Abstract
Excessive reactive oxygen species (ROS) induce apoptosis and are associated with various diseases and with aging. SIRT1 (sirtuin-1), an NAD+-dependent protein deacetylase, decreases ROS levels and participates in cell survival under oxidative stress conditions. SIRT1 modulates the transcription factors p53, a tumor suppressor and inducer of apoptosis, and the forkhead O (FOXO) family, both of which play roles for cell survival and cell death. In this study, we aimed to know which is working greatly among p53 and FOXOs transcription factors in SIRT1’s cell protective functions under oxidative stress conditions. The antimycin A-induced increase in ROS levels and apoptosis was enhanced by SIRT1 inhibitors nicotinamide and splitomicin, whereas it was suppressed by a SIRT1 activator, resveratrol, and a SIRT1 cofactor, NAD+. SIRT1-siRNA abolished the effects of splitomicin and resveratrol. p53-knockdown experiment in C2C12 cells and experiment using p53-deficient HCT116 cells showed that splitomicin and resveratrol modulated apoptosis by p53-dependent and p53-independent pathways. In p53-independent cell protective pathway, we found that FOXO1, FOXO3a, and FOXO4 were involved in SOD2’s upregulation by resveratrol. The knockdown of these three FOXOs by siRNAs completely abolished the SOD2 induction, ROS reduction, and anti-apoptotic function of resveratrol. Our results indicate that FOXO1, FOXO3a and FOXO4, are indispensable for SIRT1-dependent cell survival against oxidative stress, although deacetylation of p53 has also some role for cell protective function of SIRT1.
Highlights
Reactive oxygen species (ROS) are generated as a natural byproduct of cellular metabolism
We focused on the roles of p53 and forkhead box O (FOXO) in the anti-oxidative and anti-apoptotic function of SIRT1 in C2C12 cells treated with antimycin A, which increases and releases ROS from mitochondria by inhibiting mitochondrial respiratory chain complex III
Antimycin A significantly elevated the intracellular ROS levels measured by CM-H2DCFDA staining (Figure 1A) and increased the number of apoptotic cells identified by nuclear condensation (Figure 1B) and activated caspase-3 (Figure 1C)
Summary
Reactive oxygen species (ROS) are generated as a natural byproduct of cellular metabolism. They are produced in cells by exogenous sources, such as ionizing radiation and cytotoxic drugs. Excess amounts of ROS induce cell death, which is associated with a wide range of disorders, including cardiovascular, muscular, and neurodegenerative diseases [1,2,3]. Sirtuin-1 (SIRT1) is an NAD+-dependent protein deacetylase, the activation of which significantly decreases ROS levels and promotes cell survival [4]. Irreparable DNA damage by ROS leads to the stabilization and activation of p53 [5], resulting in the expression of pro-apoptotic proteins such as BAX and PUMA, which eventually target the mitochondria and induce apoptosis [6]. The deacetylation of p53 by SIRT1 inhibits p53’s oxidative stress-induced apoptotic activity [7,8]
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