This article is mentioned in:

Introduction

Pyrogallol (PG; benzene-1,2,3-triol) is a polyphenol compound that is commonly distributed in hard wood plants, and it has anti-fungal and anti-psoriatic properties (1). PG is a reductant that is able to generate free radicals, in particular superoxide anions (O2•−), so has frequently been used as a photographic developing agent and in the hair dying industry (1). Despite the useful effects of PG, its toxicity remains a concern for the individuals exposed to it. Multiple studies have been performed to elucidate the toxicological and pharmacological effects of PG (2–4). However, the molecular mechanisms underlying the cellular effects of PG remain only partially clarified. For example, PG induces O2•−-mediated death of various types of cell, including human lymphoma cells (5), human glioma cells (6), gastric cancer cells (7) and Calu-6 lung cancer cells (8,9). In addition, PG triggers mutagenesis, carcinogenesis and impairs the immune system (1).

O2•−, hydrogen peroxide (H2O2) and hydroxyl radicals (·•OH) are reactive oxygen species (ROS). These are involved in various cellular events, including gene expression, cell signaling, differentiation, cell growth and cell death. ROS are primarily generated during mitochondrial respiration and are specifically made by various oxidases (10). Superoxide dismutases convert O2•− to H2O2 (11). Further metabolism yields O2 and H2O via catalase or glutathione (GSH) peroxidase (12). Oxidative stress resulting from either overproduction of ROS or loss of antioxidant enzymes may initiate cellular signaling events that lead to cell death, depending on cell type. There is evidence to suggest that ROS not only affect extracellular signal regulated kinase 1/2 (ERK1/2) and mitogen-activated protein kinase kinase (MEK) activation (13) but also activate c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 (14,15). ERK1/2, JNK/SAPK and p38 are mitogen-activated protein kinases (MAPKs), which are components of signaling pathways associated with cell proliferation, differentiation and cell death (16). Each kinase has different upstream activators and specific downstream substrates (17). In general, MEK-ERK signaling is pro-survival rather than pro-apoptotic (18). JNK and p38 signaling pathways are associated with cell death (14,15,19).

The human lung is a structurally complex organ system (20). Fibroblast cells, which are primarily derived from the primitive mesenchyme, synthesize extracellular matrix components including collagen to maintain the structural and functional integrity of the lung connective tissues. Human pulmonary fibroblast (HPF) cells are involved in lung inflammation, fibrosis and cancer (21). Cultured normal human cells are frequently used in mechanistic studies of oxidative stress, being invaluable biological models (22,23). PG inhibits Calu-6 and A549 lung cancer cell growth via apoptosis (8,24,25) and depletion of GSH (24,26). In addition, MEK inhibitors, but not JNK or p38 inhibitors, have been demonstrated to slightly attenuate inhibition of cell growth, cell death and GSH depletion in PG-treated Calu-6 cells (27). The present study investigated the effect of MAPK inhibitors on PG-treated HPF cell death, in relation to ROS and GSH levels.

Materials and methods

Cell culture

HPF cells were obtained from PromoCell GmbH (Heidelberg, Germany) and were cultured in RPMI-1640 medium (GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in humidified incubator containing 5% CO2 at 37°C. HPF cells were used for experiments between passages four and eight.

Reagents

PG (Sigma-Aldrich; Merck KGaA) was dissolved in water at 100 mM as a stock solution. The MEK inhibitor (PD98059), JNK inhibitor (SP600125) and p38 inhibitor (SB203580) were obtained from Calbiochem; Merck KGaA and were dissolved in dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA). Based on a previous experiment (28), HPF cells were pretreated with 10 µM of each MAPK inhibitor for 1 h prior to PG treatment at 37°C.

Cell viability inhibition assays

Briefly, 5×103 HPF cells per well in 96-well microtiter plates (Nalge Nunc International; Thermo Fisher Scientific, Inc., Penfield, NY, USA) were exposed to 0, 50 or 100 µM PG with or without each MAPK inhibitor at 37°C for 24 h. Changes in cell viability induced by PG and/or a given MAPK inhibitor were determined by measuring the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich; Merck KGaA) dye absorbance as previously described (28).

