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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 treatment decreases SIRT1 and SIRT2 levels.BJ fibroblasts were either left untreated, C (control), or treated with D, (DMSO) or 10, 25, 50 and 100 μM of Resveratrol for 72 h and (A) used for Western blotting analysis of SIRT1 and SIRT2 expressions. β-actin was used as loading control (B) Western blots obtained as indicated in A. were densitometrically analysed and fold difference expressed as arbitrary units (a.u.). Black bars show SIRT1 and gray bars show SIRT2 levels. Expression of (C) SIRT1 (histogram with light gray bars) and (D) SIRT2 (histogram with dark gray bars) were analysed for mRNA level by Quantitative RT-PCR. Shown are means ± SD of three independent experiments. * represents p < 0, 05 vs. control, ** represents p<0,01 vs. control using the Student’s t-test.
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pone.0124837.g007: Resveratrol treatment decreases SIRT1 and SIRT2 levels.BJ fibroblasts were either left untreated, C (control), or treated with D, (DMSO) or 10, 25, 50 and 100 μM of Resveratrol for 72 h and (A) used for Western blotting analysis of SIRT1 and SIRT2 expressions. β-actin was used as loading control (B) Western blots obtained as indicated in A. were densitometrically analysed and fold difference expressed as arbitrary units (a.u.). Black bars show SIRT1 and gray bars show SIRT2 levels. Expression of (C) SIRT1 (histogram with light gray bars) and (D) SIRT2 (histogram with dark gray bars) were analysed for mRNA level by Quantitative RT-PCR. Shown are means ± SD of three independent experiments. * represents p < 0, 05 vs. control, ** represents p<0,01 vs. control using the Student’s t-test.

Mentions: Previous studies have reported resveratrol, as an activator of Sir2 enzymes in vivo and in vitro. Resveratrol was shown to increase life span in three model organisms through a Sir2-dependent pathway [1]. Furthermore different studies suggest either senescence promoting or preventing role for sirtuins in particular for SIRT1 in different cell types [13,14]. Because we found that resveratrol induce premature senescence in BJ fibroblasts, we speculated whether or not the resveratrol induced senescence was dependent on sirtuins. We analysed expression of SIRT1 and SIRT2 the two members of sirtuin family known to be involved in cellular stress responses and cell cycle, respectively. Interestingly, Western blotting analysis showed that expression of SIRT1 and SIRT2 proteins were significantly decreased upon 10 μM resveratrol treatment and also continued at higher concentrations (25, 50 and 100 μM) (Fig 7A and 7B). We confirmed these data by RT-qPCR analysis and showed that mRNA level of SIRT1 and SIRT2 was also significantly decreased starting with 10μM resveratrol treatment in BJ fibroblasts (Fig 7C and 7D).


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 treatment decreases SIRT1 and SIRT2 levels.BJ fibroblasts were either left untreated, C (control), or treated with D, (DMSO) or 10, 25, 50 and 100 μM of Resveratrol for 72 h and (A) used for Western blotting analysis of SIRT1 and SIRT2 expressions. β-actin was used as loading control (B) Western blots obtained as indicated in A. were densitometrically analysed and fold difference expressed as arbitrary units (a.u.). Black bars show SIRT1 and gray bars show SIRT2 levels. Expression of (C) SIRT1 (histogram with light gray bars) and (D) SIRT2 (histogram with dark gray bars) were analysed for mRNA level by Quantitative RT-PCR. Shown are means ± SD of three independent experiments. * represents p < 0, 05 vs. control, ** represents p<0,01 vs. control using the Student’s t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124837.g007: Resveratrol treatment decreases SIRT1 and SIRT2 levels.BJ fibroblasts were either left untreated, C (control), or treated with D, (DMSO) or 10, 25, 50 and 100 μM of Resveratrol for 72 h and (A) used for Western blotting analysis of SIRT1 and SIRT2 expressions. β-actin was used as loading control (B) Western blots obtained as indicated in A. were densitometrically analysed and fold difference expressed as arbitrary units (a.u.). Black bars show SIRT1 and gray bars show SIRT2 levels. Expression of (C) SIRT1 (histogram with light gray bars) and (D) SIRT2 (histogram with dark gray bars) were analysed for mRNA level by Quantitative RT-PCR. Shown are means ± SD of three independent experiments. * represents p < 0, 05 vs. control, ** represents p<0,01 vs. control using the Student’s t-test.
Mentions: Previous studies have reported resveratrol, as an activator of Sir2 enzymes in vivo and in vitro. Resveratrol was shown to increase life span in three model organisms through a Sir2-dependent pathway [1]. Furthermore different studies suggest either senescence promoting or preventing role for sirtuins in particular for SIRT1 in different cell types [13,14]. Because we found that resveratrol induce premature senescence in BJ fibroblasts, we speculated whether or not the resveratrol induced senescence was dependent on sirtuins. We analysed expression of SIRT1 and SIRT2 the two members of sirtuin family known to be involved in cellular stress responses and cell cycle, respectively. Interestingly, Western blotting analysis showed that expression of SIRT1 and SIRT2 proteins were significantly decreased upon 10 μM resveratrol treatment and also continued at higher concentrations (25, 50 and 100 μM) (Fig 7A and 7B). We confirmed these data by RT-qPCR analysis and showed that mRNA level of SIRT1 and SIRT2 was also significantly decreased starting with 10μM resveratrol treatment in BJ fibroblasts (Fig 7C and 7D).

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