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in vitro;

in vivo

Immune deficient mice; number per group not given

(all were immune-deficient)

• we screened a natural product library for geroprotective compounds using Werner syndrome (WS) human mesenchymal stem cells (hMSCs), a premature aging model that we recently established.
• Ten candidate compounds were identified and quercetin was investigated in detail due to its leading effects.
• Mechanistic studies revealed that quercetin alleviated senescence via the enhancement of cell proliferation and restoration of heterochromatin architecture in WS hMSCs.
• RNA-sequencing analysis revealed the transcriptional commonalities and differences in the geroprotective effects by quercetin and Vitamin C.
• Besides WS hMSCs, quercetin also attenuated cellular senescence in Hutchinson-Gilford progeria syndrome (HGPS) and physiological-aging hMSCs.
• Taken together, our study identifies quercetin as a geroprotective agent against accelerated and natural aging in hMSCs, providing a potential therapeutic intervention for treating age-associated disorders.

db/- vehicle n= 12

db/db vehicle n=15

db/- D+Q n=12

db/db D+Q n=15

(no effect was seen in D+Q vs vehicle/heterozygous)

• Obesity results in the accumulation of senescent glial cells in proximity to the lateral ventricle, a region in which adult neurogenesis occurs.
• Senescent glial cells exhibit excessive fat deposits, a phenotype termed ‘‘accumulation of lipids in senescence.’’
• Clearing senescent cells from high fat-fed or leptin receptor-deficient obese mice restored neurogenesis and alleviated anxiety-related behavior.
• Senescent cells are major contributors to obesity-induced anxiety and senolytics are a potential new therapeutic avenue for treating neuropsychiatric disorders
• BW did not change with D+Q treatment
• D+Q reduced the TAF + SABGal+ cells in the perigonadal fat 
• injection of CXCL1 was not sufficient to cause the anxiety phenotype 
• Markers for immature neurons (Dcx+), ependymal cells (CD133+), and neuronal precursor cells (Nestin+) were all significantly up-regulated after senolytic treatment with D+Q

APP/PS1 transgenic mice used in all groups

D (12 mg/kg) + Q (50 mg/kg)

9 days; once weekly for 11 weeks

D+Q 24 hours

• Aβ plaques were most abundant in patients with AD
• more than 80% of larger plaques were immunoreactive with p21
• p16 mRNA puncta were clustered in Aβ-associated cells
• aggregating Aβ may trigger oligodendrocyte progenitor cell senescence
• 10% of cells exhibited strong SA-BGal activity when exposed to Aβ
• D+ Q once daily for 9 days reduced SA-BGal activity and levels of Olig2 and p21 but had no effect on Aβ load
• no effect on microglia or astrocytes
• improved cognition in AD mice when treated once per week for 11 weeks, reduced Aβ load, IL-1, IL-6, TNFa

mutant tau mice or controls

(no benefit reported in the controls that also received D+Q)

D (5 mg/kg ) +

Q (50 mg/kg)

oral gavage; 6 sessions over 12 weeks

• Consistent with senescent cell removal, intermittent DQ treatment significantly reduced the number of NFT‐containing cortical neurons by 35% 
• gene expression of the NFT‐associated senescence gene array was reduced by DQ 
• The DQ‐dependent reduction in cortical NFTs corresponded with decreased ventricular volume pathology (28% decrease) and a reduction in cortical brain atrophy compared to controls.
• The absence of a full rescue of ventricular enlargement to that of control animals was not completely unexpected considering the severity of disease and age of the animals when treatment was initiated.
• DQ improved aberrant cerebral blood flow in tau NFT‐Mapt0/0 mice such that cerebral blood flow was no longer statistically different from controls
• DQ‐treated mice expressed significantly higher levels of neuronal proteins (NeuN: 25%, synaptophysin: 40.8%; PSD95: 38.5%)
• The astrocyte protein GFAP was unchanged, while microglia Iba1 expression was elevated (Iba1: 40%)

aged C57BL/6J mice

vehicles + D+Q

(benefit reported in aged mice)

D (5 mg/kg) + Q (10 mg/kg)

once per month for 3 months

• treatment with Dasatinib + Quercetin (via oral gavage) resulted in significant reductions in senescent cell markers (TAF+ cells) in the medial layer of the aorta from aged and hypercholesterolemic mice, but not in intimal atherosclerotic plaques 
• senolytic treatment significantly improved vasomotor function (isolated organ chamber baths) in both groups of mice, this was due to increases in nitric oxide bioavailability in aged mice and increases in sensitivity to NO donors in hypercholesterolemic mice
• intimal plaque calcification was significantly reduced in D+Q- vs. vehicle-treated mice. This was associated with reductions in protein levels of osterix.

