Biomolecular Evaluation of Piceatannol's Effects in Counteracting the Senescence of Mesenchymal Stromal Cells: A New Candidate for Senotherapeutics?

Abstract

Several investigations on senescence and its causative role in aging have underscored the importance of developing senotherapeutics, a field focused on killing senescent cells and/or preventing their accumulation within tissues. Using polyphenols in counteracting senescence may facilitate the development of senotherapeutics given their presence in the human diet, their confirmed tolerability and absence of severe side effects, and their role in preventing senescence and inducing the death of senescent cells. Against that background, we evaluated the effect of piceatannol, a natural polyphenol, on the senescence of mesenchymal stromal cells (MSCs), which play a key role in the body's homeostasis. Among our results, piceatannol reduced the number of senescent cells both after genotoxic stress that induced acute senescence and in senescent replicative cultures. Such senotherapeutics activity, moreover, promoted the recovery of cell proliferation and the stemness properties of MSCs. Altogether, our findings demonstrate piceatannol's effectiveness in counteracting senescence by targeting its associated pathways and detecting and affecting P53-dependent and P53-independent senescence. Our study thus suggests that, given piceatannol's various mechanisms to accomplish its pleiotropic activities, it may be able to counteract any senescent phenotypes.

Keywords: mesenchymal stem cells; polyphenols; senescence; senolytics.

Figure 1

Effects of piceatannol on acute senescence. MSCs at 10 DIV were treated for 30 min with 300 μM hydrogen peroxide to induce acute senescence, which was evaluated by beta-gal assay 48 h later. Next, senescent cultures were incubated with different amounts of piceatannol for another 48 h. The upper chart (A) shows the percentage of senescent cells at the end of piceatannol treatment. The lower chart (B) reports the cell viability, as detected via CCK-8 assay, of cultures treated with piceatannol. Data are expressed in arbitrary units and shown with standard deviation (SD), n = 3. *** p < 0.001 and * p < 0.05 indicate statistical significance between the control and treated samples. CT = control samples.

Figure 2

Senolytic versus senotherapeutic effects of piceatannol. Panel (A): MSCs at 10 DIV were treated for 30 min with 300 μM hydrogen peroxide to induce acute senescence, which was evaluated by beta-gal assay 96 h later. Senescence was also determined in 30 DIV cultures. The pictures show representative images of beta-gal staining in control cultures at 10 DIV (CT), in 10 DIV samples treated with hydrogen peroxide (H), and in replicative senescent cultures at 30 DIV (R). The chart on the right shows the percentage of senescent cells in healthy cells (CT) treated with ABT-737 or PCT (blue bars), in peroxide hydrogen-treated cultures (red bars) and in replicative senescent cultures (green bars). Panel (B): Apoptosis levels were detected in control cultures at 10 DIV (CT), in 10 DIV samples treated with hydrogen peroxide (H), and in replicative senescent cultures at 30 DIV (R). The picture shows the flow cytometry chart of annexin V assay to detect apoptosis. The chart on the right shows the percentage of apoptotic cells in healthy cultures (CT) treated with ABT-737 or PCT (blue bars), in peroxide hydrogen-treated cultures (red bars) and in replicative senescent cultures (green bars). Data are shown with standard deviation (SD) n = 3. The ^### p < 0.001 and ^## p < 0.01 are the statistical significances between control (CT) and peroxide hydrogen treated samples (H) or replicative senescent cultures (R). The *** p < 0.001 and ** p < 0.01 are statistical significances between control and treated samples.

Figure 3

Cell cycle profile and proliferation of cells treated with piceatannol. MSCs at 10 DIV were treated for 30 min with 300 μM hydrogen peroxide to induce acute senescence. After 48 h, senescent cultures were incubated with ABT-737 or piceatannol for another 72 h. Alternatively, MSCs at 30 DIV (i.e., replicative senescent cultures) were incubated with ABT-737 or piceatannol for 72 h. Panel (A) shows the proliferation of healthy cultures (blue lines), acute (red lines) and chronic (green lines) senescent cultures with or without ABT-737 or piceatannol. Data are expressed as 450 nm O.D. The percentage of cycling cells (i.e., Ki67 positive) and the cell cycle flow cytometry charts of cultures treated as described above are reported in Panels (B,C), respectively. In Panel (B), the pictures show representative images of Ki67 staining (i.e., in red) in control cultures at 10 DIV (CT), in 10 DIV samples treated with hydrogen peroxide (H), and in replicative senescent cultures at 30 DIV (R). The chart on the right shows the percentage of Ki67(+) cells in healthy cultures (CT) treated with ABT-737 or PCT (blue bars), in peroxide hydrogen treated cultures (red bars) and in replicative senescent cultures (green bars). Statistical data are shown with standard deviation (SD) n = 3. In panel (B,C) the *** p < 0.001, ** p < 0.01 and * p < 0.05 levels are statistical significances between control and samples with piceatannol or ABT-737. In panel (B,C), the ^### p < 0.001 are statistical significances between control (CT) and peroxide hydrogen-treated samples (H) or replicative senescent cultures (REP). In the panels, the statistically significant values refer to 72 h treatments.

Figure 4

Stemness of MSCs treated with piceatannol. MSCs at 10 DIV were treated for 30 min with 300 μM hydrogen peroxide to induce acute senescence. After 48 h, senescent cultures were incubated with ABT-737 or piceatannol for another 72 h. Alternatively, MSCs at 30 DIV were incubated with ABT-737 or piceatannol for 72 h. Following treatment with drugs, the cultures were incubated at low density in order to determine the CFU potential. Representative pictures of CFU assays performed on the control and senescent MSC cultures are shown. The histogram shows the CFU number in healthy cultures (CT) treated with ABT-737 or PCT (blue bars), in peroxide hydrogen-treated cultures (red bars) and in replicative senescent cultures (green bars). The ^### p < 0.001 indicates statistical significance between the control and senescent samples. The *** p < 0.001 indicates statistical significance between the control samples and samples treated with piceatannol or ABT-737.

Figure 5

Signaling pathways associated with senescence and the effect of piceatannol treatment. The charts show the expression of RB1/P105, RB2/P130, P107, P53, P27/KIP1, P21/CIP1, P16/INK4A and SIRT1 in control samples (CT) and in samples treated with hydrogen peroxide (H) or in replicative senescence cultures (R). The charts show the densitometric analysis of Western blot bands shown in Supplementary file S3. The expression of proteins of interest was normalized on GAPDH, chosen as the control. The ^### p < 0.001 and ^## p < 0.01 levels indicate statistical significance between the control and senescent samples. The *** p<0.01 ** p < 0.01 and * p < 0.05 indicate statistical significance between the control samples and samples treated with piceatannol or ABT-737.