Cap, DHC (purity>99%) and trypsin were purchased from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). Cell Counting Kit-8 (CCK-8), Fluo-3AM, GENMED mitochondrial permeability transition pore (MPTP) living cell fluorescence detection kit and dimethyl sulfoxide (DMSO) were purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). U251 cells were obtained from the National Platform of Experimental Cell Resources for Sci-Tech (Beijing, China). L929 cells were obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit and Cell Cycle Detection kit were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Caspase-3 activity assay kit, caspase-9 activity assay kit, Rhodamine 123 (Rh123), ROS assay kit and cytochrome C (cyto c) antibody (catalog no. AC909) were purchased from Beyotime Institute of Biotechnology (Haimen, China). UltraSensitive™ surface protein array (mouse/rabbit) immunohistochemistry (IHC) kit and 3,3′-diaminobenzidine (DAB) kit were obtained from Fuzhou Maixin Biotech Co., Ltd. (Fuzhou, China). Inverted fluorescence microscope and confocal laser scanning microscope were obtained from Nikon Corporation (Tokyo, Japan). Flow cytometry equipment was obtained from BD Biosciences (Franklin Lakes, NJ, USA).

U251 human glioma cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2 mM L-glutamine and 1% penicillin-streptomycin solution. L929 murine fibroblast cells were maintained in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 10 mM HEPES, 2 mM L-glutamine and 1% penicillin-streptomycin solution. All cultures were maintained at 37°C in a humidified chamber of 95% air and 5% CO₂. Cap and DHC were dissolved in 0.5% DMSO solution.

Cell inhibition rate and cell survival were assessed by tetrazolium salt-based colorimetric detection in the CCK-8 assay. Cells were seeded in 96-well plates at an initial density of 5×10³ cells/well. Following exposure to 50, 100, 150 and 200 µM Cap or DHC for 12 (U251 only), 24, 48 and 72 h, 10 µl CCK-8 solution was added to each well and the plate was incubated for 1 h. Cell viability was determined with a microplate reader by reading the absorbance at 450 nm (A₄₅₀). The percentage of viable cells was calculated as follows: Cell viability (%)=[A₄₅₀(treated)-A₄₅₀ (blank)]/[A₄₅₀(control)-A₄₅₀(blank)]x100%.

U251 cells (2×10⁵) were plated in 6-well plates and allowed to attach for 24 h prior to exposure to 200 µM Cap and DHC for 12 h at 37°C. Apoptosis induced by Cap or DHC was evaluated using an Annexin V-FITC Apoptosis Detection kit according to the manufacturer's protocol. Briefly, U251 cells were detached, collected and suspended in 500 µl binding buffer, then 5 µl Annexin V-FITC and 5 µl propidium iodide (PI) was added and cells were incubated for 10 min at room temperature in the dark. Apoptotic cells were acquired using a FACS-Calibur flow cytometer (BD Biosciences) and the data was analyzed using CellQuest™ Pro software (BD Biosciences) and ModFit LT™ software (Verity Software House, Inc., Topsham, ME, USE).

U251 cells (1×10⁵/ml) were exposed to 200 µM Cap or DHC for 12 h at 37°C, detached and collected, postfixed in 1% buffered OsO₄ for 2 h at 37°C, dehydrated in graded alcohol and embedded in Epon resin. Sections (60-nm thick) were cut and observed using TEM. A total of 5 slides were visualized, with 10 fields analyzed per slide.

U251 cells (2×10⁵ cells/well) were plated in 6-well plates and allowed to attach for 24 h prior to exposure to 200 µM Cap or DHC for 12 h at 37°C. Control cells were treated with DMSO only. The cell cycle was analyzed using the Cell Cycle Detection kit according to the manufacturer's protocol. The cells were harvested, washed with phosphate-buffered saline (PBS), resuspended in Reagent A (100 µl), and incubated for 10 min at room temperature. Reagent B (100 µl) was then added, and plates incubated for a further 10 min at room temperature. Cells were stained with 150 µl PI for 15 min at room temperature in the dark, then fluorescence measured with a Becton-Dickinson FACS-Calibur flow cytometer and the data was analyzed using CellQuest Pro software and ModFit LT software.

Changes in intracellular ROS levels were detected by recording the oxidative conversion of cell permeable 2′,7′,-dichlorofluorescin diacetate (DCFH-DA) to fluorescent dichlorofluorescein (DCF) by flow cytometry. Following treatment with 200 µM Cap or DHC for 12 h, 2×10⁵ cells were washed twice with PBS, then incubated with DCFH-DA at 37°C for 20 min. Cells were subsequently washed three times with serum-free DMEM, then collected and resuspended in PBS. The fluorescent signal intensity of DCF was detected using a FACS-Calibur flow cytometer and the data was analyzed using CellQuest Pro software and ModFit LT software.