in vitro

n/a

• we investigated the relationship between quercetin-induced apoptosis and the inhibition of cellular senescence and determined the mechanism of oxidative stress-induced vascular smooth muscle cell (VSMC) senescence.
• In cultured VSMCs, hydrogen peroxide (H2O2) dose-dependently induced senescence, which was associated with increased numbers of senescence-associated β-galactosidase-positive cells, decreased expression of SMP30, and activation of p53-p21 and p16 pathways.
• Along with senescence, expression of the antiapoptotic protein Bcl-2 was observed to increase and the levels of proteins related to the apoptosis pathway were observed to decrease.
• Quercetin induced apoptosis through the activation of AMP-activated protein kinase. This action led to the alleviation of oxidative stress-induced VSMC senescence.
• Furthermore, the inhibition of AMPK activation with compound C and siRNA inhibited apoptosis and aggravated VSMC senescence by reversing p53-p21 and p16 pathways.
• These results suggest that senescent VSMCs are resistant to apoptosis and quercetin-induced apoptosis attenuated the oxidative stress-induced senescence through activation of AMPK.
• Therefore, the induction of apoptosis by polyphenols such as quercetin may be worthy of attention for its anti-aging effects.

24–32-month INK‐ATTAC transgenic or wild‐type mice were randomized to either vehicle, AP20187, or D + Q treatment

(senescent cells decreased by D+Q in wild-type mice as well)

4 cycles for 3 consecutive days/cycle, 12 days apart

• In aged subjects (>70 years old), over half of the cardiac progenitor cells are senescent (p16INK4A, SA‐β‐gal, DNA damage γH2AX, telomere length, senescence‐associated secretory phenotype [SASP]), unable to replicate, differentiate, regenerate or restore cardiac function following transplantation into the infarcted heart.
• SASP factors secreted by senescent CPCs renders otherwise healthy CPCs to senescence.
• The elimination of senescent CPCs using senolytics abrogates the SASP and its debilitative effect in vitro.
• Global elimination of senescent cells in aged mice (INK‐ATTAC or wild‐type mice treated with D + Q senolytics) in vivo activates resident CPCs and increased the number of small Ki67‐, EdU‐positive cardiomyocytes.
• Therapeutic approaches that eliminate senescent cells may alleviate cardiac deterioration with aging and restore the regenerative capacity of the heart

• When aged HDFs were treated with 5 μM quercetin, isoquercetin, or rutin for 48 h, the relative ratios of stained cells between treated and untreated (control) groups were 90.6%, 97.9%, and 95.1%, respectively. Therefore, quercetin was the most effective at restoring senescent HDFs
• Quercetin directly reduced either intracellular or extracellular ROS levels in aged HDFs.
• To find the aging-related target genes by quercetin, microarray analysis was performed and two up-regulated genes LPL and KCNE2 were identified.
• Silencing LPL increased the expression levels of senescence proteins such as p16^INK4A and p53 and silencing KCNE2 reversed gene expressions of EGR1 and p-ERK in quercetin-treated aged HDFs.
• Silencing of LPL and KCNE2 decreased the expression levels of antioxidant enzymes such as superoxide dismutase and catalase.
• Also, the mitochondrial dysfunction in aged HDFs was ameliorated by quercetin treatment.
• Taken together, these results suggest that quercetin has a restoring effect on cellular senescence by down-regulation of senescence activities and up-regulation of the gene expressions of antioxidant enzymes in aged HDFs.

The murine dialysis fistula model exhibits a senescence phenotype: pathobiological mechanisms and therapeutic potential

in vivo

mice

D (5 mg/kg) and Q (50 mg/kg)

by gavage  4 and 5 days after AVF creation

• We demonstrate a senescence phenotype as indicated by increased expression of p16^Ink4a, p21^Cip1, and p53 and expected changes for certain senescence-associated microRNAs.
• RNA-sequencing analysis demonstrated differential expression of ~10,000 genes, including upregulation of proinflammatory and proliferative genes, in the vein of the AVF-CKD group.
• The vein in the AVF-CKD group exhibited telomere erosion and increased senescence-associated β-galactosidase activity and staining.
• Senescence was induced in the artery of the AVF-CKD group and in the vein of the AVF without CKD. 
• dasatinib and quercetin significantly decreased expression of p16^Ink4a at 1 wk in the vein in the AVF-CKD group

mice

ex vivo human tissue

wild-type mice 

(results seen in young damaged mice,  20-month-old non-transplanted, wild-type mice)

D (5 mg/ kg) + Q (50 mg/kg) 

by oral gavage; 2 weeks or once per month, or 3 consecutive days every two weeks

OR 1 uM + 20 uM D+Q for 48 hours

• Transplanting small numbers of senescent cells is sufficient to induce physical dysfunction in young mice (500,000) 1:7000 15,000 cells are senescent
• reduced walking speed as early as 2 weeks after transplant and persisted up to 6 months, transplanted cells survived only 40 days consistent with the possibility that senescent cells might induce senescence in normal host cells
• transplanting senescent cells in 17-month old mice led to greater impairments and lower survival with a 5.2 fold higher risk of death
• human adipose tissue, treated with 1uM+20 uM D+Q for 48 hours had significantly less TAF, p16inka, and SA-Bgal+, and more cells undergoing apoptosis 
• D+Q decreases cytokine secretion IL-6, IL-8, MCP-1, PAI-1, GM-CSF in adipose tissue from obese individuals
• treating young mice with D+Q at the same time of senescent cell transplantation attenuated the deterioration and treating them once physical dysfunction had been established resulted in its alleviation
• D+Q in 20-month-old WT mice intermittently for 4 months alleviated physical dysfunction and lowered the SASP expression
• D+Q at very old age extends remaining lifespan in WT mice by 36% with no increase in end of life morbidity

