The Cell Counting Kit-8 (CCK-8) kit and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The Annexin V-fluorescein isothiocyanate (FITC) apoptosis staining kit and antibodies used in the study were purchased from Thermo Fisher Scientific, Inc. The lysis buffer (RABLYSIS1) and propidium iodide (PI; cat. no. P4170) were purchased from Sigma-Aldrich (Merck KGaA). WZY-321, a novel analog of Evo (Fig. 1), was designed via a scaffold hopping strategy. A series of newly-designed Evo analogs, including WZY-321, were synthesized in the laboratory of Dr Shengtao Xu at the China Pharmaceutical University (Nanjing, China) (25). The chemical structure of WZY-321 was characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR, and mass spectroscopy. The purity of the compound was determined by analytical high performance liquid chromatography and the biologically evaluated compound was determined to be 98% pure.

SHG-44 glioma cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). The human glioma cell line SWO-38 was established by the Department of Neurosurgery at the Jiangning Hospital (Nanjing, China) (26). The cells were cultured at 37°C in DMEM supplemented with 10% FBS, 200 mM L-glutamine, 100 U-ml penicillin, 100 µg-ml streptomycin, 100 mM sodium pyruvate and 1% non-essential amino acids. Cells were incubated at 37°C in a humidified incubator containing 5% CO₂. The medium was changed every 2 days and the cells were passaged every 3 days. The density of the seeded cells in different biological replicates was the same.

SHG-44 and SWO-38 cells (5×10³ cells-100 µl) were seeded into each well of a 96-well plate and pre-incubated for 24 h in a humidified incubator (37°C; 5% CO₂). Total volumes of 10 µl of various concentrations of WZY-321 (0, 5, 10, 30 and 50 µM) were added to the cells and incubated for 24 or 48 h. Following incubation, 10 µl CCK-8 solution was added to each well and incubated for 1–4 h. Cell viability was proportional to the optical density and was quantitatively measured by spectrophotometry (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 450 nm. The viability of cells in the control group incubated for 48 h was defined as 100% viable.

SHG-44 cells (1×10⁵ cells-well) were cultured in complete medium in 6-well plates for 24 h, and treated in triplicate with different concentrations of WZY-321 (10, 30 and 50 µM) for 24 h. The control cells were treated with vehicle [1% dimethyl sulfoxide (DMSO) in complete medium]. Cell morphology was observed under optical microscope (magnification, ×20). The cells were subsequently harvested, washed, and stained with PI and FITC-Annexin V in the dark at 25°C for 15 min using the Annexin V-FITC apoptosis staining kit, according to the manufacturer's protocols. The percentage of apoptotic cells was determined by flow cytometry using an FC500 cytometer (Beckman Coulter, Inc., Brea, CA, USA). Annexin V-FITC binding was analyzed by flow cytometry (excitation=488 nm; emission=350 nm) using the FITC signal detector (usually FL1), and PI staining by the phycoerythrin emission signal detector (usually FL2), using FlowJo 8.8 software (Tree Star, Inc., Ashland, OR, USA).

Progression through the cell cycle was assessed by flow cytometry DNA determination with PI. SHG-44 cells were seeded in 6-well plates (1×10⁵ cells-well) and incubated at 37°C for 24 h. Cells were incubated with WZY-321 at certain concentrations (0, 10, 30 and 50 µM). Cells treated with the solvent (DMSO) were included. Following 24 h of treatment, cells were fixed with 70% ethanol at −20°C for 12 h, treated with RNase, and stained with PI at 37°C for 30 min. Cellular DNA content for the cell cycle distribution analysis was measured using a flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). The percentages of cells in different phases of cell cycle were analyzed by ModFit 4.1 software (Verity Software House, Topsham, ME, USA).

