TAN (purity ≥95%) was purchased from Aladdin Scientific Corporation. A stock solution of TAN was prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA) and was subsequently diluted in culture medium to achieve the desired concentrations, with the final DMSO concentration maintained at <0.1% in all experiments.

CaSki cells were purchased from the Korean Cell Line Bank; Korean Cell Line Research Foundation. The cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (R&D Systems, Inc.) supplemented with 10% fetal bovine serum (FBS; R&D Systems, Inc.), 10 mM HEPES (Sigma-Aldrich; Merck KGaA) and 1% antibiotic-antimycotic solution (Sigma-Aldrich; Merck KGaA). Subculturing was performed using 0.05% trypsin-EDTA (Thermo Fisher Scientific, Inc.) at a split ratio of 1:5. The cells were maintained at 37°C in a humidified incubator containing 5% CO₂.

CaSki cells were seeded in 96-well plates at a density of 4×10³ cells/well. After 24 h of incubation, the medium was replaced with RPMI-1640 supplemented with 5% FBS containing either 0.1% DMSO (control) or TAN (2.5–40 µM). After 48 h of treatment at 37°C in a humidified incubator with 5% CO₂, the culture medium was removed and 5% EZ-Cytox cell viability assay reagent (cat. no. EZ-3000; DoGenBio), a water-soluble tetrazolium salt reagent, was added and incubated for 30 min at 37°C in a humidified incubator with 5% CO₂. Absorbance was measured at 450 nm using a Synergy Neo2 Hybrid Multimode Reader (Agilent Technologies, Inc.).

CaSki cells were seeded in 6-well plates at a density of 1×10³ cells/well and incubated for 24 h. The cells were then treated with RPMI-1640 medium supplemented with 5% FBS containing either 0.1% DMSO (control) or TAN (10–40 µM) for 48 h at 37°C in a humidified incubator with 5% CO₂. Thereafter, the culture medium was replaced every 3 days with RPMI-1640 supplemented with 5% FBS, and the experiment was continued for 2 weeks. For analysis, the colonies were fixed with 4% paraformaldehyde (PFA; GeneAll Biotechnology Co., Ltd.) for 15 min at room temperature and stained with 0.5% crystal violet (Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. Colony formation was quantified by measuring the total colony area using ImageJ software (version 1.53; National Institutes of Health). Colonies with an area of ≥20 pixels² were included in the analysis. The colony formation rate was calculated by normalizing the colony area of each treatment group to that of the control group, which was set to 100%.

CaSki cells were seeded in 6-well plates at a density of 1×10⁵ cells/well and incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 48 h until they reached ~80% confluence. To minimize the contribution of cell proliferation during wound closure analysis, the cells were pretreated with mitomycin C (2 µg/ml; Roche Diagnostics) for 1.5 h at 37°C in a humidified incubator containing 5% CO₂ prior to scratch formation. A linear scratch was then generated in each well using sterile surgical forceps. After removing the culture medium and washing to eliminate cellular debris, the cells were incubated in RPMI-1640 medium supplemented with 5% FBS, containing either 0.1% DMSO (control) or TAN (1.25–5 µM). The FBS concentration (5%) was selected to maintain cell viability during the wound-healing assay (21). After treatment, the cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂. Images of the wound area were captured at 0, 24 and 48 h at ×40 magnification using an Olympus CKX41 inverted light microscope (Olympus Corporation), and wound closure was semi-quantified using ImageJ software (version 1.53).

CaSki cells (5×10⁵ cells/well) were seeded into the upper chamber of 24-well Transwell inserts (pore size, 8.0-µm). The upper chamber was filled with RPMI-1640 medium supplemented with 1% FBS containing either 0.1% DMSO (control) or TAN (10–40 µM). The lower chamber contained RPMI-1640 medium supplemented with 10% FBS as a chemoattractant. After 48 h of incubation at 37°C in a humidified atmosphere containing 5% CO₂, the non-migratory cells on the upper surface of the membrane were removed, and cells that had migrated to the lower surface were fixed with 4% PFA for 15 min at room temperature and stained with 0.5% crystal violet for 30 min at room temperature. Images were captured using an Olympus CKX41 microscope, and the number of migrated cells was semi-quantified using ImageJ software (version 1.53).

CaSki cells (3×10³ cells/well) were seeded in 6-well plates and incubated for 24 h. The culture medium was then replaced with RPMI-1640 supplemented with 5% FBS containing either 0.1% DMSO (control) or TAN (10–40 µM). After 48 h of treatment at 37°C in a humidified atmosphere containing 5% CO₂, the cells were harvested and fixed in 70% ethanol at −20°C overnight. Fixed cells were subsequently stained with Hoechst 33342 (10 µg/ml; cat. no. H3570; Thermo Fisher Scientific, Inc.) for 30 min at 37°C in a humidified atmosphere containing 5% CO₂. Hoechst 33342, a DNA-binding fluorescent dye, was used to determine cell cycle distribution based on cellular DNA content, as previously described (22). Cell cycle distribution was analyzed using a FACSymphony^™ flow cytometer (BD Biosciences) with the BV421 channel and data were analyzed using FlowJo software (version 10; BD Biosciences).

