Dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), trypsin-EDTA, fetal bovine serum (FBS), Roswell Park Memorial Institute (RPMI)-1640 medium, and penicillin-streptomycin were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). A cell cycle assay kit was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Mouse or rabbit monoclonal antibodies specific for cyclin dependent kinase 4 (CDK4; 2906S), cyclin D1 (2978), c-myc (9402S), B-cell lymphoma 2 (Bcl-2; 2876S), Bcl-2 associated X protein (BAX; 2772S) and α-actin (4967L) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Bicinchoninic acid (BCA) protein assay and Enhanced Chemiluminescence (ECL) kits were obtained from Pierce; Thermo Fisher Scientific, Inc. All other chemicals used were of the highest grade commercially available.

A. camphorata used in this study was obtained from Well Shine Biotechnology Development Co., Ltd. (Taipei, Taiwan). For preparation of the extract, 500 g A. camphorata were extracted with 70% ethanol using a refluxing method and filtered (18). The AC extract stock solution was dissolved in DMSO to a final concentration of 500 mg/ml and stored at −20°C until use. Prior to use, the stock was thawed and appropriate amounts of the AC stock solution were filter sterilized through 0.22-µm disc filters. Control cultures received the carrier solvent (0.1% DMSO).

The HCT-8 human CRC cell line was obtained from Nanjing KeyGen Biotech Co., Ltd. and maintained at 37°C in a humidified incubator at 5% CO₂. HCT-8 cells undergoing exponential growth were routinely cultured in 25 cm² plastic flasks containing 5 ml RPMI-1640 medium supplemented with 10% (v/v) FBS and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin). For AC treatment experiments, cells were incubated with 0.4, 0.8 and 1.2 mg/ml AC, by diluting the stock with DMEM, for 24 h. Control cells were cultured in medium containing an equal amount of DMSO. Cells were washed with 2 ml PBS once and harvested with 0.25% trypsin. The cells were then centrifuged at 800 × g for 3 min at 25°C, and the isolated cells were used in subsequent experiments.

Various concentrations (0, 0.4, 0.8 and 1.2 mg/ml) of AC were added to 96-well plates seeded with 8×10³ HCT-8 cells/well. Following treatment for the times indicated, 10 µl of MTT solution (5 mg/ml stock; Merck KGaA, Darmstadt, Germany) was added to each well and the plate was incubated at 37°C for 4 h. Following removal of medium, 100 µl DMSO was added to each well and the plate was gently shaken for 5 min. Absorbance was then measured at a wavelength of 570 nm using an ELISA plate reader (Model ELX800; BioTek Instruments, Inc., Winooski, VT, USA). The percentage of viable cells was determined through comparison with untreated control cells. Each experiment was replicated 3–5 times and all data are represented as mean ± standard deviation.

To assess the AC effect on cell colony formation, cells were seeded into a 6-well-plate at a density of 2×10⁵ cells/well. After 24 h, cells were exposed 0, 0.4 or 0.8 mg/ml AC or drug-free medium for 24 h. The drug-containing or drug-free media was then removed, cells were plated at a density of 1×10³/well into 60-mm dishes, and cells were incubated for 10 days under standard culture conditions as described above. Formed colonies were fixed in 4% paraformaldehyde for 30 min at 25°C and stained with 0.01% crystal violet for 15 min. Colony-forming efficiency was determined and cell survival was calculated by normalizing against control cell survival, from five random fields per well for three independent experiments.

A flow cytometer was used for cell cycle analysis (FACSCalibur; BD Biosciences, San Jose, CA, USA). HCT-8 cells were cultured in 6-well culture plates at a density of 1.5×10⁵ cells/well and treated with 0, 0.4 or 0.8 mg/ml AC for 24 h. Following trypsinization, floating and adherent cells were collected by centrifuged at 800 × g for 5 min at 25°C, washed with ice-cold PBS and then incubated with 30 µg/ml propidium iodide and 100 µg/ml RNase for 24 h in the dark at room temperature. Data acquisition and calculation of the percentage cells in the sub-G1, G0/G1, S, and G2/M phases of the cell cycle were performed using the accompanying flow cytometer software ModfitLT version 3.3.11 (CellQuest; BD Biosciences). Twenty thousand events per sample were counted and a minimum of three assessments were performed to assure each cell cycle distribution.

HCT-8 cells were plated at low density (1.5×10⁵ cells/well) into a 6-well plate and treated with AC for 24 or 48 h. At the end of the experiment, cells were washed twice with PBS at room temperature and then fixed with ice-cold 4% paraformaldehyde for 10 min. Fixed cells were then rinsed with PBS and stained with Giemsa (0.75 mg/ml) or Hoechst 33258 (10 µg/ml) for 15 min. Finally, cells were washed with PBS and analyzed under a fluorescence microscope with a UV light filter.

