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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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Expression of Ki67 antigen and induction of apoptosis in response to resveratrol treatment.BJ fibroblasts were either left untreated C control, or treated with D, DMSO or, 10, 25, 50 100 μM of Resveratrol for 72 h. (A) Immunofluorescence staining of Ki67. DAPI was used to counterstain nuclei. Bar graphs show percentage of Ki67-positive cells. BJ fibroblasts were either left untreated C control, or treated with D, DMSO or, 10, 25, 50 100, 200, 300 μM of Resveratrol for 72 h and analysed (B) for apoptosis by TUNEL staining. Bar graphs show percentage of Tunel positive cells. (C) for Western blotting analysis of cleaved caspase-3expression. β-actin was used as loading control. For Ki67 and Tunel stainings 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.g002: Expression of Ki67 antigen and induction of apoptosis in response to resveratrol treatment.BJ fibroblasts were either left untreated C control, or treated with D, DMSO or, 10, 25, 50 100 μM of Resveratrol for 72 h. (A) Immunofluorescence staining of Ki67. DAPI was used to counterstain nuclei. Bar graphs show percentage of Ki67-positive cells. BJ fibroblasts were either left untreated C control, or treated with D, DMSO or, 10, 25, 50 100, 200, 300 μM of Resveratrol for 72 h and analysed (B) for apoptosis by TUNEL staining. Bar graphs show percentage of Tunel positive cells. (C) for Western blotting analysis of cleaved caspase-3expression. β-actin was used as loading control. For Ki67 and Tunel stainings 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: Initially to determine the effects of resveratrol on proliferation of BJ cells we performed a time and concentration-response analysis via Wst-1 proliferation assay. Results from Wst-1 assay showed that resveratrol had no significant effect on BJ cells proliferation at a concentration of up to 10 μM during 72h incubation. However starting with 10 μM, increasing concentrations (25, 50, 100 μM) of resveratrol significantly reduced cell proliferation within 24 h incubation, which was further decreased at 48h time point and reached to a maximum level at 72 h time point Fig 1A). Next, in order to confirm data from Wst-1 proliferation assay we engaged BrdU incorporation assay using the same concentrations and time points. As shown in Fig 1B., similar results were obtained from BrdU assay; with increasing concentrations of resveratrol (10, 25, 50, 100 μM), Brdu incorporation in to cellular DNA was gradually decreased during 24h, 48h incubation periods and maximum level of inhibition was detected at 72h, indicating resveratrol had significant inhibitory effect on BJ cell’s proliferation in a time and dose dependent manner. We then assessed proliferation also by detection of the expression of Ki-67 antigen which is a widely used marker for measuring the growth fraction of a given cell population (Fig 2A). Since we measured the maximum inhibition of proliferation at 72h time point we stained for Ki67 expression only at this time point utilizing the same concentrations. Immunofluorescence analysis showed that Ki-67 antigen expression is significantly decreased in BJ cells treated with the increasing concentrations of resveratrol (Fig 2A and 2B). Since we found that resveratrol decreases proliferation and inhibits growth of BJ cells we asked whether apoptosis was induced. Accordingly, we treated cells with same concentrations of resveratrol and measured apoptosis after 72h and found that resveratrol did not induce apoptosis at concentrations of 10, 25, 50μM but starting with 100 μM the percentage of apoptotic cells was increased to 8,3 ±1,5 (Fig 2C). When we increased the concentrations up to 200 and 300 μM, the percentage of apoptotic cells was significantly increased and reached to (37 ±1,5) and (67±2,6) (Fig 2C), respectively. Additionally we measured apoptosis by analysing cleaved Caspase-3 expression under same conditions. As seen in Fig 2C cleaved caspase-3 was detectable in lysates of BJ fibroblasts treated with 100 to 300 μM of resveratrol. Thus, these results clearly show that in BJ fibroblasts resveratrol decreases proliferation in a time and dose-dependent manner and induce apoptosis only at higher concentrations between 100–300 μM.