Lactate dehydrogenase (LDH) assays

Necrosis in cells was evaluated using an LDH assay kit (Daeil Lab Service Co., Ltd., Seoul, Korea) according to the manufacturer's protocol. Briefly, 1×106 HPF cells in 60 mm culture plates (Nalge Nunc International; Thermo Fisher Scientific, Inc.) were incubated with 0, 50 or 100 µM PG in the presence or absence of each MAPK inhibitor at 37°C for 24 h. LDH release was expressed as the percentage of extracellular LDH activity compared with the control cells.

Annexin V/propidium iodide (PI) staining for cell death detection

Apoptosis was determined by staining HPF cells with Annexin V-fluorescein isothiocyanate (FITC; Ex/Em=488/519 nm; Invitrogen; Thermo Fisher Scientific, Inc.) and PI (Ex/Em=488/617 nm; Sigma-Aldrich; Merck KGaA) as previously described (29). Briefly, 1×106 HPF cells in 60 mm culture plates (Nalge Nunc International; Thermo Fisher Scientific, Inc.) were incubated with 0, 50 or 100 µM PG in the presence or absence of each MAPK inhibitor at 37°C for 24 h. Annexin V/PI staining was analyzed using a FACStar flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and CellQuest Pro software (version 5.1; BD Biosciences).

Measurement of mitochondrial membrane potential (MMP; ΔΨm)

MMP (ΔΨm) levels were measured using a rhodamine 123 fluorescent dye (Sigma-Aldrich; Merck KGaA; Ex/Em=485/535 nm) as previously described (28,30,31). Briefly, 1×106 HPF cells in 60 mm culture plates (Nalge Nunc International; Thermo Fisher Scientific, Inc.) were incubated with 0, 50 or 100 µM PG in the presence or absence of each MAPK inhibitor at 37°C for 24 h. Rhodamine 123 staining intensity was determined using a FACStar flow cytometer (BD Biosciences) and CellQuest Pro software (version 5.1; BD Biosciences). The absence of rhodamine 123 from cells indicated the loss of MMP (ΔΨm) in HPF cells.

Western blot analysis

Changes in apoptosis and antioxidant system-associated protein levels were determined using western blotting, as previously described (32). Briefly, 1×106 HPF cells in 60 mm culture plates (Nalge Nunc International; Thermo Fisher Scientific, Inc.) were incubated with 0, 50 or 100 µM PG in the presence or absence of each MAPK inhibitor at 37°C for 24 h. The cells were washed in PBS and suspended in ~100 µl of lysis buffer [20 mM HEPES, pH 7.9, 20% glycerol, 200 mM KCl, 0.5 mM EDTA, 0.5% NP40, 0.5 mM DTT, 1% protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA)], then centrifuged at 15,900 × g at 4°C for 20 min. Samples containing 30 µg total protein were resolved by 12.5% SDS-PAGE, transferred to Immobilon-P polyvinylidene fluoride membranes (Sigma-Aldrich; Merck KGaA) by electroblotting, and the membranes were then probed with anti-poly(ADP-ribose) polymerase (PARP; catalog no., 9542; Cell Signaling Technology, Inc., Danvers, MA, USA; dilution, 1:5,000) and anti-GAPDH antibodies (catalog no., sc-25778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; dilution, 1:5,000) at 4°C overnight without blocking. Next, the membranes were washed with TBS with Tween-20 four times and incubated with secondary antibody (anti-rabbit IgG; horseradish peroxidase-linked antibody; catalog no., 7074; Cell signaling Technology, Inc.; dilution, 1:5,000) at room temperature for 1 h.

Detection of intracellular ROS and O2•− levels

Intracellular ROS were detected using a fluorescent probe dye, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Ex/Em=495/529 nm; Invitrogen; Thermo Fisher Scientific, Inc.), as previously described (32). Dihydroethidium (DHE; Ex/Em=518/605 nm; Invitrogen; Thermo Fisher Scientific, Inc.) is a fluorogenic probe that is highly selective for O2•- among ROS. Mitochondrial O2•- levels were detected using a MitoSOX Red mitochondrial O2•- indicator (Ex/Em=510/580 nm; Invitrogen; Thermo Fisher Scientific, Inc.) as previously described (30,31,33). Briefly, 1×106 HPF cells in 60 mm culture plates (Nalge Nunc International; Thermo Fisher Scientific, Inc.) were incubated with 0, 50 or 100 µM PG in the presence or absence of each MAPK inhibitor for 24 h. DCF, DHE and MitoSOX Red fluorescence was detected using a FACStar flow cytometer (BD Biosciences) and CellQuest Pro software (version 5.1; BD Biosciences). ROS and O2•- levels were expressed as mean fluorescence intensity.

