The human melanoma cell lines, A375P and A375SM, were obtained from the Korean Cell Line Bank (Korean Cell Line Research Foundation). Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Welgene, Inc. Apigenin (Fig. 1), MTT cell lysis buffer, DAPI and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Merck KGaA). Fluoresceinisothiocyanate (FITC) Annexin V Apoptosis Detection kit was obtained from BD Pharmingen (BD Biosciences). Anti-β-actin (cat. no. 4967), anti-Bax (cat. no. 2772), anti-Bcl-2 (cat. no. 2876), anti-cleaved caspase-9 (cat. no. 9501), anti-caspase-9 (cat. no. 9502), anti-poly ADP-ribose polymerase (PARP; cat. no. 9542), anti-cleaved PARP (cat. no. 5625), anti-p53 (cat. no. 2527), anti-phosphorylated (p)-p38 (cat. no. 4631), anti-p38 (cat. no. 9212), anti-p-JNK (cat. no. 4668), anti-JNK (cat. no. 9252), anti-p-ERK (cat. no. 4376), anti-ERK (cat. no. 4695), anti-p-Akt (cat. no. 4060), anti-Akt (cat. no. 9272) and goat anti-rabbit IgG (cat. no. 7074) antibodies were obtained from Cell Signaling Technology, Inc.

Melanoma cells were maintained in MEM and DMEM supplemented with 5% FBS and 1% penicillin/streptomycin under standard culture conditions at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The culture medium was replaced every 2–3 days. For apigenin treatment, melanoma cells were seeded into a 175 cm² flask (Nalge Nunc International; Thermo Fisher Scientific, Inc.) when density reached ~80–90% and allowed to attach overnight.

The anticancer effects of apigenin were assessed using an MTT assay. A375P and A375SM cells were seeded onto a 96-well plate at a density of 2×10⁴ cells/ml and a volume of 200 µl/well. Following incubation for 24 h, cells were treated with apigenin (0, 25, 50, 75 and 100 µM) for 24 h in triplicate. After apigenin treatment, the medium was discarded and a total of 40 µl MTT solution (5 mg/ml) was added to each well followed by incubation for an additional 2 h. Subsequently, the medium was aspirated and the formazan product generated by viable cells was solubilized with the addition of 100 µl DMSO. The absorbance of the solution was determined at 595 nm using a microplate reader (Bio-Rad Laboratories, Inc.). The percentage of viable cells in the apigenin treatment group was estimated in comparison with the untreated control cells.

A375P and A375SM cells were seeded in 60-mm culture dishes at a density of 1×10⁵ cells/ml and allowed to grow for 24 h. A uniform wound was introduced by scraping the monolayers with a sterile blue-pipette tip. Subsequently, the culture medium was replaced with non-FBS fresh medium supplemented with increasing concentrations of apigenin (0, 50 and 100 µM) and cells were cultured for an additional 24 h. The rate of wound closure in apigenin-treated and untreated cells was monitored by images captured with a phase-contrast microscope (magnification, ×100) immediately after wound incision (0 h) and following 24 h. The percentage of the migrated cells was estimated compared with the cell density in the wound of the untreated control group.

Apoptotic cell death was determined by observing morphological changes using DAPI fluorescent nuclear dye. DAPI stains cells undergoing apoptosis characterized by chromatin condensation and nuclear fragmentation. A375P and A375SM cells were treated with PBS or various concentrations of apigenin (0, 50 and 100 µM) for 24 h, harvested by trypsinization and fixed in 70% ethanol overnight at 4°C. The following day, cells were stained with DAPI at room temperature for 1 min, deposited onto slides and observed under a fluorescence microscope (magnification, ×200) to detect characteristics of apoptosis. The apoptotic rate was measured using the following equation: Number of positive cells/total number of cells in three random fields from each sample.

Apoptosis rate was also determined using a FITC-Annexin V Apoptosis Detection kit (BD Pharmingen; BD Biosciences). For the Annexin V-PI staining, A375P and A375SM human melanoma cells were treated with 0, 50 and 100 µM apigenin for 24 h. Following treatment, cells were washed with PBS, suspended in trypsin-EDTA solution and centrifuged (200 × g) at 4°C for 5 min to obtain cell pellet. The harvested cells were diluted in 1X binding buffer at a density of 1×10⁶ cells/ml. Subsequently, cells were treated with 5 µl/100 µl FITC-conjugated Annexin V and phycoerythrin-conjugated PI for 15 min and apoptosis was measured by flow cytometry (BD FACSVerse™ Flow cytometer, BD Life Sciences). BD FACSuite (v1.0.6; BD Life Sciences) was used for analysis. The early and late apoptosis rate was indicated by the percentage of Annexin V positive cells.

