Glioma growth inhibition by neurostatin and O-But GD1b

doi:10.1093/neuonc/noq073

Neuro Oncol. 2010 November; 12(11): 1135–1146.

Published online 2010 July 8. doi: 10.1093/neuonc/noq073

PMCID: PMC3098019

Copyright © The Author(s) 2010. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Glioma growth inhibition by neurostatin and O-But GD1b

Beatriz Valle-Argos, Diego Gómez-Nicola, and Manuel Nieto-Sampedro

Neural Plasticity Group, Functional and Systems Neurobiology Department, Instituto Cajal, Consejo Superior de Investigaciones Científicas, Madrid, Spain (B.V.-A., D.G.-N., M.N.-S.); Experimental Neurology Unit, Hospital Nacional de Parapléjicos, SESCAM, Toledo, Spain (D.G.-N., M.N.-S.)

Corresponding Author: Diego Gómez-Nicola, PhD, Neural Plasticity Group, Functional and Systems Neurobiology Department, Instituto Cajal, Avda. Doctor Arce, 37, 28002 Madrid, Spain (Email: dgomez/at/cajal.csic.es).

Received October 7, 2009; Accepted May 5, 2010.

This article has been cited by other articles in PMC.

Abstract

In spite of their low incidence, central nervous system tumors have elevated morbidity and mortality, being responsible for 2.3% of total cancer deaths. The ganglioside O-acetylated GD1b (O-Ac GD1b; neurostatin), present in the mammalian brain, and the semi-synthetic O-butyrylated GD1b (O-But GD1b) are potent glioma proliferation inhibitors, appearing as possible candidates for the treatment of nervous system tumors. Tumoral cell division inhibitory activity in culture correlated with growth inhibition of glioma xenotransplants in Foxn1^nu nude mice and intracranial glioma allotransplants. Both O-Ac GD1b and O-But GD1b inhibited in vivo cell proliferation, induced cell cycle arrest, and potentiated immune cell response to the tumor. Furthermore, the increased stability of the butyrylated compound (O-But GD1b) enhanced its activity with respect to the acetylated ganglioside (neurostatin). These results are the first report of the antitumoral activity of neurostatin and a neurostatin-like compound in vivo and indicate that semi-synthetic O-acetylated and O-butyrylated gangliosides are potent antitumoral compounds that should be considered in strategies for brain tumor treatment.

Keywords: C6 cells, gangliosides, glioma, Neurostatin, O-But GD1b

Glioblastomas are the most frequent primary brain tumors, presenting high proliferative rates and poor prognosis. Recent data indicate that only half of the patients who received any treatment in the US survived 1 year after the tumor diagnosis.^¹,² The elevated frequency of multiform glioblastomas and astrocytomas makes the search for compounds that reduce their progression a research topic of major interest.

Gangliosides are glycosphingolipids with sialic acid in their structure,^³ located in the membrane of vertebrate cells. Gangliosides are classified in the 0, a, and b series, because of their biosynthesis and structure. In the central nervous system (CNS), gangliosides regulate morphogenesis as well as cellular growth, migration, and differentiation.^⁴ Gangliosides have been proposed as regulators of cell proliferation, with several mechanisms of action over the activation of growth factor receptors.^⁵,⁶ The presence of gangliosides in tumors of glial origin is related to malignancy degree and patient outcome. Thus, the most malignant glioma (grade IV), present an important reduction in the gangliosides of the a and b series, the ratio between simple and complex gangliosides from the b series being a prognosis marker.^⁷,⁸ The ganglioside GD1b is of particular interest, because of its use in tumor classification and prognosis.^⁹,¹⁰

O-acetylation of the sialic acid hydroxyl groups of gangliosides is one of the most common structural modifications of a ganglioside,^¹¹ generating products with physicochemical and biological properties very different from the parent compounds.^¹² When O-acetylated, ganglioside GD1b becomes a potent inhibitor of astroblast and astrocytoma division, called neurostatin, as first described by our group.^¹³,¹⁴ Although neurostatin was a potent inhibitor of glioma division, its scarcity in neural tissue and the difficulty of its isolation and purification precluded its application in vivo or its possible translation to the clinic. As an alternative to the natural compound, the use of synthetic mimetics (TS4 oligosaccharide) showed in vivo inhibitory activity of glioma growth, but at very high concentrations (in millimolar levels) generating necrotic tumor death.^¹⁵ We recently reported a method for obtaining neurostatin-related gangliosides by chemical synthesis with potent antitumoral activity on human and rat glioma cells (ID₅₀ = 0.2–2 µM), increasing the yield and simplifying the purification methods (B. Valle-Argos, unpublished data).^¹⁶ The method also permitted the preparation of butyrylated compounds, which showed potent antimitotic activity on human and rat glioma cell lines as well as increased chemical stability, thus solving a problem inherent to neurostatin structure.

