Comparison of Anticancer Effects of Histone Deacetylase Inhibitors CG-745 and Suberoylanilide Hydroxamic Acid in Non-small Cell Lung Cancer
Article information
Abstract
Background
Histone deacetylase (HDAC) inhibition offers potential anticancer effects across diverse cancers due to HDAC's significant role in cancer development and progression. Consequently, we demonstrated the therapeutic efficacy of the novel HDAC inhibitor, CG-745, in comparison with existing inhibitors such as suberoylanilide hydroxamic acid (SAHA) in non-small cell lung cancer (NSCLC) cells.
Methods
CG-745's effect on apoptosis and reactive oxygen species (ROS)-dependent mitochondrial dysfunction was investigated using annexin V assay, MitoSoX, and Western blot in human A549 and H460 cells. Additionally, HDAC expression was analyzed through real-time polymerase chain reaction. We also evaluated the inhibitory effect of CG-745 on epithelial-mesenchymal transition (EMT) induced by transforming growth factor β1 (TGF-β1) via Western blot, scratch analysis, and matrigel invasion analysis.
Results
Compared to SAHA, CG-745 inhibited cell viability and mRNA expression of HDACs such as HDAC1, HDAC2, HDAC3, and HDAC8. It also induced apoptosis, ROS, and mitochondrial dysfunction in a concentration-dependent manner. CG-745 reversed EMT triggered by TGF-β1 in A549 and H460 cells, and curtailed the migration and invasion enhanced by TGF-β1. CG-745 has demonstrably inhibited EMT and induced apoptosis in NSCLC cells.
Conclusion
CG-745 may represent a novel therapeutic strategy for NSCLC treatment.
Introduction
Histone deacetylases (HDACs) play crucial roles in regulating chromatin structure and gene expression through deacetylating histones, and their dysregulation has been implicated in several cancers [1-3]. HDACs are involved in key carcinogenic events and oncogenic protein fusion by modulating histone and non-histone proteins, which regulate cell cycle, cell death, metastasis, autophagy, and other cellular processes. Previous studies have shown high expression of HDAC1 in multiple cancers including prostate, gastric, lung, esophageal, colon, and breast cancers; overexpression of HDAC2 in colorectal, cervical, and gastric cancers; and high expression of HDAC3 in colon, gastric, and squamous cell non-small cell lung cancer (NSCLC). Additionally, HDAC1 and HDAC3 are known to be associated with poor prognosis in lung cancer, highlighting HDACs as potential targets for cancer therapy.
CG-745, a newly developed intravenous hydroxamate-based pan-HDAC inhibitor, inhibits cell growth and has been studied as an innovative anticancer treatment in various cancer cells including prostate, pancreatic, renal cell carcinoma, and colon cancers in both monoand combination therapies with other anticancer drugs. It was found to be five times more effective in acetylating histone H3 than the HDAC inhibitor vorinostat in liver cancer cell lines, and it not only induces cell cycle arrest and cancer cell death but also promotes immune activation and enhances immune cancer activity [4]. Additionally previous studies have demonstrated that CG-745 possesses anti-inflammatory and anti-fibrotic effects through epithelial-mesenchymal transition (EMT) regulation in bleomycin or polyhexamethylene guanidine (PHMG)-induced lung fibrosis models5.
In this study, we compared the effectiveness of the established HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the novel HDAC inhibitor CG-745 in NSCLC cells to evaluate their anticancer properties.
Materials and Methods
1. Materials
Roswell Park Memorial Institute medium 1640 (RPMI 1640), fetal bovine serum (FBS), and antibiotics (penicillin and streptomycin) were procured from GIBCO BRL Co. (Grand Island, NY, USA). CG-745 was synthesized by Crystal Genomics (Seongnam, Korea). SAHA, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), cycloheximide, and MG132 were procured from Sigma-Aldrich (St. Louis, MO, USA). All primary antibodies were sourced from Cell Signaling Technology (Beverly, MA, USA). Anti-rabbit immunoglobulin G (IgG)-conjugated horseradish peroxidase (HRP) antibodies and anti-mouse IgG-conjugated HRP were purchased from Bethyl (Montgomery, TX, USA).
