ORI was acquired from Aladdin Biochemical (Shanghai, China), and dissolved in 0.5% dimethyl sulfoxide (DMSO). To make sure the ORI solution was sterile it was filtered using a 0.2 µm filtration membrane before it was added to the culture medium for the in vitro assays. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), LY294002 (an inhibitor of PI3K), and DMSO were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Phosphatase inhibitor cocktail tablets were purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA). TGF-β1 was purchased from Millipore (Billerica, MA, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). All other chemicals were of analytical reagent grade. The antibodies used were as follows: Anti-E-cadherin was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Anti-vimentin was purchased from Epitomics (Burlingame, CA, USA). Anti-Snail, anti-Akt, and anti-phospho-Akt (Ser473) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-PI3K, anti-phospho-PI3K (Tyr458), anti-GSK-3β, anti-phospho-GSK-3β (Ser9) and anti-β-catenin were purchased from Abcam (Cambridge, MA, USA). Anti-β-actin, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All antibodies were diluted at 1:1,000 except where specified.

A375 human melanoma cell line obtained from ATCC (Manassas, VA, USA). B16-F10 (mouse melanoma) cell line was purchased from the Cell Culture Center of Chinese Academy of Medical Sciences (Beijing, China). The cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. All cell cultures were maintained at 37°C in an atmosphere containing 5% CO₂.

Cell viability was measured by MTT assay to evaluate the effect of ORI on cell proliferation (24). A375 or B16-F10 cells in the logarithm phase were seeded in 96-well plates at the density of 5×10³ cells/well. When the cells were adherent to the walls, the cells were treated with ORI at indicated concentrations for 24 h followed by adding 100 µl of MTT (1 mg/ml) and incubating for 4 h. Then, the medium was removed and 150 µl of DMSO was added to each well. Absorbance of each well was detected under 490 nm by microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The test was repeated three times. Inhibition rate (% of control) = (1 - absorbance of test sample/absorbance of control) × 100%.

Cell migration wounding assay was performed according to the protocol described previously (25,26). A375 or B16-F10 cells were cultured in 60 mm dishes at the density of 8×10⁵ cells/dish to 100% confluency. After wounding with pipette tip, the cells were washed with PBS and serum free medium to which indicated concentrations of ORI had been added. Then, the cells were allowed to migrate for 24 h at 37°C in 5% CO₂. At predetermined time-points (0, 3, 6, 9, 12 and 24 h), the widths of wound were measured and images of cells were taken at time 0 and 24 h with a microscope (IX50; Olympus, Tokyo, Japan). The assay was carried out double blind to eliminate the deviation induced by subjective factors. The test was repeated three times.

Cell invasion was detected by Transwell assay (26,27). After pre-treatment with indicated concentrations of ORI for 24 h, A375 or B16-F10 cells were harvested and seeded to the upper chamber which was coated with matrigel (BD Biosciences) at the density of 3×10⁴ cells/well in serum free medium. The lower chambers were filled with standard medium. The cells were allowed to invade for 24 h incubated at 37°C in 5% CO₂. The invading cells were fixed with methanol. Cell numbers were counted in five separate fields using the computer-based microcopy imaging system. The assay was carried out double blind to eliminate the deviation induced by subjective factors. The test was repeated three times.

After pre-treatment with indicated concentrations of ORI for 24 h, A375 or B16-F10 cells were harvested, re-suspended in serum free medium at the density of 2×10⁵ cells/well and seeded to the 24-well plates coated with fibronectin (10 ng/ml). After further incubations for 5, 15 and 30 min, non-adherent cells were removed by PBS washes. The adherent cells were fixed with methanol and counted in five separate fields under a light microscope (28,29). The assay was carried out double blind to eliminate the deviation induced by subjective factors. The experiment was repeated three times.

