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Cited by 364Telomeres, Telomerase, and SenescenceGenetics, Aging, and Longevity in Model OrganismsCell death mechanisms and regulation

Cardiac glycosides are broad-spectrum senolytics

Open access
Guerrero, Ana, Herranz, Nicolás, Sun, Bin, Wagner, Verena, Gallage, Suchira, Guiho, Romain, Wolter, Katharina, Pombo, Joaquim +18 more·2019·Nature Metabolism
DOI 10.1038/s42255-019-0122-zPMC PMC6887543
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Abstract

Senescence is a cellular stress response that results in the stable arrest of old, damaged or preneoplastic cells. Oncogene-induced senescence is tumor suppressive but can also exacerbate tumorigenesis through the secretion of pro-inflammatory factors from senescent cells. Drugs that selectively kill senescent cells, termed senolytics, have proved beneficial in animal models of many age-associated diseases. Here, we show that the cardiac glycoside, ouabain, is a senolytic agent with broad activity. Senescent cells are sensitized to ouabain-induced apoptosis, a process mediated in part by induction of the pro-apoptotic Bcl2-family protein NOXA. We show that cardiac glycosides synergize with anti-cancer drugs to kill tumor cells and eliminate senescent cells that accumulate after irradiation or in old mice. Ouabain also eliminates senescent preneoplastic cells. Our findings suggest that cardiac glycosides may be effective anti-cancer drugs by acting through multiple mechanism. Given the broad range of senescent cells targeted by cardiac glycosides their use against age-related diseases warrants further exploration.

AI snapshot

Written from the paper’s full text.

TL;DR

Cardiac glycosides, especially ouabain, act as broad-spectrum senolytics that preferentially kill senescent cells across oncogene-, therapy-, replicative-, and irradiation-induced models. The effect involves apoptosis partly through NOXA and Na⁺/K⁺-ATPase inhibition, and ouabain also reduces senescent cells and inflammation in old mice and preneoplastic lesions.

Key findings

  • Screening the 1,280-compound LOPAC library in IMR90 ER:RAS oncogene-induced senescence and etoposide-induced senescence identified ouabain and CGP-74514A as compounds that selectively killed both senescent models; ouabain also killed cells senesced by doxorubicin, palbociclib, or replicative exhaustion and eliminated p16INK4a-positive primary bronchial epithelial cells.
  • Ouabain’s EC50 was about 50-fold lower in senescent than normal cells; digoxin and digitoxin were also senolytic, with digitoxin active near plasma concentrations reported in cardiac patients (20–33 nM). Bufalin and aglycone forms also showed activity, arguing the glycoside chain is not required.
  • Senescent-cell killing occurred via apoptosis: cleaved caspase 3 and caspase 3/7 activity increased, and pan-caspase inhibition blocked death, whereas ferroptosis, pyroptosis, or necroptosis inhibitors did not. Mechanistically, ouabain effects were linked to Na⁺/K⁺-ATPase inhibition, K⁺ loss, and partial dependence on NOXA, with JNK, GSK3-β, and p38 signaling involved.
  • In vivo, ouabain reduced Nras-positive senescent hepatocytes in immunosuppressed mice, eliminated β-catenin-positive preneoplastic pituitary cells ex vivo, reduced lung senescent cells and Il1a/Il6 after 6 Gy irradiation, and in 24-month-old female mice lowered p16INK4a in liver, heart, and kidney, reduced liver SA-β-Gal, and reversed some age-associated blood, motor, and immune-infiltration changes; plasma ouabain was 1.24 ± 0.48 ng/ml 24 h after a treatment round.

