Sch B induces G0/G1 phase arrest by regulating the expression of cell cycle-related proteins in U87 and U251 cells

To further understand the mechanism underlying the Sch B-mediated growth-inhibitory effect in human glioma cells, we performed cell cycle analysis in U87 and U251 cells in the presence of Sch B. Sch B-mediated changes in the cell cycle of U87 and U251 cells are shown in Figure 3A and B. After treatment with Sch B for 48 h, the percentage of G0/G1-phase cells dramatically increased (from 63.2% to 82.5% for U87 cells, p<0.01; from 64.5% to 84.7% for U251 cells, p<0.01), whereas the percentage of cells in S-phase slightly decreased compared with that of control cells.

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Figure 3

Effect of Sch B on cell cycle progression in glioma cells. A. U87 and U251 cells were exposed to various concentrations of Sch B (0, 25, 50, 100 μmol/L) for 48 h and then analysed using flow cytometry. B. The percentages of the total cell population in the G0/G1, S, and G2/M phases of the cell cycle are calculated. C. Effects of Sch B on the protein levels of cyclin D1 and CDK4 in U87 and U251 cells were measured by western blot analysis, with β-actin as a loading control.

Next, we assessed the levels of the G0/G1 phase-related proteins cyclin D1 and cyclin-dependent kinase 4 (CDK4) using western blot analysis. As shown in Figure 3C, Sch B treatment reduced the expression of cyclin D1 and CDK4 in a dose-dependent manner, which is consistent with G0/G1 phase arrest induced by Sch B.

Sch B induces apoptosis in human glioma cells

To further examine whether Sch B restrained the growth of U87 and U251 cells through the induction of apoptosis, we performed annexin V/propidium iodide (PI) double staining followed by flow cytometric analysis after treating the cells with various doses of Sch B. One of the distinct phenomenon of apoptosis is the translocation of phosphatidylserine (PS) from the inner leaflet to the outer leaflet of the plasma membrane. As the human anticoagulant annexin-V has a high affinity for PS, apoptotic cells can be identified via fluorophore-labelled annexin-V. In addition, PI can detect necrotic cells due to its ability to enter disrupted cell membranes. Therefore, various cell populations can be easily distinguished using annexin V/PI staining. As shown in Figure 3A,B, The number of late apoptotic cells (from 4.2% to 65.6% for U87 cells, p<0.01; from 5.7% to 62.8% for U251 cells, p<0.01) was dramatically increased in a dose-dependent manner after Sch B treatment compared with the control group. Furthermore, morphological changes in the apoptotic cells were investigated using Hoechst 33342 staining. When U87 and U251 cells were exposed to different doses of Sch B for 48 h, the number of apoptotic cells containing condensed and fragmented nuclei increased significantly as the dose of Sch B increased (Figure 4C,D). These results indicate that the apoptotic pathway plays a pivotal role in the anti-proliferation effect of Sch B on U87 and U251 cells.

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Figure 4

Sch B induces apoptosis in U87 and U251 cells. U87 and U251 cells were exposed to various concentrations of Sch B for 48 h. A. Sch B treated cells were stained with annexin-V/propidium iodide (PI). The Q3 (annexin-V−/PI−), Q4 (annexin-V+/PI−) and Q2 (annexin-V+/PI+) quadrants represent the populations of normal, early apoptotic and late apoptotic cells, respectively. B. Percentages of surviving cells and early and late apoptotic cells are shown as the means±SD (n=3). C. Apoptotic nuclear morphological changes induced by Sch B (0, 25, 50, 100 μmol/L) for 48 h were observed using Hoechst 33342 staining in U87 and U251 cells. The arrowheads indicate apoptotic cells, which exhibited highly condensed and fragmented nuclear morphologies. D. Percentages of apoptotic cells are shown as the means ± SD (n = 3). *P<0.05, **P<0.01, ***P<0.001 compared with the control group.

Sch B decreases mitochondrial membrane potential (ΔΨm) in glioma cells

Mitochondria are key regulators of apoptosis, and apoptosis mediated by the mitochondrial pathway is often associated with decreased ΔΨm. To examine whether the mitochondrial membrane integrity is damaged by Sch B treatment, rhodamine 123 was used to determine ΔΨm in Sch-B treated glioma cells. Compared with the control cells, Sch B treatment induced a dose-dependent decrease in ΔΨm (Figure 5A,B). We found that more than 80% of U87 cells and 76% of U251 cells showed a reduction in ΔΨm after treatment with 100 μmol/L Sch B for 48 h. These findings suggest that Sch B could reduce the mitochondrial membrane potential and induce mitochondrial dysfunction in glioma cells, indicating that Sch B promotes glioma cell apoptosis through a mitochondrial-dependent apoptotic pathway.

