Sodium butyrate, TSA, mitomycin C, 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and protease inhibitor cocktail were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), RPMI-1640 medium, and Dulbecco's phosphate-buffered saline (DPBS) were from Invitrogen (Carlsbad, CA, USA). Antibodies against Rb, Bax, Bim, Bcl_XL, cyclin E, cyclin D1, cyclin-dependent kinase (CDK) 2, CDK4, p21^Waf1/Cip1, extracellular signal-regulated kinases (Erk) 1/2 and β-actin were obtained from Cell Signaling Technology (Beverly, MA, USA). MRP1, BCRP and P-gp antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and anti-glycer-aldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Calbiochem (EMD Biosciences Inc., San Diego, CA, USA). Secondary antibodies were purchased from Bio-Rad (Hercules, CA, USA). Enhanced chemiluminescence solution (ECL) was kindly donated by Detroit R&D, Inc. (Detroit, MI, USA). Propidium iodide and RNase A were from Abcam (Cambridge, UK).

The HCT116 human colon cancer cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified 5% CO₂ incubator. To establish the butyrate-resistant HCT116 colon cancer cell line (HCT116/BR), HCT116 parental cells (HCT116/PT) were initially incubated with serum-containing media supplemented with 0.2 mM sodium butyrate. Butyrate treatment induced the death of most cells while some cells survived, but they proliferated slower compared to HCT116/PT cells. When cells were 80% confluent, the media was changed to a fresh media containing the same concentration of butyrate. By continuously growing cells in the presence of the same concentration of butyrate, no more detectable death of cells was observed and then the treating concentration of butyrate increased 2-fold. After culturing cells with the increasing concentration of sodium butyrate to a maximum of 1.6 mM for 3 months, butyrate-resistant cells were established. Butyrate stock was prepared by dissolving sodium butyrate in DPBS. The treatment was carried out after overnight incubation to allow cell attachment.

Cell proliferation was determined in both cell lines according to a method described previously with slight modifications (9). HCT116/PT and HCT116/BR cells were seeded onto 96-well plates. Cells were treated with various concentrations of butyrate, paclitaxel, 5-fluorouracil (5-FU), doxorubicin and TSA. After 72-h incubation, medium was removed, 0.5% MTT stock was diluted 1:10 with medium and 100 µl added to each well, followed by incubation at 37°C for a further 2 h. Subsequently, dimethylsulfoxide (DMSO) was added to solubilize the purple formazan crystals and then absorbance at 540 nm was measured using an ELISA reader (Bio-Tek Instruments Inc., Winooski, VT, USA).

To assess cell cycle progression, HCT116/PT and HCT116/BR cells were plated onto 60-mm dishes. After overnight incubation, HCT116/PT cells were treated with 6.4 mM butyrate for 24 h. Cells were then trypsinized and centrifuged for 5 min at 500 × g at 4°C, and fixed with 70% ethanol at 4°C, followed by addition of 1 ml of propidium iodide solution (final concentration, 50 µg/ml), which contained 200 µg/ml RNase A, for 30 min in the dark (10). Flow cytometry was then performed using a FACSCalibur flow cytometer and identified using Cell Quest software (Becton-Dickinson, San Jose, CA, USA). Red fluorescence, measured at 585/542 nm, indicative of propidium iodide uptake by damaged cells, was measured by logarithmic amplification and electronic compensation for spectral overlap (11).

Cells were lysed with cell lysis buffer containing protease inhibitor cocktail, and protein concentration was determined using the bicinchoninic acid (BCA) assay. Protein samples (20–30 µg per lane) were resolved by 7.5–12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. For immunodetection, blots were incubated with 1:1,000 diluted primary antibodies in 5% bovine serum albumin (BSA) Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) at 4°C with gentle shaking overnight, followed by incubation with a secondary antibody conjugated to horseradish peroxidase at room temperature. The antigen-antibody complexes were detected using ECL reagents with an ImageQuant LAS-4000 mini (GE Healthcare Life Sciences, Piscataway, NJ, USA). GAPDH or β-actin was used as the loading control (12).

Cells were seeded onto 6-well plates to create a confluent monolayer after overnight incubation. Mitomycin C was treated (final concentration, 25 µg/ml) for 30 min and removed. A straight line was scratched in the middle of the cell monolayer. After washing with DPBS, cells were treated with chemotherapeutic drugs and incubated up to 24 h. The widths of the scratched line were measured at appropriate time intervals and compared to that at 0 h.

Activity of matrix metalloproteinases (MMPs) was detected using gelatin zymography according to a method described previously with slight modifications (13). Briefly, cells were incubated in 60-mm dishes and medium was replaced with serum-free medium to promote MMP secretion when cells reached 80% confluence. After 48-h incubation, medium was harvested and concentrated using a Speed-vac (Savant/E-C Instruments, Niantic, CT, USA). Electrophoresis was performed in 10% SDS-PAGE gels containing 0.1% gelatin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) without a reducing agent or boiling. After electrophoresis, the gel was soaked with renaturing buffer (2.5% Triton X-100, v/v) for 1 h, rinsed with distilled water three times, incubated with developing buffer (50 mM Tris-base, 150 mM NaCl, 1 µM ZnCl₂ and 50 mM CaCl₂) for 72 h at 37°C, stained with 0.5% (w/v) Coomassie Blue, and destained in destaining buffer (methanol:acetic acid:water, 10:5:85, v/v). Gel images were scanned using a Gel-Doc Imaging System (Bio-Rad) and band density was measured using the ImageJ software.

