1. Introduction

      Pharmacologically targeting fundamental mechanisms of aging is anticipated to reduce the severity or delay the onset of multiple ageassociated co-morbidities simultaneously [5–7]. One key mechanism demonstrated to drive aging is cellular senescence, whereby accumulation of DNA damage and/or other cellular stressors [1–4] cause proliferating [8,9] or terminally differentiated non-dividing cells [10–13] to enter a state characterized by profound chromatin and secretome changes, increased expression of the cell cycle inhibitor p16Ink4a, replicative arrest, and resistance to apoptosis [1,14]. Senescent cells can develop a senescence-associated secretory phenotype (SASP), consisting of pro-inflammatory cytokines, chemokines, and extracellular matrix-degrading proteins [15–18], which has deleterious paracrine and systemic effects [19–21]. Indeed, even a relatively low abundance of senescent cells is sufficient to cause tissue dysfunction [22]. Senescent cells are rare in young individuals, but increase with age in multiple tissues, including adipose tissue, skeletal muscle, kidney, and skin of all vertebrates tested [22,23].

      The role of senescent cells in age-related decline was identified by studies demonstrating the therapeutic benefits of clearing of senescent cells from progeroid or naturally-aging INK-ATTAC mice using a suicide gene expressed only in p16Ink4a expressing cells (J.L.K., T.T., J.M. van Deursen, and D.J. Baker [all Mayo Clinic] designed the INK-ATTAC strategy [19,20,24–26]). Conversely, injection of senescent cells is sufficient to drive age-related conditions such as osteoarthritis, frailty, and decreased survival [26,27]. Thus, the development of therapies that selectively kill senescent cells was anticipated to delay the onset of aging phenotypes, attenuate severity of age-related diseases, improve resiliency, and enhance survival. Importantly, it was also predicted that senolytic therapies could be administered intermittently, serving to reduce the senescent cell burden by treating quarterly or even annually, which minimizes the risk of side effects [28,29].

      We previously identified drugs that selectively kill senescent cells using a hypothesis-driven discovery paradigm [30]. Senescent cells are resistant to apoptosis due to upregulation of Senescent-Cell AntiApoptotic Pathways (SCAPs) [28,29]. Targeting SCAPs in cell culture using a combination of dasatinib and quercetin, an inhibitor of BCL-2 pro-survival pathway members, Navitoclax, or the more specific BCLxL inhibitor, A1331852, results in apoptosis of some but not all senescent cell types [30–33]. Treatment of mice with dasatinib plus quercetin (D + Q) improves cardiac ejection fraction and increases vascular reactivity in old mice after a single, 3 day treatment course [30,34]. In addition, D + Q treatment decreases vascular calcification and increases vascular reactivity in hypercholesterolemic, high fat diet fed ApoE−/−mice after three monthly 3 day treatment courses [34]. Intermittent oral D + Q treatment improves pulmonary function and reduces pulmonary fibrosis in a bleomycin-induced murine model of idiopathic pulmonary fibrosis [35], reduces high fat diet-induced liver steatosis [36], alleviates gait impairment caused by leg irradiation [30] and reduces osteoporosis in aged mice [10]. Finally, D + Q also decreases frailty, osteoporosis, loss of intervertebral disc glycosaminoglycans, and spondylosis in the Ercc1−/Δ mouse model of a human progeroid syndrome after intermittent treatment [30]. Similarly, Navitoclax, which decreases abundance of some but not all human and mouse senescent cell types in vitro [33], reduces hematologic dysfunction caused by whole body radiation [31] and reduces senescent cell-like, intimal foam cell/macrophages in vascular plaques in high fat fed LdlR−/−mice [37]. Treatment with A1331852 reduces senescent cholangiocytes and liver fibrosis in Mdr2−/− mice [38]. Taken together, these studies demonstrate that senolytic compounds can have significant effects on chronic degenerative diseases and age-related pathology.

      However, not all senescent cells are the same. Senescent cells may express different SASP factors, senescence markers, and more importantly use different mechanisms to resist apoptosis [30,39]. Furthermore, certain cancer therapeutics target SCAPs, e.g. Navitoclax, and could be repurposed as senolytics, but cause considerable toxicity including neutropenia and platelet deficiency [40,41]. Thus, new and improved senotherapeutic drugs and combinatorial approaches are needed to eliminate senescent cells safely from multiple organs or even within a single tissue [28–30,42].

      Here, we screened a panel of flavonoids for senotherapeutic activity to determine if we could improve upon quercetin. In primary murine embryonic fibroblasts induced to senescence through oxidative stress and in human fibroblasts induced to senescence with the genotoxin etoposide, fisetin was most effective at reducing senescent markers. Fisetin also reduced senescence markers in progeroid Ercc1−/Δ mice and aged WT mice, as well as human explants of adipose tissue. Fisetin treatment extended the health and lifespan in WT mice even when treatment was initiated in aged animals. This flavonoid is a natural compound present in many fruits and vegetables such as apples, persimmon, grapes, onions, cucumbers and strawberries [43,44], suggesting that it is imminently translatable. Importantly, no adverse effects of fisetin have been reported, even when given at high doses [45]. Thus, our results suggest that supplementation or even intermittent treatment with this safe, natural product could improve healthy aging, even in elderly individuals.

2. Materials and methods

2.1. Chemicals

      Chemicals were from Sigma-Aldrich (St. Louis) unless otherwise noted. The flavonoids were purchased from Selleckchem (Houston, TX): resveratrol (Cat #S1396), fisetin (Cat #S2298), luteolin (Cat #S2320), rutin (Cat #S2350), epigallocatechin gallate (EGCG, Cat #S2250), curcumin (Cat #S1848), pirfenidone (Cat #S2907), and myricetin (Cat #S2326). Apigenin, catechin, and quercetin were purchased from Sigma-Aldrich (Cat #1760595, #1096790 and 1,592,409, respectively).

2.2. Animals

      All animal studies were conducted in compliance with the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals and were approved by the Scripps Florida or Mayo Clinic Institutional Animal Care and Use Committees. Ercc1−/Δ mice were bred as previously described [46]. p16-luciferase reporter mice were obtained from Ohio State University [47] and bred to create an albino C57BL/6 p16Luc/+;Ercc1+/− and FVB/n p16+/Luc;Ercc1+/Δ strain. These mice were further crossed to create f1 p16+/Luc;Ercc1−/Δ mice with white fur for imaging. All animals were genotyped from an ear punch by TransnetYX (Cordova, TN). For diet studies, mice were fed Teklad 2020 chow (Envigo, Madison, WI) prepared with or without 500 ppm (500 mg/kg) of fisetin (Indofine Chemical Co., Hillsborough, NJ) by Envigo. Co. (Tampa, FL). For oral administration of fisetin, mice were dosed with 100 mg/kg of fisetin in 60% Phosal 50 PG:30% PEG400:10% ethanol or vehicle only by gavage. Studies in aged wildtype mice were conducted in both f1 C57BL/6;FVB/n and inbred C57BL/6 genetic backgrounds.

