The HCT-116 cell line was purchased from the China Center for Type Culture Collection. The cells were maintained in RPMI-1640 medium (HyClone; Cytiva) supplemented with 10% FBS (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.) and antibiotics (100 U/ml streptomycin and 100 U/ml penicillin) in an incubator at 37°C with 5% Co₂. A total of 24 Kunming mice, aged 4 weeks and weighing 20±2 g, were purchased from the Hubei Provincial Center for Disease Control and Prevention. The mice were housed at room temperature with 45–55% humidity and with a 10/14-h light/dark cycle under pathogen-free conditions. The health status of the mice was monitored daily. The humane endpoints were deterioration of their general condition, and the mice were sacrificed in the event of a body weight loss of >20%. All animal protocols were approved by the Hubei University of Traditional Chinese Medicine Ethics Committee.

The following five treatment groups were used in the experiments: Control (no curcumin or 5-FU); CUR(L) (10 µM curcumin); CUR(M) (20 µM curcumin); CUR(H) (30 µM curcumin); and 5-FU (500 µM 5-FU) groups. The control and 5-FU groups were used as blank and positive controls, respectively. The 5-FU injection was purchased from Tianjin Jinyao Amino Acid Co., Ltd. Curcumin (Sigma-Aldrich; Merck KGaA) was dissolved in DMSO.

The proliferation ability of HCT-116 cells that were treated with different doses of curcumin and 5-FU for 24, 36 and 48 h was analyzed using a CCK-8 assay. After the HCT-116 cells were seeded in a 96-well culture plate (Wuxi NEST Biotechnology Co., Ltd.) at a density of 5×10³ cells/well for 24 h, the cells were treated with three doses of curcumin and 5-FU for 24, 36 and 48 h. Subsequently, 10 µl CCK-8 dye (Dalian Meilun Biotechnology Co., Ltd.) was added to each well, and the cells were placed in an incubator for 3 h at 37°C. Finally, the absorbance was measured at 450 nm on a microplate reader (Bio-Rad Laboratories, Inc.). The inhibition rate of HCT-116 cell viability with different treatments was calculated according to the following equation: Inhibition rate (%)=[optical density (OD)_{control group}-OD_{treatment group}]/OD_{control group} ×100%.

Cell viability was detected using a colony formation assay. HCT-116 cells were seeded into a 6-well plate at a density of 3×10² cells/well for 24 h, followed by treatment with three different doses of curcumin and 5-FU for 12 h. HCT-116 cells were cultured with new medium for 2 weeks after the drug-containing medium was discarded. The cells were fixed with methanol-glacial acetic acid stationary solution (3:1) at room temperature for 10 min and stained with Giemsa at room temperature for 15 min. The following formula was used to calculate the colony formation inhibition rate: Colony formation inhibition rate=(control group colony number-experimental group colony number)/control group colony number ×100%.

The migration ability of HCT-116 cells was detected using a Transwell assay. Following treatment with curcumin and 5-FU for 12 h, HCT-116 cells were collected and seeded into the upper chamber of a Transwell plate at a density of 1×10⁵ cells in 200 µl serum-free medium and incubated for 24 h at 37°C. Medium containing 10% FBS (700 µl) was added to the lower chamber. Subsequently, HCT-116 cells that had migrated to the lower surface of the membrane were fixed in methanol-glacial acetic acid stationary solution (3:1) at room temperature for 10 min and stained with Giemsa at room temperature for 15 min. The number of migrated cells was counted in four random fields under an inverted microscope (Olympus CK-40; Olympus Corporation) at a magnification of ×100.

