The Second Affiliated Hospital of Soochow University Institutional Animal Care and Use Committee approved this study. U87 and U251 human glioma cell lines were obtained from the Chinese Academy of Medical Sciences (Beijing, China). Cells were cultured in Dulbeccos modified Eagles medium (DMEM; HyClone Laboratories, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) in a 5% CO₂ atmosphere at 37°C. Primary antibodies against Skp2, P21, P27, p-AKT (S473), AKT, extracellular signal-regulated kinase (ERK), p-ERK, cyclin D, p-cyclin D, cleaved caspase-3 and −9, MMP2, MMP9 and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies were from Liankebio (Hangzhou, China). Lipofectamine 3000 was from Invitrogen (Carlsbad, CA, USA). PF was from Tianjin Shilan Technology Co., Ltd., (Tianjin, China) and had a purity of 98%. PF was diluted in DMEM at a stock concentration of 400 mM. Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan).

U87 and U251 cells (5×10³) were seeded in a 96-well plate. Cells were treated with different concentrations of PF for 24 and 48 h. At the end of the treatment period, 10 µl of CCK-8 were added to each well. The plates were incubated at 37°C for 1 h and the optical density at 450 nm was then determined on a Varioskan microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

U87 and U251 cells were seeded in a 6-well plate (1–1.5×10⁵/well) and treated with 15 and 20 mM PF for 24 h. The Annexin V-phycoerythrin (PE)/7-aminoactinomycin D (7-AAD) assay was performed using a kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturers instructions. Briefly, cells were washed twice with cold phosphate-buffered saline (PBS), resuspended in 100 µl binding buffer with 7-AAD and PE-conjugated anti-Annexin V antibody, and incubated for 15 min at room temperature in the dark before analysis by flow cytometry (Beckman Coulter Inc., Brea, CA, USA).

U87 and U251 cells were seeded in a 6-well plate (1–1.5×10⁵/well) and treated with 15 or 20 mM PF for 24 h. The cells were harvested and fixed overnight at 4°C with cold 70% ethanol. Cell pellets were resuspended in PBS at a concentration of 1×10⁶ cells/ml and then incubated with propidium iodide (PI)/RNAase staining buffer (BD Biosciences) at room temperature for 15 min. DNA content was determined by flow cytometry.

To assess the effect of PF on cell migration, U87 and U251 cells were seeded in the upper chamber of a Boyden chamber (Corning Inc., Corning, NY, USA) with 200 µl of DMEM supplemented with 1% FBS; 600 µl complete medium with 20% serum were added to the lower chamber along with indicated concentration of PF. After 24 h, cells remaining in the upper chamber were removed using cotton swabs, and those in the lower chamber were fixed with methanol, stained with 0.1% crystal violet and photographed under a microscope. The invasion assay was similarly performed with a Matrigel-coated Transwell chamber (BD Biosciences). Cells in three randomly selected fields were counted.

Cells were seeded in a 6-well plate at a density of 1.5–2×10⁵/well. When they reached 80% confluence, the cells were transfected with Skp2 cDNA or short interfering (si)RNA (sense, 5-GGAGUGACAAAGACUUUGUTT-3 and antisense, 5-ACAAAGUCUUUGUCACUCCTT-3) or empty vector (Shanghai GenePharma, Co., Ltd., Shanghai, China) using Lipofectamine 3000. At 48 h post-transfection, the cells were collected for analysis of cell proliferation, migration and invasion and for western blotting.

Total RNA was extracted with TRIzol reagent (Invitrogen) and reverse transcribed into cDNA using the PrimeScript II First Strand cDNA Synthesis kit (Takara Bio, Shiga Japan). PCR was performed using the SYBR Premix Ex Taq kit (Takara Bio) and the following forward and reverse primers: Skp2, 5-GAAGGGAGTCCCATGAAACA-3 and 5-GCTGAAGAGCAAGGGAGTG-3; and glyceraldehyde 3-phosphate dehydrogenase, 5-ACCCAGAAGACTGTGGATGG-3 and 5-CAGTGAGCTTCCCGTTCAG-3. The relative expression level of Skp2 was determined with the 2^−ΔΔCt method.

Harvested cells or tumor tissue samples were lysed with radioimmunoprecipitation buffer (Beyotime Institute of Biotechnology, Nantong, China) supplemented with protease and phosphatase inhibitors (Beyotime Institute of Biotechnology). A bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology) was used to determine protein concentration, and 30–50 µg were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10–12% gel and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) that was blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature and then probed overnight at 4°C with primary antibodies. The membrane was then washed with Tris-buffered saline/0.1% Tween-20 and incubated with secondary antibodies. Protein expression was detected by enhanced chemiluminescence (Millipore) and the signal intensity of protein bands was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA) to determine the relative expression levels of target protein to β-actin. The experiment was performed three times.

Female nude mice (6 weeks old) were purchased from Shanghai Slack Experimental Animal Center (Shanghai, China). U87 glioma cells (2×10⁶) in 100 µl saline were injected into the backs of the mice. Tumor size was measured with calipers; when tumors reached 100 mm³, mice were randomly divided into experimental and control groups that were treated with PF and saline, respectively, by intragastric administration at a dose of 1 g/kg in 0.2 ml every day for 18 days. Tumor size and body weight were measured two or three times per week. Animal experiments and use of human cancer cell lines were approved by the Second Affiliated Hospital of Soochow University Institutional Animal Care and Use Committee (Suzhou, China).

Statistical analyses were carried out using Prism v.5.0 software (GraphPad Inc., La Jolla, CA, USA). Differences between the groups were evaluated by one-way analysis of variance (three or more groups) or the independent t-test (two groups). Results are presented as mean ± SD. P<0.05 was considered statistically significant.

