MTT was purchased from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). Polyclonal rabbit anti-human cleaved caspase-3 (1:1,000; cat. no. 9661S), monoclonal rabbit anti-human cleaved caspase-8 (1:1,000; cat. no. 9496S), polyclonal rabbit anti-human cleaved caspase-9 (1:1,000; cat. no. 9505S), monoclonal mouse anti-human BCL-2 (1:1,000; cat. no. 15071S), polyclonal rabbit anti-human Bax (1:1,000; cat. no. 2772S), monoclonal rabbit anti-human cyclin D1 (1:1,000; cat. no. 2978S), monoclonal rabbit anti-human CDK4 (1:1,000; cat. no. 12790S), monoclonal rabbit anti-human CDK6 (1:1,000; cat. no. 13331S) and monoclonal rabbit anti-human p21 (1:1,000; cat. no. 2947S) primary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). N-Benzyloxycarbonyl-Val-Ala-Asp (O-Me) fluoromethyl ketone (Z-VAD-FMK) was purchased from Beyotime Institute of Biotechnology (Haimen, China). The monoclonal mouse anti-human β-actin primary antibody was obtained from Abcam (1:1,000; cat. no. ab8226; Cambridge, UK). Goat anti-mouse and goat anti-rabbit secondary antibodies were purchased from Thermo Fisher Scientific, Inc., (1:5,000; cat. nos. A16072 and A16110, respectively; Waltham, MA, USA).

H. vernalis Maxim. was purchased from Shaanxi Panlong Pharmaceutical Co., Ltd. (Shangluo, China). Briefly, the dried root of H. vernalis Maxim. (10.0 kg) was extracted with 70% ethanol three times. The extracts were combined, concentrated, and dried at 80°C to obtain the HVMEE. High-performance liquid chromatography (HPLC) in tandem with mass spectrometry analysis was used to assess the main ingredients in the extracts. HPLC was conducted in tandem with mass spectrometry using an Agilent 1260 HPLC and AB SCIEX 4500Q trap triple quadrupole mass spectrometer with ESI source: Mobile phase 0.1% (v/v) (A) formic acid aqueous solution and (B) acetonitrile; injection volume 5 µl; column temperature 35°C, using a gradient elution mode. Run times from 0–10 min up to 15% B and from 11–20 min up to 27% B. The HPLC system consisted of a C18 column (3.9×300 mm, 10 µm) with 1 ml/min flow rate. The MassHunter (Agilent Technologies, Inc., Santa Clara, CA, USA) system was used.

Human CRC cell lines HT-29 and SW620 were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI-1640 (Thermo Fisher Scientific, Inc.) supplemented with 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.; cat. no. 10437-028), 100 U/ml penicillin, and 100 U/ml streptomycin in an atmosphere of 95% oxygen and 5% CO₂ at 37°C.

HT-29 and SW620 cells were seeded in 96-well plates at a density of 2×10⁴ cells/well for 24 h, then cells were treated with 0.01, 0.03, 0.1, 0.3, 1, and 3 mg/ml HVMEE for 24 h in complete medium. Following treatment, 20 µl of MTT solution (5 mg/ml) was added to each well for 4 h. The cells were then washed three times with PBS, and the resulting formazan was resuspended in 150 µl dimethyl sulfoxide. Absorbance was measured at 570 nm with a Bio-Rad ELISA reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The experiment was repeated independently three times.

Cell apoptosis was detected using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, HT-29 and SW620 cells were grown at a density of 5×10⁵ per well in 6-well plates. In accordance with the IC₅₀ values obtained from the MTT assay, the cells were treated with 0.01 or 0.05 mg/ml HVMEE for 24 h. Using the MTT assay results, the IC₅₀ values were 0.105±0.022 mg/ml for HT-29 and 0.146±0.013 mg/ml for SW620 cells. The authors identified that the minimum effective concentration of HVMEE was 0.01 mg/ml for HT-29 and SW620 cells. Therefore, five times the minimum effective concentration of HVEMEE was 0.05 mg/ml, which is a concentration with a significant effect, whilst bearing an apoptosis rate (for both HT-29 and SW620) of <50%.

Then, a total of 1×10⁶ cells were collected with centrifuge (700 × g) 3 min at room temperature, washed with ice-cold PBS and resuspended in 1X binding buffer [10 mM Hepes/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl₂] at a concentration of 1×10⁶ cells/ml. Annexin V-FITC (5 µl of a 25 µg/ml solution) and PI (5 µl of a 250 µg/ml solution) were added to 100 µl of cell suspension. The cells were then gently vortexed and incubated at room temperature in the dark for 15 min. Then, 400 µl of ice-cold binding buffer was added and mixed gently before the cell preparations were examined by flow cytometry (FACSCalibur; BD Biosciences).

To determine cell cycle distribution, cells were collected with centrifuge (700 × g) 3 min at room temperature, washed with ice-cold PBS and fixed for 2 h in 75% ethanol at −20°C. The cells were then treated with 1% RNase A for 5 min at 37°C and stained with 50 mg/ml PI for 30 min. Fluorescence intensity was examined by flow cytometry using a FACS Calibur (FACSCalibur; BD Biosciences).

Caspase-3 activity in HT-29 and SW620 cells was detected using the caspase-3 activity assay kit (Beyotime Institute of Biotechnology), as per the manufacturer's instructions. HT-29 and SW-620 cells were plated in culture dish (diameter, 10 cm) at a density of 1×10⁶ cells and allowed to grow for 24 h. The cells were then treated with HVMEE and Z-VAD-FMK (10 µM) for 24 h or 48 h in complete medium with 37°C. Following drug treatment, absorbance values were measured at 405 nm. Results were adjusted to the total protein content, and activity was expressed as nmol of p-Nitroaniline per mg of total protein.

