Today Cellular Senescence Is Considered A Common Reaction Of Almost All Types Of Proliferating Cells

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Abstract Targeted elimination of senescent cells, analysis, is one of the core trends in anti-aging therapy. Cardiac glycosides were recently proved to be broad-spectrum senolytics.Here we tested senolytic properties of cardiac glycosides towards human mesenchymal stem cells (hMSCs). Cardiac glycosides had no senolytic ability towards senescent hMSCs of various origins. Using biological and bioinformatic approaches we compare senescence development in 'cardiac glycosides-sensitive'A549 and '-insensitive'hMSCs. The absence of analysis was found to be mediated by the effective potassium import and increased apoptosis resistance in senescent hMSCs. Weakening "antiapoptotic defense" predisposes hMSCs to analysis. We revealed that apoptosis resistance, previously recognized as a common characteristic of senescence, in fact, is not a general feature of senescent cells. Moreover, only apoptosis-proline senescent cells are sensitive to cardiac glycoside-induced analysis. Thus, we can speculate that the effectiveness of analysis might depend on whether senescent cells indeed become apoptosis-resistant as compared to their proliferating counterparts.

Keywords Stem cells. Senolysis·Senescence·Apoptosis·Stress resistance

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Introduction

Today cellular senescence is considered a common reaction of almost all types of proliferating cells, including cancer and stem ones, as well as some post-mitotic cells to a variety of stressful stimuli [1-3]. The following types of cellular senescence can be distinguished accordingly to the origin of the inducing factor: replicative (due to the DNA damage at the shortened telomere ends), oncogene-induced (in response to aberrant activation of oncogenic signaling), stress-induced (mediated by the DNA damage caused by the oxidative stress, heat shock, UV and y radiation, etc.) and chemotherapy-induced (activated in cancer cells in response to chemotherapeutic drugs)[4-7]. There is heterogeneity in markers expressed by senescent cells depending on both cell type and an insult used to induce senescence. However, there are several common features typical for most types of senescent cells. The essential characteristic of senescence for any kind of dividing cell is the irreversible proliferation loss [8]. cistanche tubulosa extract The irreversibility of the cell cycle arrest is controlled by the cyclin-dependent kinase (CDK) inhibitors pl6 and p21 and is often regulated by the tumor suppressor protein p53 [8,9]. The other important features of senescent cells are the activation of a persistent DNA damage response; cell hypertrophy, which often arises as a result of impaired ribosomal biogenesis and protein synthesis; disturbance of lysosomal degradation and dysfunction of the rest degradation systems; increased activity of the specific lysosomal enzyme senescence-associated-β-galactosidase; various mitochondrial alterations; acquisition of the senescence-associated secretory phenotype(SASP), composed of pro-inflammatory factors, matrix-degrading enzymes, reactive oxygen species, etc.; epigenetic and chromatin landscape alterations, including the formation of senescence-associated heterochromatic foci and senescence-associated distension of satellites [8-10].In other words, senescent cells preserve metabolic activity and vitality, but their functioning is significantly altered compared to the origin cells.

