OR Oncology Reports 1021-335X 1791-2431 D.A. Spandidos 10.3892/or.2013.2447 or-30-01-0513 Articles MAPK inhibitors augment gallic acid-induced A549 lung cancer cell death through the enhancement of glutathione depletion PARKWOO HYUN KIMSUHN HEE Department of Physiology, Medical School, Research Institute for Endocrine Sciences, Chonbuk National University, Jeonju 561-180, Republic of Korea Correspondence to: Professor Woo Hyun Park, Department of Physiology, Medical School, Research Institute for Endocrine Sciences, Chonbuk National University, Jeonju 561-180, Republic of Korea, E-mail: parkwh71@chonbuk.ac.kr 7 2013 09 05 2013 30 1 513 519 07 02 2013 12 04 2013 Copyright © 2013, Spandidos Publications 2013 This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.

Gallic acid (GA) is involved in various biological processes such as cell growth inhibition and apoptosis through changes in reactive oxygen species (ROS). In the present study, we investigated the effects of MAPK (MEK, JNK or p38) inhibitors on cell death in GA-induced A549 lung cancer cells in relation to ROS and glutathione (GSH). Treatment with 100 μM GA inhibited the growth of A549 cells and induced apoptosis and/or necrosis, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨm). GA increased ROS levels as well as GSH depletion in A549 cells at 24 h. MEK inhibitor seemed to enhance cell growth inhibition by GA. This inhibitor also increased cell death, MMP (ΔΨm) loss and GSH depletion in GA-treated A549 cells. Both JNK and p38 inhibitors intensified growth inhibition, cell death, MMP (ΔΨm) loss and GSH depletion by GA. However, none of the MAPK inhibitors significantly altered ROS levels in GA-treated A549 cells. In conclusion, MAPK inhibitors enhanced growth inhibition and death in GA-treated A549 cells, which were correlated with GSH depletion rather than ROS levels.

 gallic acid cell death A549 mitogen-activated protein kinase reactive oxygen species glutathione Introduction

Gallic acid (GA; 3,4,5-trihydroxyl-benzoic acid) is a polyhydroxylphenolic compound, which is broadly disseminated in a variety of plants, fruits and foods (1). It is easily absorbed in humans; micromolar concentrations of free and glucuronidated forms of GA and its major metabolite 4-O-methylgallic acid have been detected in human blood plasma following the ingestion of GA-rich food (2). Diverse biological activities of GA have been reported, including anti-bacterial (3), anti-viral (4) and anti-inflammatory (5). The main focus among the properties of GA is connected to its antitumoral action. In fact, anticancer activity of GA has been reported in various cancer cells such as leukemia (6), prostate cancer (7,8), lung cancer (9,10), gastric, colon, breast, cervical and esophageal cancer (11). Apoptosis induced by GA is associated with oxidative stresses derived from reactive oxygen species (ROS), mitochondrial dysfunction and an increase in intracellular Ca2+ level (6,12). GA has both pro- and anti-oxidant properties depending on the concentrations of iron or hydrogen peroxide (H2O2) in medium and plasma (13,14).

The major ROS include H2O2, superoxide anion (O2•-) and hydroxyl radical (•OH). O2•- is metabolized to H2O2 by superoxide dismutases (15). H2O2 is further detoxified to O2 and H2O by catalase or glutathione (GSH) (16). They affect the activity of mitogen-activated protein kinases (MAPKs), which are involved in crucial signaling pathways in cell proliferation, differentiation and cell death in response to a variety of stimuli (17,18). MAPKs can sense the cellular redox status and are common targets for ROS. There are currently three well known MAPKs: the extracellular signal regulated kinase (ERK1/2), the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and the p38 (17). Each MAP kinase pathway has comparatively different upstream activators and specific substrates (19). In general, JNK and p38 are activated by ROS or a mild oxidative shift, initiating procedures related to apoptosis (20,21). ROS also induce ERK phosphorylation and activate the ERK pathway (22). In most cases, ERK signaling has pro-survival and proliferative roles rather than pro-apoptotic effects (23).

