Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, TRIzol reagent, JC-1, caspase-3 and -9 colorimetric protease assay kits were purchased from Life Technologies (Carlsbad, CA, USA). SuperScript II Reverse Transcriptase was obtained from Promega Corporation (Madison, WI, USA). Bcl-2 and Bax antibodies, and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). TUNEL assay kit was purchased from R&D Systems (Minneapolis, MN, USA). Hoechst staining kit was obtained from Beyotime Institute of Biotechnology (Jiangsu, China). All other chemicals, unless otherwise stated, were obtained from Sigma-Aldrich (St. Louis, MO, USA).

EEPS was prepared as described previously (21). For animal experiments, EEPS powder was dissolved in saline to a working concentration of 250 mg/ml. In cell-based experiments EEPS powder was dissolved in 50% dimethyl sulfoxide (DMSO) to a stock concentration of 250 mg/ml, and the working concentrations of EEPS were made by diluting the stock solution in the culture medium. The final concentration of DMSO in the medium for all cell experiments was <0.5%.

Human colon carcinoma HT-29 cells were obtained from the cell bank of the Chinese Academy of Science (Shanghai, China). The cells were grown in DMEM containing 10% (v/v) FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were cultured at 37°C in a humidified incubator with 5% CO₂.

Male BALB/c athymic (nude) mice (with an initial body weight of 20–22 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and housed under pathogen-free conditions with controlled temperature (22°C), humidity and a 12-h light/dark cycle. Food and water were given ad libitum throughout the experiment. All animal treatments were strictly in accordance with international ethical guidelines and the National Institutes of Health Guide concerning the Care and Use of Laboratory Animals, and the experiments were approved by the Institutional Animal Care and Use Committee of Fujian University of Traditional Chinese Medicine.

HT-29 (1.5×10⁶) cells mixed with Matrigel (1:1) were subcutaneously injected in the right flank to initiate tumor growth. After 5 days of xenograft implantation, mice were randomly divided into 2 groups (n=6) and given gavage-feeding with 1.93 g/kg/day of EEPS or saline daily, 5 days a week for 21 days. Body weight was recorded every 2 days throughout the study. At the end of the experiment, tumors were excised and weighed, and part of the tumor was fixed in buffered formalin and the remaining part was stored at −80°C.

The sections (4-μm) of tumor samples were analyzed by TUNEL staining using TumorTACS In Situ Apoptosis kit. Apoptotic cells were counted as DAB-positive cells (brown stained) in 5 arbitrarily selected microscopic fields at a magnification of ×400. TUNEL-positive cells were expressed as a percentage of the total cells.

Viability of HT-29 cells was examined by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay. HT-29 cells were seeded into 96-well plates at a density of 1×10⁴ cells/well in 0.1 ml medium. The cells were treated with various concentrations of EEPS for 24, 48 and 72 h respectively. 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. The absorbance was measured at 570 nm using an ELISA reader (Model ELX800; BioTek Instruments Inc., Winooski, VT, USA).

HT-29 cells were seeded into 6-well plates at a density of 2.0×10⁵ cells/well in 2 ml medium. The cells were treated with the indicated concentrations of EEPS for 24 h. Cell morphology was observed using a phase-contrast microscope (Leica, Mannheim, Germany). The images were captured at a magnification of ×200.

HT-29 cells were seeded into 12-well plates at a density of 1×10⁵ cells/well in 1 ml medium. After the treatment of EEPS for 24 h, cell apoptosis was determined by the Hoechst staining kit according to the manufacturer’s instruction. Briefly, at the end of the treatment, cells were fixed with 4% polyoxymethylene and then incubated in Hoechst solution for 5–10 min in the dark. The staining images were recorded using a phase-contrast fluorescence microscope (Leica). The images were captured at a magnification of ×400.

JC-1 is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green to red, which can be used as an indicator of mitochondrial potential. In this experiment, 1×10⁶ treated HT-29 cells were resuspended after trypsinization in 1 ml of medium and incubated with 10 μg/ml of JC-1 at 37°C in 5% CO₂, for 30 min. Both red and green fluorescence emissions were analyzed by flow cytometry after JC-1 staining.

The activity of caspase-9 and -3 was determined by a colorimetric assay kit following the manufacturer’s instructions. Briefly, after treatment with various concentrations of EEPS for 24 h, HT-29 cells were lysed with lysis buffer for 30 min on ice. The lysed cells were centrifuged at 14,000 × g for 10 min, and 100 μg of the protein was incubated with 50 μl of Asp-Glue-Val-Asp (DEAD)-pNA (specific substrate of caspase-3) or Leu-Glu-His-Asp (LEHD)-pNA (specific substrate of caspase-9) at 37°C in the dark for 2 h. Samples were read at 405 nm in an ELISA plate reader. The data were normalized to the caspase activity in control cells and represented as ‘fold of control’.

