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Introduction

Propyl gallate (PG; 3,4,5-trihydroxybenzoic acid propyl ester), a synthetic antioxidant, has been utilized for decades as an additive for processed food, cosmetics, food packing, and the pharmaceutical industry since it can be used to avoid spoilage and decomposition (1). Although PG is reported to have low toxicity, it has various beneficial properties for tissue and cell functions. For example, PG is an efficient protector of liver cells from lipid peroxidation by oxygen radicals (2). In addition, numerus studies have revealed that PG functions as an antioxidant and a chemopreventive agent in vivo and in vitro (3–5). In contrast, PG also exerts some prooxidant effects (6,7). In addition, PG mediates its cytotoxicity through the impediment of mitochondrial function in hepatocytes, endothelial cells and testicular cells (8–10). PG decreases the growth of microorganisms by constraining respiration and nucleic acid synthesis (11). Therefore, in order to clarify the discrepancies between the different effects of PG as an antioxidant or a prooxidant, further studies are required to re-evaluate its function and effects on cells and tissues.

The cell cycle is the series of events that takes place in a cell. The eukaryotic cell cycle consists of four distinct phases: The G1 phase, S phase, G2 phase and M phase (12,13). Proper progression throughout the cell cycle depends on the expression level of a family of cyclins, and the subsequent activation of cyclin-dependent kinases (CDKs) (12). Regulation of the cell cycle involves procedures crucial to cell survival, including the recognition and repair of genetic damage as well as the prevention of uncontrolled cell division (12,13). Apoptosis is a cellular response to cytotoxicological drugs (14,15). The signaling mechanism of apoptosis commonly consists of two pathways: The mitochondrial pathway and cell death receptor pathway (14–16). The commencement of apoptosis in the mitochondrial pathway is induced or accompanied by increasing BAX protein levels and decreasing Bcl-2 protein levels, causing the loss of mitochondrial membrane potential (MMP; ∆Ψm) (14). The focal point of this pathway is the efflux of cytochrome c from mitochondria to the cytosol. In the cytosol, cytochrome c forms an apoptosome complex with apoptotic protease-activating factor 1 and caspase-9, inducing the activation of a major executioner caspase, caspase-3 (15,17). The cell death receptor pathway is distinguished by the connection of cell death ligands to their death receptors with subsequent stimulation of caspase-8 and caspase-3 activities (16). Caspase-3 activation can systematically disassemble cells by severing key proteins, particularly poly(ADP-ribose) polymerase (PARP). Therefore, targeted inhibition of cell cycle progression and anti-apoptotic pathways is an attractive concept for improving cancer treatment strategies.

Lung cancer is the chief cause of cancer-related death worldwide (18). Lung cancer is classified into two main types: Small cell lung cancer (SCLC) which accounts for 10–14% of all lung cancer cases, and non-SCLC (NSCLC) which accounts for 85–90% (18,19). NSCLC is additionally sorted into three subtypes in accordance with histology: Adenocarcinoma, squamous-cell carcinoma, and large cell carcinoma (19). The anti-growth effects of PG have been confirmed in numerous cell types such as pulmonary artery and umbilical vein endothelial cells (10,20), testis cells (9), leukemia cells (21), and hepatocellular carcinoma (22). We also reported that PG inhibits the growth of HeLa cells via apoptosis and glutathione depletion (23,24). Although numerous studies have suggested that PG plays a crucial role in cell death, very little is known about the cytotoxic and anti-growth effects of PG in lung cancer cells. In the present study, human SCLC Calu-6 and NSCLC adenocarcinoma A549 cells were used to investigate the molecular mechanism involved in the anti-growth effect of PG concerning apoptosis as well as cell cycle arrest. PG inhibited the growth of these lung cancer cells via apoptosis and G1 phase arrest of the cell cycle.

Materials and methods

Cell culture

Human SCLC Calu-6 cells and NSCLC adenocarcinoma A549 cells were obtained from the American Type Culture Collection (ATCC). These cell lines were maintained in a standard humidified incubator containing 5% CO2 at 37°C. The lung cancer cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (Sigma-Aldrich; Merck KGaA) and 1% penicillin-streptomycin (Gibco BRL; Thermo Fisher Scientific, Inc.). Cells were grown in BD Falcon 100-mm plastic cell culture dishes (BD Biosciences) and harvested with trypsin-EDTA (Gibco BRL; Thermo Fisher Scientific, Inc.). Exponentially growing cells were used for the experiments.

