Rapamycin and 3-methyladenine (3-MA) were purchased from Medchem Express Co., Ltd. (Shanghai, China). Baflomycin A1 (BA) was purchased from Beijing Hua MEIKO Biotechnology Co., Ltd. (Beijing, China). The Annexin V-FITC apoptosis detection kit was purchased from BD Biosciences (San Diego, CA, USA). Cell culture media, DMEM, RPMI-1640, and McCoy's5A were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cell Counting kit-8 (CCK-8), Hoechst 33258, Ad-mCherry-GFP-LC3B, dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), penicillin, streptomycin, monoclonal antibodies of β-actin (cat. no. AA128-1), horseradish peroxidase (HRP)-labeled goat anti-rat IgG(H+L) (#A0216) and HRP-labeled goat anti-rabbit IgG(H+L) (cat. no. A0208) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). LY294002, the caspase-3 (cat. no. ab32351), caspase-9 (cat. no. ab32539) and Poly(ADP-ribose) polymerase (PARP; cat. no. ab191217) antibodies were purchased from Abcam (Cambridge, UK). LC3B (cat. no. 3868), Beclin-1 (cat. no. 3495), PI3K (cat. no. 4292), p-PI3K at Tyr458 (cat. no. 4228), Akt (cat. no. 9272), p-Akt at Ser473 (cat. no. 9271), mTOR (cat. no. 2972) and p-mTOR at Ser2448 (cat. no. 2971) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Insulin-like growth factor-I (IGF-I) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

The human colorectal carcinoma cell lines, HCT-116, SW620, HCT8, HT29 and LoVo were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were grown in RPMI-1640, DMEM or McCoy's5A (Gibco; Thermo Fisher Scientific, Inc.) culture medium containing 10% heat-inactivated FBS, 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in 5% CO₂. Cells in the logarithmic phase were routinely renewed with fresh medium every 2–3 days.

Air-dried, powdered clove was purchased from Qingdao Company of Traditional Chinese Medicine (Qingdao, Shandong, China). The isolation and extraction of clove was performed as previously described (31). Briefly, 10.0 kg powdered clove was hydrated with 95% ethanol at room temperature for 72 h. Subsequent to filtration, the solution was concentrated to generate an ethanol extract and the extract was further extracted with ethyl acetate at 60°C for 2 h, and the supernant was concentrated using a vacuum rotary evaporator (Yamato Sci., Tokyo, Japan). The ethyl acetate extract of clove (EAEC) was fractionated using silica gel column chromatography (CC; 200 and 400 mesh) and Sephadex LH-20 CC. The resulting fraction was defined as active fraction from clove (AFC). The purity was characterized by the Department of Chemistry and Molecular Engineering at Qingdao University (Qingdao) using ¹H- and ¹³C-NMR analysis, confirming that ACF contained three main compounds, including eugenol (32.32%) and oleanonic Acid (23.60). AFC underwent a comprehensive residue screen for 172 pesticides by the Pacific Agricultural Laboratory (Portland, OR, USA) and no pesticides were detected. Similarly, AFC was analyzed for heavy metal content by Avomeen Analytical Services (Ann Arbor, MI, USA) and no heavy metal was detected.

AFC was diluted with cell culture medium to final concentrations of 25, 50, 100, 200 and 400 µg/ml for the treatment of HCT-116 cells for 24, 48 or 72 h. The PI3K inhibitor, LY294002, and the autophagylysosomal inhibitor, BA, were dissolved in DMSO and diluted with culture medium. The final concentration of DMSO in the test solutions was <0.1%. This concentration of DMSO did not cause any adverse responses in cells. 3-MA was dissolved in heated sterile double distilled water to achieve a 100 mM stock solution, and was then diluted with culture medium for a final concentration of 2 mM. IGF-I (a PI3K activator) was reconstituted to 10 µg/ml in sterile PBS, and diluted with culture medium for a final concentration of 50 ng/ml. HCT-116 cells were pretreated with 10 µM LY294002, 1 nM BA, 2 mM 3-MA or 50 ng/ml IGF-I for 1 h, then further treated with 100 µg/ml AFC for 48 h. Cells were treated with fresh medium without serum as a vehicle control.

Cell viability was determined by CCK-8 assay as previously described (12). Briefly, cells at 80–90% confluency were seeded in 96-well plates at a density of 5×10³ cells/well. After a 24-h incubation 25, 50, 100, 200 or 400 µg/ml AFC, or vehicle control, was added to the wells. After 24, 48 or 72 h 10% CCK-8 reagent (10 µl/well) was added and incubated for 1 h at 37°C. The absorbance value (optical density; OD) was measured at a wavelength of 450 nm with a SpectraMAX M3 microplate spectrophotometer (Molecular Devices Corporation, Sunnyvale, CA, USA) and the cell viability ratio was calculated using the following formula: Cell viability rate = (OD of the experimental group/OD of the control group) ×100%. The half maximal inhibitory concentration (IC₅₀) values were determined using a nonlinear best fit method by GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA). All experiments were performed 3 times in triplicate.