Lung cancer is a major cause of cancer-related mortality in developed countries. The carcinogenesis of lung cancer is associated with excessive inflammation mediated by ROS of airborne and bloodborne origin. Various novel remedial strategies are still under consideration since the clinical use of conventional drugs is limited due to intrinsic or acquired resistance and toxicity (24). We recently reported that GA inhibits the growth of Calu-6 and A549 lung cancer cells (25,26). In addition, MEK inhibitor PD98059 attenuates growth inhibition and death in GA-treated Calu-6 cells (27). Since different and opposite effects of MAPKs by a variety of ROS can occur even in the same type of cells, the relationship between ROS and MAPKs requires further elucidation regarding signalings related to cell survival and cell death. In the present study, we investigated the effects of MAPK inhibitors on cell growth, death, ROS and GSH levels in GA-treated A549 lung cancer cells.

 Materials and methods Cell culture

The human pulmonary adenocarcinoma A549 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in a humidified incubator containing 5% CO2 at 37°C. A549 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco- BRL, Grand Island, NY, USA).

 Reagents

GA was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and was dissolved in ethanol at 200 mM as a stock solution. MEK inhibitor (PD98059), JNK inhibitor (SP600125) and p38 inhibitor (SB203580) were obtained from Calbiochem (San Diego, CA, USA) and were dissolved in DMSO at 10 mM as a stock solution. Cells were pretreated with each MAPK inhibitor for 30 min prior to GA treatment. Based on a previous experiment (28), 10 μM of each MAPK inhibitor was used as an optimal dose in this experiment. Ethanol (0.2%) and DMSO (0.3%) were used as a control vehicle.

 Cell growth assay

The effect of drugs on A549 cell growth was determined by trypan blue exclusion cell counting or measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye absorbance of living cells as previously described (29). In brief, 2×105 cells/well were seeded in 24-well plates (Nunc, Roskilde, Denmark) for cell counting and 5×104 cells/well were seeded in 96-well microtiter plates for an MTT assay. Following exposure to the indicated dose of GA and/or each MAPK inhibitor for 24 h, 20 μl of MTT (Sigma) solution (2 mg/ml in PBS) were added to each well of 96-well plates. The plates were incubated for an additional 4 h at 37°C. Medium in plates was withdrawn using pipetting and 200 μl DMSO was added to each well to solubilize the formazan crystals. Optical density was measured at 570 nm using a microplate reader (Spectra MAX 340, Molecular Devices Co., Sunnyvale, CA, USA).

 Sub-G1 DNA content analysis

Sub-G1 DNA content cells were determined by propidium iodide (PI; Ex/Em=488 nm/617 nm, Sigma-Aldrich) staining as previously described (30). In brief, 1×106 cells in 60-mm culture dishes (Nunc) were incubated with GA and/or each MAPK inhibitor for 24 h. Cells were then washed with PBS and fixed in 70% ethanol. Cells were washed again with PBS, then incubated with PI (10 μg/ml) with simultaneous RNase treatment at 37°C for 30 min. Cell DNA content was measured using a FACStar flow cytometer (Becton Dickinson, San Jose, CA, USA).

 Annexin V staining for cell death detection

Apoptosis was determined by staining cells with Annexin V-fluorescein isothiocyanate (FITC; Ex/Em=488 nm/519 nm, Invitrogen Corp., Camarillo, CA, USA) as previously described (30,31). In brief, 1×106 cells in 60-mm culture dishes (Nunc) were incubated with GA and/or each MAPK inhibitor for 24 h. Cells were washed twice with cold PBS and then resuspended in 500 μl of binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) at a concentration of 1×106 cells/ml. Annexin V-FITC (5 μl) was then added to these cells, which were analyzed with a FACStar flow cytometer (Becton-Dickinson).

