The human melanoma A375 cell line was obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences. The cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) at 37°C with 5% CO2. The medium was supplemented with 10% FBS (Thermo Fisher Scientific, Inc.), penicillin (100 µg/ml) and streptomycin (100 µg/ml). MTT was obtained from Sigma-Aldrich (Merck KGaA). The Dead Cell Apoptosis kit with FITC Annexin V and PI was obtained from Thermo Fisher Scientific, Inc.

The fruiting body of S. vaninii was cultivated at the Sericultural Research Institute of Zhejiang Academy of Agricultural Sciences (Hangzhou, China). The dried fruiting body powder was extracted with boiling water for 2 h. The supernatant was collected after centrifugation at 8,000 × g at room temperature for 10 min. The supernatant was lyophilised to obtain extract powders. The content of total polysaccharides and polyphenols was 5.51 and 23.00%, respectively. The major polyphenols were 3,4-dihydroxybenzalacetone, phellibaumin D and interfungin B (17).

Cell proliferation was determined by trypan blue staining and the inhibitory ratio was measured by MTT-based colorimetric assay as described by Zhong et al (16). Briefly, cells at the exponential growth phase were dispensed into a 96-well microplate at 100 µl/well. After 24 h, 100 µl of different concentrations of SH were added to prepare final concentrations of 50, 100, 200 and 400 µg/ml. Sterile water (0.1% DMSO) was used as control. The cells were cultured at 37°C for 24, 36 and 48 h for cell counting. The supernatants of each well were obtained, and 50 µl of 0.25% trypsin was added into each well. After incubation at 37°C for 5 min, the supernatants were re-added to the corresponding well to stop cell digestion. Cell suspensions (~20 µl) were transferred into a new 1.5 ml centrifuge tube with 20 µl of 0.4% trypan blue solution. After mixing, 20 µl cell suspension was added to a cell counting plate for counting with an automatic cell counter (IC1000; Countstar; ALIT Life Science Co., Ltd.). Cells cultured for 48 h were used for the MTT assay. The cells were incubated at 37°C with 50 µl of MTT solution (1 mg/ml) for 2 h, and the resulting crystals were dissolved in DMSO. The absorbance at 570 nm was recorded to assess the formation of formazan. Inhibitory ratio (%) = [1-absorbance (test)/absorbance (control)] × 100%, where the test and control correspond to treated and untreated cells, respectively.

The cell cycle phase was measured by assessing the DNA content via flow cytometry as previously described by Zhong et al (16). Briefly, A375 cells (1×10⁵ cells/well) were incubated at 37°C with SH at final concentrations of 0 (control), 200 and 400 µg/ml for 48 h. The proportion of cells at different phases was determined using a flow cytometer (Cytomics FC 500 MCL; Beckman Coulter, Inc.). MultiCycle AV software (CXP V2.3 WIN7, C30309; Phoenix Flow Systems, Inc.) was used for analysis. Apoptosis rate was measured by staining with Annexin V-FITC (5 µl) and PI (5 µl) at 4°C for 15 min. Annexin V⁻/PI⁻ (lower left quadrant) cells were alive cells, Annexin V⁺/PI⁻ (lower right quadrant) cells were early apoptotic cells, Annexin V⁺/PI⁺ (upper right quadrant) cells were late apoptotic cells and Annexin V⁻/PI⁺ (upper left quadrant) cells were necrotic cells. The proportion of cells was determined using a flow cytometer (Cytomics FC 500 MCL; Beckman Coulter, Inc.) with MultiCycle AV software (CXP V2.3 WIN7, C30309; Phoenix Flow Systems, Inc.). All experiments were performed in three biological duplicates.

Total RNA was isolated from A375 cells treated with or without SH (400 µg/ml) for 6 and 48 h using the Takara MiniBEST universal RNA extraction kit (Takara Bio, Inc.). PrimeScript RT reagent kit with gDNA Eraser (Takara Bio, Inc.) and SYBR^® Fast qPCR Mix (Takara Bio, Inc.) were used for the CFX96 real-time PCR system (Bio-Rad Laboratories, Inc.). All operations were performed in accordance with the manufacturer's instructions. The thermocycling conditions consisted of initial denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and 60°C for 30 sec. GAPDH was used as an internal reference gene, and gene expression was quantified using the comparative 2^−∆∆Cq method (18). PCR primer sequences are summarised in Table I.

A375 cells treated with or without SH (400 µg/ml) for 48 h were collected for western blot analysis as previously described (12). Briefly, the total proteins were extracted using the SD-001 buffer (Invent Biotechnologies, Inc.) and supplemented with protein phosphatase inhibitor (Beijing Solarbio Science & Technology Co., Ltd.), and the protein concentrations were determined using a BCA protein assay kit. A total of 40 µg protein/lane was separated via 4–12% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane. After blocking with 5% BSA (Merck KGaA) for 1 h at room temperature, membranes were incubated with the corresponding primary antibodies at 4°C overnight. Subsequently, membranes were washed with PBS three times and incubated with the corresponding secondary antibodies at room temperature for 1 h. Primary antibodies (1:1,000) against p21 (cat. no. ab188224), CyclinD (cat. no. ab28283), CDK2 (cat. no. ab32147), CDK4 (cat. no. ab131469), CDK6 (cat. no. ab131469), Bcl-2 (cat. no. ab32124) and β-actin (cat. no. ab8226) were obtained from Abcam. The secondary antibody was Alexa Fluor 790-conjugated goat anti-rabbit IgG H&L (1:10,000; cat. no. ab175781; Abcam). The membranes were washed with PBS three times at room temperature, and the protein signals were visualised using the ChemiDoc touch imaging system (Bio-Rad Laboratories, Inc.) with Image Lab touch software (version 1.2; Chameleon Power).