Cistanche can anti-aging

It is now clear that senescent cells can modify the surrounding microenvironment affecting both neighboring cells and cellular niches, which may lead to tissue malfunctioning and therefore may be related to the progression of aging and age-related diseases [11,12]. Keeping that in mind, today more attention is focused on the strategies for targeted "killing" of senescent cells[13-15]. To this end, a novel class of drugs termed senolytics is actively developing. Senolytics target signaling pathways that contribute to the resistance of senescent cells towards apoptosis, thus inducing apoptosis preferentially in senescent cells[16]. The list of senolytics is constantly replenishing with new agents. The most known compounds with the stated senolytic activity are navitoclax (Bcl-2 family inhibitor), a combination of dasatinib(an inhibitor of multiple tyrosine kinases) and quercetin (a natural flavonol), Hsp90 inhibitors, MDM2 inhibitors, FOXO4-p53 interfering peptide, a BET family protein degrader, uPAR-specific CAR-T, galacto-conjugated navitoclax and various senolytic natural compounds[16-24]. Recently, using high-throughput drug screening two independent research groups identified cardiac glycosides, particularly ouabain, digoxin, and bufalin, as broad-spectrum senolytics [25,26]. cistanche tubulosa reviews It is worth mentioning that almost all of the declared senolytics have limitations, such as undesirable side effects or ineffectiveness towards some cell types. Mesenchymal stem cells (MSCs), found virtually in all postnatal organs/tissues, are characterized by the capacity to self-renew (symmetric divisions) and to differentiate, thus contributing to the maintenance and regeneration of the residing tissue [27]. Due to these unique properties, along with the potent anti-inflammatory and immunosuppressive functions, MSCs are broadly applied in cell replacement therapy for the treatment of various diseases, including diabetes Mellitus, multiple sclerosis, myocardial infarction, and so on [28]. However, similar to their differentiated progenies and other unipotent cells, MSCs are able to senesce either replicative or prematurely in response to oncogenes' activation and stressful stimuli [29, 30].MSCs' senescence has plenty of undesirable aftermaths, among which are the exhaustion of the pool of stem cells, reduced tissue maintenance and regeneration, impaired differentiation capacity, SASP-mediated senescence spreading, reduced angiogenic properties, modification of stem cell niche [29,31-33]. Taking into account the biological significance of the proper MSCs' functioning, senescent MSCs might be considered the crucial target for analysis. In line with this suggestion, a number of reviews highlighting possible advantageous outcomes of senescent MSCs removal have been published over the past year [29, 31,34,35]. Nevertheless, there are only a few experimental data regarding this issue [36-40].

Within the present study, we demonstrate for the first time that cardiac glycosides, namely ouabain and bufalin, fail to display hemolytic activity towards human MSCs (hMSCs)derived from the endometrium (END-MSCs), adipose tissue (AD-MSCs), the dental pulp (DP-MSCs) and Warton jelly (WJ-MSCs).In addition, we confirm that both cardiac glycosides are able to induce apoptosis preferentially in senes-cent A549 and SK-HEP-1, as was previously described in the pilot studies [25,26]. By assessing alterations in ionic homeostasis caused by the Nat/K+-ATPase blocking and expression levels of the related genes we reveal that the absence of ouabain-induced analysis might be mediated by the enhanced effectiveness of the compensatory K+ import in senescent END-MSCs as compared to senescent A549. Furthermore, using advanced bioinformatics we demonstrate that senescence of END-MSCs, resistant to ouabain-induced analysis, is accompanied by the acquisition of apoptosis-resistant phenotype, while senescence of ouabain-sensitive A549 is not. Importantly, blocking the activity of antiapoptotic protein MCL-1 prior to cardiac glycoside treatment resulted in the analysis of apoptosis-resistant senescent END-MSCs. Therefore, we provide clear evidence that apoptosis-resist-ance is not a general feature of senescent cells. Based on the data obtained we conclude that the effectiveness of analytic approaches might depend on whether cells indeed became apoptosis-resistant during senescence development.

Materials and methods

Cells

END-MSCs, WJ-MSCs, DP-MSCs, AD-MSCs, A549, and SK-Help were obtained from the Russian Collection of Cell Cultures (Institute of Cytology, Saint-Petersburg, Russia). A549 and SK-Help lines were authenticated by karyology, tumorigenicity, isoenzyme (LDH and G6PD) tests, and STR analysis. Cells were cultured in complete medium DMEM F12(Gibco BRL)supplemented with 10% FBS (HyClone), 1% penicillin-streptomycin (Gibco BRL) and 1% glutamic (Gibco BRL).All cells were routinely tested for mycoplasma contamination.