• reduced the increase in SA-b-gal activity that was induced in HDFs and HUVECs by adriamycin treatment in a dose-dependent manner
• Q reduced the increase of p53 and p21 proteins induced by adriamycin treatment in both HDFs and HUVECs at 10 μg/mL
• Q decreased SA-b-gal activity in HDFs and HUVECs under replicative senescence with statistical significance 
• Q reduced p53 and p21 at 10 ug/ml

• Several natural compounds possess anti-aging/anti-oxidant properties. In this study, we have identified quercetin (Q) and its derivative, namely quercetin caprylate (QU-CAP) as a proteasome activator with anti-oxidant properties that consequently influence cellular lifespan, survival, and viability of HFL-1 primary human fibroblasts.
• cells treated with Q and QU-CAP exhibited a lower percentage of β-galactosidase positive staining, as compared to the DMSO- and CAP-treated cells
• Moreover, when these compounds are supplemented to already senescent fibroblasts, a rejuvenating effect is observed.
• Cells treated with the compounds exhibited a rejuvenated phenotype with more elongated morphology and lower numbers of β-galactosidase positive cells
• Finally, we show that these compounds promote physiological alterations when applied to cells (i.e. whitening effect)

D 5 uM

Q 7.5 uM

D 500 nM 

Q 50 nM  

• Fetal ASM exposed to 40% O2 for 7 days exhibited elevated levels of senescence-associated markers including β-galactosidase (β-gal), cell cycle checkpoint proteins p16, p21 and p-p53, and the DNA damage marker p-γH2A.X.
• The combination of dasatinib and quercetin, compounds known to eliminate senescent cells (senolytics), reduced the number of hyperoxia exposed β-gal, p21, p16, and p-γH2A.X positive ASM cells.
• The SASP profile of hyperoxia-exposed cells included both pro-fibrotic and pro-inflammatory mediators. Naïve ASM exposed to media from hyperoxia-exposed senescent cells exhibited increased collagen and fibronectin and higher contractility.
• Our data show that induction of cellular senescence by hyperoxia leads to the secretion of inflammatory factors and has a functional effect on naïve ASM.
• Cellular senescence in the airway may thus contribute to pediatric airway disease in the context of sequelae due to preterm birth.

• Quercetin treatment did not induce oxidative damage but protected mouse thymocytes from glucose oxidase (GO)-mediated apoptosis in a dose-dependent manner.
• Furthermore, electrophoretic mobility shift assays revealed that quercetin (50 M) treatment suppressed the GO-mediated DNA binding activity of redox state-sensitive transcription factors, such as NF-B, AP-1, and p53.
• This result suggests that quercetin has antioxidative effects on thymocytes.
• More interestingly, quercetin treatment alone (50 M) increased the DNA-binding activity of AP-1, which consisted of heterodimer of c-Jun and Fra-2.
• Finally, the antioxidant activity of quercetin was confirmed using a cell-free system of radical generation.
• Our findings suggest that quercetin protects mouse thymocytes from oxidative stress-mediated apoptosis and modulates the intracellular redox state through its antioxidant activity.

in vitro

in vivo

control, bleomycin, bleomycin + D+Q

(no non-injured group received D+Q)

• Although quercetin alone was not pro-apoptotic, it abolished the resistance to FasL- or TRAIL-induced apoptosis in IPF fibroblasts.
• Mechanistically, quercetin up-regulated Fas and caveolin-1 expression and modulated AKT activation.
• In vivo, quercetin reversed bleomycin-induced pulmonary fibrosis and attenuated lethality, weight loss, and the expression of pulmonary senescence markers p21 and p19-ARF and senescence-associated secretory phenotype (SASP) in aged mice.
• Collectively these data indicate that quercetin reverses the resistance to death ligand-induced apoptosis, by promoting FasL receptor and caveolin-1 expression, and inhibiting AKT activation, thus mitigating the progression of established pulmonary fibrosis in aged mice.

• Human embryonic kidney cell (HEK-293) was cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 100 mM (18 mg/L) for 24 h.
• The cells were treated with resveratrol (2.5, 5, 10 µm), quercetin (3, 6, 12 µm), and combination of these (R 2.5 µm, Q 3 µm) and (R 5 µm, Q 6 µm) and (R 10 µm, Q 12 µm) for 48 h, and then, cells were lysed to access RNA and lysate. 
• The analysis of data showed that beta-galactosidase enzyme gene expression as an aging marker in all treatment groups was reduced in a dose-dependent manner.
• Gene expression of Sirtuin1 and thioredoxin (Trx) in all treated groups in comparison to control group increased in a dose-dependent fashion.
• Trx interacting protein (TXNIP) gene expression decreased in a dose-dependent manner in all treated groups, especially in resveratrol and combination therapy. 

