NF-κB is a ubiquitously expressed transcription factor that is regulated by the cytoplasmic inhibitor protein IκBα, which inhibits NF-κB translocation by masking the nuclear localization signals of NF-κB proteins and keeping them sequestered in an inactive state in the cytoplasm [5]. Basal pIκBα was observed significantly in the four untreated uveal melanoma cell lines, SP 6.5, VUP, OCM1 and OM431, while the retinal pigment epithelium (RPE) cell line ARPE-19 showed weakly activation, which meant the constitutive expression and activation of NF-κB in the uveal melanoma cells (Figure 1A). To further confirm the constitutive activation of NF-κB in uveal melanoma cells, the expression and location of p65, one of the most common subunits of NF-κB, was detected by immunofluorescence. P65 was shown to be present in both cytoplasm and nucleus of the control uveal melanoma cells (Figure 1B). However, with the treatment of NF-κB specific inhibitor BAY11-7082 for 2 h, translocation of p65 in the nucleus decreased significantly as shown by the reduced staining in the nucleus, and relocated in the cytoplasm (Figure 1B). These findings indicated that NF-κB is constitutively activated in the four untreated uveal melanoma cells and the specific inhibitor BAY11-7082 could effectively block its activation.

The four uveal melanoma cell lines (SP 6.5, VUP, OCM1 and OM431) were treated with varying concentrations of BAY11-7082 for 24 h to test its effects on cell growth that were determined by CCK-8 assay as described previously [20]. As shown in Figure 2A, BAY11-7082 exhibited strong anti-proliferative effects in all four uveal melanoma cell lines in a dose-dependent manner, with approximately 50% inhibition of cell proliferation at the concentration of 5 μM.

Furthermore, BAY11-7082 treatment also significantly inhibited the colony formation capacity of the uveal melanoma cells at the concentration of 2.5 μM (Figure 2B). The number of colonies was reduced in a dose-dependent manner and fewer colonies were formed following treatment with 5 μM BAY11-7082 (Figure 2B,C). These data suggest that NF-κB is constitutively activated in uveal melanoma cells and selective blocking its activation potently inhibits cell growth.

NF-κB signaling pathway has been shown to regulate cell apoptosis. We therefore investigated whether BAY11-7082 treatment could induce apoptosis of uveal melanoma cells. A significant increase in Annexin V positive cells was determined by flow cytometry when cells were treated with BAY11-7082 (Figure 3A,B). The cleavage form of caspase 3, a specific marker for apoptosis, was greatly increased by BAY11-7082 treatment (Figure 3C), confirming that NF-κB blockade induced uveal melanoma cell apoptosis. NF-κB signaling pathway has been shown to involve in cell apoptosis by regulating the expression of a number of pro-apoptotic and anti-apoptotic genes. Corresponding to the constitutive activation of NF-κB, untreated uveal melanoma cells expressed moderate basal level of anti-apoptotic protein Bcl-2, while BAY11-7082 treatment remarkably reduced its expression (Figure 3C). However, the constitutively highly expressed Bax, one of the pro-apoptotic genes, was not influenced by BAY11-7082 treatment (Figure 3C). Thus, Bcl-2 was decreased by BAY11-7082 treatment and could help to explain why NF-κB blockade induced apoptosis in uveal melanoma cells. The change of cell cycle has also been implicated in regulating uveal melanoma cells proliferation [21–22]. We therefore further studied the effects of BAY11-7082 treatment on cell cycle distribution by flow cytometry. However, there was no significant change in the percentages in each cell cycle phase (Figure 3D). Thus, these data together suggested that BAY11-7082 inhibited the proliferation of uveal melanoma cells by inducing cell apoptosis, but not cell cycle arrest.

Liver metastases remain the leading cause of death in uveal melanoma patients [3–4]. Identifying signaling pathways that regulate uveal melanoma cell migration would provide therapeutic targets for blocking metastasis. Therefore, we explored whether NF-κB signaling pathway could regulate uveal melanoma cell migration. All the four uveal melanoma cell lines showed very low spontaneous motility, but could significantly migrate to the lower chamber in the presence of high concentrations of FBS or hepatocyte growth factor (HGF), a mesenchymal-derived or stromal-derived multifunctional growth factor which has been shown to promote the migration of uveal melanoma cell lines. SP6.5, VUP and OCM1 cells showed the most potent migration ability, while OM431 cells exhibited moderate migration ability. Treatment of BAY11-7082 at 5 μM significantly suppressed the migration of all four uveal melanoma cell lines to either high concentration FBS or HGF (Figure 4), suggesting that NF-κB pathway was also involved in the regulation of migration and may functionally contribute to liver metastasis of uveal melanoma patients.