Following 12 h treatment with 200 µM Cap or DHC, 2×10⁵ U251 cells were washed 3 times with HEPES-buffered salt solution (HBSS), then incubated with Fluo 3-AM (final concentration, 5 µM) at 37°C for 30 min in the dark. Cells were then washed 3 times with HBSS and incubated in fresh HBSS for a further 30 min to remove the remaining Fluo 3-AM. [Ca²⁺]i was then detected using a laser scanning confocal microscope, with 6 fields assessed per group.

MPTP were detected using GENMED MPTP living cell fluorescence detection kits. U251 cells (2×10⁵ cells/well) were plated in 6-well plates and allowed to attach for 24 h, then exposed to 200 µM Cap or DHC for 12 h at 37°C. Following washing with Reagent A, the cells were incubated with 500 µl Reagent B and Reagent C at 37°C for 20 min in the dark. Subsequently, cells were washed twice with Reagent A, and the fluorescence detected with an inverted fluorescence microscope using 488 nm excitation (Ex) and 505 nm emission (Em) filter settings, with 6 fields assessed per group.

Rh123 was used to estimate MMP. Treated U251 cells (1×10⁵ cells treated with 200 µM Cap or DHC for 12 h at 37°C) were harvested and washed with PBS, then incubated with Rh123 (10 µl/ml) in PBS for 30 min in the dark at 37°C. Following 2 washes with PBS, the fluorescence was measured with the laser scanning confocal microscope using 507 nm Ex and 529 nm Em filter settings.

Caspase-9 and caspase-3 activity was detected with caspase-9 and caspase-3 activity kits according to the manufacturer's protocols, based on the ability of the enzymes to hydrolyze acetyl-Asp-Glu-Val-Asp phospho (p)-nitroaniline (caspase-3) or acetyl-Leu-Glu-His-Asp-p-nitroanilide (caspase-9) into the yellow formazan product, p-nitroanilide. U251 cells (2×10⁵ cells/well) were plated in 6-well plates and allowed to attach for 24 h, followed by exposure to 200 µM Cap or DHC for 12 h at 37°C. Treated cells were washed with cold PBS, then lysed with lysis buffer (100 µl per 2×10⁶ cells) for 15 min on ice, and centrifuged for 15 min at 4°C at 16,000 × g. Supernatant (10 µl) was then mixed with 80 µl reaction buffer and 10 µl 2 mM caspase-9 and caspase-3 substrate in 96-well plates, and incubated at 37°C for 2 h. The caspase-9 and caspase-3 relative activity was evaluated with a microplate reader at 405 nm.

A total of 40 male BALB/c nude mice (weight, 18–22 g) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The use of nude mice and their treatment was approved by the Institutional Animal Care and Use Committee, Tsinghua University (Beijing, China), and all experiments were carried out in strict compliance with their regulations. Nude mouse xenograft models of human glioma were established by injecting 1.0×10⁷ U251 cells subcutaneously in the right hind leg of BALB/c nude mice, with treatment beginning after one week. Mice were divided randomly into 6 groups with 6–7 mice in each group. Cap/DHC was dissolved in 5% ethanol and 5% Tween-80. ‘Control group’ mice received PBS (0.1 ml/20 g) by oral gavage. ‘Vehicle group’ mice received the same volume (0.1 ml/20 g) of vehicle (5% ethanol+5% Tween-80). ‘Cap (5 mg/kg) group’ mice received 5 mg Cap/kg by oral gavage every three days. ‘Cap (10 mg/kg) group’ mice received 10 mg Cap/kg by oral gavage every three days. ‘DHC (5 mg/kg) group’ mice received 5 mg DHC/kg by oral gavage every three days. ‘DHC (10 mg/kg) group’ mice received 10 mg DHC/kg by oral gavage every three days. Tumors were measured by vernier calipers and tumor size was calculated using the following formula: Volume (mm³)=π(axb²)/6, where a is the long diameter and b is the short diameter. Each mouse was weighed every three days. The mice were sacrificed with CO₂ 3 weeks following treatment. The tumor, stomach, heart, spleen, lung, liver, and kidney were weighed and fixed in 10% formalin solution for histological analysis by hematoxylin and eosin (H&E) staining. Sections (5-µm thick) were stained as previously described (22). Sections were observed under a TH4-200 light microscope (Olympus Corporation, Tokyo, Japan); 10 fields were examined per mouse.

