Female C57BL/6 mice (6–8 weeks, ~25 g) were purchased from the Beijing Laboratory Animal Research Center (Beijing, China) and housed in the temperature-controlled (25°C), light-cycled (12/12) Animal Facility of Xinjiang University (Urumqi, China). All animals received pathogen-free water and food.

The human esophageal carcinoma cell line (Eca-109) was preserved by the Xinjiang Key Laboratory of Biological Resources and Genetic Engineering (College of Life Science and Technology, Xinjiang University, Urumqi, China) and cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; MRC, EN MOASAI Biological Technology Co., Ltd, Jiangsu, China), 1% L-glutamine (100 mM), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂.

CTPG-W (cat. no. SGJG20170410) was purchased from Shanghai Upbio Tech Co., Ltd. (Shanghai, China). The major compounds of CTPG were qualified and quantified by HPLC according to our previous study (24). Briefly, HPLC was conducted on a ZORBAX SB-C18 Column (250×4.6 mm; 5 µm) at 30°C and eluted with 0.2% formic acid solution and a gradient of methanol starting at 23%, as 1 ml was added every min for 45 min until reaching 31%. A total of 10 µl sample was injected and detected at 330 nm. The echinacoside standard was purchased from Shanghai Baoban Biotech Co., Ltd. (Shanghai, China), and acteoside standard was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The standards were used to analyze the components of CTPG-W.

Cell proliferation was measured with an MTT assay. Eca-109 cells were inoculated into 96-well plates at a density of 5×10³ cells in 100 µl RPMI-1640 medium/well and cultured at 37°C for 24 h, then treated by different concentrations (0, 200, 400, 600 and 800 µg/ml) of CTPG-W or 0.4% dimethyl sulfoxide (DMSO) for 24, 48 and 72 h. DMSO was used as solvent control (800 µg/ml CTPG-W containing 0.4% DMSO). Cisplatin (20 µg/ml) was used as the positive control. Subsequently, the supernatant was discarded following centrifugation at 225 x g for 5 min at room temperature and 100 µl MTT solution (0.5 mg/ml in RPMI-1640 medium without FBS) was added to each well and incubated at 37°C for 3 h. The formed formazan crystals were dissolved in 200 µl DMSO. The optical density (OD) values were measured at a wavelength of 490 nm by a 96-well microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative cell viability was calculated according to the formula: Cell viability (%)=(OD_treated/OD_untreated)×100%. The morphology of Eca-109 cells was observed with an inverted fluorescence microscope (magnification, ×200) (Nikon Eclipse Ti-E; Nikon Corporation, Tokyo, Japan).

For the proliferation of splenocytes, C57BL/6 mice were euthanized by cervical dislocation and spleens were isolated. The single cell suspension was made and splenocytes were seeded into 96-well plates at a density of 1×10⁵ cells/well in 100 µl RPMI-1640 medium, and then treated with different concentrations (0, 200, 400, 600 and 800 µg/ml) of CTPG-W for 24, 48 and 72 h at 37°C with 5% CO₂. The proliferation of splenocytes was measured by MTT assay, according to the aforementioned protocol. Stimulatory index=OD_treated/OD_untreated.

Eca-109 cells were cultured in 60 mm dishes at a density of 2.5×10⁵ cells/dish for 24 h and treated with different concentrations (0, 200, 400, 600 and 800 µg/ml) of CTPG-W or 0.4% DMSO for 24 h at 37°C with 5% CO₂. Cells were collected and stained with an Annexin V-fluorescein isothiocyanate (FITC)/Propidium iodide (PI) Apoptosis Detection kit (Shanghai Shengsheng Biotechnology Co., Ltd., Shanghai, China), according to the manufacturer's protocols. Samples were collected by flow cytometry (BD FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by FlowJo 7.6 (Tree Star, Inc., Ashland, OR, USA). To analyze the effect of CTPG-W on the cell cycle, 2.5×10⁵ Eca-109 cells were seeded in 60 mm culture dishes and treated with CTPG-W (0, 100, 200 and 400 µg/ml) or 0.4% DMSO for 24 h at 37°C with 5% CO₂. All cells were harvested and washed twice with ice-cold PBS (Gibco; Thermo Fisher Scientific, Inc.), then fixed in 70% ice-cold ethanol at 4°C for 30 min. Following washing twice with ice-cold PBS, cells were resuspended in 300 µl PI/RNase staining buffer (BD Biosciences, San Jose, CA, USA) for 10 min at room temperature. The cell cycle distribution was analyzed with the ModFit LT 3.0 software by flow cytometry (BD FACSCalibur).

