Human malignant melanoma A375 cells (cat. no. SCSP-533) were purchased from Cell Bank, Typical Culture Preservation Commission, Chinese Academy of Sciences (Shanghai, China). The normal human keratinocyte cell line HaCaT (cat. no. CL-0090; STR profiling authentication) was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). The cells were cultured with Dulbecco's modified Eagles' medium (DMEM)/high-glucose basal medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), containing 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1% sodium pyruvate (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), 0.1 U/l penicillin, and 0.1 µg/l streptomycin (Beijing Solarbio Science & Technology Co., Ltd.) and incubated in a 5% CO₂ incubator at 37°C. Fetal calf serum was inactivated in a 56°C water bath for 30 min before use.

The effect of bufotalin on the viability of A375 and HaCaT cells was determined by an MTT assay. The cells in suspension (1×10⁴ cells in 100 µl DMEM) were seeded into 96-well plates. After 24 h of incubation, different concentrations of bufotalin (cat. no. ASB-00002466-005; J&K Scientific Ltd., Beijing, China) were added, and the cells were further cultured for 24 and 48 h. Then, 10 µl MTT solution (5 mg/ml) was added into each well, and the cells were incubated for an additional 2 h. The resulting formazan crystals were dissolved using 150 µl dimethyl sulfoxide (DMSO). Absorbance was measured at a wavelength of 490 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The absorbance of the cells treated with DMEM medium containing 0.1% DMSO was regarded as the control group (survival rate 100%). IC₅₀ represents the drug concentration causing a 50% decrease in survival rate.

The A375 cells were trypsinized, collected, and diluted into 300 cells in 1 ml DMEM medium. The cell suspension was seeded into a 6-well plate at 300 cells/well. After culturing for 24 h, the cells were treated with different concentrations of bufotalin for 24 h. Then, the treated cells were incubated with DMEM complete medium at 37°C for 14 days. Finally, cells were fixed in 4% paraformaldehyde solution for 15 min at room temperature, and the cells were stained with Giemsa solution at room temperature for 10 min, then examined and images under a light microscope. The colonies containing >50 cells were counted and used to calculate the colony formation rate.

The A375 cells (6×10⁵ cells) were seeded onto sterile cover slips and placed into 6-well plates and cultured for 24 h. Then, the cells were treated with different concentrations of bufotalin. After 12 h of treatment, the cells were fixed in 4% paraformaldehyde solution for 15 min at room temperature, washed twice with PBS, and then the cells were stained with Hoechst 33258 (cat. no. C1018; Beyotime Institute of Biotechnology, Haimen, China) at room temperature for 10 min. Stained nuclei were observed, and images were captured using a fluorescence microscope. Apoptotic cells (cells exhibiting nuclear fragmentation and/or with distinct apoptotic bodies) were counted, and the percentage of apoptotic cells (number of apoptotic cells/the total number of cells × 100) was calculated.

Cell cycle distribution of the bufotalin-treated A375 cells was assessed by flow cytometry. Briefly, the bufotalin-treated A375 cells (~8×10⁵ cells/group) were collected and fixed in 70% ethanol at 4°C overnight. After fixation, the cells were centrifuged at 800 × g for 5 min to remove the ethanol. Then, the cells were suspended in 500 µl PBS containing 0.02 mg/ml propidium iodide (PI) and 0.1 mg/ml ribonuclease A (RNase A) in darkness at 37°C for 30 min. The fluorescence emitted by the PI-DNA complexes was measured using an Epics XL flow cytometry (Beckman Coulter, Inc., Brea, CA, USA). Cell distribution in different phases of the cell cycle was analyzed using WinMDI 2.8 software (The Scripps Research Institute, La Jolla, CA, USA).