Senescence- and stress-inducing conditions

For oxidative stress-induced senescence, END-MSCs/WJ-MSCs were treated with 200 uM/100 uM HO(Sigma)for 1 h. For doxorubicin-induced senescence, DP-MSCs/A549 were treated with 1 μM of doxorubicin （Veropharm） for 3 days. In each case, cells were considered senescent not earlier than 14 days after treatment. For replicative senescence AD-MSCs earlier than the 4th passage were identified as control cells and later than the 10th as senescent ones. For etoposide-induced senescence A549/END-MSCs/SK-Help were treated with 2 uM/5 uM/3 uM etoposide(Veropharm)for 3 days and analyzed not earlier than 7 days after senescence induction. In this case, the treatment design completely coincided with those described in the study from which RNA-seq data originated (Wang et al.,2017). Two cardiac glycosides we applied as senolytic compounds——Ouabain (Sigma) and Bufalin(Calbiochem).

In order to compare stress resistance between control and senescent END-MSCs or A549, cells were treated either with 400/800 uM H O2(Sigma) for 1 h or with 0.3/1 uM staurosporine (Sigma). Cell viability was analyzed 48 h after stress induction.

To block MCL-1 activity END-MSCs were pretreated with 10 uM of A-1210477(Sigma)for 3 days.

Flow cytometry analysis

Measurements of cell viability, proliferation, cell size, auto-fluorescence, apoptosis rates, caspase 3/7 cleavage, mitochondrial membrane potential, and membrane depolarization were carried out by flow cytometry. Flow cytometry was performed using the CytoFLEX (Beckman Coulter) and the obtained data were analyzed using CytExpert software version 2.0. Adherent cells were rinsed twice with PBS and harvested by trypsinization. Detached cells were pooled and resuspended in fresh medium and then counted and ana-lyzed for autofluorescence. In order to access cell viability, 50μg/ml propidium iodide （Life Technologies） was added to each sample just before analysis. The cell size was evaluated by cytometric forward light scattering of PI-negative cells. Apoptosis induction was verified using Annexin-V-APC (Invitrogen) and DAPI (Sigma) co-staining following manufacturer instructions. Caspase activity was assessed using CellEventTM Caspase-3/7 Green Flow Cytometry Assay Kit (Invitrogen) following manufactures protocol. Loss of mitochondrial membrane potential was assessed using the ratiometric dye JC-1 (Invitrogen). The staining procedure was carried out in accordance with the manufacturer's protocol. For membrane depolarization, we used a DiBAC4(3)fluorescent probe (Invitrogen).

Senescence-associated β-galactosidase staining

Senescence-associated β-Galactosidase (SA-β-Gal)staining was performed using a senescence β-galactosidase staining kit (Cell Signaling Technology) according to the manufacturer's instructions. Quantitative analysis of images was produced with the application of the MatLab package, according to the algorithm described previously[41]. For each experimental point, not less than 50 randomly selected cells were analyzed.

Western blotting

Western blotting was performed as described previously [41]. SDS-PAGE electrophoresis, transfer to a nitrocellulose membrane, and immunoblotting with ECL (Thermo Scientific) detection were performed according to standard manufacturer's protocols (Bio-Rad Laboratories). Antibodies against the following proteins were used: glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(clone 14C10), phospho-p53(Ser15), p21(clone 12D1), phospho-Rb (Ser807/811), HMGB1(clone D3E5), as well as horseradish peroxidase-conjugated goat antirabbit IgG. Dilution rates were 1:1000 for all primary antibodies and 1:7000 for secondary ones. All antibodies were purchased from Cell Signaling. The Scion Image 4.0(Scion Corporation)was used to select and determine the background-subtracted density of the bands in all the gels and blots.

Transcriptomic analysis

Samples from the following three Gene Expression Omnibus (GEO)RNA-seq datasets were used in the analysis: GSE102639(GSM2742113-GSM2742114 and GSM2742121-GSM2742122forcontrol and senescentA549cells, accordingly); GSE122081(GSM3454482-GSM3454484 and GSM3454500-GSM3454502 for control and senescent IMR-90 cells, accordingly); and our dataset GSE160702 (GSM4877895-GSM4877898 and GSM4877907-GSM4877910 for control and senescent END-MSCs, respectively). The data for all the datasets were processed in the same way.

