Chrysin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (EMD Millipore, Billerica, MA, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), 0.05% trypsin-0.02% EDTA solution, and gentamicin were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cytochrome c enzyme-linked immunosorbent assay (ELISA) and caspase-3 colorimetric assay kits were purchased from EMD Millipore. Caspase-8 and −9 colorimetric activity assay kits were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

The effects of chrysin were investigated in two human uveal melanoma cell lines (SP6.5 and M17) and compared to the effects on two distinct normal cell lines [human retinal pigment epithelial (RPE) cells and scleral fibroblasts]. The human M17 melanoma cell line, RPE cells and scleral fibroblasts were established in the Tissue Culture Center of the New York Eye and Ear Infirmary (New York, NY, USA) as previously reported (23). The SP6.5 melanoma cell line was isolated from a primary choroidal melanoma patient and was provided by Dr. Guy Pelletier (Research Center of Immunology, Quebec, Canada) (24). All melanoma cells, RPE cells and fibroblasts were cultured with DMEM, supplemented with 10% FBS and gentamicin (50 µg/ml). Cells were incubated at 37°C in a CO₂ regulated incubator in a humidified 95% air/5% CO₂ atmosphere. When cultures reached confluence, cells were detached with trypsin-EDTA solution and passaged. All tissues were obtained with premortem consent in accordance with the laws and regulations in place in the various jurisdictions.

MTT assay was performed as previously described (25). Cells (6×10³ cells/well) were plated into 96-well plates. Cells were incubated overnight at 37°C in a CO₂ regulated incubator in a humidified 95% air/5% CO₂ atmosphere. Chrysin was dissolved in DMSO at a concentration of 20 mM (stock solution). In dose-response studies, 24 h following plating, chrysin was applied to the cultures at final concentrations of 0, 10, 30 and 100 µM and cultured for 48 h. A total of 50µl of MTT solution at a concentration of 1 mg/ml was added to each well. MTT solution was aspirated following 4 h of incubation at 37°C and the produced formazan blue was dissolved in 100 µl of DMSO. Absorbance at 540 nm was measured using a microplate reader (Multiskan MCC/340; Thermo Fisher Scientific, Inc.). The control group measurement was standardized as 100% viability. The concentration at which cell growth was inhibited by 50% (the 50% inhibitory concentration, IC₅₀) was determined by linear interpolation. Experiments were performed in triplicate.

To investigate the time-effect of chrysin on uveal melanoma cells, melanoma cells (SP6.5 cell line, 6×10³ cells/well) were plated into 96-well plates and divided into two groups: Chrysin-treated group and untreated group (control group). Subsequently, chrysin was added to the cultures in the treated groups at the concentrations of 30 µM following 24 h of incubation at 37°C. MTT assay was subsequently performed in treated and untreated groups at 0, 6, 12, 24 and 48 h following the addition of chrysin. The results of each treated group were compared to the controls at the identical time point. All measurements were performed in three independent experiments, and each time point was performed in triplicate.

Cell apoptosis was determined by the terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end-labeling (TUNEL) assay (26,27) using a TACS.XL In Situ Apoptosis Detection kit (cat. no. 4828-30-DK; R & D Systems, Inc.). The assay was performed according to the manufacturer's protocol. Briefly, cells were cultured on chamber slides and analyzed 48 h following treatment with 10, 30 and 100 µM chrysin. Cells were fixed, incubated with 3% H₂O₂/methanol and permeabilized. Slides were covered by Labeling Reaction mix (R & D Systems, Inc.) and incubated for 1 h at 37°C in a dark humidified chamber, followed by incubation with anti-BrdU antibody (1:50; cat. no. 4828-30-DK; TACS.XL In Situ Apoptosis Detection kit; R & D Systems, Inc.) at 37°C for 1 h. The slides were treated with streptavidin-horseradish peroxidase solution(R & D Systems, Inc.) for 10 min, followed by staining with 3,3′-diaminobenzidine (DAB) substrate for 5 min. The samples were mounted and analyzed under a light microscope, where the apoptotic cells could be detected as highly condensed shrunken dark brown cells. A total of 200 cells were counted in each group (0, 10, 30 and 100 µM chrysin treatment groups). The experiment was repeated independently a total of three times.

MTP was measured by a Mitochondria Staining kit (cat. no. T4069; Sigma-Aldrich; EMD Millipore), which employed JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-etraethylbenzimidazol-carbocyanine iodine), a sensitive fluorescent dye, as the probe. Uveal melanoma cells (SP6.5 cell line) were plated into 96-well plates with black wells (Corning Incorporated, Corning, NY, USA) at a density of 8×10³ cells/well. Following 24 h of incubation at 37°C, cells were treated with chrysin at various concentrations (0, 10, 30 and 100 µM) at 37°C for 48 h. JC-1 (at final concentration 12.5 µg/ml) was added and incubated at 37°C in the dark for 15 min. JC-1 and culture medium were aspirated and cells were washed three times with PBS. Cells were immediately observed by fluorescence microplate reader (Spectraflour Plus; Tecan Group Ltd., Männedorf, Switzerland). Settings of 485-nm (excitation) and 590-nm (emission) were used to measure red fluorescence; 485-nm (excitation) and 535-nm (emission) were used to measure green fluorescence. The detection is based on the accumulation of the dye and its fluorescence in the mitochondria. The accumulated dye appears as red fluorescence located in the undamaged mitochondria, whereas the dye remains as monomers in the cytoplasm with diffuse green fluorescence in cells with damaged MTP (28). The results (red fluorescence/green fluorescence ratio, R/G ratio) were expressed as a percentage of the controls (cells not treated with chrysin). This experiment was performed a total of three independent times.