To study the therapeutic potential of the NF-κB inhibitor BAY11-7082 in the treatment of uveal melanoma in vivo, a xenograft nude mouse model with subcutaneous inoculation of OCM1 cells was established [23–24]. After solid tumors were established and palpable, mice were randomly grouped and 1 μM BAY11-7082 was syringed subcutaneously peri-tumor every day for 14 days. Control mice received the same volume of DMSO. BAY11-7082 treatment significantly suppressed OCM1 tumor growth and tumor volume in vivo (Figure 5A,B). Reduced tumor weight was also observed in the BAY11-7082 treated group compared with that of control mice (Figure 5C). To further examine the in vivo effects of BAY11-7082 on tumor cell apoptosis, tumor sections from both groups were prepared and examined by TUNEL staining. Remarkably, there were more apoptotic tumor cells in BAY11-7082-treated mice group, as indicated by the increased brown nuclear staining (Figure 5D,E). Therefore, blockade of NF-κB signaling pathway not only suppressed uveal melanoma tumor cell growth and survival in vitro, but also induced apoptosis and inhibited tumor growth in vivo.

BALB/c nude mice, male, four weeks of age, were purchased from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China). Mice were housed in the animal care facilities of the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine under pathogen-free conditions. All experimental procedures were approved by the Laboratory Animal Care and Use Committees of the hospital.

The previously characterized human uveal melanoma cell lines OM431, VUP, SP6.5 and OCM1 were kindly provided by Professor John F. Marshall (Tumor Biology Laboratory, John Vane Science Centre, London, UK) [20,32]. ARPE-19 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). All these established cells are composed of spindle and epithelioid cells, which were derived from primary tumors of choroidal and ciliary bodies. The specific NF-κB inhibitor BAY11-7082 (Calbiochem, San Diego, CA, USA) was reconstituted in dimethylsulphoxide (DMSO) (St. Louis, MO, USA) as a 100 mM stock solution and further diluted using PBS. Antibodies to IκBα, pIκBα, and caspase 3 were purchased from Cell Signaling Technology (Denvers, MA, USA). Antibodies to Bax, Bcl-2 and NF-κB p65 were obtained from BD Biosciences (San Diego, CA, USA). Anti-β-actin (clone AC-40) and DAPI were purchased from Sigma. IRDye 800CW goat anti-mouse secondary antibody and goat anti-rabbit secondary antibody were obtained from LI-COR Biotechnology (Lincoln, NE, USA).

Uveal melanoma cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with penicillin (100 units/mL), streptomycin (100 μg/mL) and 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Invitrogen). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. Uveal melanoma cell viability after BAY11-7082 treatment was determined with Cell Counting Kit-8 (Dojindo Molecular Technologies, Rockville, MD, USA) as described in our previous study [20]. Absorbance was measured by a microplate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA) at 450 nm and background absorbance measured at 630 nm was subtracted.

To determine the apoptosis induced by BAY11-7082 stimulation, uveal melanoma cells (2 × 10⁵ cells/well) in 6-well plates were treated with 5 μM BAY11-7082 for 24 h and the same volume of DMSO (<0.05% v/v) was added into the culture medium of the control group. All uveal melanoma cells were collected, washed, and resuspended in 100 μL binding buffer and then incubated with 4 μL FITC-conjugated annexin-V (BD Pharmingen, San Diego, CA, USA) in the dark for 15 min at room temperature. All samples were immediately analyzed by flow cytometry, and the data were processed with FlowJo software. The cell cycle was analyzed by measuring the amount of propidium iodide (PI)-labeled DNA in ethanol-fixed cells. In brief, cells were treated for 24 h, harvested by trypsinization and fixed with cold 70% ethanol. Cells were then stained for total DNA content with PI/Rase staining buffer according to the manufacturer’s instructions. Cell cycle distribution was analyzed using a flow cytometer and ModFit software.

Related protein expression in uveal melanoma cells with BAY11-7082 treatment was determined by western blot. Protein extraction, quantitation and denaturation was performed as described previously [33]. After nonspecific sites in the PVDF membrane were blocked with a solution containing 5% non-fat milk powder in TBS/Tween20 (TBS/T) for 1 h at room temperature, the membrane was probed with antibodies against β-actin, IκBα, pIκBα, caspase 3, Bax and Bcl-2 in TBS/T containing 5% bovine serum albumin (BSA) overnight at 4 °C, and then incubated with diluted IRDye 800CW goat anti-mouse secondary antibody or goat anti-rabbit secondary antibody. Antibody-antigen complexes were then detected using the Odyssey^® Infrared Imaging system (LI-COR Biosciences, Lincoln, NE, USA).

