Herein, we report the identification and validation of oxidation resistance 1 (OXR1) as a molecular target of PL in SCs. OXR1 is a cellular oxidative stress sensor that regulates the expression of a variety of antioxidant enzymes and modulates the cell cycle and apoptosis (Oliver et al., 2011 ; Yang et al., 2015 ). We found that OXR1 was upregulated in SCs induced by ionizing radiation (IR) or extensive replication. PL bound to OXR1 directly and induced its degradation through the ubiquitin‐proteasome system in an SC‐specific manner. Knocking down OXR1 selectively induced apoptosis in SCs and sensitized the cells to oxidative stress caused by hydrogen peroxide (H 2 O 2 ). These findings suggest that OXR1 is a potential senolytic target that can be exploited for the development of selective senolytic agents with improved potency and selectivity. In addition, these findings also provide new insight into the mechanism by which SCs are highly resistant to oxidative stress.

Piperlongumine (PL) is one of a few natural products identified to have the ability to selectively kill SCs (Wang et al., 2016 ; Zhu et al., 2015 , 2017 ). Compared to other known senolytic agents, PL has the advantage of low toxicity, an excellent PK/PD profile, and oral bioavailability (Raj et al., 2011 ). However, PL has a relatively higher EC 50 value against SCs compared with ABT‐263 (Wang et al., 2016 ). Moreover, its molecular target(s) and mechanism of action are unknown. To facilitate the development of PL and its analogues as senolytic drug candidates, it is critical to identify PL molecular targets, which can form a molecular basis for the rational design of new PL analogues.

However, a major challenge facing the discovery and development of effective senolytic agents is to identify and validate more senolytic targets. Since the first senolytic was published (Zhu et al., 2015 ), twelve molecular targets have been identified (Childs et al., 2017 ), including the prosurvival Bcl‐2 family proteins (Chang et al., 2016 ; Yosef et al., 2016 ; Zhu et al., 2016 ) and forkhead Box O4 (FOXO4) (Baar et al., 2017 ). These findings led to the discovery of a few senolytic agents, including ABT‐263 and ABT‐737, two Bcl‐2/xl/w inhibitors, and FOXO4‐DRI, a peptide molecule that interferes with the interaction of FOXO4 and p53. Unfortunately, the clinical application of these senolytic agents for age‐related diseases and cytotoxic cancer therapy‐induced side effects may be limited because of the toxicity of Bcl‐2/xl/w inhibitors and the difficulty of developing effective high molecular‐weight peptide therapeutics. Therefore, identification of new SC molecular targets is in urgent need for the development of novel small molecule senolytic agents.

Cellular senescence occurs when irreversible cell cycle arrest is triggered by telomere shortening or exposure to stress (Campisi, 2013 ). The induction of cellular senescence has many beneficial effects, including preventing tumorigenesis, promoting wound healing and tissue remodeling, and contributing to embryonic development (Muñoz‐Espín & Serrano, 2014 ). However, senescent cells (SCs) accumulate if they cannot be removed rapidly by the immune system due to immune dysfunction and/or a sustained, overwhelming increase in SC production. This occurs during aging or under certain pathological conditions (Childs et al., 2017 ; Muñoz‐Espín & Serrano, 2014 ). Under these circumstances, SCs can be detrimental and play a causal role in aging, age‐related diseases, and chemotherapy‐ and radiotherapy‐induced side effects, in part through the expression of the senescence‐associated secretory phenotype (Childs et al., 2017 ; Muñoz‐Espín & Serrano, 2014 ). This hypothesis is supported by recent studies demonstrating that the genetic clearance of SCs prolongs the lifespan of mice and delays the onset of several age‐related diseases and disorders in both progeroid and naturally aged mice (Baker et al., 2011 , 2016 ). Therefore, the pharmacological clearance of SCs with a small molecule, a senolytic agent that can selectively kill SCs, is potentially a novel anti‐aging strategy and a new treatment for chemotherapy‐ and radiotherapy‐induced side effects (Baar et al., 2017 ; Chang et al., 2016 ; Childs et al., 2016 ; Demaria et al., 2017 ; Jeon et al., 2017 ; Ogrodnik et al., 2017 ; Pan et al., 2017 ; Schafer et al., 2017 ; Yosef et al., 2016 ; Zhu et al., 2015 ).