In the present study, we have compared the antitumoral activity of O-Ac GD1b (neurostatin) and O-But GD1b in two models of experimental glioma growth, xenotransplants in nude mice and intracranial allotransplants in rats. Our results indicate that both compounds show potent antitumoral activity in experimental glioma models, reducing tumor cell proliferation, cell cycle progression and potentiating the antitumoral immune response, with a remarkable effect of the butyrylated compound. This is the first report of the in vivo activity of neurostatin-related gangliosides, opening the promise of an effective antiglioma treatment.

Materials and Methods

Animals

Male Sprague–Dawley rats (8–10 weeks old; Harlan Iberica) and female nude mice (Foxn1^nu/nu; 6–8 weeks old; Harlan Iberica) were maintained in the Cajal Institute (Madrid, Spain) on food and water ad libitum on a 12-hour light/dark cycle. Animals were handled complying with the European Union guidelines for care and handling of experimental animals (86/609/EEC) and the protocols approved by the Cajal Institute animal welfare committee.

Cell Culture

C6 and C6-GFP Rat Glioma Cell Lines

The rat glioma cell line C6 was cultured in DMEM complete medium (pH 7.2; Sigma-Aldrich) supplemented with fetal bovine serum (10%; GLinus), sodium bicarbonate (2 g/L; Merck), penicillin (50 IU/mL; Sigma-Aldrich) and streptomycin (50 µg/mL; Sigma-Aldrich). The cells were maintained at 37°C and 5% CO₂ in exponential growth until their use for in vitro or in vivo experiments.

Alternatively, the C6 rat glioma cells were transfected to express the green fluorescent protein GFP, using the pEGFP-C1 plasmid (Clontech) with a liposome mediated transfection system. Fugene reagent (2%; Roche) and the pEGFP-C1 plasmid (0.02 µg/µL; Clontech) in DMEM medium (Sigma-Aldrich) were applied to cell culture multiwell plates. As transfection control, some wells were incubated with DMEM medium. After 30 minutes, we added the C6 cells (10⁵) in complete DMEM medium, followed by incubation for 48 hours. Then, cells were incubated with complete DMEM medium, supplemented with Geneticin (800 µg/mL; G-418 Phosphate; Gibco), to select the plasmid transfected cells. Surviving cells (GFP-expressing positively transfected cells) were cloned and amplified. GFP-C6 cells were grown in complete DMEM medium, under the same conditions as normal C6 cells.

Perinatal Rat Astrocytes

Perinatal rat astrocytes were cultured as described by McCarthy and de Vellis,^¹⁷ with some modifications.^¹⁸ The cerebral cortices were first mechanically dissociated, centrifuged, and further cultured in DMEM/F12 medium (Sigma-Aldrich), supplemented with 10% (v/v) FBS and penicillin/streptomycin (Sigma-Aldrich) at 37°C and 5% CO₂. Culture flasks were shaken at 280 rpm overnight at 37°C (8–12 dpc), followed by washing and trypsinization (0.25% trypsin, 0.1% EDTA; Sigma-Aldrich). Dissociated cells were seeded on multiwell plates. Astrocyte cultures were around 95% pure, as estimated by GFAP immunolabeling.

Cortical Neurons from Rat Embryo

Rat cortical neurons were prepared as described by Smith et al.,^¹⁹ with modifications.^¹⁸ Cerebral cortices from E15-16 rat embryos were cut into small pieces and dissociated at 37°C with 0.25% trypsin (Sigma-Aldrich) and 20 mg/mL DNAse (Roche) in HBSS (Sigma-Aldrich). After addition of complete Neurobasal medium (GIBCO-Invitrogen) supplemented with 1% B27 (GIBCO-Invitrogen), penicillin/streptomycin (Sigma-Aldrich), and 1 mM glutamine (Sigma-Aldrich), the cells were centrifuged and cultured for 5 days in complete medium on multiwell plates.