After pre-treatment with indicated concentrations of ORI for 24 h, The total RNA of A375 or B16-F10 cells was extracted by TRIzol reagent (Roche, Suisse). Reverse transcription was carried out with fast quant RT kit (Tiangen, Beijing, China). The procedure was based on the protocol provided by Tiangen. The real-time PCR mixture volume was 25 µl including 12.5 µl SYBR Green mix, 0.2 µl cDNA, 1.5 µl primer per mix (10 µM each primer), and 10.8 µl RNAse-free H₂O. The experiment was then set up with the following PCR program on ABI 7500 (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA): 95°C for 15 min, 1 cycle; 40 cycles of 95°C for 10 sec, 60°C for 20 sec, 72°C for 30 sec. Specific primers were designed by gene runner software and were synthesized by Beijing Aoke Biotechnology Co., Ltd. (Beijing, China). The specific primers are reported in Table I. The Ct value was automatically calculated by software, the Ct values were all normalized against the quantity of the β-actin control RNA, and the relative quantification of gene expression was calculated by the 2^−ΔCt method according to the formula: ^ΔCt (target gene) = Ct (target gene) - Ct (control gene). All assays were performed in triplicate and independently repeated 3 times.

Western blotting was used for detection of expressions of E-cadherin, vimentin, Snail, PI3K, phospho-PI3K, Akt, phospho-Akt, GSK-3β, and phospho-GSK-3β, anti-β-catenin in A375 and B16-F10 cells. After pretreatment with indicated concentrations of ORI and/or LY294002 (an inhibitor of PI3K) for 24 h, cells were then stimulated by 10 ng/ml TGF-β1 for 15 min. Then the protein lysates from cultured cells were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) systems and transferred to polyvinyllidene difluoride (PVDF) membranes (Millipore). After blocking with 5% skim milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 2 h, the membranes were incubated with primary antibodies at 1:500-1:1,000 dilutions with 5% BSA in TBST overnight at 4°C. The antibodies were as follows: E-cadherin, vimentin, Snail, PI3K, phospho-PI3K, Akt, phospho-Akt, GSK-3β, phospho-GSK-3β, and anti-β-catenin. The blots were washed and incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) and incubated for 1 h at room temperature. Membranes were visualized using enhanced chemiluminescence (immobilon ECL; Millipore) and were photographed using G-BOX (Gene Company Ltd., Beijing, China). The bands were analyzed by ImageJ software.

Each experiment was repeated at least three times. All data were expressed as mean ± standard deviation (SD). Statistical Product and Service Solutions (version 19.0; SPSS, Inc., Chicago, IL, USA). P-value <0.05 was considered to indicate a statistically significant difference using one-way analysis of variance (ANOVA) test.

ORI, a diterpenoid purified from Rabdosia rubescens, has a molecular weight of 364.44 g/mol and its molecular structure is shown in Fig. 1A. The inhibitory effect of ORI on the proliferation of melanoma cell lines A375 and B16-F10 were first detected by MTT assay. As shown in Fig. 1B and C, the survival rate of ORI (5–20 µM) treated groups were >90% at 48 h, indicating that ORI at each of these concentrations alone had no anti-proliferative effect and did not cause any apparently cytotoxic effects in A375 and B16-F10 cells within 48 h. However, when cells were treated with 40 µM of ORI for 48 h, the growth of A375 and B16-F10 cells was inhibited by ORI, but the inhibition rate of 40 µM for 48 h was only 15% (Fig. 1B and C). These data indicate that 48 h of ORI (5–20 µM) exposure has no significantly influence on the cell viability of A375 and B16-F10 cells. Thus, concentrations 5 and 10 µM of ORI were used in the subsequent experiments.

Cancer progression is associated with abrogation of the normal controls that limit cell migration and invasion. Cell invasion and migration are the initial and critical events in tumor metastasis and enhance the ability of a cancer cell to enter and exit the circulation to reach distant organs (30). The effect of ORI on cellular migration was investigated using a classic in vitro wound healing model. ORI was found to be effective in reducing cellular migration in A375 and B16-F10 cells (Fig. 2). The inhibitory effect of ORI on invasion of A375 and B16-F10 cells was examined using an invasion assay with matrigel-coated filters. In the absence of ORI (control group), A375 and B16-F10 cells displayed high invasive capability as indicated by being able to completely penetrate through the matrigel-coated filters. Activity of invasion of A375 and B16-F10 cells were markedly suppressed by 24 h exposure to ORI. At concentrations of 5 and 10 µM of ORI, the number of cells able to penetrate through matrigel-coated filters was significantly decreased compared with control group (Fig. 3).