2 RESULTS

2.1 Design and synthesis of PL probes The structure of PL features two Michael acceptors (electrophiles), the C2–C3 and C7–C8 olefins (Figure 1a) (Adams et al., 2012). Our previous studies showed that both Michael acceptors were important for the senolytic activity of PL (Wang et al., 2016), indicating that PL may act as an irreversible inhibitor through the conjugated addition of the nucleophiles (e.g., cysteine residue) on its target protein(s) to the Michael acceptors. Taking advantage of the covalent interaction between PL and its target protein(s), we designed PL probes that can be “tagged” for protein purification and identification in live cells (Figure 1a). Our structure–activity relationship studies revealed that modifications on the trimethoxyphenyl group of PL were tolerated, and an affinity tag could be attached to the para‐position of the phenyl ring. We thus synthesized PL probes that contain a terminal alkyne. These probes can be used to enrich target proteins by reacting with the corresponding immobilized azide through a bio‐orthogonal copper‐catalyzed azide–alkyne cycloaddition (Click Chemistry) (Supporting Information Figure S1) (Kolb & Sharpless, 2003), which allows us to perform mass spectrometry (MS)‐based proteomic analysis to identify the target proteins. Through a cell‐based senolytic activity assay (Figure 1b), we selected a PL probe (Figure 1a) with the same senolytic activity as PL to be used in our target‐protein identification study. As a negative control, we designed a structurally related compound in which the C7–C8 double bond of the PL probe is saturated (CTL probe, Figure 1a); we modified this bond because previous studies suggested that the Michael acceptor at the PL C7–C8 olefin covalently binds target proteins, while the C2–C3 olefin can facilitate target‐protein binding after reacting with glutathione (Adams et al., 2012; Raj et al., 2011). The CTL probe was used to subtract nonspecific proteins pulled down by our PL probe. As expected, the CTL probe was ~9‐fold less potent than the PL probe and has no selective toxicity against SCs because the senolytic activity of PL depends on the presence of both Michael acceptors (Figure 1b) (Wang et al., 2016). Figure 1 Open in figure viewer PowerPoint Identification and validation of OXR1 as a target of PL. (a) The chemical structures of piperlongumine (PL), alkyne‐labeled piperlongumine (PL probe), and alkyne‐labeled negative control probe (CTL probe). The C2–C3 bonds are circled in blue, and the C7–C8 bonds are circled in red. (b) Viable cell counts of nonsenescent WI‐38 cells (NCs), IR‐induced senescent WI‐38 cells (IR‐SC), and replication‐exhausted senescent WI‐38 cells (RE‐SC) 72 hr after treatment with PL, the PL probe, or the CTL probe. Data are presented as the mean ± SE of percent cell viability from two independent experiments. (c) Schematic of the competitive pull‐down procedure by “Click” Chemistry. (d) Numbers of PL‐binding proteins identified in NCs, IR‐SCs, and RE‐SCs. The list of the proteins is presented in Supporting Information Tables S1 and S2 . (e) IR‐SC lysate was incubated with biotin‐labeled PL in the absence or presence of a fivefold excess of unlabeled PL followed by precipitation with streptavidin‐conjugated agarose beads and immunoblotting of the proteins released from the beads with anti‐OXR1 antibodies. An immunoblot of β‐actin in IR‐SC lysates was included as an input control for the pull down. (f) Human recombinant OXR1 protein was incubated with biotin‐labeled PL in the absence or presence of a fivefold excess of unlabeled PL followed by immunoblotting with antibodies against biotin to detect OXR1 bound to the biotin‐labeled PL probe. An immunoblot of OXR1 from each reaction was included as an input control

