EA.hy926 Cells and HUVECs Share Similar Senescence Phenotypes but Respond Differently to the Senolytic Drug ABT-263

Abstract

Doxorubicin (DOX) induces endothelial cell (EC) senescence, which contributes to endothelial dysfunction and cardiovascular complications. Senolytic drugs selectively eliminate senescent cells to ameliorate senescence-mediated pathologies. Previous studies have demonstrated differences between immortalized and primary EC models in some characteristics. However, the response of DOX-induced senescent ECs to senolytics has not been determined across these two models. In the present work, we first established a comparative characterization of DOX-induced senescence phenotypes in immortalized EA.hy926 endothelial-derived cells and primary human umbilical vein EC (HUVECs). Thereafter, we evaluated the senolytic activity of four senolytics across both ECs. Following the DOX treatment, both EA.hy926 and HUVECs shared similar senescence phenotypes characterized by upregulated senescence markers, increased SA-β-gal activity, cell cycle arrest, and elevated expression of the senescence-associated secretory phenotype (SASP). The potentially senolytic drugs dasatinib, quercetin, and fisetin demonstrated a lack of selectivity against DOX-induced senescent EA.hy926 cells and HUVECs. However, ABT-263 (Navitoclax) selectively induced the apoptosis of DOX-induced senescent HUVECs but not EA.hy926 cells. Mechanistically, DOX-treated EA.hy926 cells and HUVECs demonstrated differential expression levels of the BCL-2 family proteins. In conclusion, both EA.hy926 cells and HUVECs demonstrate similar DOX-induced senescence phenotypes but they respond differently to ABT-263, presumably due to the different expression levels of BCL-2 family proteins.

Keywords: ABT-263; BCL-2 family; doxorubicin; endothelial cells; senescence; senolytics.

Figure 1

Doxorubicin induces the expression of senescence markers in a concentration-dependent manner in EA.hy926 cells and HUVECs. EA.hy926 human-endothelial-derived cells and HUVECs were treated with increasing concentrations of DOX (0.1 µM, 0.2 µM, and 0.5 µM) for 24 h. Thereafter, DOX was removed and cells were incubated in DOX-free media for 72 h. Then, cells were harvested and the total protein was extracted. Expression levels of senescence markers including p53, p21, and cyclin D1 in EA.hy926 cells ((A–C), respectively) and HUVECs ((D–F), respectively) were measured via Western blotting (n = 4–6). Representative images of Western blots are shown. Values were normalized to α-tubulin and expressed relative to control cells. Values are presented as means ± SEM. Data were analyzed by one-way ANOVA followed by Dunnet’s multiple comparisons test. Note: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Doxorubicin triggers senescence in EA.hy926 cells and HUVECs, as demonstrated by the increased SA-β-gal activity and cell cycle arrest. EA.hy926 human-endothelial-derived cells and HUVECs were treated with DOX (0.5 µM) for 24 h. Thereafter, DOX was removed and cells were incubated with DOX-free media for 120 h then stained for SA-β-gal. Images of SA-β-gal staining of EA.hy926 cells (A) and HUVECs (B) are shown. The percentage of SA-β-gal positive cells were calculated. Data were analyzed via unpaired two-tailed t-test. In another set of experiments, cells were incubated with DOX-free media for 72 h and the cell cycle was analyzed by measuring the DNA content using the FACSCanto system. Percentages of each cell cycle phase in control and DOX-treated cells (n = 5–6) are shown for EA.hy926 (C) and HUVECs (D). Data are presented as means ± SEM. Data were analyzed via two-way ANOVA followed by Sidak’s post hoc test. Note: * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 3

DOX induces the gene expression of SASP factors in both EA.hy926 cells and HUVECs. EA.hy926 human-endothelial-derived cells and HUVECs were treated with DOX (0.5 µM) for 24 h. Thereafter, DOX was removed and cells were incubated with DOX-free media for 72 h. The total RNA was then extracted and the mRNA expression of SASP factors including IL-6, CXCL1, and CXCL8 in (A) EA.hy926 cells (n = 6) and (B) HUVECs (n = 4) was determined by real-time PCR. Values were normalized to B2M and expressed relative to control cells. Values are shown as means ± SEM. Data were analyzed by unpaired two-tailed t-test. Note: *p < 0.05, **** p < 0.0001.