2. Cell culture
The A549 and H460 cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were cultivated in RPMI-1640, supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, in a humidified atmosphere containing 5% CO2 at 37°C.
3. Cell viability assay
The effect of CG-745 or SAHA treatment on cell viability was assessed using the MTT assay. A549 and H460 cells were seeded at a density of 5×103 cells per well in 96-well plates in triplicate. After 24 hours, various concentrations of CG-745 or SAHA were administered, and the cells were incubated for 48 hours. To determine cell viability, MTT was introduced into the cell suspension for 4 hours. Following three washes with phosphate buffered saline (PBS; pH 7.4), the insoluble formazan product was solubilized in DMSO. The optical density (OD) of each well was subsequently measured using a microplate reader (Titertek Multiskan, Flow Laboratories, North Ryde, Australia) at 590 nm. The OD resulting from formazan production in control cells was designated as 100% viability, with all other values expressed as a percentage relative to this control value.
4. Real-time polymerase chain reaction analysis
Real-time polymerase chain reaction (PCR) was performed on complementary DNA (cDNA) samples employing the SYBR Green system (Bioneer, Daejeon, Korea). Primers used were HDAC1, sense 5’-GGCGAGCAAGATGGCGCAGA-3’ and anti-sense 5’-AATTTCCAACATCCCCGTCGTAGT-3’, HDAC2, sense 5’-TGGTGTCCAGATGCAAGCTA-3’ and anti-sense 5’-GCCACAT TTCTTCGACCTCC-3’, HDAC3, sense 5’-ACTTCGAGTACTTTGCCCCA-3’ and anti-sense 5’-GGCACGTCATGAATCTGGAC-3’, HDAC8, sense 5’-ACGTGTCTGATGTTGGCCTA-3’ and anti-sense 5’-TCCCAGCTGTAAGACCACTG-3’, tubulin, sense 5’-CAGATGCCCAGTGACAAGACC-3’ and anti-sense 5’-CAATGACCGTAGGCTC CAGAT-3’. The following general real-time PCR protocol was employed: denaturation for 10 minutes at 95°C, 40 cycles of a four segment amplification and quantification program, a melting step by slow heating from 60°C to 99°C at a rate of 0.1°C/sec with continuous fluorescence measurement, and a final cooling step down to 40°C. Crossing point values were acquired using the second derivative maximum method of the LightCycler software version 3.3 (Roche, Burlington, NC, USA). Real-time PCR efficiencies were determined by amplifying a standardized dilution series, and slopes were calculated using LightCycler software.
5. Immunoblot analysis
Cells were harvested and lysed in protein extraction solution (PRO-PREP; Intron Biotechnology, Seongnam, Korea) for 20 minutes on ice. Cell lysates were centrifuged at 15,000 rpm for 20 minutes at 4°C, and the supernatant was combined with a one-fifth volume of 5× sodium dodecyl sulfate (SDS) sample buffer. The mixture was boiled for 5 minutes and subsequently separated on a 12% SDS-polyacrylamide gel electrophoresis gel. Following electrophoresis, proteins were transferred to a membrane, which was then blocked in TBS-T (25 mM Tris [pH 7.6], 138 mM NaCl, and 0.05% Tween-20) containing 5% skim milk. Membranes were incubated with primary antibodies (at dilutions ranging from 1:1,000 to 1:5,000). After a series of washes, the membranes were further incubated with the secondary antibody (at dilutions ranging from 1:2,000 to 1:10,000) conjugated with HRP. The immunoreactive signals were detected using an ECL detection system (Amersham, England).
6. Annexin V assay for the assessment of apoptosis
A549 and H460 cells exhibiting early/late apoptosis were analyzed using annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining. Cells in the exponential growth phase (2.5×105 cells) were seeded in 35-mm2 dishes. The cells were either left untreated or incubated with specified drugs for the indicated durations at 37°C. Both adherent and floating cells were collected and processed using the annexin V assay, as per the manufacturer’s protocol. The cells were briefly washed with PBS, resuspended in annexin binding buffer, and incubated with annexin V-FITC and PI for 15 minutes at room temperature. Following incubation, the stained cells were examined using a fluorescence-activated cell sorting (FACS) Calibur system equipped with Cell Quest software (Becton Dickinson, San Jose, CA, USA). Cells without drug treatment served as controls.
