RPMI-1640 medium, fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, TRIzol reagent, and bicinchoninic acid (BCA) protein assay kit, RIPA cell lysis buffer (pierce, 89,900) were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). 5-FU, DMSO (cat. no. D5879), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; cat. no. M-2128) were obtained from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). Annexin V/propidium iodide (PI) apoptosis assay kit and DAPI were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). PrimeScript™ RT Reagent kit was purchased from Takara Bio, Inc. (Otsu, Japan). Goldview Nucleic Acid Gel stain (cat. no. G8142) was purchased from Beijing Solarbio Life Science and Technology, Ltd., (Beijing, China). Primary antibodies for β-actin (cat. no. 4967), B cell leukemia/lymphoma (Bcl-2; cat. no. 4223), Bcl-2 associated X (Bax; cat. no. 5023), cyclin D1 (cat. no. 2978), cyclin dependent kinase 4 (CDK4; cat. no. 2906), p21 (cat. no. 2947), PI3K (cat. no. 4257), p-AKT (Ser473; cat. no. 4060), AKT (cat. no. 2938) and phosphatase and tensin homolog (PTEN; cat. no. 9559), and horseradish peroxidase (HRP)-conjugated secondary antibodies (cat. no. 7074) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

HDW was purchased from a commercial supplier (Guo Yi Tang Chinese Herbal Medicine Store, Fujian, China) and the EEHDW was obtained as previously described (22). Stock solutions of EEHDW were prepared by dissolving 500 mg EEHDW powder in 1 ml DMSO to a final concentration of 500 mg/ml and stored at −20°C. Working solutions of EEHDW were made by diluting the stock solution in RPMI-1640 culture medium. The final concentration of DMSO in the medium was <0.5%.

Human colon carcinoma HCT-8/5-FU cell line was obtained from Nanjing KeyGen Biotech Co., Ltd. HCT-8/5-FU cells were cultured RPMI-1640 media supplemented with 10% FBS and 100 U/ml penicillin and 100 g/ml streptomycin in a humidified 37°C incubator supplemented with 5% CO₂.

Cell viability was determined using the MTT and colorimetric assay. Briefly, HCT-8/5-FU cells were seeded into 96-well plates at a density of 1.0×10⁴ cells/well. After 24 h, the cells were treated with different doses of EEHDW (0.5–2.0 mg/ml) for different periods of time (24 and 48 h). Treatment with 0.1% DMSO was included as the vehicle control. Following treatment, 100 µl MTT (0.5 mg/ml) were added to each well and cells were incubated for an additional 4 h at 37°C. Subsequently, the MTT formazan precipitate was dissolved in 100 µl DMSO and the absorbance was measured at 570 nm using an ELISA plate reader (Model EXL800, BioTek Instruments, Inc., Winooski, VT, USA).

HCT-8/5-FU cells from exponentially growing cultures were seeded into 12-well culture plates at a density of 1.0×10⁵ cells/well and were treated with different concentrations of EEHDW for 24 h. Cells were then harvested and seeded into 6-well plates at a final density of 1.0×10³ cells/well in 2 ml fresh RPMI-1640 medium. Following incubation for 8 days, colonies were fixed in MeOH-HAc (3:1, v/v) for 10 min in room temperature, stained with 0.1% crystal violet (Beyotime Biotech Co., Ltd. (Shanghai, China) for 10 min at room temperature and counted. Cell colony formation was calculated by showing survival of the control cells as 100%.

HCT-8/5-FU cells at a density of 2.0×10⁵ cells/well were seeded into 6-well plates and treated with different concentrations of EEHDW for 24 h. Subsequently, apoptosis of HCT-8/5-FU cells was determined by flow cytometry analysis using a fluorescence activated cell sorting (FACS) caliber (BD Biosciences, Franklin Lakes, NJ, USA) and Annexin V-fluorescein isothiocyanate/PI kit, according to the manufacturer's protocol. Annexin V-negative/PI-negative cells indicated viable cells, whereas Annexin V-positive/PI-negative and Annexin V-positive/PI-positive cells indicated cells undergoing early and late stage apoptosis, respectively.

