Cellular senescence is a stable state of proliferative arrest induced by cellular damage or stress. Persistent accumulation of senescent cells and their senescence-associated secretory phenotype can promote chronic inflammation and tissue dysfunction, whereas senolytics selectively induce senescent-cell death by disrupting their antiapoptotic networks.

Keywords: cellular senescence; senolytics; senescence-associated secretory phenotype; SASP; SCAP; dasatinib; quercetin; fisetin; Navitoclax

1 Formation and Biological Characteristics of Cellular Senescence

1.1 Concept of Cellular Senescence

Cellular senescence is a long-term and generally difficult-to-reverse state of proliferative arrest that develops after cells are exposed to replicative stress, DNA damage, or other stress stimuli. Senescent cells retain a certain degree of metabolic activity but cannot normally re-enter the cell cycle and exhibit changes in chromatin structure, organelle function, gene expression, and secretory activity. Unlike cellular quiescence, from which proliferation can resume, senescent cells are generally maintained by stable cell-cycle inhibitory networks such as p53/p21 and p16INK4a/RB.

1.2 Replicative Senescence

Continuous division of normal somatic cells causes progressive telomere shortening. When telomeres can no longer maintain chromosome-end stability, a persistent DNA-damage response may be triggered, suppressing the cell cycle through ATM/ATR, p53/p21, and p16INK4a/RB pathways. Replicative senescence is commonly studied in long-term passaging models of normal human fibroblasts and other somatic cells with limited proliferative capacity.

1.3 Stress-Induced Premature Senescence

Ionizing radiation, oxidative stress, chemical injury, mitochondrial dysfunction, disruption of protein homeostasis, and metabolic stress can induce cellular senescence without obvious telomere shortening. Stress-induced premature senescence develops relatively rapidly and is suitable for establishing in vitro models. However, the senescence-associated secretory phenotype and antiapoptotic pathway dependence generated by different stimuli are not identical.

1.4 Oncogene-Induced Senescence

Persistent activation of oncogenic signals such as RAS and BRAF can cause aberrant replication, DNA damage, and oxidative stress, driving cells into oncogene-induced senescence. This process restricts the continued proliferation of potentially malignant cells and represents an important early tumor-suppressive barrier. However, if senescent cells are not cleared promptly, their secreted factors may promote neighboring-cell growth, inflammation, and tissue remodeling.

1.5 Therapy-Induced Senescence

Chemotherapy, radiotherapy, and certain targeted therapies can induce therapy-associated senescence in tumor cells or normal tissue cells. Short-term proliferative arrest can restrict tumor expansion, but long-term persistence of senescent cells may alter the tumor microenvironment through inflammatory factors, chemokines, and matrix-remodeling molecules and may increase the risk of tissue injury and recurrence-associated effects.

1.6 Dual Roles of Cellular Senescence

Short-term and controlled cellular senescence can participate in embryonic development, wound healing, tissue remodeling, and tumor suppression, after which senescent cells are generally eliminated by macrophages, natural killer cells, and other immune cells. When senescent cells continue to form or immune clearance declines, they accumulate within tissues and exert pathological effects. Therefore, the objective of senescence intervention is to identify and eliminate persistent pathological senescent cells rather than indiscriminately removing all senescent responses.

Figure 1 Mechanism of senescent cell accumulation and senolytic-mediated selective clearance

2 Major Phenotypes and Detection Indicators of Senescent Cells

2.1 Stable Cell-Cycle Arrest

Senescent cells generally exhibit reduced DNA synthesis, decreased expression of proliferation markers such as Ki-67, and changes in cell-cycle regulators including p16INK4a, p21CIP1, and p53. Reduced EdU or BrdU incorporation reflects inhibited DNA replication but may also occur in cellular quiescence, differentiation, and nutrient deprivation and therefore cannot independently establish a senescent state.

2.2 Senescence-Associated β-Galactosidase

Increased senescence-associated β-galactosidase (SA-β-gal) activity is a commonly used marker of cellular senescence and is generally detected histochemically or fluorometrically at pH 6.0. This indicator mainly reflects increased lysosomal mass and enzymatic activity in senescent cells. However, overconfluent cultures, prolonged culture, and certain nonsenescent cells may also test positive.

2.3 DNA Damage and Chromatin Alterations

Senescent cells may exhibit persistent γ-H2AX and 53BP1 foci, telomere-associated DNA damage, senescence-associated heterochromatin foci, and reduced Lamin B1. Persistent DNA-damage foci can help distinguish stable senescence from transient stress, but the specific phenotype is influenced by cell origin, induction method, and detection time.

