Purified colchicine (98.79%) was procured from Nanjing Zelang Medical Technological Co. Ltd. (Nanjing, China). Antibodies for p38 (cat. no. 9212), phosphorylated (P)-p38 (cat. no. 4092), c-Jun N-terminal kinase (JNK) (cat. no. 9252), P-JNK (cat. no. 81E11), extracellular signal-regulated kinase (ERK) (cat. no. 9107), P-ERK (cat. no. 4370), AKT (cat. no. 9272) and P-AKT (cat. no. 587F11) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and GAPDH (cat. no. ab181602) was obtained from Thermo Fisher Scientific, Inc. (Pittsburgh, PA, USA). Antibodies for caspase-3 (cat. no. ab32499), caspase-9 (cat. no. BMS1041) and Apaf-1 (cat. no. MA515743) were purchased from Abcam (Cambridge, MA, USA) and Hoechst 33258 and an AlexaFluor 488-conjugated Annexin V Apoptosis Detection kit were obtained from Molecular Probes Life Technologies (Carlsbad, CA, USA).

The human colon cancer HT-29 cell line was obtained from Shanghai Institute of Cell Biology (Shanghai, China). Cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (Gibco Life Technologies, Carlsbad, CA, USA), 100 U/ml penicillin G and 100 μg/ml streptomycin in an incubator at 37°C, 100% humidity and an atmosphere of 5% CO₂. The growth medium was replaced every 2–3 days.

The CCK-8 (Dojindo Molecular Technologies, Inc., Shanghai, China) was used to determine cell viability according to the manufacturer's instructions. Cells were plated at a density of 5×10³ cells/well in 96-well microtiter plates and cultured overnight at 37°C in a humidified incubator with a 5% CO₂ atmosphere. Following treatment with colchicine for 24, 48 or 72 h, the culture medium was replaced with 100 μl fresh medium followed by the addition of 10 μl CCK-8 solution. The cells were incubated for a further 2 h at 37°C, and the optical density was recorded at an absorbance of 450 nm. All experiments were performed in triplicate.

Cell apoptosis was detected by an Annexin V-(f luorescein isothiocyanate) FITC/propidium iodide (PI) Apoptosis Detection kit I (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. Cells were seeded in 6-well plates at the density of 1×10⁵ cells/well. Following incubation for 24 h at 37°C, colchicine was added to the 6-well plate and incubated for another 24 h. Cells were collected and washed twice with phosphate-buffered saline (PBS) and resuspended, subsequently aliquots of 1×10⁵ cells were transferred into 5-ml culture tubes in 100 μl 1X binding buffer (BD Biosciences). Annexin V-FITC (5 μl) and 5 μl PI were added to the resuspended cells. Following incubation at room temperature for 15 min in the dark, 400 μl binding buffer was added to the resuspended cells. Flow cytometry (FACSCalibur; BD Biosciences) was used to assess the apoptotic cells. The quantitation of apoptotic cells was calculated using CellQuest Pro software version 5.1 (BD Biosciences).

The nuclei were stained using Hoechst 33342 dye (Molecular Probes Life Technologies) to visualize nuclear morphology, as well as the induction of apoptosis. Following treatment with colchicine, the cells were incubated with 10 μM Hoechst 33342 dye for 10 min at 37°C. Images were obtained using a Leica DM IRB inverted fluorescence microscope (2886; Leica Microsystems GmbH, Wetzlar, Germany; magnification, ×400).

Δψm was analyzed using a JC-1 Δψm assay kit (Cayman Chemical Company, Ann Arbor, MI, USA), according to the manufacturer's instructions. In brief, HT-29 cells were incubated with 0, 1, 10 and 20 μg/ml colchicine for 12 h, centrifuged at 1,200 × g for 5 min and resuspended in RPMI-1640 medium. Following the addition of JC-1 to each well, the cells were incubated for an additional 30 min at 37°C. After washing with PBS, the stained cells were assayed using a flow cytometer (FACSCalibur; BD Biosciences).

