Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, and BCA Protein Assay Kit were obtained from Thermo Fisher Scientific, Inc. An Annexin V-FITC Apoptosis Detection Kit was purchased from Nanjing KeyGen Biotech Co., Ltd. TRIzol Reagent and PrimeScript RT Reagent Kit were provided by Takara Bio, Inc. 5-FU, kaempferol, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and the remaining chemicals used in the present study, unless otherwise stated, were obtained from Sigma-Aldrich; Merck KGaA. Primary antibodies for Bax, Bcl-2, TS, PI3K, Akt, and phosphorylated (p)-Akt and β-actin horseradish peroxidase (HRP)-conjugated secondary antibodies were provided by Cell Signaling Technology, Inc.

The CRC cell lines HCT-8, HCT-116 and the normal human embryonic kidney cell line 293 were purchased from the Type Culture Collection of the Chinese Academy of Sciences. The cells were maintained in RPMI-1640 medium supplemented with 10% FBS, 100 unit/ml benzyl penicillin, and 100 µg/ml streptomycin in 5% CO₂ and 95% air at 37°C.

The HCT-8 cells and 293 cells (6×10³ cells/well) were seeded in 96-well plates and exposed to serial dilutions of 5-FU and/or kaempferol for 24 h. Then, MTT reagent (0.5 mg/ml in PBS) was added, and the cells were cultured for a further 4 h at 37°C prior to the addition of dimethyl sulfoxide. Absorbance at 570 nm was measured using an ELISA reader (Model ELX800; BioTek Instruments, Inc.). All assays were independently performed in triplicates. The cell inhibition ratio was calculated as follows: (A_control-A_treated)/A_control ×100%, where A_treated and A_control were the absorbance from treated and control groups, respectively. The IC₅₀ values (dose of 5-FU and kaempferol required to inhibit cell growth by 50%) were assessed using nonlinear regression analysis.

During the different dose combinations (ratios of IC₅₀ as 5-FU: kaempferol: 2:1, 1:1, 1:2, and 1:4), the HCT-8 and 293 cells were treated with various concentrations of kaempferol and 5-FU; a new concentration-dependent curve was constructed using the MTT assay. In different combination ratios, we used the chessboard concentration dilution method to design drug combinations of different concentrations and then calculated the CI according to the dose-effect curve. The Chou-Talalay method for drug combination is based on the median-effect equation (27,28). Based on these algorithms, CalcuSyn was used for determining synergism and antagonism at all doses or effect levels. The CI was analyzed by CalcuSyn where values <1, =1, and >1 indicated synergism, an additive effect, and antagonism, respectively.

The percentages of cells undergoing apoptosis with or without 5-FU and/or kaempferol were assessed by Annexin V-FITC/PI kit-based FACS (BD Biosciences). Cells were plated (3×10⁵ cells/well) in a 6-well plate and then incubated for 48 h with 100 µM kaempferol or 50 µM 5-FU alone or in combination. Subsequently, the cells were washed twice with cold PBS and stained with Annexin V/PI before being analyzed by FACS, according to the manufacturer's instructions. All assays were performed independently in triplicates.

HCT-8 cells were grown in a 6-well plate and treated with 100 µM kaempferol and/or 50 µM 5-FU for 48 h. The cells were fixed in ice-cold 4% paraformaldehyde for 10 min. Following washing with PBS, the cells were incubated with 1 µg/ml of Hoechst 33258 solution for 5 min in the dark. The cells were washed with PBS again and observed under a fluorescent microscope (DMI4000B; Leica Microsystems); the apoptotic cells appeared condensed and displayed fragmented nuclei.