INK-ATTAC mice untreated, aged + D+Q

(D+Q decreased TAF+ hepatocytes)

D (5 mg/kg) + Q (10 mg/kg)

once per month for 3 months

• D+Q treatment reduced the frequency of TAF+ hepatocytes and the average number of TAF per hepatocyte
• D+Q did not significantly change total DNA damage 
• reduction in hepatic fat deposition
• treatment with D+Q in mice with a model of Type 2 diabetes suppressed the fraction of TAF containing senescent cells and reduced liver fat accumulation
• results support the hypothesis that senescent cells are causally implicated in steatosis  
• senescence-associated mitochondrial dysfunction reduces cellular fatty acid oxidation capacity resulting in increased fat deposition
• hepatocyte senescence correlates with severity of NAFLD in analysis of liver biopsies 
• TAF and p21 were correlated with the degree of hepatic steatosis

• the maximal cytostatic doses for D and/or Q (1 + 1µM) lacked efficacy in removing doxorubicin-induced β-gal-positive senescent cells.
• D+Q did not affect doxorubicin-dependent induction of flattened morphology, activation of p16, expression of SASP-associated genes or formation of γH2AX foci.
• doxorubicin reduced tumor growth by 30% compared to control mice, while D+Q was ineffective in synergizing with doxorubicin and in clearing doxorubicin-induced HCC senescent cells
• D+Q alone appeared to have acute pro-tumorigenic effects in control mice

PBS+Veh

Bleo+Veh

Bleo+AP

Bleo+DQ

(only bleomycin treated rats received D+Q)

D (5 mg/ kg) + Q (50 mg/kg) 

by oral gavage (three treatments)

• senescence biomarkers accumulate in IPF lung
• senescent fibroblasts are eliminated by D+Q treatment
• 20 uM D + 15 uM Q eliminated >33% of senescent cells within 3 days
• D+Q reduced senescence in IMR90 fibroblasts 
• DQ treatment significantly reduced p16 transcriptional levels in the lungs and, concordantly, blunted increases in Mcp1, Il6, Mmp12 and Tgfb
• Total BAL cell counts were significantly increased in vehicle-treated bleomycin-exposed mice, and treatment with AP or DQ attenuated this increase. Differential analysis revealed a similar trend for macrophages, lymphocytes and neutrophils 
• Cytokine protein levels within the BAL fluid were highly variable; however, apparent increases in MCP1 and IL6 were diminished following AP or DQ treatment.
• Bleomycin reduced lung compliance in vehicle-treated mice by 40%. Both AP- and DQ treatment minimized this impairment, limiting bleomycin-induced reductions in lung compliance to 15%
• vehicle-treated mice lost an average of 3.6 g, relative to baseline. In comparison, both AP- and DQ-treated mice lost approximately 1.6 g of body weight
• senescent cell clearance mitigates fibrotic lung disease
• mice treated with D+Q ran, on average, >37% further to exhaustion on a graded treadmill test

• For quercetin, we did not observe any senolytic effect 
• Furthermore, none of the investigated drugs had a positive effect on culture expansion, telomere length, or epigenetic rejuvenation

D + Q to wild‐type DIO + lean mice

These effects were not seen in lean, chow‐fed control mice

cohort 1: D (5 mg/kg) + Q (50 mg/kg)

5 consecutive days monthly

cohort 2: D (5 mg/kg) + Q (50 mg/kg)

3 days with 14 days between series

• reducing senescent cell burden in obese mice, either by activating drug‐inducible “suicide” genes driven by the p16Ink4a promoter or by treatment with senolytic agents, alleviates metabolic and adipose tissue dysfunction
• senolytic interventions improved glucose tolerance, enhanced insulin sensitivity, lowered circulating inflammatory mediators, and promoted adipogenesis in obese mice.
• Elimination of senescent cells also prevented the migration of transplanted monocytes into intra‐abdominal adipose tissue and reduced the number of macrophages in this tissue.
• In addition, microalbuminuria, renal podocyte function, and cardiac diastolic function improved with senolytic therapy.
• the results implicate cellular senescence as a causal factor in obesity‐related inflammation and metabolic derangements and show that emerging senolytic agents hold promise for treating obesity‐related metabolic dysfunction and its complications

Twenty-month-old male C57BL/6 mice

vehicle or D+Q

(D+Q treated mice showed benefits)

D (5 mg/kg) + Q (50 mg/kg)

once monthly for 4 months

• In old (20–22-months) mice with established bone loss, activation of the INK-ATTAC caspase 8 in senescent cells or treatment with senolytics or the JAKi for 2–4 months resulted in higher bone mass and strength and better bone microarchitecture compared to vehicle-treated mice.
• The beneficial effects of targeting senescent cells were due to lower bone resorption with either maintained (trabecular bone) or higher (cortical bone) bone formation as compared to vehicle-treated mice.
• In vitro studies demonstrated that senescent cell-conditioned medium impaired osteoblast mineralization and enhanced osteoclast progenitor survival, leading to increased osteoclastogenesis
• p16 mRNA decreased from 0.027 to 0.013, number of senescent osteocytes 12% to 8%. p16 in adipose 0.06 to 0.04. SABGal 17% to 12%.

