Decreasing Glioma Cell Migration With Nt2-Based Bmp7 Overexpression

Glioblastoma multiforme (GBM) is the most aggressive primary intracranial tumor, well known of its high mortality rate and poor treatment outcomes. The average survival time of patients following surgery, combined with chemotherapy and/or radiotherapy, is still less than 12 months. Therefore, therapeutic approaches are truly needed to afford this devastating disease. During gliomagenesis, the tumor-suppressive activity of transforming growth factor-β (TGF-β) signaling pathway generally is perturbed leading to malignant progression. Human bone morphogenetic protein 7 (hBMP7), a member of the transforming growth factor β superfamily, plays pivotal roles in the development of bone, kidney and nervous tissues.

Interestingly, in vitro exposure to BMP7 can induce canonical BMP signaling in stem-like glioblastoma cells. The ability of BMP7 to induce differentiation of tumor stem cells is accompanied by the attenuation stem-like marker expression, reduction of self-renewal in stem-like brain tumor cells. The ccurrently used in vivo delivery methods of BMP7 are basically through multiple direct and/or intravenous injections of the recombinant hBMP7. Unlike some other growth factors, BMPs have short half-life, are not particularly soluble and seem to act locally. Thus, the aforementioned delivery routes are often ineffective[8], and need multiple injections. Unfortunately, in sensitive tissues as brain, repeated injections might lead to inevitable tissue damage.

Therefore, alternative approaches to supply BMP7 protein to glioma cells are needed to circumvent such drawbacks of conventional therapies. Amongst the promising alternatives are those based on advanced therapies based on cell/gene delivery. More specifically, human neuronal precursor NT2 cells exhibit two ideal characteristics for cancer gene therapy which are tumor-selective migratory capacity and receptivity for genetic manipulation to express selected therapeutic genes. As gene delivery systems, non-viral vectors have gained attention over the years, in cooperation with their counterparts viral based vectors. Non-viral vectors are less limited by the size of the gene to transfer, have a lower immunogenicity and oncogenic profile, and are classified as drugs rather than as biologist by the regulatory authorities. All these characteristics enhance production and regulatory issues related with their commercialization. Nevertheless, serious concerns still need to be overcome to reach clinical practice, such as the increase in transfection efficiency.

As non-viral carriers, niosomes are osmotically active self-assembled vesicles composed of cationic lipids and non-ionic surfactants. They conquer liposomes in terms of cost effectiveness and chemical stability, thus received growing attention over time as potential gene delivery vehicles. In a recently accepted study of Attia et al. (in press), we developed a novel cationic niosome gene carried based on chemical compounds depicting flattering properties for gene delivery applications (the cationic lipid 2,3-ditetradecyloxypropan-1amine, the non-ionic surfactants Poloxamer 188 and polysorbate 80). The in vitro/in vivo results obtained with reporter GFP plasmids have paved the way for the current work using NT2 cells as a model for hBMP-7 gene expression to investigate their potential “combined” antitumor effect on glioma (C6) cells.

Methods

Synthesis of niosome vesicles

The cationic lipid 2,3-di (tetradecyloxy)propan-1-amine hydrochloride (D) was synthesized by a slight modification of the experimental protocol described previously[19]. Afterwards, niosomes were manufactured by modifying the reverse phase evaporation technique as discussed earlier[20]. Briefly, the cationic lipid (5mg) was dissolved in dichloromethane (1 ml), and then emulsified in 5 ml of non-ionic surfactant “equal weight % of polysorbate 80 (P80) and Poloxamer 188 (P). The emulsion was obtained by sonication (Branson Sonifier 250®, Branson Ultrasonics Corporation, Danbury, USA) at 45 W for 30 s. After evaporation of dichloromethane, the resulting niosomes were referred to as DPP80 (Figure 1).

Synthesis of DPP80-hBMP-7 nioplexes

The niosome/DNA complexes (Nioplexes) were obtained by mixing an appropriate volume of a stock solution of pUNO1-hBMP-7 plasmid (0. 5 mg/ml) (InvivoGen, Toulouse, France) with different volumes of niosome suspensions (1mg cationic lipid/ml) to obtain different cationic lipid/DNA ratios (w/w). The mixture was incubated for 30 min to allow electrostatic interaction.

