Antibodies against caspase-3 (cat. no. 9665P), caspase-9 (cat. no. 9058P), poly ADP-ribose polymerase (PARP; cat. no. 9542) and p27 (cat. no. 3698) were supplied by Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibody against p21 (cat. no. Ab109199) was provided by Abcam (Cambridge, UK). Antibodies against Bcl-2 (cat. no. NB100-56098) and Bax (cat. no. NB100-56095) were purchased from Novus Biologicals, LLC (Littleton, CO, USA). Antibodies against p53 (cat. no. sc-6243), mouse double minute 2 homology (Mdm2; cat. no. sc-965) and GAPDH (cat. no. sc-32233) were supplied by Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The antibodies for goat anti-mouse IgG (cat. no. NCI1430KR) and goat anti-rabbit IgG (cat. no. NCI1460KR) were provided by Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Primary antibodies were diluted to 1:1,000 and secondary antibodies were diluted to 1:5,000. All chemicals, including MTT, tetramethylrhodamine methyl ester perchlorate (TMRM), and N-acetyl cysteine (NAC) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), unless otherwise indicated.

Stem and cortex of S. commixta, which was grown and collected in North Gyeongsang Province, South Korea, in 2012, were purchased from Omniherb Co., Ltd. (Daegu, South Korea). The plant was authenticated by a botanical expert working in the company. A voucher specimen is retained at the School of Korean Medicine, Pusan National University (Yangsan, South Korea). The dried plant material (50 g) was cut and extracted in a liter of distilled water at 100°C for 4 h. The extract was passed through filter paper (pore size, 6 µm; Whatman PLC; GE Healthcare Life Sciences, Little Chalfont, UK), condensed using a Eyela N-1110 rotary evaporator (Tokyo Rikakikai Co., Ltd., Tokyo, Japan) and lyophilized with a freeze dryer (Labconco Corporation, Kansas City, MO, USA). The resulting SCE powder (final yield 3.1 g) was dissolved in dimethyl sulfoxide (DMSO) to create a stock solution (100 mg/ml), and diluted with culture medium prior to use in the experiments.

Hepatocellular carcinoma Hep3B and HepG2 cells, and colon cancer HCT116 cells were provided by the American Type Culture Collection (Manassas, VA, USA). The cells were cultured with Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with L-glutamine (200 mg/l), 10% (v/v), antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin; Thermo Fisher Scientific, Inc.) and heat-inactivated fetal bovine serum (Sigma-Aldrich; Merck KGaA). The cells were incubated in a humidified cell incubator at 37°C in an atmosphere of 5% CO₂ prior to experiments.

The viabilities of the cells incubated with various concentrations of SCE were evaluated by MTT assay. Hep3B, HepG2 and HCT116 cells were incubated in 24-well plates with 0, 100, 200, 300, 400 or 500 mg/ml of SCE for 48 h at 37°C. MTT solution (2.0 mg/ml) was added to each well and the cells were incubated for 4 h at 37°C with 5% CO₂. The culture media were removed, and cells were lysed in DMSO. The amount of formazan crystals, formed by viable cells, was estimated by measuring the absorbance at 540 nm with a Spectramax M2 microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

HCT116 cells (2×10⁶ cells) were centrifuged at 2,500 × g and 4°C for 10 min, and washed twice in PBS buffer. The cell pellets were gently resuspended in 100 µl PBS, and fixed for 30 min at 4°C via serial addition of 200 µl PBS containing 10% ethanol/5% glycerol, followed by 200 µl PBS containing 50% ethanol/5% glycerol. The samples were incubated on ice for 5 min, and 100% ethanol/5% glycerol solution was added to the cell suspension to make a final volume of 1 ml PBS containing 70% ethanol/5% glycerol. The samples were stored at 4°C overnight, washed with PBS, centrifuged and resuspended with 12.5 µg of RNase (Sigma-Aldrich; Merck KGaA) in 250 µl of 1.12% sodium citrate buffer (pH 8.45). The samples were maintained at 37°C for 30 min prior to staining the cellular DNA with 250 µl of propidium iodide (50 µg/ml) at room temperature for 30 min. The relative DNA contents of the stained cells were analyzed on a flow cytometer (BD FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA) with excitation/emission wavelengths of 495/605 nm.

