Reactive oxygen species (ROS), especially hydrogen peroxide (H2O2), induce apoptosis in cancer cells by regulating mitogen-activated protein kinase (MAPK) signaling pathways. The present study investigated the effects of MAPK inhibitors on cell growth and death as well as changes in ROS and glutathione (GSH) levels in H2O2-treated Calu-6 and A549 lung cancer cells. H2O2 inhibited growth and induced death of Calu-6 and A549 lung cancer cells. All MAPK inhibitors appeared to enhance growth inhibition in H2O2-treated Calu-6 and A549 lung cancer cells and increased the percentage of Annexin V-FITC-positive cells in these cancer cells. Among the MAPK inhibitors, a JNK inhibitor significantly augmented the loss of mitochondrial membrane potential (MMP; ΔΨm) in H2O2-treated Calu-6 and A549 lung cancer cells. Intracellular ROS levels were significantly increased in the H2O2-treated cells at 1 and 24 h. Only the JNK inhibitor increased ROS levels in the H2O2-treated cells at 1 h and all MAPK inhibitors raised superoxide anion levels in these cells at 24 h. In addition, H2O2 induced GSH depletion in Calu-6 and A549 cells and the JNK inhibitor significantly enhanced GSH depletion in H2O2-treated cells. Each of the MAPK inhibitors altered ROS and GSH levels differently in the Calu-6 and A549 control cells. In conclusion, H2O2 induced growth inhibition and death in lung cancer cells through oxidative stress and depletion of GSH. The enhanced effect of MAPK inhibitors, especially the JNK inhibitor, on cell death in H2O2-treated lung cancer cells was correlated with increased O2•− levels and GSH depletion.
Superoxide anion (O2•−), hydroxyl radical (•OH) and hydrogen peroxide (H2O2) are unstable and highly reactive oxygen species (ROS). Although ROS are conventionally harmful or detrimental to cells, they specifically regulate a variety of cellular procedures such as cell proliferation, differentiation and apoptosis (
Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved signaling proteins that mediate responses to assorted stimuli. Extracellular signal regulated kinases (ERK1/2), the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPK) and the p38 kinases are the three main MAPK groups in mammalians/eukaryotes (
Lung cancer is a major cause of cancer-related mortality in developed countries. The carcinogenesis of lung cancer is closely related to tissue inflammation mediated by ROS. During inflammation, the concentration of H2O2 in tissues is anticipated to reach approximately millimolar levels (
The human lung cancer cell lines, Calu-6 and A549, were obtained from the Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI-1640 medium (GE Healthcare Life Sciences, Logan, UT, USA) with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaAA, Darmstadt, Germany) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cells were routinely cultivated in 100-mm plastic tissue culture dishes (Nalge Nunc International, Penfield, NY, USA) in a humidified incubator containing 5% CO2, at 37°C and harvested in a solution of trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.) while in a logarithmic phase of growth.
H2O2 was purchased from Sigma-Aldrich; Merck KGaAA. The MEK inhibitor (PD98059) as well as the JNK (SP600125) and p38 (SB203580) inhibitors were obtained from Calbiochem (San Diego, CA, USA). All reagents were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaAA) at 10 mM. The cells were pretreated with each MAPK inhibitor for 30 min prior to treatment with H2O2. Based on previous experiments (
The effect of the drugs on lung cancer cell growth was determined by evaluating 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich; Merck KGaAA) dye absorbance as previously described (
Sub-G1 cells were detected using propidium iodide dye (PI; Sigma-Aldrich; Merck KGaA) as previously described (
Apoptotic cell death was evaluated by measuring cell stained with Annexin V-fluorescein isothiocyanate (FITC; Molecular Probes; Thermo Fisher Scientific, Inc.) as previously described (
MMP was assessed using Rhodamine 123 mitochondrial-specific fluorescent dye (Sigma-Aldrich; Merck KGaAA) as previously described (
The intracellular ROS levels were evaluated by a fluorescent probe dye, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes; Thermo Fisher Scientific, Inc.) at 1 or 24 h as previously described (
The GSH levels were evaluated by means of a 5-chloromethylfluorescein diacetate dye (CMFDA; Molecular Probes; Thermo Fisher Scientific, Inc.) at 1 or 24 h, as previously described (
The results represent the mean of at least two independent experiments (mean ± SD). The Student's t-test or one-way ANOVA with post hoc analysis using Tukey's multiple comparison tests was used for parametric data. The results were considered statistically significant at P<0.05.
