Antibodies against Bcl-xL (1:1,000, cat. no. 54H6), Mcl-1 (1:1,000, cat. no. D5V5L), STAT3 (1:1,000, cat. no. D3Z2G), phosphorylated (p)-STAT3 (1:2,000, cat. no. Tyr705), STAT5 (1:1,000, cat. no. D206Y), p-STAT5 (1:1,000, cat. no. Tyr694), poly-(ADP-ribose) polymerase (PARP; 1:1,000, cat. no. 46D11) and GAPDH (1:1,000, cat. no. 14C10) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The secondary antibodies polyclonal goat anti-rabbit immunoglobulins (Igs) conjugated to horseradish peroxidase (HRP; 1:2,000, cat. no. P0448) and polyclonal goat anti-mouse Igs/HRP (1:2,000, cat. no. P0447) were purchased from Dako; Agilent Technologies, Inc. (Santa Clara, CA, USA). Apigenin (4′,5,7-trihydroxyflavone) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Human recombinant interleukin-6 (IL-6) and human recombinant soluble IL-6 receptor were purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). IL-6 was used supplemented with soluble IL-6 receptor (40 ng/ml).

The human colon cancer cell lines HT29, DLD-1, COLO320 and HCT116 were obtained from the American Type Culture Collection (Manassas, VA, USA). HT29 (KRAS wild-type) cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% antibiotic-antimycotic (Sigma-Aldrich; Merck KGaA). DLD-1 (KRAS mutant-type) and COLO320 (KRAS wild-type) cells were cultured in RPMI-1640 medium (Sigma-Aldrich; Merck KGaA) supplemented with 10% FBS and 1% antibiotic-antimycotic. HCT116 (KRAS mutant-type) cells were cultured in McCoy’s 5A medium (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and 1% antibiotic-antimycotic. All cell lines were maintained at 37°C in a humidified incubator containing 5% CO₂.

Colon cancer tissue samples were obtained from 40 patients (25 males and 15 females) with Stage Ⅳ colon cancer who had undergo surgery at the Nagoya City University Hospital (Nagoya, Japan) between January 2012 and December 2014. Resected specimens were staged by pathological evaluation according to the Japanese Classification of Colorectal Carcinoma (21). The mean age of the patients was 66.2±10 years (range, 37–88 years). All patients gave their written informed consent for the use of their tissue samples in the present study.

Resected specimens were fixed with formalin at room temperature for 2 days. Formalin-fixed, paraffin-embedded sections 4-µm-thick were deparaffinized in xylene and hydrated in graded alcohols. Following washing with running water, samples were soaked in soak in 10 mM citric acid buffer (9 ml 0.1 M citric acid + 41 ml 0.1 mM triso-dium citrate dihydrate + 450 ml distilled water) and boiled for 10 min. Following 3 washes with PBS (5 min/time) the slide was immersed in a mixture of 1.5 ml 30% hydrogen peroxide solution and 150 ml 100% methanol for 30 min to block endogenous peroxidase. Following washing with PBS, slides were blocked with 400 µl 4% Block Ace^® Powder (cat. no. UK-B80; DS Pharma Biomedical Co., Ltd., Osaka, Japan) for 10 min in a humidity box at room temperature. Subsequently, slides were incubated with primary antibodies against Bcl-xL (1:300) and Mcl-1 (1:100) onto at 4°C overnight. Following washing with PBS, slides were incubated with 100 µl secondary antibody (EnVision + Single Reagents HRP; cat. no. K4003; Dako; Agilent Technologies, Inc.) for 45 min at room temperature. The detection step was performed using Liquid DAB + Substrate Chromogen System (cat. no. K3467; Dako; Agilent Technologies, Inc.) for 10 min at room temperature and the sections were counterstained with hematoxylin for 30 sec at room tenmperature. Slides were analyzed using a light microscope (magnification, ×400). Ethical approval for the use of human tissue was granted by the Graduate School of Medicine, Nagoya City University (Nagoya, Japan) and all patients provided their informed consent for the use of their tissues in the current study.

Apigenin was dissolved in dimethyl sulfoxide (DMSO; Wako Chemicals GmbH, Neuss, Germany) to produce 50 mM stock solutions that were aliquoted and stored at −28°C prior to use. Stock solutions were diluted with culture medium to the indicated concentrations (final DMSO concentration, 0.1%).

