Introduction

Aging is a major risk factor for many chronic diseases, such as cardiovascular diseases, degenerative diseases, and cancer. These chronic diseases seldom exist discretely in older people, which makes it challenging for clinical strategies to treat specific diseases and resolve various symptoms resulting from comorbid conditions. Interventions that impede the aging process itself offer an alternative approach that may delay the onset of age-related chronic diseases and thus prolong healthy longevity. Cellular senescence is a process that helps to prevent damaged cells from proliferating, thereby protecting cells from transformation and maintaining tissue homeostasis [1]. Senescence is also an important trigger of tissue remodeling during embryonic development and in response to tissue damage; senescence-related factors recruit immune cells to promote tissue regeneration [2]. However, the rate of senescent cell accumulation increases with age, resulting in pervasive damage and deficient cell clearance that disturbs tissue homeostasis. An increased burden of senescent cells in various tissues is a major factor contributing to aging and age-related diseases [3, 4]. Removing senescent cells as a therapeutic strategy was first verified by killing cells with high p16Ink4a expression in INK-ATTAC and p16-3MR transgenic mice, which delayed organ dysfunction and prolonged the lifespan in mice aged both prematurely and naturally [5]. Although this opened a new approach to anti-aging, only ‘late senescence’ cells that express a high level of p16Ink4a can be killed limiting the feasibility and potential application of this approach in humans.

A pharmacological target removal approach has subsequently become the new focus. Small-molecule compounds, termed senolytics, which increase the death rate of senescent cells, can alleviate inflammation and age-related organ dysfunction by targeting diverse survival pathways. Tens of senolytics have been reported. For instance, dasatinib, either alone or combined with quercetin, inhibits tyrosine kinase activity and initiates death in certain senescent cell types [6, 7]. Fisetin and quercetin are natural flavonoids that affect the PI3K/AKT/mTOR pathway; navitoclax (ABT-263) and ABT-737 inhibit the anti-apoptotic protein family Bcl2/BCL-xL, consequently triggering mitochondrial-mediated apoptosis [8, 9], and the cardiac glycosides ouabain has a similar effect. In addition, FOXO4-related peptide inhibits the interaction of p53 with FOXO4 to promote p53-medicated apoptosis [53]. CK2 depletion reduces the formation of γH2AX foci in radiation therapy and causes cancer cell death [54]. It has also been shown that inhibition of CK2 activity improves IR-induced human mesenchymal stem cells (hMSC) senescence [55]. TBB and CX4945, as specific CK2 inhibitors, can induce apoptosis in many types of cancer cells and are promising chemotherapeutic agents for chemo-refractory tumors [56, 57]. Therefore, it is possible that ‘senolytic’ and ‘senomorphic’ effects may coexist, each with opposing activity functions on the same target, and that both ‘toxicity to senescent cells’ and ‘alleviation of cellular senescence’ could attenuate aging processes at the systemic level. Nonetheless, DNA damage severity and drug initiation timing must be carefully evaluated. CK2-based senolytic strategies merit further exploration and development especially regarding applications to healthy aging. To summarize, we have identified several DDR kinase inhibitors, exemplified by TBB, with evident senolytic potential to specifically induce apoptosis in prematurely senescent cells. TBB feeding alleviates premature aging phenotypes and extends lifespan in progeroid mice by inhibiting CK2α phosphorylation of HP1α in response to DNA damage. Together with our previous findings, this new evidence highlights CK2α as a dual target for ‘senolytic’ and ‘senomorphic’ strategies in anti-aging intervention and the senolytic potential of TBB to treat progeroid syndromes.

We propose a model (Fig. 7) that CK2α is particularly important for maintaining the DNA damage response and repair of heterochromatin. In normal cells, CK2α promotes the decondensed structure of heterochromatin by phosphorylating the HP1α in case of DNA damage, thus facilitating the DNA damage response and repair. However, due to the low activity of CK2 in progeria cells, treatment with CK2 inhibitor TBB (TBB itself does not act as an inducer of DNA damage) to further inhibit CK2 activity will lead to increased accumulation of DNA damage, thus leading to apoptosis of progeria cells, acting as a senolytics effect and delaying the phenotype of premature aging mice.

Fig. 7: Working model.
figure 7

In normal cells undergoing DNA damage, CK2α regulates heterochromatin relaxation by phosphorylating HP1α, which promotes its dynamic dissociation with H3K9me3 and relocalization at DNA double-strand break sites (DSB), which facilitates DNA damage response and repair. Reducing CK2 activity leads to the accumulation of DNA damage, which leads to cellular senescence. Since CK2 activity is low in Zmpste24-deficient cells (progeria cells), CK2 inhibition by TBB aggravates extant DDR defects in senescent cells and results in apoptosis. CK2 activity determines the fate of cells undergoing varying degrees of DNA damage.

