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).

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.

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.

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.

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).

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.

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.

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).

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).

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.

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.

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.

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).

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).

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).

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).