Phenyl 2‐pyridyl ketoxime induces cellular senescence‐like alterations via nitric oxide production in human diploid fibroblasts

Abstract

SummaryPhenyl‐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.

Nectandrin B-mediated activation of the AMPK pathway prevents cellular senescence in human diploid fibroblasts by reducing intracellular ROS levels

“…Primary HDFs were isolated and cultured as described previously [4]. Cells were maintained in DMEM containing 10% FBS and antibiotics.…”

“…Young and old HDF cells were fixed and stained for SA-β-gal activity as described previously [4]. Cells were washed twice with phosphate buffered saline (PBS) and then fixed with 2% formaldehyde/0.2% glutaraldehyde in PBS for 10 min.…”

“…Normal HDFs replicate a finite number of times in vitro and then enter a state of terminal growth arrest, termed as cellular senescence [3]. Senescent cells can be identified by the enhanced expression of senescence markers, such as senescence-associated β-galactosidase (SA-β-gal), senescence-associated secretory phenotype, senescence-associated heterochromatin foci, and increased expression of cell cycle-related proteins, including p21 waf1 , p53, p16 Ink4a , and cyclin D1/2 [4]. Cellular senescence is closely linked with aging as well as the development and progression of aging-associated diseases.…”

Downregulation of B‐myb promotes senescence via the ROS‐mediated p53/p21 pathway, in vascular endothelial cells

“…ROS can damage many cellular components and lead to oxidative stress promoting cellular senescence . ROS accumulation is considered to be one of major cause contributing to senescence or ageing and age‐related diseases.…”