Methods

  • Unbiased drug screens used IMR90 ER:RAS cells for oncogene-induced senescence and etoposide-treated IMR90 cells for therapy-induced senescence, with the LOPAC 1,280 library and viability readouts by automated microscopy; additional senescence models included doxorubicin, palbociclib, replicative exhaustion, irradiation, and multiple cancer cell lines.
  • Mechanistic work used RNA-seq, GSEA, xCell, qRT-PCR, immunoblot, siRNA/shRNA knockdown, intracellular ion probes, caspase activity assays, and inhibitors of apoptosis and signaling pathways.
  • Mouse studies included C57BL/6J mice given 6 Gy total-body irradiation, C.B17 scid/beige mice with transposon-mediated NRASG12V expression, ex vivo cultured embryonic pituitaries from a β-catenin model, and 98–103-week-old female C57BL/6J mice treated intermittently with 1 mg/kg ouabain intraperitoneally for 13 weeks, with physical tests, blood chemistry, ELISA, IHC, and RNA-seq.

Limitations

  • The paper states that it remains unclear whether Na⁺/K⁺-ATPase inhibition fully explains the senolytic effects of cardiac glycosides, how much of the benefit in old mice is specifically due to senolysis versus other effects, and whether these compounds have senolytic activity in humans.
  • The authors note that cardiac glycosides are pleiotropic, so alternative or complementary explanations—including direct killing of specific immune infiltrates—cannot be excluded; mouse resistance to ouabain due to Atp1a1 differences also complicates translation.
  • Evident from the text: several age-related endpoints were non-significant trends (for example grip strength, p21Cip1, and LINE1 ORF1), some bystander doxorubicin data are described as not shown, old mice were not littermates, and the study is preclinical with small mouse cohorts.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s42255-019-0122-z

Cites

67 of 67 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 40 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Etoposide treatment caused senescence in IMR90 cells.

    “Treatment of IMR90 cells with etoposide caused senescence (Extended data Fig 2a)”
    Quote in the paperSubstantial claim
  • Findingown data

    Cardiac glycosides eliminate senescent cells that accumulate after irradiation or in old mice.

    “eliminate senescent cells that accumulate after irradiation or in old mice.”
    • Also stated in: § Dual benefit of ouabain on therapy-induced senescence · plain
      “Treatment with ouabain resulted in a reduced presence of senescent cells”
    • Also stated in: § Ouabain treatment resets immune infiltration in old mice · plain
      “reduced p16INK4a levels when comparing ouabain-treated old mice with their age-matched counterparts”
    Quote in the paperSubstantial claim
  • Findingown data

    Ouabain selectively induced apoptosis of β-catenin+ senescent cells.

    “co-staining with antibodies recognizing cleaved caspase 3 showed that ouabain selectively induced apoptosis of β-catenin + senescent cells (Fig 4h).”
    Quote in the paperSubstantial claim
  • Findingown data

    Inhibitors of JNK, GSK3-ß, or p38 signalling confirm involvement of these pathways in induction of NOXA and apoptosis in senescent cells treated with cardiac glycosides.

    “Treatment with inhibitors of JNK, GSK3-ß or p38 signalling, confirmed the involvement of these pathways on the induction of NOXA (Fig 3j) and apoptosis in senescent cells treated with CGs (Fig 3k).”
    Quote in the paperSubstantial claim
  • Findingown data

    Ouabain preferentially kills senescent cells induced by oncogenic RAS, etoposide, doxorubicin, palbociclib, or replicative senescence.

    “Ouabain treatment not only preferentially killed cells undergoing senescence triggered by oncogenic RAS (Fig 1d), or etoposide (Fig 1i), but also those exposed to doxorubicin (Fig 2a), the CDK4/6 inhibitor palbociclib (Fig 2b) or undergoing replicative senescence (Fig 2c).”
    Quote in the paperSubstantial claim
  • May combine two claims. 35
  • Classified as probably not a substantial claim. 24

  • May combine two claims.

    Findingown data

    Senescent MEFs are not killed by high doses of ouabain, but ouabain behaves as a senolytic in BNL CL.2 murine liver cells.

    “While senescent MEFs were not killed by high doses of ouabain, ouabain behaved as a senolytic in BNL CL.2 murine liver cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Bufalin and ouabagenin display senolytic properties.

