A375, A2058, HUVEC and HaCaT cell lines were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). All the cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS). Z-VAD-FMK were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against Bcl-xL, Mcl-1, cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP, p-STAT3 and STAT3 were purchased from Cell Signaling Technology (Danvers, MA, USA). GAPDH and secondary antibodies against mouse IgG-horseradish peroxidase (HRP) and rabbit IgG-HRP were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against Ki-67, CD31, VEGF, MMP-9 and MMP-2 were obtained from Abcam (Cambridge, MA, USA).

Approximately 2 liters of n-hexane and 1 kg of EVOO (Sigma-Aldrich) were mixed, and then CH₃CN-MeOH (1 liter, 20:80) was added and shaken twice. Dried organic layer (24 g) was subjected to repeated medium pressure liquid chromatography (MPLC) in a 50×3 cm column on lipophilic Sephadex LH20 (bead size 25–100 µm; Sigma-Aldrich) using n-hexane-CH₂Cl₂ (1:9), isocratic elution, followed by MPLC (10 g, 25×1 cm column) on C-18 reversed-phase silica gel with Bakerbond octadecyl (40 µm; Mallinckrodt Baker, Inc.) to afford 13.3 mg of OC with >97% purity (HPLC) along with several other impure fractions. Identification and purity of 1 were also based on comparison of its ¹H and ¹³C NMR data with the literature (29). Generally, 1:100 ratios of mixtures to be chromatographed versus the stationary phase were used in all liquid chromatographic purifications.

Lentiviral vectors encoding the human Firefly Luciferase gene were constructed by GeneChem Corporation (Shanghai, China). The empty vector was used as a negative control. The lentiviral vectors were transfected into A375 cells with a multiplicity of infection (MOI) of 60 in the presence of polybrene (5 µg/ml). At 48 h after transfection, transfected cells were selected for 2 weeks with 2.5 µg/ml puromycin (Sigma-Aldrich). Cells, which were obtained 2 weeks after drug selection without subcloning, were used for further experiments.

Cell viability was determined using CCK-8 assay (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instruction. Melanoma, HaCaT or HUVEC cells (3×10³) were seeded into a well of a 96-well plate and cultured in 150 µl of medium supplemented with 10% FBS. After 24 h, OC (0–60 µM) was added into the culture medium. Then, after the cells were incubated at 37°C for different times (24 or 48 h), the medium was changed to 100 µl of medium and 10 µl of CCK-8 reagent. The cells were incubated for 2 h at 37°C. Finally, the optical density was measured using an EnSpire™ 2300 Multilabel Reader (PerkinElmer, Waltham, MA, USA) at 450 nm.

Ten thousand A375 cells were cultured in 6-well plates for 12 days. Fresh medium with OC was added to the plates every 3 days. At the end of treatment, colonies were stained with 0.05% crystal violet for 30 min and counted.

The cell cycle analysis kit was purchased from BD Biosciences (San Jose, CA, USA). Melanoma cells were seeded into 6-well plates at 4×10⁵/dish. After incubation with OC (20 or 40 µM) for 48 h, cells were trypsinized and washed twice with PBS. Then cells were cultured with Reagents A-C according to the manufacturer's protocol and subjected to flow cytometry.

The Annexin V-FITC apoptosis kit was purchased from BD Biosciences. Melanoma cells (4×10⁵ cells/well) were incubated with OC (20 or 40 µM) for 48 h, 1×10⁴ cells were collected and washed twice with cold PBS. Then apoptotic cells were evaluated by double staining with propidium iodide (PI) and Annexin V labeled with FITC according to the manufacturer's instruction.

Melanoma cells were seeded at a plating density of 4×10⁵/well in 6-well plates and cultured overnight. After reaching 100% confluence, the cells were gently scraped with aplastic tip to produce a wound area. After wounding cells were incubated with fresh DMEM medium with 10 µM of OC, and the cell movement throughout the wound area was examined after 24 h.

Migration assay was performed using BioCoat Chambers (BD Biosciences). DMEM containing 10% FBS was placed in the lower chamber as a chemoattractant. Cells (4×10⁵) were seeded to the upper chamber in 200 µl of serum-free medium containing different concentrations of OC, and allowed to migrate for 24 h. Then, the cells on the upper surface of the membrane were removed. Chambers were fixed in 4% paraformaldehyde for 5 min and stained with crystal violet. The cells that penetrated the filter were observed with a microscope and cells from 5 random fields were counted. Invasion assay was carried out under the same conditions as the migration assay, except that the chambers were coated with Matrigel.

Tube formation was assessed as described previously (30). Briefly, HUVECs were pretreated with various dilutions of OC for 24 h and then seeded onto the Matrigel layer in 96-well plates at a density of 1×10⁴ cells. After 8–12 h, tubular structure of endothelial cells was photographed using an inverted microscope.

