In Vitro Characterization of Doxorubicin-Mediated Stress-Induced Premature Senescence in Human Chondrocytes

Abstract

Accumulation of senescent chondrocytes is thought to drive inflammatory processes and subsequent cartilage degeneration in age-related as well as posttraumatic osteoarthritis (OA). However, the underlying mechanisms of senescence and consequences on cartilage homeostasis are not completely understood so far. Therefore, suitable in vitro models are needed to study chondrocyte senescence. In this study, we established and evaluated a doxorubicin (Doxo)-based model of stress-induced premature senescence (SIPS) in human articular chondrocytes (hAC). Cellular senescence was determined by the investigation of various senescence associated (SA) hallmarks including β-galactosidase activity, expression of p16, p21, and SA secretory phenotype (SASP) markers (IL-6, IL-8, MMP-13), the presence of urokinase-type plasminogen activator receptor (uPAR), and cell cycle arrest. After seven days, Doxo-treated hAC displayed a SIPS-like phenotype, characterized by excessive secretion of SASP factors, enhanced uPAR-positivity, decreased proliferation rate, and increased β-galactosidase activity. This phenotype was proven to be stable seven days after the removal of Doxo. Moreover, Doxo-treated hAC exhibited increased granularity and flattened or fibroblast-like morphology. Further analysis implies that Doxo-mediated SIPS was driven by oxidative stress as demonstrated by increased ROS levels and NO release. Overall, we provide novel insights into chondrocyte senescence and present a suitable in vitro model for further studies.

Keywords: ROS; SASP; SIPS; aging; chondrocytes; doxorubicin; osteoarthritis; oxidative stress; senescence; uPAR.

Figure 1

Influence of Doxo stimulation on the cell cycle and apoptosis of hAC. (A) Fluorescence intensity of the Alamar Blue assay after 7 d of Doxo-stimulation relative to the unstimulated control; TNFa + CHX served as the apoptosis control; n ≥ 6. (B) Relative caspase-3/7 activity after the addition of Doxo at different concentrations or TNF + CHX (apoptosis control); n ≥ 5. (C) Relative telomere length in untreated hAC compared to hAC treated with 0.1 µM Doxo for 7 d. (D) Exemplary histogram of the flow cytometric cell cycle analysis; blue curve = untreated hAC at passage 2; pink curve = hAC at passage 2, treated with 0.1 µM Doxo for 7 d. (E) Corresponding statistics of the cell cycle analysis; n = 3. (F) Exemplary dot plots of the cell size (FSC-H) vs. granularity (SSC-H) of unstimulated (blue) or Doxo-stimulated (pink) hAC, assessed by flow cytometry. Data are presented as scatter plot with bars, mean with standard deviation; or box plots with median, whiskers min to max. Significant differences between groups are depicted as: **** p ≤ 0.0001; ** p ≤ 0.01; * p ≤ 0.05. Statistical analysis: (A,B) one-way ANOVA, Sidak’s multiple comparisons test; (C) paired t-test; (D) multiple t-test. Ctrl = control (unstimulated cells), Doxo = doxorubicin, rel. = relative, ns = not significant.

Figure 2

Effect of Doxo stimulation on the expression of key senescence regulators CDKN1A and CDKN2A. Gene expression analysis of (A) CDKN1A (p21) and (B) CDKN2A (p16) in hAC after 10 d of Doxo stimulation at different concentrations; unstimulated hAC served as controls. (C) Exemplary images of a CDKN2A immunostaining in hAC after stimulation with 0.1 µM Doxo for 7 d. Data are presented as box plots with median, whiskers min to max. Significant differences between groups are depicted as: **** p ≤ 0.0001; *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05. Statistical analysis: (A,B) One-way ANOVA, Sidak’s multiple comparison. Scale bar = 50 μm. Ctrl = control (unstimulated cells), dapi = 4′,6-diamidino-2-phenylindole.