Assessment of DPP80-hBMP-7 nioplexes’ features

Dynamic light scattering (DLS) and Laser Doppler Velocimetry (LDV) (Zetasizer Nano ZS, Malvern Instruments, UK) were used to determine the particle size and zeta potential (ZP), respectively. Particle size was obtained by cumulative analysis where all measurements were carried out in triplicate. Nioplexes were examined by via cryo-TEM (TECNAI G2 20 TWIN), operating at an accelerating voltage of 200 KeV in a bright-field and low-dose image mode [21]. The acquired digital images were used to assess the nioplexes’ morphology. The niosomes’ potential to condense, release and protect the pUNO1-hBMP-7 plasmid DNA against enzymatic digestion was evaluated using an agarose gel retardation assay. The naked and niosome-complexed DNA samples (200 ng of plasmid/20 μl) were run on an agarose gel (0. 8% w/v). Then, the Tris–acetate–EDTA buffer-immersed gel was exposed to 120 V for 30 min. To analyze the release of DNA from nioplexes at different cationic lipid/DNA mass ratios, 20μl of an SDS solution (2%) (Sigma-Aldrich, Madrid, Spain) were added/sample. The supposed nioplexes-induced protection for DNA against DNase I (Sigma-Aldrich, Madrid, Spain) enzymatic digestion was assessed by adding 1U DNase I/2. 5μg DNA. Subsequently, mixtures were incubated at 37°C for 30 min followed by the addition of SDS solution (as above) to release DNA from nioplexes. The resulting bands were stained with GelRed™ and visualized by ChemiDoc™ MP Imaging System (Bio-Rad, Madrid, Spain).

In vitro culture and transfection of NT2 cells

NT2 cells (ATCC®−CRL,1973) were cultured in a growth medium composed of; Dulbecco’s Modified Eagle’s Medium (DMEM), 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin (Gibco®, Life Technologies S. A. , Madrid, Spain). The night before transfection, NT2 cells were seeded in 24-well plates at an initial density of 8 × 104 cells/well and allowed to grow to 70−80% confluence. Then, the medium was replaced with serum-free Opti-MEM (Gibco®, Life Technologies S. A, Madrid, Spain), and cells were exposed to the nioplexes at a concentration of 1. 25 μg of pUNO1-hBMP-7/well. After 4 h of incubation, the serum-free transfection medium was replaced with with growth medium. Cells were allowed to grow for 24h, then conditioned media (NT2-CM) was collected, filtered (0. 22 µm filters) and preserved at -80°C until ELISA was performed (R&D, UK) to determine the hBMP-7 secreted. CCK-8 viability/proliferation assay (Sigma Aldrich, Spain) was performed as previously reported[22]. Color development was read at 450 nm (Tecan M200 microplate reader), corrected with reference wavelength at 690 nm, and normalized against blank wells. The positive control, Lipofectamine® 2000 (L2K, Gibco®, Life Technologies, Madrid, Spain) was prepared following the manufacturer's transfection protocol.

In vitro culture and proliferation assay of C6 glioma cells

The rat glioma cell line (C6, CCL-107), purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), was grown in the ATCC-formulated F-12K Medium (Catalog No. 30-2004). To make the growth medium (GM), fetal bovine serum was added to a final concentration of 2. 5%, horse serum to a final concentration of 15%, 100 U/ml of penicillin, and 100 μg/ml of streptomycin. Cells were maintained in a humidified atmosphere at 37 °C with 5% CO2 and were used at the third passage.

In 24 multiple-well culture plates, C6 cells were seeded (100. 000 cells/well) overnight. Next day, GM was replaced by NT2-CM, both untransfected (+NT2) and transfected (+NT2-hBMP7). The C6-CM was prepared as mentioned in section 2. 4. After 24 h, cell proliferation was determined by CCK8 assays according to manufacturer’s instructions. The wells where NT2-CM was added were compared to “control” C6 glioma cells wells grown on C6-CM.

Co-culture and Transwell migration assays

The glioma cell migration assay was performed using uncoated Transwell® 24 well microplates with 8. 0 µm pore size (Costar-Corning, Corning, NY). About 80. 000 NT2 cells (of untransfected/transfected cultures (24h post-transfection) were seeded in the lower chambers and incubated at 37°C for 4 h to ensure cell attachment. Then, C6 glioma cells (in 200 µl serum free media) were seeded in the upper Transwell insert (100. 000 cells/insert). 16 h later, medium was aspirated, and non-migratory cells were removed by swabbing the interior of the insert wells using cotton-tipped swabs. The migrating C6 cells on the lower surface of the Transwell® membranes were fixed in methanol, stained with crystal violet (0. 09% crystal violet) and counted under an inverted optical microscope (Nikon TSM). Five random fields were counted for each membrane, and the mean values from three independent experiments, performed in triplicate, were used.

Statistical analysis

Statistical differences between groups (significance levels of ˃95%) were calculated using ANOVA and Student’s t test. P values <0. 05 were regarded as significant. Samples’ normal distribution and homogeneity of the variance were evaluated by the Kolmogorov- Smirnov and the Levene tests, respectively. All numerical data were presented as mean ± SD.