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Materials and methods

Reagents and antibodies

Rabbit anti-CK2α (ab236664), anti-ATM (ab78), anti-γH2AX (ab81299), anti-Bcl2 (ab32124), anti-Kap-1 (ab10484), anti-p-KAP-1 (Ser824, ab70369), anti-phospho-(Ser/Thr) Phe (ab17464), anti-H3K9me3 (ab8898) were obtained from Abcam (Cambridge, UK). Anti-lamin A/C (sc-20681), anti-p21 (sc-6246), and anti-p16 (sc-1661) were purchased from Santa Cruz Biotechnology. Rabbit anti-γH2AX (05-636), anti-p-ATM (Ser1981) (05-740), anti-HP1α (05–689) were sourced from EMD Millipore. Mouse anti-p-ATM (Ser1981) (#5883), anti-cleaved caspase-3 (#9661) and Rabbit anti-phospho-CK2 Substrate [(pS/pT)DXE] (#8738) were purchased from Cell Signaling Technology (Beverly, MA). Mouse anti-HA, anti-Flag were obtained from Sigma-Aldrich. Alexa Fluor 488 goat anti-rabbit IgG (#A11008), Alexa Fluor 594 goat anti-mouse IgG (#A11005), ImaGene GreenTM C12FDG Kit (#I2904) were purchased from Thermo Fisher Scientific. Mouse anti-actin, anti-GAPDH were obtained from Beyotime. Anti-pT50-HP1α monoclonal antibodies were prepared by Abmart generating from a specific phosphorylated peptide (peptide sequence C-SEEHN(pT)WEPEK). The chemical library were purchased from Targetmol Chemicals Inc. Cellular Senescence Detection Kit was purchased from Cell Signaling Technology (#9860). FITC Annexin V Apoptosis Detection Kit I (#556547), and APC Annexin V Apoptosis Detection Kit I (#550475) were purchased from BD Pharmingen.

Cell culture

The Zmpste24−/− mouse model has been described previously [22]. Primary MEFs were extracted from fetuses of 13.5-day-old pregnant female Zmpste24+/− mice (C57BL background). Cells were cultured in DMEM/HIGH glucose medium containing 10% (vol/vol) fetal bovine serum (Gibco) and 1% penicillin/streptomycin incubated at 37 °C in a 5% CO2 humidified atmosphere.

Drug preparation

All drugs (compounds in the DDR-related kinases inhibitor library- Custom compound library from Targetmol, USA.) were either prepared or provided as 10 mM stock solutions in DMSO. The compounds are list in Supplementary Table S1. They were then diluted in a culture medium to obtain a suitable working solution. Culture medium with DMSO at the same concentration was used as a negative control.

Senotherapeutics asssay

MEFs (5 × 103) at passage 6 were seeded in 96-well plates at least 6 h prior to treatment. After adding the drugs, the MEFs were incubated for 24–48 h. For fluorescence analysis of SA-β-Gal activity, cells were washed once with phosphate buffered saline, C12FDG (10 µM) was added to the culture medium, and the cells were incubated for 2 h. C12FDG fluorescence intensity was detected by Enspire 2300 Multimode Plate Reader (PerkinElmer, MA, USA). All samples were analyzed in duplicate with 3–5 fields per well and mean values and standard deviations were calculated accordingly.

Cell viability assay

MEFs were seeded on 96-well plates for 24 h, and then incubated with test compounds for another 48 h at 37 °C and 5% CO2. Cell viability was assessed from the CellTiter 96® AQueous One Solution Cell Proliferation Assay (cat#G3581, Promega, WI, USA). Pipet 20 µl of CellTiter 96® AQueous One Solution Reagent into each well of the 96-well assay plate containing the samples in 100 µl of culture medium, incubating the plate at 37 °C for 2 h in a humidified, 5% CO2 atmosphere. Record the absorbance at 490 nm using Enspire 2300 Multimode Plate Reader (PerkinElmer, MA, USA).

Plasmid construction and siRNAs

The full-length HP1α gene was subcloned into pcDNA3.1-3xFLAG vectors. CK2 was amplified from a cDNA library of HEK293 cells and cloned into pKH3-3xHA vectors. HP1α mutants were generated using a site-directed mutagenesis kit (New England Biolabs, USA). Transient transfection with these plasmids was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Small interfering RNA (siRNA) against Ck2α or mock siRNA were synthesized by GenePharma (Shanghai, China). Transfection was performed using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. The primers used are listed in Supplementary Table S2.