2.2 Identification and validation of OXR1 as a target of PL To identify the senolytic targets of PL, we conducted pull‐down experiments by incubating the PL probe or CTL probe with normal or nonsenescent WI38 cells (NCs), IR‐induced senescent WI38 cells (IR‐SCs), or replicative senescent WI38 cells (RE‐SCs) as described in method section. To further exclude proteins that bound nonspecifically to PL probe, we also carried out competitive‐binding experiments in which IR‐SCs were incubated with an excess of PL for 2 hr then with the PL probe for 3 hr or vice versa (Figure 1c). After excluding proteins that were bound by the CTL probe, we identified 436 proteins in NCs that bound the PL probe, 890 proteins in IR‐SCs, and 702 proteins in RE‐SCs (Figure 1d). Among these, 270 proteins were unique to both IR‐SCs and RE‐SCs, and 360 proteins were common to all three cell types. From the 360 common proteins, we identified 49 SC‐selective targets that had an enrichment ratio of ≥2 (i.e., the LFQ intensity of proteins in IR‐SCs and RE‐SCs was ≥2‐fold higher than those in NCs) and a PL competition ratio of ≥2 (i.e., the LFQ intensity of proteins on cells treated with the PL probe followed by PL was ≥2‐fold higher than those in the cells treated with PL followed by the PL probe). Similarly, using a PL competition ratio of ≥2 as a cutoff, 123 SC‐unique proteins were retained on the final list as SC‐specific targets (Figure 1d; for the protein list, see Supporting Information Tables S1 and S2). Overall, we identified 172 potential senolytic targets (123 SC‐specific targets plus 49 SC‐selective targets) of PL. Gene ontology (GO) analysis revealed that these PL targets were mainly involved in the regulation of protein transport and localization, autophagy, TOR signaling, protein phosphorylation, and ubiquitination (Supporting Information Table S3). KEGG pathway analysis revealed that the PL‐target proteins were mainly enriched in endocytosis, phosphatidylinositol signaling system, inositol phosphate metabolism, mTOR signaling pathway, and insulin signaling pathway (Supporting Information Table S4). We found that OXR1 was on the top of the list of the PL‐binding proteins in SCs (Supporting Information Table S2). OXR1 is crucial for protecting cells against oxidative stress by regulating the expression of several enzymes that detoxify ROS (Jaramillo‐Gutierrez, Molina‐Cruz, Kumar & Barillas‐Mury, 2010; Oliver et al., 2011; Yang et al., 2015), and it may be particularly important as a senolytic target of PL because SCs are known to produce high levels of ROS but remain resistant to oxidative stress (Supporting Information Figure S2) (Chandrasekaran, Idelchik & Melendez, 2017; Davalli, Mitic, Caporali, Lauriola & D'Arca, 2016; Lee et al., 1999; Lu & Finkel, 2008). In addition, LMD‐3, homolog of OXR1 in Caenorhabditis elegans, has been shown to protect against oxidative stress and accelerated aging in the worm (Sanada et al., 2014). Therefore, we validated whether PL could bind OXR1 directly using a biotin‐labeled PL probe (Raj et al., 2011). This PL probe pulled down OXR1 from SC lysates and bound directly to recombinant human OXR1 (rhOXR1) in a dose‐dependent manner, which was prevented with an excess of PL (Figure 1e, f). Taken together, these results demonstrate that PL binds OXR1 directly, suggesting that OXR1 may be a senolytic target of PL.

2.3 PL selectively reduces the level of OXR1 in IR‐SCs by inducing its degradation To further confirm that OXR1 is a senolytic target of PL, we examined the effect of PL on OXR1 expression in NCs and IR‐SCs. We found that IR induced a time‐dependent increase in OXR1 mRNA and OXR1 protein in WI‐38 cells, which correlated closely with the time‐dependent induction of senescence as determined by senescence‐associated β‐galactosidase staining (Figure 2a–c). PL treatment had no significant effect on the levels of OXR1 mRNA and protein in NCs (Figure 2d, e). In contrast, PL substantially reduced the levels of OXR1 protein but had no effect on OXR1 mRNA expression in IR‐SCs, suggesting that PL regulates OXR1 in IR‐SCs post‐transcriptionally, possibly by inducing its degradation. Indeed, the PL‐induced downregulation of OXR1 was attenuated by suppressing the activity of the proteasome with MG‐132 (Figure 2f). Moreover, we found that PL selectively increased the levels of poly‐ubiquitylated proteins in IR‐SCs, but not in NCs (Figure 2g); and IR‐SCs treated with PL had higher levels of both mono‐ubiquitinated OXR1 and poly‐ubiquitylated OXR1 than those treated with vehicle (Figure 2h). Collectively, these results suggest that PL can selectively reduce the levels of OXR1 in SCs, in part by inducing the proteasomal degradation of OXR1. Figure 2 Open in figure viewer PowerPoint PL selectively decreases the level of OXR1 in SCs, in part by inducing OXR1 proteasomal degradation. (a) Percentage of SA‐β‐gal–positive cells at different days after IR. (b) The levels of OXR1 mRNA in WI‐38 cells at different days after IR are presented as a fold change from un‐irradiated control cells. (c) The levels of OXR1 protein in WI‐38 cells at different days after IR. Left panel, representative immunoblot of OXR1 and β‐actin; right panel, fold changes in OXR1 expression in irradiated cells relative to un‐irradiated cells after normalization to β‐actin. (d) The levels of OXR1 mRNA in NCs and IR‐SCs after incubation with vehicle (Veh) or PL (5 μM) for 6 hr. (e) The levels of OXR1 protein in NCs and IR‐SCs after incubation with vehicle (Veh) or PL (5 μM) for 6 hr. Left panel, representative immunoblot of OXR1 and β‐actin; right panel, fold changes in OXR1 expression in cells relative to vehicle‐treated NCs after normalization to β‐actin. (f) The levels of OXR1 were determined in NCs and IR‐SCs after incubation with vehicle (Veh) or PL (5 μM) for 6 hr in the presence or absence of 5 μM MG‐132 (MG). (g) The levels of poly‐ubiquitylated (Poly‐Ub) proteins in NCs and IR‐SCs after incubation with vehicle (Veh) or PL (5 μM) for 6 hr; β‐actin was included as a loading control. (h) The levels of poly‐ubiquitylated (Poly‐Ub) OXR1 in IR‐SCs after incubation with vehicle (Veh) or PL (5 μM) for 6 hr; the cell lysates were incubated with OXR1 or IgG control and resin for overnight. After extensive washing, ubiquitinated OXR1 was eluted by SDS sample buffer and immunoblotted with ubiquitin and OXR1 antibodies. The input cell lysates were also immunoblotted with antibodies to OXR1 or β‐actin as indicated. ns indicates the nonspecific band. Data in bar graphs are the mean ± SE of two to three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. un‐irradiated cells by one‐way ANOVA. ap < 0.05 vs. NCs treated with Veh; bp < 0.05 vs. IR‐SCs treated with Veh by two‐way ANOVA