Detection of intracellular GSH

Cellular GSH levels were analyzed using a 5-chloromethylfluorescein diacetate dye (CMF; Ex/Em=522/595 nm; Invitrogen; Thermo Fisher Scientific, Inc.) as previously described (28,30,31). Briefly, 1×106 HPF cells in 60 mm culture plates (Nalge Nunc International; Thermo Fisher Scientific, Inc.) were incubated with 0, 50 or 100 µM PG in the presence or absence of each MAPK inhibitor at 37°C for 24 h. CMF fluorescence intensity was determined using a FACStar flow cytometer (BD Biosciences) and CellQuest Pro software (version 5.1; BD Biosciences). Negative CMF-staining (GSH-depleted) cells were expressed as a percentage of (−) CMF cells of total cells.

Statistical analysis

The results represent the mean ± standard deviation of at least three independent experiments. The data were analyzed using Instat software (GraphPad Prism5; GraphPad Software, Inc., La Jolla, CA, USA). Parametric data was analyzed using the Student's t-test (paired) or one-way analysis of variance following by post hoc analysis with Tukey's multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

Results

Effects of MAPK inhibitors on cell viability and necrotic cell death in PG-treated HPF cells

The effect of PG on HPF cell viability and necrotic cell death was examined. For these experiments, 0, 50 or 100 µM PG was used to differentiate the levels of cell viability inhibition or death with or without a given MAPK inhibitor. Treatment with 50 and 100 µM PG decreased HPF viability by ~40 and 65% at 24 h, respectively (Fig. 1A). Treatment with an MEK inhibitor slightly enhanced the inhibition of cell viability in 50 µM PG-treated HPF cells, whereas treatment with a p38 inhibitor mildly attenuated the inhibition of viability (Fig. 1A). In 100 µM PG-treated HPF cells, all the MAPK inhibitors increased the inhibition of viability to a certain extent (Fig. 1A), with treatment with the p38 inhibitor alone augmenting HPF control cell viability (Fig. 1A). Necrotic cell death was determined by measuring LDH release from cells. While treatment with 50 µM PG did not affect LDH release from HPF cells, 100 µM PG significantly increased LDH release (Fig. 1B). Treatment with MAPK inhibitors did not alter LDH activity in PG-treated and untreated HPF cells (Fig. 1B).

Figure 1. Effects of mitogen-activated protein kinase inhibitors on cell viability and necrotic cell death in PG-treated HPF cells. (A) Alterations in HPF cell viability were assessed using MTT assays. (B) Alterations in LDH release from the HPF cells. *P<0.05 vs. control group. #P<0.05 vs. cells treated with 50 µM PG. PG, pyrogallol; HPF, human pulmonary fibroblast; LDH, lactate dehydrogenase; MEK, mitogen-activated protein kinase kinase; JNK, c-Jun N-terminal kinase.

Effects of MAPK inhibition on necrotic and apoptotic cell death in PG-treated HPF cells

The tested doses of PG significantly increased the rate of apoptosis in HPF cells, as evidenced by Annexin V-FITC/PI staining (Fig. 2). In addition, treatment with 100 µM PG increased the number of necrotic (Annexin V- and PI+) cell death in HPF cells compared with PG-untreated control cells (Fig. 2A). Treatment with the p38 inhibitor slightly increased the number of Annexin V+ 50 µM PG-treated HPF cells compared with only 50 µM PG-treated HPF cells, and significantly increased the number of Annexin V+ 100 µM PG-treated cells compared with only 100 µM PG-treated HPF cells (Fig. 2A and C). Treatment with the other MAPK inhibitors slightly augmented the number of Annexin V+ 100 µM PG-treated HPF cells compared with only 100 µM PG-treated HPF cells (Fig. 2A and C). PARP protein levels were not altered in 50 µM PG-treated HPF cells, while it was decreased in 100 µM PG-treated cells (Fig. 2C). MEK and p38 inhibitors slightly attenuated the decrease in PARP protein levels in 100 µM PG-treated HPF cells (Fig. 2C). Apoptotic cell death is associated with a decrease in MMP (ΔΨm). Treatment with 50 and 100 µM PG significantly decreased MMP (ΔΨm) in HPF cells (Fig. 2B and D). All the MAPK inhibitors enhanced the decrease in MMP (ΔΨm) in PG-treated HPF cells, and treatment with the JNK inhibitor demonstrated a significant effect on 100 µM PG-treated HPF cells (Fig. 2B and D).