 Measurement of mitochondrial membrane potential (MMP; ΔΨ<sub>m</sub>)

MMP (ΔΨm) levels were measured by a rhodamine 123 fluorescent dye (Ex/Em=485 nm/535 nm, Sigma-Aldrich Chemical Co.) as previously described (31,32). In brief, 1×106 cells in 60-mm culture dishes (Nunc) were incubated with GA and/or each MAPK inhibitor for 24 h. Cells were washed twice with PBS and incubated with the rhodamine 123 (0.1 μg/ml) at 37°C for 30 min. Rhodamine 123 staining intensity was determined by flow cytometry. Rhodamine 123 negative cells indicated the loss of MMP (ΔΨm) in A549 cells. MMP (ΔΨm) levels in cells except MMP (ΔΨm) loss cells were expressed as mean fluorescence intensity (MFI), which was calculated by CellQuest software.

 Detection of intracellular ROS levels

Intracellular ROS such as H2O2, •OH and ONOO• were detected by means of an oxidation-sensitive fluorescent probe dye, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Ex/Em=495 nm/529 nm, Invitrogen Molecular Probes, Eugene, OR, USA) (33). H2DCFDA is poorly selective for O2•−. By contrast, dihydroethidium (DHE; Ex/Em=518 nm/605 nm, Invitrogen Molecular Probes) is highly selective for O2•− among ROS. In brief, 1×106 cells in 60-mm culture dishes (Nunc) were incubated with GA and/or each MAPK inhibitor for 24 h. Cells were then washed in PBS and incubated with 20 μM H2DCFDA or DHE at 37°C for 30 min. DCF and DHE fluorescences were detected using a FACStar flow cytometer (Becton-Dickinson). ROS and O2•− levels were expressed as MFI, which was calculated by CellQuest software.

 Detection of the intracellular GSH

Cellular GSH levels were analyzed using a 5-chloromethylfluorescein diacetate dye (CMFDA, Ex/Em=522 nm/595 nm; Invitrogen Molecular Probes) as previously described (34,35). In brief, 1×106 cells in 60-mm culture dishes (Nunc) were incubated with GA and/or each MAPK inhibitor for 24 h. Cells were then washed with PBS and incubated with 5 μM CMFDA at 37°C for 30 min. CMF fluorescence intensity was determined using a FACStar flow cytometer (Becton-Dickinson). Negative CMF staining (GSH-depleted) cells were expressed as the percentage of (−) CMF cells. CMF levels in cells except GSH-depleted cells were expressed as MFI, which was calculated by CellQuest software.

 Statistical analysis

The results represent at least three independent experiments (means ± SD). The data were analyzed using Instat software (GraphPad Prism 4, San Diego, CA, USA). One-way analysis of variance (ANOVA) with post hoc analysis using Tukey’s multiple comparison test was used for parametric data. P<0.05 was considered to indicate a statistically significant difference.

 Results Effects of MAPK inhibitors on cell growth in GA-treated A549 cells

We examined the effects of MAPK inhibitors on the growth of GA-treated A549 cells. GA dose-dependently inhibited A549 cell growth with an IC50 of ~150 μM at 24 h (25). Treatment with 100 μM GA used in this study inhibited the growth of A549 cells ~40% based on trypan blue cell counting and MTT assays at 24 h (Fig. 1A and B). It has been demonstrated that high dose usages of MAPK inhibitors may decrease their specificity (36). In fact, 20 μM concentration of each MAPK inhibitor significantly induced cell growth inhibition and death in A549 cells (data not shown). Therefore, the concentration of 10 μM each MAPK inhibitor was used in the current experiments to evade the non-specific inhibition of other kinases. While MEK inhibitor slightly enhanced the growth inhibition by GA, JNK and p38 inhibitors significantly enhanced the growth inhibition (Fig. 1A and B). Only MEK inhibitor decreased cell number in A549 control cells (Fig. 1A). The 10 μM MAPK inhibitors did not affect A549 control cell growth (Fig. 1B).

 Effects of MAPK inhibitors on cell death and MMP (ΔΨ<sub>m</sub>) in GA-treated A549 cells

GA significantly induced cell death in A549 cells, as evidenced by trypan blue positive staining (Fig. 1A) and Annexin V staining cells (Fig. 2A and C). However, GA slightly increased the number of sub-G1 DNA content cells (Fig. 2B and D). All the MAPK inhibitors increased the number of Annexin V staining cells in GA-treated A549 cells (Fig. 2A and C). However, none of the MAPK inhibitors altered the number of sub-G1 cells (Fig. 2B and D) and none of the MAPK inhibitors affected the number of Annexin V staining and sub-G1 cells in A549 control cells (Fig. 2C and D).

