Curcumin (no. A0086, CAS: 458-37-7, purity >98%) was purchased from Must Biotechnology Inc. (Chengdu, China). Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco-BRL (Grand Island, NY, USA). Dimethylsulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Triton X-100, rapamycin and anti-β-actin antibody were purchased from Sigma Chemical (St. Louis, MO, USA). Matrigel was purchased from BD Company (Franklin Lakes, NJ, USA). Propidium iodide (PI) and RNase were purchased from Takara Biotechnology Inc. (Otsu, Shiga, Japan). Lipofectamine 2000 was purchased from Life Technologies (Carlsbad, CA, USA). The antibodies for LC3-I and LC3-II were purchased from Sigma-Aldrich (Shanghai, China). The antibodies for AKT, phosphorylated-AKT (Ser473), mTOR, phosphorylated-mTOR (Ser2448), P70S6K, phosphorylated-P70S6K (Thr389) were all purchased from Cell Signaling Technology (Beverly, MA, USA).

Human malignant melanoma A375 and C8161 cell lines were obtained from Union Cell Resource Center (Beijing, China) and were cultured and maintained in DMEM supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) at 37°C and 5% CO₂ in a humid incubator. Curcumin (dissolved in DMSO) was used to treat A375 and C8161 cells. Control cells were treated with equivalent volume of DMSO.

The effect of curcumin on the viability of the cells was determined by MTT assay. Briefly A375 and C8161 cells were plated at 1×10⁴ cells/well in 200 µl culture medium into 96-well plates. Different concentrations of curcumin (15–75 µM) were added to different wells and incubated at 37°C for 24, 48, 72 and 96 h. Following incubation for the specified time, 20 µl MTT solution [5 mg/ml in phosphate-buffered saline (PBS)] was added to each well and incubated for additional 4 h. The culture medium in each well was replaced with 200 µl of DMSO to dissolve the formazan crystals that were formed from the MTT assay. Absorbance of the solution was read at a wavelength of 490 nm on a microplate reader (T17108U; Perkin-Elmer, USA).

To measure the three-dimensional movement of the cells, cell invasion assay was performed using Transwell chambers with 8-µm pore polycarbonate filter inserts (Corning, New York, NY, USA). The upper side of each insert was coated with 10 µl of Matrigel (3 mg/ml; Becton-Dickinson, Mountain View, CA, USA). A375 and C8161 cells (1×10⁶/ml) were separately cultured on Matrigel-coated Transwell inserts in DMEM supplemented with curcumin (25 and 15 µM, respectively), or DMSO. The lower chamber contained DMEM culture medium appended with 10% FBS as a chemoattractant. After 72 h of incubation, the invaded cells on the lower surface of the membranes were fixed with chilled 3.7% methanol for 15 min and stained with 0.5% crystal violet for 15 min. Cells were visualized using inverted light microscope, and invasiveness was determined by counting the number of cells appearing on the undersurface of the polycarbonate membranes in five random, non-overlapping fields at a magnification of ×200. The average number of invaded cells in the five fields was taken to represent the mean cell invasion. The experiments were performed in triplicate.

A375 and C8161 cells were treated with curcumin (25 and 15 µM, respectively) at 37°C and 5% CO₂ atmosphere for 24 h. Cells were harvested and fixed in chilled 75% alcohol for 4 h. Prior to analysis, a staining solution containing 480 µl PBS, 5 µl PI (5 mg/ml), 5 µl RNase (10 mg/ml) and 10 µl Triton X-100 (10%) was added to resuspend the cell pellet and incubated in the dark for 30 min at room temperature. Cells were analyzed by flow cytometer (Accuri C6; BD, USA) for cell cycle phase distribution.

Cells were transfected with a plasmid expressing GFP-LC3 using Lipofectamine 2000 according to the manufacturer's instructions. After 16 h post-transfection with GFP-LC3, A375 and C8161 cells were incubated with curcumin (25 and 15 µM, respectively) or rapamycin (100 nM, used as a positive control) or DMSO at 37°C for 24 h. Dot formation by GFP-LC3 was detected under a fluorescence microscope (DP73; Olympus, Japan) following drug treatment. Transfected cells were considered to have accumulated autophagosomes when five or more puncta were observed. A total of 100 transfected cells were examined/well, and the percentage of cells showing autophagy were counted in triplicate. The experiment was independently repeated three times.

Cells were harvested by scraping from the plates. They were then washed twice with PBS and fixed with 2.5% glutaraldehyde and 1% (v/v) osmic acid, followed by an increasing gradient dehydration step using ethanol and acetone. Cells were then embedded in epoxy resin and ultrathin sections were cut, and stained with 0.2% lead citrate and 1% uranyl acetate. Images were captured with a transmission electron microscope (JEM 1011CX; JEOL, Japan).

After treatment of A375 and C8161 cells with curcumin (25 and 15 µM, respectively) for 24 h, the cells were harvested and incubated in lysis buffer on ice for 30 min. Then the lysate was clarified by centrifugation at 12,000 x g for 10 min at 4°C to obtain the supernatant (total cell lysate). The total protein concentration was determined using the Coomassie brilliant blue (CBB) method. For western blotting, 25 µg of total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. After blocking of non-specific binding sites with 5% non-fat dry milk for 2 h at room temperature, membranes were incubated with respective primary antibodies at appropriate concentrations overnight at 4°C. After washing the membranes to remove unbound primary antibodies, they were incubated with either horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (1:5,000) for 1 h at room temperature. Finally the membranes were washed with PBS and chemiluminescence developed using ECL kit (APG BIO, Shanghai, China) for 1 min. Protein bands were visualized by image scanning and the optical density for each band was measured using Image Lab software (version 4.0; Bio-Rad, USA) after data were normalized to β-actin as an internal control.

