The common non-steroidal anti-inflammatory drug ibuprofen has been associated with a reduced risk of some age-related pathologies. However, a general pro-longevity role for ibuprofen and its mechanistic basis remains unclear. Here we show that ibuprofen increased the lifespan of Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster, indicative of conserved eukaryotic longevity effects. Studies in yeast indicate that ibuprofen destabilizes the Tat2p permease and inhibits tryptophan uptake. Loss of Tat2p increased replicative lifespan (RLS), but ibuprofen did not increase RLS when Tat2p was stabilized or in an already long-lived strain background impaired for aromatic amino acid uptake. Concomitant with lifespan extension, ibuprofen moderately reduced cell size at birth, leading to a delay in the G1 phase of the cell cycle. Similar changes in cell cycle progression were evident in a large dataset of replicatively long-lived yeast deletion strains. These results point to fundamental cell cycle signatures linked with longevity, implicate aromatic amino acid import in aging and identify a largely safe drug that extends lifespan across different kingdoms of life.

Aging is the greatest risk factor for many diseases, which together account for the majority of global deaths and healthcare costs. Here we show that the common drug ibuprofen increases the lifespan of yeast, worms and flies, indicative of conserved longevity effects. In budding yeast, an excellent model of cellular longevity mechanisms, ibuprofen's pro-longevity action is independent of its known anti-inflammatory role. We show that the critical function of ibuprofen in longevity is to inhibit the uptake of aromatic amino acids, by destabilizing the high-affinity tryptophan permease. We further show that ibuprofen alters cell cycle progression. Mirroring the effects of ibuprofen, we found that most yeast long-lived mutants were also similarly affected in cell cycle progression. These findings identify a safe drug that extends the lifespan of divergent organisms and reveal fundamental cellular properties associated with longevity.

Funding: This work was supported by grants to MP (National Science Foundation: MCB-0818248; IIP-1265349) and to BKK (National Institutes of Health: R01-AG043080; R01-AG025549). CH was supported by a postdoctoral fellowship from the Glenn Foundation for Medical Research. AAM was supported by grants from the Presidium of RAS (12-P-4-1005), RFBR (14-04-01596), and the President of Russian Federation (MD-1090.2014.4). The open access publishing fees for this article have been covered by the Texas A&M University Online Access to Knowledge (OAK) Fund, supported by the University Libraries and the Office of the Vice President for Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here we show that ibuprofen, a common and relatively safe non-steroidal anti-inflammatory drug, extends the lifespan of S. cerevisiae, C. elegans and D. melanogaster. We find that ibuprofen extends the replicative lifespan of yeast cells by destabilizing the high-affinity tryptophan transporter. We also show that ibuprofen causes a small size at birth and a moderate delay in initiation of cell division. Mirroring the effects of ibuprofen, we found that most long-lived yeast mutants were also moderately delayed in initiation of cell division, primarily due to a smaller size at birth. These results point to fundamental cellular properties associated with longevity, and identify a relatively safe drug that alters these properties and extends the lifespan of different species.

However, translating leads from aging screens in yeast and other model organisms to drugs that are efficacious and safe in humans represents a significant hurdle [2] . Alternatively, emphasis could be placed on relatively safe compounds that are already used in humans for some indication. One could then ask whether such compounds could extend the lifespan of model organisms. If successful, these drugs would represent excellent candidates for testing in humans for outcomes on healthspan parameters and biomarkers of longevity. They would also serve as invaluable tools to probe conserved longevity pathways, expanding and deepening our understanding of the basic biology of aging.

Studies of replicative and chronological lifespan in the budding yeast Saccharomyces cerevisiae have been driving forces in the identification of conserved genetic pathways that extend lifespan [11] , [12] . In this organism, individual cells can be tracked from birth to death [13] , with the number of divisions a cell can undergo defining its replicative lifespan (RLS) [14] . The pathways controlling yeast RLS and C. elegans lifespan exhibit significant overlap [15] . Hence, aging studies in yeast and other model systems represent invaluable platforms for the discovery of therapeutics that affect aging and a mechanistic dissection of their mode of action.

Levels of cellular and organismal dysfunction increase dramatically with old age. Aging is the greatest risk factor for numerous pathologies, including most forms of cancer, stroke, neurodegenerative disorders, heart disease and diabetes [1] . Hence, delaying aging therapeutically promises immense benefits to human health [2] . However, even with short-lived model organisms, the labor and time associated with unbiased screens for compounds that extend lifespan is a major obstacle [3] , [4] . To overcome this drawback, many studies focus on compounds that target pathways already implicated in aging, such as TOR signaling [5] , [6] , AMP kinase [7] , and Sirtuins [8] , [9] . Alternatively, phenotypes associated with aging are used as a proxy in screens for potential anti-aging therapeutics [3] . These phenotypes usually include resistance to various types of stress and mitochondrial degeneration, as well as maintenance of proteostasis and genomic stability [10] . The ultimate goal of all these approaches is to identify drugs that will delay the onset of age-related dysfunction and/or provide novel therapeutics to the diseases of aging [2] .