The cell growth and death effects of MAPK inhibitors (MEK, JNK and p38 inhibitors) were examined in H2O2-treated lung cancer cells. The same inhibitors were used to block MAPK signaling pathways in previous studies (
Using another lung cancer cell line, the A549 cells, a 100 µM H2O2-treatment induced growth inhibition to ~50% within 24 h (
Cell death is closely correlated with the collapse of MMP (
Changes in ROS levels were assessed in Calu-6 and A549 cells treated with H2O2 with or without each MAPK inhibitor. To determine whether the intracellular ROS levels in H2O2-treated lung cancer cells were altered by treatment with each MAPK inhibitor, ROS levels were assessed at the early time-point of 1 h (
As depicted in
Treatment with 75 and 100 µM H2O2 increased the intracellular ROS (DCF and DHE) levels in Calu-6 and A549 cells at 24 h (
Changes in GSH levels were assessed in Calu-6 and A549 cells treated with H2O2 with or without each MAPK inhibitor at 1 h (
Treatment with 75 µM H2O2 for 24 h resulted in an increase to ~25% in the GSH-depleted Calu-6 cells compared to the H2O2-untreated control cells (
Treatment with 75 and 100 µM H2O2 increased the number of sub-G1 and Annexin V-FITC-positive cells in Calu-6 and A549 lung cancer cells, accompanied by the downregulation of Bcl-2 and procaspase-3 proteins and activation of caspase-3 and −8 (data not shown). These results indicated that H2O2 induced cell death in these lung cancer lines via caspase-dependent apoptosis. The present study focused on evaluating the effects of MAPK inhibitors on cell growth and cell death as well as intracellular ROS and GSH levels in H2O2-treated lung cancer cells.
Usually, the ERK signaling pathway is pro-survival rather than pro-apoptotic (
ROS can augment the disturbance of redox status in cells by triggering a breakdown in MMP (
The main ROS related to cell signaling pathways are O2•− and H2O2. Intracellular ROS levels, including O2•−,were significantly increased in both lung cancer cells treated with H2O2 at 1 and 24 h. Treatment with 75 and 100 µM H2O2 directly produced O2•− by impairing the mitochondrial membrane function and both H2O2 and O2•− can be efficiently converted into toxic •OH via the Fenton reaction to destroy these cancer cells. However, H2O2 slightly increased O2•− (DHE) levels in A549 cells compared to Calu-6 cells at 1 h, indicating that it does not have a strong effect on both mitochondrial respiratory transport chain and the activity of various oxidases to generate O2•− in A549 cells within this early time-point. Only the JNK inhibitor significantly enhanced the increased ROS (DCF) levels in H2O2-treated Calu-6 and A549 cells at 1 h. Both JNK and p38 inhibitors increased O2•− (DHE) levels in H2O2-treated A549 cells at 1 h. In contrast, the MEK inhibitor decreased O2•− (DHE) levels in H2O2-treated Calu-6 cells at 1 h and the p38 inhibitor decreased ROS (DCF) levels in these cells at 1 h. In addition, none of the MAPK inhibitors significantly altered ROS (DCF) levels in H2O2-treated lung cancer cells at 24 h. However, all MAPK inhibitors, especially the JNK and p38 inhibitors augmented DHE (O2•−) levels in H2O2-treated Calu-6 and A549 cells at 24 h. Thus, it is plausible that the enhancement of H2O2-induced lung cancer cell death by MAPK inhibitors, especially the JNK inhibitor, is more related to the levels of O2•− (DHE) rather than ROS (DCF). Furthermore, although the JNK and p38 inhibitors significantly increased ROS (DCF) levels in Calu-6 control cells at 1 and 24 h, these inhibitors did not significantly provoke cell death and MMP loss. Additionally, each MAPK inhibitor had different effects on basal ROS (DCF) and O2•− (DHE) levels in H2O2-untreated Calu-6 and A549 control cells regardless of cell death. Since changes in O2•− and H2O2 levels by these MAPK inhibitors and the outcomes of their corresponding signaling pathways influenced by these types of ROS are complex in cells and different in each cell type, the detailed molecular mechanisms underlying the effects of MAPK inhibitors and their relationship between ROS and cell death require further study.
