Samantha Neveu

11/20/15

Cellular aging is influenced by the phenotypic change in cells, called senescence.

Resveratrol is a naturally occurring phytoalexin (found within the skin of grapes) and its
known to have antioxidant properties to fight against aging (Frmont 2000). Aging tissues have
been characterized by the accumulation of senescent cells. These cells have experienced a
decrease in their ability to proliferate, which has altered their phenotype. This phenotypic change
causes these cells to behave differently than normal, non-senescent cells. The presence of
senescent cells also hinders the normal cells and structural components around it, interfering with
their normal behavior and function. This ultimately leads to damaged tissues and disease
(Frmont 2000). Certain proteins and genes that play a role in cell longevity have been identified
in past studies, including AMP-activated protein kinase (AMPK), sirtuin 1 (SIRT1), and human
forkhead box O3 gene (FOXO3). Resveratrol is known to be an activator of AMPK and SIRT1.
Human primary keratinocytes were treated with hydrogen peroxide to induce aging in the
cells. While it has been suggested that resveratrol plays a role in anti-aging, the mechanisms at
which it regulates/prevents cellular senescence is unclear. The main focus of this study was to
determine the mechanisms by which AMPK, SIRT1, and FOXO3 regulate the functioning of
cells in the presence of resveratrol.
SIRT1 accompanies AMPK in the regulation of H202 induced senescence
To determine if SIRT1 and AMPK help regulate cell senescence by resveratrol, two
techniques were used: immunochemistry and western blotting. In senescent cells, a lack of
SIRT1 (brown) staining was observed (which is atypical for healthy cells). Hydrogen peroxide
was used to induce senescence, and SA-Gal (a biomarker) was used to measure the presence of

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senescence in the keratinocytes. The cells were also treated with Ex527, a SIRT1 inhibitor, and
resveratrol.
A western blot was used to analyze the treatment of the cells. Western blotting is a
technique commonly used to identify specific proteins. The proteins are separated by
electrophoresis. These results are then transferred to another medium and the target protein is
labeled using 1 and 2antibodies to visualize the results. The protein with the antibodies will
show up as a band; the thickness relates to the amount of protein present (Mahmood and Yang
2012). The control (GADH) is used to determine if the comparison of the target protein is
occurring on an equivalent basis.
This treatment did not affect the activation of AMPK by resveratrol; however it did have
an effect on resveratrols ability to prevent senescence in the cells, as seen by the presence of
SA-Gal. Lastly, cells were treated with lentivirus (a recombinant virus) that expressed SIRT1.
SIRT1 was present in the cytosol of the senesced cells, in comparison to normal (non-senesced)
cells where SIRT1 is present in the nucleus.
This evidence suggests that SIRT1 does not function properly in senescent cells. When
SIRT1 was not functional in the cell, resveratrol was not effective at preventing senescence. This
indicates that the activation of the AMPK pathway by resveratrol was not enough to prevent the
phenotypic change in the cellsSIRT1 needed to be present and functional.
FOXO3 is a common downstream target for Resveratrol activation of AMPK and SIRT1
The role of both AMPK and SIRT1 in resveratrols ability to prevent senescence suggests
that they share a common downstream target in their pathways. FOXO3 belongs to the FOXO
family of transcriptional genes. Unlike endothelial cells, however, the expression of FOXO3 was
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higher than the expression of FOXO1 in the keratinocytes. FOXO3 has a distinct fork head DNA
binding domain, involved in the protecting from oxidative stress and it increases the amount of
antioxidants to combat aging and increase longevity(Maiese et al. 2000). Immunocytocehmistry
(ICC) was used to visualize the localization of FOXO3, and it was mainly found to be present in
the cytosol.
The western blot technique was used again to test resveratrols effect on FOXO3. tAMPK
represented the total amount of the protein, and pAMK represented the phosphorylated (active)
state of the protein. As pAMPK increased so did the expression of FOXO3; kinases activate
other molecules by adding a phosphate group to that protein.
Without resveratrol, AMPK pathway is not activated (does not become phosphorylated).
Without this activation, the expression of FOXO3 is also low in the cells.
Resveratrol activated FOXO3 expression
A Fork Head Responsive Element (FHRE) reporter gene assay was ran to determine if the
resveratrol-induced increase of the FOXO3 protein was parallel to the increase rate of FOXO3
transactivation. The reporter gene assay determined the FOXO3 transcriptional activity.
Luciferase, which is an oxidative enzyme, was used as the reporter gene. When an increase of
Luciferase was observed, it represented an increase in transactivation (enzyme expression) of
FOXO3. As the reporter gene, Luciferase attached to FOXO3 and was activated by FOXO3
(which is why an increase of transactivation of FOXO3, created an increase in Luciferase).
Cells were treated with H2O, and in addition, some were treated with resveratrol. After
treatment, mRNA levels were quantified by real-time PCR. The measured mRNA indicated the
degree of expression of the genes. Real-time PCR allowed for analysis during the early stages of
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reactions, which is a more accurate measure of activity because its observed as the process is
occurring. Greater fold change correlated to a greater expression of specific genes. Therefore,
resveratrol increased FOXO3 transcriptional activity and the expression of FOXO3 target genes.
Resveratrol is an important compound in the prevention of cell senescence
FOXO3 plays a major role with the prevention of aging in cells because it is the factor of
many cellular functions such as: control of the cell cycle, proliferation, resistance to oxidative
stress, apoptosis and more (Maiese et al. 2000). Therefore, the presence and activity of FOXO3
is important for normal cell phenotype and function. For FOXO3 to have an increased
expression, resveratrol, and thereby activated AMPK, needs to be present. SIRT1 was not
observed to affect cell proliferation. However, this gene was needed in order for resveratrols
ability to prevent senescence through the activation of AMPK.

Samantha Neveu

11/20/15

References
Frmont, L. 2000. Biological effects of resveratrol. Life Sciences. 66(8): 663673.
Ido, Y., A. Duranton, F. Lan, K.Weikel, L. Breton, and N. Ruderman. 2015. Resveratrol prevents
oxidative stress-induced senescence and proliferative dysfunction by activating the
AMPKFOXO3 cascade in cultured primary human keratinocytes. PloS ONE: 10(2):1
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Mahmood, T. and P. Yang. 2012. Western blot: technique, theory, and troubleshooting. North
American Journal of Medical Sciences. 4(9): 429434.
Maiese, K., Z. Zhong Chong, J. Hou, and Y. Chen Shang. 2000. The O class: crafting clinical
care with FoxO transcription factors. Madame Curie Bioscience Database.