Results

Ibuprofen extends the lifespan of S. cerevisiae, C. elegans and D. melanogaster We decided to focus on ibuprofen for three reasons: First, it is a relatively safe over-the-counter medication. Second, ibuprofen may be associated with reduced risk of some age-related pathologies. Third, ibuprofen has not been reported to target any of the known aging pathways (e.g., the TOR or Sirtuin pathways), offering the possibility of novel insights into aging mechanisms. Ibuprofen was invented over 50 years ago. It is the prototypical 2-aryl-propionic acid NSAID. Relative to other NSAIDs, ibuprofen is arguably one of the safest [16]–[19], and is in the World Health Organization's “model list of essential medicines” (18th edition, 2013). As other NSAIDs, ibuprofen has analgesic and anti-pyretic indications. However, these indications stem from ibuprofen's well-established role as a cyclooxygenase inhibitor, interfering with prostaglandin biosynthesis [20]. With regard to age-related pathologies, long-term ibuprofen use reduced the risk of Alzheimer [21] and Parkinson [22], [23] diseases by more than 30%. However, it is unlikely that these beneficial outcomes were solely due to ibuprofen's anti-inflammatory roles because they were not necessarily shared by other NSAIDs. For example, among the NSAIDs examined, ibuprofen showed the most profound reduction in Alzheimer risk (40%), while others, such as celecoxib, had no effect [21]. Similarly, ibuprofen alone, but not other NSAIDs tested, reduced the risk of Parkinson disease [22]. To our knowledge, despite the vast number of studies dealing with ibuprofen, there are no direct measurements of ibuprofen's effects on the lifespan of organisms. Consequently, we decided to measure the effects of ibuprofen on yeast replicative lifespan. Added at 0.2 mM, we found that ibuprofen significantly extended the RLS of the standard BY4742 strain background (≈17%, p<0.0001, see Fig. 1A). To test if ibuprofen extends the lifespan of organisms other than yeast, we turned to C. elegans for three reasons: First, C. elegans is a well-established metazoan aging model, allowing us to gauge the ability of ibuprofen to extend the lifespan of organisms from different kingdoms of life [24]. Second, as in yeast, in C. elegans we could probe ibuprofen's effects independently of its role as a cyclooxygenase inhibitor because this organism lacks cyclooxygenase enzymes [25], which are targeted by ibuprofen in mammals [20]. Third, in C. elegans ibuprofen has been shown to suppress a phenotype associated with aging, inhibiting the deposition of amyloid β peptide, a marker for Alzheimer disease [26]. We found that animals exposed continuously to varying doses of ibuprofen (0.010–0.4 mM) from hatching until death had a longer lifespan (S1 Table). Note that we used UV-killed bacteria in these experiments, so it is unlikely that these effects are due to indirect effects through the action of ibuprofen on bacterial metabolism (see Materials and Methods). The concentration of ibuprofen at which the lifespan extension was maximal was 0.1 mM (Fig. 1B and S1 Table). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Ibuprofen extends the lifespan of S. cerevisiae, C. elegans and D. melanogaster. A, Ibuprofen extends yeast RLS. Survival curves for MATα (BY4742) cells treated with ibuprofen at 0.2 mM (shown in red), compared to experiment-matched untreated cells (shown in black). Mean lifespans are shown in parentheses, along with the number of cells assayed. B, Ibuprofen extends the lifespan of C. elegans. Survival curves for wild type (N2 strain) animals, treated with ibuprofen at 0.1 mM compared to experiment-matched untreated cells. Mean lifespans are shown in parentheses, along with the number of animals assayed. The data shown are from S1 Table. C, D, Ibuprofen extends the lifespan of female D. melanogaster. Survival curves for wild type male (C) and female (D) animals, treated with ibuprofen at 0.05 µM compared to experiment-matched untreated cells. Mean lifespans are shown in parentheses, along with the number of animals assayed. The data shown are from S2 Table. https://doi.org/10.1371/journal.pgen.1004860.g001 To further test the conservation of the pro-longevity effects of ibuprofen, we asked if the drug could extend the lifespan of D. melanogaster, another aging model system. Although the COX gene is absent in Drosophila, cyclooxygenase-like activity and inflammatory responses are thought to be present [27]–[29]. We found that ibuprofen (at 0.5 µM) extended the mean and the maximum lifespan of female flies (Fig. 1D and S2 Table). In males, although mean lifespan may also be extended, this was accompanied by a reduction of the maximum lifespan at all doses tested (Fig. 1C and S2 Table). The reasons for the sex-dependent differences in the longevity effects of ibuprofen are not clear. Although the effect in flies is influenced by the sex of the animal, it is nonetheless remarkable that ibuprofen promotes longevity in organisms as divergent as yeast, worms and flies. Overall, these results suggest that ibuprofen extends lifespan across different kingdoms of life. At least in the case of yeast and worms, the pro-longevity function of ibuprofen is through non-cyclooxygenase-related activity.