Celastrus orbiculatus plants (production batch no. 070510) were purchased from Guangzhou Zhixin Pharmaceutical Co., Ltd. (Guangzhou, China) in 2007 and extracted at the Department of Chinese Materia Medica Analysis, China Pharmaceutical University (Nanjing, China) as described previously (22). The chemical constituents of the stems of COE were investigated and compounds were isolated as described previously (24,25). The resultant COE micropowder was dissolved in DMSO (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and diluted to produce a range of different concentrations prior to use. The final concentration of DMSO in the cell culture did not exceed 0.1%.

The human esophageal squamous carcinoma ECA-109 cell line was obtained from the Cell Bank of Chinese Academy of Sciences, Shanghai Institute of Cell Biology (Shanghai, China). Cells were cultured in RPMI-1640 containing 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Waltham, MA, USA) in a 5% CO₂ incubator at 37°C in a humidified atmosphere.

A total of 2.0×10³ ECA-109 cells/well were seeded into 96-well plates and incubated at 37°C in a 5% CO₂ incubator for 24 h. Next, cells were treated with different concentrations of COE (320, 160, 80, 40 or 20 mg/l) for 24, 48 or 72 h. A negative control group consisting of untreated cells was also included. The plate was subjected to treatment with 5 mg/ml MTT (Sigma-Aldrich, Merck KGaA) dissolved in sterile PBS in dark for 4 h in a 5% CO₂ incubator at 37°C, and the optical density (OD) value was measured at 490 nm. The tests were independently performed ≥3 times. The cell viability rate was calculated as follows: (OD value of each concentration group/OD value of negative control group) ×100.

A total of 1×10⁶ ECA-109 cells treated with 0, 20, 40 or 80 mg/l COE for 24 h were harvested, and then fixed in 70% ethanol at 4°C for 2 h. After 24 h, the cells were washed twice with ice-cold PBS, stained with enough neat propidium iodide (PI)/RNase Staining Solution (Cell Signaling Technology, Inc., Danvers, MA, USA) at 25°C for 15 min in the dark. The measurements were performed using a FACSCaliber flow cytometer and Cell Quest Pro software version 349226 (BD Biosciences, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo 7.6 software (Tree Star, Inc., Ashland, OR, USA). The tests were performed ≥3 independent times.

A total volume of 1×10⁶ ECA-109 cells were treated with 0, 20, 40 or 80 mg/l COE for 24 h, and the harvested cells were washed twice with ice-cold PBS. Apoptotic cells were identified using the Annexin V-Fluorescein Isothiocyanate (FITC)/PI Apoptosis Detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). After centrifugation at 100 × g for 5 min at 4°C, 290 µl of 1X binding buffer, 5 µl of Annexin V-FITC and 5 µl of PI were added to the pellet, and incubated at room temperature (25°C) for 15 min in the dark. Next, 200 µl of 1X binding buffer was added prior to measurement. The data were measured and analyzed with the same machine and software as the cell cycle assay. The cell apoptosis assay was performed with 3 independent experiments.

The Mitochondrial Membrane Potential Detection kit JC-1 (Beyotime Institute of Biotechnology, Hangzhou, China) was used to measure the changes of the mitochondria membrane potential. Following treatment with 0, 20, 40 or 80 mg/l COE for 24 h, the cells were incubated with 1 ml JC-1 working solution for 20 min at 37°C in the dark, and then washed twice with JC-1 buffer. The results were observed using a fluorescence microscope at magnification, ×100.

ECA-109 cells in the logarithmic growth phase were incubated with 0, 20, 40 or 80 mg/l COE for 24 h. Cells were harvested and the supernatant was discarded. Cells were then washed twice with PBS, and 2.5% glutaraldehyde was added over 2 h at 4°C. Following a wash with 0.1 mM PBS, the cells were fixed in 1% osmium teroxide for 2 h at 4°C, and then washed, dehydrated in a graded alcohol series and acetone. Cells were embedded in different proportions of Epon 812 resin, sectioned at 50 nm and stained with uranyl acetate for 30 min and lead citrate for 15 min at room temperature, the cells were observed under a CM100 transmission electronic microscope at magnification, ×6,600 (Philips Medical Systems B.V., Eindhoven, The Netherlands).

DNA fragmentation was detected using the TUNEL technique with the In Situ Cell Apoptosis Detection kit, POD (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) performed according to the manufacturer's protocol. The cells grew prior on the glass slide were treated with COE at different concentrations for 24 h. Next, 4% (w/v) paraformaldehyde in PBS (pH 7.4) was used for fixation of cells (30 min at room temperature) and rinsed twice with PBS. The fixed cells were then incubated in permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 3 min at room temperature, and then the cells were incubated with blocking solution (3% H₂O₂ in methanol) for 10 min at 25°C and rinsed with PBS. Subsequently, 50 µl of reaction mixture containing TdT enzyme and nucleotide was added to the cells, and the cells were all incubated for 1 h at 37°C in the dark. The slides were washed with PBS and incubated with 50 µl streptavidin-horseradish peroxidase working solution for 30 min at 37°C in the dark, and rinsed three times with PBS. Finally, the cells incubated with DAB were analyzed by light microscopy.