HCT-116 cells were seeded onto coverslips in a 24-well plate at a density of 5×10⁴ cells/well and were transfected with Ad-mCherry-GFP-LC3B. Following 24 h of culture, the cells were treated with AFC (100 µg/ml) or medium (control) for 48 h then fixed with 4% polyoxymethylene and observed with an EVOS™ FL Imaging system (AMF4300; Thermo Fisher Scientific Inc.). Autophagic cells presenting ≥5 mRFP-GFP-LC3 dots were counted. All experiments were performed 3 times in triplicate.

Morphological assessment of apoptotic cells was analyzed using Hoechst 33258 staining as previously described (31). Briefly, HCT-116 cells were seeded in a 24-well plate at a density of 5×10⁴ cells/well. Following culture for 24 h, the cells were treated with 25, 50 or 100 µg/ml AFC or the same volume of vehicle control. Following incubation for another 48 h, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature, and the medium was removed and washed with PBS for 15 min. The cells were treated with 10 µg/ml Hoechst 33258 staining at room temperature for 10 min, and washed with PBS for 15 min in the dark to reduce background. The morphology of the cells was visualized and photographed under a DMR fluorescence microscope at ×400 magnification (Leica Microsystems GmbH, Wetzlar, Germany) using fluorescence excitation at 340 nm. The apoptotic index was calculated using the following formula: Apoptotic index = apoptotic cell number/total cell number ×100%. A minimum of 4 fields of view of each well, containing ≥500 cells, were required to calculate the rate of apoptosis. All experiments were performed 3 times in triplicate.

HCT-116 cells were seeded in a 24-well plate at a density of 5×10⁴ cells/well. Following 24 h of culture, the cells were treated with 25, 50 and 100 µg/ml AFC or the equivalent volume of medium (vehicle control) for 48 h. The cells were collected by trypsinization and washed twice with PBS, then fixed in 2.5% glutaraldehyde for 90 min and post-fixed in 1% osmium tetraoxide for 30 min at room temperature. Subsequent to 3 washes with PBS, the cells were progressively dehydrated in an ascending alcohol series (50, 70, 95 and 100%), and embedded in Epon resin. The ultrathin sections were contrasted with uranyl acetate and lead citrate for electron microscopy observation. The ultrastructure of the cells was then examined under a transmission electron microscope (JEM-1230; JEOL, Ltd., Tokyo, Japan). All experiments were performed 3 times in triplicate.

AFC-induced apoptosis in HCT-116 cells was also evaluated by flow cytometry with Annexin V-FITC apoptosis detection kit (cat. no. 556547BD Pharmingen; BD Biosciences), according to the manufacturer's protocol. Briefly, HCT-116 cells at 80–90% confluency were seeded in 6-well plates at a density of 4×10⁵ cells/well and cultured for 24 h. Then, the cells were treated with 100 µg/ml AFC or vehicle control for 48 h. After treatment, the cells were harvested by cryogenic centrifugation at 4°C, 1,500 × g for 5 min and washed twice with 4°C PBS. The cells were resuspended in 1× binding buffer at a concentration of 1×10⁶ cells/ml. A total of 100 µl of the solution (1×10⁵ cells) was transferred to a 5-ml culture tube, and 5 µl Annexin V-FITC and 5 µl PI were successively added to the cells and incubated at room temperature in the dark for 15 min. The quantity of stained cells was analyzed using a flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). All experiments were performed 3 times in triplicate.