All the animal experiments were carried out in strict accordance with the institutional guidelines for the ethical treatment of animals. The protocol was approved by the Ethics Committee of the Laboratory Animal of Dalian Medical University (permit no. L2015012). Six-week-old female BALB/c nude mice (Institute of Animal Center, Chinese Academy of Sciences, Shanghai, China) were housed in laminar flow rooms with stable temperature and humidity conditions. Human melanoma A375 cells (1×10⁷/ml) resuspended in PBS were injected subcutaneously into the right flank of each mouse. The mice were randomly assigned to two groups (n=5/group). Therapy was initiated on the eighth day post-inoculation with A375 melanoma cells. Mice from the control and therapeutic groups received intraperitoneal injections of DMSO or curcumin (25 mg/kg), respectively, every day for 3 weeks. Tumor size was monitored before every injection using calipers at 3 days interval, and tumor volume was calculated using the formula: Volume (mm³) = (maximal length) × (perpendicular width)²/2. All the mice were sacrificed 21 days after treatment. In addition, the tumors were resected for analyses.

All the experiments were carried out in triplicate and values are expressed as mean ± standard deviation (SD). SPSS v17.0 software (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. The Student's t-test was used for the assessment of differences between groups. A probability of ≤0.05 was deemed statistically significant. In the figures: ^*P<0.05, ^**P<0.01 and ^***P<0.001, relative to controls.

We first studied the inhibitory effect of curcumin on the growth of A375 and C8161 cells by employing MTT assay. Cells were treated with different doses of curcumin (15–75 µM) for different periods of time (24–96 h). MTT assay showed that curcumin was effective in inhibiting the proliferation of A375 and C8161 cells in a dose-dependent, as well as time-dependent manner (ranging from 15–35 and 15–25 µM, respectively, within 48 h (Fig. 1A and B). The IC₅₀ value at 24 h was estimated to be 25 and 15 µM, respectively (Fig. 1A and B).

Since curcumin treatment inhibited A375 and C8161 cell growth, we next examined whether growth inhibitory effect of curcumin was mediated through cell cycle arrest. For this purpose, the effect of curcumin on cell cycle progression in A375 and C8161 cells was determined by flow cytometry. Treatment of A375 and C8161 cells with curcumin (25 and 15 µM, respectively) resulted in a remarkable accumulation of cells in G₂/M phase and a reduction in G₀/G₁ cell population during the cell cycle (Fig. 2, Table I). This profound decrease in G₀/G₁ phase cell population suggests a G₂/M phase cell cycle arrest of melanoma cells upon exposure to curcumin and that blockage of cell cycle progression may be one of the mechanisms by which curcumin inhibits A375 and C8161 cells growth.

The Matrigel model of the basement membrane was employed as a cell invasion barrier to quantify the invasive potential of A375 and C8161 cells. The density of invaded cells on the membrane and the number of invaded cells/microscopic field are shown in Fig. 3. Treatment with curcumin (25 and 15 µM, respectively) for 72 h significantly reduced the invasive potential of A375 and C8161 melanoma cells as compared to their untreated control cells (P<0.001). When A375 cells were treated with ≥35 µM curcumin and C8161 cells treated with ≥25 µM curcumin for 72 h, no invading cells could be seen (data not shown), suggesting that curcumin may antagonize the invasive potential of melanoma cells, as evidenced on A375 and C8161 cells.

LC3 is now believed to be a reliable marker for autophagy. A375 and C8161 cells were treated with DMSO, rapamycin (100 nM) or curcumin (25 and 15 µM, respectively). As shown in Fig. 4A, autophagic vacuoles and autolysosomes accumulated in rapamycin- and curcumin-treated groups, while none was observed in DMSO-treated group (Fig. 4A). In the absence of any treatment, diffuse LC3 fluorescence was observed. Following treatment with curcumin, punctate fluorescence, which was similar to that produced by the canonical autophagy inducer rapamycin, was observed (Fig. 4B). Curcumin caused an obvious increase in the number of cells with GFP-LC3 punctate dots in both A375 and C8161 cells (Fig. 4C, P<0.001). During autophagy, LC3-II is recruited to the autophagosome. We found that LC3-II level was significantly increased with 25 and 15 µM curcumin treatment of A375 and C8161 cells as compared to DMSO treatment (Fig. 4D, P<0.001, P<0.05). These results revealed that curcumin could potently induce autophagy in both A375 and C8161 cells.

Since growth factor signaling can directly regulate autophagy through mTOR pathway, western blotting against phospho-AKT, AKT, phospho-mTOR, phospho-P70S6K and P70S6K were performed. Under the indicated concentration of curcumin treatment of A375 and C8161 cells, the expression of PI3K downstream activated proteins, p-AKT, p-mTOR and p-P70S6K, significantly decreased as compared to DMSO treated cells (Fig. 5).

BALB/c nude mice were inoculated with A375 melanoma cells to establish an explanted in vivo melanoma model. After 21 days of treatments with either curcumin or DMSO, tumors in the curcumin-treated group were generally smaller than tumors in corresponding DMSO-treated animals (Fig. 6A). The average tumor weight in curcumin-treated mice was also markedly less as compared to tumors from control mice (Fig. 6B). However, the difference between the average tumor volumes of the two groups was not statistically significant (data not shown). Curcumin, thus may suppress melanoma growth in vivo.