Mitochondrial VDAC1-based peptides: Attacking oncogenic properties in glioblastoma

Glioblastoma multiforme (GBM), a primary brain malignancy characterized by high morbidity, invasiveness, proliferation, relapse and mortality, is resistant to chemo- and radiotherapies and lacks effective treatment. GBM tumors undergo metabolic reprograming and develop anti-apoptotic defenses. We targeted GBM with a peptide derived from the mitochondrial protein voltage-dependent anion channel 1 (VDAC1), a key component of cell energy, metabolism and apoptosis regulation. VDAC1-based cell-penetrating peptides perturbed cell energy and metabolic homeostasis and induced apoptosis in several GBM and GBM-derived stem cell lines. We found that the peptides simultaneously attacked several oncogenic properties of human U-87MG cells introduced into sub-cutaneous xenograft mouse model, inhibiting tumor growth, invasion, and cellular metabolism, stemness and inducing apoptosis. Peptide-treated tumors showed decreased expression of all tested metabolism-related enzymes and transporters, and elevated levels of apoptotic proteins, such as p53, cytochrome c and caspases. Retro-Tf-D-LP4, containing the human transferrin receptor (TfR)-recognition sequence, crossed the blood-brain barrier (BBB) via the TfR that is highly expressed in the BBB to strongly inhibit tumor growth in an intracranial xenograft mouse model. In summary, the VDAC1-based peptides tested here offer a potentially affordable and innovative new conceptual therapeutic paradigm that might overcome GBM stemness and invasiveness and reduce relapse rates.


Hexokinase detachment
To visualize peptide-induced HK detachment in the cell, U-87MG or SH-SY5Y cells (4x10 4 /ml) were grown on coverslips and transfected with plasmid pEGFP-HK-I. Twenty-four hours later, the post-tranfection cells were incubated for 3 h with a solution containing 0.07% DMSO or Tf-D-LP4 or D-ΔN-Ter-Antp peptide (7 μM). The cells were fixed for 15 min using 4% paraformaldehyde prepared in PBS, rinsed with PBS, permabilized with 0.3% PBST, and stained with DAPI (1:2000). Cell imaging was carried out by confocal microscopy (Olympus 1X81).

PLGA encapsulation of the VDAC1-based peptide Retro-Tf-D-LP4
Retro-Tf-D-LP4-loaded PLGA complexes were prepared by the solvent displacement method with some modifications, as previously reported [2 3]. Twenty milligrams of Retro Tf-D-LP4 were dissolved in 40 µl of 100% DMSO and then diluted 20-fold with sterile DDW to reach a concentration of 25 mg/ml in a final DMSO concentration of 5%. PLGA (50 mg) was dissolved in acetone (1 ml). Then, 105 µl of peptide were added to the PLGA-acetone solution. The resulting peptide-PLGAacetone mixture was added drop-wise (0.5 ml/min) into 10 ml of aqueous solution containing 1% PVA (w/v). The mixtures were stirred continuously at room temperature until complete evaporation of the organic solvent. The nanoparticles were centrifuged at 15,000g (4ºC for 20 min) and the pellet was re-suspended in sterile DDW and washed two times. The resulting pellet was mixed with HBSS solution.

Xenograft and intracranial-orthotopic mouse models
U-87MG glioblastoma cells (3 x10 6 ) were inoculated s.c. into the hind leg flanks of athymic eight-week old male nude mice (Envigo). Thirteen days post-inoculation, tumor volume was measured and calculated (100-130 mm 3 ) and mice were randomized into three groups (5 mice/group). Each treatment substance was injected into the established s,c. tumors using PBS containing 0.26% DMSO or peptide in PBS, 0.26% DMSO/20 μM). The xenografts were injected 20 µl per tumor (2 points) every two days. Beginning on the day of inoculation, mouse weight and tumor volume were monitored twice a week for a period of 23 days using a digital caliper. At the end of the experiments, the mice were sacrificed, tumors were excised and ex vivo weight was determined. Half of each tumor was either fixed in 4% buffered formaldehyde, paraffin-embedded and processed for IHC or frozen in liquid nitrogen for later immunoblot analysis.
To generate an intracranial-orthotopic mouse model, U-87MG glioblastoma cells were engrafted into a nude mouse brain using a stereotactic device. The anesthetized mice were immobilized in a stereotactic-head frame (Stoelting, Wood Dale, IL). A middle incision was made on the skull and a burr hole was introduced 0.5 mm anterior to the bregma and 2.5 mm lateral to the midline using a drill (Stoelting). A 31-gauge needle loaded with 10 µL PBS was used to deliver tumor cells. The needle tip was inserted into the brain 3 mm deep, relative to the skull surface, and maintained at this depth for 2 minutes before injection of tumor cells. Under sterile conditions, a 3 µL solution containing U-87MG (8×10 4 ) cells was injected into the brain parenchyma over a period of 3 minutes using an UltraMicroPump III (World Precision Instruments, Sarasota, FL). After infusion, the needle was left in place for 1 minute before slow withdrawal. The burr hole was sealed using sterile bone wax, and the wound was closed with 5.0 nylon surgical suture. All surgical procedures were performed under sterile conditions. Forty-eight hours after surgery, the mice were randomized into three groups (6 animals/group) and treated every third day with DMSO (1.44%), Retro-Tf-D-LP4 (10 mg/kg) or Retro-Tf-D-LP4 (10 mg/kg) encapsulated by PLGA. Mice were subjected to MRI (at days 20 and 29), sacrificed, and brains were excised and processed for IHC. Tumor volume was analyzed using VivoQuant 2.10 software. These experimental protocols were approved by the Institutional Animal Care and Use Committee of Ben-Gurion University.