HCT-116 cells were seeded at a density of 4×10⁵ cells/well in 6-well plates, and the culture medium (2 ml) was replaced after 24 h in culture, with 25, 50 or 100 µl/ml AFC, or vehicle control for 24 h prior to harvesting. The cells were lysed on ice with radioimmunoprecipitation assay buffer [0.5% NP-40, 50 mM Tris-HCl, 120 mM NaCl, 1 mM EDTA, 0.1 mM Na₃VO₄, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 µg/ml leupeptin, pH 7.5] for 30 min in the presence of PhosSTOP (Roche Molecular Systems, Inc., Basel, Switzerland) with PMSF and then centrifuged at 9600 × g for 20 min at 4°C. The supernatant was collected and stored in aliquots at −80°C until analysis. Protein concentrations were determined using BCA protein assay kit (Beyotime Institution of Biotechnology, Shanghai, China) and equalized prior to loading. Equal amounts of protein (40 µg) were separated by 15% SDS-PAGE and blotted onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking with 5% non-fat dry milk or bovine serum albumin in 1× Tris-buffered saline with Tween (TBST; 20 mM Tris-HCl, 150 mM NaCl and 0.05% Tween-20) for 1 h at room temperature, the membranes were incubated with primary antibodies at a dilution of 1:1,000 (caspase-9, PARP, LC3B, Beclin-1, PI3K, p-PI3K at Tyr458, Akt, p-Akt at Ser473, mTOR, p-mTOR at Ser2448) and 1;5,000 (β-actin and caspase-3) at 4°C overnight. Then, the membranes were incubated secondary antibodies at a dilution of 1:2,000 at room temperature for 1 h. β-actin was used as a loading control. Following 3 washes in TBST, the membranes were developed by incubation with ECL Western detection reagents (Thermo Fisher Scientific Inc.). The specific protein bands were visualized using a chemiluminescence reagent (EMD Millipore) and imaged using a VersaDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA). All experiments were performed three times in triplicate. The relative protein expression was quantified by Image J software (National Institutes of Health, Bethesda, ML, USA). For each sample, the grayscale value of each band was normalized to that of corresponding β-actin and the ratio of LC3-II/I was calculated using the normalized value of LC3-II/LC3-I.

All experiments were repeated ≥3 times. Data were analyzed using SPSS 19.0 statistical software (IBM Corporation, Armonk, NY, USA) and expressed as the mean ± standard deviation. One-way analysis of variance and Tukey's test were used to compare the means among groups. P<0.05 was considered to indicate a statistically significant difference, and highly significant differences were indicated by P<0.01 and P<0.001.

We initially investigated the effects of AFC on various types of human colorectal cancer cells using a CCK-8 assay. The results demonstrated that AFC was able to significantly inhibit proliferation of HCT116, HT-29, SW620, HCT8 and LoVo cells (Fig. 1; Table I). Compared with other cells, HCT116 cells were the most sensitive to AFC. Therefore, HCT116 cells were selected for use in subsequent experiments to investigate the potential mechanisms of the effect AFC in human colorectal cancer cells.

The effects of AFC on cell viability of human colorectal cancer HCT-116 cells was investigated by CCK-8 assay. The results demonstrated that AFC inhibited the viability of HCT-116 cells in a dose- and time-dependent manner (Fig. 2A and B). Next, the effect of AFC on the induction of apoptosis of HCT-116 cells was investigated by Hoechst 33258 staining, which is commonly used to detect cell apoptosis by observation of chromatin condensation under a fluorescence microscope (31). As demonstrated in Fig. 2C, following treatment with AFC (25, 50 and 100 µg/ml) for 48 h, the cells exhibited typical apoptotic morphological features, including chromatin condensation, nuclear shrinkage and the formation of apoptotic bodies. Annexin V is a sensitive method for detection of early apoptosis of cancer cells by using fluorescein (FITC) as fluorescent probe (32). Therefore, the rate of apoptosis of HCT-116 cells was investigated by flow cytometric analysis using Annexin V-FITC/PI double staining. The results demonstrated that the percentage of apoptotic cells increased when HCT-116 cells were treated with AFC compared with control (Fig. 2D and E). To study the underlying mechanism of AFC-induced apoptosis, the cleavage of PARP, caspase-3, and caspase-9 was analyzed by western blotting. As demonstrated in Fig. 2F and G, AFC cleaved PARP, pro-caspase-3 and pro-caspase-9 into their active forms. These data indicate that AFC inhibits cell viability through inducing apoptosis of HCT-116 cells.

To study AFC-induced autophagy, the autophagic flow of LC3-puncta was firstly determined. HCT-116 cells were transfected with Ad-mCherry-GFP-LC3B and GFP-LC3 puncta were observed under a fluorescence microscope. As indicated by Fig. 3A, fluorescence of GFP-LC3 puncta was frequently observed in HCT-116 cells treated with AFC, whereas the cells treated with vehicle control exhibited a relatively homogeneous LC3 expression pattern. In addition, a large number of autophagic bodies and autophagylysosomes were observed in HCT-116 cells treated with AFC (Fig. 3B) using transmission electron microscopy.

It has been well established that the microtubule-associated protein 1A/1B-light chain 3 (LC3) is a central protein in the autophagy pathway and closely associated with autophagosome appearance. Thus, it serves as a reliable marker to monitor autophagy (33). Beclin-1 was the first gene identified to induce autophagy (34). Therefore, the effect of AFC on the expression of LC3 and Beclin-1 was next investigated. As demonstrated by Fig. 3C–E, the expression levels of LC3-II and Beclin-1 in HCT-116 cells were significantly increased by AFC in a dose- and time-dependent manner. These data indicated that AFC treatment not only results in apoptosis, but also induces autophagy in HCT-116 cells.