Reagents

PG was acquired from Sigma-Aldrich Co (Merck KGaA). PG was dissolved in ethanol at 200 mM as a stock solution. Ethanol (0.2%) was used as a control vehicle and did not influence cell growth or cell death. Stock solution was wrapped in foil and kept at 4°C or −20°C.

Cell growth inhibition assay

The influence of PG on the growth of lung cancer cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich; Merck KGaA) assays. Briefly, 5×104 cells were seeded into each well of 96-well microtiter plates (Nunc). After incubation with the designated doses of PG for 24 or 72 h, 20 µl of MTT solution [2 mg/ml in phosphate-buffered saline (PBS); Gibco BRL; Thermo Fisher Scientific, Inc.] was added to each well. The plates were incubated for 4 h at 37°C. The medium in plates was removed via pipetting, and 100–200 µl of DMSO was added to each well to solubilize formazan crystals. Optical density was measured at 570 nm with a microplate reader (Synergy™ 2, BioTekR Instruments Inc.). Each plate contained multiple wells at a given experimental condition and multiple control wells. This procedure was replicated for 2 to 4 plates per condition.

Cell cycle and sub-G1 cell analysis

Cell cycle and sub-G1 distributions in cells were determined using propidium iodide (PI, Sigma-Aldrich; Merck KGaA; Ex/Em = 488 nm/617 nm) staining. Briefly, 1×106 cells in BD Falcon 60-mm culture dishes (BD Biosciences) were incubated with the designated concentrations of PG for 24 or 72 h. Cells were washed twice with PBS and fixed in cold 70% ethanol. Cells were again washed with PBS, and then incubated with 10 µg/ml PI concurrently with RNase (Sigma-Aldrich; Merck KGaA) at a concentration of 5×105 cells/ml in PBL at 37°C for 30 min. The proportions of cells in different phases of the cell cycle or having sub-G1 DNA content were measured and analyzed with a FACStar flow cytometer (BD Sciences).

Detection of apoptosis

Apoptosis was identified via Annexin V-fluorescein isothiocyanate staining (FITC, Thermo Fisher Scientific, Inc.; Ex/Em = 488/519 nm). Annexin V-FITC is used to detect phosphatidylserine exposing cells thereby marking apoptotic cells. Briefly, 1×106 cells in BD Falcon 60-mm culture dishes (BD Biosciences) were incubated with the designated concentrations of PG for 24 or 72 h. Cells were washed twice with cold PBS and then suspended in 200 µl of binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) at a concentration of 5×105 cells/ml at 37°C for 30 min. Annexin V-FITC (2 µl) was added to the solution, and cells were analyzed with a FACStar flow cytometer (BD Sciences).

Measurement of mitochondrial membrane potential (MMP; ΔΨm)

The MMP (ΔΨm) was monitored using a Rhodamine 123 cationic fluorescent dye (Sigma-Aldrich; Merck KGaA; Ex/Em = 485/535 nm), which preferentially enters into mitochondria with high MMP (∆Ψm). Depolarization of MMP (∆Ψm) results in the loss of Rhodamine 123 from the mitochondria and reduces the intracellular fluorescence intensity of this dye. In brief, 1×106 cells in 60-mm culture dishes (Nunc) were incubated with the indicated doses of PG for 24 h. Cells were washed twice with PBS and incubated with Rhodamine 123 (0.1 mg/ml) at a concentration of 5×105 cells/ml in PBL at 37°C for 30 min. Rhodamine 123 staining intensities were determined using a FACStar flow cytometer (BD Sciences). Rhodamine 123 negative (−) cells indicated the loss of MMP (∆Ψm) in the lung cancer cells. MMP (∆Ψm) levels in cells, except MMP (∆Ψm) loss cells, were expressed as proportions compared with the control cells.