    “Bufalin also displayed senolytic properties (Fig 2g) as did ouabagenin, the aglycone of ouabain”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    In a mouse model of ACP, preneoplastic clusters of β-catenin positive cells were senescent, arrested, and enriched for senescence markers.

    “confirmed, as previously shown 38, that preneoplastic clusters of β-catenin positive cells were senescent: they were arrested (ki67 -) and enriched for markers of senescent such as p21CIP1 or Glb1”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [38] Gonzalez-Meljem, J. M. et al. Stem cell senescence drives age-attenuated induction of pituitary tumours in mouse models of paediatric craniopharyngioma. Nat. Commun. 8, 1819 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Treatment with cardiac glycosides results in cleaved caspase 3 and induces caspase 3/7 activity in senescent cells.

    “Treatment with CGs resulted in cleaved caspase 3 and induced caspase 3/7 activity in senescent cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Treatment with ABT-737 or ouabain specifically eliminated β-catenin-positive senescent cells without affecting other pituitary cell types.

    “Treatment with ABT-737 or ouabain specifically eliminated the β-catenin-positive senescent cells (Fig 4g and Extended data Fig 6a-b) without affecting other cell types in the pituitary”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ouabain activates JNK, GSK3-ß, and p38 in senescent cells.

    “We also observed an ouabain-dependent activation of JNK, GSK3-ß and p38 in senescent cells (Fig 3h).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    KCl supplementation prevents ouabain-induced death of senescent cells but not ABT-263-induced death.

    “supplementation with KCl prevented ouabain-induced (but not ABT-263-induced) death of senescent cells (Fig 3e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ouabain is a senolytic agent with broad activity.

    “Here, we show that the cardiac glycoside, ouabain, is a senolytic agent with broad activity.”
    • Also stated in: § Cardiac glycosides induce apoptosis of senescent cells · boosted
      “Taken together these results further demonstrate that ouabain behaves as a broad-spectrum senolytic agent.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Overexpression of rATP1A1 prevents the senolytic effects of ouabain but not those of ABT-263.

    “Overexpression of rATP1A1 prevented the senolytic effects of ouabain, but not those of ABT-263 (Fig 3f).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Killing of senescent cells by cardiac glycosides is prevented by pan-caspase inhibitors but not by inhibitors of ferroptosis, pyroptosis, or necroptosis.

    “the killing of senescent cells by CGs was prevented by pan-caspase inhibitors but not by inhibitors of ferroptosis, pyroptosis or necroptosis”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the liver of aged mice, the CG-resistant Atp1a1 subunit is downregulated while the CG-sensitive Atp1a3 subunit is upregulated.

    “While CG-resistant Atp1a1 is downregulated in the liver of old mice, the expression of CG-sensitive Atp1a3 increases (Supplementary Fig 6).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Ouabain levels in blood dropped to undetectable levels at the end of the holiday period.

    “levels of ouabain in blood dropped to undetectable levels at the end of the ‘holiday’ period.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    A subset of identified compounds behave as broad-spectrum senolytics while others are selective against a specific type of senescent cell.

    “A subset of the identified compounds behave as broad-spectrum senolytics while others are just selective against a specific type of senescent cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ABT-263 treatment caused a drop of platelets in blood and decreased platelet infiltration in livers of mice.

    “ABT-263 treatment that caused a drop of platelets in blood and decreased platelet infiltration in livers of mice”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In irradiated mice, ouabain treatment reduced presence of senescent cells and lower expression of cytokines such as Il1a or Il6 in lung.

    “Treatment with ouabain resulted in a reduced presence of senescent cells and lower expression of cytokines such as Il1a or Il6 (Fig 5f-h).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Intermittent ouabain treatment in 24-month-old female mice was well tolerated, with plasma ouabain levels reaching 1.24+/-0.48 ng/ml 24h after a round.

    “This regime was well tolerated in mice, with levels of ouabain in plasma reaching 1.24+/-0.48 ng/ml 24h after a round of treatment”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Blood phosphate levels were lower in old mice and significantly reversed by ouabain treatment.