Annexin V-FITC/PI double staining assay (Annexin V-FITC Apoptosis Detection Kit; cat. no. CA1020; Beijing Solarbio Science & Technology Co., Ltd.) was performed according to the manufacturer's instructions. Briefly, A375 cells (~8×10⁵ cells/group) were harvested at 24 h after bufotalin treatment and then incubated in 100 µl labeling solution, which consisted of 5 µl Annexin V-FITC, 10 µl PI, 10 µl 10X binding buffer and 75 µl H₂O in darkness at room temperature for 15 min. Then, 400 µl 1X binding buffer were added to stop the staining reaction. The stained cells were detected using FL1 and FL3 fluorescence channels on a FACSort flow cytometry system (FACSCanto II; Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and the percentages of apoptotic cells in each group were analyzed using BD FACSDiva software (version 6.1.3; BD Biosciences, Becton, Dickinson and Company).

A375 cells (1×10⁶/well) were seeded in 100-mm culture dishes and treated with different concentrations of bufotalin for 24 h. Following treatment, cells were collected and incubated in cell lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 0.1 mM phenylmethanesulfonylfluoride, 0.1 mM sodium orthovanadate, 0.5 mM dithiothreitol and 10X protease inhibitor; pH 6.8) on ice for 30 min. Cells were vortexed for 45 sec and then centrifuged at 10,000 × g at 4°C for 10 min. The supernatant was collected and stored at −20°C. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Beijing Solarbio Science & Technology Co., Ltd.). Cell lysates with a protein content of 40 mg were mixed with an equal volume of sodium dodecyl sulfate (SDS) loading dye (2% SDS, 10% sucrose, 0.002% bromophenol blue, 5% 2-mercaptoethanol and 625 mM Tris; pH 6.8) and subsequently separated by SDS-PAGE on 12.5% gels along with a rainbow-colored protein molecular marker (Beijing Solarbio Science & Technology Co., Ltd.). After running for 2 h at 110V, the proteins were transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA) using a Trans-blots SD semi-dry transfer cell (Bio-Rad Laboratories, Inc.). The membranes were blocked with 5% bovine serum albumin (BSA; cat. no. A8010; Beijing Solarbio Science & Technology Co., Ltd.) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 60 min before being probed with primary antibodies [protein kinase B (AKT), cat. no. ab38449, 1:500; phosphorylated (p)-AKT, cat. no. ab38449, 1:500; ATM serine/threonine kinase (ATM), cat. no. ab78, 1:1,000; checkpoint kinase 2 (Chk2), cat. no. ab47433, 1:1,000; cell division cycle 25C (CDC25C), cat. no. ab32444, 1:1,000; cyclin-dependent kinase 1 (CDK1), cat. no. ab18, 1:1,000; cyclin B, cat. no. ab181593, 1:1,000; BCL2-associated X, apoptosis regulator (BAX), cat. no. ab32503, 1:1,000; B cell lymphoma-2 (BCL-2), cat. no. ab32124, 1:1,000; caspase-3, cat. no. ab13585, 1:1,000; caspase-9, cat. no. ab202068, 1:1,000; all Abcam, Cambridge, UK] containing 5% BSA at 4°C overnight. Subsequently, the membranes were washed with TBS-T buffer for 45 min and then probed with appropriate secondary antibodies (goat anti-mouse IgG H&L, cat. no. ab205719, 1:20,000; or goat anti-rabbit IgG H&L, cat. no. ab6721, 1:20,000; Abcam) conjugated with horseradish peroxidase for 1 h at room temperature. Immunoreactive bands were visualized with enhanced chemiluminescence detection reagents (Invitrogen; Thermo Fisher Scientific, Inc.) using a film processor (Kodak, Rochester, NY, USA), and the gray-scale values of each image were analyzed using Image-Pro Plus 6.0 (IPP6) software.

The experiments were conducted at least three times. All the data are expressed as the mean ± standard deviation. Analyses were performed using the SPSS 21.0 software package (version 21.0; IBM Corp., Armonk, NY, USA). Student's t-test, a one-way analysis of variance (ANOVA) or a two-way ANOVA was used to calculate statistical differences, respectively. Tukey's post hoc tests were undertaken following a significant one-way ANOVA, and Bonferroni's post hoc tests were undertaken following a significant two-way ANOVA. P<0.05 was considered to indicate a statistically significant difference.