Chemical Synthesis and Purification of O-Ac GD1b and O-But GD1b Gangliosides

The gangliosides O-Ac GD1b (neurostatin; Galβ1 → 3GalNAcβ1 → 4[9-O-Ac Neu5Acα2 → 8Neu5Acα2 → 3]Galβ1 → 4Glcβ1 → 1′-ceramide) and O-But GD1b (Galβ1 → 3GalNAcβ1 → 4[9-O-But Neu5Acα2 → 8Neu5Acα2 → 3]Galβ1 → 4Glcβ1 → 1′-ceramide) were obtained by chemical O-acetylation or O-butyrylation of GD1b,^²⁰,²¹ using the method originally described by Ogura et al.^²² and further modified for gangliosides.^²³ Ganglioside GD1b (125 µg; Axxora) was dissolved in dimethylsulfoxide (DMSO; 12.5 µL) and treated either with trimethyl orthoacetate (TMOA; Sigma-Aldrich) or with trimethyl orthobutyrate (TMOB; Sigma-Aldrich) in 500 M excess, in the presence of p-toluensulfonic acid (0.0125 mg; Sigma-Aldrich) as catalyst. The reaction mixture was maintained for 8 hours in darkness at 18–21°C and acetylation was stopped by addition of methanol (1 mL). The mixture was desalted by reverse phase filtration (Sep-Pak Plus C18 cartridge; Waters) as described previously.^²⁴ O-substituted gangliosides were purified by preparative thin layer chromatography.^²⁵

Inhibition of Cell Proliferation

Exponentially growing C6 cells were seeded on 96-well plates (Beckton Dickinson), in complete DMEM medium, at a density of 5 × 10³ cells/well. The cells were allowed to attach for 6 hours, then the medium was replaced by serum-free DMEM medium and incubation was continued for 36 hours. The cells were treated with serial dilutions of O-Ac GD1b or O-But GD1b (from 5 µM to 15 nM) in DMEM, supplemented with the mitogen EGF (10 ng/mL) for 24 hours.

Cell proliferation was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich), based on the conversion of the water soluble MTT into an insoluble formazan. Briefly, the cells were treated for 4 hours at 37°C with MTT in RPMI 1640 (0.5 µg/µL; without phenol red; Sigma-Aldrich), the precipitated formazan was dissolved in SDS (0.1 g/mL in 0.01 M HCl; 4–18 hours), and the optical density of the solution was measured at 595 nm. Absorption values were referred to positive proliferation controls and cultured in DMEM supplemented with EGF. Inhibition was expressed as an ID₅₀ value, the compound concentration that reduced maximal proliferation by 50%.

Astrocyte proliferation was evaluated using the same method, but performed on poly-l-lysine (10 µg/mL; Sigma-Aldrich) coated 96-well plates (10⁴ cells/well; Beckton Dickinson) in DMEM F12 complete medium.

Neuron Toxicity Assay

The technique used for measuring proliferation was also used to monitor neuronal survival, with the following modifications. Neurons were plated on poly-l-lysine (10 µg/mL; Sigma-Aldrich) coated 96-well plates (2 × 10⁴ cells/well; Beckton Dickinson) with Neurobasal medium, supplemented with B27 (1%v/v; casa) and l-glutamine (1 mM). Neurons were treated with serial dilutions of O-Ac GD1b and O-But GD1b (from 10 µM to 30 nM) in Neurobasal complete medium for 24 hours.

Flow Cytometry

Cell cycle analysis was performed by flow cytometry of propidium iodide-stained cells. The C6 cells (5 × 10⁵ cells/well; 6-well plates) were allowed to attach for 6 hours, then the medium was replaced by serum-free DMEM medium and incubation was continued for 36 hours. The cells were treated with O-Ac GD1b and O-But GD1b (2 µM) in DMEM, supplemented with the mitogen EGF (10 ng/mL) for 24 hours. We used DMEM and DMEM + EGF-treated cells as negative and positive proliferation controls, respectively.

C6 cells were dissociated using trypsin/EDTA (Sigma-Aldrich). Adherent and detached cells were then pooled and centrifuged, before being fixed in 70% ethanol. After washing with PBS, the cell pellets were dissolved in 300 µL of PBS and stained with propidium iodide (25 µg/mL; Sigma-Aldrich), supplemented with RNAse (50 U; Sigma-Aldrich). Data were acquired using a Cytomics FC500 Flow Cytometer (Beckman Coulter).

