RPMI-1640 medium, fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and TRIzol reagent were obtained from Invitrogen (Invitrogen Life Technologies, Carlsbad, CA, USA). Bcl-2 (no. 3498), Jagged1 (no. 2155), Notch1 (no. 2495), Notch2 (no. 2420) and horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit IgG, no. 7074) were purchased from Cell Signaling Technology (Beverly, MA, USA). Fluorescein isothiocyanate (FITC)-conjugated Annexin V apoptosis detection kit was provided by Becton-Dickinson (San Jose, CA, USA). All the other chemicals, unless otherwise stated, were obtained from Sigma-Aldrich (St. Louis, MO, USA).

SB was purchased from Guo Yi Tang Chinese Herbal Medicine Store (Fujian, China). Different polar fractions of SB were prepared as previously described (11). The chloroform fraction of SB (ECSB) was dissolved in 100% dimethylsulfoxide (DMSO) to a stock concentration of 200 mg/ml and stored at −20°C. The working concentration of ECSB was obtained by diluting the stock solution in the cell culture medium. The final concentration of DMSO in the cell culture medium was ≤0.25% for all experiments.

Human colon cancer HCT-8 cells were obtained from the Nanjing KeyGen Biotech. Co. Ltd. (Nanjing, Jiangsu, China). HCT-8 cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin in a 37°C humidified incubator supplemented with 5% CO₂.

Cell viability was assessed using MTT colorimetric assay. HCT-8 cells were seeded into 96-well plates at a density of 1×10⁵ cells/ml and treated with various concentrations of ECSB for 24 or 48 h, respectively. Following removal of cell culture medium, 100 µl of MTT (0.5 mg/ml) was 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 of DMSO and the resulting absorbance was measured at 570 nm using an ELISA plate reader (model ELX800; BioTek, Winooski, VT, USA). The cell viability was determined using the formula: Cell viability (%) = sample optical density (OD)/ control OD × 100.

HCT-8 cells were seeded into 6-well plates at a density of 2×10⁵ cells/ml and treated with various concentrations of ECSB for 48 h. Cell morphology was observed using a phase-contrast microscope (Olympus, Tokyo, Japan). Photographs were taken at a magnification of ×200.

HCT-8 cells were seeded into 6-well plates at a density of 2×10⁵ cells/ml and treated with various concentrations of ECSB for 48 h. Subsequently, cells were harvested and diluted in fresh medium without ECSB, and reseeded at a density of 1,000 cells/well. The cell culture medium was replaced with fresh medium every three days. Following 12 days, cells were fixed with 10% formaldehyde, stained with 0.01% crystal violet and counted. Cell survival rate was calculated by normalizing the survival rate of control cells as 100%.

After incubation with various concentrations of ECSB for 48 h, HCT-8 cell apoptosis was determined by flow cytometry analysis using a fluorescence-activated cell sorting (FACS) caliber (Becton-Dickinson, CA, USA) and Annexin V-FITC/PI kit (Becton-Dickinson). Staining was performed according to the manufacturer's instructions. Annexin V-positive and PI-negative cells indicated presence of early apoptosis, whereas both Annexin V-positive and PI-positive cells indicated late apoptosis.

Anti-miR-34a oligonucleotide and scrambled oligonucleotide (used as negative control) were obtained from Invitrogen (Invitrogen Life Technologies). Transfection was performed using RNAiMAX kit (Invitrogen Life Technologies) according to the manufacturer's instructions. After transfection for 6 h, HCT-8 cells were treated with ECSB (150 µg/ml) for 48 h and total RNAs and protein were extracted for real-time PCR and western blot analysis.

Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies). First-strand cDNA was generated by reverse transcription of 2 µg total RNA using Oligo (dT) or special RT-miR-34a primer and SuperScript II reverse transcriptase according to the manufacturer's instructions. The mRNA levels of miR-34a, Bcl-2, Notch1, Notch2 and Jagged1 was determined with real-time PCR analysis using SYBR Premix Ex Taq II (Takara, Dalian, China) and ABI 7500 Fast PCR system, according to the manufacturer's instructions. U6 and B₂M were used as the internal controls for miR-34a and other genes, respectively. The mRNA expression was quantified by comparing the cycle threshold (Ct) values. The experimental data were analyzed using the 2^−∆∆Ct method. All experiments were performed in triplicate.

