BRAF inhibitor PLX4032 was purchased from Selleck Chemicals, and juglone was purchased from Sigma-Aldrich; Merck KGaA. They were each dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO for cell culture was <0.1% (v/v). DMSO, 3-(4′,5′-dimethylthiazol-2′-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and N-acetyl-L-cysteine (NAC) were obtained from Sigma-Aldrich; Merck KGaA. 2′,7′-dichlorofluorescein diacetate (DCFH-DA), 4% paraformaldehyde, 5-ethynyl-29-deoxyuridine (EdU) cell proliferation kit with Alexa Fluor^® 488, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-midacarbocyanine iodide (JC-1) staining kit were purchased from Beyotime Institute of Biotechnology. JNK inhibitor (SP600125) and p38 inhibitor (SB203580) were obtained from MedChemExpress. The Annexin-V-phycoerythrin (PE) Apoptosis Detection kit (cat. c559763) was supplied by BD Biosciences. The antibodies against β-actin (cat. no. ab8226), poly(ADP-ribose) polymerase (PARP; cat. no. ab191217), survivin (cat. no. ab76424), Bcl-2 (cat. no. ab32124), Bax (cat. no. ab32503), phosphorylated (P)-p38 (cat. no. ab195049), p38 (cat. no. ab170099), P-p53 (cat. no. ab33889) and p53 (cat. no. ab179477) were obtained from Abcam, and the antibody against cytochrome c (cat. no. 4280) was purchased from Cell Signaling Technology, Inc.

Human melanoma SK-MEL-5 and A375 cell lines were obtained from American Type Culture Collection and maintained in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) containing 4 mM L-glutamine, 3.7 g/l sodium bicarbonate, 4.5 g/l glucose and 10% foetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.). The cells were maintained in a 5% CO₂ humidified incubator at 37°C. Both SK-MEL-5 and A375 cells carrying the BRAFV600E mutation are sensitive to vemurafenib treatment (16,17). A SK-MEL-5 subline with acquired resistance (SK-MEL-5R) to PLX4032 was generated by continuous exposure of parental SK-MEL-5 cells to gradually increasing concentrations of PLX4032 (from 0.1 µM up to 1 µM) over a period of 3 months. The fold resistance was intermittently evaluated by cell viability assays and it was found that SK-MEL-5R showed higher cell viability compared with SK-MEL-5 when treated with different concentrations of PLX4032 (data not shown). A375 cells with acquired resistant to PLX4032 (A375R) were generated by the same method, with the concentration of PLX4032 increasing from 0.1 µM up to 2 µM, as previously described (18).

The cytotoxic effects of juglone and/or PLX4032 on SK-MEL-5R and A375R cells were assessed by MTT assays. Briefly, 4,000 cells in 200 µl cell culture medium/well were seeded into 96-well plates and incubated overnight at 37°C. After NAC (0 and 2 mM), SB203580 (0 and 10 µM) or SP600125 (0 and 10 µM) pre-treatment for 1 h at 37°C, the cells were treated with juglone (0, 5 or 7.5 µM) and/or PLX4032 (0, 5 or 10 µM) for 24 h at 37°C. DMEM containing corresponding amounts of 0.1% DMSO were used as vehicle controls. Then, 20 µl MTT solution (5 mg/ml) was added to each well, and the cells were incubated for 2 h at 37°C. Subsequently, the formazan crystals that formed were dissolved with 100 µl DMSO, and the optical density (OD) was measured at 570 nm on a microplate spectrophotometer (Infinite 200 PRO; Tecan Group, Ltd.). The cell viability was determined using the following formula: Cytotoxicity (%)=(OD treatment/OD vehicle control) ×100. For crystal violet staining, 2×10⁵ cells/well were seeded into 6-well plates, followed by exposure to juglone (0 and 5 µM) and/or PLX4032 (0 and 10 µM) for 72 h at 37°C. Then, the cells were fixed with 10% formalin for 10 min at room temperature and stained with 0.05% crystal violet solution (CV) in distilled water for 30 min at room temperature. Finally, the CV was removed, cells were washed twice with distilled water and images were captured using a Flatbed Scanner (Canon LiDE 220; Canon Inc.).

To assess the cell proliferation, an EdU assay kit was used according to the manufacturer's instructions. Briefly, 2×10⁵ cells/well were seeded in a 6-well plate and treated with juglone (0 and 5 µM) and/or PLX4032 (0 and 10 µM) for 24 h, followed by incubation with cell culture medium containing 10 µM EdU at 37°C for 2 h. Then, the cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.3% Triton X-100 in PBS for 15 min. After washing with PBS, cells were incubated with click additive solution (supplied with the kit) for 30 min in the dark at room temperature. Finally, cells were washed with 0.3% Triton X-100 and incubated with Hoechst 33342 for 10 min at room temperature. After washing with PBS 3 times for 5 min each time, the images were captured using a fluorescence microscope (Axio Vert. A1; Zeiss GmbH) at 400× magnification and the subsequent images were analyzed using ImageJ software (v1.52 g; National Institutes of Health). The data comprised three replicates.