Xenotransplant of Glioma Cells and Treatment

C6 cells, maintained in exponential growth, were trypsinized and washed (PBS) before implantation. Female Foxn1^nu/nu nude mice were subcutaneously injected in the right flank with 3 × 10⁶ C6 cells in serum-free DMEM medium. Treatment was started after the appearance of palpable tumors reaching the volume of 120 mm³. Animals were treated by intratumoral injection (40 or 80 µg/kg animal in PBS [20 µL], divided in five injections, from day 12 to 24) of O-Ac GD1b and O-But GD1b, using GD1b and PBS (vehicle) as controls. Tumors were measured every 3 days with an electronic calliper (Vogel), and tumor volume was calculated as width²×length×π/6. The tumor volume index was calculated using the difference between the measured volume and the volume measured at the beginning of the treatment. Additionally, the overall growth curve was adjusted to a linear tendency to compare the growth slope of the different treatments. The animals were sacrificed 33 days after tumor implantation to evaluate the tumor progression. Two independent experiments were carried out (n = 8).

Animals were euthanized as a “cancer death” when the tumors showed necrosis or the animals' overall health was compromised (evaluated by weight gain). Survival advantage is presented as a Kaplan–Meier survival curve using GraphPad Prism software. During the study, animals were weighed and examined frequently for clinical signs of any adverse, drug-related side effects.

Alternatively, and after the tumors reached the required volume (120 mm³), we performed an acute treatment (single injection; n = 6) with the compounds (5 µg of O-Ac GD1b or O-But GD1b, in 20 µL PBS). During the next 30 hours after treatment, mice received five sequential intraperitoneal injections of 5-bromo-2-deoxyuridine-5-monophosphate (BrdU; 7.5 mg/mL in 0.1 M Tris–HCl, pH 7.6; Sigma-Aldrich), and then were sacrificed to evaluate tumor response to the treatment.

Intracranial Glioma Implantation and Treatment

For the intracranial allotransplant tumor model, C6 or C6-GFP cells were harvested by trypsinization, washed once with serum-free DMEM medium, and resuspended in DMEM medium for implantation. A cell suspension containing 10⁶ cells/5 µL of DMEM was implanted into the brain striatal region of male Sprague–Dawley rats. Under deep isofluorane anesthesia (4% for induction and 1.5% for maintenance), rats were placed in a small-animal stereotactic frame (Stoelting). A sagittal incision was made through the skin to expose the cranium, and a hole was drilled in the skull. Cells (5 µL of cell suspension) were implanted in the striatum with a Hamilton syringe (85RN; Hamilton, Bonaduz, Switzerland) at a 0.5 µL/min rate, at the following stereotaxic coordinates from bregma: ML 3.0, AP 0.2, DV 4.5. Finally, the syringe was removed and the wound sutured. To establish the best glioma cell implantation dynamics, a pilot group of animals was sacrificed 7, 14, and 21 days after the implantation, establishing that day 7 was the most adequate to perform the treatment.

Therefore, tumor treatment started 7 days after glioma cell inoculation, following the same protocol as for the tumor implantation. Rats were treated with O-Ac GD1b and O-But GD1b, using GD1b and PBS as control treatments (single dose of gangliosides; 5 µg in 5 µL of PBS; n = 7). The animals were sacrificed 48 hours after the treatment, to evaluate the tumor progression by immunohistochemistry.

Immunohistochemistry

Rat perfusion and tissue processing were performed as previously described.^¹⁸ Briefly, coronal sections (35 µm thick) of rat brains were cut with a vibratome. After blocking nonspecific binding, the sections were incubated overnight at 4°C with mouse antinestin (BD Biosciences), rabbit antiphospho histone H3 (Ser10; Millipore), rabbit anticleaved caspase 3 (Asp174; Cell Signaling), mouse anti-CD68 (Serotec), or mouse anti-CD3 (Santa Cruz Biotechnologies). Following primary antibody incubation, the sections were incubated with the appropriate Alexa 488 or 594 conjugated secondary (1 hour 4°C; Molecular Probes). Cell nuclei of each section were labeled with Hoechst. Confocal microscopy was performed using an Leica TCS-SP5 system, coupled to an Leica DMI6000CS microscope.

