B16F0 and B16F10 cells were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China). B16F0 cells were cultured in RPMI 1640 Medium (Gibco, Grand Island, NY, USA), and B16F10 cells were maintained in Dulbecco's Modified Eagle Medium (Gibco, Grand Island, NY, USA) and supplemented with 10% FBS (Tianjin Haoyang Biological Manufacture, Tianjin, China), 100 U/mL penicillin (Shandong Sunrise Pharmaceutical, Zibo, China), and 100 μg/mL streptomycin (Shandong Sunrise Pharmaceutical, Zibo, China) at 37°C with 5% CO₂. All experiments were performed on logarithmically growing cells.

SRB (Sigma, St. Louis, MO, USA) was utilized for detecting cell viability According to the manufacturer's instructions (8). Viable cell number was determined using the trypan blue exclusion test (TBET) (9). Colony-formation assay was used to test cell survival ability (10). Cell apoptosis was analyzed using nuclear staining with Hoechst 33258 (Sigma, St. Louis, MO, USA) (9).

According to the manufacturer's instructions, cell apoptosis was assessed using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining (BD Pharmingen, San Diego, CA, USA). Cell cycle was determined with PI (0.05 mg/mL) and RNase A (0.5 mg/mL) staining. Intracellular ROS generation was measured by incubating cells with 10 mmol/L DCF-DA (Sigma, St. Louis, MO, USA) at 37°C for 30 min. ΔΨm was measured by JC-1 dye solution (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in the dark at 37°C for 20 min. The cells were analyzed using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

In order to detect melanin content, cells and the supernatant were collected, respectively. Extracellular and intracellular melanin content was measured according to the previously described method (11).

TYR activity was assayed by measuring L-3, 4-dihydroxyphenylalanine oxidase activity (12), and absorbance was measured at 405 nm using a microplate reader.

Cells were wounded by scratching with a sterile pipette tip, treated with CHB, fixed, and photographed. Images were acquired with an inverted fluorescence microscope (Zeiss, Axiovert 200, Germany) (13).

Migration and invasion were determined using transwell assay (9, 14). For adhesion assay, B16F0 cells and B16F10 cells were plated onto the 50 μg/mL matrigel precoated plate and washed at 1 and 2 h to remove non-adherent cells. The adhered cells were measured with SRB assay after washed. For migration assays, B16F0 cells and B16F10 cells were seeded in the top of the chambers with the non-coated membrane and incubated overnight. For invasion assays, cells were plated in the top chamber with matrigel-coated membrane. Medium without serum or containing 10% FBS was added to the upper chamber or the lower chamber, respectively. After 24 h, the cells were fixed in 10% neutral buffered formalin solution for 30 min and stained with Giemsa. Cells on each insert were calculated using a microscope (Zeiss, Axiovert 200, Germany).

Total RNA was extracted using TRIzol Reagent (Sangong Biotech Co. Ltd., Shanghai, China). cDNA synthesis was performed using a RevertAid First Strand cDNA Synthesis Kit (TaKaRa, Shiga, Japan), and the synthesized cDNA was amplified. The polymerase chain reaction (PCR) primers (Table 1) were set as indicated, and reaction conditions were established using 12.5 μL 2× QuantiFast SYBR (QIAGEN GmbH, Hilden, GERMANY), 3 μL cDNA template, and 0.5 μL of each primer.

The animals were prepared as previously described (15). We conducted experiments on C57BL/6 mice based on tumor suppression and tumor colony formation. Mice were divided into three treatment groups randomly (five of each group). For the tumor-suppression experiment, the mice received 1.5, and 3.0 mg/kg of CHB via intragastric administration every day for 10 days (15). For the tumor colony-formation experiment, B16F0 and B16F10 cells were pretreated with CHB (0.75, 1.5, and 3.0 mg/kg) for 24 h and injected subcutaneously into the right flank of C57BL/6 mice, and colony formation was observed 12 days after injection. The implanted melanomas were separated, weighed, and fixed in 10% formaldehyde for at least 24 h and subsequently embedded in paraffin and submitted for hematoxylin and eosin (H&E) staining (16). TUNEL assays were performed as previously described (15).

Total protein (equal amounts) were separated by SDS-PAGE, transferred onto Immobilon®-P Transfer Membrane (Millipore Corporation, Billerica, MA, USA) and blocked with 5% non-fat milk. The membranes were incubated with the respective primary antibodies against Mmp2 (Santa Cruz, CA, USA), Mmp9 (Santa Cruz, CA, USA), Bcl-2 (Santa Cruz, CA, USA), Bax (Santa Cruz, CA, USA), pro-caspase-3 (Cell Signaling Technology, Danvers, MA, United States), cleaved-caspase-3 (Cell Signaling Technology, Danvers, MA, United States), caspase-9 (Santa Cruz, CA, USA) and β-actin (Santa Cruz, CA, USA), and then incubated with the appropriate concentrations of horseradish peroxidase-conjugated secondary antibody. The blots were visualized using the SuperSignal West Pico Chemiluminescent Substrate® (Thermo Scientific, Rockford, IL, USA).