Raw reads data underwent quality filtering and adapter trimming via FilterByTile and BBDuk scripts from the BBtools package (version 38.75)using the default options. The remaining reads were additionally filtered and trimmed with the use of a trimester script from the FastqPuri pack-age (version 1.0.7). cistanche UK The trimming operation was applied for both ends of reads if they contained N's or their quality was below the quality threshold set to 27, all reads shorter than 25 bases were discarded. The quality control of trimming was held with the FastQC software (version 0.11.7) and FastqPuri scripts. The reads, having passed all operations, comprise no less than 90% of the initial data.

Transcript abundances were estimated using the Salmon lightweight mapping (version 1.1.0)running in the selective alignment mode. The list of decoys was generated based on the Gencode human reference genome GRCh38.p13 (release 33) and used further for building the index on concatenated transcriptome and genome Gencode reference files (release 33) using a kmer size of 21. Mapping operations were run with additional flags——numBootstraps 30——sequins——gcBias——validate mappings. The resulting mapping rates were around70%.

Further data processing was performed using R version 3.6.3 with the Tidyverse collection of packages (version 1.3.0). Estimated gene counts, metadata, and transcript ranges were loaded into R and summarized to a gene level using tximeta(version 1.4.5). The resulting count matrix was filtered to contain rows having at least 5 estimated counts across all samples, the resulting matrix contained 20,400 genes.

For the PCA and heatmap representation, the read counts were normalized using log transformation from the DESeq2 package (version 1.26.0). cistanche wirkung Heatmaps were constructed with the use of gene filter(version 1.38.0) and heatmap(version 1.0.12)R packages. Validation of division samples by senescence variable was conducted via subsetting normalized read counts by genes related to the Gene Ontology term "Cellular senescence"(GO 0090398). Differential expression analysis and log fold changes estimation was computed using DESeq2 for the last variable in the design formula control-ling for cells senescence status, ouabain treatment reaction, and interaction of the indicated factors. To strengthen differential expression testing, log fold change correction using a combination of adaptive shrinkage estimator from the palm package (version 1.8.0) and specifying an additional log fold change threshold equal to 0.667 was applied. The resulting shrunken estimates were used further for gene ranking and running Gene Set Enrichment Analysis using cluster profile (version 3.14.3)and fuse(version 1.12.0)R packages with p values adjusted for multiple comparisons according to the Benjamin-Hochberg method. Affymetrix microarray samples for control and senes-cent AD-MSCs were obtained from the GEO database (GSE66236) and processed with Phantasus web-application with log2 and quantile counts normalization. Gene Set Enrichment Analysis was performed with the use of a fuse (version 1.12.0)R package.

RNA extraction, reverse transcription, and real-time PCR

RNA extraction, reverse transcription, and real-time PCR were performed as described in our previous study [32]. Reagents for RNA extraction (ExtractRNA reagent), reverse transcription (MMLV RT kit), and real-time PCR (HS SYBR kit) were obtained from Evrogen. Gene expression levels were assessed using the real-time PCR BioRad CFX-96 amplifier (BioRad) with the following running program for all investigated genes: 40 cycles of melting for 10 s at 95°C, annealing for 15 s at 57.5°C, and synthesis for 15 s at 72 ℃. Melting curves analysis was applied to control the specificity of reactions. The following analysis of the obtained data was performed using the Bio-Rad CFX Manager software (BioRad) with the standard 2deltaCt quantification method with the use of GAPDH expression as the reference. Primer sequences are listed in Table 1.

Statistical analysis

To get significance in the difference between the two groups Student's t-test or Welch's t-test was applied. For multiple comparisons between groups, ANOVA with Tukey's HSD was used. Unless otherwise indicated, all quantitative data are shown as mean±SD and the asterisks indicate significant differences as follows: ns, not significant,*p<0.05, **p<0.01,****p<0.001. Statistical analysis was performed using R software.