7. Determination of reactive oxygen species
To quantify intracellular reactive oxygen species (ROS), harvested A549 and H460 cells were incubated in the dark with 10 µmol/L 5-(and-6)-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA; Molecular Probes, Eugene, OR, USA) for 30 minutes. After incubation, the cells were washed, resuspended in PBS, and maintained on ice for immediate analysis by FACS flow cytometry, utilizing an argon laser (488 nm) for excitation. Green fluorescence from intracellularly retained 2’,7’-dichlorofluorescin (DCF) was detected on the FL1 channel on a logarithmic scale. Data were collected and analyzed using the Cell Quest program (Becton Dickinson).
To measure mitochondria-derived ROS, the mitochondria-targeted, O2 − sensitive, hydroethidine analog probe MitoSOX (M36008, Invitrogen Life Technologies, Carlsbad, CA, USA) was employed to assess relative O2 − levels. Briefly, cells were incubated with 5 μM MitoSOX for 10 minutes in PBS. Following incubation, the cells were washed twice with PBS and subsequently analyzed using FACS flow cytometry.
8. Measurement of the mitochondrial membrane potential (ΔΨm)
A549 and H460 cells were harvested at specified treatment times, washed with PBS, and stained with 10 µg/mL JC-1 at 37°C for 30 minutes. After a brief rinse with PBS, the cells were immediately analyzed using a FACS Calibur system equipped with Cell Quest software. At low concentrations, JC-1 exists principally in a monomeric form, emitting green fluorescence (emission maximum at approximately 530 nm), while at higher concentrations, it forms aggregates (J-aggregates) that emit orange-red fluorescence (emission maximum at approximately 590 nm).
9. Wound healing assay
A549 and H460 cells were cultured in 6-well plates with a seeding density of 1×106 cells/well. Cells were subsequently scratched using a sterile 200 μL pipette tip. Subsequently, they were incubated for 48 hours with or without 5 ng/mL transforming growth factor β1 (TGF-β1), and with SAHA 0.5 or 1 μM, or CG200745 0.5 or 1 μM. Migration of cells into the exposed area and recovery of the monolayer were evaluated at 24-hour intervals over 48 hours using a phase contrast microscope, and digitally photographed (Nikon Diaphot 300, Nikon, Tokyo, Japan).
10. Matrigel invasion assay
Cell invasion assay kits (Chemicon International, Temecula, CA, USA) were used according to the manufacturer's instructions to evaluate cell invasion. Cells were resuspended in culture media and placed in a chemoinvasion chamber. A549 and H460 cells were seeded at a density of 2×104 per insert and cultured for 12 hours. Subsequently, the cells were maintained in wells containing the same medium supplemented with TGF-β1 (5 ng/mL), with or without SAHA or CG-745. After 48 hours, non-invading cells were removed with cotton swabs. The invasive capacity of the cells was determined following the manufacturer’s recommendations. Photomicrographs of the invasive cells were captured in five predetermined fields (magnification 200×) and quantification of stained cells was achieved by dissolving them in 10% acetic acid and measuring the OD at 540 nm.
11. Ethics statement
The study received approval from the Institutional Review Board of Wonkwang University Hospital (IRB No. WKUH NON2024-001) and was conducted in accordance with the principles outlined in the Declaration of Helsinki. Written informed consent was obtained from all participants.
12. Statistical analysis
Each experiment was conducted at least three times, with all values presented as the mean±standard deviation of triplicate experiments. Statistical significance of the results was determined using Student's t-test, with p-values less than 0.05 deemed statistically significant.