All results are expressed as mean ± standard deviation (SD). Statistical differences among groups were determined using one-way ANOVA followed by Bonferroni multiple comparison test. All analyses were done using SPSS 18.0 statistical software. The results are considered significantly different when P < 0.05 and highly significantly different when P < 0.01.

After exposure for 48 h, CHB significantly inhibited the proliferation of B16F0 and B16F10 cells in a concentration-dependent manner (Figures 1A,B). CHB did not remarkably induce cell necrosis on TBET analysis, indicating that CHB did not have a significant lethal effect on B16F0 and B16F10 cells (Figures 1A,B). In addition, there is an obvious decrease in cell density and a distinguishable morphological change in the CHB-treated group (Figures 1C,D). Colony-formation assays revealed that CHB inhibited colony formation of B16F0 cells in a dose-dependent manner (Figures 1E,F). Moreover, the colony size evidently changed following CHB exposure (Figure 1G), suggesting that CHB effectively inhibited the proliferation of B16F0 and B16F10 cells.

We examined cell cycle progression of B16F0 cells by flow cytometry (Figure 2A) to investigate the inhibitory effect of CHB. Treatment with CHB (9 μg/mL) resulted in an increase at G0-G1 phase (from 51.09 to 79.91%) and a decrease at S (from 40.22 to 18.18%) and G2-M (from 8.69 to 1.91%) phases (Figure 2B), indicating that CHB markedly caused G0/G1 phase arrest in B16F0 cells. Further, we evaluated Cdk4, cyclin D1 (Ccnd1), Pcna, and p21 expression in B16F0 cells after CHB treatment and found that compared with the control group, the CHB-treated groups had a significant decrease in Cdk4, Ccnd1, and Pcna mRNA levels and a remarkable increase in the p21 mRNA level (Figure 2C). These results suggest that CHB induced cell cycle arrest at the G0-G1 phase via decreasing the mRNA levels Cdk4, Ccnd1, and Pcna and increasing the mRNA level of p21.

We measured the melanin content of B16F0 cells to investigate the depigmentation activity of CHB. The extracellular (Figure 3A) and intracellular (Figure 3B) melanin contents increased considerably following CHB treatment. Moreover, CHB significantly increased TYR activity in a concentration-dependent manner (Figure 3C). Tyr and Tyrp1 mRNA levels dramatically increased in CHB-treated B16F0 cells (Figure 3D), indicating that CHB increased the melanin content in B16F0 cells by upregulating Tyr and Tyrp1 mRNA levels.

Invasion and migration are a vital step in the process of tumor metastasis, and it is essential to clarify whether CHB influences the migration and invasion potential of B16F0 cells. First, lower concentrations (0.5, 1.0, and 1.5 μg/mL) of CHB, with no apparent effect on B16F0 cell proliferation, were measured (Figure 4A). Next, we found that B16F0 cells exhibited low adhesion ability following CHB treatment for 1 and 2 h (Figure 4B). Wound closure assays showed a remarkable reduction of migrated cells after 24 h of CHB exposure (Figures 4C,D). In the cell migration assay, CHB significantly suppressed the migration of B16F0 cells (Figure 4E). The number of B16F0 cells migrating through the porous membrane of the transwell insert was reduced from 135.03 ± 3.21 in the control group to 44.10 ± 6.82 in the CHB group (P < 0.01). In the Matrigel invasion assay (Figure 4F), we also found that CHB significantly reduced the average number of cells through the insert membrane, from 218.04 ± 10.62 in the control group to 67.00 ± 7.14 in the CHB group (P < 0.01). Lastly, Timp1 and Timp2 mRNA levels increased, whereas Mmp2 and Mmp9 mRNA and protein levels decreased in CHB-treated cells (Figures 4G–I). Overall, the data suggested that CHB repressed the invasion and migration abilities of B16F0 cells.