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Figure 5

Sch B decreases the mitochondrial membrane potential (ΔΨm) in glioma cells. U87 and U251 cells were treated with Sch B (0, 25, 50, 100 μmol/L) for 48 h. A. Flow cytometric analysis of ΔΨm by rhodamine 123 staining. B. The corresponding histogram shows the percentages of cells with high ΔΨm (survival) and low ΔΨm (apoptosis). Values represent the means±SD (n=3). *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

Sch B-induced apoptosis via the regulation of caspase-3 and Bcl-2 family members in glioma cells

Many studies have shown that the Caspase family, Bcl-2 family and PARP play critical roles in apoptosis [16-18]. To investigate the potential molecular mechanisms responsible for Sch B-induced apoptosis of U87 and U251 cells, we detected the expression of apoptosis-related proteins (Bax, Bcl-2, cleaved caspase-3 and cleaved caspase-9) after treating the cells with various doses of Sch B for 48 h. As shown in Figure 6A, Sch B treatment leads to the upregulation of cleaved PARP, Bax, cleaved caspase-9, and cleaved caspase-3 and the downregulation of Bcl-2 in a dose-dependent manner. In addition, the Bcl-2 (antiapoptotic) to Bax (proapoptotic) ratio was significantly reduced compared with the control group (Figure 6B). In summary, Sch B promotes apoptosis through the regulation of apoptosis-related protein expression in glioma cells.

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Figure 6

Sch B induces apoptosis via the regulation of apoptosis-related proteins in glioma cells. A. After treatment with Sch B (0, 25, 50, 100 μmol/L) for 48 h, cell lysates of U87 and U251 cells were prepared, and the expression of cleaved caspase-3, cleaved PARP, Bax and Bcl-2 were analysed using western blot. β-actin was used as a loading control. B. The ratio of Bcl-2 to Bax was calculated by band density. The results were shown as the mean±SD compared with the control (designated as 1.00). *P<0.05; **P<0.01 compared with the control group.

Cell lines and culture

The human glioma U87 and U251 cell lines were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (CAS, Shanghai, China). All cells were cultured in high-glucose DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (Gibco), 100 μg/mL streptomycin, and 100 U/mL penicillin (HyClone, USA).The cells were cultured in an atmosphere containing 5% CO₂ at 37°C.

Cell viability assay

The viability of cells treated with Sch B was measured as previously described [33]. Briefly, cells were seeded into 96-well culture plates at 5,000 cells/well, incubated overnight, then treated with 0, 20, 40, 60, 80 and 160 μmol/L Sch B for 24, 48 and 72 h. Next, added 20 μL MTT solution (5 mg/mL) to each well for another 4 h after treatment. In order to solubilise the formazan crystals, 100 μL DMSO was added to each well. The absorbance was determined at 490 nm using a microplate reader (Bio-Tek, Winooski, VT, USA). The cell survival rate was measured as the absorbance compared with that of untreated controls. The results represent the average of 3 independent experiments.

Colony formation assay

Briefly, cells in the logarithmic growth phase were seeded into 6-well plates (Corning, Corning, NY, USA) at a density of 400 cells/well and allowed to grow for 24 h. The cells were then treated with Sch B (0, 4, 8 and 16 μmol/L) for another 48 h. The Sch B-containing medium was then discarded, and the cells were washed with PBS and cultured to form colonies in complete medium for another 14 days. Next, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and stained with 0.1% crystal violet (Sigma-Aldrich) for 30 min. After washing, the total number of colonies (>50 cells/colony) was counted manually and the stained colonies were photographed using a microscope (Leica, Wetzlar, Germany). The results were expressed as the average of three independent experiments.