Foxn1^nu/nu nude mice were perfused with saline buffer and tumor xenografts were dissected and then postfixed for 4 hours (buffered 4% paraformaldehyde). Tumors were cryoprotected with sucrose solution (30% w/v in 0.1 M phosphate buffer) and 25 µm-thick coronal sections cut in a cryostat. Detection of BrdU incorporation in nude mice tumors was performed following the immunohistochemical method previously described, with some modifications. Prior to the blocking step, sections were treated with boiling sodium citrate (10mM; 5 minutes), to unveil intracellular antigens. After washing, sections were then treated with HCl (2 N; 30 minutes; 37°C), to facilitate the antibody access to the BrdU. We used mouse anti-BrdU as a primary antibody (Developmental Studies Hybridoma Bank, University of Iowa), using biotinylated goat antimouse antibody (Jackson ImmunoResearch) as secondary link. After washing steps, the sections were incubated with Vectastain ABC complex (Vector Labs) and revealed with diaminobenzidine (DAB). Sections were visualized in an Olympus Provis AX70 microscope coupled to an Olympus DP50 image acquisition system.

Quantification Techniques

For the intracranial glioma model, the tumor volume was measured on sections from C6 or C6-GFP implanted animals, using GFP or antinestin immunohistochemistry to detect the tumor presence. Images were quantified with the help of an image analysis system (AIS, Imaging Research Inc.), using a ×4 lens. The proportional area stained for GFP or nestin (epitope-positive area/scan area) was determined in each visible ipsilateral striatum in control animals (PBS) and animals treated with GD1b, O-Ac GD1b, or O-But GD1b. Measurements were carried out on 7 animals per group in all the sections with tumor. Tumor volume was calculated using the proportional stained area/scanned area × section thickness × number of sections, using PBS-treated controls as reference.

Quantification of the phh3 or caspase-3-positive cell number in the intrastriatal gliomas was performed on 4 sections/animal (7 animals/group), in the GFP- or nestin-labeled region. In each slide, labeled cells were counted in 3 different ×20 fields.

Macrophage and T-cell quantification (CD68 or CD3 labeled images; ×10 lens) in the intrastriatal gliomas were performed on 4 sections/animal (7 animals/group), in the GFP- or nestin-labeled region.

Quantification of the BrdU-positive cell number for each group was performed on 10 random slices taken from the tumors (n = 6) obtained from mice injected with BrdU as described previously. In each slide, labeled nuclei were counted in 3 different ×40 fields.

In the cell quantification methods, ImageJ software was used to quantify the field area (ImageJ 1.38x^²⁶).

Western Blotting

Processing of Foxn1^nu/nu nude mice tumoral tissue and analysis of protein expression were performed as described previously.^¹⁸ Tissue samples (20 µg protein/lane) were electrophoresed and transferred to nitrocellulose membranes (Whatman GmbH) and the blots blocked and then incubated with rabbit antiphospho histone H3 (Ser10; Millipore), mouse anti-p21^waf1/cip1 (Cell Signaling), rabbit anti-p27^kip1 (Cell Signaling), mouse anticyclin D1 (Cell Signaling), and mouse anti-CDK6 (Cell Signaling), using mouse antiglyceraldehyde phosphate dehydrogenase (GAPDH; Chemicon) as a loading control. Blots were incubated with HRP-conjugated goat antirabbit or goat antimouse antibodies (Jackson Immunoresearch) and then protein bands were detected using Supersignal west pico chemiluminiscent substrate (Pierce). Image densitometry was performed with Quantity One 4.2 software (BIO-RAD Labs) referring sample intensity to GAPDH expression.

Statistical Analysis

Quantification of BrdU immunolabeling was performed in 6 animals per group (n = 6). The analysis of GFP- or nestin-labeled area (tumor area) and pHH3 or caspase-3-positive cells was performed using 6 animals per group (n = 6). The quantification of macrophage or T-cell infiltration was performed on 6 animals per group (n = 6). Data were expressed as mean ± SEM and analyzed with the STATISTICA 6.0 software package from Statsoft Inc. (Tulsa, OK). For all data sets, normality and homoscedasticity assumptions were reached, validating the application of the 1-way ANOVA, followed by the Tukey post hoc test for multiple comparisons. Multiparameter statistics for the Kaplan–Meier survival curves were performed by a log-rank test using GraphPad Prism software. Differences were considered significant for P < 0.05.