Tumor cells with or without 5 μM BAY11-7082 treatment were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X100 in PBS containing 1% BSA for 10 min. The cells were washed and blocked with 5% BSA for 1 h at room temperature. Cells were then incubated overnight at 4 °C with NF-κB p65 primary antibody (1:1000) (BD Biosciences), washed with PBS and incubated with Alexa Fluor^® 568 Goat Anti-Mouse IgG (H+L) (1:2000) (Invitrogen) for 1 h at room temperature. After washing, the cells were mounted with mounting medium containing DAPI (Sigma). Negative controls were run using the same protocol while replacing primary antibody with mouse IgG antibody. Stained cells were evaluated under a fluorescence microscope with a magnification of 200×.

Cells were seeded into 6-well plates in triplicate at a density of 300 cells/well in 2 mL of medium containing 10% FBS. After 24 h, cultures were replaced with fresh medium containing 0.5% FBS (control) or the same medium containing 2.5 μM or 5 μM BAY11-7082 in a 37 °C humidified atmosphere containing 95% air and 5% CO₂ and grown for three weeks. The cell clones were stained for 15 min with a solution containing 0.5% crystal violet and 25% methanol, followed by three rinses with tap water to remove excess dye. Colonies consisting of >50 cells were counted under a microscope with a magnification of 200×.

To measure cell migration in vitro, 8-μm pore size-culture inserts (Transwell, Corning, Lowell, MA, USA) were placed into 24-well culture plates, separating the upper and the lower chambers. The upper chamber contained cells in DMEM plus 1% FBS with or without 5 μM BAY11-7082, and the lower chamber contained DMEM plus 20% FBS or recombinant human hepatocyte growth factor (HGF) (20 ng/mL; R & D Systems, Minneapolis, MN, USA) in DMEM containing 1% FBS. Cells were incubated for 12 h at 37 °C in a humidified atmosphere containing 5% CO₂. Non-migrated cells were removed from the upper surface of the transwell membrane with a cotton swab. Migrated cells remaining on the bottom surface were fixed and stained with a solution containing 0.5% crystal violet and 25% methanol, and then five independent visual fields under a light microscope were counted at a magnification of 25×. Results were expressed as the mean + SD. For each migration condition, three identical replicates were performed.

Animal protocols were approved by the Animal Care and Use Committee at Shanghai Jiao Tong University School of Medicine. For xenograft implantation, a total of 5 × 10⁶ OCM1 cells/mouse were injected subcutaneously into the back next to the right hind limb, and permitted to grow until palpable. Then mice were then randomly assigned into the control or treatment group and treatment was initiated. For the control group, 100 μL vehicle PBS containing the same volume of DMSO injection per mouse was injected subcutaneously peri-tumor every day for 14 days. For the treatment group, 1 μM BAY11-7082 in 100 μL PBS/mouse was injected in the same way as that in the control group. The day of cell inoculation was designated Day 0. Tumors were measured every 3 days with vernier calipers and tumor volumes were calculated according to the following formula:

where a is the longest diameter and b is the shortest diameter. Body weight of the mice was also recorded. At the end of the experiments, tumor-bearing mice were sacrificed, and tumors were weighed after being separated from the surrounding muscles and dermis. Finally, the tumors were fixed with 4% phosphate-buffered paraformaldehyde and embedded in paraffin.

For TUNEL staining, paraffin-embedded tumors from nude mice were assayed for DNA fragmentation according to the manufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis, IN, USA) as described previously [33]. According to the manufacturer’s instructions of the in situ Cell Death Detection Kit supplied by Roche Applied Science, apoptotic cells were indicated by brown stains in the nucleus. Thus, we counted the apoptotic cells under light microscopy. Therefore, increased numbers of nucleus brown-stained cells suggested that more tumor cells underwent apoptosis. A total of 10 tissue sections were analyzed for each animal. All sections were examined by light microscopy and a total of 10 tissue sections were analyzed for each animal.

A two-way repeated-measures analysis of variance (ANOVA) was used to test differences in tumor growth. One-way ANOVA was used to test differences in the mean growth inhibition, apoptosis rate and tumor weight. All results are expressed as mean ±/+ SD. All statistical tests were two-sided, and p-values less than 0.05 were considered statistically significant.