Results

H2O2-treated END-MSCs enter the premature senescence

Within the present study, we used human mesenchymal stem cells isolated from desquamated endometrium (END-MSCs), which satisfy the minimum criteria suggested by the ISCT for defining hMSCs [41]. Previously, we have developed a reliable experimental model to study various aspects of the premature senescence of END-MSCs [30]. Namely, we have shown that END-MSCs subjected to sublethal oxidative stress gradually acquired all the typical features of senescent cells, including persistent DNA dam-age foci and active DNA damage response, irreversible cell cycle arrest mediated by the classical p53/p21/Rb pathway, proliferation loss, cell hypertrophy, appearance of SA-β-Gal staining and development of senescence-associated secretory phenotype [30, 32, 42]. Here we applied the designed model to study senolytic effect of ouabain as well as to reveal the underlying intracellular alterations in ion homeostasis. Initially, by estimating the most common parameters we confirmed that single-dose（1 h， 200 μM）HO treatment was sufficient to induce senescence in END-MSCs. Importantly, stressed END-MSCs were considered senescent two weeks after the oxidative stress; therefore, all the senescence markers were assessed not earlier than 14 days after H-Oz treatment. Indeed, H-Oz treatment of END-MSCs led to proliferation block, cell hypertrophy as indicated by the increased cell size, accumulation of the lipofuscin detected by the elevation of autofluorescence, appearance of the SA-β-Gal, activation of the p21/Rb pathway and loss of HMGB1 together supporting senescence establishment under the chosen experimental conditions (Fig. a-c,e,f).

The most harmful effects of senescent cells at tissue and organismal levels are believed to be the consequence of their prolonged vitality. In line with this point, H-O?-treated END-MSCs retained high viability even at the late stages of senescence development (viability of senescent END-MSCs was assessed 17 days (14 days＋3 days) after the initial oxidative stress)(Fig.1d). To sum up, sublethal H-O2-treatment of END-MSCs is the appropriate model of the premature senescence and is relevant to investigate the effects of senolytic compounds on END-MSCs.

Cardiac glycoside ouabain has no senolytic activity towards senescent END-MSCs in a wide concentration range

Recent evidence suggests that cardiac glycosides, including ouabain, digoxin, and bufalin, represent a family of compounds with hemolytic activity [25,26]. Even though today cardiac glycosides are considered the broad-spectrum senolytics, the data regarding their effects on senescent hMSCs are lacking. Therefore, here we tested whether ouabain has the potential to induce death selectively in senescent END-MSCs. To do so, we assessed the viability of control and senes-cent END-MSCs after treatment with ouabain at the wide concentration range (from 10-' to 10~ M). Interestingly, neither concentration applied led to a noticeable decrease in the viability of both control and senescent cells on the first day after ouabain application (Fig.2 and Supplemental Fig.S1).

However, on the third day after ouabain treatment, we revealed a significant dose-dependent decline in the viability of control END-MSCs (Fig. 2a and Supplemental Fig. S1). Unexpectedly, senescent cells turned out to be more resistant to ouabain at each concentration tested. In line with this result,10-M ouabain led to more prone apoptotic death in control cells(20.75% An+/PI-and 36.47% An+/PI+)as compared to senescent ones(15.01% An＋/PI-and 12.15%An+/PI+)(Fig.2b). Obtained results clearly demonstrate that in context of senescent END-MSCs ouabain has no senolytic activity.

To exclude possible effects associated with variability of senescence-inducing stimuli on senolytic action of ouabain, we performed an additional set of experiments. Namely, we treated END-MSCs with etoposide instead of oxidative stress to induce premature senescence. citrus bioflavonoids Etoposide-treated END-MSCs displayed all the features typical for senescent cells(Fig.3a-e).

Importantly, ouabain had no hemolytic action towards etoposide-treated senescent END-MSCs (Fig. 3f and Supple-mental Fig. S2). These data provide additional confirmation for the above results and evidence that the lack of ouabain-induced analysis is not the consequence of the concrete senescence trigger used to induce senescence.