Vehicle radiated

Vehicle non-radiated

D+Q radiated

D+Q non-radiated

(no difference between vehicle and D+Q nonradiated groups)

D (5 mg/kg)

Q (50mg/kg)

F (50mg/kg) 

or  D (5 mg/kg) + Q (50 mg/kg)

administered on day 0 and on day 14 post-FRT by oral gavage

• tested the efficacy of three senolytic regimens, including Dasatinib (D), Quercetin (Q) and Fisetin (F), where the treatments were given on days 0 and 14 post-FRT and the bones were analyzed at 42 days post-FRT
• none of the senolytic compounds singly were able to completely rescue the phenotype and failed to reach significance in any bone parameter measured, including BV/TV [D (35.8%, n.s.), Q (34.5%, n.s.), and F (9.52%, n.s.)], connectivity density (Conn.Dens.)[D (45.05%, p=0.051), Q (47.61%, n.s., and F(43.60%, n.s.)], structural model index (SMI) [D (-14.38%, n.s.), Q (- 14.19%, n.s.), and F (-9.57%, n.s.)] and Tb.Th [D (8.02%, n.s.), F (-6.76%, n.s.), and Q (5.22%,n.s.), compared to vehicle-treated R-femurs.
• Intriguingly, the senolytic cocktail of D+Q resulted in significant improvements in the radiated bones, with an increase in BV/TV (74.2%, p=0.0046), increase in the Conn. Dens. (91.29%, p=0.018), a non-significant decrease in SMI (-24.19%, n.s.), and a non-significant increase in Tb.Th (12.88%, p=0.096) compared to the vehicle Rfemurs 
• D+Q treatment resulted in a significant 66% increase in N.Ob/B.Pm, a 4-fold decrease in N.Ad/T.Ar, and a nonsignificant 25.6% increase in N.Ot/B.Ar compared to vehicle-treated R-bones
• In D+Q-treated R-femurs, a 1.95-fold increase in MS/BS and a 4.5-fold increase in BFR/BS were observed when compared to vehicle-treated R-bones
• Interestingly, the bone formation marker N-terminal propeptide of type I procollagen (P1NP) in the serum, was significantly elevated in D+Q-treated mice as This article is protected by copyright. All rights reserved. compared to the vehicle-treated mice, but no change in the resorption marker, serum C-terminal telopeptide of type I collagen (CTX), was observed post-D+Q treatment
• Examining the senescence profile in bone tissue from untreated and D+Q-treated animals at 42 days post-FRT, we observed that D+Q significantly suppressed p21 and p16Ink4a gene expression
• Strikingly, intermittent (and infrequent) dosing of D+Q reduced TIF+ senescent osteocytes, TIF+ senescent bone lining cells including osteoblasts and TIF+ senescent bone marrow cells to levels found in NR femurs

Dasatinib: Control + IOP

Control: Control + IOP

(no difference between control control and dasatinib control)

• dasatinib treatment prevented the loss of RGC similar to what was observed in GCV-treated animals.
• Most importantly, VEP analysis revealed that senolytic drug treatment successfully prevented vision loss upon IOP elevation

ex vivo

human

• After in vitro exposure to TNF-alpha, MSC demonstrated an upregulation of SASP components, including interleukins-6 and -8 and MCP-1.
• Staining of the subcutaneous adipose tissue sections revealed a greater inflammatory response in preeclampsia, based on the higher levels of both TNF-alpha and MCP-1 compared to normotensive pregnancies
• MSC isolated from PE demonstrated a lower percentage of live MSC cells, lower proliferation, and higher migration.
• At baseline, PE-MSC demonstrated a senescent phenotype, reflected by more abundant staining for SABGal (p < 0.001), upregulation of senescence markers and SASP components, as well as lower angiogenic potential, compared to NP-MSC.
• Treatment with dasatinib increased significantly the number of apoptotic PE-MSC compared to NP-MSC and decreased the gene expression of p16 and six SASP components.
• The mechanistic link between senescence and impaired angiogenesis in PE was confirmed by the improved angiogenic potential of PE-MSC after dasatinib treatment.

• We found that quercetin caused cell death in non-senescent endothelial cells at a concentration that has been reported to selectively remove senescent cells and that Q3G was not cytotoxic to either young or senescent cells.
• Thus, in primary adult human endothelial cells, quercetin and Q3G are not senolytics.
• Earlier work reporting positive results was done with HUVECs, and given their origin and the disparate findings from the current study, these may not be the best cells for evaluating potential senolytics in clinically relevant endothelial cells

chow-fed vehicle

chow-fed Q

high fat vehicle

high fat Q

(no difference between chow-fed vehicle and chow-fed Q)

50 mg/kg

5-days biweekly via oral gavage for another 10-weeks

• Renal function was studied in vivo using magnetic resonance imaging, and renal senescence and histology were evaluated ex vivo.
• Mice fed a HFD developed obesity and hypercholesterolemia, whereas renal size remained unchanged.
• Murine obesity impaired renal function and cortical oxygenation and induced glomerulomegaly.
• Renal markers of senescence (eg, expression of p16, p19, and p53) and its secretory phenotype were upregulated in the obese hypercholesterolemic compared to lean mice in renal tubular cells, but attenuated in quercetin-treated murine kidneys, as was renal fibrosis.
• Quercetin treatment also increased renal cortical oxygenation and decreased plasma creatinine levels in obese mice, whereas body weight and cholesterol levels were unaltered.
• Therefore, murine obesity and dyslipidemia induce renal tissue senescence and impairs kidney function, which is alleviated by chronic senolytic treatment.