Western blot analysis

Protein expression levels were evaluated via western blotting. Briefly, 5×106 cells in BD Falcon 100-mm culture dishes (BD Biosciences) were incubated with the indicated concentrations of PG for 24 h. Cells were washed with PBS and 4 volumes of lysis buffer (Intron Biotechnology) was added. Total proteins (30 µg) were resolved via 8–15% SDS-PAGE gels and then transferred to Immobilon-P PVDF membranes (Millipore) by electroblotting. Membranes were probed with anti-Bcl-2 (cat. no. 2872, 1:1,000 dilution), anti-caspase-3 (cat. no. 9662, 1:1,000 dilution), anti-PARP (cat. no. 9542, 1:1,000 dilution), anti-cleaved PARP (cat. no. 9541, 1:1,000 dilution) antibodies (Cell Signaling Technology, Inc.), and anti-β-actin antibody (sc-81178, 1:1,000 dilution, Santa Cruz Biotechnology, Inc.). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (sc-2004 or sc-2005, 1:1,000 dilution, Santa Cruz Biotechnology, Inc.). Western blots were developed using an EZ-Western Lumi Pico ECL solution kit (DoGen, Korea).

Quantification of caspase-3 and caspase-8 activities

The activities of caspase-3 and caspase-8 were evaluated using Caspase-3 and Caspase-8 Colorimetric Assay Kits (R&D Systems, Inc.). In brief, 1×106 cells were incubated with the specified concentrations of PG for 24 h. Cells were washed with PBS and 4 volumes of lysis buffer (Intron Biotechnology) was added. Samples containing 50 µg of total protein were added to 2X Reaction buffer containing DEVD-pNA for caspase-3 activity or IETD-pNA for caspase-8 activity in 96-well microtiter plates (Nunc) and incubated at 37°C for 1 h. Optical density was measured at 405 nm using a microplate reader (Synergy™ 2). Each plate contained multiple wells of a given experimental condition as well as multiple control wells. Caspase activity is expressed in arbitrary absorbance units (absorbance at a wavelength of 405 nm).

Statistical analysis

The results represent the mean of at least three independent experiments (mean ± SD). Data were analyzed using Instat software (GraphPad Prism 5.0; GraphPad Software, Inc.). One-way analysis of variance with post hoc analysis using Tukey's multiple comparison test was applied to judge statistical significance which was defined at P<0.05.

Results

Effects of PG on the growth of Calu-6 and A549 lung cancer cells

The effect of PG on the growth of Calu-6 and A549 lung cancer cell types was observed using MTT assays. A dose-dependent reduction in cell growth was observed in Calu-6 cells with a half maximal inhibitory concentration (IC50) of approximately 800 µM following treatment with PG for 24 h (Fig. 1A). Additionally, a 50 µM concentration of PG appeared to significantly reduce the growth of Calu-6 cells by approximately 60% at 72 h (Fig. 1A). Thus, the IC50 of PG in Calu-6 cells at 72 h was less than 50 µM. The growth of A549 cells was also reduced with an IC50 of ~800 µM after a 24-h incubation with PG (Fig. 1B). In addition, the IC50 of PG in A549 cells at 48 h was approximately 150 µM (data not shown). Treatment with 100 µM PG decreased the growth of A549 cells by approximately 50% at 72 h (Fig. 1B).

Figure 1. Effects of PG on cell growth in Calu-6 and A549 lung cancer cells. Exponentially growing cells were incubated in the presence of the designated concentrations of PG for 24 or 72 h. Cell growth was evaluated by MTT assays. Graphs show cell growth in (A) Calu-6 and (B) A549 cells. *P<0.05 compared with the PG-untreated control cells. PG, propyl gallate.

Effects of PG on the cell cycle distribution of lung cancer cells

Because the growth inhibition of Calu-6 and A549 cells by PG could be explained by an arrest during cell cycle progression, allocations of cells in different stages of the cell cycle were observed after a 24- or 72-h incubation period with PG. DNA flow cytometric analysis indicated that tested doses of PG induced a G1 phase arrest of the cell cycle in Calu-6 cells at 24 and 72 h (Fig. 2A and B). Particularly, concentrations of 200 and 400 µM PG showed a significant increase in the G1 phase at 24 h (Fig. 2A and B). In addition, the tested doses of PG induced a G1 phase arrest of the cell cycle in A549 cells at 24 h (Fig. 2C and D). Treatment with 100 and 200 µM PG also increased the proportion of cells at the G1 phase at 72 h (Fig. 2C and D).

