Background/Purpose: Inflammatory diseases such as rheumatoid arthritis (RA) are associated with oxidative stress as a result of elevated levels of reactive oxygen species (ROS). Oxidative stress leads to the formation of malondialdehyde (MDA) and acetaldehyde (AA), both of which react to form malondialdehyde-acetaldehyde (MAA) protein adducts. Our previous in vitro experiments have demonstrated that methotrexate (MTX) significantly decreases MAA-adduction of proteins (human albumin). Furthermore, the ROS generated by the reaction of MDA+AA to form MAA-adducted proteins are directly scavenged by MTX. However, the biological relevance of MTX’s ability to scavenge ROS and inhibit the generation of MAA-adducted proteins remains unclear. Therefore, we tested the hypothesis that MTX inhibits intracellular redox signaling as well as the formation of MAA-adducted proteins.

Methods:  To test our hypothesis, we examined the activation of a well-described redox-sensitive intracellular signaling pathway in which nuclear factor erythroid 2-related factor (NRF2), a redox-sensitive transcription factor, is stabilized and translocates to the nucleus. In the nucleus, NRF2 binds to antioxidant response elements (ARE) by utilizing a NRF2/ARE Luciferase reporter cell line in which luciferase expression is under control NRF2. Cells were incubated with vehicle, Human Serum Albumin (ALB), AA, MDA, MDA+AA (unbound), MAA-Alb for 24 hours at 37^oC in the absence or presence of MTX (2μM).  In addition, to further evaluate the scavenging of ROS by MTX, tert-butylhydroquinone (TBHQ, 5 mM), a known inducer of NRF2, in the presence of MTX was added to NRF2/ARE cells and incubated for 24 hours. NRF2 activation was quantified by measuring the amount of luciferase via luminescence the cell line produced in response to various stimuli.

Results:  As shown in Figure 1. MDA+AA significantly (p<0.001) increased NRF2 activation compared to cells treated with vehicle or Alb controls and MTX significantly attenuated (p<0.001) this redox-sensitive response. NRF2 activation with AA, MDA, or MAA-Alb in isolation demonstrated no increase in NRF activation.  Furthermore, as a positive control MTX significantly (p<0.001) inhibited the well-characterized TBHQ-dependent activation of NRF2 by almost 3-fold.    

Conclusion: This study demonstrates that when MDA+AA are mixed in the culture, a biologically relevant redox-sensitive signaling pathway is induced, that activates NRF2.  Interestingly, the MAA-adduct (MAA-Alb) is unable to activate NRF2, indicating the response is activated through the metabolites of ROS.  Furthermore, the ability of MTX to inhibit this redox-dependent cellular response corroborates our previous observation that MTX scavenges ROS. Together, these data strongly indicate a novel mechanism of MTX and suggest a new therapeutic benefit of MTX, which is its antioxidant potential.