Tempol has been shown to protect cells and tissues from oxidative damage 23–27. In contrast, it has also been reported that Tempol enhances inflammation and oxidative damage in numerous cell and tissue models 21,24. According to our unpublished data, Tempol at 0.5 ~ 4 mM inhibits the growth of lung cancer and normal cells with an IC50 of approximately 1 ~ 2 mM at 48 h, and induces apoptosis in these cells. Thus, the current study has focused on investigating changes in cellular redox status and antioxidant enzymes in Tempol-treated lung cancer and normal 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 significantly increased in A549 cells treated with 0.5 ~ 4 mM Tempol and in WI-38 VA-13 cells treated with 2 and 4 mM Tempol. However, ROS (DCF) were significantly reduced in Calu-6 cells treated with 0.5 ~ 4 mM Tempol and in WI-38 VA-13 cells treated with 0.5 mM Tempol. In addition, Tempol at 0.5 and 1 mM significantly decreased ROS (DCF) in primary HPF cells. These results imply that Tempol can raise or lower ROS (DCF) levels depending on the cell type without significant differences between lung cancer and normal lung cells. Tempol at 1 mM significantly increased the activity of catalase in Calu-6 cells, possibly resulting in a decrease in intracellular H2O2 level due to the high conversion from H2O2 to O2 and H2O. However, the increased activity of catalase in Tempol-treated A549 cells did not reduce ROS (DCF) levels and the decreased activity of catalase in Tempol-treated WI-38 VA-13 cells did not increase ROS (DCF). In addition, Tempol did not apparently alter the expression of catalase in A549 and WI-38 VA-13 cells and decreased catalase in Calu-6 cells. Therefore, the expression levels of catalase in lung cancer and normal cells did not correlate with its enzymatic activity.
Intracellular O2•− (DHE) levels were increased in A549, Calu-6, WI-38 VA-13, and primary HPF cells treated with 2 or 4 mM Tempol. Tempol also increased O2•− levels in Calu-6 and primary HPF cells at 0.5 and 1 mM but these doses did not augment O2•− levels in A549 and WI-38 VA-13 cells. Relatively high concentrations of Tempol appeared to increase O2•− in each of the cell lines. Tempol is a mimic of SOD which metabolizes O2•− to H2O2 23,30. Similarly, 1 mM Tempol up-regulated the activity of SOD in Calu-6 cells at 48 h. However, Tempol did not affect the activity of SOD in WI-38 VA-13 cells. Tempol at 1 mM even reduced the activity of SOD in A549 cells. Treatment with Tempol increased SOD1 protein levels in Calu-6 cells but not in A549 cells. Contrastingly, at 1 mM it decreased the levels in WI-38 VA-13 cells. Therefore, the expression levels of SOD1 in A549 and WI-38 VA-13 cells were not related to its enzymatic activity. It was speculated that the increased amount of intracellular O2•− 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 33,34,41, suggesting that Tempols effects on mitochondria may result in excessive production of O2•−, causing oxidative stress and consequently apoptosis. In fact, 2 and 4 mM of Tempol strongly increased mitochondrial O2•− levels (as determined by MitoSOX™ Red agent) in WI-38 VA-13 cells at 48 h, by approximately 6-fold and 22-fold, respectively (data not shown). In lung cancer and normal cells, death from Tempol was more correlated with changes in O2•− levels than ROS (DCF) levels. The apparently paradoxical effects of Tempol in different conditions and different cells may be explained by assuming that each cell has a different basal activity of mitochondria and a variety of antioxidant enzymes.
The Trx system consists of Trx, TrxR, and NADPH and is a fundamental enzymatic complex that preserves cellular redox homeostasis 14. Trx1 and/or TrxR1 are overexpressed in various pulmonary diseases including cancer 16,42−44. Hence, it is plausible that the Trx system is a promising target for treatment of lung cancer. Tempol at 1 mM significantly decreased the activity of TrxR1 in A549, Calu-6, and Wi-38 VA-13 cells. However, TrxR1 protein levels were increased in all these lung cells. Thus, the increased activity of TrxR1 in Tempol-treated lung cells is more likely to be attributed to other factors such as Trx and Trx-interacting proteins rather than simple expression levels. In addition, Tempol treatment increased Trx1 protein expression in A549 cells and WI-38 VA-13 cells but not in Calu-6 cells. Further research is needed on the mechanism underlying alterations in the Tempol-mediated Trx system.