2.3. MEF isolation

      The Ercc1−/− MEFs were isolated from pregnant females at embryonic day 13 (E13) and cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F10 with 10% fetal bovine serum, 1× nonessential amino acids, penicillin, and streptomycin and incubated at 3% O2 initially, followed by a shift to 20% for 5 passages to induce senescence [48]. Cells were genotyped by TransnetYX (Cordova, TN) and routinely tested for mycoplasma contamination using the MycoAlert PLUS mycoplasma detection kit (Lonza, Walkersville, MD).

2.4. Assays to identify senotherapeutics

      Ercc1−/− MEFs were passaged 5 times at 20% O2 to induce senescence then seeded at 5000 cells per well in 96 well plates at least 6 h prior to treatment. Following the addition of drugs, the MEFs were incubated for 24–48 h at 20% O2. Subsequently SA-β-gal activity was measured in three independent experiments, as previously described [49]. Briefly, cells were washed with PBS then 10 μM C12FDG added in fresh culture medium and incubated for 2 h. Ten min prior to analysis, 2 μg/mL Hoechst dye was added. An IN Cell Analyzer 6000 was used to quantitate total number of viable cells (Hoechst+) and the number of senescent cells (C12FDG+). All samples were analyzed in duplicate with 3–5 fields per well and reported as the mean ± S.D. Senotherapeutic activity was confirmed in human fibroblasts (IMR90). The cells were obtained from American Type Culture Collection (ATCC) and cultured in EMEM medium with 10% FBS and antibiotics. To induce senescence, the cells were treated for 24 h with 20 μM etoposide. Two days after etoposide removal, ~70% of the cells were SA-β-gal+. Cells were treated for 48 h with different concentrations of fisetin (1–15 μM) and the percentage of SA-β-gal+ cells was determined using C12FDG, as described above.

2.5. IVIS in vivo imaging detection of luciferase activity

      Isoflurane-anesthetized mice (n = 2–10 mice per group) were injected intraperitoneally with 10 μL per gram body weight D-luciferin substrate (Caliper Life Sciences, Hopkinton, MA; 15 mg/mL diluted in sterile PBS) and were imaged using an IVIS Lumina (PerkinElmer, Billerica, MA), as previously described [47,50].

2.6. Measurement of lipid peroxidation

      Levels of 4-hydroxynonenal-protein adducts of liver lysates (n =4–6 mice per group) prepared in RIPA buffer were measured in the livers of mice using the OxiSelect HNE Adduct Competitive ELISA kit (Cell Biolabs, San Diego, CA), as described [50].

2.7. Measurement of glutathione

      Murine livers (n= 4–7 mice per group) fixed in 5% sulfosalicylic acid were prepared and analyzed for the concentration of reduced (GSH) and oxidized (GSSG) glutathione using the Glutathione Assay Kit (Cayman Chemical, Ann Arbor, MI), as described [50]. Sample absorbance was measured at 405 nm using a plate reader and the ratio of GSH: GSSG was reported for each sample.

2.8. Clinical chemistries

     Whole blood (n = 3–6 mice per group) was collected immediately following animal euthanasia via cardiac puncture into heparinized tubes for analysis of clinical chemistries utilizing VetScan Comprehensive Diagnostic Profile rotors on a VestScan VS2 (Abaxis, Union City, CA).

2.9. Serum MCP-1

      Serum concentrations of MCP-1 (n = 5 mice per group) were measured using a mouse-specific MCP-1 ELISA (Raybiotech, Norcross, GA), as described [51].

2.10. Histopathology

      Mouse tissues were collected at necropsy (n = 3–8 mice per group) and placed in 10% neutral buffered formalin for 48 h, transferred to 70% alcohol, and subsequently processed into paraffin blocks for sectioning and hematoxylin and eosin staining. Tissue sections were scored for the presence and severity of a well-defined panel of age-related lesions by a veterinary pathologist to create a composite tissue lesion score for each animal that reflects healthspan, as previously described [52].

2.11. RNA isolation and qPCR

      Tissues were harvested from mice (n = 4–10 mice per group) and snap frozen in liquid nitrogen. Total RNA was harvested from tissues and the expression of several markers of senescence was measured, as previously described in [51]. Total RNA was quantified using a Nanodrop spectrophotometer (Thermo Fisher, Waltham, MA) and 1 μg of total RNA was used to generate cDNA using the Transcriptor First Strand cDNA synthesis kit (Roche, Basel, Switzerland). Gene expression changes were carried out in 20 μL reactions using the Universal SYBR Green master mix with ROX (Roche) and a StepOne thermocycler (Thermo Fisher). Primers for the genes of interest are as follows: Cdkn1a (p21Cip1) Fwd 5’-GTCAGGCTGGTCTGCCTCCG-3′, Cdkn1a (p21Cip1) Rev. 5’-CGGTCCCGTGGACAGTGAGCAG-3′; Cdkn2a (p16Ink4a) Fwd 5′- CCCAACGCCCCGAACT-3′, Cdkn2a (p16Ink4a) Rev. 5′- GCAGAA GAGCTGCTACGTGAA-3′; Gapdh Fwd 5’-AAGGTCATCCCAGAGCTGAA-3′, Gapdh Rev. 5’-CTGCTTCACCACCTTCTTGA-3′; Il6 Fwd 5’-CTGGGAAATCG TGGAAT-3′, Il6 Rev. 5’-CCAGTTTGGTAGCATCCATC-3′; Mcp1 Fwd 5’- GCATCCACGTGTTGGCTCA-3′, Mcp1 Rev. 5’-CTCCAGCCTACTCATTGGG ATCA-3′. Data were analyzed by ΔΔCt method and gene expression was normalized to Gapdh.