Fig.1 Validation of oxidative stress-induced END-MSCs premature senescence model. Senescent END-MSCs a loose proliferation, b undergo hypertrophy, c acquire elevated autofluorescence, retain high cell viability d and e display SA-β-Gal activity as compared to the control ones. f Phosphorylation levels of p53 and Rb and expression levels of p21 and HMGBI proteins in control and senescent END-Ouabain has no hemolytic action towards human mesenchymal stem cells of various origins To broaden our observations regarding the absence of ouabain-induced analysis in END-MSCs, we analyzed ouabain effects on hMSCs isolated from other sources including adipose tissue, dental pulp, and Wharton's jelly. To additionally strengthen our data, we applied different models of senescence——replicative senescence for AD-MSCs, doxorubicin-induced senescence for DP-MSCs, and oxidative stress-induced senescence for WJ-MSCs (Fig.4a-f).

As shown in Fig.4g, various types of hMSCs differed in the viability upon ouabain treatment, for example, both control and senescent DP-MSCs were much more resistant to ouabain action than WJ-MSCs (Fig.4g and Supplemental Fig. S3b, c). Nevertheless, ouabain was not able to induce senolysis in either type of senescent hMSCs (Fig. 4g and Supplemental Fig. S3). Together, the data obtained demonstrate the absence of ouabain-induced analysis is a common feature for various types of hMSCs. MSCs. The values presented are mean±SD. For multiple group comparisons at a and d, one-way ANOVA was applied, n=3, ns not significant,***p<0.001. For pair comparisons at b,c and e Welch's t-test was used,n=3 for b and c,n=50 for e,***p<0.001. Scale bars for images are 500 um.

GAPDH was used as a loading control

Cardiac glycoside bufalin fails to kill senescent END-MSCS

To verify the absence of senolytic action of cardiac glycosides towards hMSCs, we applied bufalin, another compound with the stated senolytic activity belonging to the cardiac glycosides family [25]. Bufalin had almost no effect on the viability of control and H2O2-treated senes-cent END-MSCs in a wide concentration range(from 10-7 to 10-5 M) (Fig. 5a and Supplemental Fig. S4a).

Similar to what we observed for ouabain treatment, the absence of analysis upon bufalin was independent of senescence-inducing stimuli, since the viability of ESCs that senesced either in response to oxidative stress or to etoposide was unaffected (Fig. 5a, b, Supplemental Fig. S4a, b). The results described above confirm that cardiac glycosides turned out to be ineffective for targeted death induction in senescent END-MSCs. MSCs. The values presented are mean±SD. For multiple group comparisons at a and d, one-way ANOVA was applied, n=3, ns not significant,***p<0.001. For pair comparisons at b,c and e Welch's t-test was used,n=3 for b and c,n=50 for e,***p<0.001. Scale bars for images are 500 um. GAPDH was used as a loading control

Cardiac glycoside bufalin fails to kill senescent END-MSCS

To verify the absence of senolytic action of cardiac glycosides towards hMSCs, we applied bufalin, another compound with the stated senolytic activity belonging to the cardiac glycosides family [25]. Bufalin had almost no effect on the viability of control and H2O2-treated senes-cent END-MSCs in a wide concentration range(from 10-7 to 10-5 M) (Fig. 5a and Supplemental Fig. S4a).

Similar to what we observed for ouabain treatment, the absence of analysis upon bufalin was independent of senescence-inducing stimuli, since the viability of ESCs that senesced either in response to oxidative stress or to etoposide was unaffected (Fig. 5a, b, Supplemental Fig. S4a, b). The results described above confirm that cardiac glycosides turned out to be ineffective for targeted death induction in senescent END-MSCs.

Fig.2 Ouabain has no hemolytic activity towards H-O2-treated senes-cent END-MSCs in a wide concentration range. Relative cell viability (%) of control and senescent END-MSCs in 3 days after treatment with 1077,10~6,10-5 M ouabain. b Apoptosis induction in END-MSCs upon 10-6 M ouabain assessed by Annexin V/DAPI double staining. n=3 independent experiments. All data correspond to the mean±SD. Statistical significance was assessed by the Welch's t-test:ns not significant,***p<0.05

Both cardiac glycosides ouabain and bufalin are able to induce cell death selectively in senescent A549 and SK-Hep1 cells

