisplatin with glutathione were examined. Enzymes encoded by all three alleles tested, GSTP1a (I(104)A(113)), GSTP1b (V(104)A(113)), and GSTP1c (V(104)V(113)), increased the formation rate of the mono-platinum/glutathione derivative of cisplatin with relative catalytic activities of 1.0 (GSTP1a-1a variant) and 1.8 to 1.9 (GSTP1b-1b and GSTP1c-1c variants). Although these data are consistent with the idea that very low level resistance to cisplatin may be conferred by GSTP1-1-mediated cisplatin/glutathione conjugation, two observations indicate that such catalysis plays a minor role in the protection from cisplatin toxicity. First, the rates of GSTP1-1-mediated conjugation are extremely slow (1.7-2.6 h(-1) at 25 degrees C). Second, despite an 80% to 90% increase in catalysis of cisplatin conjugation by GSTP1b-1b or GSTP1c-1c over GSTP1a-1a, we observed no discernable differences in relative resistances conferred by these alternative variants when expressed in MCF7 cells. We conclude that high-level cisplatin resistance attributed to GSTP1-1 in other studies is not likely due to catalysis of cisplatin conjugation but rather must be explained by other mechanisms, which may include GSTP1-mediated modulation of signaling pathways.

Expression of MRP1 and GSTP1-1 modulate the acute cellular response to treatment with the chemopreventive isothiocyanate, sulforaphane.

A major component of the anticarcinogenic activity of the dietary chemopreventive agent sulforaphane (SFN) is attributed to its ability to induce expression of phase II detoxification genes containing the antioxidant response element (ARE) within their promoters. Because SFN is a reactive electrophile--readily forming conjugates with glutathione (GSH)--we asked whether expression of glutathione S-transferase (GST) P1-1 and the GSH conjugate efflux pump, multidrug resistance or resistance-associated protein (MRP) 1, would significantly modify the cellular response to SFN exposure. This was investigated using GST- and MRP1-poor parental MCF7 cells and transgenic derivatives expressing GSTP1-1 and/or MRP1. Compared with parental cells, expression of GSTP1-1 alone enhanced the rate of intracellular accumulation of SFN and its glutathione conjugate, SFN-SG--an effect that was associated with increased ARE-containing reporter gene induction. Expression of MRP1 greatly reduced SFN/SFN-SG accumulation and resulted in significant attenuation of SFN-mediated induction of ARE-containing reporter and endogenous gene expression. Coexpression of GSTP1-1 with MRP1 further reduced the level of induction. Depletion of GSH prior to SFN treatment or the substitution of tert-butylhydroquinone for SFN abolished the effects of MRP1/GSTP1-1 on ARE-containing gene induction-indicating that these effects are GSH dependent. Lastly, analysis of NF-E2-related factor 2 (Nrf2)--a transcription factor operating via binding to the ARE--showed that the increased levels of Nrf2 following SFN treatment were considerably less sustained in MRP1-expressing, especially those coexpressing GSTP1-1, than in MRP1-poor cells. These results suggest that the regulating effects of MRP1 and GSTP1-1 expression on SFN-dependent induction of phase II genes are ultimately mediated by altering nuclear Nrf2 levels.

Multidrug resistance protein 1 (MRP1, ABCC1) mediates resistance to mitoxantrone via glutathione-dependent drug efflux.

Based upon several previous reports, no consistent relationship between multidrug resistance protein 1 (MRP1, ABCC1) expression and cellular sensitivity to mitoxantrone (MX) toxicity can be ascertained; thus, the role of MRP1 in MX resistance remains controversial. The present study, using paired parental, MRP1-poor, and transduced MRP1-overexpressing MCF7 cells, unequivocally demonstrates that MRP1 confers resistance to MX cytotoxicity and that resistance is associated with reduced cellular accumulation of MX. This MRP1-associated reduced accumulation of MX was partially reversed by treatment of cells with 50 microM MK571 [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid]-an MRP inhibitor that increased MX accumulation in MRP1-expressing MCF7 cells but had no effect on MRP-poor MCF7 cells. Moreover, in vitro experiments using inside-out membrane vesicles show that MRP1 supports ATP-dependent, osmotically sensitive uptake of MX. Unlike ABCG2 (breast cancer resistance protein, mitoxantrone-resistant protein), MRP1-mediated MX transport is dependent upon the presence of glutathione or its S-methyl analog. In addition, MX stimulates transport of [3H]glutathione. Together, these data are consistent with the interpretation that MX efflux by MRP1 involves cotransport of MX and glutathione. The results suggest that MRP1-like the alternative MX transporters ABCG2 and ABCB1 (MDR1, P-glycoprotein)-can significantly influence tumor cell sensitivity to and pharmacological disposition of MX.

5-Oxo-ETE analogs and the proliferation of cancer cells.