Figure 4

DOX induces the protein expression of SASP factors in conditioned media of EA.hy926 cells and HUVECs. EA.hy926 human-endothelial-derived cells and HUVECs were treated with DOX (0.5 µM) for 24 h. Thereafter, DOX was removed and cells were incubated with DOX-free media for 72 h. Conditioned media were collected and the expression of SASP factors including IL-6, TNF-α, IL-8, IL-1B, and MCP-1 in (A) EA.hy926 cells (n = 6) and (B) HUVECs (n = 5) was determined by Luminex. Values were normalized to the protein (ptn) content of the cells determined by BCA and the results are expressed as the fold expression relative to control cells. Values are shown as means ± SEM. Data were analyzed by unpaired two-tailed t-test. Note: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Assessment of the activity of multiple senolytics in EA.hy926 cells and HUVECs. Both EA.hy926 human-endothelial-derived cells and HUVECs were treated with DOX (0.5 µM) for 24 h to establish the senescence phenotype or left untreated. Three days post DOX exposure, DOX-treated and untreated cells were incubated with increasing concentrations of different senolytics including (A) dasatinib, (B) quercetin, (C) fisetin, and (D) ABT-263 for 24 h. Thereafter, the cell viability was measured using MTT assays in both cell lines. The cell viability was calculated relative to control wells and expressed as a percentage. Values are presented as means ± SEM.

Figure 6

EA.hy926 and HUVEC cell lines respond differently to the senolytic drug ABT-263. Both EA.hy926 cells and HUVECs were treated with DOX (0.5 µM) for 24 h or left untreated (control cells). Three days post DOX exposure, DOX-treated and control cells were treated with ABT-263 (0.1 µM) for 6 h. Thereafter, expression levels of the apoptotic markers cleaved caspase-3 and cleaved PARP in EA.hy926 cells (A,B, respectively) and HUVECs (C,D, respectively) were measured via Western blotting (n = 4–6). Values are presented as means ± SEM. Data were analyzed by one-way ANOVA followed by Dunnet’s multiple comparisons test. Note: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 7

EA.hy926 and HUVECs demonstrate differential protein expression of the BCL-2 family following DOX treatment. Both EA.hy926 cells and HUVECs were treated with 0.5 µM DOX for 24 h. Three days post DOX exposure, cells were harvested and total protein was extracted. Thereafter, protein expression levels of the BCL-2 family including anti-apoptotic members (BCL-2, BCL-xL, and BCL-W) and pro-apoptotic members (BAK and BAX) in (A) EA.hy926 cells and (B) HUVECs were measured via Western blotting (n = 4–6). Representative images of Western blots are shown. Values were normalized to α-tubulin and expressed relative to control cells. Values are presented as means ± SEM. Data were analyzed by unpaired two-tailed t-test. Note: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 8

EA.hy926 cells and HUVECs respond differently to the BCL-2-selective inhibitor venetoclax. Both EA.hy926 cells and HUVECs were treated with DOX (0.5 µM) for 24 h to establish the senescence phenotype or left untreated. Three days post DOX exposure, DOX-treated and untreated cells were incubated with 10 µM venetoclax for 24 h. Thereafter, the cell viability was measured using MTT assays in both cell lines. The cell viability was calculated relative to control wells and expressed as a percentage. Note: * p < 0.05, compared to untreated treatment of the same cell line; # p < 0.05, compared to EA.hy926 cells of same treatment by two-way ANOVA with Tukey’s post hoc analysis. Values are presented as means ± SEM.