RPMI 1640 medium, fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA and TRIzol reagent were obtained from Invitrogen Life Technologies (Grand Island, NY, USA). Primary antibodies against P-gp (#13342) and ABCG2 (#4477), and a horseradish peroxidase (HRP)-conjugated secondary antibody (#4967) were provided by Cell Signaling Technology, Inc. (Beverly, MA, USA). The BCA Protein Assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA) and the PrimeScript RT reagent kit was provided by Takara Biotechnology Co., Ltd. (Dalian, China). All the other chemicals, unless otherwise stated, were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Authentic plant material was purchased from a commercial supplier (Guo Yi Tang Chinese Herbal Medicines, Fuzhou, China), and the EEHDW was prepared as previously described (21). Stock solutions of EEHDW were prepared by dissolving the EEHDW powder in 40% dimethyl sulfoxide (DMSO) to a reach a final concentration of 400 mg/ml. The stock solutions were stored at −20°C. Working concentrations of EEHDW were produced by diluting the stock solution in the culture medium. The final concentration of DMSO in the medium was <0.5%.

Human colon carcinoma HCT-8 cells and 5-FU-resistant HCT-8/5-FU cells were purchased from Nanjing KeyGen Biotech. Co. Ltd. (Nanjing, China). Cells were grown in RPMI 1640 medium, supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin, at 37°C in a humidified chamber with 5% CO₂. The HCT-8/5-FU cells were cultured in the supplemented media with the addition of 15 µg/ml 5-FU.

Cell viability was determined using an MTT colorimetric assay. Briefly, HCT-8 and HCT-8/5-FU cells were seeded into 96-well plates at a density of 1.0×10⁴ cells/well in 0.1 ml media. After 24 h, the cells were treated with various concentrations of 5-FU or EEHDW for indicated periods of time. Treatment with 0.1% DMSO was included as the vehicle control. At the end of the treatment, 100 µl MTT (0.5 mg/ml) in phosphate-buffered saline (PBS) was added to each well, and the samples were incubated for an additional 4 h at 37°C. The purple-blue MTT formazan precipitate was dissolved in 100 µl DMSO and absorbance was measured at 570 nm using an ELISA reader (EXL800; BioTek Instruments, Inc., Winooski, VT, USA). The resistance index (RI) of the HCT-8/5-FU cells to 5-FU was calculated by dividing the drug concentration required to inhibit growth by 50% (IC₅₀) for HCT-8/5-FU cells by the IC₅₀ value for the parental cells (HCT-8). IC₅₀ values were determined using nonlinear regression analysis.

HCT-8/5-FU cells were seeded into six-well plates at a density of 2.5×10⁵ cells/well in 2 ml media. The cells were treated with various concentrations of EEHDW for 24 h. Cell morphology was observed with a phase-contrast microscope (Leica Camera AG, Wetzlar, Germany), and images were photographed at a magnification of ×200.

Retention of a P-gp transporter substrate and the subsequent reversal of the drug resistance phenotype were investigated using a Rh-123 exclusion assay. HCT-8/5-FU cells were treated with different concentrations of EEHDW for 24 h. The cells were collected, and a total of 10⁶ cells/ml were incubated with 5 µl Rh-123 (1 mM in PBS) at 37°C for 10 min. The cells were washed twice with chilled PBS and resuspended in 0.5 ml PBS, followed by incubation for an additional 30 min at 37°C. Fluorescence intensity was measured at 488 nm to determine the intracellular content of Rh-123 and quantitated using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

HCT-8/5-FU cells (4×10⁵) were seeded into six-well plates in 2 ml culture media and treated with the indicated concentrations of EEHDW for 24 h. Total RNA from the HCT-8/5-FU cells was isolated using TRIzol reagent (Invitrogen). Oligo(dT)-primed RNA (1 µg) was reverse-transcribed using the PrimeScript RT reagent kit, according to the manufacturer's instructions. The obtained cDNA was used to determine the mRNA expression levels of P-gp and ABCG2 by RT-PCR. GAPDH was used as an internal control. The RT-PCR conditions for 30 cycles were performed as follows: Denaturation at 94°C for 40 sec, annealing for 40 sec and extension at 72°C for 45 sec. The primers used for the amplification of ABCG2, P-gp and GAPDH transcripts were as follows: ABCG2 forward, 5′-GCCGTGGAACTCTTTGTGGTAG-3′ and reverse, 5′-ACAGCAAGATGCAATGGTTGT-3′; P-gp forward, 5′-TGACATTTATTCAAAGTTAAAAGCA-3′ and reverse, 5′-TAGACACTTTATGCAAACATTTCAA-3′; and GAPDH forward, 5′-GTCATCCATGACAACTTTGG-3′ and reverse, 5′-GAGCTTGACAAAGTGGTCGT-3′.

