Abstract
SummaryThe selective depletion of senescent cells (SCs) by small molecules, termed senolytic agents, is a promising therapeutic approach for treating age‐related diseases and chemotherapy‐ and radiotherapy‐induced side effects. Piperlongumine (PL) was recently identified as a novel senolytic agent. However, its mechanism of action and molecular targets in SCs was unknown and thus was investigated. Specifically, we used a PL‐based chemical probe to pull‐down PL‐binding proteins from live cells and then mass spectrometry‐based proteomic analysis to identify potential molecular targets of PL in SCs. One prominent target was oxidation resistance 1 (OXR1), an important antioxidant protein that regulates the expression of a variety of antioxidant enzymes. We found that OXR1 was upregulated in senescent human WI38 fibroblasts. PL bound to OXR1 directly and induced its degradation through the ubiquitin‐proteasome system in an SC‐specific manner. The knockdown of OXR1 expression by RNA interference significantly increased the production of reactive oxygen species in SCs in conjunction with the downregulation of antioxidant enzymes such as heme oxygenase 1, glutathione peroxidase 2, and catalase, but these effects were much less significant when OXR1 was knocked down in non‐SCs. More importantly, knocking down OXR1 selectively induced apoptosis in SCs and sensitized the cells to oxidative stress caused by hydrogen peroxide. These findings provide new insights into the mechanism by which SCs are highly resistant to oxidative stress and suggest that OXR1 is a novel senolytic target that can be further exploited for the development of new senolytic agents.
Highlights
Cellular senescence occurs when irreversible cell cycle arrest is triggered by telomere shortening or exposure to stress (Campisi, 2013)
PL bound to oxidation resistance 1 (OXR1) directly and induced its degradation through the ubiquitin-proteasome system in an senescent cells (SCs)-specific manner
Data in (a) and (b) are the mean Æ SE of fold changes in mRNA expression compared with shCtrl-transfected cells from two to three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. shCtrl by unpaired t test. (c) Percent viability of nonsenescent WI38 cells (NCs) and ionizing radiation (IR)-SCs transfected with shCtrl or shOXR1 presented as the mean Æ SE of two independent experiments. *p < 0.05 vs. IR-SCs transfected with shCtrl by unpaired t test. (d) Levels of intracellular ROS in NCs and IR-SCs transfected with shCtrl or shOXR1 in the presence or absence of 2 mM N-acetyl-L-cysteine (NAC)
Summary
Cellular senescence occurs when irreversible cell cycle arrest is triggered by telomere shortening or exposure to stress (Campisi, 2013). Senescent cells (SCs) accumulate if they cannot be removed rapidly by the immune system due to immune dysfunction and/or a sustained, overwhelming increase in SC production This occurs during aging or under certain pathological conditions (Childs et al, 2017; Mun~oz-Espın & Serrano, 2014). Knocking down OXR1 selectively induced apoptosis in SCs and sensitized the cells to oxidative stress caused by hydrogen peroxide (H2O2) These findings suggest that OXR1 is a potential senolytic target that can be exploited for the development of selective senolytic agents with improved potency and selectivity. These findings provide new insight into the mechanism by which SCs are highly resistant to oxidative stress
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