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Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation.

Kilic Eren M, Kilincli A, Eren Ö - PLoS ONE (2015)

Bottom Line: Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis.Interestingly DNA damaging agent doxorubicin also induced senescence in BJ fibroblasts associated with decreased SIRT1/2 levels.In conclusion our data reveal that resveratrol induced premature senescence is associated with SIRT1 and SIRT2 down regulation in human dermal fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biology, Adnan Menderes University Medical School, Aydın, Turkey; ADU-BILTEM (Adnan Menderes University, Science and Technology Research and Application Center), Aydin, Turkey.

ABSTRACT
The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene) has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol's anti-aging effects both in vitro and in vivo attributed to activation of a (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. In mammals seven members (SIRT1-7) of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ) and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated β-galactosidase (SA-β-gal) activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21CIP1 and p16INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-β-gal activity, γ-H2AX phosphorylation and p53, p21CIP1 and p16INK4A levels. Interestingly DNA damaging agent doxorubicin also induced senescence in BJ fibroblasts associated with decreased SIRT1/2 levels. In conclusion our data reveal that resveratrol induced premature senescence is associated with SIRT1 and SIRT2 down regulation in human dermal fibroblasts. Here we suggest that the concomitant decline in SIRT1/2 expression in response to resveratrol treatment may be a cause for induction of senescence, which is most likely mediated by a regulatory mechanism activated by DNA damage response.

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Resveratrol induces premature senescence in BJ fibroblasts.Cells were either left untreated, C (control), or treated with D, (DMSO) or 10, 25, 50 and 100 μM of Resveratrol for 72 h (A) used for assessment of SA-β-gal activity. SA-b-gal staining increased with RV doses in BJ cells (B) The percentage of SA-β-gal positive senescent cells in RV-treated and control or DMSO treated cells is presented as mean ± s.d of three independent experiments. The data represent the average and standard deviation of three independent counts of 100 cells each. Mean ± s.d. of three independent experiment of is shown, ** represents p<0,01 using the Student’s t-test.
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pone.0124837.g003: Resveratrol induces premature senescence in BJ fibroblasts.Cells were either left untreated, C (control), or treated with D, (DMSO) or 10, 25, 50 and 100 μM of Resveratrol for 72 h (A) used for assessment of SA-β-gal activity. SA-b-gal staining increased with RV doses in BJ cells (B) The percentage of SA-β-gal positive senescent cells in RV-treated and control or DMSO treated cells is presented as mean ± s.d of three independent experiments. The data represent the average and standard deviation of three independent counts of 100 cells each. Mean ± s.d. of three independent experiment of is shown, ** represents p<0,01 using the Student’s t-test.

Mentions: Since we found that resveratrol decreases proliferation in BJ cells and apoptosis was not the main response at these concentrations, we investigated whether or not resveratrol treatment induces premature senescence in BJ cells. Increased SA-β-gal activity is a well-known marker of senescence [32], hence we measured senescence via SA-β-gal staining. As shown in “Fig 3A”, the number of SA-β-gal positive senescent cells was significantly increased in resveratrol-treated cells compared to control or DMSO treated cells. Furthermore, the percentage of SA-β-gal positive cells increased with the concentrations of resveratrol indicates that resveratrol induces premature senescence in a dose-dependent manner (Fig 3A and 3B).


Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation.

Kilic Eren M, Kilincli A, Eren Ö - PLoS ONE (2015)

Resveratrol induces premature senescence in BJ fibroblasts.Cells were either left untreated, C (control), or treated with D, (DMSO) or 10, 25, 50 and 100 μM of Resveratrol for 72 h (A) used for assessment of SA-β-gal activity. SA-b-gal staining increased with RV doses in BJ cells (B) The percentage of SA-β-gal positive senescent cells in RV-treated and control or DMSO treated cells is presented as mean ± s.d of three independent experiments. The data represent the average and standard deviation of three independent counts of 100 cells each. Mean ± s.d. of three independent experiment of is shown, ** represents p<0,01 using the Student’s t-test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414559&req=5

pone.0124837.g003: Resveratrol induces premature senescence in BJ fibroblasts.Cells were either left untreated, C (control), or treated with D, (DMSO) or 10, 25, 50 and 100 μM of Resveratrol for 72 h (A) used for assessment of SA-β-gal activity. SA-b-gal staining increased with RV doses in BJ cells (B) The percentage of SA-β-gal positive senescent cells in RV-treated and control or DMSO treated cells is presented as mean ± s.d of three independent experiments. The data represent the average and standard deviation of three independent counts of 100 cells each. Mean ± s.d. of three independent experiment of is shown, ** represents p<0,01 using the Student’s t-test.
Mentions: Since we found that resveratrol decreases proliferation in BJ cells and apoptosis was not the main response at these concentrations, we investigated whether or not resveratrol treatment induces premature senescence in BJ cells. Increased SA-β-gal activity is a well-known marker of senescence [32], hence we measured senescence via SA-β-gal staining. As shown in “Fig 3A”, the number of SA-β-gal positive senescent cells was significantly increased in resveratrol-treated cells compared to control or DMSO treated cells. Furthermore, the percentage of SA-β-gal positive cells increased with the concentrations of resveratrol indicates that resveratrol induces premature senescence in a dose-dependent manner (Fig 3A and 3B).

Bottom Line: Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis.Interestingly DNA damaging agent doxorubicin also induced senescence in BJ fibroblasts associated with decreased SIRT1/2 levels.In conclusion our data reveal that resveratrol induced premature senescence is associated with SIRT1 and SIRT2 down regulation in human dermal fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biology, Adnan Menderes University Medical School, Aydın, Turkey; ADU-BILTEM (Adnan Menderes University, Science and Technology Research and Application Center), Aydin, Turkey.

ABSTRACT
The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene) has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol's anti-aging effects both in vitro and in vivo attributed to activation of a (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. In mammals seven members (SIRT1-7) of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ) and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated β-galactosidase (SA-β-gal) activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21CIP1 and p16INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-β-gal activity, γ-H2AX phosphorylation and p53, p21CIP1 and p16INK4A levels. Interestingly DNA damaging agent doxorubicin also induced senescence in BJ fibroblasts associated with decreased SIRT1/2 levels. In conclusion our data reveal that resveratrol induced premature senescence is associated with SIRT1 and SIRT2 down regulation in human dermal fibroblasts. Here we suggest that the concomitant decline in SIRT1/2 expression in response to resveratrol treatment may be a cause for induction of senescence, which is most likely mediated by a regulatory mechanism activated by DNA damage response.

Show MeSH
Related in: MedlinePlus