2.12. Isolation of peripheral blood CD3+ T lymphocytes

      Blood was obtained from mice n = 4–10 mice per group) postmortem by cardiac puncture, immediately placed into 1/10th volume of 0.5 M EDTA, and gently mixed to prevent coagulation. Samples were centrifuged at 300 g for 10 min in a table top centrifuge. The supernatant was discarded and the cell pellet was resuspended in 1 mL ACK buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4) and incubated at room temperature for 10 min to lyse red blood cells. The cells were spun down and ACK lysis repeated. The cells were spun down, washed with PBS, and resuspended in PBS containing 0.5% FBS and 2 mM EDTA. Fifty μL of CD3-Biotin conjugate (Miltenyi Biotech, San Diego, CA) were added to the cell suspension and incubated for 30 min on ice. The cells were centrifuged at 100 g for 10 min and washed twice in resuspension buffer. The cell pellet was resuspended in 500 μL of resuspension buffer and 100 μL of anti-biotin microbeads were added followed by a 15 min incubation on ice. The cells were washed twice and then resuspended in 500 μL of resuspension buffer and applied to a MACS column attached to a magnet. The cells were washed with 3 column volumes of resuspension buffer before elution. The cells were centrifuged and RNA isolation conducted using an RNeasy kit (Qiagen, Germantown, MD) according to the manufacturer's specifications. qPCR analysis of senescence markers was performed as indicated above.

2.13. Senescence-associated β-galactosidase (SA-β-gal) staining of tissue

      Fresh fat tissues (n = 6–7 mice per group) were fixed and stained to detect senescence-associated β-galactosidase activity, as described [53].

2.14. Mass cytometry/CyTOF in adipose tissue

      This high dimensional single-cell proteomics technique combines time-of-flight mass spectrometry with metal-labelling technology to detect up to 40 protein targets per cell [54,55]. A panel of antibodies based on cell surface markers and transcription factors (see Supplemental Table 1) was designed for CyTOF analysis of adipose tissues. Each antibody was tagged with a rare metal isotope and its function verified by mass cytometry according to the factory manual (Multi Metal labeling Kits, Fluidigm, CA). A CyTOF-2 mass cytometer (Fluidigm, South San Francisco, CA) was used for data acquisition. Acquired data were normalized based on normalization beads (Ce140, Eu151, Eu153, Ho165, and Lu175). One gram of subcutaneous fat tissue (n = 6–9 mice per group) was dissociated into a single-cell suspension using an adipose tissue dissociation kit (Adult Adipose Tissue Dissociation Kit, Miltenyi Biotec Inc.CA). Collected cells were incubated with metal-conjugated antibodies for cell surface markers and intracellular proteins. Fixation and permeabilization were conducted according to the manufacturer's instructions (Transcription Factor Staining Buffer Set, eBioscience, San Diego, CA). CyTOF data were analyzed by Cytobank (Santa Clara, CA).

2.15. Human adipose tissue explants

      The protocol was approved by the Mayo Clinic Foundation Institutional Review Board for Human Research. Informed consent was obtained from all subjects (n = 3). Human greater omental adipose tissue was resected during surgery from 2 lean (BMI 25.5 and 26.2) and 1 obese (BMI 45.6) female subjects, ages ranging from 55 to 66 years. No subject was known to have a malignancy. The adipose tissue was cut into small pieces and washed with PBS 3 times. The adipose tissue was then cultured in medium containing 1 mM sodium pyruvate, 2 mM glutamine, MEM vitamins, MEM non-essential amino acids, and antibiotics with 20 μM of fisetin or DMSO. After 48 h, the adipose explants were washed 3 times with PBS and was then maintained in the same media without drugs for 24 h to collect conditioned medium (CM) for multiplex protein analysis. The adipose explants then were fixed and stained to detect senescence-associated β-galactosidase activity [85].

2.16. Multiplex protein analyses

      Pro-inflammatory cytokine and chemokine protein levels in CM from the adipose tissue explants (n = 3) were measured using Luminex xMAP technology. The multiplexing analysis was performed using the Luminex™ 100 system (Luminex, Austin, TX) by Eve Technologies Corp. (Calgary, Alberta, Canada). Human multiplex kits were from Millipore (Billerica, MA). The secreted protein levels in CM were nor malized to the tissue weights and plotted as a percent relative to the vehicle control.

3. Results

      We previously demonstrated that the flavonoid quercetin, an antioxidant, which also targets PI3 kinase delta as well as certain BCL-2 family members, reduces senescence in primary human umbilical vein endothelial cells (HUVECs) and murine embryonic fibroblasts (MEFs), especially when used in combination with the tyrosine kinase inhibitor dasatinib [30]. To determine if other flavonoids might have more potent senotherapeutic activity than quercetin, a panel of flavonoids was screened for effects on senescence induced by oxidative stress [49]. Primary MEFs from Ercc1−/− mice were used. These cells undergo premature senescence if grown at atmospheric oxygen [56]. Ercc1−/− MEF cultures were established at 3% O2 then shifted to 20% O2 for three passages to induce senescence. To quantify senescent cells, SA-ß-gal activity was measured using the fluorescent substrate C12FDG [57] using an IN Cell Analyzer 6000 confocal imager. At a dose of 5 μM, fisetin was most effective in reducing the fraction of SA-ß-gal positive MEFs (Fig. 1A). Luteolin and curcumin also showed weak activity at a dose where quercetin was ineffective. In addition, fisetin reduced senescence in MEFs and IMR90 cells in a dose-dependent manner (Fig. 1B and C).These results are consistent with our previous finding that fisetin selectively reduces the viability of senescent HUVECs without affecting proliferating cells [32]. In HUVECs, fisetin induces apoptosis as measured by caspase3/7 activity, whereas in MEFs, fisetin suppressed markers of senescence without evidence of cell killing [32].

      To test the senotherapeutic activity of fisetin in vivo, initially progeroid Ercc1−/Δ mice carrying a p16Ink4a-luciferase reporter transgene were used [47,50]. These mice show accelerated accumulation of senescent cells compared to WT mice, but the overall level of senescence never exceeds that of naturally aged mice [50]. Ercc1−/Δ; p16Ink4a-luciferase mice were fed a standard Teklad 2020 chow diet with or without supplementation with 500 ppm (500 mg/kg) of fisetin, ad libitum (approximately 60 mg/kg fisetin per day). The mice were exposed to a fisetin diet intermittently from 6 to 8 then 12–14 wks of age. Whole body luciferase activity was measured before starting the fisetin diet then weekly thereafter. Animals in the two treatment groups had an equivalent luciferase signal prior to administration of the experimental diet. Dietary fisetin suppressed the luciferase signal of Ercc1−/Δ; p16Ink4a-luciferase mice significantly (Fig. 2A-B). The luciferase signal was lower at every time point after initiation of the fisetin diet (Fig. 2B-C). Notably, the level of p16Ink4a expression remained significantly lower in the fisetin-treated mice throughout the 4 week period when the animals were not exposed to fisetin (8–12 wks of age, Fig. 2B). This is consistent with a mechanism of action where senescent cells are cleared (senolytic) or senescence is reversed (senomorphic) but not a mechanism in which fisetin must be chronically present to suppress senescence.

