HMG-CoA Reductase Inhibition Causes Increased Necrosis and Apoptosis in an In Vivo Mouse Glioblastoma Multiforme Model

Materials and Methods

Animal model and surgery. A subclone of mouse glioblastoma (GL-26) cell line was adapted to grow in C5LKa mice. GL 26 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, Gaithersburg, MD, USA) with 1% penicillin/streptomycin and 10% fetal calf serum (Gibco). These tumor cells had a 98.5% plating efficiency with a duplicating time of 31.34±3.72 hours. In vivo, these tumor cells formed solid tumor with the same morphology and histology resembling that of human glioblastoma multiforme (GBM), an orthotopic brain tumor model as previously reported (11). Mice were anesthetized with an intramuscular injection of ketamine (22-44 mg/kg), xylazine (2.5 mg/kg), and acepromazine (0.75 mg/kg). A total of 1×10⁵ GL-26 cells in 5 μl were implanted stereotactically into the left striatum of the animal using a mouse stereotactic frame (Kopf Instruments, Tujunga, CA, USA) under a surgical protocol approved by the Administrative Panel on Laboratory Animal Care at Stanford University, USA. Twenty-six animals were used for the survival study. Animals were euthanized when they were moribund according to the protocol approved by our Administrative Panel on Laboratory Animal Care.

Simvastatin treatment. Mice were divided into three treatment groups: control (PBS-treated), low-dose simvastatin (1 mg/kg/day; CalBiochem, La Jolla, CA, USA) and high-dose simvastatin (10 mg/kg/day) as previously described (12, 13); 1 ml solution was administered daily via oral gavage, starting on postoperative day (POD) one. Upon becoming moribund, animals were perfused, their brains excised and immunohistochemistry was performed on brain sections. Imaging analysis (necrotic cavity measurement) was performed using Simple PCI software. For frozen section preparation, prepared perfused brains (as previously described) were cut at 12 μm thickness on a cryostat (Leica CM 3000; Leica, Germany) at −15°C. Tumor size, vascular density, vessel caliber and survival in treatment and non-treatment groups were measured as previously described (14). Statistical significance was determined by using two-way ANOVA and Student's t-test.

Immunohistochemistry. For immunofluorescence staining and epifluorescence microscopy, animals were perfused intracardially with cold PBS (Invitrogen, USA) and 0.01 M EDTA (Invitrogen), followed by 4% neutral paraformaldehyde at 4°C. Brains were stored in 4% paraformaldehyde for 3 days, and subsequently dehydrated in 30% sucrose for 3 days. Brains were then cut into 20 μm serial sections on a freezing microtome (Microm HM450; Thermo Scientific, Waltham, MA, USA). Sections were stored at −20°C in tissue cryoprotectant solution and removed for immunohistochemistry. Sections were stained for endothelial cells with CD31 (primary antibody, rat anti-mouse, 1:100; Jackson ImmunoResearch, USA) and Cy3-labled secondary antibody (donkey anti-rat, 1:250; Chemicon, CA, USA). Sections were mounted and counterstained with Vectashield with DAPI (H-1500; Vector Laboratories, CA, USA) and examined with Nikon epifluorescent microscope (Eclipse 80i; Nikon, Tokyo, Japan). Images were digitally recorded with Advanced PCI software (Compix Inc., PA, USA) under ×100 and ×200 power. Four sections were examined per animal, and four quadrants were imaged per section. Vascular density was determined by analyzing 280 slide sections and 836 vessels, and expressed as number of vessels/mm² (mean±SD). The diameter of vessels was defined as the smallest vector measurement between two edges of an outlined blood vessel. To determine tumor size, sections were counterstained with crystal violet in anatomical order. The area of the sections was determined using Advanced PCI software. The sum of the sectional areas was then multiplied by the sectional thickness (20 μm), and the resulting tumor size was expressed in mm³. Statistical analysis was performed using Graph Prism software (San Diego, CA, USA). Pericytes were visualized and counted by immunostaining with NG-2 (1:50, Chemicon) as the pericytic marker. The vascular tree in this part of the study was visualized by using FITC—conjugated tomato lectin (1:200; Vector Laboratories).