    “blood phosphate levels were lower in old mice and this decrease was significantly reversed upon ouabain treatment”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    GSEA showed upregulation of gene signatures associated with senescence, inflammation, and aging in old mice, significantly reversed by ouabain treatment.

    “Using GSEA analysis, we observed the upregulation of gene signatures associated with senescence, inflammation and aging in old mice, a trend that significantly reversed upon ouabain treatment”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Immune infiltration in liver changed in old mice, and many changes were significantly reversed in ouabain-treated old mice.

    “Using xCell, we observed that the immune infiltration in the liver changed in old mice. Interestingly, many of these changes were significantly reversed in ouabain-treated old mice”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The senolytic properties of cardiac glycosides could be related to inhibition of their canonical target, Na+,K+-ATPase, although other mechanisms cannot be excluded.

    “suggesting that the senolytic properties of CGs could be related to the inhibition of their canonical target, Na+, K+-ATPase, although we cannot formally exclude that CGs induce senolysis via other cellular mechanisms.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Senescent cells accumulate in old tissues and are associated with an increasing list of pathologies.

    “Senescent cells are present in pre-neoplastic and fibrotic lesions, they accumulate in old tissues and are associated with an increasing list of pathologies 8.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [8] McHugh, D. & Gil, J. Senescence and aging: Causes, consequences, and therapeutic avenues. J. Cell Biol. 217, 65–77 (2018). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing).

    Premisecitation

    A two-step combination of senescence-inducing anti-cancer agents followed by senolytics could be an improved strategy to treat cancer.

    “It has been proposed that a two-step combination of senescence-inducing anti-cancer agents followed by senolytics could be an improved strategy to treat cancer 43.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [43] Wang, L. & Bernards, R. Taking advantage of drug resistance, a new approach in the war on cancer. Front. Med. 12, 490–495 (2018). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Bcl2 family inhibitors are the most widely used senolytics and are effective at killing a range of senescent cells in vivo.

    “Currently Bcl2 family inhibitors are the most widely used senolytics, having been shown effective at killing a range of senescent cells in vivo”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23] Ovadya, Y. & Krizhanovsky, V. Strategies targeting cellular senescence. J. Clin. Invest. 128, 1247–1254 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Therapy-induced senescence plays an important role in determining the outcome of anti-cancer treatment and is responsible for some side effects.

    “Therapy-induced senescence plays an important role in determining the outcome of anti-cancer treatment 40, whilst also being responsible for some of its side effects 41.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [40] Schmitt, C. A. et al. A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy. Cell 109, 335–346 (2002). source

      Not checked in the free run.

    • cites [41] Demaria, M. et al. Cellular senescence promotes adverse effects of chemotherapy and cancer relapse. Cancer Discov. 7, 165–176 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Irradiation, chemotherapy, and some targeted anti-cancer drugs, including aurora kinase inhibitors, induce senescence.

    “Irradiation, chemotherapy and some targeted anti-cancer drugs, including aurora kinase inhibitors, all induce senescence 40,42.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [40] Schmitt, C. A. et al. A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy. Cell 109, 335–346 (2002). source

      Not checked in the free run.

    • cites [42] Wang, L. et al. High-throughput functional genetic and compound screens identify targets for senescence induction in cancer. Cell Rep. 21, 773–783 (2017). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Oncogene-induced senescence protects against tumor initiation.

    “Oncogene-induced senescence protects against tumor initiation 34–37.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [34] Collado, M. et al. Tumour biology: senescence in premalignant tumours. Nature 436, 642 (2005). source

      Not checked in the free run.

    • cites [35] Michaloglou, C. et al. BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436, 720–724 (2005). source

      Not checked in the free run.

    • cites [36] Braig, M. et al. Oncogene-induced senescence as an initial barrier in lymphoma development. Nature 436, 660–665 (2005). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Preneoplastic senescent cells are subject to active immune surveillance and failure of this process results in increased tumor risk.