It has been documented that autophagy could facilitate cell survival in adverse microenvironments and that inhibition of autophagy leads to increased cytotoxicity and induction of apoptosis (35). The effect of inhibition of autophagy on apoptosis induced by AFC in HCT-116 cells was investigated using 2 inhibitors, 3-MA and BA. 3-MA suppresses class III phosphatidylinositol 3-kinase (PI3K), essential for the initiation of the early stages of autophagy (36), while BA, an inhibitor of the vacuolar-type ATPase, inhibits the fusion of autophagosomes with lysosomes, preventing autophagic degradation (37). As demonstrated in Fig. 4A, cell viability was significantly decreased in HCT-116 cells treated with the combination of AFC (100 µg/ml) and 3-MA (2 mM) or BA (1 nM). The resulting cell viability was 40.33±2.52 and 54.34±4.04%, respectively, compared with 73.25±5.13% for cells treated with AFC alone. This result indicated that the inhibitory effect of AFC on HCT-116 cell proliferation was enhanced by autophagic inhibitors of 3-MA and BA.

Recent studies suggest that increased LC3 expression could reflect either increased autophagosome formation, due to increased autophagic activity, or reduced autophagosome turnover (33). Therefore, the effects of AFC on LC3 expression in the presence of 3-MA or BA were also studied. As demonstrated in Fig. 4B, 2 mM 3-MA decreased the expression of LC3, but 1 nM BA increased the level of LC3 expression. The opposite effects of 3-MA and BA on LC3 are associated with blocking autophagy at different stages: 3-MA inhibits autophagosome formation, whereas BA prevents degradation of LC3 in autophagolysosomes and in turn increases the LC3 expression level (38). Additionally, 3-MA significantly decreased the LC3 expression induced by AFC, whereas BA increased the LC3 expression induced by AFC treatment (Fig. 4B). These data suggested that 3-MA and BA inhibited autophagy induced by AFC at different stages.

Furthermore, the effect of AFC on the expression of apoptotic genes in the presence of 3-MA or BA was determined. The expression of cleaved-PARP and cleaved-caspase-3 were increased significantly with combined treatment of AFC and 3-MA or BA compared with either AFC, 3-MA or BA alone in HCT-116 cells (P<0.05; Fig. 4C and D). Similar effects were evident in the Annexin V-FITC/PI double staining assay, in which combined treatment of AFC and 3-MA or BA increased apoptosis compared with AFC, 3-MA or BA alone in HCT-116 cells (Fig. 4E and F). These data suggest that autophagy induced by AFC exerted a suppressive effect on the apoptotic pathways of HCT-116 cells.

It has been well documented that the PI3K/Akt/mTOR signaling pathway serves a key role in regulating both apoptosis and autophagy through divergent pathways (39). Therefore, the effect of AFC on the PI3K/Akt/mTOR signaling pathway in HCT-116 cells was investigated by western blotting. Treatment with AFC inhibited PI3K phosphorylation, decreased the levels of phospho-Akt, and downregulated phospho-mTOR in a dose-dependent manner (Fig. 5A). The ratios of p-Akt/Akt and p-mTOR/mTOR were significantly decreased following AFC treatment (Fig. 5B). LY294002 is a well-characterized inhibitor of PI3K (40), and the effect of combined AFC (100 µg/ml) and LY294002 (10 µM) on the expression of p-Akt/Akt and p-mTOR/mTOR on HCT-116 cells was investigated by western blotting. The results revealed that the combination of AFC and LY294002 treatment was more effective in decreasing of the ratios of p-Akt/Akt (P<0.001) and p-mTOR/mTOR (P<0.05) compared with AFC or LY294002 alone (Fig. 5C and D). Insulin-like growth factor-I (IGF-I), a PI3K activator, is capable of upregulating PI3K expression, as well as its downstream targets, Akt and mTOR (41). IGF-I treatment (50 ng/ml) significantly increased the phosphorylation of Akt and mTOR (Fig. 5E and F). When cells were pretreated with IGF-I (50 ng/ml), the effect of AFC was significantly attenuated. Furthermore, significant differences in the expression of the LC3-II/LC3-I, cleaved-PARP/PARP and cleaved-caspase-3/procaspase-3 ratios were observed between AFC treatment alone and the combination of AFC and IGF-I (Fig. 5G and H). These data indicate that the effect of AFC on induction of apoptosis and autophagy is associated with inhibition of the PI3K/Akt/mTOR signaling pathway.