Flow cytometry can distinguish early apoptotic cells, late apoptotic cells and normal cells. The cells were seeded in 6-well plates at a density of 1×10⁶ cells/well, followed by a 24-h incubation at 37°C. The cells were then treated with different concentrations of curcumin and 5-FU for 24 and 48 h. The assay was performed using the Annexin V-FITC apoptosis detection kit (BestBio; Nanjing Fengfeng Biological Medicine Technology Co., Ltd.) according to the manufacturer's protocol. Subsequently, the cells were analyzed by flow cytometry (BD Accuri C6; BD Biosciences), and the data were analyzed using FlowJo™ software (version 10; FlowJo LLC).

Normal, early apoptotic, late apoptotic and necrotic cells may be detected by fluorescence microscopy with green fluorescence. The HCT-116 cells were seeded at a density of 5×10⁵ cells/well in 6-well plates in which a round cover slide was placed, followed by a 24-h incubation at 37°C. Subsequently, the cells were treated with different concentrations of curcumin and 5-FU for 24 h. The cover slides to which the cells attached were stained with 20 µl AO/EB (Sigma-Aldrich; Merck KGaA) and observed under a green fluorescence microscope (Nikon 80i; Nikon Corporation) at a magnification of ×200. A total of 1,000 cells in each group were counted, and the apoptosis rate was calculated according to the following equation: Apoptosis rate=(early apoptotic cells + late apoptotic cells)/1,000 ×100%.

The HCT-116 cell culture, drug treatment and cell collection methods were as described for the apoptosis assay based on Annexin V-FITC/PI flow cytometry. The cells were fixed and incubated with 400 µl PI dye solution and 100 µl RNase (100 µg/ml) at 4°C for 30 min. The cells were filtered using a 300 mesh (70 µm) cell strainer and analyzed by flow cytometry. The experimental results were analyzed using Modfit LT software (version 3.1; Verity Software House).

MMP-9 expression in HCT-116 cells was analyzed using a zymography assay. Following drug treatment for 12 h, the medicated medium was discarded, and the cells were washed twice with PBS. Subsequently, the cells were cultured with serum-free medium for another 24 h. The medium was harvested, and a 20-µl medium sample was analyzed in a zymography assay. The zymography assay was performed as previously described (18). The results were obtained using a gel imaging system (ChemiDoc XRS⁺; Bio-Rad Laboratories, Inc.).

Following curcumin and 5-FU treatment for 12 h, HCT-116 cells were collected and washed with PBS. The cells were suspended in PBS to a density of 4×10⁶/ml. The cancer cells were then labeled with 5 µM carboxyfluorescein succinimidyl amino ester (CFSE) in a 37°C incubator for 10 min. After washing with serum-free medium and PBS, the cancer cells were again suspended in PBS to a density of 2.5×10⁶/ml. The 4-week-old Kunming mice were divided into three groups (n=8 per group), and 5×10⁵ CFSE-labeled HCT-116 cells were injected via the tail vein. At 6 and 24 h after injection, the 24 mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg/kg body weight) and immediately sacrificed by cervical dislocation. The lungs were then harvested, dissected and fixed with 4% paraformaldehyde for 6 h at 4°C. Subsequently, the mouse lung samples were dehydrated in 20% sucrose. Frozen mouse lung sections were observed under a fluorescence microscope (Nikon 80i; Nikon Corporation; magnification, ×100) at 488 nm. Additionally, 20 fields of view of lung tissue sections were randomly selected, and the number of fluorescent nodules observed in each section was counted (19).