Cells were treated with various concentrations of apigenin (0, 50 and 100 µM) for 24 h, total proteins were extracted using PRO-PREP protein extraction solution (Intron Biotechnology, Inc., cat. no. 17081), and then the protein concentration was determined using a Bradford protein assay (Bio-Rad Laboratories, Inc.). Total proteins (30 µg per lane) in each cell lysate were resolved via SDS-PAGE on 6–14% gels, and subsequently electrotransferred onto nitrocellulose membranes. Following blocking with 5% non-fat dry milk in Tris-buffered saline with 0.5% Tween-20 (TBST) buffer for 1 h at room temperature, membranes were incubated with specific primary antibodies diluted in blocking solution at 4°C overnight. After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Following washing, bands were visualized using an enhanced chemiluminescence detection reagent (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The following antibodies were used: β-actin (1:1,000), Bax (1:1,000), Bcl-2 (1:1,000), caspase-9 (1:1,000), cleaved-caspase-9 (1:1,000), PARP (1:1,000), cleaved-PARP (1:1,000), p38 (1:1,000), p-p38 (1:1,000), JNK (1:1,000), p-JNK (1:1,000), ERK (1:1,000), p-ERK (1:1,000), Akt (1:1,000) and p-Akt (1:1,000) as primary antibodies and rabbit IgG (1:1,000) as the secondary antibody. Protein intensity was semi-quantified by the ImageJ software (National Institutes of Health, v1.8.0).

A total of 15 male BALB/c nude (nu/nu) mice (age, 5 weeks, body weight, 17–19 g) were purchased from Orient Bio Inc. Animal experiments were performed in accordance with the Guidelines for the Kongju National University Institutional Animal Care and Use Committee (Chungcheongnam, Korea) and approved by the Ethics Committee of Kongju National University (approval no. KNU_2018-5). Equipment was provided by the Laboratory Animal Resource Center (Gwangju, Korea). Mice were maintained under a 12 h light/dark cycle and housed under a controlled temperature of 23±3°C and humidity of 40±10%. Mice were allowed ad libitum access to laboratory pellet food and water. A375SM cells at 80–90% density were maintained in DMEM and MEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO₂. A375SM cells were harvested from cultures using 0.25% trypsin. Trypsinization was stopped using a solution containing 10% FBS, cells were then rinsed twice and resuspended in DMEM and MEM. Subsequently, a total of 2×10⁷ cells in 0.2 ml culture medium were injected subcutaneously into the right flank of donor nude mice. On day 7 following injection, A375SM cells growing under the skin of nude mice developed tumors. When the tumors became palpable, mice were assigned randomly into three groups (n=5), namely the vehicle-treated control group and the apigenin-treated groups (25 or 50 mg/kg body weight). Apigenin was orally administrated five times/week for 3 weeks at a dose of 25 or 50 mg/kg body weight, while control mice were treated with the vehicle only. Oral administration was performed using an oral zonde needle. Animal health and behavior were monitored daily. Body weight and tumor volume were monitored twice weekly. The tumor volumes were calculated using the following equation: Tumor volume (mm³)=0.5 × length × width². Then, three weeks after the start of apigenin injection, the final tumor size was measured. All mice were sacrificed using CO₂ gas (30% per min, 3 min) and tumors were excised to measure tumor weight. A section of the tumor tissue was embedded in paraffin and fixed with 10% formalin at room temperature for 12 h was subsequently used for TUNEL and immunohistochemistry (IHC) assays. The criteria used to determine when an animal should be euthanized were set as follows: i) Mice showed a weight loss of ≥20% of its normal weight; ii) tumor grew to ≥10% of its normal weight; iii) mice developed ulcers or infections in the tumor area; or iv) erosion of surrounding tissues.

