Gyp was extracted from Gynostemma pentaphyllum Makino that was provided by Professor Jung-Chou Chen (China Medical University, Taichung, Taiwan) as described previously (18). Dimethyl sulfoxide (DMSO), Tris-HCl, propidium iodide (PI), trypan blue, Triton X-100, ribonuclease-A, penicillin-streptomycin and trypsin-EDTA were all purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). DiOC₆ and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco; Thermo Fisher Scientific, Inc.

The human oral squamous cell carcinoma HSC-3 cell line was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin (100 U/ml penicillin, 100 µg/ml streptomycin) in 75T tissue culture flasks, dispensed into new flasks every 2 to 3 days and all cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂, as described previously (20–22).

HSC-3 cells (5×10⁴ cells/well) were maintained in 12-well plates for 24 h at 37°C in a humidified atmosphere containing 5% CO₂ and subsequently incubated with 0, 60, 90, 120, 150 or 180 µg/ml Gyp for 12, 24, 48 or 72 h. Control cells were treated with DMSO alone. Following incubation, cells were harvested and stained with propidium iodide (PI, 5 µg/ml). Cell viability was assessed using CellQuest™ (version 5.2.1; BD Biosciences, San Jose, CA, USA) and flow cytometry (BD Biosciences) following a previously described protocol (20,21).

HSC-3 cells (5×10⁴ cells/well) in the 12-well plate were incubated with 0 or 120 µg/ml Gyp for 6, 12, 24, 48 and 72 h at 37°C in a humidified atmosphere containing 5% CO₂ and subsequently collected for cell cycle distribution assays. The percentage of cells in the sub-G1 (apoptosis), G0/G1, S- and G2/M phases were measured using ModFit LT software (version 3.0; BD Biosciences) and flow cytometry (BD Biosciences), as described previously (20,21,23).

HSC-3 cells (5×10⁴ cells/well) in a 12-well plate were treated with 0, 60, 90, 120, 150 or 180 µg/ml Gyp at 37°C for 24 h, stained with DAPI (37°C, 15 min) and assessed using fluorescence microscopy as described previously (20,21).

HSC-3 cells (5×10⁴ cells/well) were treated with 0, 60, 90, 120, 150 or 180 µg/ml Gyp at 37°C for 24 h and subsequently harvested to examine the DNA damage with the Comet assay kit (Trevigen, Inc., Gaithersburg, MD, USA) as described previously (20,21).

HSC-3 cells (5×10⁴ cells/well) were treated with 120 µg/ml Gyp at 37°C for 0.25, 0.5, 1, 3, 6, 12, 24 and 48 h. Cells were collected from each treatment, washed twice with PBS, re-suspended in 500 µl DiOC₆ (4 mol/l) and incubated at 37°C for 30 min. Levels of ΔΨ_m were assessed using CellQuest™ (version 5.2.1; BD Biosciences) by flow cytometry (BD Biosciences) as described previously (20,21).

HSC-3 cells (5×10⁴ cells/well) were maintained in a 12-well culture plate in DMEM medium for 24 h and subsequently incubated with 0 or 120 µg/ml Gyp for 24 h at 37°C. Following incubation, cells were collected from the control and Gyp treated-groups and total RNA was extracted using the Qiagen RNeasy Mini kit (P/N 74104; Qiagen Inc., Valencia, CA, USA) and quantity and purity were assessed at 260 and 280 nm using a spectrophotometer (Nanodrop 1000; Thermo Fisher Scientific, Inc.) (24). Total RNA was used to perform cDNA reverse transcription, synthesis, amplification, fragmentation and terminal labeling with the GeneChip WT Sense Target Labeling and Control reagents (Qiagen, Inc.). Labeling and microarray hybridization were performed on the chip (Affymetrix GeneChip Human Gene 1.0 ST array; Affymetrix, Inc., Santa Clara, CA, USA) as previously described (24). The resulting localized concentrations of fluorescent molecules on the chip were further detected and quantified using an Affymetrix GeneChip^® Scanner 3000 (Affymetrix; Thermo Fisher Scientific, Inc.). The data were further analyzed using Expression Console software version 1.1.2 (Affymetrix; Thermo Fisher Scientific, Inc.) with default RMA parameters (24). Upregulated and downregulated gene expression in HSC-3 cells following exposure to Gyp were examined and changes of ≥2-fold were recorded, with +signifying upregulation and -signifying downregulation.

The list containing the 2,992 unique Gyp, complete with Affymetrix transcript identifiers, was uploaded onto GeneGo MetaCore™ software (version 5.0; GeneGo, Inc., St. Joseph, MI, USA). GeneGo recognizes the Affymetrix identifiers and maps the Gyp to the MetaCore™ data analysis suite, generating maps to describe common pathways or molecular connections between Gyp on the list. Graphical representations of the molecular relationships between genes were generated using the GeneGo pathway analysis (24).