Taking into account the fact that our results do not correspond with the recently published evidence regarding the broad-spectrum senolytic action of cardiac glycosides, we decided to reproduce this effect using the cellular model described in the relevant studies [25,26]. Thus, we performed a series of experiments using control and senescent A549 lung carcinoma cells. Senescence in A549 cells was induced by etoposide treatment. Etoposide-induced senescence of A549 cells is a frequently used and thus well-characterized model of therapy-induced senescence [4]. Also, ouabain was shown to selectively kill etoposide-treated senescent A549 cells[25].To prove senescence in A549, we assessed proliferation rate, cell size, accumulation of lipo-fine, SA-β-Gal staining, the activation status of the p53/p21/Rb pathway, and expression level of HMGB1 (Fig. 6a-e).

To verify hemolytic activity of ouabain towards senescent cancer cells, we first estimated dose-dependent cell viability. In line with our results described above, we were not able to detect any significant decline in the number of viable control or senescent A549 cells within 24 h after ouabain application (Supplemental Fig. S5a). However, in 3 days after treatment ouabain significantly reduced the viability of senescent A549 cells in a dose-dependent manner, while the number of control A549 cells decreased to a much lesser extent (Fig.7a and Supplemental Fig. S5a). Namely, approximately 90%of control cells preserved viability at 10- M ouabain compared to 50% of senescent A549 cells treated with the same dose (Fig.7a). Moreover, senescent A549 cells were more prone to bufalin-induced cell death than their control counterparts (Fig.7b)(Fig.5b and Supplemental Fig.S5b).

According to the published data, ouabain triggered caspase-3-dependent apoptosis in senescent A549 cells [26]. Indeed, we revealed a significant increase in the double positive and/or PI-fraction in senescent cancer cells treated with 10-6M ouabain (Fig.7c). Furthermore, using fluorescent assay we observed activation of caspase-3 in ouabain-treated senescent A549 cells (Fig.7d). Taken together, these results are completely consistent with the data described by other authors and confirm the hemolytic activity of ouabain towards A549.

Also, we utilized doxorubicin instead of etoposide to cause senescence in A549 (Fig.8a-e). As expected,doxorubicin-treated senescent A549, as well as etoposide-treated demonstrated higher sensitivity both to ouabain and bufalin compared to their control counterparts (Fig. 8g and Supplemental Fig. S6). The latter confirms that senolytic effects of cardiac glycosides are independent of senescence inducing stimuli. Thus, cardiac glycosides indeed have senolytic

Fig.3 Ouabain is unable to induce senolysis in etoposide-treated senescent END-MSCs. Validation of the episode-induced senescence model for END-MSCs: a proliferation ability, b cell size, c autofluorescence levels and d SA-β-Gal activity, e phosphorylation level of Rb and expression levels of p21 and HMGBI proteins. f Relative cell viability (%) of control and senescent cells after treatment with 10-6 ouabain. Values are mean±SD. For multiple groups comparisons at a one-way ANOVA were applied,n=3, ns not significant,***p<0.001.For pair comparisons at b,c,d,f Welch's t-test was used, n=3 for b, c,f n=50 for d, ns not significant, *p<0.05, ***p<0.001. Scale bars for images are 500 μm. GAPDH was used as loading control activity towards A549, but these compounds turned out to be ineffective for targeted death induction in senescent hMSCs.

Finally, we decided to reproduce analysis experiments on etoposide-treated liver cancer cells SK-Help, another cell model with proven senolytic effects of cardiac glycosides according to Guerrero et al. study [25] (Fig.9a-e). Etoposide-treated senescent SK-Help demonstrated higher sensitivity to both agents compared to their control counterparts, proving the hemolytic action of cardiac glycosides towards this cell type (Fig.9f and Supplemental Fig. S7).

Together these data evidence that the selectivity of hemolysis induced by cardiac glycosides relies more on cell nature than on the concrete senescence trigger.

This article is extracted from Cellular and Molecular Life Sciences (2021) 78:7757–7776 https://doi.org/10.1007/s00018-021-03980-x