TUNEL staining was performed in paraffin-embedded 5-µm-thick tumor sections using the DeadEnd™ Colorimetric TUNEL System (Promega Corporation), according to the manufacturer's protocol. Briefly, sections were deparaffinized in xylene, dehydrated via a series of graded alcohol rinses (100, 95, 85, 70 and 50% ethanol (v/v) in double-distilled H₂O) and rehydrated in PBS (pH 7.5). Subsequently, the tissue samples were permeabilized with a proteinase K solution following refixing in 4% paraformaldehyde solution at room temperature for 15 min. Slides were treated with the rTdT reaction mix and incubated at 37°C for 1 h. Reactions were terminated by immersing the slides in 2X SSC solution for 15 min at room temperature. Following blocking of endogenous peroxidase activity with 0.3% hydrogen peroxide, slides were washed with PBS, and then incubated with streptavidin HRP solution for 30 min at room temperature. After washing, slides were incubated with a 3,3-diaminobenzidine (DAB; substrate) solution until a light brown background appeared (10 min) and rinsed several times in deionized water. Following mounting, slides were observed under a light microscope. The number of positive cells in three random fields from each sample was counted indicating the number of apoptotic cells.

The paraffin-embedded sections were deparaffinized and dehydrated by sequential immersion in xylene and graded alcohol solutions, respectively. Sections were blocked using 1X Animal-Free Blocking Solution (Cell Signaling Technology, Inc., cat. no. 15019) at room temperature for 1 h. The sections were incubated with an antibody against p-ERK (1:100) at 4°C overnight, and subsequently incubated with a HRP-conjugated goat anti-rabbit antibody for 1 h at room temperature. The tumor sections were visualized using a DAB solution, treated with mounting reagent and observed under a routines light microscope (magnification, ×200). Finally, p-ERK positive cells were counted in three random fields from each sample.

The excised liver and kidney specimens were immediately fixed in 10% neutral buffered formalin at room temperature for 72 h, embedded in paraffin and cut into 5-µm-thick sections. Following hematoxylin and eosin (H&E) staining at room temperature (hematoxylin for 5 min, eosin for 1 min), the sections were examined under a light microscope (magnification, ×200).

Results are presented as the mean ± standard error of the mean for tumor volume, tumor weight, and body weight in vivo, while others are presented as the mean ± standard deviation. Differences in the mean values between control and apigenin-treated groups were assessed by one-way ANOVA followed by a Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of apigenin on A375P and A375SM cell viability, cells were treated with different concentrations (0, 25, 50, 75 and 100 µM) of apigenin and viability was assessed using an MTT assay. When A375P and A375SM cells were treated with 0, 25, 50, 75 and 100 µM of apigenin for 24 h, the cell survival rates were 100, 90, 71, 55 and 35%, and 100, 89, 78, 69 and 55% for A375P and A375SM cells, respectively (Fig. 2). The decrease in cell viability was dose-dependent, as it decreased with increasing concentrations of apigenin. Significant effects were observed at concentrations ≥50 µM.

Subsequently, to explore the effect on apigenin on A375P and A375SM cell migration, melanoma cells were treated with various concentrations of apigenin (0, 50 and 100 µM) that significantly affected the viability of A375P and A375SM cells as demonstated by the MTT assay. The results revealed that cell migration was attenuated in a dose-dependent manner compared with that noted in the control group (Fig. 3A and C). The migration rates in A375P and A375SM cells treated with 0, 50 and 100 µM apigenin were 100, 53 and 25%, and 100, 34 and 4%, respectively (Fig. 3B and D).

To determine whether the decreased cell viability observed in apigenin-treated A375P and A375SM cells was mediated by apoptosis, the morphological changes and chromatin condensation in the nucleus of apigenin-treated cells were observed following DAPI staining. Therefore, A375P and A375SM cells were treated with 0, 50 and 100 µM of apigenin for 24 h, co-treated with DAPI staining and observed under a fluorescence microscope (Fig. 4A). Consistent with the MTT assays, the results revealed that apigenin exerted inhibitory effects on cancer cell growth by decreasing total cell count. The inhibitory effect of apigenin on cancer cell growth was mediated by apoptosis as confirmed by the presense of morphological features of apoptosis, such as apoptotic bodies and chromatin condensation. To quantitatively analyze apoptosis, DAPI-positive cells were counted (Fig. 4B) and the morphology of the apoptotic bodies was observed under a fluorescence microscope. The apoptosis rates of A375P and A375SM cells treated with 0, 50 and 100 µM apigenin were 3.8, 24.8 and 44.8%, and 6.6, 20.0 and 36.4%, respectively. Thus, this indicated that apigenin increased apoptosis in a dose-dependent manner.