      To validate the imaging data, Ercc1−/Δ mice were treated with the 500 ppm fisetin diet for 10 wks beginning at 10 wks of age, then tissues collected for measurement of multiple markers of senescence including p16Ink4a and p21Cip1 and SASP factors. Expression of p21Cip1 was included since not all senescent cells are p16Ink4a positive: some are p21Cip1 positive, but p16Ink4a negative. As shown in Fig. 3A-D, p16Ink4a and p21Cip1 mRNA, as well as SASP markers, were significantly elevated in the fat, spleen, liver, and kidney of Ercc1−/Δ mice compared to age-matched WT mice. Fisetin reduced expression of senescence and SASP markers significantly in all tissues. Similarly, there was a reduction in the expression of p16Ink4a, p21Cip1 and the SASP factors in peripheral blood CD3+ T cells (Fig. 3E), a cell type that demonstrates a robust increase in p16INK4a expression as humans age [58]. In addition, fisetin reduced oxidative stress in the liver as determined by measuring the lipid peroxidation product 4-hydroxynonenal (HNE) adducts and an increase in the ratio of reduced to oxidized glutathione (Fig. 3F-G), consistent with data indicating fisetin has antioxidant activity as well as increasing intracellular glutathione [45].

 

      To confirm further the data obtained in progeroid mice, we employed naturally aged C57BL/6 mice and different methods of detecting senescence in tissue. 22–24-month-old mice were treated with 100 mg/kg fisetin for 5 consecutive days by oral gavage, or vehicle only. Mice were sacrificed 3 days after the last dose and the number of SA-ß-gal+ cells present in inguinal fat was determined by staining tissue sections to measure SA-β-gal activity. Fat tissue was chosen for the analysis since there is a clear upregulation of senescence markers including SASP in our mouse models, the tissue has a significant increase in the fraction of senescent cells including senescent immune cells, such as T and endothelial cells and macrophages, and injection of senescent pre-adipocytes is sufficient to induce frailty in young mice [26,50,59,60]. Short-term treatment with fisetin significantly reduced the fraction of senescent cells in white adipose tissue (WAT). To determine which cells become senescent in WAT and which cell types are cleared by fisetin, CyTOF analysis was performed on subcutaneous adipose tissue from aged INK-ATTAC mice expressing a Flag-tagged FKBPCasp8 protein from the p16Ink4a promoter (Fig. 4B). The Flag tag enabled identification of senescent (p16Ink4a-expressing) cells using an anti-Flag antibody. CyTOF analysis revealed a significantly elevated fraction of senescent cells in fat from old mice compared to young and identified these cells as mesenchymal stem/progenitor cells, T lymphocytes, natural killer cells, and endothelial cells (Fig. 4C). The short-course treatment with fisetin resulted in a significant reduction in the fraction of senescent cells in each of these populations (Fig. 4C). Fisetin reduced the fraction of p16Ink4a-expressing, c-Kit+ stem/progenitor cells, CD4+and CD8+ T cells, NK-1.1+ NK cells, and CD146+CD31+ endothelial cells (Fig. 4C). These data are also shown in Spanning-tree Progression Analysis of Density-normalized Events (SPADE) analysis (Supplemental Fig. 1). To confirm senescence in these cell populations, CENP-B protein was measured by CyTOF (Fig. 4D). CENP-B binds centromeric satellite DNA [61], which becomes distended in senescent cells. The fraction of CENP-B+ cells in WAT was significantly increased in old mice compared to young and suppressed by treating the mice with a short-course of fisetin, in the same manner as p16Ink4a-expressing/FLAG+ cells. In contrast, p21Cip1 (another cell-cycle regulator that is often up-regulated in senescent cells) expression was not significantly elevated in these cell populations (Supplemental Fig. 2). While FLAG+ dendritic cells and macrophages were increased in WAT from old mice consistent with a previous report [62], fisetin treatment had no substantial effect on the fraction of macrophages or dendritic cells with high p16Ink4a, CENP-B, or p21Cip1 (Supplemental Fig. 3). Taken together, these data demonstrate that a short-course of fisetin reduces the number of p16Ink4aexpressing cells in subcutaneous WAT including mesenchymal stem/ progenitor, immune, and endothelial cells. This is the first time a senotherapeutic has been demonstrated to differentially affect senescent cells of different lineages in vivo.

      To determine if fisetin also reduces senescence in human adipose tissue, greater omental adipose explants resected during surgery were treated with fisetin ex vivo. The tissue explants were treated for 48 h with 20 μM fisetin, washed, and cultured for an additional 24 h before measuring SASP factors by multiplex protein analysis [26]. Fisetin treatment caused a significant reduction in the percent of SA-ß-gal positive cells (Fig. 4E) as well as in expression of the SASP factors IL-6, IL-8, and MCP-1 in human WAT (Fig. 4F). These data support the translational potential of fisetin to reduce senescent cell burden and associated inflammation.

      To determine if fisetin-mediated clearance of senescent cells impacts the health or lifespan of mice, WT f1 C57BL/6:FVB mice were fed a diet containing 500 ppm fisetin beginning at 85 wks of age, roughly equivalent to age 75 years in humans. This resulted in an extension of median as well as maximal lifespan (Fig. 5A-B). Amylase and alanine aminotransferase (ALT) were significantly lower in serum of aged WT mice fed the diet supplemented with fisetin, consistent with improved pancreatic and liver homeostasis (Fig. 5C). Brain, kidney, liver, lung, and forepaw tissue sections were stained with hematoxylin and eosin and evaluated by a veterinary pathologist. Using the Geropathology Grading Platform to score age-related lesions [63], several tissues had reduced age-related pathology in the fisetin diet group compared to the control diet (Fig. 5D). An example of this is illustrated in a representative image from renal sections in Fig. 5E. Similar to the progeroid mice, fisetin reduced the expression of senescence and SASP markers in multiple tissues of aged WT mice exposed to oral fisetin (Fig. 5F-I). Furthermore, there was a reduction in senescence and SASP factor expression in peripheral CD3+ T cells (Fig. 5J). There was also a reduction in levels of circulating MCP-1 (Fig. 5K), a SASP factor [51]. Finally, fisetin reduced oxidative stress in the liver of old WT mice (Fig. 5L-M).