In order to investigate the effect of PF on glioma cell growth, the viability of U87 and U251 cells treated with different concentrations of PF for 24 and 48 h was evaluated with the CCK-8. The cell growth was decreased in a time- and dose-dependent manner by the PF treatment in both U87 and U251 cells (Fig. 1A). After the treatment with PF for 24 h, the half-maximal inhibitory concentration of U87 and U251 cells was approximately 20 mM.

PF has been shown to inhibit cancer cell growth. We investigated whether apoptosis is involved in this process with the Annexin V/PE/7-AAD assay. U87 and U251 cells were treated with 15 or 20 mM PF for 24 h. The rate of apoptosis increased from 7.4 to 22.5% in U87 cells and from 3.7 to 23.7% in U251 cells upon PF treatment (Fig. 1B), indicating that PF induces glioma cell apoptosis as reported in previous studies (19).

The effect of PF on cell cycle progression was evaluated by PI staining and flow cytometry. Treatment with PF for 24 h caused cells to arrest in G2/M phase, with the fraction increasing from 23.48 to 34.07% in U87 cells and from 27.34 to 52.11% in U251 cells (Fig. 1C).

To clarify the effects of PF on glioma cell proliferation and apoptosis, ERK, p-cyclin D1, cyclin D, and cleaved caspase-3 and −9 expression was evaluated by western blotting. PF treatment decreased p-ERK and cyclin D and increased cleaved caspase-3 and −9 levels in a dose-dependent manner (Fig. 1D), confirming the results of CCK-8 and flow cytometric analyses.

Since PF at concentrations of 5 and 10 mM did not inhibit the proliferation of U87 and U251 cells (Fig. 1), we chose these concentrations to study the effects of PF on glioma cell motility with the migration and invasion assays. The number of migrating U87 and U251 cells (Fig. 2A) and the number of glioma cells infiltrating the membrane (Fig. 2B) were decreased by PF treatment relative to the control group. These results demonstrate that PF inhibits glioma cell invasion.

To confirm the effects of PF on tumor cell migration and invasion, MMP2 and MMP9 levels were evaluated by western blotting. We found that the expression of both proteins was downregulated by treatment with PF (Fig. 2C); a similar trend was observed for Skp2 in the presence of 5 and 10 mM PF (Fig. 2D). Thus, PF likely inhibits glioma cell migration and invasion via modulation of Skp2 signaling.

Skp2 is known to play an oncogenic role in tumorigenesis. To test whether Skp2 is involved in the anti-glioma effects of PF, we evaluated the Skp2 expression in glioma cells treated with PF. Skp2 mRNA (Fig. 3A) and protein (Fig. 3B and C) levels were downregulated by PF treatment in U87 and U251 cells. We then examined whether Skp2 inactivation by PF influences the expression of downstream factors such as P21, P27 and p-AKT. PF inhibited the expression of p-AKT and induced that of P21 and P27 in both U87 and U251 cells (Fig. 3B and C). These results suggest that the antitumor activities of PF are mediated by Skp2 signaling.

To clarify the role of Skp2 in the antitumor activities of PF in glioma cells, U87 and U251 cells were transfected with Skp2 cDNA and treated with PF for 24 h, and cell growth and apoptosis were evaluated with the CCK-8 and Annexin V/PE/7-AAD assays, respectively. Cell proliferation was increased in cells overexpressing Skp2 (Fig. 4A); moreover, inhibition of cell growth by PF treatment was abolished (Fig. 4A), suggesting that the antitumor effects of PF are exerted at least in part via negative regulation of Skp2. This was confirmed by the finding that overexpressing Skp2 decreased the number of apoptotic U87 glioma cells and abrogated the pro-apoptotic effect of PF (Fig. 4B).

To evaluate the role of Skp2 in glioma cell motility, U87 and U251 cells were transfected with Skp2 cDNA and treated with PF for 24 h, and migration and invasion assays were performed. Skp2 overexpression increased the number of cells that infiltrated through the Transwell membrane and abrogated the inhibitory effect of PF on glioma cell invasion (Fig. 4C and D). Similar results were obtained for the migration assay (Fig. 5A). Skp2 overexpression increased Skp2 levels in glioma cells and reversed the inhibition of Skp2 and p-AKT and upregulation of P21 induced by PF (Fig. 5B and C). These results confirm that PF exerts antitumor effects via downregulation of Skp2 in glioma cells.

To examine the role of Skp2 in glioma in greater detail, RNA interference was carried out to suppress Skp2 expression in U87 and U251 cells. The cells were transfected with Skp2 siRNA and then treated with PF for 24 h. The growth rate decreased (Fig. 6A) whereas apoptosis was increased in U87 cells upon Skp2 knockdown (Fig. 6B), indicating that Skp2 deficiency increases the sensitivity of the cells to PF. We next investigated whether Skp2 is involved in the regulation of U87 and U251 cell invasion. The inhibitory effects of PF on glioma cell invasion were enhanced in cells transfected with Skp2 siRNA as compared to those treated with PF or Skp2 siRNA only (Fig. 6C and D). We also observed that Skp2 expression was suppressed and P21 expression was increased to a greater degree in Skp2-depleted cells treated with PF as compared to those subjected to either treatment alone (Fig. 6E and F).

We established a U87 cell mouse xenograft model to evaluate the in vivo effects of PF. Tumor volume was lower in PF-treated as compared to control mice (P<0.05; Fig. 7), whereas no difference in body weight was observed between the two groups. Consistent with our in vitro observations, Skp2 and p-AKT levels were lower while that of P21 was higher in the tumor tissue of mice treated with PF relative to that of control animals.