Total RNA was extracted from HCT-116 cells treated with curcumin (20 µM) and 5-FU (500 µM) for 24 h using TRIzol^® reagent (Thermo Fisher Scientific, Inc.). cDNA was synthesized from 1 µg RNA using the Bestar™ qPCR RT kit (DBI^® Bioscience). mRNA expression levels were assessed with a qPCR assay using SYBR Green Realtime PCR Master mix on a Bio-Rad CFX96 Real-Time PCR system (Bio-Rad Laboratories, Inc.). The assay was performed according to the manufacturer's protocol. The housekeeping gene used for normalization was β-actin. The primers (Tsinke Biological Technology Co., Ltd.) used were as follows: Fas forward, 5′-TCTGGTTCTTACGTCTGTTGC-3′ and reverse, 5′-CTGTGCAGTCCCTAGCTTTCC-3′; Fas-associated via death domain (FADD) forward, 5′-GGGAGTCACTGAGAATCTGGAA-3′ and reverse, 5′-GGCCTGCTGAACCTCTTGTAC-3′; caspase-8 forward, 5′-TTTCTGCCTACAGGGTCATGC-3′ and reverse, 5′-GCTGCTTCTCTCTTTGCTGAA-3′; caspase-3 forward, 5′-CATGGAAGCGAATCAATGGACT-3′ and reverse, 5′-CTGTACCAGACCGAGATGTCA-3′; and β-actin forward, 5′-TGCTGTCCCTGTATGCCTCT-3′ and reverse, 5′-TTTGATGTCACGCACGATTT-3′. The following thermocycling conditions were used: 95°C for 60 sec, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. Relative mRNA expression levels were determined using the 2^−∆∆Cq method (2). The experiments were performed with four repeats for each sample.

The effects of curcumin and 5-FU on the expression levels of Fas, FADD, caspase-8, caspase-3, NF-κB, E-cadherin and claudin-3 were analyzed using western blotting. Protein samples were obtained from HCT-116 cells that were treated with different concentrations of curcumin and 5-FU for 24 h using cell lysis buffer (Wuhan Servicebio Biotechnology Co., Ltd.). The protein concentration was detected using the BCA method (Beyotime Biotechnology Co., Ltd.). Total protein (20 µg) was separated using SDS-PAGE (12% gel) and transferred onto a nitrocellulose membrane (Millipore; Merck KGaA). Following blocking with Tris-buffered saline containing 1% Tween-20 and 5% fat-free milk powder, the membrane was incubated with the corresponding antibodies. The assay protocol has been previously described in detail (2). The following antibodies were used: CD95/Fas (cat. no. bs-0215R; rabbit polyclonal; 1:1,000 dilution; Beijing Biosynthesis Biotechnology Co., Ltd.), FADD (cat. no. SC-271520; mouse polyclonal; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.), caspase-8 (cat. no. bsm33190M; rabbit polyclonal; 1:1,000 dilution; Beijing Biosynthesis Biotechnology Co., Ltd.), cleaved caspase-8 (cat. no. Asp384; mouse polyclonal; 1:1,000 dilution; Cell Signaling Technology, Inc.), caspase-3 (cat. no. BS1067; rabbit polyclonal; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.), cleaved caspase-3 (cat. no. Asp175; rabbit polyclonal; 1:1,000 dilution; Cell Signaling Technology, Inc.), NF-κB (cat. no. bs-19789R; rabbit polyclonal; 1:1,000 dilution; Beijing Biosynthesis Biotechnology Co., Ltd.), E-cadherin (cat. no. 3195S; rabbit polyclonal; 1:1,000 dilution; Cell Signaling Technology, Inc.), claudin-3 (cat. no. 3195S; rabbit polyclonal; 1:1,000 dilution; Bioworld Technology, Inc.), β-actin (cat. no. SC-47778; mouse polyclonal; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.) and horseradish peroxidase immunoglobulin G antibody (cat. no. SE134, goat anti-rabbit, 1:1,000 dilution; and cat. no. SESE131, goat anti-mouse, 1:1,000 dilution; Beijing Solarbio Science & Technology Co., Ltd.). Human β-actin was used for normalization. Following incubation with secondary antibody and chemiluminescence reagent (Wuhan Servicebio Biotechnology Co., Ltd.), the blots of the proteins of interest were scanned using a gel imaging system (ChemiDoc XRS⁺; Bio-Rad Laboratories, Inc.) and the analysis was performed using Image Lab™ software (version 5.0; MCM DESIGN). The relative protein expression levels were calculated as follows: Relative expression of protein=(gray value of the band in the experimental group)/(gray value of the band in the control group).