Bcl-xL siRNA (5 nM, ID#:s1922; forward, 5′-GGAACUCUAUGGGAACAAUTT-3′ and reverse, 5′-AUUGUUCCCAUAGAGUUCCAC-3′), Mcl-1 siRNA (5 nM, ID#:s8583; forward, 5′-CCAGUAUACUUCUUAGAAATT-3′ and reverse, 5′-UUUCUAAGAAGUAUACUGGGA-3′), negative control siRNA (5 nM, cat. no. 4390843, Silencer Select Negative Control #1) and STAT3 siRNA (5 nM, ID#:s744, sequence: forward, 5′-GGCUGGACAAUAUCAUUGATT-3′ and reverse, 5′-UCAAUGAUAUUGUCCAGCCAG-3′) were purchased from Thermo Fisher Scientific, Inc. On the day before siRNA transfection, DLD-1 and HCT116 cells (1.0×10⁴ cells) were seeded to 60–80% confluence in their respective growth mediums without antibiotics. siRNAs and Lipofectamine RNAiMAX were diluted in Opti-MEM medium (both from Thermo Fisher Scientific, Inc.), mixed gently and incubated for 10–20 min at room temperature. Cells were transfected by adding the siRNA-Lipofectamine RNAiMAX complex dropwise to the medium to achieve a siRNA final concentration of 100 nM. Cells were incubated at 37°C in a CO₂ incubator and knockdown efficiency was evaluated 24 h post-transfection.

Cells (5×10⁵/well) were seeded into 6-well culture plates and incubated at 37°C overnight. Medium was replenished with serum-free medium and incubated for a further 4 h. Cells were then treated with apigenin at various doses 0, 5, 15 and 50 µM or transfected to target Bcl-xL, Mcl-1 or Bcl-xL and Mcl-1 together. DNA fragmentation was analyzed using a Cell Death Detection ELISA^Plus kit (Sigma-Aldrich; Merck KGaA) following the manufacturer’s protocol. The degree of apoptosis was evaluated using an enrichment factor (mU of treated cells/mU of non-treated cells).

The effect of apigenin or siRNA on cell proliferation was measured using WST-1 and BrdU assays. Each assay was performed at least three times. The WST-1 assay was performed using a Premix WST-1 cell proliferation assay system (Takara Bio, Inc., Tokyo, Japan). Cells (5×10³–5×10⁴/well) were seeded into 96-well tissue culture plates and incubated at 37°C overnight. Cells were treated with 0, 5, 15 and 50 µM apigenin or transfected to target Bcl-xL, Mcl-1 or Bcl-xL and Mcl-1 together in serum-free medium. After 48 h, the medium was removed and replaced with fresh medium (90 µl/well) containing Premix WST-1 (10 µl/well). Cells were then incubated at 37°C for 2 h. Absorbance was measured using a plate reader at 450 nm. The BrdU assay was performed using BrdU Cell Proliferation ELISA kit (colorimetric; cat. no. ab126556; Abcam, Cambridge, MA, USA), following the manufacturer’s protocol. Cells (5×10³–5×10⁴/well) were seeded into 96-well tissue culture plates and incubated at 37°C overnight. Cells were treated with 0, 5, 15 and 50 µM apigenin or transfected to target Bcl-xL, Mcl-1, or Bcl-xL and Mcl-1 together in serum-free medium for 32 h at 37°C. A total of 20 µl 1/500 diluted BrdU was added and incubated for 16 h at room temperature. Subsequently, fixing solution was added for 30 min at room temperature to fix and permeabilize cells, and denature the DNA. Subsequently, anti-BrdU antibody was added to each well for 1 h and peroxidase-conjugated goat anti-mouse immunoglobulin G antibody was added for 30 min. Incubation with primary and secondary antibodies was performed at room temperature. Finally, 3,3′,5,5′-tetra-methylbenzidine peroxidase substrate was added to each well and following incubation in the dark for 30 min, stop solution was added to each well. Absorbance was measured at 450 nm using a microplate reader.

Western blotting was performed to identify the mechanism of action of apigenin. Cells (DLD-1, HCT116: 1×10⁶/dish) were seeded in 4 dishes and incubated at 37°C overnight. The following day, cells were treated with 0, 5, 15 and 50 µM IL-6 in serum-free medium for 48 h. Proteins were the extracted and expression of p-STAT3 was evaluated by western blotting.