Young controls: 3-month-old mice given placebo treatment (YC; n=10)

Young treatment: 3-month-old mice given dasatinib plus quercetin (D+Q) treatment (YT; n=10)

Old controls: 18-month-old mice given a placebo (OC; n=10); and 4) Old treatment: 18-month-old mice given D+Q (OT; n=10)

(no difference in treatment compared to control)

• Uterine levels of microRNAs (miR34a, miR34b, miR34c, miR146a, miR449a, miR21a, miR126a, and  miR181b) were evaluated.
• Aging promoted down-regulation of genes of the Pi3k/Akt1/mTor 35 signaling pathway 
• as well as a reduction in the expression of miR34c, miR126a, and miR181b.
• D+Q treatment increased p53 gene expression and decreased levels of miR34a.
• Our results demonstrate a role for the Pi3k/Akt1/mTor signaling pathway in uterine aging and suggest for the  first time a possible anti-fibrotic effect of D+Q senolytic therapy in the uterus

TIG-1

HUVE

• 3-Hydroxyflavone, luteolin, and apigenin were more toxic toward TIG-1 cells than the other flavonoids, and luteolin, 3-hydroxyflavone, and quercetin were more toxic toward HUVE cells. HUVE cells were more vulnerable to flavonoid cytotoxicity than TIG-1 cells.
• The ROS level increased significantly in the presence of the flavone apigenin or luteolin or the flavonol 3-hydroxyflavone, quercetin, or kaempferol.
• These results suggest that these flavones and flavonols exert cytotoxicity through increasing intracellular ROS levels.
• Further, the incorporation of apigenin, 3-hydroxyflavone, luteolin, and quercetin, which are more toxic, into TIG-1 cells during 24-h incubation was examined.
• These flavonoids were incorporated into them and the order of their incorporation efficiency was similar to that of their cytotoxicity.
• For TIG-1 cells, quercetin was significantly toxic at an LC50 value 303 uM
• For HUVE cells, quercetin was highly toxic at an LC50 value of 64 uM
• In conclusion, some flavonoids are cytotoxic at higher concentrations toward human normal cells. Further, it is suggested that they are incorporated into cells, increase intracellular ROS levels, and then exert cytotoxicity.

The Achilles' heel of senescent cells: from transcriptome to senolytic drugs.

in vitro

in vivo mice

aged, or irradiated

(improvements were seen in treated group)

D  (5 mg/kg) +

Q (50 mg/kg)

single dose   

oral gavage

• D preferentially reduced viability and caused cell death of senescent human preadipocytes, but was much less effective on senescent HUVECs
• by day 3, proliferating preadipocytes increased by 2-5-fold in number vs. day 0 in the presence of D. The viability of nondividing, senescent preadipocytes from the same subjects decreased by 30–40% in the presence of 50 nM or greater D, indicating a selective reduction in the viability of senescent cells
• Q reduced the viability and caused cell death of senescent HUVECs to a greater extent than proliferating cells, but was less effective on preadipocytes
• at 10 lM Q, nonsenescent HUVECs achieved a 2-3-fold increase in cell number between days 0 and 3, while parallel cultures of senescent cells were reduced by 50%, indicating selective killing of senescent cells
• The combination of D+Q afforded the selective killing of both senescent preadipocytes and endothelial cells. By day 3, the viability of nondividing senescent preadipocytes exposed to D+Q was reduced by ~70% compared to day 0, while nonsenescent, proliferating cells had increased by 2 - 4-fold. By day 3, the viability of senescent HUVECs treated with 10 lM Q and 100 nM D was reduced by ~50% compared to day 0. Parallel cultures of nonsenescent, proliferating HUVECs increased in number by 1.5-fold over the same period of time. This suggests that the combination of D+Q selectively targets a broader range of senescent cell types than either agent alone
• the combination of D+Q led to a significant reduction in the number of senescent, C12FDG-positive, primary mouse embryonic fibroblasts (MEFs) compared to either drug alone
• Q alone or D+Q caused a significant reduction in the number of senescent bone marrow-derived murine mesenchymal stem cells
• reduced SA-bgal+ cells (Fig. 3C) and p16 mRNA in fat from old mice within 5 days. D+Q also reduced p16- positive cells in the liver from old mice
• not all senescent cells were removed by D+Q., yet functional improvement was seen
• Following irradiation of one leg of wild-type mice, a single treatment with D+Q reduced p16 expression in muscle and SA-bGal+ cells in fat at the site of localized ionizing radiation exposure in these mice
• a single dose of D+Q significantly improved left ventricular ejection fraction and fractional shortening, effects that were mediated by reductions in end-systolic cardiac dimensions but not cardiac preload
• D+Q yielded physiologically important and consistent improvements in vascular smooth muscle sensitivity to nitroprusside and acetylcholine
• following a single dose of D+Q, exercise time, distance, and total work performed to exhaustion on the treadmill were greater in the mice treated with D+Q compared to vehicle.
• Senescent markers were reduced in muscle and inguinal fat 5 days after treatment. At 7 months after the single treatment, exercise capacity was significantly better in the mice that had been irradiated and received a single dose of D+Q than in vehicle-treated controls. D+Q-treated animals had endurance essentially identical to that of sham-irradiated controls
• significant reduction in a composite score of age-related symptoms, including kyphosis, dystonia, tremors, loss of grip strength, coat condition, ataxia, urinary incontinence, impaired gait, hind limb paralysis, and poor body condition.
• This reduction in symptoms indicates an extension of healthspan due to both the delay in onset of symptoms and attenuation of their severity. In particular, the mice showed reduced dystonia and delayed onset of ataxia and gait disorders