Figure 2. Effects of mitogen-activated protein kinase inhibitors on apoptosis and MMP (ΔΨm) in PG-treated HPF cells. (A) Representative graphs depicting the results of Annexin V-FITC/PI staining. (B) Representative graphs depicting the results of rhodamine 123 staining. M1 regions indicate rhodamine 123− cells, with decreased MMP (ΔΨm). (C) PARP and GAPDH protein levels were assessed in PG-treated HPF cells by western blot. The graph depicts the percentage of Annexin V+ cells from A. (D) The percentage of rhodamine 123− cells from B. *P<0.05 vs. control group. #P<0.05 vs. cells treated with 100 µM PG. MMP (ΔΨm), mitochondrial membrane potential; PG, pyrogallol; HPF, human pulmonary fibroblast; FITC, fluorescein isothiocyanate; PI, propidium iodide; PARP, poly(ADP-ribose) polymerase; MEK, mitogen-activated protein kinase kinase; JNK, c-Jun N-terminal kinase.

Effects of MAPK inhibitors on ROS levels in PG-treated HPF cells

To assess the level of intracellular ROS in HPF cells treated with PG, DHE, H2DCFDA and MitoSOX Red fluorescent dyes were used (Fig. 3). The level of DHE fluorescent dye, which reflects the accumulation of O2•- in cells, increased in HPF cells treated with PG (Fig. 3A and D). All the MAPK inhibitors were likely to increase O2•- level in 100 µM PG-treated HPF cells (Fig. 3A and D). ROS (DCF) level in HPF cells was increased by 50 µM PG treatment but not 100 µM PG treatment (Fig. 3C). All the MAPK inhibitors decreased ROS levels in HPF cells treated with 50 µM PG, and treatment with p38 and JNK inhibitors also decreased the level of ROS in 100 µM PG-treated HPF cells (Fig. 3C). Furthermore, MitoSOX Red fluorescence levels, indicating the presence of mitochondrial O2•-, were markedly increased in PG-treated HPF cells (Fig. 3B and E). Treatment with a p38 inhibitor increased mitochondrial O2•- levels in PG-treated HPF cells, whereas treatment with an MEK inhibitor slightly decreased mitochondrial O2•- levels (Fig. 3B and E). Treatment with a JNK inhibitor reduced the mitochondrial O2•- level in HPF control cells (Fig. 3E).

Figure 3. Effects of mitogen-activated protein kinase inhibitors on ROS levels in PG-treated HPF cells. ROS levels were measured using a FACStar flow cytometer. Representative graphs of (A) DHE (O2•−) and (B) mitoSOX (mitochondrial O2•−) levels in PG-treated HPF cells. (C) The graph indicates the percentage of ROS (as determined by H2DCFDA) levels compared with the control cells. The graphs indicate the percentage of (D) DHE (O2•−) levels from (A and E) mitoSOX (mitochondrial O2•−) levels from (B) compared with the control cells. *P<0.05 vs. control group. #P<0.05 vs. cells treated with 50 µM PG. ROS, reactive oxygen species; PG, pyrogallol; HPF, human pulmonary fibroblast; DHE, dihydroethidium; H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; MEK, mitogen-activated protein kinase kinase; JNK, c-Jun N-terminal kinase.

Effects of MAPK inhibitors on GSH levels in PG-treated HPF cells

Changes in intracellular GSH levels in HPF cells treated with PG and/or each MAPK inhibitor were assessed using a CMFDA dye. Treatment with 50 or 100 µM PG significantly increased the number of GSH-depleted cells in HPF cells compared with the negative control (Fig. 4). None of the MAPK inhibitors significantly altered GSH depletion in PG-treated or untreated HPF cells (Fig. 4).

Figure 4. Effects of mitogen-activated protein kinase inhibitors on GSH levels in PG-treated HPF cells. GSH levels were measured with a FACStar flow cytometer. The graph depicts the percentage of CMF− (GSH-depleted) cells. *P<0.05 vs. control group. GSH, glutathione; PG, pyrogallol; HPF, human pulmonary fibroblast; CMF, chloromethylfluorescein diacetate; MEK, mitogen-activated protein kinase kinase; JNK, c-Jun N-terminal kinase.