Results

O-Ac GD1b and O-But GD1b Inhibit C6 Glioma Cell Growth

The antitumoral activity of the synthesized compounds had been previously assayed in human and rat glioma cells (ID₅₀ = 0.2–2 µM; B. Valle-Argos, unpublished data).^¹⁶ As a necessary step before its application to in vivo models of tumor growth, we tested the antiproliferative activity of the compounds on C6 and C6-GFP cells (Fig. 1A). O-Ac GD1b and O-But GD1b presented similar inhibitory activities on both cell types (ID₅₀ for O-Ac GD1b = 0.23 ± 0.12 µM and 0.33 ± 0.15 µM, on C6 and C6-GFP, respectively; ID₅₀ for O-But GD1b = 0.32 ± 0.17 µM and 0.40 ± 0.16 µM, on C6 and C6-GFP, respectively). The two compounds also inhibited astroblast growth with ID₅₀ = 4.95 ± 0.98 and 4.56 ± 0.87 µM, respectively, for the acetylated and butyrylated compounds (Fig. 1A), corroborating previously reported data.^¹³ However, the compounds did not present any inhibitory or toxic activity for cultured neurons at the maximal tested concentration (10 µM; Fig. 1A).

Fig. 1.

Inhibitory activity of C6 growth by neurostatin-related compounds. (A) Activity of neurostatin (O-Ac GD1b, black bars) and the neurostatin-related compound, O-But GD1b (grey bars), on the proliferation of C6, C6-GFP, astrocytes, and the survival of neurons, (more ...)

Next, we analyzed the activity of the compounds on the proliferation of C6 cells by flow cytometry (Fig. 1B). The treatment with EGF, a mitogenic control, caused a marked change in the cell cycle profile of the C6 cells, with an increase in the proportion of cells in G₂–M phase (41.1%) and a decrease in the proportion of cells in G₀–G₁ (47.3%; Fig. 1B), in relation to control conditions (DMEM; G₂–M = 19.8% and G₀–G₁ = 65.3%). When the cells were additionally treated with O-Ac GD1b, a significant change of the cell cycle profile was observed, showing phase rates similar to the resting control (Fig. 1B), with most of the cells arrested in the G₀–G₁ phase of the cycle (58.6%) and a reduced number of cells with a proliferating behavior (G₂–M = 26.5%). Treatment with O-But GD1b caused a similar effect to that observed with neurostatin, arresting cells in the G₀–G₁ phase (59.7%) with a reduced proportion of cells in G₂–M (22.6%), as indicative of reduced proliferative activity.

These results point to an elevated antitumoral activity of O-Ac GD1b and O-But GD1b, with no toxic effect on normal neural cells, making possible their application in in vivo models of glioma.

O-Ac GD1b and O-But GD1b Inhibit Tumor Growth in Nude Mice

To determine the compounds' (O-Ac GD1b and O-But GD1b) in vivo antitumoral activity, we continuously administrate the gangliosides to glioma xenografts in Foxn1^nu/nu nude mice. The tumor volume increase was measured, observing that the parental ganglioside GD1b did not have any inhibitory activity on the tumor growth, acting as the PBS control, nor did it promote tumor growth (Fig. 2A; 80 µg/kg). The chemically substituted compounds (O-Ac GD1b and O-But GD1b), administered at a low dose (40 µg/kg), inhibited tumor growth (Fig. 2A), appearing as statistically significant from day 27 after cells inoculation until the end of the experiment (day 33), when compared with the vehicle (PBS) and parental compound (GD1b) controls. O-But GD1b presented the most potent activity (Fig. 2A and B), reducing tumor growth at day 33 by 49% compared with the PBS control, and by 45% when compared with the unmodified ganglioside (GD1b). O-Ac GD1b also inhibited the tumor growth, but in a less effective manner than O-But GD1b (O-Ac GD1b vs. O-But GD1b nonsignificant), significantly reducing tumor growth by 32% compared with PBS control and by 27% when compared with GD1b control (Fig. 2A and B). When the gangliosides were administered at a higher dose (80 mg/kg), we observed a more potent antitumoral activity over the tumor (Fig. 2A). O-Ac GD1b reduced tumor growth by 44% and 50% when compared with the PBS and GD1b (80 µg/kg) controls, respectively (Fig. 2A). O-But GD1b reached a higher reduction of 62% and 66% when compared with the PBS and GD1b (80 µg/kg) controls, respectively (Fig. 2A). The overall growth behavior was also assessed by comparing the slopes of the time-course of growth plots, showing that both compounds significatively reduced the tumor growth (Fig. 2A), when compared with both controls (PBS and GD1b), at the 2 doses assayed.