HCT-8 cells were lysed with mammalian cell lysis buffer (Pierce Chemical Co., Rockford, IL, USA) containing protease and phosphatase inhibitor. The protein concentrations were quantified using the bicinchoninic acid protein assay (Pierce Chemical Co.), and 50 µg of proteins were separated on 12% SDS-PAGE gels and transferred onto PVDF membranes (Millipore Corp., Billerica, MA, USA). The membranes were blocked with 5% non-fat dry milk and probed with primary antibodies against Bcl-2, Notch1, Notch2, Jagged1 and GAPDH (1:1,000) overnight at 4°C. The membranes were washed three times with Tris-buffered saline with Tween-20 (TBST) prior to incubation with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Following washing in TBST, the protein bands were detected using enhanced chemiluminescence. GAPDH was used as an internal control.

Each experiment was performed three times independently. Data were expressed as mean ± standard deviation. Statistical analysis was analyzed using SPSS package for Windows (version 13.0; SPSS Inc., Chicago, IL, USA) by the Student's t-test. P-values <0.05 were considered as statistically significant.

We first examined the effect of ECSB on HCT-8 cell viability using MTT assay. ECSB treatment significantly inhibited cell viability in a dose- and time-dependent manner (Fig. 1A). After 24 h, ECSB treatment decreased HCT-8 cell viability from 98.4 to 36.7%. Similarly, after 48 h, ECSB treatment decreased HCT-8 cell viability from 86.4 to 23.5%. We further examined the effect of ECSB on HCT-8 cell morphology using phase-contrast microscopy. Untreated control cells appeared healthy and had a high rate of confluence, whereas ECSB treatment significantly decreased the confluence and state of HCT-8 cells, in a dose-dependent manner (Fig. 1B). Moreover, ECSB treatment resulted in distinctive rounding of cells, indicative of cellular apoptosis. Taken together, these data revealed that ECSB significantly inhibited the growth of HCT-8 cells.

We next examined whether ECSB inhibited cell growth through inducing cell apoptosis. HCT-8 cells were stained with Annexin V-FITC/PI and analyzed using flow cytometry analysis (Fig. 2). Following treatment with 0, 100, 150 and 200 µg/ml ECSB, the percentage of cells undergoing either early or late apoptosis were 10.42, 16.68, 19.46 and 23.25%, respectively, which demonstrated that ECSB promoted HCT-8 cell apoptosis in a dose-dependent manner. Furthermore, we performed colony formation assays to determine the proliferation of HCT-8 cells following ECSB treatment. Treatment with 100, 150 and 200 µg/ml ECSB for 48 h significantly decreased the survival rate of HCT-8 cells by 27.13, 49.24 and 62.96%, respectively (P<0.05), which demonstrated that ECSB inhibited the proliferation of HCT-8 cells in a dose-dependent manner (Fig. 3).

miR-34a is a member of the miR-34 family which functions as a tumor suppressor in numerous cancers including colon cancer, through inhibition of the genes involved in multiple oncogenic signaling pathways. In addition, genes which are involved in cancer cell growth, such as Bcl-2, Notch1, Notch2 and Jagged1 are candidate target genes of miR-34a. We therefore examined whether ECSB suppresses cancer cell growth through regulation of miR-34a, using real-time PCR analysis. ECSB treatment (150 µg/ml) for 48 h significantly increased the level of miR-34a mRNA expression in HCT-8 cells (Fig. 4A). Furthermore, ECSB treatment also significantly decreased both the mRNA and protein expression levels of miR-34a target genes Bcl-2, Notch1/2 and Jagged1 (Fig. 4). These results suggest that the inhibitory effect of ECSB on cancer cell growth is likely mediated by upregulation of miR-34a expression and the inhibition of its downstream target genes.

To further verify that ECSB inhibited cancer cell growth through directly regulating miR-34a and its downstream target genes (Bcl-2, Notch1, Notch2 and Jagged1), we treated HCT-8 cells with the combination of ECSB and anti-miR-34a oligonucleotide (AMO-34a) comparing to AMO-34a only and a scrambled oligonucleotide was used as the negative control. Expression of miR-34a was significantly decreased following transfection of AMO-34a, which was rescued by ECSB treatment (Fig. 5A), suggesting that ECSB can directly mediate the expression of miR-34a. We next examined the mRNA and protein expression of miR-34a downstream target genes following transfection of AMO-34a in HCT-8 cells. The mRNA expression of Notch1/2, but not Bcl-2 or Jagged1 was significantly upregulated whereas the protein expression of Notch1/2, Bcl-2 and Jagged1 was significantly upregulated following transfection of AMO-34a in HCT-8 cells. However, following co-treatment with ECSB, the expression of Notch1, Notch2, Bcl-2 and Jagged1 was drastically decreased compared to AMO-34a transfected cells (Fig. 5B-D). Collectively, these findings reveal that ECSB-induced suppression of cancer cell growth via directly targeting miR-34a and downregulating its downstream target genes.