Cells (DLD-1, HCT116: 1×10⁶/dish) were seeded in 4 dishes and incubated at 37°C overnight. The following day, cells were treated with or without 50 µM (DLD-1), 15 µM (HCT 116) apigenin for 2 h and then with or without 50 µM IL-6 for a further 48 h. Proteins were extracted and expression of p-STAT3, Bcl-xL and Mcl-1 was evaluated by western blotting.

Protein samples were prepared in radioimmunoprecipitation lysis buffer with Protease Inhibitor Single Use Cocktail and Phosphatase Inhibitor Cocktail (all from Thermo Fisher Scientific, Inc.). Protein concentrations were measured using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of protein extract were denatured by boiling at 90°C for 5 min. Proteins (20 µg) were fractionated on 4–15% Mini-PROTEAN TGX gels and transferred to nitrocellulose membranes (both from Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primary and secondary antibody reactions were performed using an iBind Flex Western System (Thermo Fisher Scientific, Inc.). Membranes were incubated with iBind Flex Solution (iBind Flex Buffer, iBind Flex Additive and distilled water) for 10 min at room temperature to block nonspecific binding. Primary (Bcl-xL, Mcl-1, STAT3, p-STAT3, STAT5, p-STAT5, PARP and GAPDH) and secondary (polyclonal goat anti-rabbit IGs conjugated to HRP) antibody reactions were performed at room temperature for 2.5 h, following the manufacturer’s protocol. Protein-antibody complexes were visualized using SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.). The immunoreactive protein band was detected and band density was quantified by densitometry using an Amersham Imager 600 (GE Healthcare Life Sciences, Little Chalfont, UK).

All statistical analyses were performed using EZR software (Easy R) version 1.27 (Saitama Medical Center, Jichi Medical University, Saitama, Japan). All data are presented as the mean ± standard deviation. Statistical comparisons with paired observations were determined using the Student’s t-test. Comparisons of more than two groups were made by a one-way analysis of variance followed by Dunnett’s test. P<0.05 was considered to indicate a statistically significant difference.

To investigate whether apigenin inhibits the proliferation of colon cancer cells, four colon cancer cell lines (HT29, DLD-1, COLO320 and HCT116) were treated with 0, 5, 15 and 50 µM apigenin for 48 h. Cell proliferation was measured using WST-1 (Fig. 1A) and BrdU (Fig. 1B) assays. The results demonstrated that apigenin significantly reduced the proliferation of all four colon cancer cell lines in a dose-dependent manner.

To investigate whether the inhibition of cell proliferation by apigenin was due to the induction of apoptosis, four colon cancer cell lines were exposed to various doses of apigenin for 24 h and the degree of DNA fragmentation in the cells was then measured. The results demonstrated that apigenin induced DNA fragmentation in a dose-dependent manner in all four colon cancer cell lines (Fig. 2A).

Subsequently, it was determined whether the effect of apigenin on apoptosis was mediated by caspase activation. The four colon cancer cell lines were treated with various doses of apigenin for 24 h and the cleavage of PARP was evaluated by western blotting. The results demonstrated that apigenin induced the cleavage of PARP in a dose-dependent manner (Fig. 2B). In HCT116 cells, the apoptosis-inducing effect of 50 µM apigenin was weaker than that of 15 µM apigenin. However, as presented in Fig. 1, the suppression of cell proliferation in HCT116 occurred in a dose-dependent manner, with 50 µM apigenin inducing the most marked inhibition. Thus, it was considered that this phenomenon may involve other processes, as well as apoptosis, such as necrosis. Therefore, only 5 and 15 µM apigenin were used to treat HCT116 cells in subsequent experiments investigating the effects of apigenin on the apoptosis of colon cancer cells.

The anti-apoptotic Bcl-2 family of proteins serves an important role in protecting against DNA damage-induced apoptosis. Therefore, the present study investigated the effect of apigenin on the expression of anti-apoptotic Bcl-2 family proteins. The four colon cancer cell lines were exposed to various doses of apigenin for 24 h and the expression of Bcl-xL and Mcl-1 were measured by western blotting. The results demonstrated that apigenin significantly downregulated the expression of Bcl-xL and Mcl-1 in all colon cancer cells lines in a dose-dependent manner (Fig. 3).

Apigenin downregulated the expression of Bcl-xL and Mcl-1; therefore, it was hypothesized that the anti-apoptotic Bcl-2 family proteins Bcl-xL and Mcl-1 are involved in apigenin-induced apoptosis. To determine whether this was the case, the DLD-1 and HCT116 cell lines were used. As a preliminary experiment, immunostaining of clinical specimens of colon cancer was performed. The results demonstrated that expression of Bcl-xL was very strong in colon cancer and that the expression of Mcl-1 was relatively weak (data not presented). Since these data were in accordance with the results of western blotting regarding Bcl-xL and Mcl-1 expression in the DLD-1 and HCT116 cell lines, the following experiments were performed on these two cell lines.

