Enhanced Longevity by Ibuprofen, Conserved in Multiple Species, Occurs in Yeast through Inhibition of Tryptophan Import

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.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

Ibuprofen extends the lifespan of <i>S. cerevisiae</i>, <i>C. elegans</i> and <i>D. melanogaster</i>.

<p><b>A</b>, 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>B</b>, Ibuprofen extends the lifespan of <i>C. elegans</i>. 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004860#pgen.1004860.s011" target="_blank">S1 Table</a>. <b>C, D,</b> Ibuprofen extends the lifespan of female <i>D. melanogaster</i>. Survival curves for wild type male (<b>C</b>) and female (<b>D</b>) 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004860#pgen.1004860.s012" target="_blank">S2 Table</a>.</p

LL mutant birth size and fitness are more constrained than those of NLL mutants.

<p><b>A</b>, The birth size values (y axis) from all 3,842 NLL mutants from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004860#pgen.1004860.s010" target="_blank">S1 Dataset</a> were plotted against their corresponding fitness values (x axis). Because many data points were overlapping, they were binned using the hexbin function of the R software package. The displayed colors represent the number of strains within each bin, as indicated by the color key to the right. <b>B</b>, The birth size values (y axis) from all 137 LL mutants from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004860#pgen.1004860.s010" target="_blank">S1 Dataset</a> were plotted against their corresponding fitness values (x axis), as in A. The number of strains in each bin (counts) are shown on the right.</p

Stabilization of Tat2p increases birth size and suppresses replicative lifespan extension in <i>hxk2Δ</i> and <i>rpl20bΔ</i> cells.

<p><b>A</b> and <b>B</b>, The cell size of the indicated haploid cell populations cultured in YPD (2% Dextrose) medium was measured using a channelyzer. Cell numbers are plotted on the y axis and the x axis indicates size (in fL). <b>C</b> and <b>D</b>, Experiment-matched survival curves of the same strains shown in A and B. Mean lifespans are shown in parentheses, along with the number of cells assayed.</p

G1 phase cell cycle parameters of LL and NLL strains.

<p><b>A</b>, The birth size values (y axis) of the 14 LL and 13 NLL strains shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004860#pgen.1004860.s014" target="_blank">S4 Table</a>. The filled black squares correspond to the values of the wild type control. The data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004860#pgen.1004860.s014" target="_blank">S4 Table</a> were used to generate the graphs shown. <b>B</b>, The specific rate of cell size increase (<i>k</i>, shown on the y axis) for the LL and NLL strains shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004860#pgen.1004860.s014" target="_blank">S4 Table</a> was calculated from synchronous, elutriated cultures (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004860#s4" target="_blank">Materials and Methods</a>). <b>C</b>, The critical size (y axis) of the LL and NLL strains shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004860#pgen.1004860.s014" target="_blank">S4 Table</a> was calculated from the same experiments shown in B. <b>D</b>, LL mutants have efficient cell size control mechanisms. On the x axis is the logarithm of the normalized birth size values of the LL mutants shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004860#pgen.1004860.s014" target="_blank">S4 Table</a>, plotted against their relative growth in size during the G1 phase of each strain (<i>k</i>T_G1, y axis). The dashed line is a linear fit obtained with the regression function of Microsoft Excel. The filled square is the wild type strain.</p

Ibuprofen destabilizes Tat2p without triggering other TOR pathway outputs.

<p><b>A</b>, Steady-state Tat2p-TAP levels are reduced upon ibuprofen treatment. Exponentially proliferating cells expressing from its chromosomal location TAP-tagged Tat2p were exposed to ibuprofen or rapamycin at the indicated concentration and for the times shown. Tat2p levels were evaluated by SDS-PAGE and immunoblotting. From the same samples, steady-state levels of untagged Cdc28p are shown for comparison. <b>B</b>, Ibuprofen destabilizes Tat2p-TAP. The experiment was performed as in A, except that the cells were treated with cycloheximide at time 0. Tat2p-TAP band densities were quantified with Image J software. These values were then normalized for loading and fitted on an exponential decay function to obtain the half-life values shown on the right of each blot. <b>C</b>, Ibuprofen does not trigger gene expression of downstream effectors of the TOR pathway. <i>Left</i>, we examined three sets of targets of the TOR pathway, whose expression is known to be affected by rapamycin. <i>Right</i>, expression of the targets genes shown was evaluated by qPCR (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004860#s4" target="_blank">Materials and Methods</a>) from exponentially growing cells after addition of ibuprofen (at 0.2 mM) or rapamycin (at 50 ng/ml) at the indicated times. The targets were grouped based on the diagram shown on the left. We also included <i>TAT1</i> and <i>TAT2</i> expression in this experiment. The average values from at least three experiments in each case were log₂-transformed and displayed on the heatmap shown using the open-source <i>pheatmap</i> package for the R language. <b>D</b>, Ibuprofen does not trigger Gln3p dephosphorylation. Cells expressing TAP-tagged variants of two downstream effector of TOR, Npr1p and Gln3p, were treated with ibuprofen or rapamycin for 60 min, at the same concentrations as in A. Npr1p-TAP and Gln3p-TAP levels and mobility, evaluated by immunobloting. <b>E</b>, Ibuprofen does not extend the RLS of a strain that carries the stable <i>2HA-TAT2-5KR</i> allele. Survival curves of the strains shown treated with ibuprofen at 0.2 mM compared to experiment-matched untreated cells. For comparison, wild type cells carrying a similarly tagged wild type <i>TAT2</i> allele (<i>2HA-TAT2</i>) were also included in this experiment and they were either untreated (shown in black) or treated (shown in red) with ibuprofen. Mean lifespans are shown in parentheses, along with the number of cells assayed.</p

Ibuprofen at low doses moderately delays G1, primarily through a reduction in birth size.

<p><b>A</b>, Schematic of the variables that determine the length of the G1 phase. <b>B</b>, The birth size of BY4743 cells exposed at different doses of ibuprofen was measured from three independent experiments in each case, similar to the ones shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004860#pgen.1004860.s004" target="_blank">S4 Figure</a>. Asterisks indicate statistically significant differences compared to the untreated samples (p<0.05, from Student's <i>t</i> tests). <b>C</b>, The specific rate of cell size increase constant <i>k</i> (in h⁻¹) was measured from the elutriation experiments shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004860#pgen.1004860.s005" target="_blank">S5 Figure</a>, assuming exponential growth. <b>D</b>, The critical cell size of the indicated strains (shown in fl), was measured from the same elutriation experiments shown in C and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004860#pgen.1004860.s005" target="_blank">S5 Figure</a>.</p