Total protein extracts were obtained using lysis buffer and concentrations were determined by the BCA assay (both from Pierce Chemical Co.; Thermo Fisher Scientific, Inc.). Equal amounts of protein (50 µg) from each sample were separated by 12% SDS-PAGE gels and transferred to PVDF membranes (EMD Millipore). The membranes were blocked with 5% skimmed milk for 1 h at room temperature and probed with specific a primary antibody Bax (1:1,000; cat. no. 5023, Cell Signaling Technology, Inc.), Bcl-2 (1:1,000; cat. no. 4223; Cell Signaling Technology, Inc.), TS (1:1,000; cat. no. 5449; Cell Signaling Technology, Inc.), β-actin (1:1,000; cat. no. 4967, Cell Signaling Technology, Inc.), PI3K (1:1,000; cat. no. 4257; 1:1,000), AKT (1:1,000; cat. no. 2938; Cell Signaling Technology, Inc.), p-Akt (1:1,000; ser473; cat. no. 4060, Cell Signaling Technology, Inc.), PTEN (1:1,000; cat. no. 4257; Cell Signaling Technology, Inc.) overnight at 4°C. After being washed three times with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:25,000; cat. no. 31460; Thermo Fisher Scientific, Inc.) or rabbit anti-mouse immunoglobulin G secondary antibody (1:25,000; cat. no. 27025; Thermo Fisher Scientific Inc.) at room temperature for 2 h. Following washing again in TBST, protein signals were visualized by an enhanced chemiluminescence reaction system (Bio-Rad Laboratories, Inc.) and quantified using the ImageQuant software (Version 3.0; Bio-Rad Laboratories, Inc.). β-actin served as the loading control. All protein quantifications were normalized to their respective β-actin expression levels.

Three independent experiments were performed in triplicate. Data are presented as the mean ± standard deviation (SD). Differences between two groups were analyzed using unpaired Student's t-test. Datasets that involved more than two groups were assessed with one-way analysis variance (ANOVA), along with the Tukey-Kramer test. A P-value <0.05 was regarded as statistically significant. Regular analysis was carried out using the SPSS package for Windows (version 17.0; SPSS, Inc.).

Firstly, the cell viability inhibition potential of each drug was examined in the HCT-8 and HCT-116 cells. As anticipated, the growth of the cells was significantly decreased by treatment with kaempferol and 5-FU in a dose-dependent manner. The IC₅₀ values of 5-FU and kaempferol were 177.78 and 350 µM, respectively, in HCT-8 cells and 77.63 and 184.33 µM, respectively, in the HCT-116 cells. The IC₅₀ concentrations were then used to generate fixed ratios for subsequent combination studies and to calculate the CI. Among them, 50 µM of 5-FU combined with 100 µM of kaempferol exhibited synergistic anticancer effects on HCT-8 cells (CI value, 0.351; Table I), when compared with the effects of the two compounds used alone. Consistent results were found in the HCT-116 cell line (CI, 0.621; Table II).

The combination of 100 µM of kaempferol with 50 µM of 5-FU did not exhibit greater cytotoxic effects on the 293 cells (CI, >1) when compared with either of the two agents used alone (Table III).

Microscopy was used to observe the cell morphology. Cells treated with 5-FU and kaempferol were crenulated, and the nuclei were dim (Fig. 2A). Cell viability was significantly lower in cells subjected to treatment with 5-FU and kaempferol alone when compared with that of the untreated control cells (Fig. 2B). These results indicated that combined treatment with kaempferol and 5-FU could inhibit the growth of HCT-8 cells.

To assess if kaempferol plus 5-FU could induce apoptosis, Hoechst staining and flow cytometric analysis were performed to detect cell apoptosis (Fig. 3A). Kaempferol combined with 5-FU treatment significantly induced cancer cell apoptosis in HCT-8 cells when compared with either of the two compounds alone. Although 50 µM of 5-FU and 100 µM of kaempferol induced 10.13 and 3.31% apoptosis, respectively, in HCT-8 cells, a combination of these two induced 31.41% apoptosis, which is almost three times that induced by 50 µM 5-FU (Fig. 3B).

Apoptosis-related gene expression of Bax, Bcl-2, and 5-FU metabolic enzyme TS was detected by western blotting. The expression levels of Bax were higher in cells subjected to combination treatment when compared with those with either agent alone (Fig. 4). Conversely, the expression levels of Bcl-2 were decreased in the combination group when compared with single-agent treatment; TS expression levels were significantly decreased in HCT-8 cells when treated with kaempferol and 5-FU (Fig. 4). These results indicated that kaempferol combined with 5-FU exhibits synergistic anticancer effects by inducing CRC cell apoptosis and altering the expression levels of TS.

To determine whether AKT activation was involved in the synergistic effects of kaempferol and 5-FU, the levels of PI3K, PTEN, AKT, and p-AKT in HCT-8 cells were examined by western blotting. The p-AKT levels were attenuated in cells treated with kaempferol and increased after 5-FU treatment. However, PI3K and p-AKT levels were significantly lower in cells subjected to combination treatment when compared with 5-FU alone (Fig. 5). Thus, kaempferol and 5-FU may have synergistically suppressed CRC cell growth by inhibiting the activation of the PI3K/Akt pathway.