2.4 Cellular Morphology and Organelle Changes

Senescent cells are generally enlarged and flattened, with increased cytoplasmic granularity, lysosomal abundance, mitochondrial dysfunction, reactive oxygen species accumulation, and altered autophagic flux. Senescent cells may retain substantial metabolic activity; therefore, normal or increased results in metabolic assays such as CCK-8 do not exclude cellular senescence.

2.5 Multimarker Identification

Reliable identification of cellular senescence should include independent evidence such as long-term proliferative arrest, increased SA-β-gal activity, altered p16 or p21 expression, persistent DNA damage, reduced Lamin B1, and a senescence-associated secretory phenotype. Because no single senescence marker is applicable to all tissues and cell types, at least three categories of indicators should generally be combined in experimental studies.

3 Senescence-Associated Secretory Phenotype and Its Tissue Effects

3.1 Composition of the SASP

The senescence-associated secretory phenotype (SASP) includes inflammatory cytokines such as IL-6, IL-8, and members of the IL-1 family; chemokines of the CCL and CXCL families; multiple growth factors; matrix metalloproteinases; protease inhibitors; extracellular matrix components; and bioactive lipids. The SASP is not a fixed secretory profile and varies with cell type, senescence-inducing stimulus, duration of senescence, and tissue environment.

3.2 Physiological Functions of the SASP

Short-term SASP signaling can promote immune-cell recruitment, tissue remodeling, angiogenesis, and wound repair and can help the immune system identify and clear senescent cells. Moderate SASP activity can also regulate regenerative responses in stem cells and neighboring cells and restrict the continued proliferation of damaged cells.

3.3 Pathological Functions of the SASP

Persistent SASP activity can induce DNA damage, inflammatory activation, or paracrine senescence in neighboring cells and alter the extracellular matrix, stem-cell niche, and immune-cell composition. Even when senescent cells represent only a small proportion of a tissue, they may continuously impair tissue function through paracrine signaling and inflammatory amplification.

3.4 SASP and Age-Related Diseases

Accumulation of senescent cells and SASP factors is associated with atherosclerosis, osteoarthritis, pulmonary fibrosis, kidney disease, metabolic abnormalities, neurodegenerative disorders, and remodeling of the tumor microenvironment. The major sources of senescent cells, SASP composition, and antiapoptotic pathway dependence differ among diseases, requiring intervention targets to be selected according to the specific tissue and cell population.

4 Senescent-Cell Antiapoptotic Pathways and Senolytics

4.1 Formation of SCAPs

Senescent cells remain exposed to DNA damage, oxidative stress, inflammatory stimuli, and proteostatic pressure for prolonged periods but do not immediately die. An important reason is the enhancement of senescent-cell antiapoptotic pathways (SCAPs). SCAPs may involve BCL-2, BCL-XL, BCL-W, PI3K/AKT, receptor tyrosine kinases, p53 regulatory networks, HSP90, and other survival signals.

4.2 Mechanisms of Action of Senolytics

Senolytics transiently inhibit survival pathways on which senescent cells are highly dependent, thereby restoring their sensitivity to mitochondrial apoptosis, oxidative damage, and proteostatic stress. Nonsenescent cells may depend less strongly on the same pathways, creating a potential selectivity window at appropriate doses and treatment durations. However, this selectivity is influenced by cell origin, senescence-inducing stimulus, and tissue environment.

4.3 Strategies Targeting the BCL-2 Family

Some senescent cells resist mitochondrial apoptosis by increasing BCL-2, BCL-XL, or BCL-W activity. Compounds such as Navitoclax, ABT-737, A1331852, and A1155463 can remove this antiapoptotic protection. However, BCL-XL also supports platelet and normal-cell survival, so target-associated toxicity and the therapeutic window must be evaluated.

4.4 Strategies Targeting Tyrosine Kinases and PI3K/AKT

Dasatinib inhibits multiple tyrosine kinases, whereas quercetin affects PI3K/AKT, oxidative-stress, and inflammatory signaling. Their combination can cover certain senescent-cell populations with different sensitivities to each single agent and is a commonly used senolytic combination. However, its activity is highly dependent on cell type and senescence model.