    “These preneoplastic senescent cells are the subject of active immune surveillance and failure of this process results in increased tumor risk 5.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] Kang, T. W. et al. Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature 479, 547–551 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Clearing senescent cells from progeroid or naturally aging mice improves healthspan, increases lifespan, and benefits multiple pathologies.

    “clearing senescent cells from progeroid or naturally aging mice improves healthspan 10, increases lifespan 12 and benefits an array of pathologies”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [10] Baker, D. J. et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479, 232–236 (2011). source

      Not checked in the free run.

    • cites [12] Baker, D. J. et al. Naturally occurringp16(Ink4a)-positive cells shorten healthy lifespan. Nature 530, 184–189 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    In the in vivo experiments, mice were allocated to treatment groups at random.

    “For in vivo studies, mice were randomly assigned to treatment groups.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Therapy-induced senescence also contributes to the adverse effects of chemotherapy and radiotherapy.

    “whilst also contributing to their side effects 41.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] Demaria, M. et al. Cellular senescence promotes adverse effects of chemotherapy and cancer relapse. Cancer Discov. 7, 165–176 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The broad senolytic specificity of cardiac glycosides and the causal role of senescence in age-related disease suggest that these drugs may have benefits beyond their anti-cancer effects.

    “Moreover, the broad specificity of CGs as senolytics, and the causal role of senescence in age-related disease suggest that CGs might have benefits that extend beyond their anti-cancer properties.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Senescence has a causal role in age-related disease.

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationown data

    The paper's data suggest that the senolytic effects of cardiac glycosides are due to on-target inhibition of Na+/K+-ATPase, but it needs clarification whether this inhibition fully explains the senolytic properties.

    “Although our data suggest that the senolytic effects of CGs are due to on-target inhibition of the Na+/K+-ATPase, it needs to be clarified whether this inhibition (fully) explains the senolytic properties of CGs.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A follow-up senolytic treatment has been proposed to improve cancer therapy.

    “Given that chemotherapy, radiotherapy and some targeted anti-cancer drugs rely on senescence induction, a follow-up senolytic treatment has been proposed as a way to improve cancer therapy 42.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Wang, L. et al. High-throughput functional genetic and compound screens identify targets for senescence induction in cancer. Cell Rep. 21, 773–783 (2017). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    Depending on the indication, cardiac glycosides could be administered locally rather than systemically.

    “However, their senolytic action may require intermittent rather than continuous usage, and depending on the indication, administration could be local rather than systemic.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Accumulation of senescent cells in ageing organisms contributes to many diseases, including cancer.

    “The accumulation of senescent cells in ageing organisms contributes to many diseases, including cancer 46.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [46] Childs, B. G., Durik, M., Baker, D. J. & van Deursen, J. M. Cellular senescence in aging and age-related disease: from mechanisms to therapy. Nat. Med. 21, 1424–1435 (2015). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Oncogene-induced senescence acts as a strong tumor suppressor mechanism.

    “OIS is a powerful tumor suppressor mechanism 34–37.”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [34] Collado, M. et al. Tumour biology: senescence in premalignant tumours. Nature 436, 642 (2005). source

      Not checked in the free run.

    • cites [35] Michaloglou, C. et al. BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436, 720–724 (2005). source

      Not checked in the free run.

    • cites [36] Braig, M. et al. Oncogene-induced senescence as an initial barrier in lymphoma development. Nature 436, 660–665 (2005). source

      Not checked in the free run.

    • cites [37] Chen, Z. et al. Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature 436, 725–730 (2005). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Senescent cells are associated with cancer, accumulate with age, and are present in many pathologies.

    “Senescent cells are associated with cancer, accumulate with age and are present in many pathologies 9.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Munoz-Espin, D. & Serrano, M. Cellular senescence: from physiology to pathology. Nat. Rev. Mol. Cell Biol. 15, 482–496 (2014). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The results are consistent with the Collado group's identification of cardiac glycosides as senolytic in bleomycin-induced senescence of cancer cells.