2.4 More intracellular PL in IR‐SCs than NCs To understand the mechanism by which PL selectively induced the degradation of OXR1 in IR‐SCs, we measured the levels of PL in NCs and IR‐SCs by taking advantage of our alkyne‐bearing PL probe. Specifically, NCs and IR‐SCs were incubated with the PL probe, and its levels were determined by a Click Chemistry reaction with Alexa Fluor 488 azide after the free probe was removed. With fluorescence microscopy, we found that IR‐SCs appeared to have a higher level of the PL probe than NCs (Figure 3a). To quantify the difference in the intracellular levels of the PL probe between NCs and IR‐SCs, we measured the mean fluorescence intensity (MFI) of the Alexa Fluor 488‐PL probe with flow cytometry after the MFI was normalized by cell size according to the forward scatter (FSC) measurement because SCs are bigger than NCs (Figure 3b). We found that IR‐SCs had a significantly higher level of intracellular PL probe than NCs. This finding was also confirmed by analysis with an ImageStream flow cytometer after considering the difference in cell size measured by the average area of cells between NCs and IR‐SCs (Figure 3c). These results suggest that IR‐SCs may uptake and retain more PL or degrade less PL than NCs, which may contribute to the selective induction of OXR1 degradation and cell death in SCs by PL. Figure 3 Open in figure viewer PowerPoint IR‐SCs have higher levels of PL than NCs. NCs and IR‐SCs were incubated with vehicle (Veh) or alkyne‐labeled PL probe (5 μM) for 5 hr, and probe uptake was detected with Alexa Fluor azide 488. (a) PL probe uptake was visualized by microscopy, (b) measured by flow cytometry, and (c) quantified by ImageStream flow cytometry. (a) DAPI was used for nuclear staining. (b) Left and middle panels, representative histograms of flow cytometric analysis; right panel, relative MFI of Alexa Fluor 488‐PL staining, normalized by cell size. (c) Left and middle panels, representative brightfield and Alexa Fluor 488 fluorescent images of NCs and IR‐SCs after incubation with the PL probe; right panel, normalized relative Alexa Fluor 488 fluorescence intensity in NCs and IR‐SCs according to cell size. Data in the bar graphs are the mean ± SE of two to three independent experiments. *p < 0.05 vs. NCs by unpaired t test