SHG-44 cells were incubated at 37°C in the presence or absence of WZY-321 at a concentration of 0, 10, 30 and 50 µM for 24 h. 3-MA was used as an autophagy inhibitor and cells were pretreated with 2.5 mM 3-MA at 37°C for 12 h prior to treatment with WZY-321. Following incubation, cells were collected, centrifuged at 13,000 x g at 4°C for 15 min and washed twice with ice cold PBS. The pellets were re-suspended in lysis buffer. The cells were lysed on ice for 20 min and the lysates were centrifuged at 13,000 × g at 4°C for 15 min. The protein concentration in the supernatant was determined using bicinchoninic acid protein assay reagents. Equal amounts of protein (20 µg-well) were separated via SDS-PAGE on a 10% gel and transferred to a polyvinylidene fluoride Hybond-P membrane. Membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated with primary antibodies against: Beclin1 (cat. no. sc-48341); LC3 (cat. no. sc-271625); apoptosis regulator Bcl-2 (Bcl-2; cat. no. sc-509); cyclin-dependent kinase 1 (cdc2; cat no. sc-54); cyclin D1 (cat. no. sc-70899); poly (ADP-ribose) polymerase (cat. no. sc-136208); and β-actin (cat. no. sc-58673) or GAPDH (cat. no. sc-293335) (1:1,000 dilution; all Santa Cruz Biotechnology, Inc., Dallas, TX, USA) by gentle rotation overnight at 4°C. β-actin and GAPDH were used as loading controls. The membranes were subsequently washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:20,000 dilution; cat. no. sc-2489; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature and visualized using enhanced chemiluminescent reagent (EMD Millipore, Billerica, MA, USA). The densitometric analysis was performed using Image J 1.48 software (National Institutes of Health, Bethesda, MD, USA).

Values are expressed as the mean ± standard error of the mean calculated from three independent experiments. Statistical analysis was performed using Student's t-test (for two groups) or one-way analysis of variance followed by Duncan's multiple-range test (for three or more groups) Analysis was performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

SHG-44 and SWO-38 glioma (26) cells were pretreated with various concentrations of WZY-321 (5, 10, 30 or 50 µM) for 24 or 48 h. The number of viable cells was determined using the CCK-8 assay. As presented in Fig. 2, WZY-321 significantly reduced the viability of SHG-44 and SWO-38 cells in a dose-dependent manner. Treatment with 10 µM WZY-321 for 48 h reduced the cell count to ~30% compared with the control group. In addition, incubation with WZY-321 for 24 h significantly decreased the viability of SHG-44 glioma cells. Prolonged incubation of cells with WZY-321 for up to 48 h displayed little benefit. SWO-38 cells were less sensitive to WZY-321 compared with SHG-44 cells. The histological subtypes of SHG-44 and SWO-38 cells may explain this sensitivity (27). Therefore, in the subsequent studies, SHG-44 glioma cells were incubated with WZY-321 for 24 h prior to processing the samples for subsequent pharmacological tests.

SHG-44 cells treated with WZY-321 for 24 h displayed marked alterations in cell morphology, exhibiting chromatin condensation, which suggested the cytostatic effect of WZY-321 may be associated with cell apoptosis (Data not shown). To confirm whether WZY-321 is able to induce apoptosis in SHG-44 cells, vehicle- or WZY-321-treated SHG-44 cells were stained with Annexin V-PI for flow cytometry analysis. Externalization of phosphatidylserine (PS) from the inner leaflet to the outer leaflet of the plasma membrane is a distinct phenomenon observed in early apoptotic cells. Annexin V has high affinity for PS, and fluorochrome-labeled Annexin V may be used for the detection of apoptotic cells. By contrast, PI is used to detect necrotic cells due to its ability to permeate the damaged cell membrane. In the present study, flow cytometric analysis identified four groups of cells: i) Viable cells (Annexin V-, PI-); ii) early apoptotic cells (Annexin V+, PI-); iii) late apoptotic cells (Annexin V+, PI+); and iv) necrotic cells (Annexin V-, PI+). The percentage of apoptotic SHG-44 cells detected by Annexin V-FITC and PI double staining following incubation with various concentrations of WZY-321 (0, 10, 30 or 50 µM) for 24 h is presented in Fig. 3. The number of early-stage apoptotic SHG-44 cells was increased in the 50 µM WYZ-321-treated group (11.3%) compared with that in the control group (1.6%), in a dose-dependent manner. The upper left quadrants representing the necrotic cells were comparable in each group. However, the increase in the number of advanced stage apoptotic cells following treatment with WZY-321 was not significant. These results demonstrated that WZY-321 may dose-dependently induce apoptosis in SHG-44 cancer cells.