Results
1. CG-745 is more effective than SAHA in inhibiting cell viability and HDACs expression in human lung carcinoma cell lines
To evaluate the effectiveness of CG-745 and HDAC inhibitor SAHA in NSCLC cells, A549 and H460 cells were treated with various doses of CG-745 and SAHA for 48 hours. Compared to SAHA, cell viability was lower in the CG-745 treated group than in the SAHA treated group in a dose-dependent manner (Figure 1A). To determine if CG-745 is involved in HDAC regulation, the mRNA expression of HDAC was analyzed using real-time PCR. Compared to the control group, SAHA reduced the expression of HDAC3 and HDAC8 at doses of 0.5 and 1 μM and reduced the expression of HDAC1 and HDAC2 at a dose of 1 μM. Compared to the control group, CG-745 reduced the expression of HDAC1, HDAC2, HDAC3, and HDAC8 at doses of 0.5 and 1 μM. CG-745 also decreased the expression of HDAC1, HDAC2, HDAC3, and HDAC8 more than SAHA (Figure 1B).
CG-745 is more effective than suberoylanilide hydroxamic acid (SAHA) in inhibiting cell viability and Histone deacetylases (HDACs) expression in human lung carcinoma cell lines. (A) A549 and H460 cells were treated with different concentration of CG-745 or SAHA for 48 hours, and viability was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The viability of control (CTL) cells was set at 100%, and cell survival relative to CTL is presented. (B) The mRNA levels of class I HDAC family were measured by real-time polymerase chain reaction analysis in CG-745 or SAHA treated A549 and H460 cells. *p<0.05 vs. CTL; †p<0.05 SAHA vs. CG-745.
2. CG-745 is more effective than SAHA in inducing apoptosis in human lung carcinoma cell lines
To investigate if the cell death occurred via enhanced apoptosis, apoptosis-related markers were assayed by Western blotting. Consequently, the expression of acetylated histone H3 (Ac-H3), cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase -3, -8, -9 was higher in NSCLC cells treated with CG-745 than in those treated with SAHA in a dose-dependent manner (Figure 2A). Moreover, annexin V staining indicated that CG-745 treatment eliminated viable cells more effectively than SAHA treatment in a dose-dependent manner (Figure 2B).
CG-745 is more effective than suberoylanilide hydroxamic acid (SAHA) in inducing apoptosis in human lung carcinoma cell lines. A549 and H460 cells were treated with indicated concentration of CG-745 or SAHA for 48 hours. (A) Cells were subjected to Western blot for acetylated histone H3 (Ac-H3), H3, cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-9, -8, and -3. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels were monitored as a loading control (CTL) whole-cell extracts. (B) The apoptosis in A549 and H460 was evaluated by fluorescein isothiocyanate (FITC)-annexin V+propidium iodide. *p<0.05 vs. CTL; †p<0.05 SAHA vs. CG-745.
3. CG-745 is more effective than SAHA in inducing ROS and mitochondrial dysfunction in human lung carcinoma cell lines
We investigated whether ROS is involved in CG-745-induced apoptosis by utilizing H2DCFDA as the total ROS marker and MitoSOX Red to identify mitochondrial superoxide over 48 hours. The results demonstrated an increase in ROS production following treatment with CG-745 and SAHA, with CG-745 showing a significantly greater increase compared to SAHA in a dose-dependent manner, assessed via H2DCFDA (Figure 3A). Additionally, using MitoSOX to measure intracellular O2 levels linked with total ROS production onset, it was found that O2 levels increased more following CG-745 treatment than with SAHA (Figure 3B). Analysis of ROS effects on membrane potential differences using JC-1 revealed that, compared to SAHA, JC-1 monomer levels increased after CG-745 treatment (Figure 3C).
CG-745 is more effective than suberoylanilide hydroxamic acid (SAHA) in inducing reactive oxygen species (ROS) and mitochondrial dysfunction in human lung carcinoma cell lines. A549 and H460 cells were treated with indicated concentration of CG-745 or SAHA for 48 hours. (A) The generation of ROS was evaluated by 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA). (B) MitoSOX and (C) JC-1 as described in Materials and Methods. Fluorescence measurements were carried out using a flow cytometry. *p<0.05 vs. control (CTL); †p<0.05 SAHA vs. CG-745. DCF: 2’,7’-dichlorofluorescin; ΔΨm: mitochondrial membrane potential.