HCT-8/5-FU cells were seeded into 25 cm² flasks at a density of 1.0×10⁶ cells/flask in 5 ml media. The HCT-8/5-FU cells were treated with the various concentrations of EEHDW for 24 h, and lysed with mammalian cell lysis buffer containing different protease inhibitors. The total protein concentration in each sample was determined using a bicinchoninic acid assay. Equal amounts of protein from each cell lysate were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 2 h with 5% nonfat dry milk and the membrane was incubated with primary monoclonal rabbit anti-human antibodies against P-gp (1:1,000), ABCG2 (1:1,000) and β-actin (1:1,000) at 4°C overnight. Then, the membranes were incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:5,000) at room temperature. Blots were visualized using an electrochemiluminescence western blotting kit and images were captured and analyzed using ChemiDoc XRS+ imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Data are presented as the mean ± standard deviation for the indicated number of independently performed experiments. The data were analyzed using the SPSS package for Windows (version 17.0; SPSS, Inc., Chicago, IL, USA). Statistical analyses were performed using the Student's t-test and analysis of variance, where P<0.05 was considered to indicate a statistically significant difference.

To verify the 5-FU resistance profiles of the CRC cell lines used in the study, HCT-8 and HCT-8/5-FU cells were exposed to different concentrations of 5-FU for 48 h, and the cell viability was measured using an MTT assay. The viability of the HCT-8 cells was significantly decreased following treatment with 5-FU, whereas the viability of the HCT-8/5-FU cells did not significantly change compared with the parental cells (Fig. 1A and B). The IC₅₀ values of 5-FU in the HCT-8 and HCT-8/5-FU cell lines were 145.16 µM and 4.2668 mM, respectively. In addition, the RI for 5-FU was 29.39. These results support previously obtained data, demonstrating the resistance properties of HCT-8/5-FU cells to 5-FU treatment (27).

In order to evaluate the effect of EEHDW on the HCT-8/5-FU cell line, the viability of the treated cells was determined by an MTT assay. Treatment with 0.5–2 mg/ml EEHDW for 24 or 48 h reduced the cell viability by 13.78–66.61 or 18.02–83.76%, respectively, when compared with the untreated control cells (P<0.05; Fig. 2). Furthermore, the cell viability was reduced to 33.38 and 16.23% at the highest concentration of EEHDW (2 mg/ml) after 24 or 48 h of treatment, respectively. These results indicated that EEHDW inhibited HCT-8/5-FU cell viability in a concentration- and time-dependent manner.

To determine the effects of EEHDW on cell morphology, the appearance of the treated versus untreated HCT-8/5-FU monolayers were compared by phase-contrast microscopy. Untreated HCT-8/5-FU cells appeared as a crowded and disorganized monolayer after 24 h (Fig. 3). However, the cell density was reduced in the confluent monolayers that had been treated with EEHDW, with the attached cells exhibiting a round appearance and condensed nuclei. Therefore, these observations demonstrated that EEHDW inhibited the growth of HCT-8/5-FU cells.

Rh-123 is a substrate for the ABC transporter, P-gp, and its accumulation and efflux are predictive of the levels of P-gp in the cells exhibiting the MDR-associated phenotype (28,29). To determine whether EEHDW affects the activity of P-gp transporters, the intracellular accumulation of Rh-123 was measured in EEHDW-treated HCT-8/5-FU cells. Compared with the untreated controls, there was a significant increase in the accumulation of intracellular Rh-123 in the HCT-8/5-FU cells treated with EEHDW for 24 h (Fig. 4). These results indicated that EEHDW may inhibit the efflux activity of P-gp.

To further investigate which drug resistance mechanisms are targeted by EEHDW, the mRNA and protein expression levels of P-gp and ABCG2 in EEHDW-treated HCT-8/5-FU cells were analyzed. EEHDW treatment was shown to significantly reduce the mRNA and protein expression levels of P-gp and ABCG2 in the HCT-8/5-FU cells in a concentration-dependent manner (Fig. 5A and B). These observations indicated that EEHDW may modulate the MDR phenotype in HCT-8/5-FU cells via the regulation of P-gp and ABCG2 expression.