Our results demonstrate that non-toxic concentrations of 2′,5′-DHC induce a burst of ROS (), which plays a crucial role in triggering an increase in iGSH levels, as shown using the catalytic antioxidant MnTDE-1,3-IP5+
(). Moreover, ROS are involved in triggering the AP-1 transcriptional response, as shown using the inhibitor of the JNK pathway SP600125 (), immunoblotting of phospho-c-Jun () and c-Jun responsive reporter activity (). However, while JNK and p38MAPK were important mediators of 2′,5′-DHC-induced Nrf2/ARE transcriptional activity and the increase in iGSH levels (), activation of this pathway appeared independent from ROS production (). Our findings suggest that 2′,5′-DHC induces an increase in iGSH levels through at least two transcriptional pathways: 1) a ROS-dependent and JNK-mediated activation of the AP-1 transcriptional response, and; 2) a ROS-independent and JNK- and p38MAPK-mediated activation of the Nrf2/ARE transcriptional response. Previous studies suggest that induction of HO-1 expression by 2′-HC and by synthetic chalcone and curcumin analogs involves the PI3K pathway [24
]. Our results also indicate that this pathway is involved in the 2′,5′-DHC-induced increase in iGSH levels, but to a lesser extent than the JNK and p38MAPK pathways ().
The 2′,5′-DHC-induced increase in iGSH levels ( and ) was accompanied by a small (~20%) but significant increase in GCL enzymatic activity (). Surprisingly, while the relative levels of the GCL subunits are a major determinant of cellular GCL activity and both GCL subunits are highly regulated at the transcriptional level [16
], 2′,5′-DHC-induced Nrf2/ARE and AP-1 transcriptional activity was not accompanied by an increase in GCL subunit expression levels (). Increased GCL holoenzyme formation could also account for the increased GCL activity. Indeed, we and others have demonstrated that oxidative stress can increase GCL holoenzyme formation and/or GCL activity in the absence of increased GCL subunit protein expression [41
]. However, no increase in GCL holoenzyme was detected in response to 2′,5′-DHC treatment (). Such findings suggest that 2′,5′-DHC-induced GCL activity may involve a post-translational regulatory mechanism [16
]. We have recently demonstrated that 4-hydroxy-2-nonenal (4-HNE), an α,β-unsaturated aldehyde produced during lipid peroxidation, can activate GCLC via direct covalent modification [37
]. However, it remains to be determined whether 2′,5′-DHC-induced ROS production is associated with increased lipid peroxidation and formation of 4-HNE. 2′,5′-DHC may also increase GCL activity by altering GCL enzyme kinetics by a mechanism independent of GCL holoenzyme formation, such as GSH negative feedback regulation or increased cysteine availability. Consistent with this hypothesis, 2′,5′-DHC induces a transient reduction in iGSH (), which could increase GCL activity by relieving the negative feedback regulation of GCL by GSH [44
]. Nrf2 also regulates the expression of the Xc- glutamate/cystine antiporter [45
] and 2′,5′-DHC-induced Nrf2/ARE activity could increase Xc- expression and indirectly enhance GSH biosynthesis via increased cysteine uptake and availability. Irrespective of the molecular mechanism(s) mediating 2′,5′-DHC-induced activation of GCL, it is likely that additional mechanisms contribute to the increase in iGSH. In this regard, increased iGSH could result from increased glutathione synthetase (GS) activity and/or decreased utilization or reduced extrusion of iGSH. Interestingly, many enzymes involved in the synthesis, metabolism, and export of GSH are regulated by Nrf2 [45
]. Thus, while it is unclear why increased Nrf2/ARE and/or AP-1 activity in response to 2′,5′-DHC does not result in increased GCL subunit expression in this cell system, increased GCL activity in conjunction with other molecular mechanisms are likely to account for the increase in iGSH.
Several structure-activity relationship studies have been previously carried out on the ability of HCs and synthetic analogs to: 1) inhibit the synthesis of the inducible nitric oxide (NO) synthase and induce the expression of heme oxygenase-1 (HO-1) [23
]; 2) inhibit P-glycoprotein- and BCRP-mediated xenobiotic efflux [47
], and; 3) induce GSH efflux [8
]. However, while antioxidant adaptive responses have been examined for curcumin, some flavonoids and other polyphenols [21
], the ability of 2′,5′-DHC to increase iGSH levels was never previously examined. Chrysin and other flavonoids are known to trigger GSH efflux through MRP1 and to inhibit BCRP-mediated drug efflux [11
]. In contrast, 2′,5′-DHC-induced GSH efflux is mediated by BCRP/ABCG2 and naturally occurring chalcones are poor inhibitors of BCRP-mediated drug efflux [12
]. Another relevant difference between 2′,5′-DHC and chrysin is that only 2′,5′-DHC is able to induce a significant rebound in iGSH levels, as shown in this study. The evidence associating aging-related diseases with oxidative stress is mounting, renewing the interest in understanding the mechanisms of the antioxidant adaptive responses [53
]. However, as the data increases, so does the complexity of the system. The Nrf2/ARE transcriptional pathway is most studied by its known triggers, which include oxidative stress, electrophiles and multiple protein kinase signaling pathways, yet ROS may or may not be required [17
]. Cross-talk between pathways is also at play, since JNK- and ERK-mediated pathways have been associated with both Nrf2/ARE and AP-1 transcriptional responses [17
]. Recent results showed a synergistic effect between several natural compounds, and that such effects rely on several signaling pathways [21
]. Further studies are needed to determine whether hydroxychalcones can contribute to similar synergistic effects.