For IHC analysis of cyto c, sections (5 µm thick) were cut, dewaxed, rehydrated and immersed in a 10 nM sodium citrate buffer (pH 6.0) and boiled for 30 min in a pressure cooker while maintaining the pressure of 181 kpa, then cooled and washed in PBS. Endogenous peroxidase activity was blocked with Immunol Staining Blocking buffer (Beyotime Institute of Biotechnology) for 1 h at room temperature to reduce nonspecific background staining. Subsequently, cyto C primary antibody diluted 1:1,000 in Immunol Staining Primary Antibody Dilution buffer (Beyotime Institute of Biotechnology) was added to the slides, incubated for 1 h at room temperature, then washed in PBS. Biotinylated anti-mouse immunoglobulin diluted 1:1,000 in Immunol Staining Secondary Antibody Dilution buffer (Beyotime Institute of Biotechnology) and streptavidin conjugated to horseradish peroxidase, from the cyto C antibody kit, were subsequently applied for 30 min at room temperature. Finally, DAB was used for color development according to the manufacturer's protocol and sections were counterstained with hematoxylin. Negative control slides in the absence of primary antibody were included for each staining. Sections were observed under a TH4-200 light microscope; 6 fields were examined per mouse.

Data are presented as the mean ± standard error for the indicated number of independently performed experiments. Data analyses were performed using SPSS v.20.0 (IBM SPSS, Armonk, NY, USA). Statistical significance was evaluated by one-way analysis of variation, followed by the least significant difference post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

The effects of Cap and DHC on the viability of U251 cells were examined by CCK-8 assay. Cap and DHC both significantly inhibited the proliferation of U251 cell lines in a dose- and time-dependent manner (Fig. 1A). Treatment of U251 cells for 72 h with 200 µM Cap and 200 µM DHC, resulted in 3.08±1.60 and 3.49±1.50% survival, respectively (P<0.001 and P<0.001, respectively; Fig. 1A), indicating that the majority of cells were killed by exposure to high concentrations of Cap and DHC. Cell viability of vehicle-treated cells (0.5% DMSO) remained >94.17%, indicating that 0.5% DMSO is not toxic to U251 cells (Fig. 1A). However, the viability of L929 normal mouse fibroblasts was not significantly affected by exposure to the equivalent concentrations of Cap and DHC that were highly cytotoxic to cancer cells (Fig. 1B).

Following exposure to Cap or DHC (200 µM) for 12 h, the proportion of U251 cells in the G₂/M phase significantly decreased from 16.50% in the control group to 5.78% with Cap (P=0.001; Fig. 2A) and 7.43% with DHC (P=0.001; Fig. 2A). However, the proportion of cells in the G₀/G₁ phase significantly increased from 71.47% in the control group to 75.78% with Cap (P=0.006; Fig. 2A) and 76.32% with DHC (P=0.003; Fig. 2A), while the proportion of cells in S phase also significantly increased from 12.02% in the control group to 18.43% with Cap (P=0.001; Fig. 2A) and 16.25% with DHC (P=0.006; Fig. 2A). The anti-tumor effect of Cap and DHC was, therefore, associated with G₀/G₁ and S phase cell-cycle arrest.

Annexin V-FITC/PI double staining assays were also performed to analyze cell apoptosis following 12 h treatment with 200 µM Cap or DHC. The proportion of apoptosed cells significantly increased from 14.16% in the DMSO-treated control group to 50.81% with Cap (P<0.001; Fig. 2B) and 42.79% with DHC (P<0.001; Fig. 2B). Necrosis was also significantly increased from 3.45% in the control group to 7.17% with Cap (P=0.001; Fig. 2B) and 11.15% with DHC (P<0.001; Fig. 2B).

TEM also revealed that treatment with either 200 µM Cap or 200 µM DHC for 12 h resulted in condensed cytoplasm, nuclei displaying peripheral chromatin condensation, which had broken up into rounded bodies, and cells developing into apoptotic bodies, compared with the integrated cellular morphology of the untreated cells (Fig. 2C).

ROS can initiate the loss of MMP, mitochondrial translocation of pro-apoptotic proteins, including Bcl-2 associated protein X apoptosis regulator (Bax) and Bcl-2 associated agonist of cell death (Bad), and release of cyto c (23). Intracellular ROS levels in U251 cells were, therefore, evaluated by DCFH-DA assays and flow cytometry detection. DCFH-DA is intracellularly hydrolyzed to DCFH, which is oxidized by ROS to generate fluorescent DCF. Following 12 h exposure to Cap or DHC, mean DCF fluorescence intensity in U251 cells increased from 3.45 in the control group to 5.84 with Cap and 6.41 with DHC (Fig. 3A).