HCT-8/5-FU cell morphology and apoptosis was monitored following staining with DAPI. HCT-8/5-FU cells were seeded on 12-mm diameter round, glass cover slips in 24-well plates and treated with EEHDW (0.5, 1.0, 2.0 mg/ml) for 24 h. The cover slips were then washed with PBS, fixed with 4% paraformaldehyde for 10 min and stained with DAPI (4 µg/ml) for 10 min at room temperature, then observed under fluorescence microscopy. Cells with clear condensed nuclei were identified as apoptotic cells.

HCT-8/5-FU cells were seeded into 6-well plates at a density of 4×10⁵ cells/well and treated with different concentrations of EEHDW for 24 h. RNA was extracted from HCT-8/5-FU cells using TRIzol reagent (Takara Bio, Inc.). cDNA was obtained using reverse transcription with PrimeScript RT reagent kit, according to the manufacturer's protocol. PCR was performed to determine the mRNA expression levels of Bax, Bcl-2, cyclin D1, CDK4 and p21. GAPDH was used as an internal control. The primers used for amplification of Bax, Bcl-2, cyclin D1, CDK4 and p21 transcripts are as follows: GAPDH forward (F) 5′-GTCATCCATGACAACTTTGG-3′ and reverse (R) 5′-GAGCTTGACAAAGTGGTCGT-3′; Bcl-2 F 5′-CAGCTGCACCTGACGCCCTT-3′ and R 5′-GCCTCCGTTATCCTGGATCC-3′; Bax F 5′-TGCTTCAGGGTTTCATCCAGG-3′ and R 5′-TGGCAAAGTAGAAAAGGGCGA-3′; CDK4 F 5′-CATGTAGACCAGGACCTAAGC-3′ and R 5′-AACTGGCGCATCAGATCCTAG-3′; cyclin Dl F 5′-TGGATGCTGGAGGTCTGCGAGGAA-3′ and R 5′-GGCTTCGATCTGCTCCTGGCAGGC−3′; p21 F 5′-GAGCGATGGAACTTCGACTTTGTC-3′ and R 5′-GGCGTTTGGAGTGGTAGAAATCTG−3′. The PCR was repeated 3 independent times. A BIO-RAD S1000 Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to perform the experiment under the following conditions: 95°C for 30 sec, annealing at the 60°C for 30 sec and extension at 72°C for 30 sec for 30 cycles. Samples were analyzed by 1.5% agarose gel electrophoresis. The DNA bands were examined using a gel documentation system (Gel Doc 2000; Bio-Rad Laboratories, Inc.). PCR results were calculated based on 3 independent experiments.

Western blot analysis was used to observe HCT-8/5-FU cell apoptosis induced by EEHDW treatment and expression of PI3K/AKT signaling pathways. HCT-8/5-FU cells were seeded into 25 cm² flasks at a density of 1.0×10⁶ cells/flask in 5 ml RPMI-1640 media and treated with different concentrations of EEHDW for 24 h. Cells were lysed using RIPA cell lysis buffer containing protease inhibitors, and the resulting protein concentration in each sample was determined by BCA assay. Equal quantities of protein (50 µg) were then separated on 10% SDS-PAGE gel and transferred onto PVDF membranes. The membranes were blocked for 2 h with 5% non-fat dry milk at room temperature, then incubated with β-actin, Bax, Bcl-2, Cyclin D1, CDK4, p21, PI3K, p-AKT, AKT and PTEN primary antibodies (1:1,000) overnight at 4°C. Following washing and subsequent incubation with HRP-conjugated secondary antibodies (1:2,000) for 2 h at room temperature, protein bands of interest were detected using enhanced chemiluminescence by SuperSignal West Pico Chemiluminescent Substrate. Image Lab™ software version 3.0 (Bio-Rad Laboratories, Inc.) was used for densitometric analysis and quantification of western blots.