Fig. 2.

O-Ac GD1b and O-But GD1b inhibit experimental glioma growth in nude mice. (A) Time-course of the effect of O-Ac GD1b and O-But GD1b on the growth of C6 glioma xenograft (n = 8). When tumors reached the required size (120 mm³; day 12), they were treated (more ...)

Additionally, we evaluated the survival response to the treatment with the gangliosides (80 µg/kg; Fig. 2C). The treatment with either PBS or GD1b caused a similar survival rate in the mice with C6 cells xenografts. However, when the animals were treated either with O-Ac GD1b or O-But GD1b, we observed a significant increase in the survival rate (Fig. 2C).

O-Ac GD1b and O-But GD1b Reduce Cell Proliferation and Induce Cell Cycle Arrest in C6 Tumors

The inhibitory activity of the antitumoral compounds was studied in detail using an acute treatment paradigm of C6 tumors implanted in Foxn1^nu/nu nude mice, to evaluate the induced changes in cell proliferation markers. When the proliferation rate was assessed by BrdU incorporation, we observed that both O-Ac GD1b (Fig. 3B) and O-But GD1b (Fig. 3C) significatively inhibited tumor cell division (Fig. 3D) when compared with the PBS control, as observed by immunohistochemical analysis (Fig. 3A). The number of BrdU-positive cells decreased by 48.5% in tumors treated with O-Ac Gd1b and by 65.6% in tumors treated with O-But GD1b when compared with the PBS control (Fig. 3D). The observed reduction in the proliferative rate was correlated with the expression of the mitotic marker phospho-histone H3 (pHH3). When tumors were treated with both modified gangliosides, pHH3 expression was reduced 3-fold, as determined by Western blotting analysis (Fig. 3E and F).

Fig. 3.

O-Ac GD1b and O-But GD1b inhibit tumor cell proliferation and cell cycle progression in nude mice. (A–D) Quantification of tumor cell proliferation in nude mice in response to the acute treatment with PBS (vehicle, control; A), O-Ac GD1b (B), (more ...)

The results obtained with cultured C6 cells led us to consider the possible action of the compounds on the cell cycle progression. When this effect was analyzed on treated tumors by Western blotting, we observed an overexpression of the cell cycle progression inhibitors p27 and p21, when compared with the PBS control (Fig. 3E, G, and H). O-Ac GD1b and O-But GD1b, respectively, caused a 4.9- and 6.4-fold increase in the p27 levels. A 5-fold increase in p21 levels was observed by the treatment with O-Ac GD1b, causing 10-fold overexpression of O-But GD1b. Consequently, we evaluated the expression of the cell cycle progression markers Cyclin D1 and CDK6 (Fig. 3E). The treatment with either O-Ac GD1b or O-But GD1b significatively reduced their expression, evaluated by Western blotting (Fig. 3E, E,3I,3I, I,3J),3J), and compared to control treatment (PBS). O-Ac GD1b reduced CDK6 expression by 64.22% and Cyclin D1 expression by 41.05% (Fig. 3I, 3J). O-But GD1b reduced CDK6 expression by 70.76% and Cyclin D1 expression by 66.42% (Fig. 3I, 3J). When the effect of both inhibitors (O-Ac GD1b and O-But GD1b) was compared, no statistically significant differences were observed, although O-But GD1b seemed more inhibited.

Therefore, mono-acetylated or mono-butyrylated GD1b derivatives were effective as inhibitors of xenotransplanted glioma in nude mice, blocking cell cycle progression, thus reducing tumor cell proliferation.

Intracranial C6 Tumor Growth Was Reduced by the Treatment with O-Ac GD1b or O-but GD1b

Taking into consideration the previous results, we evaluated the effect of the GD1b-derived compounds (O-Ac GD1b and O-But GD1b) in a model of intracranial glioma in rats (C6 or C6-GFP), to better study the antitumor response in a complete system.

As observed by immunohistochemical quantification of the tumoral volume, the unsubstituted parental compound GD1b promoted tumor growth, when compared with the PBS treated tumors (Fig. 4E). The administration of low doses of O-Ac GD1b or O-But GD1b significatively reduced the tumor volume (Fig. 4A–D). O-Ac GD1b reduced tumor growth by 59.26% and 73.92%, when compared to the effect of PBS or GD1b, respectively (Fig. 4E). O-But GD1b reduced the tumoral volume by 40.57% and 61.93%, when compared with the effect of PBS and GD1b, respectively (Fig. 4E). When these results were compared with those obtained when treating tumors allotransplanted in nude mice, no significant differences were observed in the inhibitory activities of O-Ac GD1b and O-But GD1b.