4. CG-745 is more effective than SAHA in suppressing TGF-β1 induced EMT in human lung carcinoma cell lines
The effect of CG-745 on cell proliferation in NSCLC cells was investigated using a cell counting kit-8 assay. CG-745 treatment reduced the increase in cell proliferation induced by TGF-β1 compared to SAHA treatment (Figure 4A). To explore the impact of CG-745 on TGF-β1-induced EMT, we assessed the expression of EMT markers. CG-745 and SAHA treatment resulted in increased expression of E-cadherin and occludin, which are typically reduced by TGF-β1; CG-745 demonstrated greater expression than SAHA. Additionally, N-cadherin, vimentin, snail, slug, and zinc finger E-box binding homeobox 1 (ZEB1), which are upregulated by TGF-β1, showed reduced expression with CG-745 treatment compared to SAHA (Figure 4B). The role of CG-745 in inhibiting lung cancer cell migration was evaluated using a wound healing assay. CG-745 effectively in-hibited migration of A549 and H460 cells, which was enhanced by TGF-β1, in a time and dose-dependent manner relative to SAHA (Figure 5). CG-745 also proved effective in inhibiting TGF-β1 induced lung cancer migration. The impact of CG-745 on lung cancer cell invasion was assessed using a matrigel invasion assay. While TGF-β1 treatment increased cell invasion compared to untreated cells, CG-745 treatment effectively inhibited lung cancer cell invasion in a dose-dependent manner compared to SAHA (Figure 6).
CG-745 is more effective than suberoylanilide hydroxamic acid (SAHA) in suppressing cell proliferation and transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transition (EMT) in human lung carcinoma cell lines. A549 and H460 cells were treated with CG-745 or SAHA at the specified concentrations for 48 hours with or without TGF-β1 (5 ng/mL). (A) Cell proliferation was measured using the cell counting kit-8 assay. The proliferation of control cells was set at 100%, and cell proliferation relative to control is presented. (B) Cells were subjected to Western blot for acetylated histone H3 (Ac-H3), H3, EMT markers (E-cadherin, occludin, N-cadherin, vimentin, snail, slug, zinc finger Ebox binding homeobox 1 [ZEB1]). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels were monitored with loading control whole-cell extract. *p<0.05 vs. control; †p<0.05 vs. TGF-β1; ‡p<0.05 SAHA vs. CG-745.
CG-745 is more effective than suberoylanilide hydroxamic acid (SAHA) in inhibiting transforming growth factor β1 (TGF-β1)-induced cell migration in human lung cancer cell lines. Cell migration was measured using a wound healing assay. (A) A549 and (B) H460 cells were treated with a specific concentration of CG-745 or SAHA with TGF-β1 (5 ng/mL), and observed at 0, 24, and 48 hours. CTL: control.
CG-745 is more effective than suberoylanilide hydroxamic acid (SAHA) in inhibiting transforming growth factor β1 (TGF-β1)-induced cell invasion in human lung cancer cell lines. Cell invasion was measured using the matrigel invasion assay (×100). A549 and H460 cells were treated with a specific concentration of CG-745 or SAHA with TGF-β1 (5 ng/mL) and observed at 48 hours. (A) Matrigel invasion of A549 and H460 cells, as counted in five random views. (B) The data represent the mean±standard deviation of three independent experiment. *p<0.05 vs. control (CTL); †p<0.05 vs. TGF-β1; ‡p<0.05 SAHA vs. CG-745.
Discussion
HDACs deacetylate histone lysine for condensation and subsequently inhibit chromatin [6], thereby regulating the expression of oncogenes and exhibiting anticancer effects [1]. Elevated HDAC levels have been observed in various malignant tumors such as stomach, lung, liver, pancreatic, and colon cancer, with high expression of HDAC associated with poor outcomes in cancers [7,8]. HDAC plays a role in cancer progression by regulating cellular functions including differentiation, cell cycle progression, DNA damage response, apoptosis, and angiogenesis [9].