Data were expressed as the mean ± standard deviation and analyzed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA) by using Student's t-test or one-way AVOVA analysis. LSD and Dunnet's were used as post-hoc tests. P<0.05 was considered to indicate a statistically significant difference.

HCT-8/5-FU cell viability was measured using MTT assay following exposure to different concentrations of EEHDW for 24 h and 48 h. As presented in Fig. 1, treatment with 0.5–2.0 mg/ml of EEHDW for 24 and 48 h significantly reduced cell viability of HCT-8/5-FU cells by 22.22–47.58% and 26.67–78.27%, respectively compared with untreated control cells (P<0.01; Fig. 1). This suggests that EEHDW inhibited HCT-8/5-FU cell viability in a concentration- and time-dependent manner.

In order to fully evaluate the effect of EEHDW on HCT-8/5-FU cells, the number of colonies formed by HCT-8/5-FU cells following EEHDW treatment was examined using a colony formation assay. As presented in Fig. 2, treatment with 0.5, 1.0 and 2.0 mg/ml EEHDW for 24 h significantly reduced the cell survival rate of HCT-8/5-FU cells when compared with untreated control cells in a dose-dependent manner (P<0.05).

Due to the innate resistance of the cancer cells to apoptosis, clinical treatment for various cancers such as CRC is frequently insufficient. The present study examined the possibility that EEHDW may overcome drug-resistance and induce apoptosis of HCT-8/5-FU cells. Annexin-V/PI staining and FACS analysis demonstrated that EEHDW treatment significantly increased the percentage of HCT-8/5-FU cells undergoing early-stage (lower right quadrant) and late-stage apoptosis (upper right quadrant) in a dose-dependent manner when compared with the untreated control cells (Fig. 3A and B; P<0.05). The cellular and nuclear morphology of HCT-8/5-FU cells was examined using DAPI staining. As presented in Fig. 3C, EEHDW treated cells showed more intense staining and apparent DNA condensation when compared with untreated control cells, suggesting that EEHDW treatment promoted HCT-8/5-FU cell apoptosis.

The proliferation of the majority of animal cells is primarily regulated in the G1/S transition, one of the two main checkpoints in the cell cycle which is mediated by the pro-proliferative cyclin D1 and CDK4. Apoptosis is tightly regulated by Bcl-2 family proteins, including anti-apoptotic members such as Bcl-2 and pro-apoptotic members such as Bax (28–30). In order to investigate the underlying mechanisms of EEHDW and how it overcomes the drug resistance of cancer cells, the present study examined the mRNA and protein expression levels of cyclin D1, CDK4, p21, Bax and Bcl-2 following EEHDW treatment in HCT-8/5-FU cells. As presented in Fig. 4, EEHDW treatment significantly reduced cyclin D1, CDK4 and Bcl-2 mRNA and protein expression; however, p21 and Bax mRNA and protein expression levels in HCT-8/5-FU cells were increased when compared with untreated control cells (P<0.05). This suggests that EEHDW likely modulates the drug resistance of CRC HCT-8/5-FU cells by regulating the expression of cyclin D1, CDK4, p21, Bcl-2 and Bax.

The PI3K/AKT signaling pathway has a crucial role in numerous cellular systems and its activation is closely associated with the prevention of cellular apoptosis. AKT is activated by phospholipid binding and activation loop phosphorylation at Thr308 by pyruvate dehydrogenase kinase 1 and by phosphorylation within the carboxy terminus at Ser473. In order to determine the mechanism behind EEHDW's ability to overcome drug resistance of CRC, the present study examined the expression of key proteins involved in the PI3K/AKT signaling pathway, including PI3K, p-AKT, AKT and PTEN following EEHDW treatment. As presented in Fig. 5, EEHDW treatment significantly reduced PI3K and p-AKT expression and the ratio of p-AKT to total AKT; however, led to increased PTEN expression in HCT-8/5-FU cells, in a dose-dependent manner when compared with untreated control cells (P<0.05). This suggests that EEHDW may also modulate the drug-resistance of CRC HCT-8/5-FU cells via regulation of the PI3K/AKT signaling pathway.