In addition, GA significantly triggered the loss of MMP (ΔΨm) in A549 cells (Fig. 3A and B). All the MAPK inhibitors intensified the MMP (ΔΨm) loss in GA-treated A549 cells (Fig. 3A and B). In relation to MMP (ΔΨm) levels, GA reduced MMP (ΔΨm) levels in A549 cells (Fig. 3A and C). Only p38 inhibitor significantly decreased MMP (ΔΨm) levels in GA-treated A549 cells (Fig. 3A and C). Although MEK or JNK inhibitor alone increased MMP (ΔΨm) levels in A549 control cells, p38 inhibitor reduced the MMP (ΔΨm) levels (Fig. 3A and C).

 Effects of MAPK inhibitors on ROS and GSH levels in GA-treated A549 cells

Next, we determined whether intracellular ROS and GSH levels in GA-treated A549 cells were altered by treatment with each MAPK inhibitor. ROS (DCF) level such as H2O2 was increased in GA-treated A549 cells (Fig. 4A and C). The MAPK inhibitors did not significantly change ROS level in GA-treated A549 cells (Fig. 4A and C). Red fluorescence derived from DHE reflecting intracellular O2•− level was also increased in A549 cells (Fig. 4B and D). None of the MAPK inhibitors altered O2•− levels in GA-treated A549 cells (Fig. 4B and D). The MAPK inhibitors did not alter ROS level in A549 control cells (Fig. 4C and D).

GA increased the number of GSH-depleted cells in A549 cells (Fig. 5A and B). MEK and p38 inhibitors significantly increased GSH-depleted cell number in GA-treated A549 cells and JNK inhibitor showed a light effect on that (Fig. 5A and B). In addition, GA increased GSH level in A549 cells (Fig. 5A and C). All the MAPK inhibitors seemed to intensify the increased GSH level by GA (Fig. 5A and C).

 Discussion

Since GA inhibited the growth of A549 cells and induced their death, we focused on elucidating the toxicological effect of GA on cell growth and death in A549 cells pretreated with MAPK inhibitors in relation to ROS and GSH. GA increased Annexin V-FITC positive cells in A549 cells, which indirectly indicated that GA-induced A549 cell death occurred via apoptosis. However, GA did not increase sub-G1 cell number as much as Annexin V positive cell number by it. In addition, 200 μM GA did not increase sub-G1 cell number compared with the number in 100 μM GA-treated cells (data not shown). Therefore, GA seemed to induce growth inhibition in A549 cells via necrosis as well as apoptosis.

ERK activation has a pro-survival function rather than pro-apoptotic effects (23). Similarly, MEK inhibitor, which presumably decreased ERK activity, slightly enhanced the growth inhibition by GA and significantly intensified cell death by it. In addition, MEK inhibitor alone significantly reduced the number of A549 control cells. Therefore, this result indirectly suggested that the inhibition of ERK signaling by MEK inhibitor plays a pro-apoptotic role in GA-treated A549 cells and has an anti-growth function in A549 control cells. However, MEK inhibitor attenuates growth inhibition and death in GA-treated Calu-6 cells (27), indicating that MEK inhibitor differently affects cell growth and death in GA-treated A549 and Calu-6 lung cancer cells. Moreover, MEK inhibitor did not affect cell growth inhibition and death in GA-treated human pulmonary fibroblast cells (37). Therefore, the targeted therapy related to ERK signaling should be carefully considered in lung cancer treatment. Considerable evidence demonstrates that JNK or p38 signaling is related to cell death (20,21). In fact, GA induces PC12 rat pheochromocytoma cell death through the activation of JNK and its inhibitor protects PC12 cells against GA-induced cell death (38). In addition, p38 inhibitor prevented anisomycin-induced macrophage death (39) and its inhibitor decreased the death of pyrogallol-induced calf pulmonary artery endothelial cells (28). Moreover, JNK and p38 inhibitors do not significantly alter cell death in GA-treated human pulmonary fibroblast cells and Calu-6 cells (27,37). However, results of the present study demonstrated that JNK or p38 inhibitor enhanced growth inhibition and death in GA-treated A549 cells. It is also demonstrated that JNK and p38 inhibitors enhance cell growth inhibition and death in GA-treated HeLa cells (40). Therefore, the inhibition of JNK or p38 signaling by each inhibitor seemed to be a pro-apoptotic function in GA-treated A549 cells. Collectively, anti- or pro-apoptotic effects of JNK or p38 inhibitor depend on cell types or co-treated agents. Markedly, none of the MAPK inhibitors increased the number of sub-G1 cells in GA-treated A549 cells. These results indicated that the inhibition of each MAPK signaling by its inhibitor was involved in the enhancement of apoptosis rather than necrosis.

