Phenyl 2‐pyridyl ketoxime induces cellular senescence‐like alterations via nitric oxide production in human diploid fibroblasts |
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| Authors: | Kyeong Eun Yang Hyun‐Jin Jang In‐Hu Hwang Young‐Ho Chung Jong‐Soon Choi Tae‐Hoon Lee Yun‐Jo Chung Min‐Seung Lee Mi Young Lee Eui‐Ju Yeo Ik‐Soon Jang |
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| Affiliation: | 1. Drug & Disease Target Group, Division of Bioconvergence Analysis, Korea Basic Science 2. Institute, Daejeon, Korea;3. Department of Physiology, Korea University College of Medicine, Seoul, Korea;4. Department of Oral Biochemistry, Dental Science Research Institute, Chonnam National University, Gwangju, Korea;5. Center for University‐Wide Research Facilities, Chonbuk National University, Jeonju, Korea;6. Department of Biochemistry, College of Medicine, Gachon University, Inchon, Korea;7. KM Convergence Research Division, Korea Institute of Oriental Medicine, Daejeon, Korea |
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| Abstract: | Phenyl‐2‐pyridyl ketoxime (PPKO) was found to be one of the small molecules enriched in the extracellular matrix of near‐senescent human diploid fibroblasts (HDFs). Treatment of young HDFs with PPKO reduced the viability of young HDFs in a dose‐ and time‐dependent manner and resulted in senescence‐associated β‐galactosidase (SA‐β‐gal) staining and G2/M cell cycle arrest. In addition, the levels of some senescence‐associated proteins, such as phosphorylated ERK1/2, caveolin‐1, p53, p16ink4a, and p21waf1, were elevated in PPKO‐treated cells. To monitor the effect of PPKO on cell stress responses, reactive oxygen species (ROS) production was examined by flow cytometry. After PPKO treatment, ROS levels transiently increased at 30 min but then returned to baseline at 60 min. The levels of some antioxidant enzymes, such as catalase, peroxiredoxin II and glutathione peroxidase I, were transiently induced by PPKO treatment. SOD II levels increased gradually, whereas the SOD I and III levels were biphasic during the experimental periods after PPKO treatment. Cellular senescence induced by PPKO was suppressed by chemical antioxidants, such as N‐acetylcysteine, 2,2,6,6‐tetramethylpiperidinyloxy, and L‐buthionine‐(S,R)‐sulfoximine. Furthermore, PPKO increased nitric oxide (NO) production via inducible NO synthase (iNOS) in HDFs. In the presence of NOS inhibitors, such as L‐NG‐nitroarginine methyl ester and L‐NG‐monomethylarginine, PPKO‐induced transient NO production and SA‐β‐gal staining were abrogated. Taken together, these results suggest that PPKO induces cellular senescence in association with transient ROS and NO production and the subsequent induction of senescence‐associated proteins . |
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| Keywords: | cellular senescence human diploid fibroblast nitric oxide phenyl 2‐pyridyl ketoxime reactive oxygen species |
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