HT-29 and SW-620 cells were plated in culture dish (diameter, 10 cm) at a density of 10×10⁶ cells and allowed to grow for 24 h. Then, HT-29 cells and SW620 cells were treated with 0.01 and 0.05 mg/ml HVMEE for 24 and 48 h. The cells were then collected with a centrifuge (700 × g) 3 min at room temperature, and resuspended in lysis buffer (Beyotime Institute of Biotechnology) for 30 min in an ice bath. Following centrifugation at 14,000 × g at 4°C for 30 min, the supernatant was collected. Protein concentrations were determined using the Bradford method. Equal protein amounts (30 µg) were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred electrophoretically to nitrocellulose membranes (Pall Corporation, New York, NY, USA) and blocked with TBS buffer containing 0.05% Tween-20 and 10% non-fat milk for 2 h at room temperature. The membranes were then incubated overnight at 4°C with various primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies at room temperature for 1.5 h. Following washing the membranes three times for 5 min in TBS buffer containing 0.05% Tween-20. Immunoreactive proteins on the membrane were detected using the Immobilon Western HRP substrate (EMD Millipore, Billerica, MA, USA). The bands were quantified using Multi Gauge version 3.2 software (Fujifilm Holdings Corporation, Tokyo, Japan). Experiments were repeated independently 3 times, and the relative expression of the target protein was normalized to the level of β-actin in the same sample.

SPSS software (version, 13.0; SPSS, Inc., Chicago, IL, USA) was used for the statistical analysis. Data were expressed as the mean ± standard deviation. Statistical analyses were carried out using a one-way analysis of variance and Fisher's least significant difference (LSD) t-test to compare differences between groups. P<0.05 was considered to indicate a statistically significant difference.

Ion flow results from HPLC analysis of the HVMEE are demonstrated in Fig. 1. The alkaloid content of the HVMEE was as high as 89.67%. The principal alkaloids observed were allocryptopine, protopine, berberine, sanguinarine, chelerythrine, tetrahydroberberine, and coptisine; their relative contents were 13.48, 55.33, 0.16, 14.86, 5.81, 0.002, and 0.02%, respectively (Fig. 1).

The effect of HVMEE on cell viability was assessed by MTT assay. As demonstrated in Fig. 2, HVMEE effectively decreased cell viability in HT-29 and SW620 cells in a concentration-dependent manner. The IC₅₀ values were 0.105±0.022 mg/ml for HT-29 and 0.146±0.013 mg/ml for SW620 cells (Fig. 2).

Apoptosis of HVMEE colorectal cancer cells was assessed by flow cytometry analysis. As demonstrated in Fig. 3, the percentages of apoptotic cells (the sum of B2 and B4 quadrants of the flow cytometry plots) in the untreated control HT-29 (Fig. 3A) and SW620 cells (Fig. 3B) were 3.7±0.3% (Fig. 3A and C) and 4.9±0.2% (Fig. 3B and C), respectively. When exposed to 0.01 mg/ml HVMEE for 24 h, the percentage of apoptotic cells increased to 15.3±1.4% in the HT-29 cells (P<0.01; Fig. 3A and C) and 9.9±1.8% in the SW620 cells (P<0.05; Fig. 3B and C). When exposed to 0.05 mg/ml of HVMEE for 24 h, the percentages of apoptotic cells increased to 27.7±1.3% in the HT-29 cells (P<0.01; Fig. 3A and C) and 19.7±0.7 in the SW620 cells (P<0.01; Fig. 3B and C). In addition, treatment of HT-29 and SW620 cells with 0.01 and 0.05 mg/ml HVMEE resulted in a significant increase in caspase-3 activation in both cell lines compared with untreated control (Fig. 4). The HVMEE-induced caspase-3 activity was significantly blocked by pretreatment with a general caspase inhibitor, Z-VAD-FMK (Fig. 4).

The cell cycle phase distribution in HT-29 (Fig. 5A) and SW620 (Fig. 5B) cells was examined following treatement with 0.01 and 0.05 mg/ml HVMEE for 24 h. Flow cytometric analysis of nuclear DNA distribution demonstrated a concentration-dependent increase of G₁ proportions in HVMEE-treated cells, compared with untreated control cells (Fig. 5).

Following 24 and 48 h of 0.01 and 0.05 mg/ml HVMEE treatment in HT-29 and SW620 cells, expression levels of several apoptosis-related proteins were examined by western blotting (Fig. 6A and B, respectively). Expression levels of pro-apoptotic proteins cleaved caspase-3, cleaved caspase-9, and Bax increased in HVMEE-treated cells compared with untreated cells (Fig. 6). Expression levels of cleaved caspase-8 presented no significant change (Fig. 6). By contrast, expression levels of Bcl-2, an apoptosis inhibitor and a central regulator of caspase activation, significantly decreased in the HVMEE-treated cells compared with untreated cells (Fig. 6).

To test the mechanisms of HVMEE-induced cell cycle arrest, expression of cell cycle-related proteins was further assessed by western blotting. Following treatment of HT-29 and SW620 cells with 0.01 and 0.05 mg/ml HVMEE for 24 and 48 h, the expression levels of cyclin D1, cyclin dependent kinase (CDK) 4, and CDK6 decreased significantly in HVMEE-treated cells compared with the untreated cells (Fig. 7). By contrast, protein expression levels of cyclin dependent kinase inhibitor 1A (known as p21) were significantly upregulated in the HVMEE-treated cells compared with the untreated cells (Fig. 7). Since induction of p21 leads to CDK inhibition and cell cycle arrest at the G₁/S checkpoint (9–11), these data indicate that the HVMEE-induced G₁ phase arrest may be due to inhibition of CDK4/6-cyclin D1 complexes by p21 upregulation.