TUNEL apoptosis assay. In separate experiments, human umbilical vein endothelial cells (HUVECs) and GL-26 cells of each type were grown on coverslips in 6-well plates for 3 days to produce a 80-90% confluent layer in media containing DMEM with 1% penicillin/ streptomycin, and 10% fetal calf serum (Gibco). Three days later, when the confluent layer was obtained, cells were washed from old media twice by adding 1 × PBS to the coverslips, then fresh media were added with either 1 or 10 μM purified active simvastatin (sodium salt; CalBiochem), or with vehicle (PBS only). Cells were incubated for 24, 48 and 72 hours and at each time point 3 coverslips were taken from each treatment and washed with PBS twice. Terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeling (TUNEL) assay was performed on these slides using ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Millipore Corp., Bedford, MA, USA) according to the manufacturer's protocol. Slides were then counter stained with hematoxylin QS (Vector Laboratories), mounted on slides and screened with a Nikon epifluorescent microscope (Nikon Eclipse 80i).

MTT cell proliferation assay. HUVECs were grown in 24 well plates in culture conditions as described above and each tested condition was done in triplicates. Cells were treated daily with PBS, 1 mM and 10 mM simvastatin for 3 and 5 days. On the final treatment day, 80 μL of 5 mg/mL thiazolyl blue tetrazolium bromide (MTT) were added to each well. Cells were incubated for 3.5 hours at 37°C in culture hood. Media was carefully removed, and 600 μL MTT solvent (4 mM HCl, 0.1% NP40 in isopropanol) were added to each well. Well plates were covered with tinfoil and placed on an orbital shaker for 15 minutes. Media and cells from each well were carefully transferred to clear glass vials and final absorbance was spectrophotometrically measured at 570 nm absorbance to determine cell proliferation.

³H-Thymididne incorporation assay. GL-26 cells were cultured in 6-well plates for 3 days to produce a 80-90% confluent layer in media containing DMEM with 1% penicillin/streptomycin, and 10% fetal calf serum (Gibco). Three days later, when the confluent layer was obtained, cells were washed from old media twice by adding 1 × PBS, then fresh media were added with either 1 or 10 μM purified active simvastatin (sodium salt; CalBiochem) or with vehicle (PBS only). 1 μCi/ml of ³H-thymidine (Amersham, Arlington Heights, IL, USA) was added to into media of the plated cells for six hours. The media was then aspirated and cells were rinsed with PBS and subsequently lysed with 5% trichloroacetic acid (Aldrich Chemical Company, Milwaukee, WI, USA) at 4°C. Cells were fixed with 70% ethanol and solubilized with 0.2 N NaOH. The solute was collected in InstaGel (Packard, Meriden, CT, USA) prior to counting.

Qualitative 3-D angiogenesis. HUVECs were seeded onto gelatin-coated cytodex microcarriers (MCs) to a final density of approximately 30 cells/bead and maintained in large volume of M199 medium with 4 mM glutamine, 50 mg/ml heparin and 2.5 μg/ml of endothelial cell growth supplement-ECGS for 2-4 days at 37°C. Prior to placing the cells into 24-well plates, cell-coated MCs were then added into fibrin gel and allowed to set with the addition of thrombin (0.625 U/ml). After clotting was complete, the fibrin gel was equilibrated with M199 medium for 2 hours. Fresh media was changed after 2 hours, and embedded cells on the fibrin gel were cultured up to 3 days to allow angiogenic development. Cells were treated either 1 μM purified active simvastatin and/or 50 ng/ml recombinant human VEGF₁₆₅ (Intergen Co., NY, USA). The vessel formation was scored at day 8.