Luciferase reporter assay was performed as described previously (30). Luciferase activity was determined with the Promega luciferase assay kit.

Melanoma cells were plated at a density of 4×10⁵/well on 6-well plates. After incubation with different concentrations of OC, the cells were washed twice with ice-cold PBS and treated with 120 µl sample buffer on ice for 30 min. The cell lysate was centrifuged at 12,000 rpm for 10 min at 4°C. Protein lysates (30 µl) were electrophoresed on a 12 or 10% SDS gel. Then the proteins were electrotransferred to a PVDF membrane and the membrane was blocked for 30 min with blocking solution (5% non-fat dry milk in PBS-0.5% Tween-20). The membrane was then incubated overnight at 4°C with primary antibodies (1:1,000). After that the membrane was washed in PBST for 30 min, exposed to HRP-conjugated secondary antibody (diluted 1:2,000), and washed again in PBST for 30 min. Final detection was performed using enhanced chemi-luminescence solution for 5 min.

The tumor tissues were isolated to perform hematoxylin and eosin staining and immunohistochemical staining. The tumor tissues were immediately fixed with 4% paraformaldehyde, sectioned, and stained using hematoxylin and eosin for light microscopy. For immunohistochemical staining, the non-specific binding was blocked with 1% (w/v) bovine serum albumin at room temperature for 30 min. The sections were then incubated with anti-Ki-67 and anti-CD31 antibodies. The slides were incubated with biotinylated secondary antibody for 1 h at room temperature. Finally, the sections were developed with DAB color solution (50 µl/section) for 5 min at room temperature. The number of positive cells were mounted and examined under a light microscope.

Male BALB/c athymic nude mice (5 weeks old) were obtained from the experimental animal center of Shanghai Institute for Biological Sciences (Shanghai, China) and housed under standard conditions and cared for according to the institutional guidelines for animal care. All procedures and care administered to the animals were approved by the Institutional Ethics Committee. We used six mice in each group to establish subcutaneous xenograft model. A375 cells (4×10⁶) in 200 µl of PBS were subcutaneously injected into the flanks of nude mice. After the tumor volume reached 100 mm³, OC (10 mg/kg/day) was administered i.p. for 3 weeks. At the end of treatment, tumors were excised and tumor volume was calculated by using the following equation: Tumor volume = length × (width)² × π/6

For the lung metastasis model, A375 cells (5×10⁶) in 200 µl of PBS were injected into the nude mice through the tail vein (n=6/group). Animals were randomized to receive either OC (15 mg/kg/day) or DMSO at 1 week after injection. Tumor metastasis was imaged and quantified by bioluminescence every two weeks. After 6 weeks, the mice were sacrificed, and their lungs were collected for further experiment.

Data are presented as mean values ± standard deviation (SD). Comparisons between multiple groups were performed using the one-way analysis of variance (ANOVA) followed by Dunnetts t-test. A value of P<0.05 was considered statistically significant.

CCK-8 assay was first performed to detect the anti-proliferative effect of OC on melanoma cells. Melanoma cells (A375 and A2058 cells) were cultured in different concentrations (0–60 µM) for 24 h, and then cell viability was measured. OC significantly inhibited proliferation of melanoma cells in a dose-dependent manner (Fig. 1A). Extending drug exposure to 48 h resulted in additional cytotoxicity, indicating that OC also suppressed proliferation of melanoma cells in a time-dependent manner (Fig. 1B). In addition, OC treatment showed lesser toxicity on HaCaT cells (a keratinocyte cell line from adult human skin), which suggested that OC was more potent to cancer cells than normal cells (Fig. 1A and B). Colony formation ability was then investigated to determine the long-term impact of OC on melanoma cell growth. As is shown in Fig. 1C and D, the colony formation ability of A375 cells was significantly inhibited by OC in a dose-dependent manner.

To determine whether anti-proliferation effect induced by OC involved apoptosis, flow cytometric analysis with Annexin V-PI staining was performed. OC induced apoptosis of A375 and A2058 cells in a dose-dependent manner after OC treatment for 48 h (Fig. 2A). To investigate the signaling cascade which mediated OC-induced apoptosis, key proteins of capase pathway were detected. Results showed that OC significantly increased the expression of cleaved-caspase-9, cleaved-caspase-3 and cleaved-PARP in a dose-dependent manner (Fig. 2B). To explore the dependence of OC-induced apoptosis on the caspase pathway, melanoma cells were pretreated with a pan-caspase inhibitor, Z-VAD-FMK (10 mmol/l), before treatment with OC. The pretreatment with Z-VAD-FMK partially reduced OC-induced apoptosis as determined by Annexin V-PI staining (Fig. 2C). Western blotting was performed to determin whether Z-VAD-FMK suppressed OC-induced activation of caspase-3. Data showed that OC-induced activation of caspase-3 was partially reversed by pretreatment of Z-VAD-FMK (Fig. 2D). Then, flow cytometric analysis was performed to evaluate the effect of OC on cell cycle progression. However, there was no apparent change in the distribution of the cell cycle after OC treatment (data not shown). These data showed that OC induce apoptosis in melanoma cells, partially in a caspase-dependent manner.