2.5 Knocking down OXR1 selectively kills IR‐SCs by downregulating the expression of antioxidant enzymes and sensitizing the cells to oxidative stress OXR1 is an important antioxidant protein that protects cells from oxidative stress by regulating the expression of enzymes that detoxify ROS, such as glutathione peroxidase 2 (GTX2), heme oxygenase 1 (HO‐1), and catalase (CAT) (Jaramillo‐Gutierrez et al., 2010; Oliver et al., 2011; Yang et al., 2015). SCs are known to produce high levels of ROS, but they are highly resistant to oxidative stress (Chandrasekaran et al., 2017; Davalli et al., 2016; Lee et al., 1999; Lu & Finkel, 2008). This resistance may be due to the increased expression of OXR1 and its downstream targets in SCs; therefore, OXR1 may be a genuine senolytic target. To test this hypothesis, we used two different OXR1 shRNAs (shOXR1‐1 and shOXR1‐2) to knock down the expression of OXR1 in both NCs and IR‐SCs (Figure 4a). We found that shOXR1‐1 was slightly more effective than shOXR1‐2 at knocking down OXR1 in both cell types; thus, we used shOXR1‐1 (referred to as shOXR1) for the rest of the study. Figure 4 Open in figure viewer PowerPoint Knocking down OXR1 selectively kills IR‐SCs by downregulating the expression of antioxidant enzymes to sensitize the cells to oxidative stress. (a) The levels of OXR1 mRNA (top) and OXR1 protein (bottom) in NCs and IR‐SCs transfected with either scramble control shRNA (shCtrl) or two different shRNA constructs targeting OXR1 (shOXR1‐1 or shOXR1‐2). (b) Expression of glutathione peroxidase 2 (GPX2), heme oxygenase 1 (HO‐1), catalase (CAT), superoxide dismutase 1 (SOD1), and SOD2 mRNA in NCs and IR‐SCs transfected with shCtrl or shOXR1. Data in (a) and (b) are the mean ± SE of fold changes in mRNA expression compared with shCtrl‐transfected cells from two to three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. shCtrl by unpaired t test. (c) Percent viability of NCs and IR‐SCs transfected with shCtrl or shOXR1 presented as the mean ± SE of two independent experiments. *p < 0.05 vs. IR‐SCs transfected with shCtrl by unpaired t test. (d) Levels of intracellular ROS in NCs and IR‐SCs transfected with shCtrl or shOXR1 in the presence or absence of 2 mM N‐acetyl‐ l ‐cysteine (NAC). Data are presented as the mean ± SE of mean fluorescent intensity (MFI) of dihydrorhodamine 123 (DHR) from two independent experiments. *p < 0.05 vs. IR‐SCs transfected with shOXR1 without NAC by unpaired t test. (e) Percent apoptotic cells in NCs and IR‐SCs transfected with shCtrl or shOXR1 in the presence or absence of 2 mM NAC are presented as the mean ± SE of two independent experiments. *p < 0.05 vs. IR‐SCs transfected with shOXR1 without NAC by unpaired t test. (f) Percent viable cells in NCs and IR‐SCs transfected with either shCtrl or shOXR1 after incubation with 100 μM H 2 O 2 for 3 day. Data are presented as the mean ± SE of percent viable cells compared with their respective controls without H 2 O 2 treatment from two independent experiments. *p < 0.05 vs. IR‐SCs transfected with shCtrl by unpaired t test. (g) Percent apoptotic cells in NS and IR‐SCs transfected with shCtrl or shOXR1 after incubation with 100 μM H 2 O 2 in the presence or absence of 2 mM NAC. Data are presented as the mean ± SE of two to three independent experiments. *p < 0.05 vs. IR‐SCs transfected with shOXR1 with H 2 O 2 alone by unpaired t test As expected, IR‐SCs expressed significantly higher levels of the known downstream targets of OXR1, that is, GTX2 and HO‐1 and CAT, and other antioxidant enzymes such as superoxide dismutase 1 (SOD‐1) and SOD2 than NCs (Figure 4b). Knocking down OXR1 slightly reduced the expression of HO‐1 and CAT mRNA but not GTX2 mRNA, in NCs. In contrast, IR‐SCs showed a greater reduction in GTX2, HO‐1, and CAT mRNA expression after OXR1 knockdown. Knocking down OXR1 also moderately reduced the expression of SOD‐1 and SOD‐2 mRNA in NCs and SOD‐1 mRNA in IR‐SCs. Importantly, knocking down OXR1 significantly reduced the viability of IR‐SCs, but not NCs (Figure 4c). These findings suggest that OXR1 may be a novel senolytic target. To determine whether OXR1 knockdown selectively killed IR‐SCs through oxidative stress, we measured ROS production and apoptosis in IR‐SCs and NCs after transfection with control shRNA (shCtrl) or shOXR1 in the presence or absence of the antioxidant N‐acetyl‐cysteine (NAC). The IR‐SCs produced significantly higher levels of ROS than NCs, and IR‐SCs produced even more ROS after knockdown of OXR1 (Figure 4d). This increase in ROS was associated with a significant increase in apoptosis in IR‐SCs after knocking down OXR1 (Figure 4e). Importantly, the increases in ROS and apoptosis in IR‐SCs induced by shOXR1 were abrogated by NAC. In contrast, knocking down OXR1 in NCs only slightly increased ROS production, but this had no significant effect on NC apoptosis. These results suggest that knocking down OXR1 selectively kills IR‐SCs by sensitizing them to oxidative stress and inducing apoptosis. This is supported by our finding that knocking down OXR1 also sensitized IR‐SCs but not NCs to H 2 O 2 ‐induced cell death and apoptosis, and this was abrogated by NAC (Figure 4f, g).