Specimens of gliomas
This study was carried out at the First Affiliated Hospital of Sun Yat-sen University and the Guangdong General Hospital (Guangzhou, China). Archival formalin-fixed, paraffin-embedded specimens from 76 Chinese patients who underwent surgery from 2001 to 2009 were recruited. All patients had intracranial gliomas and no history of other malignancies. Histological sections of the primary resected surgical specimens were reviewed by authoritative pathologists according to the criteria of the WHO histological classification . All experimental protocols were carried out with the approval of the Committee on Use of Human & Animal Subjects in Teaching and Research of Sun Yat-sen University according to the Helsinki Declaration.
The patients were 52 males and 24 females with a median age of 47 years (range 8–76). Of these, 41 patients had high-grade gliomas, WHO grade III − IV, including 23 glioblastoma multiformes, 15 anaplastic astrocytomas and 3 anaplastic oligoastrocytomas. The other 35 had low-grade gliomas, WHO grade I–II, including 21 fibrillary astrocytomas, 5 pilocytic astrocytomas, 5 oligodendrogliomas, 3 ependymomas and 1 pleomorphic xanthoastrocytoma. Since gliomas may present as ill-defined lesions, various magnetic resonance imaging (MRI) sequence combinations do not provide a unique contour for tumor delineation. The extension of surgical resection was conducted by preoperative imaging. For presumed low-grade gliomas, manual segmentation was performed with region of interest analysis to measure tumor volumes (cm3) on the basis of FLAIR or T2 axial slices. For high-grade gliomas, a similar segmentation was made using the volume of contrast-enhancing tissue seen on T1-weighted MRI. After surgery, patients were followed up for a mean of 25.6 months (range, 3–58 months). None of the patients had received chemotherapy or radiotherapy before surgery.
After surgery, patients with high-grade gliomas, including glioblastoma multiforme and anaplastic astrocytoma, underwent conventional external-beam radiotherapy with a total dose of 60 Gy and continuous daily temozolomide (75 mg per m2 of body-surface area per day, 7 days per week from the first to the last day of radiotherapy), followed by six cycles of adjuvant temozolomide (150–200 mg per m2 for 5 days during each 28-day cycle). The patients with astrocytoma (WHO grade II) were treated with temozolomide 1 month after the initial surgery only. The patients with oligodendrogliomas and oligoastrocytomas underwent PCV chemotherapy [procarbazine, methyl-1-(2-chloroethyl)-1-nitrosourea (CCNU), and vincristine] every 6 weeks (42-day cycles) for two to five cycles [23–25].
Immunohistochemistry and scoring
The sections were subjected to immunostaining using a ChemMate Envision/HRP Kit (DAKO, Denmark). Slides were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol and rinsed in phosphate-buffered saline. After blocking with normal serum for 10 min, the slides were incubated with a 1:100 dilution of rabbit polyclonal HGF antibody, a 1:500 dilution of the mouse monoclonal ki-67 antibody or a 1:100 dilution of mouse monoclonal CD34 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60 min, respectively. Slides were detected by ChemMate Envision/HRP Kit for 30 min at room temperature, followed by developing with diaminobenzidine (DAB) for visualization. Negative controls were provided by substituting non-immune serum for the primary antibodies. The immunostaining results were evaluated and scored semiquantitatively by two pathologists without knowledge of the clinical data of patients. Evaluation of HGF expression was calculated by a double scoring system (stain intensity times stain area) as previously described . Stain intensity was scored as 0 for no staining, 1 for weak staining, 2 for moderate staining and 3 for strong staining. The staining area was scored as 1 for less than 35%, 2 for 35–75% and 3 for >75% of tumor cells. High expression of HGF was defined when the immunostaining score was ≥ 4, whereas low expression of proteins was defined as a score <4. The proliferation index (PI) of gliomas was determined according to a previous description. Briefly, the percentage of ki-67-positive nuclei was counted in ten high-power fields in the areas with the highest density of labeled nuclei. The PI of each sample was the mean of the independent percentage of ki-67-positive signals by two observers. The high PI was defined as a value greater than 5%, whereas low PI of tumor was defined as a value ≤5% . The counting of microvessels in gliomas was evaluated by the previously reported method . In brief, intratumoral microvessel density (IMD) was observed in areas of the most intense neovascularization or hotspots in the tumor by light microscopy. After the area of the highest neovascularization had been determined, single microvessels were manually counted on a × 200 field. Any brown-stained endothelial cell or cell cluster that was clearly separated from the adjacent microvessels was considered as a single, countable microvessel, and the IMD value of each sample was the mean of the independent microvessels counted by two observers.
Cell culture and reagents
In this study, the human U87MG cell line (HGF high-expressed glioma cell line ) was used. U87MG cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal calf serum (FCS) and antibiotics (50 U/ml penicillin and 100 mg/ml streptomycin, Gibco/Invitrogen) at 37°C in a humidified incubator with 5% CO2.