For detecting morphological changes associated with apoptosis, HCT116 cells (1×10⁵ cells) were cultured in the 0, 50, or 100 mg/ml of SCE for 48 h at 37°C. The cells were washed with PBS and fixed with 3.7% paraformaldehyde/PBS solution for 10 min at room temperature. The samples were washed with PBS and stained with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA) for 10 min at room temperature. The stained cells were washed twice with PBS and analyzed by confocal microscopy (magnification, ×200; ZEISS LSM700 laser; Carl Zeiss AG, Oberkochen, Germany) with excitation/emission wavelengths of 360/460 nm. Four randomly chosen fields of view were captured from each slide.

HCT116 cells were treated with the 0, 50, or 100 mg/ml of SCE and total proteins were then extracted from these cells using radioimmunoprecipitation assay buffer (Cell Signaling Technology, Inc.) containing a protease inhibitor cocktail (cat. no. 5871, Cell Signaling Technology, Inc.). The samples were centrifuged at 450 × g for 1 min and the protein concentrations were estimated by Bio-Rad Protein Assay Kit II (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts (20 µg) of protein from each sample were fractionated using 10% SDS-PAGE. The proteins were transferred to nitrocellulose membrane filters (Hybond ECL; GE Healthcare Life Sciences) by electrophoresis. The filters were blocked with 5% non-fat dry milk or 5% bovine serum albumin at room temperature for 1 h. The membranes were then incubated with specific primary antibodies against target proteins at 4°C overnight. The filters were rinsed twice with TBS containing 0.1% Tween 20, and incubated with appropriate secondary horseradish peroxidase-conjugated antibodies at room temperature for 1 h. The bands for proteins of interest were detected using ECL Plus Western Blotting Detection Reagent (GE Healthcare Life Sciences).

Total RNA was extracted from HCT116 cells treated with 0, 50, or 100 mg/ml of SCE using the GeneJET RNA Purification kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The concentration of total RNA was validated using a NanoDrop spectrophotometer (Thermo Fischer Scientific, Inc.). Equal amounts of total RNA (2 µg) were used for synthesis of complementary DNA at 70°C for 5 min, 42°C for 60 min, and 94°C for 5 min using oligo-dT primers and AccuPower RT-PreMix (Bioneer Corporation, Daejeon, Korea). Subsequently, 2 µg of cDNA was amplified by PCR with the following primers: P53, 5′-GAAACTACTTCCTGAAAACAACGT-3′ (sense) and 5′-GCCTCACAACCTCCGTCAT-3′ (antisense); GAPDH (control), 5′-GGAGCCAAAAGGGTCATCAT-3′ (sense) and 5′-GTGATGGCATGGACTGTGGT-3′ (antisense). The amplification reactions were performed with the AccuPower PCR-PreMix (Bioneer Corporation), under the following conditions: An initial denaturation at 95°C for 5 min; followed by 30 cycles of denaturation for 30 sec at 95°C; annealing for 30 sec at 60°C; and extension for 30 sec at 72°C with a final extension for 10 min at 72°C. Amplified PCR products were electrophoresed in 1.0% agarose gels, stained with ethidium bromide and detected with GelDoc-It TS Imaging System (UVP, Inc., Upland, CA, USA). The intensities of the bands were quantified using ImageJ software (Version 1.48; National Institutes of Health, Bethesda, MD, USA).

For measuring MMP, HCT116 cells were incubated with 250 nM TMRM for 30 min at 37°C. The cells were mounted with cover slides in a chamber filled with complete culture medium, and incubated at 37°C for 30 min in cell culture incubator. The fluorescent images of samples were observed and were captured using a ZEISS LSM700 confocal laser scanning microscope (magnification, ×200; Carl Zeiss AG) with excitation/emission at 573/600 nm. Four randomly chosen fields of view were captured from each sample. The samples were also analyzed by flow cytometry using a BD FACSCanto II flow cytometry system at excitation/emission wavelengths of 488/560 nm.

Production of intracellular ROS by each cell was detected with 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Briefly, the cells were incubated with 100 µM carboxy-H2DCFDA in culture medium and incubated at 37°C for 30 min. The samples were washed with PBS and their fluorescence was measured using a BD FACSCanto II flow cytometry system at excitation/emission wavelengths of 488/525 nm.

