The human skin melanoma cell line CHL-1 used in the present study was purchased from the American Type Culture Collection (Manassas, VA, USA). ZER, dimethyl sulfoxide (DMSO) and Hoechst^® 33342 were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan) and the fetal bovine serum (FBS) was purchased from Hyclone (GE Healthcare Life Sciences, Logan, UT, USA). The CHL-1 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS and 1% penicillin-streptomycin, and incubated at 37°C in a humidified atmosphere containing 5% CO₂. The culture medium was replaced between 2 and 3 times/week.

Melanoma tumor cell viability was evaluated using the CCK-8 assay. CHL-1 cells were seeded in 96-well plates at a density of 1×10⁵ cells/well and allowed to adhere for 24 h. Then culture media was removed and cells were treated with 0, 1, 2, 4, 8 and 16 µg/ml ZER dissolved in DMSO for 48 h. Control cultures were treated with 0.1% DMSO. Subsequently, 10 µl CCK-8 was added to each well and the plates were incubated at 37°C for 2 h. Optical density (OD) was measured at a wavelength of 450 nm using a multimode plate reader (Tecan Group, Ltd., Zürich, Switzerland). Values are represented as the fold change in OD value compared with the control cells. Cell proliferation assays were also performed by detecting 5-bromo-20-deoxyuridine (BrdU)-labeled DNA using an anti-BrdU monoclonal antibody conjugated to peroxidase (Cell Proliferation ELISA; Roche Applied Science, Penzberg, Germany). The amount of BrdU-labeled DNA was quantified using the ELISA, according to the manufacturer's protocol.

Cell migration was assessed using a Transwell migration assay. A total of 1×10⁵ cells in 200 µl serum-free media were seeded into the upper chamber of Transwell^® plates (BD Biosciences, Franklin Lakes, NJ, USA) and 200 µl DMEM containing 10% FBS was added to the lower chamber. Following treatment with ZER for 48 h at 37°C in 5% CO₂, the non-invasive cells in the upper chamber were removed. The invasive cells in the lower chamber were stained with Hoechst 33342 for 10 min, imaged and counted using an upright fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Intracellular ROS production was determined using the 2′,7′-dichlorofluorescin-diacetate (DCFH-DA) oxidation kit (Beyotime Institute of Biotechnology, Haimen, China), according to the manufacturer's protocol. A total of 1×10⁵ cells/well were seeded in 96-well plates and following treatment with ZER, were incubated with DCFH-DA at 37°C for 20 min. DCFH-DA passively diffused into the cells and was deacetylated by esterases to form DCFH, which reacted with any ROS present to form the fluorescent product DCF within the cells (15). The fluorescence was read at a wavelength of 485 nm (excitation) and 530 nm (emission) using a monochromator microplate reader (Tecan Group, Ltd.).

the JC-1 kit (Beyotime Institute of Biotechnology) was used to measure the MMP of CHL-1 cells, according to the manufacturer's protocol. Following treatment with ZER, 1×10⁵ cells were cultured in 24-well plates and incubated with 5 µg/ml JC-1 staining solution for 20 min at 37°C. Cells were subsequently rinsed twice with JC-1 staining buffer, and the fluorescence intensities of mitochondrial JC-1 monomers (λ_excitation, 514 nm; λ_emission, 529 nm) and aggregates (λ_excitation, 585 nm; λ_emission, 590 nm) were detected using a monochromator microplate reader. The MMP of CHL-1 cells in each treatment group was calculated as the red/green fluorescence ratio.

Cellular ATP levels were measured using a firefly luciferase-based ATP assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Following treatment with ZER for 48 h, cells were centrifuged at 12,000 × g for 5 min at 4°C. In 24-well plates, 100 µl supernatant was mixed with 100 µl ATP detection solution (working dilution) that was part of the kit. Luminescence was measured using a monochromator microplate reader.

The quantitative polymerase chain reaction (qPCR) was used to evaluate mtDNA levels as previously described (16–18). A total of 1×10⁶ cells/well were seeded into six-well plates. Following treatment with ZER, total cellular DNA was extracted using the E.Z.N.A.^® Tissue DNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA), according to the manufacturer's protocol. As previously described, relative amounts of mtDNA were compared with nuclear DNA copy numbers. The mtDNA amplicons were generated from two distinct segments of the mtDNA genes: Mitochondrially encoded cytochrome c oxidase I (COX1), encoded for on the heavy strand of mtDNA; and mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 6 (ND6), encoded for on the light strand of mtDNA. The nuclear amplicon was generated by amplification a segment of the β-actin gene, which was selected as the internal control. The following mtDNA primers were used: COX1 forward, 5′-CCACTTCGCCATCATATTCGTAGG-3′ and reverse, 5′-TCTGAGTAGCGTCGTGGTATTCC-3′ (fragment length, 90 bp); and ND6 forward, 5′-TCACCCAGCTACCACCATCATTC-3′ and reverse, 5′-CACTGAGGAGTACCCAGAGACTTG-3′ (fragment length, 250 bp). The primers for β-actin were: Forward, 5′-CCACACCCGCCACCAGTTC-3′ and reverse, 5′-CCTTCTGACCCATTCCCACCATC-3′ (fragment length, 173 bp). The qPCR was performed using the Real Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the SYBR-Green I detection method, according to the manufacturer's protocol.