Mitochondria were isolated from the untreated HT-29 cells to investigate the effect of colchicine on ROS production and the possibility of the mitochondria as a target of colchicine. Cells were washed twice in cold PBS, resuspended in hypotonic buffer (1 mM EDTA, 5 mM Tris-HCl (pH 7.5), 210 mM mannitol, 70 mM sucrose, 10 μM Leu-pep, 10 μM Pep-A and100 μM phenylmethanesulfonylfluoride; Molecular Probes Life Technologies), homogenized, then centrifuged at 500 × g for 5 min at 4°C to pellet the nuclear fraction. The supernatant was centrifuged at 12,000 rpm for 15 min at 4°C. The cytosolic supernatant was disposed of and the pellet (containing mitochondria) was resuspended in cold reaction buffer and treated with 10 μM colchicine. Amplex Red (Molecular Probes Life Technologies) in combination with horse-radish peroxidase was used to assess mitochondrial ROS production. Fluorescence (excitation, 560 nm; emission, 590 nm) was measured using a spectrofluorometer (SpectraMax Gemini XS; Molecular Devices, LLC, Sunnyvale, CA, USA) every 5 min for 5 h. The readings were analyzed using GraphPad Prism 6.0 (GraphPad Inc., La Jolla, CA, USA) and expressed as relative fluorescence units per microgram of protein. The data are representative of three independent experiments.

All experiments were repeated at least three times and values are expressed as the mean ± standard error. Statistical analysis was performed using two-way analysis of variance with GraphPad Prism 6.0 software. P<0.05 was considered to indicate a statistically significant difference between values.

Colchicine inhibits the proliferation of colon cancer cells in vitro. HT-29 cells were incubated with increasing doses of colchicine (0, 1, 10 and 20 ug/ml) or a vehicle control for 48 h, and cell viability was measured using a CCK-8 assay. As shown in Fig. 1, colchicine significantly inhibited the proliferation of HT-29 cells when the cells were treated with >20 μg/ml colchicine, compared with the control, and the cell viability decreased significantly in a dose-dependent manner. In addition, the result revealed that colchicine inhibited HT-29 cell proliferation in a time-dependent manner.

Colchicine inhibited cell proliferation and induced cellular death in colon cancer cells; however, the underlying mechanisms have not been investigated in detail. To establish whether this treatment could also induce apoptosis in HT-29 cells, flow cytometry was used to assess the percentage of apoptotic cells induced by colchicine treatment. Similar to the results of the cell viability assay, colchicine concentrations of >20 μg/ml significantly increased cell apoptosis compared with the respective control cells (Fig. 2A), and the increase was dose- and time-dependent. Treatment of colchicine at doses of 10 and 20 μg/ml for 24 h significantly increased the number of early apoptotic cells (Fig. 2A: Q1-LR) in a dose-dependent manner compared with control cells. The significant induction of apoptosis indicated the anticancer effect of colchicine against HT-29 cells. The colchicine-induced apoptosis in the HT-29 cells was morphologically identified using fluorescence staining with Hoechst 33258 (Fig. 2B); chromosomal condensation and nuclear fragmentation were observed in the colchicine-treated cells.

During the apoptotic process, mitochondrial membrane pores open and Δψm is disrupted. As shown in Fig. 3, the JC-1 staining data demonstrates that colchicine-treated cells exhibited a significant decrease in the Δψm in a dose-dependent manner, when compared with the control cells.

Mitochondria are critical in the initiation and progression of cell death processes and, therefore, may serve as targets for numerous chemotherapeutic agents. To determine whether the mitochondria are involved in colchicine-induced cell death, mitochondria were isolated from HT-29 cells and treated with 0, 1, 10 or 20 μg colchicine for 12 h. Staining with Amplex Red in combination with horse-radish peroxidase followed by flow cytometric analysis of the fluorescence showed that colchicine increased ROS production of HT-29 cells in a dose-dependent manner (Fig. 4).

The expression of apoptosis-associated proteins and the phosphorylation of kinases was detected by western blot and hybridization to clarify the mechanism of HT-29 cell apoptosis induced by colchicine treatment. As shown in Fig. 5, treatment with colchicine (0, 1, 10 and 20 μg/ml) for 12 h increased p38 phosphorylation dose-dependently and decreased AKT phosphorylation. However, colchicine treatment did not significantly affect phosphorylation of the JNK and ERK proteins. Furthermore, treatment with colchicine for 12 h evidently increased the protein expression of caspase-3 and Bax, and decreased Bcl-2 expression in a dose-dependent manner when compared with the control group (Fig. 5).