Data are presented as means ± standard deviation (SD). Statistical analysis was performed using the GraphPad Prism 5 software. Data were compared using an unpaired two-tailed t-test or one-way analysis of variance (ANOVA), followed by Dunnett's post hoc test. P-values <0.05 were considered to indicate statistical significance (14).

HCT116/BR cells were derived from the HCT116 human colon cancer cell line by chronic exposure to butyrate for 3 months. Cell morphology was altered slightly (Fig. 1). HCT116/PT cancer cells had cuboidal and sharp-pointed shapes, but HCT116/BR cells were more rounded and expanded. In addition, vacuolization was observed in HCT116/BR cells. The growth rate of the cells was lower than that of HCT116/PT cells.

Whether resistance to other chemotherapeutic agents accompanied resistance to butyrate was assessed by determining cell viability by MTT assay. HCT116/PT and HCT116/BR cells were treated with butyrate, paclitaxel, 5-FU, doxorubicin and TSA, and the dose-response curve and the concentrations that inhibited cell growth by 50% (IC₅₀) values are shown in Fig. 2 and Table I, respectively. The inhibition of cell proliferation was increased in a concentration-dependent manner in both cell lines, but HCT116/BR cells exhibited greater chemoresistance. The IC₅₀ values of HCT116/BR cells for butyrate, paclitaxel, 5-FU, doxorubicin and TSA were 10.8, 2.42, 2.36, 4.31 and 11.3-fold higher, respectively, than those of HCT116/PT cells.

Drug efflux pumps are related to drug resistance (8). One possible mechanism of chemoresistance to butyrate is regulation of the expression of drug efflux pumps. Therefore, we determined P-gp, BCRP and MRP1 protein levels by immunoblotting (Fig. 3). There was no significant difference in P-gp and BCRP expression between the cell lines, and MRP1 was not detected in HCT116/PT or HCT116/BR cells. Thus, drug efflux pumps did not affect the chemoresistance of HCT116/BR cells.

Butyrate induces cell cycle arrest and apoptosis in cancer cells (15), but our findings indicated no such effects in HCT116/BR cells. We performed flow cytometry to assess the effect of butyrate on cell cycle progression in HCT116/BR cells (Fig. 4). We also treated HCT116/PT cells with 6.4 mM butyrate (4-fold of 1.6 mM) for 24 h (HCT116/AT_6.4) and compared the results with those of HCT116/PT and HCT116/BR cells. The proportion of subG₁-phase cells was increased by treatment with 6.4 mM butyrate compared to untreated HCT116/PT cells (6.69 vs. 0.47%) suggesting that apoptosis was triggered. The proportion of subG₁-phase HCT116/BR cells was similar (0.68%). The proportion of cells in S phase, which is a DNA-replication phase of the cell cycle, was elevated by 62.0% in HCT116/BR compared to HCT116/PT cells. In addition, the proportion of HCT116/AT_6.4 cells in G₂ phase was >2-fold greater than that of HCT116/PT cells (51.4 vs. 21.6%), but was restored to the control level in HCT116/BR cells (29.1%).

We conducted an immunoblotting analysis to examine the expression of proteins involved in the cell cycle and apoptosis in HCT116/PT and HCT116/BR cells. We treated HCT116 cells with 1.6 and 6.4 mM butyrate for 24 h (HCT116/AT_1.6 and HCT116/AT_6.4, respectively) to determine the effects of acute butyrate treatment.

Expression of p21^Waf1/Cip1 was greatly increased in HCT116/AT_1.6 and HCT116/AT_6.4 cells, but the increase was less marked in HCT116/BR cells compared to HCT116/PT cells (Fig. 5). HCT116/BR cells showed greater cyclin D1 expression compared to HCT116/PT cells. CDK4 protein expression was minimally changed. Rb expression was decreased in HCT116/BR cells. Cyclin E expression in HCT116/BR cells was slightly increased by butyrate; however, CDK2 expression was greatly reduced.

The Bcl-2 family, which includes Bcl-xL, Bim and Bax, is involved in the regulation of apoptosis, and their expression is shown in Fig. 6. The expression of Bcl-xL, which is an important anti-apoptotic protein, was decreased in HCT116/AT_1.6 and HCT116/AT_6.4 cells by the acute butyrate treatment, but was significantly increased in HCT116/BR cells. Bim and Bax are pro-apoptotic proteins, the expression of which was decreased in HCT116/AT_1.6, HCT116/AT_6.4 and HCT116/BR cells by both chronic and acute butyrate treatment. Moreover, activation of Erk reduces the expression of Bim, and phospho-Erk1/2 expression was increased in HCT116/BR cells.

We performed gelatin zymography and a wound healing assay to evaluate cell invasiveness (Fig. 7) and migratory activity (Fig. 8). The wound healing rate was generally slower in HCT116/BR than HCT116/PT cells. MMP-9 plays a role in cancer cell metastasis, and there was no significant difference in its expression between HCT116/PT and HCT116/BR cells (Fig. 7). However, following short-term butyrate treatment, MMP-9 expression was significantly increased in both HCT116/AT_1.6 and HCT116/AT_6.4 cells. MMP-2 was not detected in HCT116/PT and HCT116/BR cells. There was no significant difference in wound healing rate between HCT116/PT and HCT116/BR cells following treatment with butyrate, paclitaxel and TSA; however, doxorubicin significantly inhibited wound healing in both cell types.