Figure 2. Effects of PG on cell cycle phase distribution in lung cancer cells. Exponentially growing cells were incubated in the presence of the designated concentrations of PG for 24 (0–1,600 µM) or 72 h (0–200 µM). Cell cycle phase distribution was evaluated by DNA flow cytometry. (A and C) Each histogram shows the cell cycle distributions in PG-treated (A) Calu-6 and (C) A549 lung cancer cells at 24 and 72 h. M1 indicates sub-G1 cells. G1, S, and G2 represent each phase of the cell cycle. (B and D) Graphs show cell cycle phase distribution derived from G1, S, and G2 phases in A and C in (B) Calu-6 and (D) A549 cells, respectively. *P<0.05 compared with the PG-untreated control cells. PG, propyl gallate.

Effects of PG on cell death in lung cancer cells

Whether PG induces cell death was evaluated using sub-G1 cells and Annexin V-staining cells. As shown in Fig. 3A, the tested doses of PG increased the quantity of sub-G1 cells in Calu-6 cells at 24 or 72 h, but the effects were not dose-dependent. PG also increased the quantity of sub-G1 cells in A549 cells at 24 or 72 h (Fig. 3B). Furthermore, 100–400 µM PG did not increase the percentage of Annexin V-stained cells in both Calu-6 (Fig. 4A and B) and A549 cell lines (Fig. 4A and C) and 800 µM PG slightly increased the percentage of Annexin V-staining cells in these cells (Fig. 4). However, the percentage of Annexin V-stained cells in Calu-6 and A549 cells was significantly increased after treatment with 1,600 µM PG at 24 h (Fig. 4).

Figure 3. Effects of PG on sub-G1 cells in lung cancer cells. Exponentially growing cells were incubated in the presence of the designated concentrations of PG for 24 or 72 h. Sub-G1 cells were measured with FACStar flow cytometer. (A and B) Graphs show the proportions of sub-G1 cells derived from M1 in Fig. 2A and C in (A) Calu-6 and (B) A549 cells, respectively. *P<0.05 compared with the PG-untreated control cells. PG, propyl gallate.

Figure 4. Effects of PG on cell death in lung cancer cells. Exponentially growing cells were incubated in the presence of the designated concentrations of PG for 24 h. Annexin V-stained cells were assessed with FACStar flow cytometer. (A) Representative histograms for Annexin V-FITC staining in PG-treated Calu-6 and A549 cells. M1 indicates Annexin V-FITC-positive cells. (B and C) Graphs show the percentages of M1 regions in A in (B) Calu-6 and (C) A549 cells. *P<0.05 compared with the PG-untreated control cells. PG, propyl gallate.

Effects of PG on mitochondrial membrane potential (MMP; ∆Ψm) in lung cancer cells

As apoptosis is closely related to the collapse of MMP (∆Ψm), loss of MMP (∆Ψm) in PG-treated cells was evaluated using Rhodamine 123 dye. Loss of MMP (∆Ψm) in both lung cancer cell types was dose-dependently induced by PG at concentrations of 100–1,600 µM at 24 h (Fig. 5A-C). After exposure to 800 µM PG, the proportions of cells with MMP (∆Ψm) loss in Calu-6 and A549 cell lines were approximately 30 and 45%, respectively (Fig. 5A-C). In relation to the levels of MMP (∆Ψm) in both lung cancer cell lines at 24 h, 100 µM PG increased the MMP (∆Ψm) level in Calu-6 cells whereas PG at 200–1,600 µM significantly decreased the MMP (∆Ψm) level in these cells (Fig. 5A and D). Treatment with 100–1,600 µM PG also reduced MMP (∆Ψm) levels in A549 cells (Fig. 5A and E). The levels of MMP (∆Ψm) were approximately 30 and 20% in Calu-6 and A549 cell lines treated with 800 µM PG, respectively (Figs. 5A, D and E).

Figure 5. Effects of PG on mitochondrial membrane potential (MMP; ∆Ψm) in lung cancer cells. Exponentially growing cells incubated in the presence of the designated concentrations of PG for 24 h. MMP (∆Ψm) in lung cancer cells was measured using FACStar flow cytometer. (A) Representative histograms for Rhodamine 123 staining in PG-treated Calu-6 and A549 cells. M1 indicates Rhodamine 123-negative [MMP (∆Ψm) loss] cells. M2 indicates cells without MMP (∆Ψm) loss. (B and C) Graphs show the percentages of M1 regions in A in (B) Calu-6 and (C) A549 cells. (D and E) Graphs indicate the proportions of MMP (∆Ψm) levels derived from M2 regions in A in (D) Calu-6 and (E) A549 cells. *P<0.05 compared with the PG-untreated control cells. PG, propyl gallate.