Data from the cell viability, TMRM and ROS production assays were expressed as percentages relative to the control cells (set at 100%), and are presented as the mean ± standard error of mean (SEM). Data from the cell cycle analysis are shown as a percentage of the total cells analyzed and presented as the mean ± SEM. The half-maximal inhibitory concentration (IC₅₀) values were calculated according to the ‘log(inhibitor) vs. response’ equation using GraphPad Prism software (version 5.01; GraphPad Software, Inc., La Jolla, CA, USA). The statistical significance of the differences between the mean values of each group were evaluated by one-way analysis of variance with a Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. All experiments were independently performed at least three times.

Initially, the cytotoxic effect of SCE was examined in three different cancer cell lines (Hep3B, HepG2 and HCT116). The results showed that SCE treatment produced dose-dependent cytotoxicity in all three cell lines (Fig. 1). The IC₅₀ values for SCE in the Hep3B, HepG2 and HCT116 cells were 300.1 (P<0.0001), 237.8 (P<0.0001) and 77.7 µg/ml (P<0.0001), respectively. As the cytotoxic effect of SCE was most potent in the colon cancer HCT116 cells, the HCT116 cell line was selected for subsequent experiments. To elucidate whether the cytotoxic effect of SCE was due to apoptotic cell death, the cell cycle of SCE-treated HCT116 cells was investigated. The data demonstrated that the population of sub-G1 phase cells was increased by SCE treatment in a dose-dependent manner (P<0.0001; Fig. 2A and B). In addition, nucleic DNA was degraded following SCE treatment, as measured using DAPI staining (Fig. 2C). These results suggest that SCE-induced cell death involves apoptosis.

To examine the mechanism underlying SCE-induced apoptosis, the levels of certain proteins involved in the apoptosis cascade were assessed. The results demonstrated that SCE reduced the levels of the proforms of caspase-9, caspase-3 and PARP, whereas the cleaved forms of caspase-3 and PARP were increased by SCE treatment (Fig. 2D) Cleaved and pro-PARP were detected using the same antibody and analyzed according to their molecular weights. To evaluate the effect of SCE on the mitochondrial membrane stability, a TMRM MMP assay was performed. The results from the fluorescent microscopic observation and fluorescence-activated cell sorting analysis demonstrated that MMP was reduced by SCE treatment in a dose-dependent manner (Fig. 3; P=0.0028 for panel A and P<0.0001 for panel B).

To examine the effect of SCE on the stability of mitochondria, the balance of Bax and Bcl-2 was examined. The data showed that SCE increased the amount of proapoptotic Bax and reduced that of antiapoptotic Bcl-2 (Fig. 4A). In addition, the protein expression levels of p21 and p27 were dose-dependently increased by SCE treatment (Fig. 4B). RT-PCR and western blotting were then used to confirm whether the expression of p53 was altered by SCE treatment. The results showed that SCE increased the expression of p53 at the mRNA and protein levels (Fig. 4C). Furthermore, Mdm2 protein, an important negative regulator of p53, was cleaved by SCE treatment (Fig. 4D). From these results, it was hypothesized that SCE increases the expression of the tumor suppressor p53 through transcriptional and posttranslational regulation.

It was examined whether the production of ROS was increased by SCE treatment. The data showed that SCE dose-dependently increased ROS production in HCT116 cells. Furthermore, the addition of a ROS scavenger, NAC (5 mM), significantly reduced the SCE-stimulated intracellular ROS levels (Fig. 5A; P=0.0006). To elucidate whether ROS production was involved in the p53-mediated apoptosis of HCT116 cells, cell viability was assessed. The results revealed that the SCE-induced death of HCT116 cells was attenuated by NAC (P=0.0001; Fig. 5B). In addition, the SCE-stimulated expression of proapoptotic proteins, including p53, p21 and Bax, was reduced by NAC treatment. By contrast, the expression of anti-apoptotic protein, Bcl-2, was recovered by NAC (Fig. 5C). From these results, it was suggested that SCE induces apoptotic cell death via ROS-mediated mitochondrial damage (Fig. 6).