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KMID : 1239920070010020105
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Nutrition Research and Practice
2007 Volume.1 No. 2 p.105 ~ p.112
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Ascorbic acid extends replicative life span of human embryonic fibroblast by reducing DNA and mitochondrial damages
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Hwang Won-Sang
Kim Min-Ju
Oh Soo-Jin
Park Seong-Hoon
Kang Hong-Jun
Lee Jae-Yong
Kim Jae-Bong
Park Jae-Bong
Kim Hyun-Seok
Kim Sung-Chan
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Abstract
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Ascorbic acid has been reported to extend replicative life span of human embryonic fibroblast (HEF). Since the detailed molecular mechanism of this phenomenon has not been investigated, we attempted to elucidate. Continuous treatment of HEF cells with ascorbic acid (at 200 μM) from 40 population doubling (PD) increased maximum PD numbers by 18% and lowered SA-β-gal positive staining, an aging marker, by 2.3 folds, indicating that ascorbic acid extends replicative life span of HEF cells. Ascorbic acid treatment lowered DCFH by about 7 folds and Rho123 by about 70%, suggesting that ascorbic acid dramatically decreased ROS formation. Ascorbic acid also increased aconitase activity, a marker of mitochondrial aging, by 41%, indicating that ascorbic acid treatment restores age-related decline of mitochondrial function. Cell cycle analysis by flow cytometry revealed that ascorbic acid treatment decreased G1 population up to 12%. Further western blot analysis showed that ascorbic acid treatment decreased levels of p53, phospho-p53 at ser 15, and p21, indicating that ascorbic acid relieved senescence-related G1 arrest. Analysis of AP (apurinic/apyrimidinic)
sites showed that ascorbic acid treatment decreased AP site formation by 35%. We also tested the effect of hydrogen peroxide treatment, as an additional oxidative stress. Continuous treatment of 20 μM of hydrogen peroxide from PD 40 of HEF cells resulted in premature senescence due to increased ROS level, and increased AP sites. Taken together, the results suggest that ascorbic acid extends replicative life span of HEF cells by reducing mitochondrial and DNA damages through lowering cellular ROS.
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KEYWORD
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Ascorbic acid, human embryonic fibroblasts, replicative life span, mitochondrial damage, DNA damage, reactive oxygen species
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