Gel electrophoresis and immunoblotting
Cells or tumor tissue were lysed using lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl 2 , 10% glycerol, 1% Triton X-100, supplemented with a protease inhibitor cocktail (Calbiochem, UK). Cell lysates were then centrifuged at 600xg (10 min at 4°C) and samples (10-40 μg of protein) were subjected to SDS-PAGE and immunoblotting using various primary antibodies (sources and dilutions as detailed in supplementary Table 2), followed by the appropriate HRP-conjugated secondary antibodies (i.e., anti-mouse, anti-rabbit or anti-goat). Blots were developed using enhanced chemiluminescence (Biological Industries). Band intensities were analyzed by densitometry using Multi Gauge software (Fujifilm) and the values were normalized to the intensities of the appropriate β-actin signal that served as a loading control.

IHC of tumor tissue sections
IHC staining was performed on 5 μm-thick formalin-fixed and paraffin-embedded tumor tissue sections. The sections were deparaffinized by placing the slides at 60°C for 1 h and using xylene. Thereafter, the tissue sections were rehydrated with a graded ethanol series (100%-50%). Antigen retrieval for some proteins (ATP synthase 5a, AIF, caspase 3, citrate synthase, Cyto c, cytochrome c oxidase subunit IVc, GAPDH, Glut1, HK-II, Klf4, LDH, Nestin, NGFR, P53, SMAC/Diablo, Sox2, S100b, VDAC1) was performed in 0.01 M citrate buffer (pH 6.0). For CD31, HK-I and Ki-67, antigen retrieval was performed in 10 mM Tris-EDTA (pH 9) and 0.5 M Tris (pH 10), for 30 minutes each at 95-98°C. After washing sections in PBS containing 0.1% Triton-X100 (pH 7.4), non-specific antibody binding was reduced by incubating the sections in 10% normal goat serum for 2 h. After decanting excess serum, sections were incubated overnight at 4°C with primary antibodies (sources and dilutions used detailed in Table S2) and washed with PBST. For IHC, endogenous peroxidase activity was blocked by incubating the sections in 3% H 2 O 2 for 15 min. After washing thoroughly with PBST, the sections were incubated with the appropriate secondary antibodies for 2 h. For IHC, anti-mouse, anti-goat, or anti-rabbit secondary antibodies conjugated to HRP were used. Sections were washed five times in PBST and the peroxidase reaction was subsequently visualized by incubating with DAB.
After rinsing in water, the sections were counter-stained with hematoxylin, and mounted with mounting medium. Finally, the sections were observed under a microscope (Leica DM2500) and images were collected at 20× magnification with the same light intensity and exposure time. Non-specific control experiments were carried out using the same protocols but omitting incubation with the primary antibodies. Hematoxylin-eosin (H&E) staining was performed as described previously [4]. Cells were pelleted and fixed in Karnovsky's fixative (2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4) for 10 minutes at room temperature and then in fresh fixative for 3.5 days in the cold. Following buffer rinses, the cells were incubated with 1% osmium tetroxide in 0.1 M cacodylate buffer, dehydrated in an ethanol series, incubated with epoxy propane and embedded in Araldite resin. Ultrathin sections were cut using an UltracutUCT microtome (Leica, Austria), mounted on formvar-coated copper grids, doubly stained with uranyl acetate and lead citrate and viewed in a