4.5 Strategies Targeting HSP90

HSP90 maintains the stability of multiple kinases and survival proteins, and some senescent cells depend on HSP90 to alleviate proteotoxic and apoptotic stress. HSP90 inhibitors such as Geldanamycin, Tanespimycin, and Alvespimycin can disrupt this protective network and are used to study clearance of HSP90-dependent senescent cells.

4.6 Strategies Targeting Redox and Ionic Homeostasis

Some senescent cells remain under high levels of oxidative and ionic stress and may be more sensitive to further increases in reactive oxygen species or changes in membrane potential. Piperlongumine disrupts redox balance, whereas cardiac glycosides such as Ouabain and Digoxin inhibit Na⁺/K⁺-ATPase and alter ionic homeostasis. The selectivity window and toxicity toward normal cells must be independently evaluated for these compounds.

4.7 Senolytics and Senomorphics

The principal objective of senolytics is to selectively induce senescent-cell death. Senescence phenotype-modifying agents, also called senomorphics or senostatics, mainly reduce the SASP or attenuate the harmful effects of senescent cells and do not necessarily eliminate the cells. Rapamycin, metformin, resveratrol, curcumin, and luteolin are more commonly used in studies of mTOR, AMPK, NF-κB, oxidative stress, and SASP regulation and should not be classified as senolytics solely on the basis of anti-inflammatory or antioxidant activity.

5 Representative Senolytics and Their Characteristics

5.1 Dasatinib and Quercetin

Dasatinib is a multitarget tyrosine kinase inhibitor, whereas quercetin is a natural flavonol. Dasatinib shows relatively strong activity against certain senescent preadipocytes, whereas quercetin has activity in some senescent endothelial-cell and other models. Their combination can broaden coverage across different senescent-cell populations but cannot eliminate all types of senescent cells.

5.2 Fisetin

Fisetin is a natural flavonol that can reduce senescent-cell burden, regulate the SASP, and affect antiapoptotic signaling in certain cellular and animal models. Because fisetin also has antioxidant, anti-inflammatory, and metabolic regulatory activities, its experimental effects cannot all be attributed to senescent-cell clearance. Selectivity should be validated through parallel toxicity comparisons between senescent and nonsenescent cells.

5.3 Navitoclax

Navitoclax (ABT-263) inhibits BCL-2, BCL-XL, and BCL-W and removes the dependence of certain senescent cells on BCL-2-family proteins. It has relatively well-defined clearance activity in multiple senescence models but may affect platelets and other normal cells that depend on BCL-XL. It is therefore suitable for mechanistic research, dosing optimization, and targeted-delivery studies.

5.4 BCL-XL-Selective Inhibitors

A1331852 and A1155463 primarily target BCL-XL and can be used to determine whether senescent cells depend on BCL-XL for survival. These compounds are useful for dissecting senescent-cell antiapoptotic networks, but because BCL-XL has important physiological functions, effects on platelets and normal tissues require particular attention.

5.5 Piperlongumine

Piperlongumine can increase oxidative stress and weaken antioxidant defenses in some senescent cells. Because senescent cells frequently exist under elevated reactive oxygen species pressure, they may be more sensitive to further redox disruption. Selectivity should nevertheless be verified by measuring reactive oxygen species, glutathione levels, and toxicity toward nonsenescent cells.

5.6 HSP90 Inhibitors

Geldanamycin, Tanespimycin, and Alvespimycin inhibit HSP90 chaperone activity and destabilize multiple survival-associated client proteins. HSP90 inhibitors can eliminate certain senescent cells but may also affect normal proliferating cells. Dose gradients, treatment duration, and intermittent dosing should therefore be optimized to establish a selectivity window.

5.7 Cardiac Glycosides

Cardiac glycosides such as Ouabain and Digoxin inhibit Na⁺/K⁺-ATPase and alter membrane potential, ionic gradients, and intracellular acid-base homeostasis. Certain senescent cells show increased sensitivity because of altered ion-regulatory capacity. However, cardiac glycosides have narrow therapeutic windows, and their senolytic effects should not be concluded solely from a single cell-viability assay.

5.8 Procyanidin C1

Procyanidin C1 (PCC1) is a natural polyphenol that may exhibit SASP-modulating or senescent-cell-clearing activity depending on concentration and treatment conditions. Its antioxidant effects, senomorphic effects, and selective cytotoxicity toward senescent cells should be evaluated separately.

6 Research Basis for the Dasatinib and Quercetin Combination

6.1 Cellular Studies

The rationale for combining dasatinib and quercetin is that different senescent cells depend on different SCAPs. The two compounds show different activity profiles in certain senescent preadipocytes, senescent endothelial cells, and other models. Combination treatment can broaden the range of affected senescent cells, indicating that senolytic screening should not rely on a single cell source or a single method of senescence induction.