Cells were transfected with siRNA targeting Bcl-xL, Mcl-1, or Bcl-xL and Mcl-1 together. Transfection with the siRNAs downregulated Bcl-xL and MCl-1 expression in DLD-1 and HCT116 cells (Fig. 4A). To investigate whether transfection with siRNA inhibits colon cancer cell proliferation, DLD-1 and HCT116 cells were transfected with siRNA targeting Bcl-xL, Mcl-1, or Bcl-xL and Mcl-1 together for 48 h. Cell proliferation was measured using WST-1 (Fig. 4B) and BrdU assays (Fig. 4C). Downregulation of Bcl-xL alone or Mcl-1 alone reduced cell proliferation. However, the simultaneous downregulation of Bcl-xL and Mcl-1 significantly reduced the proliferation of the two cell lines.

Subsequently, it was determined whether transfection with siRNA induces apoptosis in colon cancer cells. DLD-1 and HCT116 cells were transfected with siRNA targeting Bcl-xL, Mcl-1, or Bcl-xL and Mcl-1 together for 24 h. Subsequently, the degree of DNA fragmentation in the cells was measured. Downregulation of Bcl-xL or Mcl-1 alone induced DNA fragmentation in DLD-1 and HCT116 cells. However, the simultaneous downregulation of Bcl-xL and Mcl-1 significantly induced DNA fragmentation (Fig. 4D).

Subsequently, it was determined whether the effect of the simultaneous downregulation of Bcl-xL and Mcl-1 on apoptosis was mediated by the activation of caspases. DLD-1 and HCT116 cells were transfected with siRNA targeting Bcl-xL, Mcl-1 or Bcl-xL and Mcl-1 together for 24 h and the degree of PARP cleavage was evaluated using western blotting. Downregulation of Bcl-xL or Mcl-1 alone slightly increased the cleavage of PARP in DLD-1 and HCT116 cells. However, the simultaneous downregulation of Bcl-xL and Mcl-1 significantly increased the cleavage of PARP in the two cell lines (Fig. 4E), which was consistent with the results of the cell proliferation and DNA fragmentation studies. These results suggest that the simultaneous downregulation of Bcl-xL and Mcl-1 strongly induces the apoptosis of colon cancer cells.

To further investigate the mechanism by which apigenin induces apoptosis, the effect of apigenin on the expression of p-STAT3 and STAT5 in DLD-1 and HCT116 cells was investigated. DLD-1 and HCT116 cells were exposed to various doses of apigenin for 24 h, then the expression of p-STAT3, p-STAT5 and total STAT3, STAT5 were measured by western blotting. Levels of p-STAT3 significantly decreased in a dose-dependent manner; however, apigenin treatment did not affect the total amount of STAT3 expression (Fig. 5A). Furthermore, apigenin did not affect the expression of p- and total STAT5 (Fig. 5B). Subsequently, it was determined whether Bcl-xL and Mcl-1 were down-regulated by STAT3 knockdown. DLD-1 and HCT116 cells were transfected with siRNA targeting STAT3 for 24 h and subsequently the expression of Bcl-xL and Mcl-1 were measured using western blotting. The results demonstrated that levels of Bcl-xL and Mcl-1 were significantly downregulated in DLD-1 and HCT116 cells following transfection with siSTAT3 (Fig. 5C).

It has been reported that IL-6 leads to STAT3 phosphorylation by activating janus kinase (22). DLD-1 and HCT116 cell lines were administered various doses of IL-6 for 48 h and the expression of p-STAT3 was measured using western blotting. The results indicated that p-STAT3 expression increased in a dose-dependent manner following treatment with IL-6 (Fig. 6A). Subsequently, 50 µM IL-6 was administered to cells 2 h following treatment with apigenin. After 48 h, levels of p-STAT3, Bcl-xL and Mcl-1 were measured and it was demonstrated that the expression of p-STAT3, Bcl-xL and Mcl-1 significantly increased following stimulation with IL-6; however, this increase was almost completely reversed following administration of apigenin (Fig. 6B). Therefore, it was suggested that apigenin suppresses the expression of Bcl-xL and Mcl-1 via STAT3 signaling (Fig. 6C).