To investigate whether the formation of apoptotic bodies observed with DAPI staining was mediated by apoptosis, Annexin V-PI staining was used. Therefore, A375P and A375SM cells were treated with 0, 50 and 100 µM apigenin for 24 h and apoptosis was confirmed by flow cytometry (Fig. 5A and B). The results showed that the apoptosis rates (percentage of Annexin V positive cells) of apigenin-treated melanoma cells increased in a dose-dependent manner. As shown in Fig. 5C and D, apoptosis rates of A375P and A375SM cells treated with 0, 50 and 100 µM apigenin were 8.7, 40.3 and 59.6%, and 11.6, 38.5 and 47.5%, respectively.

DAPI and Annexin V-PI staining showed that apoptosis was significantly increased in A375P and A375SM cell lines treated with 50 and 100 µM of apigenin. Therefore, the expression levels of proteins that regulate apoptosis were measured using western blot analysis. To detect the expression levels of Bax, Bcl-2, PARP, p53 and caspase-9, which are known to regulate apoptosis, A375P and A375SM cells were treated with apigenin (0, 50 and 100 µM), and subsequently western blotting was performed. As the concentration of apigenin increased, the expression levels of PARP and caspase-9 decreased, and the expression levels of pro-apoptotic proteins Bax, p53, cleaved PARP and cleaved caspase-9 significantly increased, whereas the expression of Bcl-2, known to inhibit apoptosis, was downregulated (Fig. 6A and B).

The phosphorylation status of specific proteins in cancer cells is widely recognized as an important process in determining cell fate, including apoptosis. Various kinases of the MAPK pathway, an intracellular signaling system, are involved in intercellular and intracellular reactions in response to changes in the intracellular environment (34). ERK, JNK and p38, which belong to the MAPK pathway, are known to regulate several biological functions including cell proliferation, differentiation and apoptosis. Furthermore, Akt plays a central role in cell growth and proliferation, and is active in the majority of tumor tissues (35,36). However, Akt expression significantly differs in response to different active cellular materials, biological functions and environment. Therefore, western blot analysis was performed to reveal the effects of apigenin (0, 50 and 100 µM) on the protein expression of members of the Akt and MAPK pathways in A375P and A375SM cells (Fig. 7A and B). The expression levels of the MAPK pathway-related proteins, p-JNK and p-p38, significantly decreased and increased, respectively, in A375P cells. Furthermore, expression of p-ERK and p-Akt decreased following treatment with 100 µM apigenin. However, the increase in p-p38 expression was not associated with the concentration of apigenin and p-ERK was not downregulated in a dose-dependent manner. In A375SM cells, the expression levels of p-JNK and p-ERK in the MAPK pathway increased, whereas those of p-p38 and p-Akt decreased, depending on the concentration of apigenin. However, the increase in p-ERK expression was not associated with the concentration of apigenin. The aforementioned results indicated that proteins in the signal transduction pathways were differentially expressed in the two melanoma cell lines.

Based on the in vitro experiments, a A375SM cell line-derived xenograft mouse model was established. A375SM melanoma cells were transplanted into nude mice and the effect of apigenin on tumor growth was investigated. Apigenin was diluted in PBS and orally administered at concentrations of 25 and 50 mg/kg five times per week for 3 weeks. Tumor size was measured twice per week. Tumor size was decreased in the apigenin-treated group after two weeks of treatment compared with that observed in the control group (Fig. 8A). The tumor inhibition rate was 35.0% on the 21st day post-administration in the low-concentration group (25 mg/kg) and 15.4% in the high-concentration group (50 mg/kg), as shown in Table I. Tumor weights were 0.64±0.1, 0.46±0.1 and 0.57±0.07 g in the control group, low-concentration group and high-concentration group, respectively. Therefore, tumor weight was decreased in both groups following treatment with apigenin compared with the control group (Fig. 8B). However, there were no statistically significant changes in body weight following apigenin administration (Fig. 8C).

A TUNEL assay was performed to determine whether the inhibitory effect of apigenin on the growth of A375SM melanoma cells isolated from a melanoma xenograft model was mediated by apoptosis. The results revealed that the number of TUNEL-positive cells was increased in the apigenin-treated group compared with the control group (Fig. 8D and E).

The in vitro experiments demonstrated that p-ERK was upregulated in apigenin-treated melanoma cells. Therefore, melanoma tumor samples were isolated from the melanoma mouse xenograft model 3 weeks following apigenin administration, and IHC revealed an increase in p-ERK expression compared with the control group (Fig. 9).

Subsequently, to assess apigenin-induced organ toxicity, liver and kidney tissues derived from tumor-xenografted mice were histologically examined by H&E staining under a light microscope (Fig. 10). However, no histopathological abnormalities were observed, indicating that apigenin had no detectable toxic effects.