Western blot analysis

Total protein was isolated with modified RIPA buffer (0.1 M Tris-HCl, pH 7.5, 0.1 M NaCl, 1 mM EDTA, 1 mM DTT), phosphatase inhibitor, and protease inhibitor cocktail (Roche, Germany), quantified with a BCA protein assay kit (Invitrogen) and separated by SDS polyacrylamide gel electrophoresis followed by western blotting with the respective antibodies.

Co-immunoprecipitation assay

For co-immunoprecipitation, cells that were subjected to the indicated treatments were lysed in lysis buffer (250 mM NaCl, 20 mM Tris-HCl pH 8.0, 2 mM EDTA, 10% glycerol, and 0.1% NP-40), supplemented with EDTA-free protease inhibitor (Roche). Whole-cell extracts were then incubated with the indicated antibody overnight at 4 °C. Bead-bound immunoprecipitates were washed with lysis buffer, boiled in a loading buffer, then analyzed by western blotting analysis.

Flow cytometry

Senescent MEF cells from passage 6 were seeded at 70–80% confluence in 6-well culture plates and cultured for 24 h at 37 °C. For MEF, 5 × 105 cells/well were used. Following the addition of the drugs, lysosomal alkalinization was induced by pretreating cells with 100 nM bafilomycin A1 for 1 h in a fresh cell culture medium at 37 °C. C12FDG (10 μM) solution was then added to the cell culture medium for 2 h. The cells were harvested by trypsinization and resuspended in 1× Annexin V buffer containing 5 µl of Annexin V-APC and 5 µl 7-AAD per 1 × 105 cells/100 µl. The cells were analyzed by flow cytometry within 1 h. To estimate relative SA-β-Gal activity, a two-parameter display of forward scatter versus side scatter was set up, excluding subcellular debris. The events within this region were shown in a green fluorescence histogram, where the Y-axis indicates cell number and the X-axis indicates C12FDG fluorescence intensity on a log scale.

Immunofluorescence staining

Cells were mounted on glass slides and fixed in 4% paraformaldehyde. The cells were then permeabilized with PBS containing 0.1% Triton X-100 for 15 min, and blocked with 1% bovine serum in PBS for 30 min at room temperature. The cells were then incubated with various primary antibodies overnight at 4 °C, and detected by Alexa Fluor conjugated secondary antibodies (Alexa −488, −594; 1: 500, Life Technologies) for 1 h at room temperature in the dark. The cells were then co-stained with 4’, 6-diamidino-2-phenylindole (DAPI) (1:10000 dilution) to visualize the nuclei. Images were captured under a confocal laser scanning microscope system (CarlZeiss). A representative image for each condition is shown.

RNA isolation and quantitative RT-PCR

Total RNA was isolated using Trizol reagent (Invitrogen, 15596-026). RNA (2 μg) and reverse transcribed into cDNA using 5× Primescript RT Master Mix (Takara). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using 2× SYBR Green Mix (Takara) in a Bio-Rad detection system. Each sample was run in triplicate and gene expression levels were normalized to Gapdh. The primers are listed in Supplementary Table 2.

TUNEL staining

TUNEL staining was assessed with a DeadEnd™ Fluorometric TUNEL System (G3250, Promega, WI, USA) according to the manufacturer’s instructions, and then images were captured under a confocal laser scanning microscope system (CarlZeiss).

SA-β-Gal staining and apoptosis assays

SA-β-Gal staining was assessed with a cellular senescence assay kit according to the manufacturer’s instructions, and then photographed. Blue-stained MEFs were counted in more than 300 randomly chosen cells. Apoptosis was analyzed by flow cytometry as described previously [58].

Animal studies

The Zmpste24−/− mice used have been described elsewhere [22]. Zmpste24+/+ and Zmpste24−/− mice were assigned to the vehicle or the TBB treatment groups, which were fed TBB mixed into their drinking water at a final concentration of 50 μM/L. Survival of TBB-treated or vehicle-treated Zmpste24−/− mice and wild-type controls was recorded, and their body weight was monitored weekly. The survival curves of untreated and TBB-fed mice were compared by Kaplan–Meir analysis.

For bone density analysis, 4-month-old male mice were euthanized by decapitation. The thigh bone was fixed in 4% PFA at 4 °C overnight. The relevant data were collected by micro-CT (Scanco Medical, CT100). Each experimental group included at least three mice. All experiments using mice were performed in accordance with protocols approved by the Committee on the Use of Live Animals in Teaching and Research of Shenzhen University.

Statistical analysis

All experiments were carried out with at least three replicates. All data values are expressed as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). For multiple comparison, one-way or two-way ANOVA were applied as indicated. Statistical analysis was done by Student’s t test, with a p < 0.05 considered statistically significant.