To detect the release of mitochondrial cytochrome c, uveal melanoma cells (SP6.5 cell line, 4×10⁵ cells/well) were plated into 6-well plates. A total of 24 h later, chrysin was added at various concentrations (0, 10, 30 and 100 µM). Following 2 h of incubation at 37°C, the cells were harvested. The cells were washed with cold PBS three times, treated with 0.02% EDTA solution and scraped from the well. Subsequently, cells were counted and centrifuged at 400 × g at 4°C for 5 min, and the cell pellets were collected. Following cell lysis and centrifugation at 1,000 × g at 4°C for 10 min, the supernatant (cytosol and mitochondria) was collected and centrifuged at 10,000 × g at 4°C for 20 min. The pellets (mitochondria) and the supernatant (cytosol) were then collected separately. A cytochrome c ELISA kit (EMD Millipore) was used to measure the level of cytochrome c in the cytosol and the procedure was performed according to the manufacturer's protocol. The cytochrome c level was demonstrated as a percentage of the controls (cells not treated with chrysin).

Melanoma cells (SP6.5 cell line, 4×10⁵ cells/well) were plated into 6-well plates, and chrysin was added at various concentrations (0, 10, 30 and 100 µM) following 24 h of incubation at 37°C. A total of 2 h later, cells were washed with cold PBS and collected. Subsequently, the cells were counted and centrifuged at 400 × g at 4°C for 5 min, and the cell pellets were collected. The cells were lysed using cell extraction buffer (BioSource; Thermo Fisher Scientific, Inc.) with protease inhibitor cocktail (Sigma-Aldrich; EMD Millipore) and phenylmethane sulfonyl fluoride (BioSource; Thermo Fisher Scientific, Inc.), incubated on ice for 30 min, and vortexed for 30 sec. The lysates were centrifuged at 15,000 × g for 10 min at 4°C. The supernatant was stored at −70°C until analysis. The levels of caspase-3, −8,and −9 in cell lysates were measured by specific colorimetric kits in accordance with the manufacturer's protocol. A microplate reader at 405 nm was used to measure the optical density. Caspase-3, caspase-8 and caspase-9 activities were demonstrated as a percentage of the controls (cells not treated with chrysin). This measurement was performed independently three times.

Data was analyzed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Significance was evaluated using one-way analysis of variance and the least significant difference method in comparison of data of more than two groups and Student's t-test for of comparison data between two groups. P<0.05 was considered to indicate a statistically significant difference.

Chrysin induced cell death in cultured human uveal melanoma cells in a dose-dependent manner (Figs. 2 and 3). There was a significant difference in cell viability between melanoma cells (M17 and SP6.5) treated or untreated with chrysin (P<0.001) at 30–100 µM levels. The IC₅₀ dose of chrysin for M17 and SP6.5 at 48 h was 35.8 and 28.3 µM, respectively. Chrysin at 10–100 µM levels did not affect the cell viability of cultured human RPE cells or fibroblasts. These results demonstrated that chrysin at 30–100 µM selectively reduced the viability of melanoma cells without affecting RPE cells or fibroblasts.

Time-effect study demonstrated that chrysin at 30 µM decreased the viability of uveal melanoma cells (SP6.5) in a time-dependent manner from 6 to 48 h (data not shown). There was a significant difference in cell viability at various time points between the treated and untreated group (P<0.001).

TUNEL analysis was used for the detection of cell apoptosis. Normal cells were stained by methyl green and were green in color. The nuclei of apoptotic cells were stained by DAB and were dark-brown in color. Chrysin significantly increased the apoptotic rate of uveal melanoma cells in all treated cultures compared to the control (P<0.001; Fig. 4).

For cells cultured with JC-1, red fluorescence was due to a potential-dependent aggregation of JC-1 in the mitochondria. Green fluorescence observed in the cytosol following mitochondrial membrane depolarization reflected the monomeric form of JC-1. Chrysin significantly decreased the R/G ratio in a dose-dependent manner (compared to the control; P<0.001 at all tested levels), which revealed that chrysin caused damage to the MTP in uveal melanoma cells (Fig. 5).

Chrysin caused an increase of cytosol cytochrome c levels in melanoma cells in a dose-dependent manner (compared to the control; P=0.25 at the levels of 10 µM; P<0.001 at the levels of 30–100 µM) (Fig. 6).

Chrysin increased the activities of caspase-3 and −9 in melanoma cells in a dose-dependent manner (Fig. 7A and B). Chrysin at 30–100 µM levels resulted in a significant increase of caspase-3 and −9 activities in melanoma cells (P<0.001). In uveal melanoma cells treated with 100 µM chrysin, an ~4.11-fold and 4.58-fold increase in caspase-3 and caspase-9 activities was noted, respectively. There was no significant change in caspase-8 activity following treatment with chrysin at 10–100 µM levels (Fig. 7C).