Discussion

PG is known to trigger the collapse of MMP (ΔΨm) and O2•−-mediated cell death via apoptosis in various types of cancer cell (7,8,24,25,34). In the present study, PG increased O2•− levels, particularly mitochondrial O2•− levels, in HPF cells and induced decreased MMP (ΔΨm). The high production of mitochondrial O2•− in PG-treated HPF cells resulted in cell death. In particular, treatment with 100 µM PG induced apoptosis as well as necrosis in HPF cells.

In general, the activation of ERK is pro-survival rather than pro-apoptotic (18). The results of the present study demonstrated that treatment with an MEK inhibitor, which resulted in decreased ERK activity, enhanced the inhibition of cell viability, cell death and MMP (ΔΨm) loss in PG-treated HPF cells, suggesting that ERK signaling in PG-treated HPF cells is involved in HPF cell survival. In addition, treatment with an MEK inhibitor increased ROS levels in 100 µM PG-treated HPF cells. However, this inhibitor decreased ROS levels in 50 µM PG-treated HPF cells and decreased the mitochondrial O2•− level in PG-treated 50 and 100 µM HPF cells. These results suggested that the different effects of MEK inhibition on ROS levels in HPF cells are dependent on the incubation doses of PG.

The JNK and p38 signaling pathways have been suggested to be associated with cell death (14,19,35). Previous data have also demonstrated that JNK and p38 inhibitors significantly prevent the inhibition of cell growth, cell death and MMP (ΔΨm) loss in PG-treated As4.1 juxtaglomerular cells (36). In contrast, treatment with a JNK inhibitor enhanced the inhibition of cell growth, cell death and MMP (ΔΨm) loss in PG-treated calf pulmonary arterial endothelial cells, whereas treatment with a p38 inhibitor significantly attenuated these effects in the same cells (28). According to the results of the present study, treatment with JNK and p38 inhibitors increased the inhibition of HPF cell viability, cell death and MMP (ΔΨm) loss following treatment with 100 µM PG, implying that the JNK and p38 signaling pathways in PG-treated HPF cells are pro-survival rather than pro-apoptotic. In addition, treatment with JNK and p38 inhibitors slightly increased O2•− levels in 100 µM PG-treated HPF cells, whereas these same inhibitors decreased ROS (DCF) levels in 50 and 100 µM PG-treated HPF cells. These results suggested that it was the JNK or p38 MEK inhibitor-induced altered O2•− levels rather than ROS (DCF) levels that influenced PG-induced cell death. Furthermore, treatment with a p38 inhibitor partially attenuated the inhibition of 50 µM PG-treated HPF cell viability, and this inhibitor alone significantly increased cell viability and ROS levels, including O2•−, in HPF control cells without the induction of cell death. Treatment with a JNK inhibitor alone also specifically affected mitochondrial O2•− levels independent to HPF cell viability and death. These results indicated that JNK and p38 inhibition differently influences ROS levels, cell viability and cell death in HPF cells, which are altered depending on the concentration of PG.

PG induces GSH depletion in a variety of cells (9,26,28,37). The results of the present study also demonstrated that PG treatment increased the number of GSH-depleted HPF cells in a dose-dependent manner. However, none of the MAPK inhibitors, which demonstrated a partial effect on HPF cell death, altered the number of GSH-depleted cells following treatment with 50 and 100 µM PG. Therefore, the intracellular GSH content was at least partially associated with PG-induced HPF cell death.

In conclusion, PG induced apoptosis as well as necrosis in HPF cells. MAPK inhibitors slightly promoted cell death in PG-treated HPF cells. HPF cell death following treatment with PG and/or MAPK inhibitors was partially associated with the O2•− level and changes in GSH content. The results of the present study enhance understanding of PG-induced cell death on normal lung cells in association with MAPK signaling pathways and ROS levels.

Acknowledgements

The present study was supported by a grant from the National Research Foundation of Korea, funded by the Korean government (Ministry of Science, ICT and Future Planning; grant nos., 20080062279 and 2016R1A2B4007773).

Glossary

Abbreviations

Abbreviations:

References