Abstract
A high-throughput anti-aging drug screen was developed that simultaneously measures senescence-associated β-galactosidase activity and proliferation. Applied to replicatively pre-aged fibroblasts, this screen yielded violuric acid (VA) and 1-naphthoquinone-2-monoxime (N2N1) as its top two hits. These lead compounds extended the replicative life spans of normal and progeroid human cells in a dose-dependent manner and also extended the chronological life spans of mice and C. elegans. They are further shown here to function as redox catalysts in oxidations of NAD(P)H. They thus slow age-related declines in NAD(P)+/NAD(P)H ratios. VA participates in non-enzymatic electron transfers from NAD(P)H to oxidized glutathione or peroxides. N2N1 transfers electrons from NAD(P)H to cytochrome c or CoQ10 via NAD(P)H dehydrogenase (quinone) 1 (NQO1). Our results indicate that pharmacologic manipulation of NQO1 activity via redox catalysts may reveal mechanisms of senescence and aging.
Highlights
Aging and senescence are accompanied by systemic changes in the structural integrity of cells that are caused by alterations in metabolic and signal transduction pathways.[1]
Screen for anti-aging agents The classic method for senesce associated β-galactosidase (SAβG) activity[4] utilizes ferricyanide/ ferrocyanide to amplify the X-gal development in formaldehyde fixed cells
Β-galactosidase was released into a solution compatible with its enzymatic activity by adding Triton X100 and a catalyst, nitro blue tetrazolium salt (NBT), to shorten assay processing times
Summary
Aging and senescence are accompanied by systemic changes in the structural integrity of cells that are caused by alterations in metabolic and signal transduction pathways.[1] The proximal events which initiated the deterioration of complex cellular systems result in dramatic cell cycling deceleration and establishment of metabolically perplexed, irreversible non-dividing state. Early observations have indicated that normal cells are characterized by a limited replicative lifespan (RLS).[2,3] The senesce associated β-galactosidase (SAβG) activity is considered one of the classic hall-marks of cell senescence.[4] Among the others are telomere deterioration, multiple epigenetics changes in histones and DNA, metabolic perturbations caused by tendency of aging cells to rely more on glycolysis, skewed mitochondrial dynamic toward more segregated, less respiring mitochondria.[5].
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