4. Discussion

      Aging is a complex process involving numerous pathways and both genetic and environmental components [64–69]. The biological processes that drive the aging process contribute to the etiology of most chronic diseases including: 1) chronic, “sterile” inflammation; 2) macromolecular changes in proteins, carbohydrates, lipids, mitochondria, and DNA; 3) stem cell and progenitor dysfunction; and 4) increased cellular senescence [5,70]. These processes are linked in that interventions that target one appear to attenuate others. For example, senescent cells accumulate with age and at sites of pathogenesis in chronic diseases [5,70]. Reducing senescent cell burden can lead to reduced inflammation, decreased macromolecular dysfunction, and enhanced function of stem/progenitor cells [1,3,19]. Adult stem cells also become dysfunctional with age, displaying evidence of senescence [71]. We previously demonstrated that the combination of dasatinib and quercetin function as a senolytic in vivo, alleviating many age-related diseases [26,30]. Because of the senotherapeutic activity of quercetin, we examined other natural flavonoids for effects on senescent cells in hopes of improving therapeutic efficacy.

      Here, we demonstrate that when tested against a panel of other flavonoids, fisetin had the most potent senotherapeutic effects in several cell types in vitro and showed strong anti-geronic effects in vivo. We demonstrated that acute (oral) or chronic (dietary) treatment of progeroid and WT mice with fisetin reduces markers of senescence and senescence-associated secretory phenotype in multiple tissues (Fig. 3A-D, 4A, 5F-I). These data were collected in two labs using progeroid and WT mice in two distinct genetic backgrounds. Specifically, p16Ink4a-expressing or CENP-B+ mesenchymal stem/progenitor, T lymphocytes, natural killer and endothelial cells were removed from subcutaneous fat of old mice, but not activated senescent-like macrophages or dendritic cells. The effect of fisetin was greater on p16Ink4aexpressing cells than on p21CIP1-expressing cells, at least in subcutaneous fat (Fig. 4). Our findings reveal that fisetin targets multiple, but not all types of senescent cells in vivo. Furthermore, by reducing the percent of senescent cells, fisetin reduces expression of senescence markers in multiple organs as measured by qPCR. This results in improved tissue homeostasis and reduction in multiple age-related pathologies, consistent with effects on a fundamental aging process.

      The fact that fisetin reduced the fraction of senescent T and NK cells could help amplify the beneficial effects of fisetin, since healthy immune cells are important for clearing senescent cells [72,73]. Similarly, fisetin reduces markers of inflammation and oxidative stress (Figs. 3F-G and 5L-M), consistent with prior literature [45]. This too could contribute to the reduction in senescence markers. The decrease in these markers was observed in tissues harvested several days after completion of fisetin administration. Since the rapid and terminal half-lives of fisetin are 0.09 and 3.1 h respectively [74], these improvements did not depend on continued presence of circulating fisetin. This is more consistent with fisetin causing removal of senescent cells, which take days to weeks to form after an insult (at least in culture), than with effects exerted by continued occupancy of a receptor or effects on an enzyme. However, it is important to note that given the multiple reported activities of flavonoids like fisetin, it is also possible that the extension of healthspan is due to mechanisms in addition to the reduction in senescence, such as altering the gut microbiome [75].

      Fisetin extends the replicative lifespan of S. cerevisiae by 55% [76] and the lifespan of D. melanogaster by 23% [77]. Here, we show for the first time a similar effect in vertebrate animals. Chronic exposure to fisetin improves healthspan and extends the median and maximum lifespan of mice. Importantly, in our study fisetin supplementation was initiated in mice N20 months old. This increases the translational potential of our study since senotherapeutic interventions are most feasibly administered in elderly humans after the onset of age-related diseases, rather than in younger asymptomatic subjects, in whom any sideeffects would be unacceptable.

      Fisetin is a member of the flavonoid family, a family of naturally occurring polyphenolic compounds. Fisetin, a high Trolox-equivalent antioxidant, is present in low concentrations in many fruits and vegetables such as apples, persimmon, grapes, onions, and cucumbers and at higher concentrations in strawberries [43,44]. The average dietary intake of naturally occurring fisetin in Japan is approximately 0.4 mg/day [78,79], apparently without any adverse effects. Fisetin has anti-cancer activity and appears to block the PI3K/AKT/mTOR pathway [80]. We previously found that transiently disrupting the PI3K/AKT pathway by RNA interference leads to death of senescent cells [30], as with other SCAPs that defend senescent cells from their own proapoptotic SASP [28,29]. Fisetin, like some other flavonoids, is a topoisomerase inhibitor, which may also contribute to its anti-cancer activity [81]. It increases the catalytic activity of hSIRT1 at least in vitro [76]. Also in vitro, fisetin inhibits the activity of several pro-inflammatory cytokines, including TNFα, IL-6, and the transcription factor NF-κB [82]. Fisetin has direct activity as a reducing agent, chemically reacting with and neutralizing reactive oxygen species [83,84]. Fisetin scavenges free radicals as a result of its electron donating capacity, which is due to the presence of two hydroxyl groups on one ring and a third hydroxyl group on another ring. Fisetin also upregulates synthesis of glutathione, an endogenous antioxidant [43,82]. Other biological activities include anti-hyperlipidemic [84–86], anti-inflammatory [85], and neurotrophic [87] effects, some of which could be mediated through a reduction in the senescent cell burden, particularly since when administered intermittently, fisetin alleviated dysfunction despite its short elimination half-life, more consistent with elimination of senescent cells than action on a receptor or enzyme requiring continuous drug presence.

      The chemical structure of fisetin is almost the same as quercetin except for a hydroxyl group in position 5. Thus, it is highly likely that these two closely related compounds exert many similar effects. Interestingly, preliminary medicinal chemistry on fisetin has identified analogues with enhanced senotherapeutic activity, suggesting that even more effective flavonoids can be developed for extending healthspan with minor alterations in the structure of fisetin.

      Given that fisetin is a natural product found in common foods and available as an oral dietary supplement and has no reported adverse side effects [45], our pre-clinical data suggest that fisetin should be imminently translatable and could have a significant benefit to the health of elderly patients. Based on these mouse studies, clinical trials to evaluate the short-term benefits of intermittent fisetin treatment on certain aspects of aging such as frailty are currently underway.

Author contributions

      M.J.Y. and J.I.K. performed the analyses of senescence and SASP markers in the tissues of the treated mice; Y.Z. performed the CyTOF analyses with oversight and analysis by E.A.A.; S.J.M., K.M., E.A.W., C.M. and L.A. performed the mouse treatment studies at The Scripps Research Institute; T.P. and C.L.I. performed the mouse studies at Mayo Clinic; H.F., D.G; and Y.Y.L. performed the testing of flavonoids in cell culture; M.X. performed the analysis in human adipose tissue explants; W.L.L performed histopathology analyses; C.E.B. provided the p16INK4aLuc+/− mice and assisted with analyses; T.T., J.L.K., E.A.A., P.D.R., and L.J.N. oversaw all experimental design, data analysis, and manuscript preparation. 