Expression levels of B-cell lymphoma 2 (BCL2; Epitomics, Burlingame, California, USA; cat. no. S0820; dilution, 1:1,000), Bcl-2-associated X [Bax; Cell Signaling Technology (CST), Inc. Danvers, MA, USA; cat. no. 2772; dilution, 1:1,000], PI3K (CST; cat. no. 4249; dilution, 1:1,000), AKT (CST; cat. no. 4691; dilution, 1:1,000), phosphorylated (p)-AKT(Ser473) (CST; cat. no. 4058; dilution, 1:1,000), mTOR (CST; cat. no. 2983; dilution, 1:1,000), phosphorylated mTOR (Ser2448; CST; cat. no. 5536; dilution, 1:1,000) in ECA-109 cells treated with 0, 20, 40 or 80 mg/l COE were detected by western blot analysis. β-actin (CST; cat. no. 4970; dilution, 1:1,000) was used as a marker for cytosolic proteins. Lysate proteins were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked in blocking buffer (5% not-fat dry milk and 1% Tween-20 in PBS) for 2 h at room temperature, and then incubated with appropriate primary antibodies in blocking buffer at 4°C overnight. Subsequently, the membranes were washed with TBS containing Tween-20 to remove the residual primary antibodies mentioned above, and incubated with horseradish peroxidase conjugated goat anti-rabbit immunoglobulin G secondary antibodies (Huaan Biotechnology company, Hangzhou, Zhejiang, China; cat. no. HA-1001-100; dilution, 1:2,000) for 2 h at room temperature. Enhanced chemiluminescence was used to detect signals, using the Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) on a Molecular Imager Chemi Doc XRS System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The experiments were repeated 3 times. The bands from western blotting were quantified using Quantity One analysis software version 4.62 (Bio-Rad Laboratories, Inc.).

To investigate the effect of mTOR inhibition on cell growth, apoptosis and the cell cycle in ECA-109 cells further, confluent cell cultures were pretreated with 100 nM rapamycin (Sigma-Aldrich, Merck KGaA; cat. no. R0395) for 1 h and then incubated in the presence or absence of COE (80 mg/l) for 24 h. The cells were then subjected to the cell apoptosis assay, cell cycle analysis, evaluation of mitochondrial membrane depolarization, TUNEL assay, transmission electron microscopy and western blotting, as aforementioned.

Each experiment was repeated ≥3 times. The experimental results were analyzed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation. Comparison between two groups was performed using the Student's t-test. Statistical comparisons of more than two groups were performed by one-way analysis of variance with Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference. The graphs were obtained using GraphPad Prism 5.0 software (GraphPad, Inc., La Jolla, CA, USA).

To assess the anticancer activity of COE on ESCC, the effect of COE on the proliferation of the ESCC ECA-109 cell line was investigated. Morphologically, with increasing concentrations of COE, cells were increasingly shrunk and detached from the coverslip (Fig. 1A). As a result, the viability of cells decreased with COE treatment in a time- and dose-dependent manner, as assessed by MTT assay (Fig. 1B). The inhibitory effects on proliferation were not significant at 24 and 48 h in the 20 mg/l group.

The cell cycle assay demonstrated that COE produced a significant decrease in the number of cells in the G₂/M phase, and a significant accumulation in the number of cells in the G₀/G₁ phase. These findings clearly demonstrated that COE triggered G₀/G₁ cell cycle arrest in ECA-109 cells in a dose-dependent manner (Fig. 1C).

Next, whether apoptosis was responsible for the anticancer activity of COE was assessed. The results demonstrated that COE treatment led to the significant accumulation of cells in early-(Annexin V⁺/PI⁻) and late-stage (Annexin V⁺/PI⁺) apoptosis in a dose-dependent manner (Fig. 2A). The proportion of cells in early apoptosis significantly increased when COE was added, whereas the percentage of late apoptosis cells treated with 20 mg/l COE did not significantly change.

COE induced the loss of mitochondrial membrane potential (Fig. 2B), a classical marker of the activation of intrinsic apoptosis, which indicated that COE triggered mitochondrial apoptosis. This was indicated by the transition of JC-1 fluorescence from red to green gradually with the increasing concentration gradient.

To determine whether COE induces the DNA damage response, a TUNEL assay was performed to detect apoptotic nuclei in ECA-109 cells (Fig. 2C). The TUNEL staining revealed gradual and significant increases in the rate of apoptosis in the groups treated with 20, 40, and 80 mg/l COE.

To verify apoptosis, transmission electron microscopy was used to observe organelles in ECA-109 cells treated with COE (Fig. 2D). The results revealed that untreated cells possessed normal mitochondria and cytoplasm, whereas COE-treated cells exhibited the appearance of apoptotic bodies of various sizes. Swollen and transparent mitochondria were also observed by electron microscopy, which occurred in a DOE dose-dependent manner.

Western blot analysis demonstrated that treatment with COE significantly increased the ratio of Bax to Bcl-2 protein, whereby the effect was larger in the 40 mg/l compared with 80 mg/l treatment group (Fig. 3A). As presented in Fig. 3B, levels of PI3K, phosphorylated Akt, mTOR, phosphorylated mTOR were significantly lower in groups treated with COE compared with the control group, with no significant changes in Akt expression overall.

The combined treatment of rapamycin and COE significantly induced cell cycle arrest (Fig. 4A), and apoptosis (Fig. 4B) compared with treatment with COE alone. In addition, the effects of combination treatment on the decrease of mitochondrial membrane potential were significantly more evident compared with treatment with COE alone (Fig. 5A). Furthermore, apoptotic bodies induced by combination treatment were markedly more evident compared with COE alone (Fig. 5B). Western blot analysis indicated that the combination treatment significantly enhanced the observed changes that occurred following treatment with COE or rapamycin alone, including changes to the Bax/Bcl-2 ratio (Fig. 6A), and PI3K, Akt, p-Akt, mTOR and p-mTOR protein expression levels (Fig. 6B).