• Dasatinib increased PGC-1α mRNA expression up to 6-fold in 3T3-F442A adipocytes, primary adipocytes, and epididymal white adipose tissue from lean and diet-induced obese mice.
• Importantly, gene expression translated into increased PGC-1α protein content analyzed in melanoma cells and isolated mitochondria from adipocytes.
• However, dasatinib treatment had an adverse effect on glucose tolerance in diet-induced obese and Ob/Ob mice. This correlated with increased hepatic PGC-1α expression and the gluconeogenesis genes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase.
• Despite the beneficial effects of increased PGC-1α content in adipose tissue, dasatinib significantly impaired glucose tolerance in obese but not lean mice. 

• Recently, most TKIs are found to be substrates of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP).
• However, little information is available regarding the Pgp- or BCRP-mediated interaction of TKIs with coadministered drugs/food/beverages.
• Our objective was to evaluate the effect of the major ingredients of grapefruit juice (GFJ), orange juice (OJ), apple juice (AJ), and green tea on P-gp and BCRP-mediated dasatinib efflux.
• Among the 14 ingredients screened, only tangeretin and nobiletin moderately inhibited P-gp-mediated dasatinib efflux.
• In contrast, four ingredients in GFJ [i.e., bergamottin, 6 ,7 -dihydroxybergamottin (DHB), quercetin, and kaempferol], two ingredients in OJ (tangeretin and nobiletin), and one ingredient in AJ (i.e., hesperetin) greatly inhibited BCRP-mediated dasatinib efflux at the concentration of 50 M (p < 0.001).
• Further concentration-dependent studies revealed that bergamottin, DHB, tangeretin, and nobiletin are potent BCRP inhibitors, with IC50 values 3.19, 5.2, 1.19, and 1.04 M, respectively.
• Further in vivo investigations are warranted to evaluate the BCRP-mediated FJ–TKI interaction. 

• The tyrosine kinase inhibitors (TKIs), nilotinib, ponatinib, and dasatinib (but not bosutinib or imatinib), are associated with vascular adverse events (VAEs) in chronic myeloid leukemia (CML).
• Though the mechanism is inadequately understood, an effect on vascular cells has been suggested.
• We investigated the effect of imatinib, nilotinib, dasatinib, bosutinib, and ponatinib on tube formation, cell viability, and gene expression of human vascular endothelial cells (HUVECs).
• We found a distinct genetic profile in HUVECs treated with dasatinib, ponatinib, and nilotinib compared to bosutinib and imatinib, who resembled untreated samples.
• However, unique gene expression and molecular pathway alterations were detected between dasatinib, ponatinib, and nilotinib.
• Angiogenesis/blood vessel-related pathways and HUVEC function (tube formation/viability) were adversely affected by dasatinib, ponatinib, and nilotinib but not by imatinib or bosutinib.
• These results correspond to the differences in VAE profiles of these TKIs, support a direct effect on vascular cells, and provide direction for future research.

D (10 mg/kg)

daily for 8 weeks

• Pleural ultrasonography revealed that rats chronically treated with dasatinib developed pleural effusion after 5 weeks.
• Consistent with these in vivo observations, dasatinib led to a rapid and reversible increase in paracellular permeability of human pulmonary endothelial cell monolayers as reflected by increased macromolecule passage, loss of vascular endothelial cadherin and zonula occludens-1 from cell-cell junctions, and the development of actin stress fibers.
• These results were replicated using human umbilical vein endothelial cells and confirmed by decreased endothelial resistance.
• Interestingly, we demonstrated that this increased endothelial permeability is a reactive oxygen species (ROS)-dependent mechanism in vitro and in vivo using a cotreatment with an antioxidant agent, N-acetylcysteine.
• This study shows that dasatinib alters pulmonary endothelial permeability in a ROS-dependent manner in vitro and in vivo leading to pleural effusion.