    “The results presented here are consistent with those of the Collado group, that also identified CGs as senolytic in the context of bleomycin-induced senescence of cancer cells (crossreference).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites crossreference

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Because cardiac glycosides eliminate incipient preneoplastic cells, they should lower the rate of cancer initiation.

    “In particular, the fact that CGs will eliminate incipient preneoplastic cells, should reduce the rate of cancer initiation and could suggest a prophylactic use to diminish cancer incidence.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Cardiac glycosides can be combined with anti-cancer therapies to enhance elimination of cancer cells and avoid accumulation of bystander senescent cells responsible for some side effects.

    “The above results suggest that CGs can be combined with anti-cancer therapies to both enhance the elimination of cancer cells and avoid the accumulation of bystander senescent cells responsible of some of the side effects of these treatments.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Digoxin has a long clinical history and its side effects can be monitored and managed.

    “Given that digoxin has a long history of use in the clinic and its side effects can be monitored and managed 56”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [56] Li, B. et al. Ouabain ameliorates bleomycin induced pulmonary fibrosis by inhibiting proliferation and promoting apoptosis of lung fibroblasts. Am. J. Transl. Res. 10, 2967–2974 (2018).

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Genetic and pharmacological strategies have demonstrated benefits of eliminating senescent cells in aging and disease.

    “Genetic and pharmacological strategies have demonstrated the benefits of eliminating senescent cells in the context of aging and disease 10,12,51.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [10] Baker, D. J. et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479, 232–236 (2011). source

      Not checked in the free run.

    • cites [12] Baker, D. J. et al. Naturally occurringp16(Ink4a)-positive cells shorten healthy lifespan. Nature 530, 184–189 (2016). source

      Not checked in the free run.

    • cites [51] Childs, B. G. et al. Senescent cells: an emerging target for diseases of ageing. Nat. Rev. Drug Discov. 16, 718–735 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Ouabain can be used as a senolytic beyond cancer and reduces local inflammation and immune infiltration.

    “In conclusion, the above results suggest that ouabain can be used as a senolytic beyond cancer and that besides eliminating senescent cells, ouabain reduced the levels of local inflammation and immune infiltration”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Both sexes were used throughout the study.

    “Both sexes were used throughout the study.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Ouabain is not just a senolytic but also has a synthetic lethal interaction with RAS, potentially making it a cancer therapy with a dual mechanism of action.

    “This result suggests that ouabain is not just a senolytic compound but it also has a synthetic lethal interaction with RAS, which potentially makes it a cancer therapy with a useful dual mechanism of action.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The canonical activity of cardiac glycosides is to inhibit Na+, K+-ATPase, but other activities have been suggested to explain their pleiotropic effects.

    “The canonical activity of CGs is to inhibit the Na+, K+-ATPase 25 but other activities have been suggested to explain their pleiotropic effects 26.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [25] Therien, A. G. & Blostein, R. Mechanisms of sodium pump regulation. Am. J. Physiol. Cell Physiol. 279, C541–C566 (2000). source

      Not checked in the free run.

    • cites [26] Prassas, I. & Diamandis, E. P. Novel therapeutic applications of cardiac glycosides. Nat. Rev. Drug Discov. 7, 926–935 (2008). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    The broad-spectrum senolytic action of cardiac glycosides suggests they might target a common vulnerability induced by senescence.

    “The broad-spectrum senolytic action of CGs suggest that they might target a common vulnerability induced by senescence.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Mice are more resistant than humans to the canonical effects of cardiac glycosides because of differences in the Atp1a1 protein.

    “Mice have been described more resistant than human to the canonical effects of CGs due to differences in the Atp1a1 protein 32.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [32] Price, E. M. & Lingrel, J. B. Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme. Biochemistry 27, 8400–8408 (1988). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Every replicate in the study came from a different mouse.

    “All replicates in this study represent different mice.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Mice in the experiments were randomly assigned to control or treatment groups.