Effects of PG on apoptosis-related protein levels and activities of caspases in lung cancer cells

Since PG induced cell death in both lung cancer cell types, the levels of apoptosis-related proteins were assessed by western blot analysis. Examination of Bcl-2 regulation in PG-treated cells revealed that Bcl-2 protein levels were reduced following treatment with 200–800 µM PG in both lung cancer cell types (Fig. 6A and B). Caspase-3 plays an essential role as an executioner in apoptosis (25). Whether PG activates caspase-3 in PG-treated lung cancer cells was also examined. Levels of procaspase-3 (32 kDa precursor) were visibly reduced following treatment with 400–800 µM PG in the Calu-6 cells (Fig. 6A) and following treatment with 200–800 µM PG in the A549 cells (Fig. 6B). Cleavage of PARP provides one of the most recognizable markers of apoptosis (26). While the intact 116 kDa moiety of PARP was reduced in the 400–800 µM PG-treated Calu6 cells, the cleaved form of PARP increased in these cells (Fig. 6A). PG at 200–800 µM also reduced the intact form of PARP in the A549 cells (Fig. 6B). Furthermore, 400–800 µM PG treatment significantly increased caspase-3 activities in the Calu-6 cells (Fig. 6C). Caspase-8, which is correlated with the cell death receptor pathway (25), was also significantly activated by treatment with 800 µM PG in Calu-6 cells (Fig. 6C). However, 400 µM PG downregulated the activity of caspase-8, compared with basal activity of the control cells (Fig. 6C).

Figure 6. Effects of PG on apoptosis-related protein levels in lung cancer cells. Exponentially growing cells were incubated in the presence of the designated concentrations of PG (0–800 µM) for 24 h. Protein extracts were resolved via 8–15% SDS-PAGE gel, transferred to PVDF membranes, and immunoblotted with the designated antibodies. Western blot analysis shows the levels of Bcl-2, caspase-3, PARP and β-actin in the PG-treated (A) Calu-6 and (B) A549 cells. (C) Graphs show the activities of caspase-3 and caspase-8 in Calu-6 cells. *P<0.05 compared with the PG-untreated control cells. PG, propyl gallate; PARP, poly(ADP-ribose) polymerase.

Discussion

Although propyl gallate (PG) is used to preserve and stabilize medicinal provisions included on the US Food and Drug Administration list (27), the latent toxicity of PG has been inspected to assess various in vivo or in vitro toxicological properties (2,28–30). In the present study, the anti-growth effects of PG were examined in Calu-6 and A549 lung cancer cell lines in relation to apoptosis as well as cell cycle arrest. Treatment with PG decreased the growth of both lung cancer cell types with an IC50 of approximately 800 µM at 24 h. Similarly, 800 µM of PG was found to reduce the growth of HeLa cells by approximately 50% at 24 h (23). However, 800 µM PG was found to strongly reduce the growth of umbilical vein and pulmonary artery endothelial cells by more than 50% at 24 h and the susceptibility of pulmonary artery endothelial cells to PG was higher than that of umbilical vein endothelial cells (20). In addition, 400 µM PG did not significantly inhibit the growth of primary human pulmonary fibroblasts at 24 h (31). Because PG can damage mitochondria which leads to ATP depletion (9,20,23,32), the varying susceptibility to PG is perhaps owing to the different basal activities of mitochondria and antioxidant enzymes that each cell type has. Thus, the toxicity of PG should be carefully studied and interpreteted in vivo and in vitro depending on PG concentration, incubation time, and experimental target cells.