in vitro

in vivo

D (50 mg/kg)

for 30 days

• Liver dysfunction is a common side effect associated with the treatment of dasatinib and its mechanism is poorly understood.
• Autophagy has been thought to be a potent survival or death factor for liver dysfunction, which may shed the light on a novel strategy for the intervention of hepatotoxicity caused by dasatinib.
• In this study, we show for the first time that autophagy is induced, which is consistent with the formation of liver damage.
• Autophagy inhibition exacerbated dasatinib-induced liver failure, suggesting that autophagy acted as a self-defense mechanism to promote survival.
• Oxidative stress has been shown to be an important stimulus for autophagy and hepatotoxicity.
• Interestingly, dasatinib increased the activity of p38, which is a critical modulator of the oxidative stress related to liver injury and autophagy.
• p38 silencing significantly blocked LC3-II induction and p62 reduction by dasatinib, which was accompanied by increased caspase-3 and PARP cleavage, indicating that autophagy alleviated dasatinib-induced hepatotoxicity via p38 signaling.
• Finally, the p38 agonist isoproterenol hydrochloride (ISO) alleviated dasatinib-induced liver failure by enhancing autophagy without affecting the anticancer activity of dasatinib.
• Thus, this study revealed that p38-activated autophagy promoted survival during liver injury, which may provide novel approaches for managing the clinical applications of dasatinib.

D (1.25-5 mg/kg)

over 4-48 hours

• Here, we show that dasatinib weakly affects platelet activation by thrombin or adenosine diphosphate but is a potent inhibitor of platelet signaling and functions initiated by collagen or FcRIIA cross-linking, which require immunoreceptor tyrosine-based activation motif phosphorylation by SFKs.
• Accordingly, dasatinib treatment rapidly decreases the volume of thrombi formed under arterial flow conditions in whole blood from patients or mice perfused over a matrix of collagen.
• Moreover, treatment of mice with dasatinib increases the tail bleeding time in a dose-dependent manner. Interestingly, these effects are rapidly reversible after the interruption of the treatment.
• dasatinib affects platelet functions in vitro and in vivo, which has important implications in the clinic and could explain increased risks of bleeding observed in patients.

in vivo

in vitro

cardiomyocytes

dogs

• Dasatinib at 0.03 and 0.3 mg/kg was intravenously administered to the halothane-anesthetized dogs for 10 min with an interval of 20 min between the dosing (n=4).
• Meanwhile, 0.1, 0.3, and 1 μM were cumulatively applied to the human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) (n=7).
• In the dogs, the low and high doses provided peak plasma concentrations of 40±5 (0.08) and 615±38 ng/mL (1.26 μM), respectively.
• The low dose decreased the heart rate, impaired the left ventricular mechanical function, and prolonged the ventricular effective refractory period.
• The high dose prolonged the repolarization period, induced hemorrhagic tendency, and increased plasma cardiac troponin I level in addition to enhancement of the changes observed after the low dose, whereas it neither affected the cardiac conduction nor induced ventricular arrhythmias.
• In the hiPSC-CMs, dasatinib prolonged the repolarization and refractory periods like in dogs, while it did not induce apoptotic or necrotic process, but that it increased the conduction speed. 

• Treatment with second-generation dasatinib is often complicated by hemorrhagic events. Previous lumi-aggregometry studies have shown impaired platelet function in patients on dasatinib therapy.
• Dual agonist activated platelets (coated-platelets) are also sensitive indicators of platelet function. We hypothesized that dual activation with convulxin and thrombin of platelets in a flow cytometric assay could be a more sensitive method for detecting platelet dysfunction as compared to single agonist studies used in lumi-aggregometer.
• Platelets of healthy volunteers incubated with dasatinib as well as platelets from patients on dasatinib therapy were investigated. Low therapeutic plasma level dasatinib concentrations at which a considerable reduction in coated-platelet generation was observed in vitro, did not cause a detectable change in platelet aggregation response.
• Coated-platelet assay and lumi-aggregometry were also investigated at 0, 1 and 4 hours after drug administration in dasatinib treated CML patients. A significant decrease was observed at 1 hour in maximal aggregation by collagen. Although the aggregation curves became normalized by 4 hours, coated-platelet generation was still inhibited in dasatinib treated patients. 

Non-irradiated

Irradiated

Irradiated + D+Q

(no D+Q on healthy animals)

oral mucositis mouse model

skin ulcer rat model

human cells 

D (5 mg/kg) +

Q (50 mg/kg)

5 consecutive days by oral gavage (mice); i.p. injection (rats)

• They showed senescent cells accumulate after radiation exposure, which can induce cell and tissue dysfunction.
• Increased expression of p16 (a senescence biomarker) occurs in human radiation ulcers after radiotherapy
• Radiation-induced persistent cell senescence in animal ulcer models has been demonstrated.
• Senescent cells secreted a senescence-associated secretory phenotype (SASP) and induced cell senescence in adjacent cells, which was alleviated by JAK inhibition.
• DQ treatment eliminates senescent cells through the intrinsic apoptotic pathway (increased caspase 3, PARP)
• In vitro, a single dose of DQ eliminated 40-60% of senescent HOK and 10-20% senescent skin fibroblasts within 24 hours.
• DQ treatment largely prevented the development of ulcers in mice with downregulated DNA damage markers and maintained proliferative capacity.
• The clearance of senescent cells following treatment with a senolytics cocktail, Dasatinib plus Quercetin (DQ), mitigated radiation ulcers in mice and rats.
• DQ induced tumor cell apoptosis and enhanced radiosensitivity in vitro