Apoptosis was measured using flow cytometry. Cells were seeded into 6-well culture plates at a density of 2×10⁵ cells/well, and then treated with juglone (0 and 5 µM) and/or PLX4032 (0 and 10 µM) for 24 h at 37°C. Next, cells were harvested, washed with PBS twice and resuspended in 1X binding buffer (supplied with kit). Finally, the cells were incubated with Annexin V-PE and 7-AAD in the dark at room temperature for 15 min, and analysed using a flow cytometer (Attune NxT; Thermo Fisher Scientific, Inc.). The data were analysed using FlowJo software V6.0 (Tree Star, Inc.).

Cells were harvested following the pre-treatment with NAC (0 and 2 mM), SB203580 (0 and 10 µM) or SP600125 (0 and 10 µM) for 1 h at 37°C and treated with juglone (0 and 5 µM) and/or PLX4032 (0 and 10 µM) for 6 h at 37°C. The cells were then lysed in RIPA buffer (Beyotime Institute of Biotechnology). The mixture was centrifuged at 12,000 × g at 4°C for 15 min, and the supernatants were collected. The protein concentrations were determined using a bicinchoninic acid protein assay kit (Bio-Rad Laboratories, Inc.). Next, 30 µg cellular proteins were separated on a 10 or 12% SDS-polyacrylamide gel and electroblotted onto a PVDF membrane. Membranes were blocked with 5% milk in Tween-20/Tris-buffered saline (TBST) for 1 h at room temperature, prior to incubation overnight at 4°C with 1:1,000 dilution of primary antibodies against β-actin, PARP, survivin, Bcl-2, Bax, p-p38, p38, p-p53, p53, JNK, p-JNK and cytochrome c. Membranes were washed with TBST, followed by 1 h incubation at room temperature in 1:4,000 dilution of horseradish peroxidise-conjugated secondary antibodies (cat. nos. 7074 and 7076; Cell Signaling Technology, Inc.). After washing again, the blots were developed using Supersignal West Femto Chemiluminescent substrate (Thermo Fisher Scientific, Inc.). Images were acquired by the ChemiDoc™ MP Imaging system (Bio-Rad Laboratories, Inc.). The band intensities were semi-quantified using ImageJ software and normalized to β-actin as the loading control. The western blot assays were repeated three times.

ROS were detected using the fluorescent probe DCFH-DA. Briefly, the cells were seeded into 6-well culture plates at a density of 2×10⁵ cells/well, and then treated with juglone (0 and 5 µM) and/or PLX4032 (0 and 10 µM) for 2 h. The cells were then washed with PBS, incubated in serum-free medium, and loaded with DCFH-DA (10 µM). Following incubation in the dark at 37°C for 30 min, the cells were washed with PBS and vortexed in 700 µl lysis buffer (90% DMSO:10% PBS)/well for 15 min. The cell lysis buffer was transferred into a black 96-well Immuno Plate (Thermo Fisher Scientific, Inc.), and fluorescence was detected using a fluorescence microplate reader (Infinite 200 PRO; Tecan Group, Ltd.) at an excitation wavelength of 485 nm, and an emission wavelength of 535 nm.

ΔΨ_M was evaluated using a JC-1 kit. Briefly, 1×10⁶ cells seeded in a 6-well plate were cultured with juglone (0 and 5 µM) and/or PLX4032 (0 and 10 µM) for 3 h. Then, the cells were labelled with JC-1 and incubated for 20 min at 37°C, according to the manufacturer's instructions. Cells were washed twice, resuspended and detected by flow cytometer (Attune NxT; Thermo Fisher Scientific, Inc.); the PE and FITC detection channels were selected at emission wavelengths of 530 and 585 nm, and an excitation wavelength of 488 nm. The data were analysed by FlowJo software V6.0 (Tree Star, Inc.). Mitochondrial carbonyl cyanide 3-chlorophenylhydrazone (supplied in kit) was used as a positive control. The percentage of depolarized mitochondrial was calculated as previously described (19).

All results are presented as the mean ± standard deviation of at least three independent experiments. Statistical analysis was performed by SPSS Statistics 21.0 software (IBM Corp.). Student's t-test was used for comparisons between two groups, and one-way ANOVA and subsequent Tukey's post-hoc analysis was applied for comparison of more than two independent groups. P<0.05 was considered to indicate a statistically significant difference.

MTT was used to determine the cytotoxic effects of juglone and/or PLX4032 on melanoma cells. As shown in Fig. 1A and C, the combination of juglone and PLX4032 in SK-MEL-5R and A375R melanoma cells induced a much higher cytotoxicity than juglone or PLX4032 alone (P<0.01). In addition, according to the MTT results, the combination index (CI) of juglone and PLX4032 cotreatment in SK-MEL-5R cells was determined using the Chou-Talalay method (20). The CI values varied from 0.34–3.65, which suggested a strong synergistic effect between juglone and PLX4032 (Fig. 1B). Crystal violet staining confirmed that the combination of juglone and PLX4032 further induced cytotoxicity to melanoma cells, compared with either juglone alone or PLX4032 alone (Fig. 1D). EdU assays also suggested that cell proliferation was further inhibited after treatment with a combination of PLX4032 and juglone (Fig. 1E and F).