Competing financial interests

      J.L.K, T.T., Y.Z., M.X., and T.P. have a financial interest related to this research. Patents on senolytic drugs (PCT/US2016/041646) are held by Mayo Clinic. This research has been reviewed by the Mayo Clinic Conflict of Interest Review Board and was conducted in compliance with Mayo Clinic Conflict of Interest policies. None of the other authors has a relevant conflict of financial interest.

Appendix A. Supplementary data

      Supplementary data to this article can be found online at https://doi. org/10.1016/j.ebiom.2018.09.015.

References

[1] Tchkonia T, Zhu Y, van Deursen J, Campisi J, Kirkland JL. Cellular senescence and the senescent secretory phenotype: therapeutic opportunities. J Clin Invest 2013;123 (3):966–72.

[2] Lebrasseur NK, Tchkonia T, Kirkland JL. Cellular senescence and the biology of aging, disease, and frailty. Nestle Nutr Inst Workshop Ser 2015;83:11–8.

[3] Zhu Y, Armstrong JL, Tchkonia T, Kirkland JL. Cellular senescence and the senescent secretory phenotype in age-related chronic diseases. Curr Opin Clin Nutr Metab Care 2014;17(4):324–8.

[4] Swanson EC, Manning B, Zhang H, Lawrence JB. Higher-order unfolding of satellite heterochromatin is a consistent and early event in cell senescence. J Cell Biol 2013;203(6):929–42.

[5] Kirkland JL. Translating the science of aging into therapeuticiInterventions. Cold Spring Harb Perspect Med 2016;6(3):a025908.

[6] Justice J, Miller JD, Newman JC, Hashmi SK, Halter J, Austad SN, et al. Frameworks for proof-of-concept clinical trials of interventions that target fundamental aging processes. J Gerontol A Biol Sci Med Sci 2016;71(11):1415–23.

[7] Newman JC, Milman S, Hashmi SK, Austad SN, Kirkland JL, Halter JB, et al. Strategies and challenges in clinical trials targeting human aging. J Gerontol A Biol Sci Med Sci 2016;71(11):1424–34.

[8] Campisi J. Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell 2005;120:513–22.

[9] Campisi J. d'Adda di Fagagna F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol 2007;8(9):729–40.

[10] Farr JN, Xu M, Weivoda MM, Monroe DG, Fraser DG, Onken JL, et al. Targeting cellular senescence prevents age-related bone loss in mice. Nat Med 2017;23(9):1072–9.

[11] Jurk D, Wang C, Miwa S, Maddick M, Korolchuk V, Tsolou A, et al. Postmitotic neurons develop a p21-dependent senescence-like phenotype driven by a DNA damage response. Aging Cell 2012;11(6):996–1004.

[12] Jurk D, Wilson C, Passos JF, Oakley F, Correia-Melo C, Greaves L, et al. Chronic inflammation induces telomere dysfunction and accelerates ageing in mice. Nat Commun 2014;2:4172.

[13] Sapieha P, Mallette FA. Cellular senescence in postmitotic cells: beyond growth arrest. Trends Cell Biol 2018(8):595–607.

[14] Wang E. Senescent human fibroblasts resist programmed cell death, and failure to suppress bcl2 is involved. Cancer Res 1995;55(11):2284–92.

[15] Coppé JP, Patil C, Rodier F, Sun Y, Muñoz DP, Goldstein J, et al. Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol 2008;6:2853–68.

[16] Kuilman T, Peeper DS. Senescence-messaging secretome: SMS-ing cellular stress. Nat Rev Cancer 2009;9:81–94.

[17] Acosta JC, Banito A, Wuestefeld T, Georgilis A, Janich P, Morton JP, et al. A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nat Cell Biol 2013;15(8):978–90.

[18] Coppe JP, Desprez PY, Krtolica A, Campisi J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu Rev Pathol 2010;5:99–118.

[19] Xu M, Tchkonia T, Ding H, Ogrodnik M, Lubbers ER, Pirtskhalava T, et al. JAK inhibition alleviates the cellular senescence-associated secretory phenotype and frailty in old age. Proc Natl Acad Sci U S A 2015;112(46) E6301-E10.

[20] Xu M, Palmer AK, Ding H, Weivoda MM, Pirtskhalava T, White TA, et al. Targeting senescent cells enhances adipogenesis and metabolic function in old age. Elife 2015. https://doi.org/10.7554/eLife.12997.

[21] Nelson G, Wordsworth J, Wang C, Jurk D, Lawless C, Martin-Ruiz C, et al. A senescent cell bystander effect: senescence-induced senescence. Aging Cell 2012;11(2):345–9.

[22] Herbig U, Ferreira M, Condel L, Carey D, Sedivy JM. Cellular senescence in aging primates. Science 2006;311(5765):1257.

[23] Tchkonia T, Morbeck DE, von Zglinicki T, van Deursen J, Lustgarten J, Scrable H, et al. Fat tissue, aging, and cellular senescence. Aging Cell 2010;9:667–84.

[24] Baker DJ, Childs BG, Durik M, Wijers ME, Sieben CJ, Zhong J, et al. Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan. Nature 2016;530(7589):184–9.

[25] Baker DJ, Wijshake T, Tchkonia T, Lebrasseur NK, Childs BG, van de Sluis B, et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 2011;479(7372):232–6.

[26] Xu MP, T.; Farr, J.N.; Weigand, B.M.; Palmer, A.K.; Weivoda, M.M.; Inman, C.L.; Ogrodnik, M.B.; Hachfeld, C.M.; Fraser, D.G.; Onken, J.L.; Johnson, K.O.; Verzosa, G.C.; Langhi, L.G.; Weigl, M.; Giorgadze, N.; Lebrasseur, N.K.; Miller, J.D.; Jurk, D.; Singh, R.J.; Allison, D.B.; Ejima, K.; Hubbard, G.B.; Ikeno, Y.; Cubro, H.; Garovic, V.D.; Hou, X.; Weroha, S.J.; Robbins, P.D.; Niedernhofer L.J.; Khosla, S.; Tchkonia, T.; Kirkland, J.L. Senolytics Enhance Physical Function in Old Age and Extend PostTreatment Survival. Nat Med 2018.