A375 cells on a slide were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, and then washed with PBS for 3 times. Cells were blocked with 3% BSA (Merck KGaA) for 30 min at room temperature, and then primary antibodies against CyclinD (cat. no. ab28283) and Bcl-2 (cat. no. ab32124) (both 1:200; Abcam) were added at 4°C overnight. Subsequently, the slides were washed with PBS and incubated with DyLight 488-conjugated goat anti-rabbit IgG (1:200; cat. no. BA1127; Wuhan Boster Biological Technology, Ltd.) for 1 h at room temperature. The slides were mounted with DAPI-containing medium (C1002; Beyotime Institute of Biotechnology) for counterstaining of nuclei and then observed under a fluorescence microscope (Nikon Eclipse 80i; Nikon Corporation) at ×100 magnification.

Data were expressed as the mean ± SD. Statistical analysis was performed using SPSS 16.0 software (SPSS, Inc.). One-way ANOVA was used to analyse statistical differences between groups under different conditions, followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

To examine the effects of SH on melanoma cells, A375 cells were treated with different concentrations of SH (0, 50, 100, 200 and 400 µg/ml) for 24, 36 and 48 h (Fig. 1). As shown in Fig. 1A and B, treatment of A375 cells with SH for 36 and 48 h significantly decreased cell viability compared with the control group in a dose-dependent manner. Particularly, 400 µg/ml SH effectively inhibited proliferation of A375 cells, along with a significant inhibitory ratio of >43% (Fig. 1B and C). Hence, SH suppressed the proliferation of A375 cells in vitro, suggesting that SH may be used as a cell proliferation inhibitor for the treatment of melanoma.

For further investigations on the inhibitory mechanism of SH on A375 cells, flow cytometry was conducted to assess whether the anti-proliferative effect of SH on A375 cells was mediated by cell cycle arrest. Under the higher inhibitory concentrations of 200 and 400 µg/ml, SH significantly increased the number of cells in S phase (Fig. 2). At 200 µg/ml, SH significantly decreased the number of cells at the G2/M phase, while the number of cells at the G0/G1 phase significantly decreased with 400 µg/ml SH (Fig. 2). Hence, SH mainly induced cell cycle arrest of A375 cells at the S phase.

Flow cytometry with Annexin V-FITC and PI staining was then used to examine the contribution of apoptosis to the SH-mediated inhibition of cell proliferation. SH treatment significantly increased early apoptotic, late apoptotic and necrotic cells compared with the control group (Fig. 3). Hence, S-phase arrest and apoptosis may be mainly involved in the anti-proliferative effect of SH on A375 cells.

To further elaborate the molecular mechanism underpinning the anti-proliferative action of SH, RT-qPCR analysis was performed using A375 cells treated with 400 µg/ml SH for 6 and 48 h, since 400 µg/ml SH exhibited an increased ability of S phase arrest. A number of key genes involved in cell cycle were analysed. As shown in Fig. 4A, the expression levels of p21 were significantly upregulated, whereas those of p27, CyclinsA, C, D and E, and CDK1, 2, 4, 5 and 6 were significantly downregulated after 48 h of SH treatment. No significant difference was observed in the expression levels of TGFB, Smad2, Smad4, p53, CyclinB, CDK3, CDK7 and Rb. Additionally, transcription factors E2F1 and DP1 were significantly downregulated after 48 h of SH treatment.

To confirm the difference in gene expression, western blot and immunofluorescence analyses were conducted. As shown in Fig. 4B, the expression levels of the proteins encoded by some of the aforementioned key genes, such as p21, CyclinD, CDK2, CDK4 and CDK6, were consistent with their gene expression levels after 48 h of SH treatment. Immunofluorescence analysis of CyclinD-stained cells revealed that the green fluorescence signals in nuclei were strong without SH treatment, while after treatment with 400 µg/ml SH for 48 h, the number of cells decreased markedly compared with the control, and the green fluorescence signals were weaker (Fig. 4C). Therefore, SH may inhibit A375 cell proliferation by inducing cell cycle arrest at the S phase by upregulating p21 expression, which inhibits the expression levels of cyclin-CDK complexes.

Several key genes involved in apoptosis were analysed by RT-qPCR analysis using A375 cells treated with 400 µg/ml SH for 6 and 48 h, since 400 µg/ml SH strongly induced apoptosis. The relative mRNA expression levels of pro-apoptotic genes (Bad, Bik, Bid, Bim and Bak) decreased under SH treatment. The expression levels of Bik and Bim decreased at 6 h significantly, Bid and Bak were downregulated at 48 h significantly, and the relative mRNA expression levels of Bad decreased at 6 and 48 h significantly (Fig. 5A). No significant difference was observed in the expression levels of MAPK signalling pathway-associated genes (Ras, Raf, MEK1 and ERK1; Fig. 5A). The anti-apoptotic gene Bcl-2 was significantly downregulated after SH treatment at 48 h (Fig. 5A), consistent with the western blot analysis indicating significantly decreased Bcl-2 protein expression (Fig. 5B). Immunofluorescence analysis of Bcl-2-stained cells indicated that the cell density decreased markedly after treatment with 400 µg/ml SH for 48 h, and the green fluorescence signals were weaker than in the control group (Fig. 5C). The current results suggested that SH may induce apoptosis by downregulating Bcl-2 expression.