All experiments were repeated three times. Data were analyzed using GraphPad Prism software (version 6.0; GraphPad Software, Inc.). Student's t-test and ANOVA followed by Tukey's post hoc test were used to compare different groups. P<0.05 was considered to indicate a statistically significant difference. Data are presented as the mean ± standard deviation (n=3).

The results of the CCK-8 assay revealed that the proliferation of cells in the curcumin and 5-FU groups differed compared with that in the blank control group (Fig. 1A). The inhibitory effect increased gradually with increasing curcumin concentration in a dose- and time-dependent manner (P<0.05). The cell proliferation inhibition rates of the CUR(H) group at 24, 36 and 48 h were not significantly different from those of the 5-FU group. Additionally, according to the colony formation assay results (Fig. 1B and C), the cell viability in each group was significantly inhibited compared with the blank control group (P<0.05), and the inhibition rate of CUR(H) (49.4±3.15%) was higher compared with that of 5-FU (48.8±2.50%). These data indicated that curcumin inhibited the viability and proliferation of HCT-116 cells, and its effect was comparable to that of 5-FU.

The results of the Transwell assay (Fig. 2A) revealed that numerous cells in the control group migrated into the membrane of the upper chamber, whereas curcumin treatment markedly reduced the cell migration rate (Fig. 2B). Notably, the inhibitory effect on migration increased gradually with increasing curcumin concentration in a dose-dependent manner (P<0.05). The inhibition rate in the Transwell assays was markedly increased from that of the control group to 44.64±2.60% in the CUR(L), 61.59±2.13% in the CUR(M), 86.53±0.72% in the CUR(H) and 70.15±7.04% in the 5-FU treatment groups. Furthermore, the zymography assay demonstrated that curcumin treatment decreased MMP-9 expression in HCT-116 cells (Fig. 2C and D). Overall, the data demonstrated that curcumin markedly inhibited the migration of HCT-116 cells. Notably, the inhibition of migration and MMP-9 expression in HCT-116 cells following curcumin treatment reached the levels observed with 5-FU treatment.

As shown in Fig. 3A and B, early and late apoptotic cells may be selected by flow cytometry. The apoptosis rate of each group was significantly different from that of the blank control group (P<0.05; Fig. 3C). The proportion of apoptotic cells increased from 3.6±0.60% in the control group to 23.7±1.01% in the CUR(L), 27.2±2.00% in the CUR(M), 34.6±3.00% in the CUR(H) and 34.9±3.01% in the 5-FU experimental groups following treatment for 24 h. The proportion of apoptotic cells increased from 22.07±2.00% in the control group to 37.08±2.51% in the CUR(L), 58.80±2.01% in the CUR(M), 89.20±2.03% in the CUR(H) and 90.02±4.51% in the 5-FU experimental groups following treatment for 48 h. At 24 and 48 h, the apoptosis rate of the CUR(H) group was not significantly different from that of the 5-FU group. Therefore, curcumin induced apoptosis of the HCT-116 cells in a dose- and time-dependent manner (P<0.05), and the ability of curcumin to induce the apoptosis of HCT-116 cells was comparable to that of 5-FU.