HT-29 cells were seeded into 6-well plates at a density of 2.0×10⁵ cells/well in 2 ml medium. The cells were treated with indicated concentrations of EEPS for 24 h. Total RNA was isolated with TRIzol reagent. Oligo(dT)-primed RNA (1 μg) was reverse-transcribed with SuperScript II Reverse Transcriptase according to the manufacturer’s instructions. The obtained cDNA was used to determine the mRNA amount of Bcl-2 and Bax by PCR. GAPDH was used as an internal control. The sequences of the primers used for amplification of Bcl-2, Bax and GAPDH transcripts are as follows: Bcl-2 forward, 5′-CAGCTGCACCT GACGCCCTT-3 and reverse, 5′-GCCTCCGTTATCCTGGAT CC-3′; Bax forward, 5′-TGCTTCAGGGTTTCATCCAGG-3′ and reverse, 5′-TGGCAAAGTAGAAAAGGGCGA-3′; GAPDH forward, 5′-GTCATCCATGACAACTTTGG-3′ and reverse, 5′-GAGCTTGACAAAGTGGTCGT-3′.

HT-29 (1×10⁶) cells were seeded into culture flasks in 5 ml medium and treated with various concentrations of EEPS for 24 h. The treated cells were lysed with mammalian cell lysis buffer containing protease and phosphatase inhibitor cocktails, and the lysates were separated by 12% SDS-PAGE gels using 80 V for 2 h. The proteins were then electrophoretically transferred onto PVDF membranes. Membranes were blocked for 2 h with blocking solution at room temperature, washed in TBS with 0.25% Tween-20 (TBS-T) and exposed to primary antibodies against Bcl-2 and Bax (1:1,000) overnight at 4°C. β-actin (1:1,000) was measured as an internal control for protein loading. After the membranes were washed in TBS-T, secondary HRP-conjugated antibodies (anti-rabbit or anti-mouse) were added at 1:2,000 dilutions for 1 h at room temperature and the membranes were washed again in TBS-T followed by enhanced chemiluminescence detection.

Data are presented as means ± SD for the indicated number of independently performed experiments and analyzed using the SPSS package for Windows (version 16.0). Statistical analysis of the data was performed with the Student’s t-test and ANOVA. Differences with P<0.05 were considered to be statistically significant.

The anticancer activity of EEPS in vivo was determined via examining tumor weight in CRC xenograft mice, whereas its side-effects were determined by measuring body weight changes. As shown in Fig. 1A and B, EEPS treatment caused a 45% decrease in tumor weight compared with the control (0.728±0.064 or 0.397±0.062 g/mouse in control or EEPS-treated group, P<0.05), whereas EEPS did not affect body weight in the experimental animals (Fig. 1C). To determine the in vitro inhibitory effects of EEPS on the growth of CRC cells, we examined the viability of human colon carcinoma HT-29 cells using the MTT assay. As shown in Fig. 2, EEPS treatment reduced HT-29 cell viability in both a dose- and time-dependent manners (P<0.05). We further verified these results by determining the effect of EEPS on HT-29 cell morphology that represents the healthy status of cells in culture. As shown in Fig. 3, untreated HT-29 cells appeared as densely packed and disorganized multilayers, whereas after incubation with various concentrations of EEPS for 24 h many of the cells became rounded and shrunken, and detached from each other or floated in the medium. Taken together, this suggests that EEPS is effective in suppressing colorectal tumor growth both in vivo and in vitro, without apparent adverse effects.

Cell apoptosis in CRC tumor tissues was determined by immunohistochemical (IHC) staining for TUNEL. As shown in Fig. 4A, the percentage of TUNEL-positive cells in the control and the EEPS-treated mouse group was 37.00±16.70 and 84.33±23.25%, respectively, indicating the pro-apoptotic effect of EEPS in vivo. The apoptosis of HT-29 cells was evaluated by observation of nuclear morphological changes by cell nuclear staining with DNA-binding dye Hoechst. As shown in Fig. 4B, EEPS-treated cells showed condensed chromatin and fragmented nuclear morphology, typical apoptotic morphological features, whereas the untreated cell nuclei were homogeneously stained and less intense.

To explore the mechanism of the pro-apoptotic activity of EEPS, we examined its effect on the expression of Bcl-2 family proteins that are important regulators of apoptosis. As shown in Fig. 5A and C, EEPS significantly reduced the anti-apoptotic Bcl-2 mRNA level both in HT-29 cells and the CRC tumor tissues, whereas that of pro-apoptotic Bax was profoundly increased after EEPS treatment. The protein expression patterns of Bcl-2 and Bax were similar to their respective mRNA levels (Fig. 5B and D), suggesting that EEPS promotes CRC cell apoptosis both in vivo and in vitro through an increase in the pro-apoptotic Bax/Bcl-2 ratio.

The effect of EEPS on the change in mitochondrial membrane potential in HT-29 cells was examined via JC-1 staining followed by FACS analysis. The membrane-permeable JC-1 dye displays potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~525 nm) to red (~590 nm). Therefore, collapse of mitochondrial potential during apoptosis is indicated by a decrease in the ratio of red/green fluorescence intensity. As shown in Fig. 6, after treatment with 0, 0.5, 1 and 2 mg/ml of EEPS the JC-1 red/green fluorescent ratio in HT-29 cells was 5.34±0.84, 3.12±0.75, 2.93±0.41, 1.69±0.30 (P<0.05) respectively, suggesting that EEPS dose-dependently induced the loss of mitochondrial membrane potential in the CRC cells.

To identify the downstream effectors in the apoptotic signaling pathway, the activation of caspase-3 and -9 was examined by a colorimetric assay using specific chromophores, DEVD-pNA (specific substrate of caspase-3) and LEHD-pNA (specific substrate of caspase-9). As showed in Fig. 7, EEPS treatment significantly and dose-dependently induced activation of both caspase-3 and -9 in HT-29 cells (P<0.05).