6.2 Senescent-Cell Transplantation Models

Transplantation of senescent cells into animals can reduce mobility, endurance, and tissue function and induce senescence-associated changes in host tissues. Intermittent treatment with dasatinib plus quercetin can reduce senescent-cell burden and improve physiological function in certain models, supporting a causal relationship between senescent-cell accumulation and tissue dysfunction.

6.3 Natural Aging and Disease Models

Dasatinib plus quercetin has been studied in models of natural aging, pulmonary fibrosis, kidney disease, skeletal dysfunction, and metabolic disorders. Drug exposure, senescent-cell origin, and SCAP dependence differ substantially among tissues. Effective conditions in one model therefore cannot be directly applied to another disease or cell system.

6.4 Boundaries of Research Conclusions

Senolytics can improve age-related pathological and functional indicators in certain animal models. However, aging also involves genomic instability, epigenetic alterations, loss of proteostasis, mitochondrial dysfunction, stem-cell exhaustion, and altered intercellular communication. Eliminating senescent cells addresses only part of these mechanisms and should not be interpreted as comprehensively reversing human aging.

7 Experimental Design and Evaluation of Senolytics

7.1 Establishment of Senescence Models

Common models include replicative senescence generated through long-term passaging, premature senescence induced by radiation or DNA-damaging agents, oncogene-induced senescence, oxidative stress-induced senescence, and anticancer therapy-induced senescence. SASP composition and SCAP dependence differ markedly among these models. The induction conditions, recovery period, and stage of senescence maturation should therefore be clearly defined.

7.2 Confirmation of Senescence Models

Before drug treatment, stable senescence should be confirmed using indicators such as SA-β-gal, EdU or BrdU incorporation, p16, p21, Lamin B1, γ-H2AX, and the SASP. If cells are merely undergoing transiently reduced proliferation or acute injury, drug-induced cytotoxicity cannot be interpreted as senolytic activity.

7.3 Evaluation of Selective Clearance

Senolytic activity should be evaluated in parallel in senescent cells and corresponding nonsenescent cells by comparing cell viability, apoptosis, half-maximal effective concentration, and maximal effect. A reduction in senescent-cell number alone does not exclude broad cytotoxicity. The selectivity window between senescent and nonsenescent cells should be calculated.

7.4 Validation of Apoptotic Mechanisms

Senolytics generally induce cell death by removing antiapoptotic protection. Annexin V, caspase-3/7 activity, cleaved caspase-3, cleaved PARP, cytochrome c release, and mitochondrial membrane-potential changes can be assessed. Caspase inhibitors or knockdown of key SCAP proteins can further verify whether the compound clears senescent cells through apoptotic pathways.

7.5 Evaluation of Senescent-Cell Burden

After treatment, SA-β-gal positivity, p16, p21, Lamin B1, DNA-damage foci, and SASP changes should be measured and interpreted together with total cell number and cell-death indicators. Reduced senescence-marker signals may result from senescent-cell clearance, phenotype suppression, or altered assay signals and should not be interpreted as clearance on the basis of a single marker.

7.6 SASP Detection

IL-6, IL-8, chemokines, and matrix metalloproteinases can be measured using ELISA, immunoblotting, multiplex assays, or transcriptional analysis. Reduced SASP levels do not necessarily indicate that senescent cells have died and may instead result from senomorphic activity, reduced cellular metabolism, or a decrease in total cell number.

7.7 Endpoints in Animal and Translational Studies

Animal studies should evaluate senescent-cell burden, tissue inflammation, fibrosis, muscle strength, exercise capacity, organ function, and drug toxicity. Senescent-cell clearance has a clearer functional significance only when reduced senescence markers coincide with improved tissue function and no substantial injury occurs in normal tissues.

8 Products

Senolytic studies should demonstrate that candidate compounds preferentially eliminate senescent cells rather than merely reducing senescence markers or producing broad cytotoxicity. Experiments should include nonsenescent-cell controls, apoptosis validation, SASP measurements, and tissue-function evaluation to define selectivity and the boundaries of activity.

References

[1] Xu M, et al. Senolytics improve physical function and increase lifespan in old age. Nature Medicine. 2018;24(8):1246-1256.

[2] van Deursen JM. Senolytic therapies for healthy longevity. Science. 2019;364(6441):636-637.

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