Dulbecco's modified Eagle's medium (DMEM), RPMI-1640 medium, fetal bovine serum (FBS), penicillin-streptomycin, 0.25% trypsin-EDTA, DreamTaq Green PCR Master Mix and Pierce Bicinchoninic Acid (BCA) Protein Assay kit were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). MTT was purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) Cell Proliferation and Tracking kit and Annexin V-Fluorescein Isothiocyante (FITC) Apoptosis Detection kit were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). RNAiso Plus and PrimeScript RT Reagent kit with gDNA Eraser (Perfect Real Time) was purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Radio immunoprecipitation assay (RIPA) lysis buffer and blocking buffer were purchased from Beyotime Institute of Biotechnology (Haimen, China). Rabbit polyclonal antibodies against β-actin (20536–1-AP), Survivin (0508-1-AP), protein kinase B (AKT; 10176-2-AP) and extracellular-signal-regulated kinase (ERK; 16443-1-AP) were purchased from Proteintech Wuhan Sanying Biotechnology (Wuhan, China). Rabbit polyclonal antibodies against Bcl-2-associated X-protein (Bax; D220073), B-cell lymphoma 2 (Bcl-2; D160117), cyclin-dependent kinase 4 (CDK4; D220396) and proliferating cell nuclear antigen (PCNA; D120014) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Rabbit polyclonal antibodies against Cyclin D1 (sc-753), phospho-AKT (p-AKT; sc-135650) and phospho-ERK (p-ERK; sc-16982-R) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (E030120-01) was purchased from Earthox LLC (Millbrae, CA, USA). Culture flasks and plates were purchased from NEST Biotechnology Co., Ltd. (Wuxi, Jiangsu, China). All other chemicals used, unless otherwise stated, were purchased from Sigma Aldrich; Merck KGaA (Darmstadt, Germany).

HDW was purchased from the Guo Yi Tang Chinese Herbal Medicine Store (Fujian, China). Using a procedure described previously (18), 500 g HDW was extracted. A series of solvents were applied including chloroform, petroleum ether, n-butanol and ethyl acetate resulting in the chloroform extract of HDW (CEHDW), the petroleum ether extract of HDW (PEEHDW), the n-butanol extract of HDW (NBEHDW) and the ethyl acetate extract of HDW (EAEHDW), respectively. All extracts were separately evaporated on a rotary evaporator. Subsequently, powders of the extracts were dissolved in 100% dimethyl sulfoxide (DMSO) to a stock concentration of 100 mg/ml and stored at −20°C. The final concentration of DMSO in the medium for all experiments was <0.5%.

The human colorectal cancer cell lines SW620, HT-29, HCT116 and HCT-8 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). SW620 and HT-29 cells were cultured in DMEM supplemented with 10% (v/v) FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. HCT116 and HCT-8 cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. All of the cell lines were cultured at 37°C in a humidified incubator containing 5% CO₂.

An MTT assay was used to assess the cell viability. SW620, HT-29, HCT116 and HCT-8 cells were incubated in 96-well plates at a density of 1×10⁵ cells/ml in 100 µl culture medium for 12 h and treated with various concentrations (0, 150, 300 and 500 µg/ml) of HDW extract for 24 h at 37°C. In addition, SW620 cells were treated with various concentrations (0, 12.5, 25, 50, 75 and 100 µg/ml) of CEHDW for 24 h at 37°C in an additional MTT assay. Subsequently, the medium was replaced with 100 µl MTT (0.5 mg/ml in PBS) and cells were incubated at 37°C. After 4 h, the MTT solution was removed and 100 µl DMSO was added to solubilize the purple-blue formazan precipitate. The resulting absorbance was measured at 570 nm using an ELISA reader (model ELX800; BioTek Instruments, Inc., Winooski, VT, USA).

SW620 cells were seeded into 6-well plates at a density of 3×10⁵ cells/ml in 2 ml culture medium. Following treatment with various concentrations (0, 50, 75 and 100 µg/ml) of CEHDW for 24 h at 37°C, cells were harvested and diluted with fresh medium without CEHDW, and subsequently reseeded in 6-well plates at a density of 1,000 cells/well in 2 ml. The medium was replaced with fresh medium every 4 days. After 10 days, colonies were fixed with 10% formaldehyde for 15 min at room temperature, stained with 0.01% crystal violet for 10 min at room temperature and photographed with digital camera.

The CFDA-SE probe was used to determine the cell proliferation (19). Briefly, SW620 cells were stained with 5 µM CFDA-SE for 30 min at 37°C in the dark then seeded in 12-well plates at a density of 2.5×10⁵ cells/ml in 1 ml medium according to the manufacturer's protocol. Subsequently, cells were exposed to a series of concentrations of CEHDW for 24 h. CFDA-SE fluorescence was detected using a FACSCaliber instrument (BD Biosciences, San Jose, CA, USA).

SW620 cells were treated with various concentrations (0, 50, 75 and 100 µg/ml) of CEHDW for 24 h at 37°C. Cell apoptosis was determined using flow cytometry. Annexin V/PI staining was performed prior to analysis using a FACSCaliber instrument, according to the manufacturer's protocol. In this assay, the annexin V/PI double-negative population indicates viable cells, and the annexin V-positive/PI-negative or annexin V/PI double-positive population represents cells undergoing early or late apoptosis, respectively.