Figure 3

Influence of Doxo stimulation on the release of SASP factors in hAC. Secretion of (A) MMP-13, (B) IL-6, and (C) IL-8 into the culture media after 5 d and 10 d of Doxo stimulation was quantified by means of specific ELISAs. Data are presented as scatter plot with bars, mean with standard deviation. Significant differences between groups are depicted as: **** p ≤ 0.0001; *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05. Statistical analysis: (A–C) Multiple t-test. Ctrl = control (unstimulated cells), rel. = relative.

Figure 4

Effects of Doxo stimulation on oxidative stress markers in hAC. (A) Gene expression of iNOS, NOX2, and NOX4 as well as CAT, SIRT1, SOD1, and SOD2 after Doxo stimulation for 10 d. (B) Exemplary western blot analysis of SIRT1 and SOD2 after Doxo stimulation (n = 2); a-TUB = alpha tubulin. NO release into culture media of Doxo-treated hAC after 5 d and 10 d, respectively. (D) Representative images of DCFDA staining of unstimulated and Doxo-stimulated hAC after 7 d and corresponding quantification of the corrected total cell fluorescence (CTFC). (E) Exemplary co-staining of mitochondrial superoxide (MitoSOX; red) and cytoplasmic ROS (DCFDA; green) of unstimulated (Ctrlol) and Doxo-stimulated hAC after 7 d. Ctrl = unstimulated hAC. Data are presented as scatter plot with bars, mean with standard deviation; or box plots with median, whiskers min to max. Significant differences between groups are depicted as: **** p ≤ 0.0001; ** p ≤ 0.01. Statistical analysis: (A) One-way ANOVA, Sidak’s multiple comparison; (C) multiple t-test; (D) paired t-test. Ctrl = control (unstimulated cells), rel. = relative.

Figure 5

Impact of Doxo stimulation on uPAR expression and SA-β-gal activity in hAC. (A) Outline of the experimental setup. (B) Histogram of cytometric analysis of uPAR (CD87) on hACs unstimulated (blue) or stimulated with 0.1 µM Doxo for 7 d (pink). (C) Corresponding statistics of cytometric analysis of uPAR w/or w/o Doxo stimulation. (D) Exemplary phase contrast microscopy images of SA-β-gal staining in hACs (w/or w/o Doxo stimulation). (E) Corresponding quantification of SA-β-gal positive cells. Data are presented as scatter plot with median. Significant differences between groups are depicted as: **** p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01. Statistical analysis: (C,E) one-way ANOVA, Sidak’s multiple comparisons test. Ctrl = control (unstimulated cells), ns = not significant.

Figure 6

Continuing effect of Doxo stimulation on the gene expression of SA markers. Gene expression levels of (A) CDKN1A, (B) CDKN2A, (C) CXCL1, (D) IL6, (E) IL8, and (F) MMP13 were analyzed in hAC stimulated with 0.1 µM Doxo for 7 d. Doxo = analysis directly after 7 d of Doxo stimulation; Doxo depr. = analysis 7 d after deprivation of Doxo. Data are presented as box plots with median, whiskers min to max. Significant differences relative to unstimulated hAC are depicted as: cc = p ≤ 0.01; c = p ≤ 0.05. Statistical analysis: (A–F) one-way ANOVA, Sidak’s multiple comparisons test. Ctrl = control (unstimulated cells), rel. = relative, ns = not significant.

Figure 7

Overview of Doxo-mediated SIPS in hAC. Doxo treatment leads to oxidative stress by enhanced expression of ROS-generating NADPH oxidases (NOX2/4) and disturbance of mitochondrial function. Subsequent DNA damage results in upregulation of CDKN1A and CDKN2A, which initiate cell cycle arrest. ROS accumulation further triggers the expression of NO-generating iNOS and SASP factors. These paracrine mediators create a pro-inflammatory and catabolic microenvironment, not only fueling the progression of cartilage degeneration but also the spread of senescence. Moreover, Doxo-treated hAC express high levels of membrane-associated uPAR and intracellular β-gal, which are both well-described markers in chondrosenescence. Morphologically, senescent hAC are characterized by a flattened, fibroblast-like cell shape and an increase in granularity.