To analyze the Δψm, Eca-109 cells were treated with CTPG-W (0, 400, 600 and 800 µg/ml) or 0.4% DMSO for 24 h at 37°C with 5% CO₂, and stained with the Mitochondrial membrane potential assay kit with JC-1 (Beyotime Institute of Biotechnology, Shanghai, China), according to the manufacturer's protocol, for 20 min at 37°C. Following washing twice with JC-1 washing buffer (Beyotime Institute of Biotechnology), samples were resuspended with 300 µl JC-1 washing buffer and analyzed by flow cytometry (BD FACSCalibur). The fluorescence of JC-1 dye in Eca-109 cells was also observed with an inverted fluorescence microscope (magnification, ×200; Nikon Eclipse Ti-E). For analysis of ROS, Eca-109 cells were treated with CTPG-W (0, 400, 600 and 800 µg/ml) for 2, 4, 6, 12 and 24 h, and stained with Reactive Oxygen Species Assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol, for 20 min at 37°C. Following washing three times with ice-cold PBS, samples were collected by flow cytometry (BD FACSCalibur) and analyzed by FlowJo 7.6 software.

The free radical scavenging activity of CTPG-W was determined with a DPPH free radical assay according to the published protocol with a minor modification, as methanol was substituted with ethanol to dissolve DPPH (25,26). For steady state measurements, 150 µl DPPH (100 mmol/l) in ethanol was mixed with different concentrations of CTPG-W (25, 50, 75, 100, 250, 300, 400, 500 and 600 µg/ml) in 50 µl PBS, and incubated in the dark for 30 min at room temperature. The absorbance at 517 nm was detected in the presence and absence of CTPG-W. A total of 50 µl Vitamin C was used as the positive control. The DPPH radical scavenging activity was calculated using the formula: Scavenging (%)=[1-(A_sample-A_blank)/A₀] ×100, where A_blank is the absorbance of the control (without DPPH), A_sample is the absorbance of the sample and A₀ is the absorbance of PBS with DPPH.

Eca-109 cells were treated with CTPG-W (0, 200, 600 µg/ml) or 0.4% DMSO for 24 h at 37°C with 5% CO₂. Following washing twice with PBS, cells were lysed in Radioimmunoprecipitation Assay Lysis buffer (Beijing ComWin Biotech Co., Ltd., Beijing, China) for 20 min on ice. After centrifugation at 10,000 x g for 10 min at 4°C, the supernatants were collected, and protein concentrations were detected with a bicinchoninic acid kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Western blot analysis was conducted according to our previous description (24). The antibodies against caspase-7 (cat. no. D120077), caspase-8 (cat. no. D155240), caspase-9 (cat. no. D220078), B-cell lymphoma-2 (Bcl-2)-associated X (Bax) (cat. no. D220073) and Bcl-2 (cat. no. D260117), and anti-mouse IgG-horseradish peroxidase (HRP) (cat. no. D111050) and anti-rabbit IgG-HRP (cat. no. D110058) were purchased from BBI Life Sciences (Shanghai, China). The antibodies against caspase-3 (cat. no. E-AB-10050) and active caspase-3 (cat. no. E-AB-22115) were bought from Elabscience (Wuhan, China). Other antibodies against caspase-7 (cat. no. 9492), poly (ADP-ribose) polymerase (PARP) (cat. no. 9542), cytochrome c (cat. no. AC909), c-Jun NH₂-terminal kinase (JNK) (cat. no. 9252S) and β-actin (cat. no. 58169) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). All primary and secondary antibodies were diluted at 1:1,000. The primary antibodies were incubated at 4°C overnight and the secondary antibodies were incubated at 37°C for 1 h. The target proteins were detected using enhanced chemiluminescence assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol.

Statistical significance was calculated by one-way analysis of variance with Tukey's post hoc test and results were analyzed using GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA) among the treatment and control groups. All data were presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

The components of CTPG-W were qualified and quantified by HPLC (Fig. 1), which were compared with the standards of echinacoside and acteoside. According to the peak retention times and the peak areas, CTPG-W contained 39.16% of echinacoside and 2.44% of acteoside. Firstly, the effect of CTPG-W on the viability of Eca-109 cells was determined with an MTT assay. CTPG-W was dissolved in DMSO at 200 mg/ml and diluted with RPMI-1640 medium containing 10% heat-inactivated FBS to indicated concentrations. Eca-109 cells were treated with CTPG-W and cell viability was analyzed with an MTT assay at the indicated time points. CTPG-W treatment significantly reduced the viability of Eca-109 cells in a dose- and time-dependent manner (P<0.001; Fig. 2A). The morphology of Eca-109 cells was observed with an inverted fluorescence microscope (magnification, ×200) following CTPG-W treatment for 24 h, which changed notably in a dose-dependent manner, with the cells shrinking and becoming round following CTPG-W treatment (Fig. 2B). These results indicate that CTPG-W suppresses the growth of Eca-109 cells. The effect of CTPG-W on the proliferation of splenocytes was also detected with an MTT assay. CTPG-W significantly promoted the proliferation of splenocytes in a dose-dependent manner (Fig. 2C), indicating that it has no cytotoxic effect on splenocytes.