Cell death induced by GA is associated with mitochondrial dysfunction (12). Accordingly, GA induced the loss of MMP (ΔΨm) in A549 cells and reduced MMP (ΔΨm) levels. All the MAPK inhibitors intensified MMP (ΔΨm) loss in GA-treated A549 cells. Therefore, the cell death by GA and/or MAPK inhibitors seemed to be tightly connected with the loss of MMP (ΔΨm). In addition, p38 inhibitor significantly reduced MMP (ΔΨm) levels in GA-treated A549 cells. This result likely clarifies the strong growth inhibition of A549 cells by co-treatment with GA and p38 inhibitor since MTT reduction is considered to be an indirect measurement of mitochondrial activity (41). However, since all the MAPK inhibitors affected the basal MMP (ΔΨm) level in A549 control cells without changes in cell growth, the changes in MMP (ΔΨm) levels are not fully associated with those of MTT reduction in cells. Nevertheless, the basal activity of MAPK signalings seemed to be involved in the maintenance of MMP (ΔΨm) in A549 control cells.

Increasing evidence suggests that apoptosis induced by GA is associated with oxidative stresses derived from ROS (6,42). Similarly, ROS levels including O2•− were significantly increased in GA-treated A549 cells. The MAPK inhibitors did not significantly alter ROS levels in GA-treated A549 cells. These data suggest that changes in ROS levels by MAPK inhibitors in GA-treated A549 cells are not closely related to cell death. GSH is a main non-protein antioxidant and eliminates the O2•− by providing electrons for enzymes such as GSH peroxidase, which reduce H2O2 to H2O. It has been reported that the intracellular GSH content has a decisive effect on anticancer drug-induced apoptosis, indicating that apoptotic effects are inversely comparative to GSH content (34,43,44). Similarly, GA increased the number of GSH-depleted cells in A549 cells. MEK and p38 inhibitors significantly enhanced GSH depletion in GA-treated A549 cells, and JNK inhibitor slightly increased the number. These results might be correlated with the results derived from Annexin V and MMP (ΔΨm) assays. These results support the hypothesis that the intracellular GSH content has a decisive effect on cell death (32,34,45,46). It is of note that CMF (GSH) level in A549 cells was increased by GA. The increased GSH level was likely to occur against the increasing ROS level by GA. It may be that some cells which could not defend oxidative stress resulting from GA treatment underwent cell death pathway. All the MAPK inhibitors enhanced GSH level in GA-treated A549 cells. These results suggest that MAPK inhibitors are involved in the upregulation of GSH levels in GA-treated A549 cells, consequently affecting ROS levels and the proportions of GSH-depleted cells. The mechanism underlying the effect of MAPKs on intracellular GSH level in cells requires further clarification.

In conclusion, GA induced growth inhibition and death in A549 cells, which was accompanied by intracellular ROS increase and GSH depletion. All the MAPK inhibitors enhanced growth inhibition and death in GA-treated A549 cells, which were related to GSH depletion rather than ROS level.

 Acknowledgements

The present study was supported by a grant from the Ministry of Science and Technology (MoST)/Korea Science and Engineering Foundation (KOSEF) through the Diabetes Research Center at Chonbuk National University (2012-0009323) and the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (2010-0021808).