CaSki cells (3×10³ cells/well) were seeded in 6-well plates and incubated for 24 h. The culture medium was then replaced with RPMI-1640 supplemented with 5% FBS containing either 0.1% DMSO (control) or TAN (10–40 µM). After 48 h of treatment at 37°C in a humidified atmosphere containing 5% CO₂, the cells were harvested and resuspended in 100 µl 1X Annexin-binding buffer. Subsequently, 5 µl Alexa Fluor^™ 488 Annexin V and 1 µl PI (100 µg/ml) from the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit (Invitrogen; Thermo Fisher Scientific, Inc.) were added to each sample and incubated for 15 min at room temperature in the dark according to the manufacturer's instructions. Following incubation, 400 µl 1X Annexin-binding buffer was added and the samples were immediately analyzed using a FACSymphony flow cytometer and the percentage of apoptotic cells was quantified using FlowJo software (version 10).

CaSki cells were seeded in 96-well plates at a density of 6×10³ cells/well and incubated for 24 h at 37°C in a humidified atmosphere containing 5% CO₂. The culture medium was then replaced with RPMI-1640 supplemented with 5% FBS containing either 0.1% DMSO (control) or TAN (10–40 µM). After 48 h of treatment at 37°C in a humidified atmosphere containing 5% CO₂, the medium was removed, and the cells were incubated with MitoSOX^™ Red Mitochondrial Superoxide Indicator (5 µg/ml; cat. no. M36008; Thermo Fisher Scientific, Inc.) and Hoechst 33342 (5 µg/ml; cat. no. H3570; Thermo Fisher Scientific, Inc.) for 10 min at 37°C. Fluorescence images were acquired using a Lionheart^™ FX Automated Imaging System (BioTek; Agilent Technologies, Inc.), with Hoechst 33342 detected in the DAPI channel and MitoSOX Red detected in the RFP channel. Images were acquired using a Lionheart FX Automated Microscope (BioTek; Agilent Technologies, Inc.) and analyzed using Gen5 software (version 3.05; BioTek; Agilent Technologies, Inc.) under identical image acquisition settings. Background fluorescence was automatically corrected using the built-in background subtraction function of the instrument. MitoSOX Red fluorescence intensity was normalized to the number of Hoechst 33342-positive nuclei in each field.

CaSki cells were seeded in black 96-well plates at a density of 5×10³ cells/well and incubated for 24 h. The culture medium was then replaced with RPMI-1640 supplemented with 5% FBS containing either 0.1% DMSO (control) or TAN (10–40 µM). After 24 h of treatment at 37°C in a humidified atmosphere containing 5% CO₂, the medium was removed and JC-1 dye (cat. no. ab113850; Abcam) was added to each well at a final concentration of 3 µg/ml. The cells were incubated for 30 min at 37°C and fluorescence images were acquired using a Lionheart FX Automated Imaging System, with JC-1 monomers detected in the GFP channel and JC-1 aggregates detected in the RFP channel. Images were acquired using a Lionheart FX Automated Microscope and fluorescence intensity was quantified using Gen5 software (version 3.05) under identical image acquisition settings. Background fluorescence was automatically corrected using the built-in background subtraction function of the instrument. Mitochondrial membrane potential (MMP) was evaluated based on the ratio of JC-1 aggregate (RFP) fluorescence to JC-1 monomer (GFP) fluorescence.

Cells in the control group were cultured in RPMI-1640 supplemented with 5% FBS containing 0.1% DMSO (vehicle control), and TAN-treated cells were cultured in RPMI-1640 supplemented with 5% FBS containing TAN (20 or 40 µM) for 48 h. Total cellular proteins were extracted using PRO-PREP™ protein extraction solution (Intron Biotechnology, Inc.) supplemented with a protease inhibitor cocktail (ATTO Corporation). Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay following the Sigma-Aldrich BCA protein assay protocol, using BCA solution (cat. no. B9643; Sigma-Aldrich; Merck KGaA) and 4% copper(II) sulfate solution (cat. no. C2284; Sigma-Aldrich; Merck KGaA), with bovine serum albumin (BSA; cat. no. A9418; MilliporeSigma) used as the protein standard. Western blot analysis was performed using 30 µg total protein per lane. Total proteins were separated by SDS-PAGE on 8% gels for E-cadherin and 10% gels for Bax and p53, followed by transfer onto PVDF membranes (Amersham^™ Hybond^™ P; cat. no. 10600023; Cytiva). The membranes were blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween-20 for 1 h 30 min at room temperature. After blocking, the membranes were incubated overnight at 4°C with primary antibodies against Bax (1:1,000; cat. no. 2772; Cell Signaling Technology, Inc.), E-cadherin (1:1,000; cat. no. 3195; Cell Signaling Technology, Inc.), p53 (1:1,000; cat. no. bs-2090R; BIOSS) and GAPDH (1:5,000; cat. no. ABS16; MilliporeSigma) diluted in 5% BSA (MilliporeSigma) in Tris-buffered saline containing 0.1% Tween-20 (cat. no. 11332465001; Roche Diagnostics). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:3,000; cat. no. 1706515; Bio-Rad Laboratories, Inc.) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection reagent (Amersham^™ ECL^™ Select Western Blotting Detection Reagent; cat. no. RPN2235; Cytiva) and images were captured using a LuminoGraph II imaging system (Cytiva). Band intensities were semi-quantified using ImageJ software (version 1.53).