To investigate whether CTPG-W suppressed the growth of Eca-109 cells through the induction of apoptosis or necrosis, cells were treated with different concentrations of CTPG-W. After 24 h, the apoptosis and necrosis of Eca-109 cells were detected with Annexin V/PI staining. As depicted in Fig. 3A, Annexin V⁻/PI⁺ cells were gated as necrotic cells, and Annexin V⁺/PI⁺ and Annexin V⁺/PI⁻ cells were gated as apoptotic cells. CTPG-W primarily induced the apoptosis of Eca-109 cells in a dose-dependent manner, although the necrotic Eca-109 cells also increased significantly under the treatment of 600 and 800 µg/ml CTPG-W (P<0.001 at 600 µg/ml and P<0.05 at 800 µg/ml). Consistently, the levels of pro-apoptotic Bax and anti-apoptotic Bcl-2 in Eca-109 cells were upregulated and downregulated, respectively, upon CTPG-W treatment (Fig. 3B). The results indicated that CTPG-W primarily inhibited the growth of Eca-109 cells through the induction of apoptosis.

Disturbance of the cancer cell cycle will suppress cell growth and promote apoptosis (27). The distribution of the cell cycle in Eca-109 cells was detected with PI staining following CTPG-W treatment for 24 h. It was observed that cells in the S phase increased and cells in the G₀/G₁ phases indicated an overall significant decrease upon CTPG-W treatment (P<0.05; Fig. 4), indicating that CTPG-W arrests the Eca-109 cell cycle at the S phase.

CTPG-W decreases Δψm and induces the release of cytochrome c. Apoptosis can be induced by a mitochondrial-dependent pathway (28,29). The pro- and anti-apoptotic members of the BCL-2 protein family serve important roles in the regulation of mitochondrial membrane integrity (30,31). Following CTPG-W treatment for 24 h, the Δψm was assessed using JC-1 staining. JC-1 aggregate (red fluorescence detected in FL-2) will disintegrate into monomer (green fluorescence detected in FL-1) when Δψm is reducing (32). As depicted in Fig. 5A, the frequencies of FL-1⁺FL-2^−/+ cells increased significantly in a dose-dependent manner, indicating that the Δψm of Eca-109 cells decreased. The fluorescence changes in Eca-109 cells were also observed with an inverted fluorescence microscope (Fig. 5B). With the increasing concentrations of CTPG-W, the red fluorescence decreased and the green fluorescence increased, which is consistent with the data from flow cytometry. It was also observed that the level of cytochrome c in cytosol was notably increased (Fig. 5C), which is a result of a reduction of Δψm. This reinforces the conclusion drawn from the increased count of FL-1⁺FL-2^−/+ cells that Δψm decreased as a result of CTPG-W treatment. It has reported that JNK can regulate the activation of the BCL-2 protein family causing the release of cytochrome c (33–35). It was also determined that the level of JNK was notably upregulated following CTPG-W treatment (Fig. 5C). The results indicated that CTPG-W may induce the apoptosis of Eca-109 cells through a mitochondrial-dependent pathway.

ROS could reduce Δψm to induce apoptosis (36). To investigate whether CTPG-W can increase ROS production, Eca-109 cells were treated with different concentrations of CTPG-W. Cells were collected at the indicated time points and stained with DCFH-DA. The production of intracellular ROS in Eca-109 cells was determined by flow cytometry. As depicted in Fig. 6A, 800 µg/ml CTPG-W significantly increased ROS production from 2–6 h, and decreased from 12–24 h. Additionally, 400 µg/ml CTPG-W significantly increased ROS production from 12–24 h. Furthermore, 200 µg/ml CTPG-W did not notably alter ROS production. The dynamic changes of ROS production may be associated with Eca-109 cell apoptosis. It was also determined that CTPG-W had free radical scavenging activity (Fig. 6B), which may be associated with the decreased ROS production in Eca-109 cells treated with 800 µg/ml CTPG-W after 12 h.

The release of cytochrome c due to Δψm reduction could activate the caspase proteases to induce apoptosis (29,30,37). Following CTPG-W treatment for 24 h, the activation of caspase-3, 7, 8, 9 and PARP was detected by western blot analysis. Compared with the control, the levels of cleaved-caspase-9, cleaved-caspase-7, cleaved-caspase-3 and cleaved-PARP, but not the levels of cleaved-caspase-8, were upregulated by CTPG treatment in a dose-dependent manner (Fig. 7). These results indicated that CTPG-W reduced Δψm and promoted cytochrome c release to activate caspases that induce the apoptosis of Eca-109 cells.