GSH is an important tripeptide (non-protein) antioxidant in cells. GSH content has a vital effect on cell death (
In conclusion, exogenous H2O2 induced growth inhibition and cell death in Calu-6 and A549 lung cancer cells by increasing intracellular ROS and depleting GSH. All MAPK inhibitors generally amplified H2O2-induced lung cancer cell death. Especially, the enhanced cell death and MMP loss effects of the JNK inhibitor in H2O2-treated lung cancer cells were related to increased O2•− (DHE) levels and GSH depletion.
This study was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP; 2016R1A2B4007773) and supported by the ‘Research Base Construction Fund Support Program’ funded by Chonbuk National University in 2017.
hydrogen peroxide
reactive oxygen species
glutathione
mitogen-activated protein kinase
MAP kinase or ERK kinase
extracellular signal-regulated kinase
c-Jun N-terminal kinase
mitochondrial membrane potential
3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide
fluorescein isothiocyanate
2′,7′-dichlorodihydrofluorescein diacetate
dihydroethidium
5-chloromethylfluorescein diacetate
propidium iodide
Effects of MAPK inhibitors on cell growth in H2O2-treated lung cancer cells. After 30 min of preincubation with the respective MAPK inhibitors, the exponentially growing lung cancer cells were treated with 75 or 100 µM H2O2 for 24 h. (A and B) The graphs demonstrate the growth changes in Calu-6 and A549 cells, as assessed by MTT assays. *P<0.05 compared to the control group. #P<0.05 compared to cells treated only with H2O2.
Effects of MAPK inhibitors on cell death in H2O2-treated Calu-6 cells. Followed by 30 min preincubation with the respective MAPK inhibitors, the exponentially growing Calu-6 cells were treated with 75 µM H2O2 for 24 h. (A) The images show representative cell cycle analysis, as analyzed by FACStar flow cytometer. M1 regions show sub-G1 cells. (B) The images show representative Annexin V-FITC staining cells, as analyzed by FACStar flow cytometer. M1 regions show Annexin V-FITC positive cells. (C and D) Graphs indicate the percentages of sub-G1 cells in M1 regions of (A) and Annexin V-FITC-positive cells in M1 regions of (B), respectively. *P<0.05 compared to the control group. #P<0.05 compared to cells treated only with H2O2.
Effects of MAPK inhibitors on cell death in H2O2-treated A549 cells. After a 30 min preincubation with the respective MAPK inhibitors, exponentially growing A549 cells were treated with 100 µM H2O2 for 24 h. (A) The images show representative cell cycle analysis, as analyzed by FACStar flow cytometer. M1 regions show sub-G1 cells. (B) The images show representatives Annexin V-FITC staining cells, as analyzed by FACStar flow cytometer. M1 regions show Annexin V-FITC positive cells. (C and D) Graphs indicate the percentages of sub-G1 cells in M1 regions of (A) and Annexin V-FITC positive cells in M1 regions of (B), respectively. *P<0.05 compared to the control group. #P<0.05 compared to cells treated only with H2O2.
