The human glioblastoma (GBM) cell lines U87 and LN229 were purchased from the Chinese Academy of Sciences Cell Bank. The cells were maintained at 37°C in a 5% CO₂ incubator in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Temozolomide (TMZ) was purchased from Sigma (USA). TMZ was dissolved in 10% dimethyl sulfoxide (DMSO; Sigma) to make a stock solution (TMZ, 100 mg/ml). DMSO concentration was maintained below 0.1% in all the cell cultures and did not exert any detectable effect on cell growth or cell death.

Target sequences for Src (5′-GGGCGAACCACCUGAA CAA-3′) and the control (5′-UUCUCCGAACGUGUCACGU-3′) were purchased from Sigma Genosys. Rabbit anti-human polyclonal antibodies against AKT, p-AKT, FAK, P-FAK and GAPDH; and the horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

For the cell viability assay, cells were plated on 96-well plates in triplicate and incubated for 24 h at 37°C in 5% CO₂. After incubation, cells were washed, serum and antibiotic-free medium were added and treated with the control or Src siRNA. After 6 h, cells were washed and incubated in serum-containing medium overnight. Afterwards, cells were washed and either regular or docetaxel-containing medium was added. After 72 h, the cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

U87 and LN229 cells were plated in 6-well plates and transfected with oligonucleotide. The apoptosis ratio was analyzed 48 h post-transfection using the Annexin V-FITC Apoptosis Detection kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. Annexin V-FITC and propidium iodide (PI) double staining was used to evaluate the percentage of apoptotic cells. Annexin V⁻/PI⁻ cells were used as the controls. Annexin V⁺/PI⁻ cells were evaluated as apoptotic and Annexin V⁺/PI⁺ cells were evaluated as necrotic. Tests were repeated in triplicate.

For western blotting, protein was abstracted from the treated cells. After the protein concentration was determined, equivalent amounts of proteins were boiled in sample buffers with DL-dithiothreitol (DTT) and then centrifuged at 12,000 rpm for 15 min at 4°C. The protein was separated by SDS-PAGE (PAGE, 15% gel for MAP1LC3; 10% gel for Beclin 1). Separated proteins on the gel were transferred onto PVDF membranes by an electroblot apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Filters were blocked for 2 h in 5% low-fat milk and incubated with the rabbit anti-human antibody for Src, AKT, p-AKT, FAK, p-FAK and GAPDH at 4°C for 12 h. Membranes were then washed with PBS-T and incubated with HRP-conjugated anti-goat antibody for 1 h. Specific signals were detected from the quantitative gel and western blot imaging system (Becton-Dickinson, Franklin Lakes, NJ, USA).

Female nude mice, 5-weeks old, were subcutaneously injected with LN229 and U87 glioma cells. When the tumor volumes reached 50 mm³, the mice were randomly divided into groups: the control groups, Src siRNA group, TMZ group, Src siRNA plus TMZ group, AKT siRNA group, AKT siRNA plus TMZ group and Src siRNA, TMZ plus AKT group and received the relevant treatment with a local injection in the xenograft tumor in multiple sites. The treatment was performed once every 2 days for 20 days, and the tumor volume was measured with a caliper every 2 days, using the formula: Volume = length × width²/2. At the end of the 20-day observation period, the mice bearing xenograft tumors were sacrificed.

Statistical analyses were performed using SPSS version 13.0 software (SPSS Inc., Chicago, IL, USA). Multiple groups were compared using analysis of variance (ANOVA) followed by post hoc Fisher’s least significant difference (LSD) testing when appropriate. Data were considered statistically significant at P<0.05. All data are expressed as the means ± standard deviation (SD).

To determine the expression of Src in glioblastoma and low-grade glioma tissue, the mRNA and protein expression of 10 GBM and 10 low-grade gliomas was determined using RT-PCR and immunohistochemistry (IHC). Both mRNA and protein expression of Src was overexpressed in glioblastoma relative to that in the low-grade gliomas (Fig. 1). Thus, Src is overexpressed in GBM, and may contribute to its metastatic progression.

As demonstrated, Src was overexpressed in the GBM samples. Therefore, we subsequently evaluated the in vivo biological effect of Src gene silencing. U87 and LN229 cells were harvested 48 h after transfection with Src siRNA, and the expression of total Src was determined by RT-PCR and western blot assay. Both mRNA and protein expression of Src was significantly reduced both in the U87 and LN229 cells (Fig. 2). As expected, Src silencing resulted in decreased phosphorylation of downstream proteins such as FAK and AKT (Fig. 2B). Next, we examined the effects of Src gene silencing on cell viability. In both cell lines, Src silencing resulted in lower cell viability compared with the untreated and control siRNA groups (P<0.05; Fig. 3A).

To measure the effect of Src expression on tumor cell apoptosis, we transfected U87 and LN229 cells with Src siRNA or a scrambled control. After 48 h of transfection, apoptosis was measured by flow cytometry. Statistically significant increases in Annexin V⁺ apoptotic cells were observed in the Src siRNA-treated group compared to the untreated or scramble controls (Fig. 3B).

Based on prior in vitro data, we next assessed the in vivo therapeutic efficacy of Src silencing. Seven days after i.p. injection of tumor cells, mice were randomly allocated to 1 of 4 treatment groups. Mice were sacrificed when animals in any group became moribund. As expected, tumor growth was significantly reduced in the control siRNA TMZ group (Fig. 4A). Src siRNA also resulted in significant growth inhibition compared with the control siRNA group (Fig. 4). Combination of Src siRNA plus TMZ resulted in the greatest tumor reduction compared with the control siRNA grop (Fig. 4).

To determine the potential mechanisms responsible for the therapeutic effects of Src silencing and TMZ, we examined the in vivo therapeutic efficacy of AKT suppression. Tumor growth was significantly reduced in the AKT siRNA group. Combination of AKT siRNA and TMZ resulted in the greatest tumor reduction compared with the control siRNA (Fig. 5A and B). In addtion, we found that enhanced expression of AKT could recover the survival of glioma cells after Src silencing and TMZ (Fig. 5C and D). Thus, we concluded that Src enhances the cytotoxic effect of temozolomide by AKT regulation in glioma.