[27] Xu M, Bradley EW, Weivoda MM, Hwang SM, Pirtskhalava T, Decklever T, et al. Transplanted Senescent Cells Induce an Osteoarthritis-like Condition in mice. J Gerontol A Biol Sci Med Sci 2017;72(6):780–5.

[28] Kirkland JL, Tchkonia T, Zhu Y, Niedernhofer LJ, Robbins PD. The clinical potential of senolytic drugs. J Am Geriatr Soc 2017;65(10):2297–301.

[29] Kirkland JL, Tchkonia T. Cellular Senescence: a Translational Perspective. EBioMedicine 2017;21:21–8.

[30] Zhu Y, Tchkonia T, Pirtskhalava T, Gower A, Ding H, Giorgadze N, et al. The Achilles' heel of senescent cells: from transcriptome to senolytic drugs. Aging Cell 2015;14: 644–58.

[31] Chang J, Wang Y, Shao L, Laberge RM, Demaria M, Campisi J, et al. Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice. Nat Med 2016;22(1):78–83.

[32] Zhu Y, Doornebal EJ, Pirtskhalava T, Giorgadze N, Wentworth M, FuhrmannStroissnigg H, et al. New agents that target senescent cells: the flavone, fisetin, and the BCL-XL inhibitors, A1331852 and A1155463. Aging (Albany NY) 2017;9 (3):955–63.

[33] Zhu Y, Tchkonia T, Fuhrmann-Stroissnigg H, Dai HM, Ling YY, Stout MB, et al. Identification of a novel senolytic agent, navitoclax, targeting the Bcl-2 family of antiapoptotic factors. Aging Cell 2016;15(3):428–35.

[34] Roos CM, Zhang B, Palmer AK, Ogrodnik MB, Pirtskhalava T, Thalji NM, et al. Chronic senolytic treatment alleviates established vasomotor dysfunction in aged or atherosclerotic mice. Aging Cell 2016:Feb:10 [doi: .1111/acel.12458. Epub ahead of print].

[35] Schafer MJ, White TA, Iijima K, Haak AJ, Ligresti G, Atkinson EJ, et al. Cellular senescence mediates fibrotic pulmonary disease. Nat Commun 2017;8:14532.

[36] Ogrodnik M, Miwa S, Tchkonia T, Tiniakos D, Wilson CL, Lahat A, et al. Cellular senescence drives age-dependent hepatic steatosis. Nature Commun [In press].

[37] Childs BG, Baker DJ, Wijshake T, Conover CA, Campisi J, van Deursen JM. Senescent intimal foam cells are deleterious at all stages of atherosclerosis. Science 2016;354 (6311):472–7.

[38] Moncsek A, Al-Suraih MS, Trussoni CE, O'Hara SP, Splinter PL, Zuber C, et al. Targeting senescent cholangiocytes and activated fibroblasts with B-cell lymphoma-extra large inhibitors ameliorates fibrosis in multidrug resistance 2 gene knockout (Mdr2(−/−) ) mice. Hepatology 2018;67(1):247–59.

[39] Niedernhofer LJ, Robbins PD. Senotherapeutics for healthy ageing. Nat Rev Drug Discov 2018;17(5):377.

[40] Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S, et al. ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res 2008;68(9):3421–8.

[41] Zhang H, Nimmer PM, Tahir SK, Chen J, Fryer RM, Hahn KR, et al. Bcl-2 family proteins are essential for platelet survival. Cell Death Differ 2007;14(5):943–51.

[42] Kirkland JL, Tchkonia T. Clinical strategies and animal models for developing senolytic agents. Exp Gerontol 2015;68:19–25.

[43] Khan N, Syed DN, Ahmad N, Mukhtar H. Fisetin: a dietary antioxidant for health promotion. Antioxid Redox Signal 2013;19(2):151–62.

[44] Adhami VM, Syed DN, Khan N, Mukhtar H. Dietary flavonoid fisetin: a novel dual inhibitor of PI3K/Akt and mTOR for prostate cancer management. Biochem Pharmacol 2012;84(10):1277–81.

[45] Maher P. How fisetin reduces the impact of age and disease on CNS function. Front Biosci (Schol Ed) 2015;7:58–82.

[46] Ahmad A, Robinson AR, Duensing A, van Drunen E, Beverloo HB, Weisberg DB, et al. ERCC1-XPF endonuclease facilitates DNA double-strand break repair. Mol Cell Biol 2008;28(16):5082–92.

[47] Burd CE, Sorrentino JA, Clark KS, Darr DB, Krishnamurthy J, Deal AM, et al. Monitoring tumorigenesis and senescence in vivo with a p16(INK4a)-luciferase model. Cell 2013;152(1–2):340–51.

[48] Niedernhofer LJ, Odijk H, Budzowska M, van Drunen E, Maas A, Theil AF, et al. The structure-specific endonuclease Ercc1-Xpf is required to resolve DNA interstrand cross-link-induced double-strand breaks. Mol Cell Biol 2004;24(13):5776–87.

[49] Fuhrmann-Stroissnigg H, Ling YY, Zhao J, McGowan SJ, Zhu Y, Brooks RW, et al. Identification of HSP90 inhibitors as a novel class of senolytics. Nat Commun 2017;8(1):

[50] Robinson AR, Yousefzadeh MJ, Rozgaja TA, Wang J, Li X, Tilstra JS, et al. Spontaneous DNA damage to the nuclear genome promotes senescence, redox imbalance and aging. Redox Biol 2018;17:259–73.

[51] Yousefzadeh MJ, Schafer MJ, Noren Hooten N, Atkinson EJ, Evans MK, Baker DJ, et al. Circulating levels of monocyte chemoattractant protein-1 as a potential measure of biological age in mice and frailty in humans. Aging Cell 2018;17(2).

[52] Ladiges W. Pathology assessment is necessary to validate translational endpoints in preclinical aging studies. Pathobiol Aging Age Relat Dis 2016;6:31478.

[53] Zhu Y, Tchkonia T, Pirtskhalava T, Gower AC, Ding H, Giorgadze N, et al. The Achilles' heel of senescent cells: From transcriptome to senolytic drugs. Aging Cell; 2015.

[54] Giesen C, Wang HA, Schapiro D, Zivanovic N, Jacobs A, Hattendorf B, et al. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat Methods 2014;11(4):417–22.

[55] Spitzer MH, Nolan GP. Mass Cytometry: Single Cells, many Features. Cell 2016;165 (4):780–91.

[56] Niedernhofer LJ, Garinis GA, Raams A, Lalai AS, Robinson AR, Appeldoorn E, et al. A new progeroid syndrome reveals that genotoxic stress suppresses the somatotroph axis. Nature 2006;444(7122):1038–43.