Total RNA from treated HCT-8 cells was extracted using a phenol/chloroform method with RNAiso reagent (Takara Biotechnology Co., Ltd., Dalian, China). The absorbance ratio at 260/280 nm of all samples was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc.). Reverse transcription was performed using 1 µg total RNA with the PrimeScript™ II First-Strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. qPCR was performed using SYBR Premix Ex Taq II (Takara Biotechnology Co., Ltd.) with an ABI 7500 Fast instrument according to the manufacturer's instructions. The reaction contained 10 µl 2X SYBR Premix Ex Taq II, 0.8 µl forward primer (10 µM), 0.8 µl reverse primer (10 µM), 0.4 µl 50X ROX dye II, 2.0 µl cDNA and 6 µl dH₂O in a total volume of 20 µl. The qPCR reaction conditions were as follows: 30 sec at 94°C, followed by 40 cycles of 3 sec at 94°C and 30 sec at 60°C. The primers used for qPCR are listed in Table I. Relative amount of target mRNA was calculated using the 2^−∆∆Cq method (19).

Following treatment as indicated above, HCT-8 cells were harvested and incubated in ice-cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.) with freshly added protease and phosphatase inhibitor cocktails (Roche Applied Science, Penzberg, Germany) on ice for 30 min. The cells were scraped from the plate and the lysate was collected in a centrifuge tube and centrifuged at 12,000 × g for 20 min at 4°C. The supernatant containing protein was collected, aliquoted, and stored at −80°C. The lysate protein concentrations were measured using a BCA protein assay kit, following the manufacturer's instructions. Total cell lysates (50 µg) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk for 2 h at 25°C and incubated with primary antibodies (1:1,000 dilution) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:5,000 dilution) at room temperature. Membranes were subsequently visualized using an ECL detection kit.

All data are presented as means ± standard deviation of three independent experiments and analyzed by one-way analysis of variance followed by Tukey's post hoc test, using SPSS Statistics version 20 software (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate the potential effects of AC treatment on proliferation of HCT-8 cells, the cells were exposed to 0–1.2 mg/ml AC for 24 and 48 h. Fig. 1A demonstrates that AC inhibited cell proliferation in a dose- and time-dependent manner (P<0.05). When treated with 0.4, 0.8 and 1.2 mg/ml AC for 24 and 48 h, HCT-8 cell viabilities were 91 and 85%, 65 and 57%, and 31 and 27%, respectively, compared with control untreated cells. Morphological changes in HCT-8 cells following AC treatment for 24 h were also evident (Fig. 1B). Based on phase-contrast inverse microscopy, the typical morphological apoptotic characteristics were observed post-treatment, including decreased cell number and an increase in apoptotic body formation, accompanied by an increased abundance of dead floating cells.

The ability of HCT-8 cells to form colonies in the presence of AC was analyzed using a flat plate colony formation assay. The results illustrated in Fig. 2A suggest that AC effectively inhibited colony formation in a dose-dependent manner. Control untreated cells demonstrated increased colony formation ability compared with cells treated with AC (P<0.05; Fig. 2B). This evidence suggests that AC may have significantly affected HCT-8 cell proliferation.

As AC appeared to inhibit HCT-8 cell proliferation in vitro, the hypothesis that AC may influence cell cycle progression was tested via flow cytometry. Following treatment with AC for 24 h, the percentage of cells in the S phase in the 0.8 mg/ml AC treatment group was significantly decreased compared with the control group (21.5 and 39.4%, respectively; Fig. 3). In addition, the percentage of cells in the G0/1 phase was 56.4% in the control group and 66.0% in the 0.8 mg/ml AC treatment group. AC treatment significantly increased the percentage of cells in the G0/G1 phase (P<0.05; Fig. 3). These results suggested that AC arrested the cell cycle at the G0/G1 phase.

To visualize apoptotic body formation and nuclear morphological alterations characteristic of apoptosis, Hoechst 33258 staining was performed after 24 h treatment with 0.4 and 0.8 mg/ml AC in HCT-8 cells. Hoechst 33258 reagent is a membrane-permeable blue fluorescent dye that stains the cell nucleus and allows visualization of nuclear morphology. Control untreated cells exhibited characteristics of healthy cells with intact oval shape and uniform nuclei that stained less brightly with Hoechst (Fig. 4). However, cells treated with AC exhibited more brightly stained nuclei as treatment concentration increased (Fig. 4).

Having demonstrated that AC affects colon cancer cell proliferation and apoptosis, the effect of AC treatment on associated gene expression was examined. The results demonstrated that the mRNA expression levels of cyclin D1, CDK4, cMyc and Bcl-2 were markedly downregulated in the AC treatment groups compared with the control group (P<0.05; Fig. 5). In contrast, the mRNA expression levels of Bax were increased following treatment with AC for 24 h compared with the control group (P<0.05; Fig. 5). These findings suggested that AC may inhibit proliferation and apoptosis of HCT-8 cells by regulating expression of cell division and apoptosis-associated genes.

To characterize the differential expression of Bax, cMyc, Bcl-2, cMyc, cyclin D1 and CDK4 proteins, western blot analysis was performed. As demonstrated in Fig. 6, incubation of HCT-8 cells with AC reduced Bcl-2 protein expression levels, a potent cell-death inhibitor, whereas it increased Bax protein levels, compared with untreated cells. These findings suggested that the AC-induced apoptosis may be associated with an AC-induced dysregulation of the Bcl-2/Bax ratio in HCT-8 cells. In addition, the results demonstrated that AC treatment downregulated the protein expression levels of cyclin D1, CDK4 and cMyc in HCT-8 cells following AC treatment, compared with untreated cells (Fig. 6).