To confirm apoptosis induction in B16F0 cells following CHB treatment, fluorescence microscopy was used to examine the morphological changes, which demonstrated a notable change in nuclei with typical characteristics (Figure 5A). Annexin V-FITC apoptosis assay was performed to further distinguish the inhibition of cell proliferation, and the apoptotic rates were increased in B16F0 cells following CHB treatment (Figure 5B). The data demonstrated that the maximum concentration (9 μg/mL) of CHB induced a higher percentage (67.77 ± 3.30%) of apoptosis cells in the CHB group (8.95 ± 1.20%) (Figure 5C). Furthermore, mRNA levels of apoptosis-associated genes were examined by quantitative PCR (qPCR) assay to determine the molecular mechanism of CHB-induced apoptosis. The Bax, caspase-3, and caspase-9 mRNA levels were significantly increased, whereas the Bcl-2 mRNA level was remarkably decreased (P < 0.01) in a dose-dependent manner in the CHB treatment (Figure 5D). Moreover, CHB decreased the expression of antiapoptotic Bcl-2 protein, increased expressions of proapoptotic Bax and caspase-9 proteins and induced caspase-3 cleavage in B16F0 cells (Figures 5E,F). In order to clarify whether CHB-induced apoptosis was through mitochondrial pathway, ΔΨm changes were tested by JC-1 dye following CHB treatment for 2, 8, and 12 h. As shown in Figures 5G,H, CHB broke down mitochondrial integrity. We also examined intracellular ROS level in B16F0 cells following exposure to CHB for 2, 8, 12, and 24 h and found that the ROS level was increased in a dose- and time-dependent manner (Figures 5I,J). The results indicated that CHB induced B16F0 cell apoptosis via the mitochondrial-dependent apoptosis pathway.

Considering that CHB induces mouse melanoma B16F0 cell apoptosis in vitro, we used B16F0 tumor-bearing mice to study whether CHB could suppress tumor progression in vivo. First, the mice were treated with CHB every 2 days after inoculation for 7 days, and there was an obvious difference in tumor-bearing mice following CHB treatment (Figure 6A). Tumor growth inhibition rates were significantly increased in the CHB-treated groups (69.25% for 1.5 mg/kg and 90.38% for 3.0 mg/kg) (Figure 6B). Next, C57BL/6 mice were subcutaneously injected with CHB in the right flank, and colony formation was observed, indicating that CHB inhibited tumor colony formation (Figures 6C,D). In addition, we examined cell morphology of melanoma tumors with H&E staining and observed that the tumor cells had become more irregular following CHB treatment (Figure 6E). Furthermore, the TUNEL assay demonstrated that CHB increased TUNEL-positive cells in tumor tissue in a dose-dependent manner (Figure 6F). The results confirmed that CHB inhibited mouse melanoma B16F0 cell in vitro and vivo.

In order to ascertain the effect of CHB on melanoma, we supplement studies on B16F10 cells. CHB treatment (9 μg/mL) caused an increase at G0-G1 phase (from 53.86 to 70.98%) and a decrease at S phase (from 37.03 to 20.78%) and G2-M phase (from 9.11 to 8.14%) (Figure 7A), indicating that CHB markedly caused G0/G1 phase arrest in B16F10 cells. Compared with B16F0 cells, B16F10 cells are metastatic or have higher metastatic potential. Transwell assays examined the effect of CHB on migration and invasion; the cell migration assay indicated that CHB significantly suppressed B16F10 cell migration (Figure 7B). The number of B16F10 cells migrating through the porous membrane of the transwell cell culture insert was reduced from 145.1 ± 9.85 in the control group to 48.27 ± 5.21 in the CHB-treated group (P < 0.01). The Matrigel invasion assay revealed that CHB significantly reduced the average number of migrating cells from 112.95 ± 5.72 in the control group to 30.41 ± 6.22 in the CHB-treated groups (P < 0.01) (Figure 7C), demonstrating that CHB considerably decreased the invasion and migration abilities of B16F10 cells. In addition, the maximum concentration (9 μg/mL) of CHB induced a higher percentage (51.08 ± 2.70%) of apoptosis in B16F10 cells than the control group (4.86 ± 0.91%). The apoptotic rates were significantly increased in B16F10 cells following CHB treatment (Figure 7D). These results implied that the inhibitory effect of CHB might be kind of “universal” to various types of melanoma cells.

Lastly, we used tumor-bearing mice models to investigate the effect of CHB on B16F10 tumor progression in vivo. There was a notable difference in tumor-bearing mice after CHB treatment (Figure 8A). The B16F10 tumor growth inhibition rates were 60.38% (1.5 mg/kg) and 72.94% (3.0 mg/kg) in the CHB-treated groups (Figure 8B). In addition, the results of the tumor colony-formation experiment indicated that CHB remarkably inhibited tumor colony formation (Figures 8C,D). Taken together, these results indicated that CHB inhibited mouse melanoma B16F0 and B16F10 cells in vitro and vivo.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.