Treatment of Tempol at 1 ~ 5 mM inhibits cell growth and induces apoptosis in various cancer cells 33–36,41,45. 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. Moreover, Tempol at 1 mM also significantly increased the amounts of annexin V-positive cells in scramble siRNA-transfected A549, Calu-6, and Wi-38 VA-13 cells by about 30%, 24%, and 22% at 48 h, respectively. Therefore, Tempol is likely to induce lung cell death via apoptosis and/or necrosis. There was no significant difference in the susceptibility to Tempol among these lung cancer and normal cells. TrxR1 silencing itself did not influence the growth of Calu-6 and Wi-38 VA-13 cells compared with scramble siRNA-transfected control cells but it did inhibit the growth of A549 control cells by about 20%. Transfection of TrxR1 siRNA did not significantly alter the growth rates of Tempol-treated lung cancer and normal cells. Thus, decreased TrxR1 protein level was involved in growth inhibition of A549 cells and did not affect growth changes in response to Tempol in these lung cells. In addition, TrxR1 siRNA alone did not increase the amounts of annexin V-positive cells in A549, Calu-6, and Wi-38 VA-13 control cells. Furthermore, knock-down of TrxR1 slightly decreased cell death in Tempol-treated A549 cells and significantly attenuated cell death in Tempol-treated Wi-38 VA-13 cells. These results suggest that down-regulated TrxR1 levels are not closely related to cell survival in Tempol-naive lung cells but in part to cell survival in Tempol-treated cells.
A549 cells transfected with TrxR1 siRNA showed slightly decreased ROS (DCF) levels at 48 h while transfected Calu-6 cells showed slight increases. TrxR1 siRNA reduced the increased levels of ROS (DCF) in Tempol-treated A549 cells whereas silencing partially recovered the decreased levels of ROS (DCF) in Tempol-treated Calu-6 cells. In Wi-38 VA-13 cells, TrxR1 siRNA did not alter ROS (DCF) levels regardless of 1 mM Tempol treatment. It has been reported that Trx or TrxR1 could regulate the basal cellular metabolism of glycolysis, tricarboxylic acid cycle, and mitochondria to produce adenosine triphosphate (ATP) or NAD(P)H 46. In addition, there are potentially harmful interactions between Tempol and enzyme complexes, which are inolved in glucose transport, glutamine metabolism and mitochondiral ATP production 21,24,34. Therefore, further studies are needed on the difference in cellular ROS levels in lung cancer and normal cells following downregulation of TrxR1 and treatment with Tempol.
Depletion of GSH promotes cell death 47–49 and this has been reported to occur in Tempol-treated leukemia and cervical cancer cells 41,50. Similarly, the present study demonstrated an increased proportion of GSH-depleted cells in A549, Calu-6, and Wi-38 VA-13 cells treated with 2 and 4 mM Tempol. Lower doses of Tempol (0.5 and 1 mM) induced fewer GSH-depleted cells in Calu-6 cells. These results support the notion that the induction of cell death was inversely proportional to GSH content 47–49. However, 1 mM Tempol, which showed cell growth inhibition and cell death in A549 and WI-38 VA-13 cells, did not significantly augment the number of GSH-depleted cells in these cells. GSH content is a crucial factor or indicator of cell death induced by Tempol, but its concentration alone is not sufficient to accurately predict cell death.
In summary, the present results have shown that Tempol at 0.5 ~ 4 mM could either increase or decrease ROS (DCF) levels in lung cancer and normal cells and this drug specifically increased O2•− levels and GSH depletion. Tempol at 1 mM inhibited the growth of lung cancer and normal cells and also induced their cell death. There was no significant difference in Tempol sensitivity between lung cancer and normal cells. In addition, Tempol had other effects on the expression and activity of antioxidant enzymes, especially TrxR1 in lung cancer and normal cells. Furthermore, down-regulation of TrxR1 partially affected cell growth and death as well as ROS (DCF) levels in both Tempol-untreated or -treated cells, depending on the cell type. The current results provide valuable information for understanding the cellular effects of Tempol on lung cancer and normal cells with regard to cell growth inhibition and cell death, as well as redox states (ROS and GSH levels) and various antioxidant pathways.