[57] Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O. Protocols to detect senescence-associated beta-galactosidase (SA-[beta]gal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protocols 2009;4(12):1798–806.

[58] Liu Y, Sanoff HK, Cho H, Burd CE, Torrice C, Ibrahim JG, et al. Expression of p16 (INK4a) in peripheral blood T-cells is a biomarker of human aging. Aging Cell 2009;8(4):439–48.

[59] Hall BM, Balan V, Gleiberman AS, Strom E, Krasnov P, Virtuoso LP, et al. Aging of mice is associated with p16(Ink4a)- and beta-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells. Aging (Albany NY) 2016;8(7):1294–315.

[60] Prattichizzo F, Bonafe M, Olivieri F, Franceschi C. Senescence associated macrophages and "macroph-aging": are they pieces of the same puzzle? Aging (Albany NY) 2016;8(12):3159–60.

[61] Khan WA, Chisholm R, Tadayyon S, Subasinghe A, Norton P, Samarabandu J, et al. Relating centromeric topography in fixed human chromosomes to alpha-satellite DNA and CENP-B distribution. Cytogenet Genome Res 2013;139(4):234–42.

[62] Hall BM, Balan V, Gleiberman AS, Strom E, Krasnov P, Virtuoso LP, et al. p16(Ink4a) and senescence-associated beta-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli. Aging (Albany NY) 2017;9 (8):1867–84.

[63] Ladiges W, Snyder JM, Wilkinson E, Imai DM, Snider T, Ge X, et al. A new preclinical paradigm for testing anti-aging therapeutics. J Gerontol A Biol Sci Med Sci 2017;72 (6):760–2.

[64] Kirkwood TB. Understanding ageing from an evolutionary perspective. J Intern Med 2008;263(2):117–27.

[65] Passos JF, von Zglinicki T, Kirkwood TB. Mitochondria and ageing: winning and losing in the numbers game. BioEssays 2007;29(9):908–17.

[66] Vijg J, Campisi J. Puzzles, promises and a cure for ageing. Nature 2008;454(7208): 1065–71.

[67] Campisi J, Vijg J. Does damage to DNA and other macromolecules play a role in aging? If so, how? J Gerontol A Biol Sci Med Sci 2009;64((2):175–8.

[68] Hoeijmakers JH. DNA damage, aging, and cancer. N Engl J Med 2009;361(15): 1475–85.

[69] Franceschi C, Bonafe M, Valensin S, Olivieri F, De Luca M, Ottaviani E, et al. Inflammaging. An evolutionary perspective on immunosenescence. Ann N Y Acad Sci 2000; 908:244–54.

[70] Krishnamurthy J, Torrice C, Ramsey MR, Kovalev GI, Al-Regaiey K, Su L, et al. Ink4a/ Arf expression is a biomarker of aging. J Clin Invest 2004;114(9):1299–307.

[71] Lavasani M, Robinson AR, Lu A, Song M, Feduska JM, Ahani B, et al. Muscle-derived stem/progenitor cell dysfunction limits healthspan and lifespan in a murine progeria model. Nat Commun 2012;3:608.

[72] Xue W, Zender L, Miething C, Dickins RA, Hernando E, Krizhanovsky V, et al. Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature 2007;445(7128):656–60.

[73] Iannello A, Thompson TW, Ardolino M, Lowe SW, Raulet DH. p53-dependent chemokine production by senescent tumor cells supports NKG2D-dependent tumor elimination by natural killer cells. J Exp Med 2013;210(10):2057–69.

[74] Touil YS, Auzeil N, Boulinguez F, Saighi H, Regazzetti A, Scherman D, et al. Fisetin disposition and metabolism in mice: Identification of geraldol as an active metabolite. Biochem Pharmacol 2011;82(11):1731–9.

[75] Gurau F, Baldoni S, Prattichizzo F, Espinosa E, Amenta F, Procopio AD, et al. Antisenescence compounds: a potential nutraceutical approach to healthy aging. Ageing Res Rev 2018;46:14–31.

[76] Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu S, Wood JG, et al. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 2003; 425(6954):191–6.

[77] Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar M, et al. Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature 2004;430(7000): 686–9.

[78] Kimira M, Arai Y, Shimoi K, Watanabe S. Japanese intake of flavonoids and isoflavonoids from foods. J Epidemiol 1998;8(3):168–75.

[79] Arai Y, Watanabe S, Kimira M, Shimoi K, Mochizuki R, Kinae N. Dietary intakes of flavonols, flavones and isoflavones by Japanese women and the inverse correlation between quercetin intake and plasma LDL cholesterol concentration. J Nutr 2000;130 (9):2243–50.

[80] Syed DN, Adhami VM, Khan MI, Mukhtar H. Inhibition of Akt/mTOR signaling by the dietary flavonoid fisetin. Anticancer Agents Med Chem 2013;13(7):995–1001.

[81] Salerno S, Da Settimo F, Taliani S, Simorini F, La Motta C, Fornaciari G, et al. Recent advances in the development of dual topoisomerase I and II inhibitors as anticancer drugs. Curr Med Chem 2010;17(35):4270–90.

[82] Gupta SC, Tyagi AK, Deshmukh-Taskar P, Hinojosa M, Prasad S, Aggarwal BB. Downregulation of tumor necrosis factor and other proinflammatory biomarkers by polyphenols. Arch Biochem Biophys 2014;559:91–9.

[83] Ishige K, Schubert D, Sagara Y. Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms. Free Radic Biol Med 2001;30(4):433–46.

[84] Prasath GS, Subramanian SP. Antihyperlipidemic effect of fisetin, a bioflavonoid of strawberries, studied in streptozotocin-induced diabetic rats. J Biochem Mol Toxicol 2014;28(10):442–9.

[85] Zheng LT, Ock J, Kwon BM, Suk K. Suppressive effects of flavonoid fisetin on lipopolysaccharide-induced microglial activation and neurotoxicity. Int Immunopharmacol 2008;8(3):484–94.

[86] Prasath GS, Subramanian SP. Fisetin, a tetra hydroxy flavone recuperates antioxidant status and protects hepatocellular ultrastructure from hyperglycemia mediated oxidative stress in streptozotocin induced experimental diabetes in rats. Food Chem Toxicol 2013;59:249–55.

[87] Sagara Y, Vanhnasy J, Maher P. Induction of PC12 cell differentiation by flavonoids is dependent upon extracellular signal-regulated kinase activation. J Neurochem 2004; 90(5):1144–55.

This article  is excerpted from M.J. Yousefzadeh et al. / EBioMedicine 36 (2018) 18–28 by Wound World.