    “The mice used for all experiments were randomly assigned to control or treatment groups.”
    • Also stated in: § Mouse models and drug treatments · plain
      “The mice were randomly assigned to control or treatment group.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Differences in senolytic specificity open possibilities for choosing senolytics depending on intended clinical use.

    “These differences in specificity open up possibilities for the choice of senolytic depending on their intended clinical use.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Broad-spectrum senolytics such as cardiac glycosides will also eliminate other senescent cell subsets, including preneoplastic cells, bystander senescent cells caused by anti-cancer therapy, and age-associated senescent cells that drive chronic inflammation.

    “Our data suggest that broad-spectrum senolytics such as CGs, will also eliminate other subsets of senescent cells such as preneoplastic cells, bystander senescent cells induced by anti-cancer therapy and age-associated senescent cells which contribute to chronic inflammation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    The grip strength protocol is preferred because low scores often reflect failure to grip the bar rather than muscular weakness.

    “This protocol is preferred because lower scores are often due to the mouse failing to grip the bar effectively, rather than reflecting muscular strength.”
    Quote in the paperNot a substantial claim
53 of 53

  • The paper relies on this without support.

    How senolytics affect cancer initiation, progression, and treatment is poorly defined or understudied.

    “Despite the many roles that senescence plays in cancer initiation, progression and treatment, how senolytics can affect these processes is poorly defined.”
    “Despite the multiple roles that senescence holds during cancer initiation, progression and treatment, how senolytics impact on these processes is understudied.”

  • The paper relies on this without support.

    There is a need to identify further compounds with senolytic properties because of side-effects of known senolytics.

    “Due to the important side-effects associated to known senolytic compounds, there is a need to identify further compounds with senolytic properties.”

  • The paper relies on this without support.

    Senescence occurs at different stages of cancer evolution and treatment.

    “Senescence occurs at different stages of cancer evolution and treatment.”

  • The paper relies on this without support.

    Chemotherapy, radiotherapy and some targeted anti-cancer drugs rely on senescence induction.

    “Given that chemotherapy, radiotherapy and some targeted anti-cancer drugs rely on senescence induction”

  • The paper relies on this without support.

    Cardiac glycosides have been used in the clinic for a long time.

    “CGs have been used in the clinic for a long time.”

  • The paper relies on this without support.

    Digoxin and digitoxin are used for heart failure and atrial arrhythmia.

    “Digoxin and digitoxin are used for the treatment of heart failure and atrial arrhythmia.”

  • The paper relies on this without support.

    Senescence has a causal role in age-related disease.

    “the causal role of senescence in age-related disease”

  • The paper relies on this without support.

    A factor limiting the use of cardiac glycosides has been their potential for side effects.

    “a factor limiting their use has been the potential for side effects.”
8 of 8

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Cardiac glycosides, including digoxin, selectively killed TIS cancer cells in culture and in vivo.

    “Cardiac glycosides, including the widely prescribed digoxin, were able to selectively kill TIS cancer cells in culture and invivo 208,209 .”

    From Senescence and cancer — role and therapeutic opportunities (2022) · cites this paper as [209]

    The Results section, “Dual benefit of ouabain on therapy-induced senescence” (page unavailable), reports that sequential treatment with ouabain or digoxin killed therapy-induced senescent cancer cells in culture. The in vivo Results describe ouabain eliminating preneoplastic senescent cells and treatment-associated bystander senescent cells in mice, but do not establish selective killing of therapy-induced senescent cancer cells in vivo. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • Lung fibroblasts and prostate stromal cells are classified as SID5 by SenCID in two independent datasets.