Total RNA was isolated from cortical neurons using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The first-strand cDNA was generated from total RNA (1 µg) using a High-Capacity cDNA Reverse Transcription kit (Invitrogen; Thermo Fisher Scientific, Inc.). Following equalization of the RNA quantity in each group, the mRNA levels were quantitated using a Bio-Rad iQ5 Gradient Real-Time PCR system (Bio-Rad Laboratories, Inc.), and GAPDH was used as an endogenous control. Primers used for all RT-qPCR experiments were as follows: TFAM forward 5′-GGTGTATGAAGCGGATTT-3′ and reverse, 5′-CTTTCTTCTTTAGGCGTTT-3′; GAPDH forward 5′-AAGGTGAAGGTCGGAGTCAA-3′ and reverse 5′-AATGAAGGGGTCATTGATGG-3′. qPCR was performed using the Real Time PCR Detection System (Bio-Rad Laboratories, Inc.) and the SYBR-Green I (Applied Biosystems; Thermo Fisher Scientific, Inc.) detection method, according to the manufacturer's protocol. The PCR system contained the following: 2.5 µl 10X PCR buffer, 1.5 µl MgCl₂ solution, 0.5 µl forward primer, 0.5 µl reverse primer, 2.5 µl Taq polymerase and 1 µl cDNA. Thermocycling conditions for qPCR were as follows: 95°C for 15 min, followed by 40 cycles of 95°C for 30 sec, 60°C for 20 sec and 72°C for 30 sec. RT-qPCR products were detected using 1.5% agarose gel electrophoresis and quantified using the 2^−ΔΔCq method (19).

Values are presented as the mean ± standard error of the mean for triplicate experiments. Statistical analysis was performed using SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA). Data were statistically analyzed using the one-way analysis of variance or Student's t test. P<0.05 was considered to indicate a statistically significant difference.

The chemotherapeutic effect of treatment with ZER in human melanoma cells was investigated. Following treatment with 2–16 µg/ml ZER, CHL-1 cell viability was significantly decreased in a dose-dependent manner compared with the control cells (F=199.12; P<0.001; Fig. 2A). Consistently, the results of the BrdU assay demonstrated significantly decreased cell proliferation following treatment with ZER compared with the control cells (F=258.38; P<0.001; Fig. 2B). Furthermore, the effect of treatment with ZER on CHL-1 cell migration was determined. Results of the Transwell migration assay indicated that treatment with ZER significantly inhibited CHL-1 cell migration compared with the untreated cells in a dose-dependent manner (F=152.22; P<0.001; Fig. 3).

It is well-documented that the induction of cell death by certain antitumor agents is associated with ROS generation and the perturbation of mitochondrial function (20,21). To understand the underlying mechanism of inhibited cell proliferation and migration, ROS generation and the MMP were investigated in CHL-1 cells following treatment with ZER. Following treatment with ZER, ROS production was significantly increased in CHL-1 cells compared with the control cells in a dose-dependent manner (F=23.96; P<0.001; Fig. 4A). Furthermore, following treatment with ZER, the MMP of CHL-1 cells was significantlydecreased compared with the control cells (F=99.37; P<0.001; Fig. 4B).

To determine whether ZER affects mitochondrial biogenesis, cells were treated with ZER for 48 h. Mitochondrial ATP synthesis was measured, and the results demonstrated that treatment with ZER significantly decreased ATP content compared with the untreated cells (F=25.13; P<0.001; Fig. 5A). The level of mtDNA was also decreased following treatment with ZER compared with the untreated cells (F=19.52; P<0.001; Fig. 5B). In addition, mRNA expression of mitochondrial transcription factor A (TFAM), a mitochondrial biogenesis factor, was evaluated using RT-qPCR (Fig. 6). The results demonstrated that treatment with 4 and 16 µg/ml ZER significantly decreased TFAM mRNA expression compared with the untreated cells (F=19.48; P=0.002; Fig. 6). These results suggest that ZER inhibits mitochondrial biogenesis.