One of the mechanisms by which chemical antitumor agents inhibit cell proliferation is through the induction of cell cycle arrest (28). To determine this mechanism involved in the suppression of cell growth by WZY-321, the DNA content of the cell nuclei was detected by flow cytometry. SHG-44 cells were treated with varying concentrations of WZY-321 (0, 10, 30 and 50 µM). As presented in Fig. 4A and B, the number of cells which arrested at the G2-M phase increased from 45.9 to 51.6% in a dose-dependent manner, along with a concomitant decrease in the percentage of cells in the S and G1 phases. Western blot analysis was performed to determine the expression levels of cell cycle regulatory proteins. As displayed in Fig. 4C, treatment of SHG-44 cells with WZY-321 resulted in a decrease in the protein expression levels of cdc2 and cyclin D1, in a dose-dependent manner. These results suggest an association between WZY-321-induced apoptosis and the accelerated entry of cells into G2-M arrest.

The cytotoxicity of certain anti-tumor agents is induced by autophagy (29–31). To assess the association of autophagy with the pro-apoptotic effects of WZY-321, a number of autophagic markers were analyzed. Beclin1, a novel Bcl-2-interacting coiled-coil protein has structural similarity to yeast autophagy-related protein 6. The proven importance of Beclin1 in tumor suppression triggered research into the role of autophagy in tumor suppression. LC3 is the mammalian homolog of yeast autophagy-related gene 8. LC3-II is derived from LC3-I following cleavage from the C-terminal and serves an important role in the formation of autophagosomes, as it localizes to the outer and inner membranes. Beclin1 and LC3 are frequently-used markers for the evaluation of tumorigenesis in brain tumors. In the present study, SHG-44 glioma cells were incubated with varying concentrations of WZY-321 for 24 h and the expression levels of Beclin1 and LC3 were evaluated. As presented in Fig. 5, incubation with various concentrations of WZY-321 increased the expression levels of Beclin1 and LC3-II. Accordingly, statistical analysis of the immunoblotting confirmed the above findings (Fig. 5). Notably, while expression levels of Beclin1 and LC3-II in cells incubated with 30 µM WZY-321 were comparable with those treated with 10 µM WZY-321, levels in the 50 µM group were slightly higher compared with the untreated group for LC3-II-LC3-I (*P<0.05), and significantly higher for Beclin-1 (**P<0.01), indicating a dose-dependent induction of autophagy by WZY-321 in SHG-44 cells.

A complex association exists between autophagy and apoptosis. The activation of autophagy may potentiate cytotoxicity, or impede autophagic cell death. In order to probe whether enhanced autophagy contributed to the anti-cancer potential of WZY-321, or impeded the therapeutic effects of WZY-321, 3-MA was used as an autophagy inhibitor and the expression levels of Bcl-2 and Beclin1 were analyzed (Fig. 6). In WZY-321-treated SHG-44 cells, a marked decrease was observed in the protein expression of Bcl-2, confirming the apoptotic effect of WZY-321. However, this apoptotic effect was blocked when cells were pretreated with 3-MA prior to treatment with WZY-321. As presented in Fig. 6B, the expression level of Beclin1 was lower in 3-MA-pretreated SHG-44 cells treated with WZY-321 compared with cells treated with WZY-321 alone. In addition, the expression levels of Bcl-2 were higher in 3-MA-pretreated SHG-44 cells treated with WZY-321 compared with cells treated with WZY-321 alone.