Transfection of glioma cells with small interfering RNA (siRNA) targeting HGF
Human HGF siRNA (siHGF) and nontargeting siRNA control (siControl) were designed and synthesized by Ribo Biotechnology Co. (Beijing, China). The sequence was identified to be specific to the human HGF gene by using the BLAST search of the NCBI database. siControl was used for negative control purposes. siRNAs were reconstituted, and subsequent transfections were conducted in six-well plates using TurboFect siRNA Transfection reagent (Fermentas, USA), according to the manufacturer’s instructions. In brief, U87MG cells were seeded into six-well plates with a density of 2 × 105 cells/well. Once the cells reached 80% confluence, they were treated with either siHGF or siControl (50 nM) complexed with TurboFect according to the manufacturer’s instructions. Ten percent FCS was added 4 h after transfection, and fresh DMEM with 10% FBS was added as needed after 24 h. Cells were collected at 48 h after transfection to assess HGF protein levels by Western blot and the other functional assays listed in the following sections.
Polymerase chain reaction (PCR) for the detection of HGF mRNA expression levels
U87MG cells were transfected for 48 h, and total RNA was isolated from cells using Trizol reagent (Sigma-Aldrich, USA) according to the manufacturer’s instructions. First-strand cDNA (20 ul) was synthesized from 2 ug total RNA using oligo (dT) primers. An aliquot (2 ul) of cDNA was used as template for PCR amplification with primers specific for HGF (sense primer: 5´CCACACGAACACAGCTATCGGGG -3’; antisense primer: 5´-TGGGAGCAGTAGCCAACTCGGA-3´, Invitrogen, USA). PCR was performed in an automatic thermal cycler (Perkin-Elmer-Cetus, Norwalk, CT). Samples were amplified through 35 consecutive cycles with annealing temperature of 57°C. A 10-ul volume of each PCR product was analyzed by electrophoresis on 1.5% agarose gel containing 0.5 ug/ml ethidium bromide, and the bands were visualized under ultraviolet light.
Western blotting assay
Transfected U87MG cells were lysed as described previously . Equal protein samples were subjected to 12% SDS-PAGE electrophoresis, followed by the transfer to a polyvinylidene fluoride (PVDF) membrane, blocking in 5% fat-free milk and incubation with HGF (at 1:500 dilution) or GADPH antibody (Abcam, USA) at 4°C overnight. Detection was performed using horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence reagents from Amersham (Amersham Life Sciences, UK). The relative optical density (ROD, ratio to GADPH) of each blot band was quantified by NIH Image software (Image J 1.36b).
Transfected U87MG cells were plated on coverslips, fixed with 4% paraformaldehyde/phosphate-buffered saline (PBS) for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 2 min, and then incubated in PBS containing 5% skim milk for 1 h at room temperature. Cells were incubated with anti-HGF antibody (1:100, Abcam, USA) for 1 h at room temperature, followed by incubation with goat anti-rabbit IgG/Cy3 (1:500, Invitrogen, USA) for 1 h and nuclear counterstaining with DAPI.
The effect of siHGF on the viability of U87MG glioma cells was measured by 3-[4, 5-dimethylthiazol-2-thiazolyl]-2, 5-diphenyltetrazolium bromide (MTT) assay as described previously . Briefly, U87MG cells were seeded at 1 × 105 cells/well into 96-well plates in quintuplicate, and transfected with siHGF or siControl for 24, 48 and 72 h, respectively. Four hours before the desired time points, 20 ul of 5 mg/ml MTT was added into each sample. The plates were then incubated at 37°C in a 5% CO2/95% air atmosphere for 4 h. Thereafter, the medium was discarded, and 150 ul of DMSO was added into each well. The absorbance was determined at 490 nm by an enzyme-linked immunosorbent assay reader. Results represented the OD ratio between the siRNA-treated and untreated cells at the same indicated time points.
In vitro wound healing assay
Migratory ability of U87MG cell was measured using the in vitro wound healing assay. Cells were seeded in six-well plates and transfected with siHGF or siControl for 48 h. Transfected cells were grown to 100% confluence. Wounds were created by scraping monolayer cells with a sterile pipette tip. At 0, 12, 24 and 48 h after the creation of wounds, wound distances were measured at each time point and expressed as the average percent of wound closure by comparing the zero time.
Cisplatin cytotoxicity assay
For the acute cytotoxicity assay, U87MG cells were seeded at 1 × 104 cells per well in 96-well plates, and were transfected by siHGF or siControl for 48 h, and subsequently exposed to cisplatin at final concentrations of 0.5, 1.0, 2.0, 4.0 or 8.0 ug/ml for 24 h in triplicate wells. Cell survival was determined using a previously described colorimetric MTT assay. Then 20 ul of 5 mg/ml MTT was added to each well, and the plates were incubated for 4 h at 37°C. The absorption was read at 490 nm using an automated microplate reader. Mean cell viability was calculated by the ratio of absorbance units of transfected cell samples to the mean absorbance units of the control cell samples. All the experiments were repeated at least three times. The IC50 value is defined as concentration of cisplatin that is required for a 50% reduction in absorbance calculated from the growth curves.
All statistical analysis was carried out by SPSS 13.0 software for Windows. The chi-square test was used to assess HGF expression and PI with clinicopathological characteristics. Vascular density and data derived from experiments in vitro were given as the mean ± SD as indicated. Data were analyzed by one-way ANOVA with Dunnett’s post hoc test and Tukey’s post hoc test for multigroup comparisons. The survival curve of patients was determined by the Kaplan-Meier method and Cox regression, and statistical evaluation was performed using the log rank test. A value of P < 0.05 was considered statistically significant.