The altered adhesiveness of tumor cells plays an important role in the formation of distal foci (31). The extracellular matrix (ECM) is a powerful regulator of cancer progression, which promotes transformation and regulates the tumor metastasis (32). Hence, we used fibronectin as the basement membrane to mimic the adhesion of A375 and B16-F10 cells. After the pre-treatment with indicated concentrations of ORI, the number of cells adhering to the fibronectin significantly decreased compared with the control group. Additional quantitative data are shown in Fig. 4. These data indicate that ORI inhibits the adhesiveness of A375 and B16-F10 cells adhering to fibronectin.

EMT plays a key role in cancer progression. Enhanced cell migration and invasion properties are important consequences of EMT (33). The most well characterized factor responsible for induction of EMT is TGF-β1. Snail as one of the transcriptional factors has been reported to be involved in the regulation of E-cadherin (34). The accumulation of cytoplasmic β-catenin and its subsequent nuclear translocation is also a key event in EMT (35). Here we investigated the effect of ORI on TGF-β1-mediated EMT in A375 and B16-F10 cells by qRT-PCR and western blotting. As shown in Fig. 5, TGF-β1 reduced the expression of the epithelial marker E-cadherin and enhanced the expression of the mesenchymal marker vimentin. Snail and β-catenin expression levels were also enhanced, induced by TGF-β1. Our data demonstrate that the effects of ORI are dose dependent increasing the expression of E-cadherin and decreasing the expression of vimentinin, Snail and β-catenin in A375 and B16-F10 cells compared with control group.

Recent studies demonstrated that aberration of PI3K/Akt/GSK-3β signaling pathway is a very common mechanism for many human cancers including melanoma, since it can mediate survival, apoptosis, migration and invasion pathways (36). A growing number of studies showed that inhibition of PI3K/Akt/GSK-3β pathway could inhibit the migration and invasion of cancer cells (37,38). Hence, we investigate the effects of ORI on the PI3K/Akt/GSK-3β signaling pathway in A375 and B16-F10 cells. As shown in Fig. 6, TGF-β1 significantly increased the protein expression levels of p-PI3K, p-Akt and p-GSK-3β in both types of cells. Our studies have demonstrated that ORI has a significant inhibitory effect on TGF-β1-mediated EMT, but the detailed effect mechanism is unclear. In the present study, we firstly examined the inhibitory activity of ORI against TGF-β1-induced PI3K/Akt/GSK-3β pathway phosphorylation. The data demonstrated that ORI could downregulate the levels of p-PI3K, p-Akt and p-GSK-3β. It is necessary to verify whether ORI could inhibit the EMT through TGF-β1 mediated PI3K/Akt/GSK-3β pathway. To verify this hypothesis, PI3K inhibitor LY294002 was used to block the PI3K/Akt pathway, then ORI and TGF-β1 were added. The data demonstrated that, LY294002 and ORI abolished the TGF-β1-induced decrease in E-cadherin and increase in vimentin, p-PI3K, p-Akt, p-GSK-3β, Snail and β-catenin expression. ORI had little effect on E-cadherin, Vimentin, p-PI3K, p-Akt, p-GSK-3β and β-catenin (Fig. 7). There was no difference between their effects. ORI combined with LY294002 synergistically suppressed the related protein expression. The above data suggest that ORI may regulate the expression of E-cadherin and vimentin may work through PI3K/Akt/GSK-3β signaling pathway, thereby in inhibiting TGF-β1-induced EMT, which may be one of the mechanisms through which ORI inhibited the A375 and B16-F10 cells migration and invasion.