All results are expressed as the mean ± standard deviation. Statistical analysis was performed using an unpaired Student's t-test and SigmaPlot version 10.0 (Systat Software, Inc., San Jose, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Following incubation with various concentrations of Gyp (0, 60, 90, 120, 150 and 180 µg/ml) for 12, 24, 48 and 72 h, HSC-3 cells were collected for PI staining and to measure cell viability using flow cytometry. The results indicated that cell viability decreased in a time- and dose-dependent manner compared with control (untreated) cells (Fig. 1).

The results indicated that 48–72 h Gyp treatment (120 µg/ml) induced a decrease in the percentage of cells in the G0/G1 (enhanced G0/G1 peak) and S-phases and an increase in the percentage of cells in the G2/M phase (Fig. 2A). Cells treated with 120 µg/ml Gyp for 6–72 h contained a significantly higher percentage of apoptotic cells in the sub-G1 phase compared with the control group (P<0.01; Fig. 2B). Cells in the sub-G1 phase are apoptotic (25); therefore treatment with 120 µg/ml Gyp induced apoptosis in HSC-3 cells.

HSC-3 cells were treated with various concentrations of Gyp (0, 60, 90, 120, 150 and 180 µg/ml) for 24 h and stained with DAPI. The results demonstrated that Gyp markedly induced chromatin condensation (cell apoptosis) in HSC-3 cells in a dose dependent manner based on the images obtained via fluorescent microscopy (Fig. 3). Gyp induced nuclear condensation and the incorporation of labeled nucleotide into the DNA, indicating apoptosis, whereas control cells were negative for DAPI staining.

HSC-3 cells were treated with various concentrations of Gyp (0, 60, 90, 120, 150 or 180 µg/ml) for 24 h and DNA damage was assessing using the Comet assay. In cells with damaged DNA, a Comet assay will show longer comet tails (26). The results indicated that Gyp induced marked DNA damage in HSC-3 cells based on the production of the comet tail (Fig. 4). Higher concentrations of Gyp led to greater DNA damage, as indicated by the presence of longer comet tails (Fig. 4).

HSC-3 cells were treated with 120 µg/ml Gyp for 0, 0.25, 0.5, 1, 3, 6, 12, 24 and 48 h. The levels of ΔΨ_m production were analyzed and quantified using flow cytometry. Levels of ΔΨ_m were significantly decreased in HSC-3 cells treated with Gyp (P<0.05; Fig. 5) and this decrease occurred in a time-dependent manner.

HSC-3 cells were treated with or without 120 µg/ml Gyp and subsequently underwent cDNA microarray analysis of gene expression. A total of 953 genes upregulated ≥2-fold, 20 genes were upregulated ≥10-fold and 118 genes were upregulated between 4 and 10-fold (Table I). By contrast, 2,039 genes were downregulated ≥2-fold, 23 genes were downregulated >10-fold and 276 genes were downregulated between 4 and 10-fold (Table I). Genes in HSC-3 cells that were highly influenced by Gyp treatment in vitro are listed in Table II. Among those affected genes, 10 were upregulated >13-fold as follows: GTP binding protein overexpressed in skeletal muscle (GEM), serpin peptidase inhibitor clade E member 1 (SERPINE1), ras homolog family member B (RHOB), kelch repeat and BTB domain containing 8 (KBTBD8), interleukin (IL)11, activating transcription factor (ATF)3, cytochrome P450 family 1, subfamily A, member 1 (CYP1A1), ADP-ribosylation factor-like 14 (ARL14), transfer RNA selenocysteine 2 (TRNAU2), and syntaxin 11 (STX11). However, the following 10 genes were downregulated >14-fold: Six transmembrane epithelial antigen of prostate family member 4 (STEAP4), γ-aminobutyric acid A receptor ε (GABRE), serpin peptidase inhibitor clade B, member 13 (SERPINB13), transcriptional-regulating factor 1 (TRERF1), apolipoprotein L1 (APOL1), follistatin (FST), LOC100506718, microRNA (MIR)205, neuregulin (NRG)1 and G protein-coupled receptor (GPR)110.

GeneGo analysis is presented in Figs. 6–8. Experimental data were mapped on the processes. Upregulation was marked as red and downregulation was marked as blue circles of different intensities, which indicated the different levels of inhibition in HSC-3 cells following incubation with Gyp (120 µg/ml) in vitro. Fig. 6 represents the top scored network from Gyp (120 µg/ml) vs. control using the Analyze Networks (AN) algorithm, Fig. 7 represents second scored AN network from Gyp (120 µg/ml) vs. control and Fig. 8 represents the third scored AN network from Gyp (120 µg/ml) vs. control.