To examine the antiproliferative effect of bufotalin on human malignant melanoma A375 cells, the cells were treated with or without various concentrations of bufotalin for 24 and 48 h, then the viability of bufotalin-treated cells was determined by the MTT assay. Bufotalin (10–80 ng/ml) significantly inhibited A375 cell proliferation, with an IC₅₀ value of 40 ng/ml after 24 h treatment (Fig. 1A). Furthermore, the proliferation rate was significantly lower in A375 cells after 48 h treatment with bufotalin than that in A375 cells after 24 h treatment with the same concentration (10–80 ng/ml) of bufotalin (Fig. 1A). Subsequently, according to the IC₅₀ value of bufotalin (40 ng/ml), we selected 30, 40 and 50 ng/ml as the concentrations of bufotalin used in the next studies. Additionally, light microscopy indicated changes in cell number and cell morphology. There was a marked decrease in the number of A375 cells after treatment with bufotalin, and some cells began to shrink and deform, and even detach from the dish surface (Fig. 1B). In addition, bufotalin significantly inhibited A375 cell colony formation at concentrations of 30, 40 and 50 ng/ml (Fig. 1C and D). Bufotalin also effectively inhibited HaCaT cell proliferation at 50–80 ng/ml after 24 h treatment, whereas the cytotoxicity of bufotalin on HaCaT cells is significantly lower than that on A375 cells (Fig. 1E).

To explore the mechanism of action underlying the bufotalin-mediated antiproliferative effect, the cell cycle distribution profile of bufotalin-treated A375 cells was investigated. After exposure of A375 cells to bufotalin for 24 h, a marked accumulation of cells at the G2/M phase compared with the control group was observed (Fig. 2A and B). The protein levels of G2/M phase regulatory proteins, CDK1 and cyclin B, were subsequently analyzed by western blot analysis. The results demonstrated that the CDK1 protein expression levels in bufotalin-treated A375 cells significantly decreased compared to the control group, whereas no change in cyclin B protein expression levels in bufotalin-treated A375 cells was detected (Fig. 2C and D). These results indicated that bufotalin effectively inhibited the expression of CDK1, but not cyclin B.

Considering the cell cycle arrest at the G2/M phase and the decreased ratio of CDK1/cyclin B in bufotalin-treated A375 cells, changes in the levels of the components of the ATM/Chk2/CDC25C signaling pathway, which regulates cell transmission through the G2/M phase (18), were examined by western blot analysis. The results demonstrated that the protein levels of ATM and Ckh2 were significantly higher, and that of CDC25C was significantly lower in bufotalin-treated A375 cells compared to the control group (Fig. 3A and B).

In the above experiment, bufotalin effectively inhibited A375 cell proliferation through G2/M cell cycle arrest. Additionally, the changes in A375 cell morphology that were induced by bufotalin were similar to those observed during apoptosis. To explore whether this proliferation inhibition is associated with cell apoptosis, the bufotalin-treated A375 cells were stained with Hoechst 33258 and examined by fluorescence microscopy. The typical apoptosis characteristics, nuclear shrinkage, DNA condensation, and apoptotic bodies, were observed in bufotalin-treated A375 cells (Fig. 4A and B). Furthermore, the apoptotic rate of bufotalin-treated A375 cells was determined using an Annexin V FITC/PI double staining assay and flow cytometry analysis. The percentage of apoptotic cells was significantly increased with increasing bufotalin concentration (Fig. 4C and D). These results suggested that bufotalin induced cell apoptosis in A375 cells, leading to an antiproliferative effect.

The levels of cell apoptosis-associated proteins, BCL-2, BAX, caspase-3 and −9, were detected in A375 cells by western blot analysis. The level of BCL-2 was significantly downregulated and the levels of BAX, caspase-3 and −9 were markedly upregulated in bufotalin-treated A375 cells compared with the control group (Fig. 5A and B). Additionally, considering the regulatory effect of the activation of AKT on caspase-3/-9, the levels of AKT and p-AKT in bufotalin-treated A375 cells. Bufotalin markedly decreased p-AKT levels and the ratio of p-AKT/AKT, whereas no significant change in AKT expression levels was observed in A375 cells following bufotalin treatment (Fig. 5C and D).