In total, five HDAC inhibitors, namely vorinostat (SAHA), romidepsin, belinostat, tucidinostat, and panobinostat, have been approved as anticancer drugs by the U.S. Food and Drug Administration. Vorinostat (SAHA) is classified as a pan-HDAC inhibitor and has demonstrated significant antitumor effects in NSCLC [10] . SAHA induces antiproliferative effects by inhibiting telomerase activity, upregulating the cyclin-dependent kinase inhibitor p21, suppressing the G0–G1 cell cycle, and decreasing the expression of C-myc and bcl-2 [11].
CG-745 has demonstrated a novel anticancer effect in various cancer cells, including prostate, pancreatic, renal cell carcinoma, and colon cancer, both as a mono-therapy and in combination with other anticancer therapies. It also has therapeutic effects on immunity, high fat diet induced hypertension, and kidney fibrosis [4,5,12-16]. Accordingly, we compared the established HDAC inhibitor, SAHA, with the new HDAC inhibitor, CG-745, to evaluate their anticancer effects in NSCLC using apoptotic factors. CG-745 exhibited higher expression of cleaved PARP, cleaved caspase-9, -8, and -3 compared to SAHA in A549 cells and H460 cells among NSCLC cell lines. ROS produced in mitochondria inhibit cell growth and induce cell cycle arrest and apoptosis. ROS are known to play a crucial role as regulators of cell death and multidrug resistance in cancer cells [17-20]. CG-745 increased ROS and mitochondrial dysfunction, showing a greater increase compared to SAHA, indicating that ROS is implicated in the enhanced apoptosis induced by CG-745.
SAHA has been shown to decrease the expression of extracellular signal-regulated kinase 1/2 (Erk1/2) and matrix metalloproteinase-9 (MMP-9), enhance p53 expression, thereby influencing cell proliferation and apoptosis, and increase the acetylation of histones H3 and H4 in ovarian carcinoma cells [21]. Additionally, entinostat-induced cytotoxic effects are partially dependent on p53. Conversely, Chun et al. [22] found that the antitumor effects of CG-745 on NSCLC cells are not influenced by the mutation status of the p53-encoding genes, indicating that different HDAC inhibitors have varying dependencies on p53 [23].
EMT is involved in various cellular processes including cell plasticity, metastasis, migration, invasion, mod-ification of the extracellular matrix, and cell death. It also regulates cancer progression, metastasis, and the induction of drug resistance [24,25]. Several well-established biomarkers are associated with EMT, including transcription factors (SNAIL, TWIST), growth factors (TGF-β, Wnts), cytoskeletal proteins (vimentin), and adhesion molecules (cadherin) [26]. TGF-β is recognized as the most significant signaling factor [27]. Compared with SAHA, CG-745 effectively inhibited TGF-β1 induced EMT along with migration and invasion, demonstrating its efficacy in EMT inhibition.
Previous clinical studies have shown that HDAC inhibitors have limited effectiveness in solid tumors and are linked to unfavorable adverse effects [28]. In contrast, CG-745 exhibited a favorable safety profile during its initial human trial, with no dose-limiting toxicities observed. Only grade 3/4 hematologic toxicities, such as anemia and neutropenia, were reported; these resolved within a week. Moreover, administration of CG-745 proved safe at doses effective in inhibiting HDAC activity in both peripheral blood mononuclear cells and tumor tissues, highlighting its potential as a therapeutic agent [29].
In conclusion, this study demonstrated that CG-745 can induce apoptosis in NSCLC cells and inhibit their proliferation, migration, and invasion by regulating EMT expression. This suggests that CG-745 could be a novel treatment strategy for lung cancer. However, more research is required to elucidate the mechanisms behind its anticancer effects.
Notes
Authors’ Contributions
Conceptualization: Kim HJ, An UR, Kim HR. Methodology: Kim HJ, Kim YS, Kim HR. Formal analysis: Kim HJ, An UR, Kim YS, Kim HR. Writing - original draft preparation: Kim HJ, An UR, Hwang KE, Kim HR. Writing - review and editing: An UR, Hwang KE, Kim YS, Kim HR. Approval of final manuscript: all authors.
Conflicts of Interest
No potential conflict of interest relevant to this article was reported.
Funding
This study was supported by a grant from Wonkwang University in 2023.