Gossypol (>98% pure) was purchased from LKT Laboratories, Inc. (Hanzhou, China) and reconstituted in DMSO (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). RPMI-1640 medium and fetal bovine serum (FBS) were obtained from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The Cell counting Kit-8 (CCK-8), 5,5′,6,6′-tetra-chloro-1,1′,3,3′-tetra-ethylbenzimidalyl-carbocyanineiodide (JC-1) and Annexin V/PI apoptosis detection kits were purchased from Beyotime Institute of Biotechnology (Nanjing, China). Antibodies against Caspase-3, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), microtubule-associated protein light chain 3 (LC3), Beclin-1, Cytochrome c (Cyt-c) and β-actin were commercially available from MBL International Co. (Boston, MA, USA).

The HT-29 human colon cancer cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere in a 5% CO₂ incubator at 37°C.

The growth-inhibitory effect of gossypol was determined using a CCK-8 assay. Briefly, the HT-29 human colon cancer cells were seeded at a density of 1×104 cells/100 µl/well in 96-well plates and were cultured overnight for attachment. Following 24 h of incubation, the HT-29 cells were incubated with gossypol (5, 10, 20, 40 or 80 µM/l) in 96-well plates for 24 h at 37°C. Following incubation, 10 µl CCK-8 solution was added to each well and incubated at 37°C for 4 h, and the absorbance was measured using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 450 nm.

Apoptosis kits were used to detect the effect of gossypol on the HT-29 cells. The cells (5×104 cells/well) were treated with gossypol (20 and 40 µM/l) for 24 h at 37°C. The cells were harvested and washed with ice-cold PBS, resuspended with binding buffer, and simultaneously stained with Annexin-V-FITC and PI for 5–15 min. The cells were then analyzed immediately using flow cytometry.

JC-1 staining was used to detect changes in ΔΨm. The cells (2×105 cells/well) were seeded in 6-well plates and treated with or without gossypol (20 and 40 µM/l) for 24 h. Following incubation; the collected cells were incubated with 5 µg/ml JC-1 in culture medium for 15 min at 37°C. The staining solution was removed and the cells were washed twice with JC-1 staining buffer. The cell-associated fluorescence was analyzed using a FACSCalibur flow cytometer.

The HT-29 cells were seeded at a density of 1×105 cells/well in 6-well plates and, following incubation for 24 h, the cells were treated with gossypol (20 and 40 µM/l) for 24 h and then harvested. The expression levels of Bcl-2, Caspase-3, Bax, LC3 and Beclin-1, and the release of mitochondrial Cyt-c were then assessed using western blot analysis. Proteins were extracted from HT-29 cells using a total protein extraction kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the instructions of the manufacturer. Protein concentrations were determined using a BCA protein assay kit (Beyotime Institute of Biotechnology). Aliquots of cell lysates containing 20 µl of protein were separated on a 12% SDS-PAGE gel, and transferred onto PVDF membranes. The membranes were blocked with TBST buffer containing 10 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.05% Tween-20, containing 5% skimmed milk, and were then incubated with polyclonal antibodies against Bcl-2 (cat. no. 2876; 1:500), Bax (cat. no. sc-493; 1:500), caspase-3 (sc-16647; 1:1,000), Cyt-c (cat. no. 250621; 1:1,000), LC3 (cat. no. 4108; 1:1,000), beclin-1 (cat. no. 4122; 1:500) and β-actin (cat. no. 4967; 1:1,000) overnight at 4°C, respectively. The membrane was then incubated with either HRP-conjugated goat anti-rabbit IgG (cat. no. sc-2005; 1:10,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or goat anti-mouse IgG (cat. no. sc-2004; 1:10,000; Santa Cruz Biotechnology, Inc.) secondary antibodies for 1.5 h at room temperature. Peroxidase activity was detected via ECL visualization of the bands. The images were analyzed and quantified using a Quantity One software version 4.6.2 (Bio-Rad Laboratories, Inc.).

Statistical analyses were performed using SPSS version 10.0 (SPSS Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation. Statistical comparisons were performed using Student's t-test. P<0.05 was considered significant a statistically significant difference between groups.

A CCK-8 assay was performed to assess the inhibitory effects of gossypol on the HT-29 cells. The cells were cultured with 5–80 µM/l gossypol for 12, 24 and 48 h. As shown in Fig. 1, gossypol inhibited the growth of HT-29 cells in a time- and dose-dependent manner, with a 12 h IC₅₀ of 31.20 µM/l, 24 h IC₅₀ of 23.60 µM/l and 48 h IC₅₀ of 17.97 µM/l. The two concentrations of 20 and 40 µM/l were selected for the following experiments.

To determine the induction of cell apoptosis by gossypol, an Annexin V and PI staining assay based on flow cytometry was used. As shown in Fig. 2, there were significant changes in the proportions of early and late apoptotic, or necrotic HT-29 cells following exposure to gossypol for 24 h. Compared with the control group, the proportion of early apoptotic cells increased from 5.88 to 46.24% and the proportions of late apoptotic cells increased from 4.67 to 28.55%.

A loss of ΔΨm was detected by the fluorescent dye, JC-1. As shown in Fig. 3, treatment with gossypol (20 and 40 µM/l) for 24 h induced a loss in the ΔΨm of the HT-29 cells, suggesting damage to the mitochondria.

To investigate the mechanism underlying gossypol-induced apoptosis, the effects of gossypol on the modification of apoptosis-associated proteins were examined. The results of the western blot analysis demonstrated that gossypol led to an increase in the protein levels of Bax, and decrease in the levels of Bcl-2 in the HT-29 cells following treatment with gossypol at concentrations of 20 and 40 µM/l (Fig. 4A and B). Gossypol also promoted the activation of caspase-3 (Fig. 4C).

The activity of autophagy in HT-29 cells was evaluated following treatment with gossypol. Alterations in the levels of autophagy marker proteins, LC3-I and LC3-II, in HT-29 cells were detected using western blot analysis. As shown in Fig. 5, the ratio of LC3-II/LC3-I and Beclin-1 were enhanced in a dose-dependent manner following exposure to gossypol (20 and 40 µM/l) for 24 h.