Human malignant melanoma A375 and C8161 cell lines were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). All cells were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C and 5% CO₂. Andrographolide (MF: C₈H₈O₄, MW: 168.15, purity >98%) was purchased from Shanghai Yuanye Biotechnology, Co., Ltd. (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 80 mM. Antibodies against cleaved-PARP, cleaved-caspase-3, phospho-JNK, JNK, p38, phospho-p38, and GAPDH were all obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Inhibition of cell proliferation by Andro was detected using the 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) assay. Briefly, cells were trypsinized and plated into 96-well plate at a density of 5×10³ cells/well and incubated overnight at 37°C in a humidified incubator, then treated with fresh medium containing various concentration of Andro (5, 10, 20, 40, and 80 µM). And the cells were incubated for 24, 48 h. Subsequently, final concentration of 0.5 mg/ml MTT was added directly to and incubated for 4 h at 37°C. The plates were depleted and a total of 100 µl of DMSO was added to each well, and the optical density was measured at 490 nm using microplate reader iMark (Molecular Devices, LLC, Sunnyvale, CA, USA). Data represented the mean of five replicates. Three independent experiments were carried out in triplicate.

To examine whether Andro affects cell cycle distribution, we used flow cytometer with PI/RNase staining buffer (BD Biosciences, Mountain View, CA, USA). In brief, 5×10⁵ cells were seeded in 6-well plates and incubated with Andro for 24 h at 37°C. And then the cells were collected and fixed with 70% ethanol at −20°C overnight. The cells were washed and resuspended in cold PBS and followed by staining with 10 mg/ml RNase and 1 mg/ml propidium iodide for 30 min at 37°C. Next, the samples were detected on Accuri C6 (BD Biosciences) and the data were analyzed by ModFit LT software (FACSCalibur).

To evaluate apoptosis induction, a FITC-Annexin apoptosis detection kit (BD Biosciences, Sparks, MD, USA) was used. Cells were seeded in 6-well plates and treated with or without Andro for 24 h. Apoptotic cells were measured by using Annexin V-FITC/PI double staining. Briefly, cells were collected and washed with ice-cold PBS, and then resuspended in 500 µl of binding buffer. These cells were stained with 5 µl of Annexin V/FITC solution and 10 µl propidium iodide (PI) (1 g/ml). Cells were then incubated for 15 min at 37°C in the dark. The samples were detected by the Accuri C6.

Cells were cultured in 6-well plates and treated with as indicated for 24 h. The cell were washed twice with cold PBS solution and then resuspended in radioimmunoprecipitation assay (RIPA) lysis buffer with phosphorylase and protease inhibitor. The protein concentration was determined using using BCA Protein Assay kit and equalized before loading. Equivalent amounts of total protein (25–50 µg) were separated by SDS-PAGE and and transferred onto a PVDF membrane (0.45 mm; Millipore, Bedford, MA, USA). Following blocking with 5% fat-free milk for 60 min, membrane was blotted with the following primary antibodies as follows: Cleaved-PARP, cleaved-caspase-3, phospho-JNK, JNK, p38, phospho-p38, and GAPDH at 4°C overnight. Horseradish peroxidase-linked anti-mouse or anti-rabbit IgG was then used as secondary antibody. And cells were incubated with horseradish peroxidase-linked anti-mouse or anti-rabbit IgG (1:5,000 dilution) for 1 h. Specific antibody binding was visualized using ECL (Millipore, Plano, TX, USA) and digitalized by scanning.

All data represent at least three independent experiments and were expressed as mean ± standard deviation (SD). The statistical differences were calculated by one-way ANOVA analysis of variance with Dunnett's test or unpaired Student's t-test. All statistical analyses were performed using SPSS 19.0 software and significant difference was analyzed by Duncan's multiple-range test. (SPSS Inc., Chicago, IL, USA). P-values <0.05 were considered to indicate a statistically significant difference.

To investigate the antiproliferative activity of Andro, A375 and C8161 cells were treated with different concentration of Andro for 24 and 48 h (Fig. 1). The cell viability was measured by MTT assay. The IC₅₀ values of Andro for A375 were 23.08 µM (24 h), 12.07 µM (48 h), while the IC₅₀ values for C8161 were 20.31 µM (24 h), 10.92 µM (48 h). These results suggest that Andro exhibits potent anti-proliferation effects in malignant melanoma cells in a dose- and time-dependent manner.

To gain insights into the mechanism by which Andro inhibits cell proliferation, we examined the effect of Andro on the cell cycle progression. As shown in Fig. 2, in comparison to DMSO-treated cells, a significant increase of cells in G2/M phase was observed after Andro treatment, while a corresponding decrease in G0/G1 phases was observed after Andro treatment. These data showed that Andro induces G2/M phase arrest.

To examine whether the cell growth inhibition induced by Andro is also dependent on apoptosis, Andro-treated cells were analysed by Annexin V-FITC/PI double staining. As shown in Fig. 3A and B, in comparison to DMSO-treated cells, Andro resulted in a significant increase of the proportion of apoptosis the number of apoptotic cells in a dosedependent manner. Western blot analyses showed that cleaved poly(ADP-ribose) polymerase (PARP) and cleaved caspase-3 were increased after treatment with Andro for 24 h (Fig. 3C). Overall, these results clearly indicate that Andro provoks caspase-dependent apoptosis in melanoma cell lines.

Recent studies have suggested JNK signaling pathway plays important roles in many cellular events, such as regulating cell cycle, cell apoptosis and autophagy (19,20). To further elucidate the effect of Andro on JNK signal pathway, we measured the expression of JNK an p-P38 genes. As shown in Fig. 4, phosphorylation of JNK and p-P38 were induced in a dose-dependently manner after Andro treatment. These results clearly indicate that Andro activated the JNK and p-P38 signaling pathway.