In the present study, PG induced the apoptosis of Calu-6 and A549 cells, as demonstrated by the proportions of sub-G1 and Annexin V-stained cells. In addition, PG treatment clearly decreased the Bcl-2 levels, along with an increase in the cleavage form of PARP. DNA flow cytometry indicated that PG induced arrest at the G1 phase of the cell cycle in both lung cancer cell types at 24 and 72 h. Similarly, PG was previously found to induce G1 phase arrest of the cell cycle along with an increase in p27, a cyclin-dependent kinase (CDK) inhibitor (CDKI), in HeLa cells (23). In the present study, treatment with 800 µM PG inhibited the growth of lung cancer cells by approximately 50% but this dose of PG induced cell death by approximately 10% in view of the percentages of the sub-G1 cells and Annexin V-stained cells. Thus, the significant G1 phase arrest by PG is another conceivable underlying mechanism for the inhibition of cell growth. It is worthwhile to study whether PG affects the expression levels of cyclin, CDKs, and CDKIs to induce cell cycle arrest, especially at the G1 phase in lung cancer cells. Collectively, PG inhibits the growth of lung cancer cells via G1 phase arrest of the cell cycle as well as apoptosis.

Apoptosis is closely related to the failure of mitochondrial membrane potential [MMP (∆Ψm)] (33), and PG can cause a breakdown in MMP (∆Ψm) (9,20,23,32). In the present study, likewise, PG dose-dependently induced the loss of MMP (∆Ψm) and reduced the MMP (∆Ψm) level in lung cancer cells. The degree of cells with MMP (∆Ψm) loss in the PG-treated Calu-6 (SCLC; small cell lung cancer) cells was lower than that in the PG-treated A549 (NSCLC; non-small cell lung cancer) cells. For example, 800 µM of PG increased the percentage of cells with MMP (∆Ψm) loss in the Calu-6 and A549 cell lines by approximately 30 and 45%, respectively. While the level of MMP (∆Ψm) was approximately 30% in the 800 µM PG-treated Calu-6 cells, the MMP (∆Ψm) level in A549 cells was approximately 20%. Interestingly, the degree of MMP (∆Ψm) loss in the PG-treated lung cells was higher than that of the Annexin V-stained cells. Furthermore, the proportions of Annexin V-stained cells in the PG-treated Calu-6 cells were lower than those in the A549 cells. These results imply that PG initially influences the mitochondrial membrane, especially in adenocarcinoma A549 (NSCLC) cells, which precedes the next step in apoptosis.

Apoptosis involves the mitochondrial (intrinsic) and cell death receptor (extrinsic) pathways (15). Caspase-3 plays a critical role as an executioner of apoptosis. The levels of procaspase-3 (32 kDa precursor) were reduced in the PG-treated lung cancer cells, which suggests that activation of caspase-3 occurred in these cells. In fact, PG treatment upregulated the activity of caspase-3 in Calu-6 cells. In particular, the activation of caspase-8 was observed in apoptosis of Calu-6 cells induced by PG. However, 400 µM PG that showed a slightly decreased amount of Annexin V-stained cells lessened caspase-8 activity, compared with the basal activity of the control cells. Further research is required to elucidate the exact mechanism that is involved. Caspase-8 activation is linked with the cell death receptor pathway of apoptosis (25,34). PG-induced apoptosis in lung cancer cells involved both extrinsic and intrinsic pathways.

In conclusion, PG treatment inhibited the growth of lung cancer cells, especially Calu-6 cells via caspase-dependent apoptosis as well as G1 phase arrest of the cell cycle (Fig. 7). The presented data provides valuable information for understanding the cytotoxicological effects of PG in lung cancer cells in view of apoptosis and cell cycle arrest. Since PG has been reported to exert prooxidant properties (6,7), it is worth studying whether PG induces apoptosis of Calu-6 and A549 lung cancer cells through oxidative stress in the future.

Figure 7. Schematic diagram of the anti-growth effects of PG on lung cancer cells, with a focus on SCLC Calu-6 cells. PG, propyl gallate; SCLC, small cell lung cancer; MMP (∆Ψm), mitochondrial membrane potential.

Acknowledgements

Not applicable.

Funding

The present study was supported by a grant (2019R1I1A2A01041209) of the Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Education, Republic of Korea.

Availability of data and materials

Data collected during the present study are available from the corresponding author upon reasonable request.

Authors' contributions

WHP conceived and designed the study, performed the experiments, and wrote the manuscript. WHP agrees to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Authors' information

Professor Woo Hyun Park: ORCID: 0000-0003-4341-5188.

Glossary

Abbreviations

Abbreviations:

References