Assessment of MMP in H2O2-treated lung cancer cells in the presence and absence of each MAPK inhibitor. Followed by 30-min preincubation with the respective MAPK inhibitors, exponentially growing lung cancer cells were treated with 75 or 100 µM H2O2 for 24 h. MMP in lung cancer cells was assessed by estimating incorporated Rhodamine 123 intensity using FACStar flow cytometer. (A and D) The images show the representative Rhodamine 123 staining of each cell line. M1 regions show Rhodamine 123-negative (MMP loss) cells in Calu-6 (A) and A549 cells (D). M2 regions show cells excluding MMP-loss cells. (B and E) Graphs show the percentage of Rhodamine 123-negative cells in M1 regions in Calu-6 (A) and A549 cells (D), respectively. (C and F) Graphs represent the percentages of mean MMP levels in M2 regions in Calu-6 (A) and A549 cells (D), respectively. *P<0.05 compared to the control group. #P<0.05 compared to cells treated only with H2O2.
Effects of MAPK inhibitors on ROS levels in H2O2-treated lung cancer cells at 1 h. Exponentially growing cells were treated with 75 or 100 µM H2O2 for 1 h after 30 min of preincubation with the respective MAPK inhibitor. The ROS levels in lung cancer cells were assessed using a FACStar flow cytometer. (A and B) Graphs indicate DCF (ROS) and DHE (O2•−) levels (%) in Calu-6 cells. (C and D) Graphs designate DCF (ROS) and DHE (O2•−) levels (%) in A549 cells. *P<0.05 compared to the control group. #P<0.05 compared to cells treated with H2O2 only.
Measurement of ROS levels upon MAPK inhibitor pretreatment in H2O2-treated lung cancer cells at 24 h. Exponentially growing cells were treated with 75 or 100 µM H2O2 for 24 h after 30 min of preincubation with MAPK inhibitors. ROS levels, including O2•−, were assessed in lung cancer cells using a FACStar flow cytometer. (A and B) The graphs indicate DCF (ROS) and DHE (O2•−) levels (%) in Calu-6 cells. (C and D) The graphs show DCF (ROS) and DHE (O2•−) levels (%) in A549 cells. *P<0.05 compared to the control group. #P<0.05 compared to cells treated with H2O2 only.
Estimation of GSH levels in H2O2-treated lung cancer cells prior to treatment with each MAPK inhibitor at 1 h. Exponentially growing cells were treated with 75 or 100 µM H2O2 for 1 h after 30 min of preincubation with MAPK inhibitors. GSH levels in lung cancer cells were calculated from the CMF intensity values obtained from FACStar flow cytometer analysis. (A and B) Graphs show the mean CMF (GSH) levels (%) in Calu-6 and A549 cells, respectively. *P<0.05 compared to the control group. #P<0.05 compared to cells treated only with H2O2.
Evaluation of GSH levels in H2O2-treated lung cancer cells prior to treatment with MAPK inhibitors at 24 h. Exponentially growing cells were treated with 75 or 100 µM H2O2 for 24 h after 30 min preincubation with MAPK inhibitors. GSH levels in lung cancer cells were calculated from the CMF intensity values obtained from FACStar flow cytometer analysis. (A and D) The images demonstrate representative cells of each CMF-staining state. M1 regions illustrate CMF-negative stained (GSH-depleted) cells in Calu-6 (A) and A549 cells (D). M2 regions show cells excluding GSH-depleted cells. (B and E) Graphs show the percentage of Rhodamine 123-negative cells in M1 regions in Calu-6 (A) and A549 cells (D), respectively. (C and F) Graphs represent the percentages of mean CMF (GSH) levels in M2 regions in Calu-6 (A) and A549 cells (D), respectively. *P<0.05 compared to the control group. #P<0.05 compared to cells treated only with H2O2.