To investigate the effect of OC on melanoma cell migration, wound-healing assay was performed by using 10 µM of OC to treat melanoma cells. OC at such doses did not affect cell proliferation, so its anticancer metastasis effect can be further studied unambiguously at noncytotoxic doses. Data showed that OC treatment significantly decreased the migration of A375 and A2058 cells (Fig. 3A and B). Matrigel-coated Transwell was then used to explore the effect of OC on the invasion ability of melanoma cells. Data showed that the invasive ability of A375 and A2058 cells was significantly suppressed after OC treatment (Fig. 3C and D).

Angiogenesis plays a critical role in the growth and metastasis of malignant tumors as the newly formed tumor vasculature serve initially as feeding tubes providing nutrients and oxygen for the tumor mass (31). Therefore, we next investigated the anti-angiogenic ability of OC. We firstly detected its effect on the motility of HUVECs using the Transwell assay. Results showed that OC significantly decreased the migration and invasion abilities of HUVECs in a dose-dependent manner (Fig. 4A and B). For further insight into the role of OC on angiogenesis, tube formation assay of HUVECs was performed. We found that OC significantly suppressed tube formation ability of HUVECs in a dose-dependent manner (Fig. 4C and D). The process of angiogenesis also requires the proliferation of endothelial cells, so the effect of OC on proliferation of HUVECs was also detected. Results showed that OC suppressed the cell viability of HUVECs in a dose-dependent manner (Fig. 4E).

Constitutive activation of STAT3 signaling pathway has been reported to play a key role in the growth, angiogenesis, and metastasis of melanoma (8). Thus, we investigated if OC suppressed the constitutive phosphorylation of STAT3. OC treatment inhibited the phosphorylation of STAT3 at the tyrosine 705 (Tyr705) site in a dose-dependent manner in A375 and A2058 cells, with little impact on the expression of total STAT3 (Fig. 5A). STAT3 is recognized to be activated by upstream tyrosine kinases such as JAK2 and Src in melanoma (32). Therefore, we explored if OC suppressed the activation of JAK2 and Src. OC inhibited the constitutive phosphorylation of JAK2 and Src in A375 and A2058 cells (Fig. 5A). We next investigated whether OC could suppress the nuclear localization of STAT3. After OC treatment, the cytoplasmic and nuclear proteins of melanoma cells were extracted and expression of STAT3 in both fractions were determined by western blotting. The levels of STAT3 in nuclear fractions were decreased in a dose-dependent manner after OC treatment, while those in cytoplasmic fractions were slightly increased (Fig. 5B). To detect whether OC affected STAT3-mediated transcriptional activity, a STAT3-luciferase reporter construct harboring four copies of STAT3 binding sites was transfected into melanoma cells. Then, melanoma cells were treated with indicated concentrations of OC for 24 h before testing the transcriptional activity by luciferase assay. OC inhibited the STAT3-luciferase reporter activity in a dose-dependent manner (Fig. 5D). Furthermore, OC also downregulated the STAT3-regulated gene products, including anti-apoptotic proteins (Bcl-xL and Mcl-1), invasion-related proteins (MMP-2 and MMP-9), and angiogenesis protein (VEGF) (Fig. 5C).

We established a subcutaneous xenograft model in nude mice using A375 cells to determine the effects of OC on melanoma growth in vivo. Compared with the control group, OC treatment resulted in a significant decrease of tumor size (Fig. 6A). We further investigated the effect of OC on the expression of Ki-67 (a marker of proliferation) and CD31 (a marker of angiogenesis) in melanoma tumor tissues by immunohistochemical analysis and found that OC significantly decreased the number of Ki-67-positive tumor cells and the microvessel density, compared with the control group (Fig. 6B and C). Furthermore, we investigated expression of tumor-related proteins in nude mice by using western blotting, and the results were consistent with in vitro data (Fig. 6D). The effect of OC on the metastatic ability of melanoma was explored in vivo by injecting luciferase-expressing A375 cells into the tail vein of nude mice to establish a lung metastasis model. We detected stronger illumination signals in control group than that in the OC-treated group (Fig. 6E). At the end of treatment, lungs were excised to perform hematoxylin and eosin staining, and lung micrometastases were microscopically evaluated. Fewer and smaller metastatic foci were detected in the OC-treated group, compared with the control group (Fig. 6F).