To further demonstrate the synergistic effect of juglone and PLX4032, flow cytometry analysis was performed to measure the percentages of both Annexin V⁺/7-AAD⁻ (early apoptotic cells) and Annexin V⁺/7-AAD⁺ (late apoptotic/necrotic cells) cells. In SK-MEL-5R cells, PLX4032 alone induced only minor apoptosis. However, the combination of juglone and PLX4032 exhibited a much higher rate of apoptosis than either PLX4032 or juglone alone (Fig. 2A and C; P<0.01). Similar results were observed in A375R cells (Fig. 2B and C; P<0.01).

PARP is one of the terminal pro-apoptotic proteins. The cleaved forms of PARP are its active forms (21). Survivin, on the other hand, inhibits apoptosis (22,23). As demonstrated by western blotting, the protein expression of cleaved PARP in the juglone and PLX4032 combination group was the highest, while the protein expression of survivin was the lowest, as compared with levels in either the PLX4032 or juglone alone groups (Fig. 2D-F).

ΔΨ_M decline is one of the earliest indicators of intrinsic apoptosis. In the present study, the fluorescent probe JC-1 was used to determine the effect of juglone on ΔΨ_M in SK-MEL-5R cells. When the mitochondrial potential is normal, JC-1 forms a polymer in the mitochondrial matrix and exhibits red fluorescence with an emission of 590 nm; however, when the ΔΨ_M is downregulated, JC-1 changes into a monomeric form that yields green fluorescence with an emission of 530 nm, so the PE and FITC channel were selected to measure the fluorescence intensity of JC-1 dye in the cells (24). As shown in Fig. 3A and B, juglone caused a marked decrease in ΔΨ_M compared with the vehicle control. Moreover, the combination of juglone and PLX4032 further decreased the ΔΨ_M, and the percentage of cells with low potential was increased from 44.5% in the juglone alone group to 55.8% when cotreated with juglone and PLX4032.

The pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 serve central roles in the mitochondria-dependent apoptotic pathway; Bax can be translocated to the outer mitochondrial membrane following a death signal, where it promotes a permeabilization that favors the release of different apoptogenic factors, such as cytochrome c (25). In addition, the ability of Bax to form channels on the mitochondrial membrane can be inhibited by Bcl-2 (26). As demonstrated by western blot analysis (Fig. 3C), the protein expression of Bax was notably increased after cotreatment with juglone and PLX4032, while no changes were seen in the juglone or PLX4032 only groups. A reduction in the Bcl-2/Bax ratio was observed after the combination drug treatment in SK-MEL-5R cells (Fig. 3D). The combination of PLX4032 and juglone also increased the protein expression of cytochrome c (Fig. 3C and E).

Since mitochondrial apoptosis is associated with the generation of ROS (27), the production of intracellular ROS was examined using the oxidant-sensitive fluorescent probe DCFH-DA. The amount of ROS was analysed in cells treated with juglone and/or PLX4032 using a fluorescence microplate reader. Both PLX4032 or juglone alone increased the level of ROS. However, the level of ROS was significantly higher in the juglone and PLX4032 cotreatment group compared with the juglone or PLX4032 alone groups (Fig. 4A).

Previous evidence has indicated that ROS can also mediate cell death via key modulators, such as p38 mitogen-activated protein kinase (MAPK) and p53 (28,29). To test whether the p38-p53 pathway was activated by juglone and PLX4032 cotreatment, western blotting was used to measure the protein expression of p38, p53 and their phosphorylated counterparts. As demonstrated in Fig. 4B, in SK-MEL-5R cells, juglone and PLX4032 cotreatment markedly increased p38 and p53 phosphorylation compared with treatment of PLX4032 or juglone alone. JNK (Thr183/Tyr185) activation was also observed after juglone and PLX4032 cotreatment.

After cells were pre-treated with NAC (a ROS scavenger), or SB203580 (a p38 inhibitor), juglone and PLX4032 cotreatment-induced cytotoxicity was partially reversed (Fig. 4C and D). Furthermore, as demonstrated by western blotting, the protein expression of cleaved PARP in the NAC or p38 inhibitor pre-treatment group was lower than the protein expression in the juglone/PLX4032 combination group without NAC or p38 inhibitor pre-treatment (Fig. 4E-H; P<0.05). However, pre-treatment with SP600125 (a JNK inhibitor), failed to reverse the cytotoxicity induced by juglone and PLX4032 cotreatment (Fig. 4D). This was consistent with the inability of JNK inhibitor to reduce the levels of cl-PARP (Fig. 4F and H). These results suggested that ROS production and p38 activation play important roles in juglone-induced PLX4032 sensitization.