    “We also find that lung fibroblasts and prostate stromal cells, commonly used as models for senolytic drug development, are classified as SID5 by SenCID (Figure S3H) in two independent datasets85”

    From Single-cell senescence identification reveals senescence heterogeneity, trajectory, and modulators (2024) · cites this paper as [85]

    The Results section (‘A drug screen identifies ouabain as a broad-spectrum senolytic’; page unavailable) uses IMR90 cells as a senescence model for senolytic screening, supporting part of the sentence’s model context. The paper does not identify prostate stromal cells or report a SenCID/SID5 classification; the citing sentence presents that classification as its own finding in Figure S3H. The cited paper therefore does not substantiate the full claim about both cell types in two datasets. (LLM, conf=0.78)

    Experimental — accuracy not yet measured

  • The cardiac glycoside ouabain shows senolytic activity, partly by inducing the proapoptotic BCL-2 family protein NOXA.

    “Recently, the cardiac glycoside ouabain demonstrated senolytic activity, at least in part, through the induction of NOXA, a proapoptotic BCL-2 family protein203,204.”

    From Cellular senescence in ageing: from mechanisms to therapeutic opportunities (2021) · cites this paper as [203]

  • Another group independently obtained similar results using other unrelated chemical libraries.

    “similar results have been obtained by another group performing independently cell-based screenings and using other unrelated chemical libraries”

    From Identification and characterization of Cardiac Glycosides as senolytic compounds (2019) · cites this paper as [50]

  • Other senolytic compounds, including cardiac glycosides, are widely used clinically and cooperate with prosenescence therapies to kill cancer cells.

    “Other senolytic compounds, including cardiac glycosides, are widely used clinically and cooperate with prosenescence therapies to kill cancer cells (Guerrero et al. 2019).”

    From Senescence and the SASP: many therapeutic avenues (2020) · cites this paper as Guerrero et al. 2019

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Extended Figure 5 defines n as both cells and mice

    Minor

    The figure legend specifies n = 200 cells for the digoxin experiment, but its closing statement says n represents the number of mice. These are different sample units.

1 of 1

Cites

Flagged first, then by citations

4 of the 71 references couldn’t be matched to a known paper.

Cited by

Sorted by citations

Related

Authors
Guerrero, A., Herranz, N., Sun, B., Wagner, V., Gallage, S., Guiho, R., Wolter, K., Pombo, J., Irvine, E. E., Innes, A. J., Birch, J., Glegola, J., Manshaei, S., Heide, D., Dharmalingam, G., Harbig, J., Olona, A., Behmoaras, J., Dauch, D., Uren, A. G., Zender, L., Vernia, S., Martínez-Barbera, J. P., Heikenwalder, M., Withers, D. J., & Gil, J.
Affiliations
  • MRC London Institute of Medical Sciences (GB)
  • German Cancer Research Center (DE)
  • University College London (GB)
  • Universitätsklinikum Tübingen (DE)
  • Hammersmith Hospital (GB)
Corresponding author
Gil, Jesús
ORCID
Venue
Nature Metabolism
Publisher
Springer Science and Business Media LLC
Published
2019-10-21
Volume
1
Issue
11
Pages
1074-1088
ISSN
2522-5812 (ISSN-L)
DOI
10.1038/s42255-019-0122-z
PMC
PMC6887543
OpenAlex
W2980638238
Cited by
359
References
71
Citations by year
2026: 36 · 2025: 60 · 2024: 58 · 2023: 49 · 2022: 52 · 2021: 52 · 2020: 48 · 2019: 4
FWCI
26.75
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Metabolism (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • UCL Discovery (University College London) (submittedVersion)
  • Spiral (Imperial College London) (submittedVersion)
Funders
  • RCUK | Medical Research Council (MC-A652-5PZ00)
  • RCUK | MRC | Medical Research Foundation (MC-A652-5PZ00)
Topics
  • Health Sciences › Medicine › Physiology › Telomeres, Telomerase, and Senescence
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
Keywords
cardiac glycosides, ouabain, senolytics, cellular senescence, oncogene-induced senescence, Noxa, apoptosis, radiation-induced senescence, age-related diseases
MeSH terms
Animals, Antineoplastic Agents, Cardiac Glycosides, Humans, Ouabain, Quercetin, Cellular Senescence, Apoptosis, Mice, Rats

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-05.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-05.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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