Cell cycle analysis

U87 and U251 cells were treated with various concentrations of Sch B for 48 h. Both adherent and floating cells were harvested, washed twice in cold PBS, and fixed in 70% ethanol at 4°C overnight. After fixation, the cells were washed and incubated in staining buffer (10 mg/mL RNase and 1 mg/mL propidium iodide (Sigma-Aldrich)) at 37°C in the dark for 30 min. Subsequently, the samples were analysed using flow cytometry (BD Biosciences, San Diego, CA, USA). The percentages of cells in the G0/G1, S, and G2/M phases were calculated using Cell Quest acquisition software (BD Biosciences).

Cell apoptosis assay

Apoptosis was analysed using the annexin V/PI apoptosis kit according to the manufacturer’s recommendations (BD Biosciences, San Diego, CA, USA). Briefly, Two hundred thousand cells (2×10⁵) were first seeded into 6-well plates and treated with Sch B (0, 25, 50, and 100 μmol/L) for 48 h. The cells were then harvested and resuspended at a density of 1×10⁶ cells/mL in binding buffer. Then 5 μL annexin V-FITC and 5 μL PI (100 μg/mL) were added to the suspension cells. After incubation for 15 min in the dark at room temperature, 400 μL of the binding buffer was added to the cell suspension. The stained cells were then immediately analysed using flow cytometry (BD Biosciences, San Diego, CA, USA). Each sample was assayed in duplicate, and the experiment was repeated three times.

Hoechst 33342 staining assay

Nuclear fragmentation was determined by staining apoptotic nuclei with Hoechst 33342. U87 and U251 cells were treated with various concentrations of Sch B for 48 h, the cells were harvested, washed in cold PBS and fixed with acetic acid:ethanol (1:3) for 15 min at room temperature. The fixed cells were then washed and stained with 5 μg/mL Hoechst 33342 for 10 min. Digital images were captured under a fluorescence microscope (Leica, Germany).

Mitochondrial membrane potential (ΔΨm) assay

Rhodamine 123 (Rho123) was used to determine the mitochondrial membrane potential (ΔΨm) as previously described [34]. Briefly, after treatment with various concentrations of Sch B for 48 h, U87 and U251 cells were collected and washed twice with cold PBS. The cells were then stained with Rhodamine 123 (Sigma-Aldrich) for 30 min at 5% CO₂ and 37°C in the dark. The samples were then analysed using flow cytometry (BD Biosciences, San Diego, CA, USA). Each assay was carried out in triplicate, and the results were expressed as the mean±SD.

Western blot analysis

U87 and U251 cells were treated with Sch B (0, 25, 50 and 100 μmol/L) for 48 h, total protein was extracted using RIPA buffer (Beyotime Institute of Biotechnology, Beijing, China) supplemented with protease inhibitor (Roche Applied Science, Indianapolis, IN, USA) at 4°C for 5 min. The protein concentration was determined using the bicinchoninic acid (BCA) assay kit (Beyotime). Total protein samples (40 μg/lane) were separated using 10% SDS-PAGE and then electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% skim milk for 1–2 h at room temperature, the membranes were then incubated with the indicated primary antibodies against Bcl-2, Bax, cleaved PARP, cleaved caspase-9, cleaved caspase-3, CDK4, cyclin D1, and β-actin (1:1000) at 4°C overnight. The membranes were then washed with TBST buffer for 3×5 minutes and incubated with the secondary antibodies (HRP-conjugated goat anti-rabbit IgG) for 1 h at room temperature. The bands were detected using enhanced chemiluminescence and visualised by a Gel Doc 2000 (BioRad, Hercules, CA, USA).

In vivo tumour xenograft study

BALB/c homozygous (nu/nu) nude mice (6–8 weeks old, 18–20 g body weight) that had been bred in house were maintained in a specific pathogen-free environment. U87 cells in log-phase growth (2.5×10⁶) were suspended in 100 μL PBS and then subcutaneously injected into the left flank of the nude mice. Twenty-four hours post inoculation, the mice were randomly divided into three groups (6 mice/group). The control group was administered PBS intraperitoneally and the other two treatment groups were administered Sch B (25 and 100 mg/kg) intraperitoneally in a volume of 0.2 mL every 2 days for up to 20 days. On day 22, all mice were sacrificed, and the tumours were excised and weighed. The experimental procedure was conducted according to the institutional animal care guideline and approved by the Laboratory Animal Ethics Committee of Wenzhou Medical University with the reference number wydw2014-0026.

This work was supported by grants from the Zhejiang medical and health science and technology plan project (No.2012RCA042) and the Wenzhou science and technology bureau (No. Y20120008).