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)

Expression of Ki67 antigen and induction of apoptosis in response to resveratrol treatment.BJ fibroblasts were either left untreated C control, or treated with D, DMSO or, 10, 25, 50 100 μM of Resveratrol for 72 h. (A) Immunofluorescence staining of Ki67. DAPI was used to counterstain nuclei. Bar graphs show percentage of Ki67-positive cells. BJ fibroblasts were either left untreated C control, or treated with D, DMSO or, 10, 25, 50 100, 200, 300 μM of Resveratrol for 72 h and analysed (B) for apoptosis by TUNEL staining. Bar graphs show percentage of Tunel positive cells. (C) for Western blotting analysis of cleaved caspase-3expression. β-actin was used as loading control. For Ki67 and Tunel stainings 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.g002: Expression of Ki67 antigen and induction of apoptosis in response to resveratrol treatment.BJ fibroblasts were either left untreated C control, or treated with D, DMSO or, 10, 25, 50 100 μM of Resveratrol for 72 h. (A) Immunofluorescence staining of Ki67. DAPI was used to counterstain nuclei. Bar graphs show percentage of Ki67-positive cells. BJ fibroblasts were either left untreated C control, or treated with D, DMSO or, 10, 25, 50 100, 200, 300 μM of Resveratrol for 72 h and analysed (B) for apoptosis by TUNEL staining. Bar graphs show percentage of Tunel positive cells. (C) for Western blotting analysis of cleaved caspase-3expression. β-actin was used as loading control. For Ki67 and Tunel stainings 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: Initially to determine the effects of resveratrol on proliferation of BJ cells we performed a time and concentration-response analysis via Wst-1 proliferation assay. Results from Wst-1 assay showed that resveratrol had no significant effect on BJ cells proliferation at a concentration of up to 10 μM during 72h incubation. However starting with 10 μM, increasing concentrations (25, 50, 100 μM) of resveratrol significantly reduced cell proliferation within 24 h incubation, which was further decreased at 48h time point and reached to a maximum level at 72 h time point Fig 1A). Next, in order to confirm data from Wst-1 proliferation assay we engaged BrdU incorporation assay using the same concentrations and time points. As shown in Fig 1B., similar results were obtained from BrdU assay; with increasing concentrations of resveratrol (10, 25, 50, 100 μM), Brdu incorporation in to cellular DNA was gradually decreased during 24h, 48h incubation periods and maximum level of inhibition was detected at 72h, indicating resveratrol had significant inhibitory effect on BJ cell’s proliferation in a time and dose dependent manner. We then assessed proliferation also by detection of the expression of Ki-67 antigen which is a widely used marker for measuring the growth fraction of a given cell population (Fig 2A). Since we measured the maximum inhibition of proliferation at 72h time point we stained for Ki67 expression only at this time point utilizing the same concentrations. Immunofluorescence analysis showed that Ki-67 antigen expression is significantly decreased in BJ cells treated with the increasing concentrations of resveratrol (Fig 2A and 2B). Since we found that resveratrol decreases proliferation and inhibits growth of BJ cells we asked whether apoptosis was induced. Accordingly, we treated cells with same concentrations of resveratrol and measured apoptosis after 72h and found that resveratrol did not induce apoptosis at concentrations of 10, 25, 50μM but starting with 100 μM the percentage of apoptotic cells was increased to 8,3 ±1,5 (Fig 2C). When we increased the concentrations up to 200 and 300 μM, the percentage of apoptotic cells was significantly increased and reached to (37 ±1,5) and (67±2,6) (Fig 2C), respectively. Additionally we measured apoptosis by analysing cleaved Caspase-3 expression under same conditions. As seen in Fig 2C cleaved caspase-3 was detectable in lysates of BJ fibroblasts treated with 100 to 300 μM of resveratrol. Thus, these results clearly show that in BJ fibroblasts resveratrol decreases proliferation in a time and dose-dependent manner and induce apoptosis only at higher concentrations between 100–300 μM.

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