MDA-MB-231, MCF7, and SKOV3 cancer cells, but not HEK-293 cells, expressed mRNA for the leukocyte G protein-coupled 5-oxo-eicosatetraenoate (ETE) OXE receptor. 5-Oxo-ETE, 5-oxo-15-OH-ETE, and 5-HETE stimulated the cancer cell lines but not HEK-293 cells to mount pertussis toxin-sensitive proliferation responses. Their potencies in eliciting this response were similar to their known potencies in activating leukocytes and OXE receptor-transfected cells. However, high concentrations of 5-oxo-ETE and 5-oxo-15-OH-ETE, but not 5-HETE, arrested growth and caused apoptosis in all four cell lines; these responses were pertussis toxin-resistant. The same high concentrations of the oxo-ETEs but again not 5-HETE also activated peroxisome proliferator-activated receptor (PPAR)-gamma. Pharmacological studies indicated that this activation did not mediate their effects on proliferation. These results are the first to implicate the OXE receptor in malignant cell growth and to show that 5-oxo-ETEs activate cell death programs as well as PPARgamma independently of this receptor.

Glutathione S-transferases (GSTs) inhibit transcriptional activation by the peroxisomal proliferator-activated receptor gamma (PPAR gamma) ligand, 15-deoxy-delta 12,14prostaglandin J2 (15-d-PGJ2).

15-Deoxy-Delta(12,14)prostaglandin J(2) (15-d-PGJ(2)), a terminal metabolite of the J-series cyclopentenone prostaglandins, influences a variety of cellular processes including gene expression, differentiation, growth, and apoptosis. As a ligand of peroxisomal proliferator-activated receptor gamma (PPAR gamma), 15-d-PGJ(2) can transactivate PPAR gamma-responsive promoters. Previously, we showed that multidrug resistance proteins MRP1 and MRP3 attenuate cytotoxic and transactivating activities of 15-d-PGJ(2) in MCF7 breast cancer cells. Attenuation was glutathione-dependent and was associated with formation of the glutathione conjugate of 15-d-PGJ(2), 15-d-PGJ(2)-SG, and its active efflux by MRP. Here we have investigated whether the glutathione S-transferases (GST) can influence biological activities of 15-d-PGJ(2). MCF7 cells were stably transduced with human cytosolic GST isozymes M1a, A1, or P1a. These GSTs had no effect on 15-d-PGJ(2) cytotoxicity when expressed either alone or in combination with MRP1. However, expression of any of the three GSTs significantly inhibited 15-d-PGJ(2)-dependent transactivation of a PPAR gamma-responsive reporter gene. The degree of inhibition correlated with the level of GST expressed. Under physiologic conditions, the nonenzymatic rate of 15-d-PGJ(2) conjugation with glutathione was significant. Of the three GST isozymes, only GSTM1a-1a further stimulated the rate of 15-d-PGJ(2)-SG formation. Moreover, GSTM1a-1a rate enhancement was only a transient burst that was complete within 15 s. Hence, catalysis plays little, if any, role in GST inhibition of 15-d-PGJ(2)-dependent transactivation. In contrast, inhibition of transactivation was associated with strong GST/15-d-PGJ(2) interactions. Potent inhibition by 15-d-PGJ(2) and 15-d-PGJ(2)-SG of GST activity was observed with K(i) in the 0.15-2.0 microM range for the three GST isozymes, results suggesting avid associations between GST and 15-d-PGJ(2) or 15-d-PGJ(2)-SG. Electrospray ionization mass spectrometry (ESI/MS) studies revealed no stable adducts of GST and 15-d-PGJ(2) indicating that GST/15-d-PGJ(2) interactions are primarily noncovalent. These results are consistent with a mechanism of GST-mediated inhibition of transactivation in which GST binds 15-d-PGJ(2) and 15-d-PGJ(2)-SG thereby sequestering the ligands in the cytosol away from their nuclear target, PPAR gamma.

Role of multidrug resistance protein 2 (MRP2, ABCC2) in alkylating agent detoxification: MRP2 potentiates glutathione S-transferase A1-1-mediated resistance to chlorambucil cytotoxicity.

Our previous studies have shown that the glutathione S-transferases (GSTs) can operate in synergy with the efflux transporter multidrug resistance protein 1 (MRP1, ABCC1) to confer resistance to the cyto- and genotoxicities of some anticancer drugs and carcinogens. The current study was designed to determine whether the alternative efflux transporter, MRP2 (ABCC2), can also potentiate GST-mediated detoxifications in HepG2 cells. HepG2 cells, which express high-level MRP2 but not MRP1, were stably transduced with GST expression vectors under tetracycline-repressible transcriptional control. MRP2 was able to support GSTA1-1-mediated resistance to chlorambucil (CHB) cytotoxicity in HepG2 cells. Resistance was GST isozyme-specific in that GSTP1a-1a and GSTM1a-1a failed to confer protection from CHB toxicity. Moreover, inhibition of MRP2 with sulfinpyrazone completely reversed GSTA1-1-associated resistance, indicating that MRP2-efflux function is required to potentiate GSTA1-1-mediated resistance. Relative transport by MRP1 versus MRP2 of monoglutathionyl-CHB (CHB-SG) was examined using inside-out plasma membrane vesicles derived from MCF7 cells transduced with MRP1 or MRP2 expression vectors. Both MRP1 and MRP2 transported CHB-SG efficiently, at the levels of protein expressed, with similar Vmax and with Km of 0.39 and 10 microM, respectively. We conclude that detoxification of CHB by GSTA1-1 requires the removal of the glutathione conjugate formed and that either MRP1 or MRP2 can serve this efflux function. These findings have implications for the role of MRP2 in detoxification of alkylating agents in the apical epithelium of liver and kidney where it is highly expressed as well as the role of MRP2 in the emergence of alkylating drug resistance in cancer cells.