SW620 cells were treated with various concentrations (0, 50, 75 and 100 µg/ml) of CEHDW for 24 h at 37°C. RNA from cell samples was isolated with RNAiso Plus. Oligo-dT-primed RNA (1 µg) was reverse-transcribed using PrimeScript RT Reagent kit with gDNA Eraser (Perfect Real Time), according to the manufacturer's protocol. The resultant cDNA was used to determine the amount of Survivin, PCNA, Cyclin D1, CDK4, Bcl-2 and Bax mRNA using PCR with DreamTaq Green PCR Master Mix. PCR was performed using the 3-step method, with a denaturation stage at 95°C for 30 sec, an annealing stage at an appropriate temperature (55°C for Survivin, CDK4, Bcl-2 and Bax, and 58°C for PCNA, Cyclin D1 and GAPDH) for 30 sec and an extension stage at 72°C for 30 sec for 30 cycles. GAPDH was used as an internal control. The primers were synthesized by Invitrogen; Thermo Fisher Scientific, Inc., and the sequences are listed in Table I.

SW620 cells were treated with various concentrations (0, 50, 75 and 100 µg/ml) of CEHDW for 24 h at 37°C. Cells were washed with PBS three times and lysed with RIPA lysis buffer containing EASYpack protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and PhosSTOP (Roche Diagnostics). The concentrations of resultant protein were quantified using the BCA Protein Assay kit. Proteins (50 µg) were separated by SDS-PAGE and transferred onto nitrocellulose membranes (EMD Millipore Corporation, Darmstadt, Germany). The membranes were blocked with blocking buffer for 2 h at room temperature and incubated with antibodies against Survivin, PCNA, Cyclin D1, CDK4, Bcl-2, Bax, AKT, ERK, p-AKT, p-ERK or β-actin (all 1:1,000 dilution) for 16 h at 4°C, and then washed three times with Tris-buffered saline with Tween-20 (TBST), respectively. Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibodies for 1 h at room temperature. Cells were washed again in TBST and membranes were visualized using a BeyoECL Plus instrument. Image Lab™ software (version 3.0; Beyotime Institute of Biotechnology) was used for densitometric analysis and quantification of western blots.

Data were analyzed using the SPSS package for Windows (version 17.0; SPSS Inc., Chicago, IL, USA) using one-way analysis of variance. Fisher's least significant difference and Dunnett's test were used as post-hoc tests. P<0.05 was considered to indicate a statistically significant difference.

The inhibitory effects of CEHDW, PEEHDW, NBEHDW and EAEHDW on the viability of the CRC cell lines were determined using an MTT assay. As presented in Fig. 1A-D, the four cell lines were exposed to various concentrations of the four extracts for 24 or 48 h. CEHDW exhibited the most antitumor activity in all cell lines. NBEHDW exhibited no significant inhibitory effects. PEEHDW decreased the cell viability of SW620 and HT-29 cells, but exhibited a limited effect on HCT116 and HCT-8 cells. Similarly, EAEHDW significantly inhibited the viability of SW620, HT-29 and HCT116 cells, but not HCT-8 cells.

Following CEHDW treatment, SW620 demonstrated the most drug sensitivity as presented in Fig. 1A. Therefore, the SW620 cell line was selected for further study. In order to evaluate the effect of CEHDW on SW620 cells, viability was determined using an MTT assay. Treatment with between 12.5 and 100 µg/ml CEHDW for 24 h decreased cell viability by between 7.3 and 60.23%, when compared with untreated cells (Fig. 2A). To observe the effects of CEHDW on cell morphology, the appearance of the treated and untreated SW620 monolayers were compared using phase-contrast microscopy. Untreated SW620 cells appeared as a crowded and disorganized monolayer after 24 h (Fig. 2B). The cell density was decreased in the confluent monolayers that had been treated with CEHDW, with the attached cells exhibiting a round appearance (Fig. 2B). To estimate the effect of CEHDW on cell survival, SW620 cells were examined by performing the colony formation assay. The number of colonies was decreased following CEHDW treatment, and almost no colonies were observed following treatment with CEHDW at concentrations of 50, 75 and 100 µg/ml for 24 h (Fig. 2C). To determine the effect of CEHDW on cell proliferation, a CFDA-SE assay was performed. As presented in Fig. 2D and E, the ratio of fluorescence intensity of cells significantly increased following treatment with CEHDW (50, 75 and 100 µg/ml) for 24 h was 1.73, 2.27 and 2.77, respectively, compared with that of the control group. These results suggest that CEHDW is potent in suppressing the proliferation of SW620 cells.

In order to investigate the underlying molecular mechanism of the proliferation suppressing activity of CEHDW, its effect on apoptosis in SW620 cells was assessed using annexin V/PI staining followed by FACS analysis. As presented in Fig. 3A and B, the proportion of cells undergoing either early apoptosis or late apoptosis following treatment with 0, 50, 75 and 100 µg/ml CEHDW was 6.58, 16.86, 28.75 and 79.47%, respectively, suggesting that CEHDW treatment induces apoptosis in SW620 cells in a dose-dependent manner.

To further explore the underlying molecular mechanism of the proliferation inhibition effect of CEHDW, RT-PCR and western blot analysis were performed to determine the expression of Survivin, PCNA, Cyclin D1, CDK4, Bcl-2 and Bax at the mRNA and protein levels. RT-PCR results revealed that CEHDW treatment decreased mRNA expression of the pro-proliferative PCNA, Cyclin D1 and CDK4 and anti-apoptotic Bcl-2 and Survivin, while also increasing expression of the pro-apoptotic Bax (Fig. 4A and B). The protein expression patterns of Survivin, PCNA, Cyclin D1, CDK4, Bcl-2 and Bax were similar to that observed for the respective mRNA (Fig. 4C and D).

To gain further insight into the association between CEHDW and the proliferation and apoptosis of CRC cells, AKT and ERK signaling molecules were investigated. As presented in Fig. 5A and B, AKT and ERK phosphorylation were decreased following treatment with 50, 75 and 100 µg/ml CEHDW for 24 h, indicating that the AKT and ERK signaling pathways may be involved in CRC cell apoptosis.