All in vitro experiments were performed using technical triplicates under identical experimental conditions unless otherwise specified. Data are presented as the mean ± standard deviation and were analyzed using GraphPad Prism software (version 10; Dotmatics). Statistical significance was evaluated using one-way analysis of variance followed by Dunnett's post hoc test for multiple comparisons against the control group. As the data were obtained from technical triplicates, the sample size was insufficient for reliable assessment of normality and homogeneity of variance; therefore, these tests were not consistently performed. P<0.05 was considered to indicate a statistically significant difference.

Previous studies have reported that TAN suppresses cell viability in various types of cancer (23–25). In the present study, TAN treatment reduced the viability of CaSki cells (Fig. 1A). Based on the cell viability assay results, 10, 20 and 40 µM TAN were selected for subsequent experiments because these concentrations showed greater inhibitory effects on cell viability than lower concentrations and allowed the evaluation of concentration-dependent biological responses. In addition, the anti-proliferative effect of TAN in CaSki cells was evaluated using a colony formation assay (Fig. 1B), which revealed a significant reduction in colony formation area in the TAN-treated group compared with that in the control group (Fig. 1C). These results indicated that TAN may reduce cell viability by inhibiting CaSki cell proliferation.

TAN has been reported to suppress cancer cell proliferation primarily through the induction of G₁ phase arrest (26,27). In the current study, Hoechst 33342 staining was employed to examine cell cycle alterations associated with TAN-mediated inhibition of CaSki cell proliferation (Fig. 2A). The assay showed that TAN treatment increased the proportion of cells in the G₁ phase compared with that in the control group (Fig. 2B). These findings suggested that TAN-associated inhibition of CaSki cell proliferation may be related to G₁ phase arrest.

Effective cancer treatment requires not only the inhibition of cancer cell proliferation but also the suppression of cancer cell migration and metastasis (28). The effects of TAN on cell migration were first investigated using a wound-healing assay (Fig. 3A). Notably, TAN treatment at 5 µM significantly reduced wound closure compared with the control group at both 24 and 48 h (Fig. 3B). In addition, a Transwell migration assay was conducted to evaluate the effect of TAN on chemotactic migration (Fig. 3C). The number of migratory cells was significantly decreased in cells treated with 20 and 40 µM TAN compared with the control group (Fig. 3D). Western blot analysis was subsequently performed to detect E-cadherin (Fig. 3E). The protein expression levels of E-cadherin were increased in the TAN-treated groups compared with those in the control group (Fig. 3F). These results suggested that TAN not only inhibits the proliferation of CaSki cells, but is also associated with reduced migratory capacity and migration-associated phenotypic changes.

Previous studies have reported that TAN suppresses cancer cell proliferation by inducing apoptosis, a form of programmed cell death (19,26). In the present study, apoptotic cells were analyzed by flow cytometry using Annexin V and PI staining (Fig. 4A). The proportion of apoptotic cells was significantly increased in the 20 and 40 µM TAN-treated groups compared with in the control group (Fig. 4B). Subsequently, the expression levels of the pro-apoptotic protein Bax were examined by western blotting (Fig. 4C). Bax expression was significantly upregulated in the 40 µM TAN-treated group compared with in the control group (Fig. 4D). These results suggested that TAN treatment may be associated with apoptosis-related changes and increased Bax expression in CaSki cells, potentially involving the intrinsic apoptosis-associated signaling pathway.

TAN has been reported to exhibit various anticancer effects by increasing ROS levels in various cancer cell lines (18). The present study performed MitoSOX staining to evaluate whether TAN increases mitochondrial ROS (Fig. 5A), and observed elevated intramitochondrial ROS levels in the 20 and 40 µM TAN-treated groups compared with in the control group (Fig. 5B). Based on reports that excessive mitochondrial ROS in cancer cells induces changes in MMP (29), the current study further evaluated changes in MMP using JC-1 staining (Fig. 5C). The decrease in MMP was confirmed in the TAN-treated groups compared with in the control group (Fig. 5D). These findings suggested that TAN treatment is associated with increased mitochondrial ROS accumulation and decreased MMP in CaSki cells.

The tumor suppressor protein p53 is frequently inactivated or dysregulated in various cancer cell lines, and previous studies have reported that phytochemicals can modulate the expression of tumor suppressor proteins, including p53 (30–32). To evaluate the effect of TAN on p53 expression in CaSki cells, p53 protein levels were analyzed by western blotting (Fig. 6A). TAN treatment at 40 µM significantly increased p53 protein expression compared with that in the control group (Fig. 6B). These findings suggested that TAN treatment may be associated with increased p53 expression in CaSki cells.