High levels of Ca²⁺ can open mitochondrial permeability transition pores, depolarize mitochondrial membrane potential, activate caspase-9 and caspase-3, initiate the mitochondrial apoptosis pathway, to induce cell apoptosis (24). Fluo 3-AM was, therefore, used to observe the intracellular concentration of Ca²⁺. The mean fluorescence intensity in U251 cells increased from 333.79 in the control group to 1,116.46 (P<0.001) with Cap and 1,240.66 (P<0.001) with DHC, respectively (Fig. 3B), indicating an increase in the concentration of intracellular Ca²⁺.

MPTP formation is one of the principle regulators of the mitochondrion during cell apoptosis. The opening of MPTPs can lead to matrix swelling, rupture of the outer membrane of mitochondrion and a release of intermembrane space proteins into the cytoplasm, ultimately resulting in a loss of MMP. The presence of MPTPs was, therefore, detected using a GENMED MPTP living cell fluorescence detection kit. Following exposure to Cap or DHC for 12 h, the fluorescence intensity in U251 cells was decreased compared with the control group (Fig. 3C), indicating the formation of MPTPs.

Depolarization of the MMP induces release of cyto c and apoptosis-inducing factor (AIF) from the intermembrane space into the cytoplasm, resulting in fragmentation of nucleus and cell apoptosis (25). The mean fluorescence intensity in U251 cells decreased from 980.90 in the control group to 216.13 following 12 h exposure to 200 µM Cap (P<0.001) and 222.48 with 200 µM DHC (P<0.001), respectively (Fig. 3D), indicating the loss of MMP.

Apoptotic cell death is inevitable following caspase-3 activation, and is a vital factor in mitochondrial pathway-induced apoptosis. Caspase-9 (Fig. 4A and B) and −3 (Fig. 4C and D) activity were dose-dependently increased in U251 cells exposed to Cap or DHC for 12 h, compared with control group, indicating that Cap- and DHC-induced apoptosis is associated with the activation of caspase-9 and −3.

In vitro experiments are valuable for quick, convenient, and large-scale screening of latent anti-tumor agents, however the effects observed in cell-based assays require further investigation in suitable pre-clinical in vivo models prior to clinical trials. Tumor xenografts in nude mice is an accepted animal model for pre-clinical evaluation of potential anti-tumor agents. The effect of Cap and DHC on the development of U251 glioma tumor xenografts was, therefore, evaluated in nude mice. The rate of U251 glioma tumor xenograft development was attenuated in mice administered with Cap or DHC in both the low dose group (5 mg/kg, every 3 days) and high dose group (10 mg/kg, every 3 days; Fig. 5A). The mean tumor volume at 23 days in control mice (2316.48±211.28 mm³) was ~2.8-fold larger than tumors from the Cap (10 mg/kg)-treated group (821.15±215.63 mm³; P<0.001) and ~2.95-fold larger than tumors from the DHC (10 mg/kg)-treated group (785.83±218.31 mm³; P<0.001; Fig. 5A). Similar results were obtained with mice treated with 5 mg/kg Cap (P<0.001) and DHC (P<0.001) treated mice compared with control mice (Fig. 5A). The mean wet weight of tumors from control mice was 2.30±0.44 g, compared with 0.92±0.33 g in Cap (10 mg/kg)-treated mice (P<0.001; Fig. 5B) and 0.71±0.34 g in DHC (10 mg/kg)-treated mice (P<0.001; Fig. 5B). A similarly significant anti-tumor effect was demonstrated in mice treated with 5 mg/kg Cap or DHC compared with control mice (P<0.001 and P<0.001, respectively; Fig. 5B). However, the differences between high-dose and low-dose treatments with the same drug were not statistically significant (Fig. 5B). As previous reported, alcohol may certain antitumor effects (26). In addition, no difference in average body weight was observed between the control group, Cap- and DHC-treated mice throughout the experiment (data not shown). The liver index (liver weight/body weightx100) and spleen index (spleen weight/body weightx100) in Cap- and DHC-treated mice were also not significantly different compared with the control group (data not shown), indicating that Cap and DHC are not toxic to mice. Histological analysis of the tumors by H&E staining revealed liquefaction and necrosis in tumors from Cap- and DHC-treated mice, compared with vigorous growth in control and vehicle-treated mice (Fig. 5C). No ulcers or erosion, were observed in the stomachs of any mice, nor were any apparent side effects observed during the histological analysis of the hearts, spleens, lungs, livers and kidneys of the mice (data not shown). IHC analysis of cyto c expression revealed numerous cyto c-positive cells in the tumors of Cap- and DHC-treated mice, but few in the control or vehicle-treated groups (Fig. 5D), indicating that cyto c was released from mitochondria to the cytoplasmic matrix, inducing U251 cell apoptosis. Cap and DHC have, therefore, been demonstrated to significantly reduce U251 human glioma tumor development with no apparent negative side effects on the organs.