 Abbreviations GA

gallic acid

 ROS

reactive oxygen species

 MAPK

mitogen-activated protein kinase

 MEK

MAP kinase or ERK kinase

 ERK

extracellular signal-regulated kinase

 JNK

c-Jun N-terminal kinase

 SOD

superoxide dismutase

 MMP (ΔΨm)

mitochondrial membrane potential

 FBS

fetal bovine serum

 FITC

fluorescein isothiocyanate

 H2DCFDA

2′,7′-dichlorodihydrofluorescein diacetate

 DHE

dihydroethidium

 GSH

glutathione

 CMFDA

5-chloromethylfluorescein diacetate

 PI

propidium iodide

 MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

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Effects of MAPK inhibitors on cell growth in GA-treated A549 cells. Exponentially growing cells were treated with 100 μM GA for 24 h following a 30-min pre-incubation of 10 μM MEK, JNK or p38 inhibitor. (A) Cell number and (B) cell growth were assessed by trypan blue exclusion cell counting and MTT assays, respectively. *P<0.05 compared with the control group. #P<0.05 compared with cells treated with GA only.

Effects of MAPK inhibitors on cell death in GA-treated A549 cells. Exponentially growing cells were treated with 100 μM GA for 24 h following a 30-min pre-incubation of 10 μM MEK, JNK or p38 inhibitor. Annexin V-FITC and sub-G1 cells were measured with a FACStar flow cytometer. (A and B) Each histogram as a representative indicates Annexin V and sub-G1 cells, respectively. M1 in A indicates Annexin V staining cells. M1 in B indicates sub-G1 cells. (C and D) Graphs show the percentage of Annexin V positive staining cells (M1 region in A) (C) and sub-G1 cells (M1 region in B) (D). *P<0.05 compared with the control group. #P<0.05 compared with cells treated with GA only.

Effects of MAPK inhibitors on MMP (ΔΨm) in GA-treated A549 cells. Exponentially growing cells were treated with 100 μM GA for 24 h following a 30-min pre-incubation of 10 μM MEK, JNK or p38 inhibitor. MMP (ΔΨm) in A549 cells was measured with a FACStar flow cytometer. (A) Each histogram as a representative indicates MMP (ΔΨm). M1 regions indicate rhodamine 123 negative [MMP (ΔΨm) loss] cells. M2 regions indicate cells except rhodamine 123 negative [MMP (ΔΨm) loss] cells. (B and C) Graphs show the percentage of rhodamine 123 negative [MMP (ΔΨm) loss] cells from M1 regions of A (B) and MMP (ΔΨm) levels (%) from M2 regions of A (C). *P<0.05 compared with the control group. #P<0.05 compared with cells treated with GA only.

Effects of MAPK inhibitors on ROS levels in GA-treated A549 cells. Exponentially growing cells were treated with 100 μM GA for 24 h following a 30-min pre-incubation of 10 μM MEK, JNK or p38 inhibitor. ROS levels in A549 cells were measured using a FACStar flow cytometer. (A and B) Each histogram as a representative indicates DCF and DHE levels in each cell group, respectively. (C and D) Graphs indicate DCF (ROS) levels (%) compared with GA-untreated control cells (C) and DHE (O2•−) levels (%) compared with GA-untreated control cells (D). *P<0.05 compared with the control group. #P<0.05 compared with cells treated with GA only.

Effects of MAPK inhibitors on GSH levels in GA-treated A549 cells. Exponentially growing cells were treated with 100 μM GA for 24 h following a 30-min pre-incubation of 10 μM MEK, JNK or p38 inhibitor. GSH levels in A549 cells were measured using a FACStar flow cytometer. (A) Each histogram as a representative shows CMF intensities in cells. M1 indicates (−) CMF (GSH-depleted) cells. M2 indicates cells except (−) CMF cells. (B and C) Graphs show the percentage of (−) CMF (GSH-depleted) cells (M1 region in A) (B) and mean CMF (GSH) levels compared with GA-untreated control cells (M2 region in A) (C). *P<0.05 compared with the control group. #P<0.05 compared with cells treated with GA only.