Tempol inhibits growth of As4.1 juxtaglomerular cells via cell cycle arrest and apoptosis

Abstract

Abstract.A stable nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-osyl (Tempol) is widely used as an antioxidant in vitro and in vivo. In this study, we investigated the effects of Tempol on the growth of As4.1 juxtaglomerular cells in relation to cell cycle and cell death. Tempol dose-dependently decreased the growth of As4.1 cells with an IC 50 of ~1 mM at 48 h. DNA flow cytometry analysis and BrdU staining indicated that Tempol induced S phase arrest, which is accompanied by a downregulation of cyclin A. Tempol also induced apoptotic cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; ∆Ψm), an activation of caspase-3 and cleavage of poly(ADP-ribose)polymerase-1 (PARP-1). Furthermore, Tempol increased reactive oxygen species (ROS) levels, and the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). MEK and JNK inhibitors significantly attenuated a growth inhibition in Tempol-treated As4.1 cells. In conclusion, Tempol inhibited the growth of As4.1 cells via cell cycle arrest and apoptosis. Tempol also activated ERK and JNK signaling, which was responsible for cell growth inhibition. Our present data provide useful information for the toxicological effects of Tempol in juxtaglomerular cells in relation to cell growth inhibition and cell death. IntroductionTempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl or 4-hydroxy-tempo) is a stable piperidine nitroxide, which is widely employed in electron spin resonance spectroscopy (1). It is also known that Tempol protects cells from oxidative damage in vitro and in vivo (2). Several antioxidant mechanisms have been proposed to account for these protective effects. It may act as a mimic of superoxide dismutase (SOD) and it can reduce the formation of hydroxyl radicals either by scavenging superoxide anion or by reducing intracellular concentration of Fe(II) (3). Usually, such beneficial effects are observed up to a few µM. However, the prospect of moving it from the experimental to the clinical arena has prompted more detailed studies on their possible toxicity. Much evidence suggests that Tempol impairs ferritin synthesis and Fe metabolisms, consequently leading to cell death (4). In addition, mutagenic effects has been reported in different bacterial strains (5). Of note, Tempol as a pro-oxidant induces the cell cycle arrest and apoptosis depending on cell types (6). Therefore, in order to clarify these discrepancies among the different effects of Tempol, further study needs to be performed for re-evaluating its real biological functions and roles.The mitogen-activated protein kinases (MAPKs) are a large family of serine/threonine kinases, which are major comp onents of signaling pathways in cell proliferation, differentiation, and cell death (7). There are currently three known MAPKs: the extracellular signal-regulated kinase (ERK1/2), the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and the p38 (7). Each MAPK pathway has relatively different upstream activat...

Tempol differently affects cellular redox changes and antioxidant enzymes in various lung-related cells

“…In contrast, it has also been reported that Tempol enhances inflammation and oxidative damage in numerous cell and tissue models 21 , 23 . In addition, millimole concentrations of Tempol or extended exposure to it increases ROS levels in juxtaglomerular cells 32 , breast cancer cells 34 , and ovarian cancer cells 33 , 35 . The current study has focused on investigating changes in cellular redox changes and antioxidant enzymes in Tempol-treated lung cancer and normal cells.…”

“…It was speculated that the increased amount of intracellular O 2 •− in cells in response to Tempol may have been originated from mitochondria. Numerous reports have demonstrated that Tempol mediates its toxicity via the disruption of mitochondrial function 32 , 33 , 41 , suggesting that Tempol’s effects on mitochondria may result in excessive production of O 2 •− , causing oxidative stress and consequently apoptosis. In fact, 2 and 4 mM of Tempol strongly increased mitochondrial O 2 •− levels in WI-38 VA-13 cells at 48 h, by approximately sixfold and 22-fold, respectively (Supplementary Fig.…”

“…It has been reported that Tempol at millimole concentrations inhibits cell growth and induces apoptosis, depending on cell types and origins 31 – 33 . Moreover, high concentrations of, or extended exposure to Tempol increases ROS levels in juxtaglomerular cells 32 , breast cancer cells 34 , and ovarian cancer cells 33 , 35 . Thus, in order to clarify the different effects of Tempol as pro-oxidant or antioxidant in cells and tissues, further in depth studies need to be undertaken to elucidate its biological mechanisms.…”

Tempol Differently Affects Cellular Redox Status and Antioxidant Enzymes in Various Lung-related Cells

“…It is reported that millimole concentrations of Tempol or extended exposure to it increases ROS levels in juxtaglomerular cells 33 , breast cancer cells 35 , and ovarian cancer cells 34,36 . Results from the present study showed that ROS (DCF) levels were signi cantly increased in A549 cells treated with 0.…”

“…Treatment of Tempol at 1 ~ 5 mM inhibits cell growth and induces apoptosis in various cancer cells [33][34][35][36]41,45 . Likewise, 1 mM Tempol reduced the growth of scramble siRNA-transfected A549 and Calu-6 lung cancer cells, and Wi-38 VA-13 normal cells by about 55%, 50%, and 55% at 48 h, respectively.…”