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. 2022 Oct 28;27(21):7341.
doi: 10.3390/molecules27217341.

Tempol Inhibits the Growth of Lung Cancer and Normal Cells through Apoptosis Accompanied by Increased O2•- Levels and Glutathione Depletion

Woo Hyun Park  1
Affiliations

Tempol Inhibits the Growth of Lung Cancer and Normal Cells through Apoptosis Accompanied by Increased O2•- Levels and Glutathione Depletion

Woo Hyun Park. Molecules. .

Abstract

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a stable, cell-permeable redox-cycling nitroxide water-soluble superoxide dismutase (SOD) mimetic agent. However, little is known about its cytotoxic effects on lung-related cells. Thus, the present study investigated the effects of Tempol on cell growth and death as well as changes in reactive oxygen species (ROS) and glutathione (GSH) levels in Calu-6 and A549 lung cancer cells, normal lung WI-38 VA-13 cells, and primary pulmonary fibroblast cells. Results showed that Tempol (0.5~4 mM) dose-dependently inhibited the growth of lung cancer and normal cells with an IC50 of approximately 1~2 mM at 48 h. Tempol induced apoptosis in lung cells with loss of mitochondrial membrane potential (MMP; ∆Ψm) and activation of caspase-3. There was no significant difference in susceptibility to Tempol between lung cancer and normal cells. Z-VAD, a pan-caspase inhibitor, significantly decreased the number of annexin V-positive cells in Tempol-treated Calu-6, A549, and WI-38 VA-13 cells. A 2 mM concentration of Tempol increased ROS levels, including O2•- in A549 and WI-38 VA-13 cells after 48 h, and specifically increased O2•- levels in Calu-6 cells. In addition, Tempol increased the number of GSH-depleted cells in Calu-6, A549, and WI-38 VA-13 cells at 48 h. Z-VAD partially downregulated O2•- levels and GSH depletion in Tempol-treated these cells. In conclusion, treatment with Tempol inhibited the growth of both lung cancer and normal cells via apoptosis and/or necrosis, which was correlated with increased O2•- levels and GSH depletion.

Keywords: cell death; glutathione; human pulmonary fibroblast; lung cancer cells; mitochondrial membrane potential; reactive oxygen species; tempol.

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Conflict of interest statement

The author declares no conflict of interest.

Figures

Figure 1
Figure 1
Effects of Tempol on the growth of normal and lung cancer cells. Exponentially growing cells were incubated with Tempol at indicated concentrations for 48 h. Cell growth was evaluated by MTT assays. Graphs show the growth of Calu-6 cancer cells (A), A549 cancer cells (B), WI-38 VA-13 normal cells (C), and primary HPF normal cells (D). Student’s t-test was used. * p < 0.05 compared to Tempol-untreated control cells.
Figure 2
Figure 2
Effects of Tempol on sub-G1 cells of normal and lung cancer cells. Cells in exponential growth phase were incubated with Tempol at indicated concentrations for 48 h. Cells in sub-G1 phase were measured with a FACStar flow cytometer. Graphs show proportions of sub-G1 cells in Calu-6 cells (A), A549 cells (B), WI-38 VA-13 cells (C), and primary HPF cells (D). Student’s t-test was used. * p < 0.05 compared to Tempol-untreated control cells.
Figure 3
Figure 3
Effects of Tempol on annexin V-positive lung cancer and normal cells. Cells in exponential growth phase were incubated with Tempol at indicated concentrations for 48 h. Annexin V-FITC positive cells were evaluated with a FACStar flow cytometer. Graphs show the proportion of annexin V-positive Calu-6 cells (A), A549 cells (B), WI-38 VA-13 cells (C), and primary HPF cells (D). Student’s t-test was used. * p < 0.05 compared to Tempol-untreated control cells.
Figure 4
Figure 4
Effects of Tempol on MMP (∆Ψm) levels in lung cancer and normal cells. Exponentially growth of cells incubated with Tempol at indicated concentrations for 48 h. MMP (∆Ψm) in lung cells was measured using a FACStar flow cytometer. Graphs show the proportion of rhodamine 123-negative [MMP (∆Ψm) loss] Calu-6 cells (A), A549 cells (B), WI-38 VA-13 cells (C), and primary HPF cells (D). Student’s t-test was used. * p < 0.05 compared to Tempol-untreated control cells.
Figure 5
Figure 5
Effects of Tempol and/or Z-VAD on caspase-3 activity and cell death in Calu-6, A549, and WI-38 VA-13 cells. Exponentially growing cells were pretreated with Z-VAD for 1 h and then treated with 2 mM Tempol for 48 h. (A): Graph shows the activities of caspase-3 in Calu-6, A549, and Wi-38 VA-13 cells, measured via Colorimetric Assay. (B–D): Graphs show the proportion of annexin V-positive Calu-6 cells (B), A549 cells (C), and WI-38 VA-13 cells (D), measured with a FACStar flow cytometer. ANOVA test was used. * p < 0.05 compared to Tempol-untreated control cells. # p < 0.05 compared to cells treated with Tempol only.
Figure 6
Figure 6
Effects of Tempol and/or Z-VAD on ROS levels in Calu-6, A549, and WI-38 VA-13 cells. Exponentially growing cells were pretreated with Z-VAD for 1 h and then treated with 2 mM Tempol for 48 h. Intracellular DCF (ROS) and DHE (O2•−) levels in lung cells were measured using a FACStar flow cytometer. (A–C): The graphs indicate the mean DCF (ROS) levels (%) in Calu-6 (A), A549 cells (B), and WI-38 VA-13 cells (C). (D–F): The graphs indicate the mean DHE (O2•−) levels (%) in Calu-6 (D), A549 cells (E), and WI-38 VA-13 cells (F). ANOVA test was used. * p < 0.05 compared to Tempol-untreated control cells. # p < 0.05 compared to cells treated with Tempol only.
Figure 7
Figure 7
Effects of Tempol and/or Z-VAD on intracellular GSH depletion in Calu-6, A549, and WI-38 VA-13 cells. Exponentially growing cells were pretreated with Z-VAD for 1 h and then treated with 2 mM Tempol for 48 h. The intracellular CMF (GSH) levels in lung cells were measured using a FACStar flow cytometer. The graphs indicate the percentages of (-) CMF (GSH-depleted) Calu-6 cells (A), A549 cells (B), and WI-38 VA-13 cells (C). ANOVA test was used. * p < 0.05 compared to Tempol-untreated control cells. # p < 0.05 compared to cells treated with Tempol only.
Figure 8
Figure 8
Schematic diagram of Tempol-induced growth inhibition of lung cells.
See this image and copyright information in PMC

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