Following staining with AO/EB, normal cells emitted green or yellow-green fluorescence, early apoptotic cells emitted orange fluorescence, and late apoptotic cells emitted red fluorescence, with nuclear debris and apoptotic bodies. As shown in Fig. 3D, the morphology of cells in each group was markedly altered. The HCT-116 cells became smaller and more rounded following curcumin and 5-FU treatment compared with cells in the control group. Not only did the cell density decrease with the increase in drug concentration, but the proportion of early and late apoptotic cells also increased gradually. The apoptosis rates were as follows: Control, 4.30±0.86%; CUR(L), 23.576±1.01%; CUR(M), 33.16±1.02%; CUR(H), 43.2±1.08%; and 5-FU, 43.1±0.95%. The apoptosis rate of each group was significantly different from that of the blank control group (P<0.05), and the apoptosis rate of the CUR(H) group was not significantly different from that of the 5-FU group (Fig. 3E). Curcumin induced apoptosis in the HCT-116 cells in a dose-dependent manner (P<0.05). The percentages of early and late apoptotic cells increased gradually with an increase in drug concentration. These results were consistent with the flow cytometry results, in that curcumin induced apoptosis of HCT-116 cells, and its effect was comparable to that of 5-FU treatment.

As shown in Fig. 4A, the proportion of cells that were arrested in the S-phase in each experimental group was significantly different from that in the blank control group (P<0.05), and this proportion increased in a dose-dependent manner (P<0.05; Fig. 4B). The proportions of cells that were arrested in the G₀/G₁ and G₂/M phase were lower compared with those in the blank control group. The proportion of cells in S-phase increased from 17±0.60% in the control group to 23±0.81% in the CUR(L), 26.4±1.00% in the CUR(M), 45.83±0.40% in the CUR(H) and 42.8±3.00% in the 5-FU treatment groups. These data indicated that curcumin induced S-phase arrest in HCT-116 cells, and this effect reached the level of 5-FU treatment.

Tumor cells migrate through the blood to the lungs, where they accumulate by penetrating through the wall of the vessels. HCT-116 cells were fluorescence-labeled with CFSE and were injected into the mice via the tail vein. When the lungs were surgically removed from the mice, the ability of cells to aggregate and invade into the lungs were evaluated by counting the number of fluorescent nodules in the lungs. At 6 h after HCT-116 cells were injected (Fig. 4C), the numbers of cancer cells that had invaded the lungs of the treatment groups (20 µM curcumin and 500 µM 5-FU) were markedly decreased compared with those in the control group. As shown in Fig. 4D, the number of fluorescent nodules decreased from 255±2% in the control group to 150±2% (CUR 20 µM) and 160±2% (5-FU) in the experimental groups. At 24 h after injection, the body's immune system quickly cleared a large number of HCT-116 cells (Fig. 4C and D), and the numbers of HCT-116 cells in lung tissue samples from each group were reduced compared with those at 6 h. Only a small percentage of HCT-116 cells invaded the lung tissue, and their behavior determined the number of metastases. These data indicated that curcumin inhibited CRC cell metastasis in vivo.

The present study aimed to explore the possible molecular mechanisms of apoptosis through western blot analysis. Compared with the blank control group, the expression levels of Fas, FADD, caspase-8 and caspase-3 were found to be significantly upregulated (P<0.05) following curcumin (20 µM) and 5-FU (500 µM) treatment for 24 h (Fig. 5A).

Compared with the blank control group, the expression levels of Fas, FADD, cleaved caspase-8 and cleaved caspase-3 in each drug treatment group were significantly upregulated (P<0.05). As shown in Fig. 5B and C, the expression levels of Fas, FADD, cleaved caspase-8 and cleaved caspase-3 increased gradually with increasing curcumin concentrations (P<0.05). These data indicated that curcumin promoted the activation of the Fas death receptor signaling pathway to induce apoptosis in HCT-116 cells. Additionally, the relative protein expression levels of NF-κB and claudin-3 decreased gradually with increasing curcumin concentrations (Fig. 5B and C). Conversely, curcumin treatment upregulated the relative expression levels of E-cadherin. These data indicated that curcumin treatment not only regulated the apoptosis, but also the metastasis of CRC cells. Notably, the relative expression levels of these proteins in the CUR(H) group were significantly different from those in the 5-FU group (P<0.05). The data indicated that the regulatory effect of curcumin on the Fas death receptor signaling pathway and metastasis of HCT-116 cells could match or even exceed that of 5-FU treatment.