Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen are commonly used to relieve pain, fever, and inflammation. These drugs inhibit cyclooxygenase (COX) enzymes that produce prostaglandin, a lipid compound that triggers the body’s inflammatory response. 

NSAID applications extend beyond the routine pulled muscle, however. Scientists explore these drugs as treatments for diseases involving inflammation, including rheumatoid arthritis and various cancers (1,2). In these settings, different NSAID drugs have shown drastically different effects. For example, the NSAID diclofenac has emerged as a promising treatment for arthritis, while other NSAID drugs that target the same enzymes did not show equivalent activity (3). 

“From a clinical perspective, we've known for a long time that even NSAIDs that target the same COX isozymes seem to have different responses in people with the same disease,” said Andrew Wang, an immunologist at the Yale School of Medicine. “It wasn't entirely intuitive to me how that would exactly work.”

In a new study in Cell Immunity, Wang and his colleagues report that NSAIDs act through an additional, non-COX mechanism, providing a starting point for explaining discrepancies in their behavior and guiding NSAID use for treating disease (4). 

While looking for clues to guide their research, the team uncovered a 1996 study suggesting that NSAIDs induce expression of the growth/differentiation factor 15 (GDF15) gene in colorectal cancer cells that do not have COX enzymes. This led them to explore a potential mechanism involving GDF15 (5). Wang’s team treated bone marrow-derived macrophages with two NSAIDs, ketoprofen and indomethacin, and measured GDF15 protein levels. Indomethacin induced GDF15 expression, while ketoprofen did not. 

The researchers repeated the experiment in mice and systematically removed signaling proteins and molecules needed for GDF15 expression. They discovered that to induce GDF15, indomethacin required another protein called NRF2, a transcription factor that regulates protection against oxidative stress. They then evaluated indomethacin in mouse models of gout and endotoxemia in which NRF2 had been deleted. They observed that the drug lost its anti-inflammatory activity, demonstrating that its efficacy depends on NRF2. “We were not expecting to discover a novel mechanism of action of commonly used drugs,” Wang said. 

The picture is not complete yet. "The two limitations of the study are the fact that they couldn't downregulate COX-1 and COX-2 completely,” said Antonio Cuadrado, a biochemist at the University Autonoma of Madrid who was not involved in the study. Because of this, the authors can’t claim that the effects they observed with NRF2 were entirely independent of COX activity. The second limitation is that they were unable to isolate the reaction product of the suggested NRF2 activation. ­"The formal proof is still missing,” said Cuadrado. Hence, the exact chemistry behind the NSAID-NRF2 interaction that activated GDF15 remains unknown. 

Wang agrees that further uncovering how NSAIDs engage NRF2 could inform NSAID development in the future. “The path forward would be understanding exactly the chemistry in which this occurs and then leveraging that understanding to make designer NSAIDs not based on COX, but on NRF2,” he said. 

The fact that NSAIDs can exert effects on multiple targets is an exciting avenue to explore, according to Wang. “The way that the biopharmaceutical industry designs drugs is usually by looking at a single target,” he said. “Yet it's only specific for that target insofar as you’ve actually looked at everything else.”

References

1. Crofford, L.J. Use of NSAIDs in treating patients with arthritis. Arthritis Res Ther  15, S2 (2013).
2. Wong R. S. Y. Role of Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) in Cancer Prevention and Cancer Promotion. Advances in pharmacological sciences, 3418975 (2019). 
3. da Costa, B.R., Reichenbach, S., Keller, N., Nartey, L., Wandel, S., Juni, P., and Trelle, S. Effectiveness of non-steroidal anti-inflammatory drugs for the treatment of pain in knee and hip osteoarthritis: a network meta-analysis. Lancet   390, e21–e33 (2017).  
4. Eisenstein, A., Hilliard, B.K., Pope, S.D., et al. Activation of the transcription factor NRF2 mediates the anti-inflammatory properties of a subset of over-the-counter and prescription NSAIDs. Immunity.  55, 1082-1095 (2022).
5. Hanif, R., Pittas, A., Feng, Y., et al. Effects of nonsteroidal anti-inflammatory drugs on proliferation and on induction of apoptosis in colon cancer cells by a prostaglandin-independent pathway. Biochem Pharmacol.  52, 237 - 245 (1996).