Fig. 4.

Neurostatin-related compounds reduce the experimental intracranial tumor growth in rats. (A–E) Evaluation of neurostatin-related compounds activity on intracranial C6 tumor growth in rats, analyzed by immunohistochemistry. Seven days after intracranial (more ...)

Treatment of Intracranial C6 Tumors with O-Ac GD1b or O-but GD1b Reduces Proliferation and Induces Apoptosis of Tumoral Cells

The time-course of the inhibitory effect of both O-Ac GD1b and O-But GD1b on intracranial C6 tumors was evaluated by immunohistochemical analysis of the mitosis marker pHH3 and the cell apoptosis marker cleaved caspase-3 (Fig. 5).

Fig. 5.

O-Ac GD1b and O-But GD1b reduce proliferation and induce apoptosis of intracranial tumors. (A, D, G, and J) Evaluation of the effect of neurostatin-related compounds on the proliferation of intracranial gliomas, evaluated by pHH3 immunohistochemistry (more ...)

When tumors were treated with PBS as a control, we observed an elevated number of pHH3 positive nuclei in the tumoral cells (nestin-positive; Fig. 5A, A,5J),5J), with absence of apoptotic cells (caspase-3; Fig. 5B, B,5C,5C, C,5K).5K). Animals with intracranial C6 tumors treated with O-Ac GD1b showed tumoral cells with a significantly reduced number of pHH3-labelled nuclei (65% reduction; Fig. 5D, D,5J),5J), when compared with the control conditions (PBS). This reduction in the tumor mitosis was also observed when treating with O-But GD1b (65% reduction; Fig. 5G, G,5J).5J). Treatment with either O-Ac GD1b or O-But GD1b induced a significant increase in the number of apoptotic tumoral cells, as observed by cleaved caspase-3 immunolabeling (Fig. 5E, F, H, I, and K).

Treatment of Intracranial Gliomas with O-Ac GD1b Or O-but GD1b Promoted T-Cell and Macrophage Recruitment

One of the main differences between the animal models we used to evaluate the compounds activity is the participation of the immune system in the antitumoral response. The tumor implantation in nude mice is developed under a generally depressed immune response, while the intracranial allotransplant of C6 cells count with the participation of the immune system. Thus, we evaluated by immunohistochemistry the immune cell infiltration (T-cells, CD3 + ; macrophages, CD68 + ) within the intracranial glioma.

When tumors were treated with PBS as a control, we observed a reduced immune cell infiltration within the tumor parenchyma (nestin; green), both with CD68 and CD3 labelling (Fig. 6A, B, G, and H). Treatment with O-Ac GD1b induced a significant increase in the infiltration of macrophages within the tumor (Fig. 6B and G), with a similar effect of O-But GD1b (Fig. 6C and G). T-cell infiltration in the tumor was also significantly elevated with the treatment with O-Ac GD1b (Fig. 6E and H), with a more robust effect of O-But GD1b (Fig. 6F and H), when compared with the control conditions (PBS).

Fig. 6.

Intracranial glioma treatment with neurostatin-related compounds induces the infiltration of immune cells. (A–H) Analysis of the effect of O-Ac GD1b or O-But GD1b on the macrophage (CD68-positive, red; A–C and G) and T-cell (CD3-positive, (more ...)

In summary, both O-Ac GD1b and O-But GD1b were effective in limiting the growth of intracranial tumors, reducing cell proliferation, inducing tumor cell death and facilitating immune cell infiltration within the tumor parenchyma.

Discussion

The identification and characterization of neurostatin began with the evidence of specific astroblast mitogen inhibitory activity in the rat brain.^²⁷,²⁸ The inhibitor was first designated ERI (EGFR-related inhibitor) due to the sharing of epitopes with the EGF receptor (EGFR), to be later identified as the O-acetylated GD1b-derived ganglioside called neurostatin.^¹³,¹⁴ O-acetyl-ganglioside neurostatin (Galβ1 → 3GalNAcβ1 → 4[9-O-Ac Neu5Acα2 → 8Neu5Acα2 → 3] Galβ1 → 4Glcβ1 → 1'-ceramide) was purified from brain extracts and was effective in inhibiting growth of rat and human glioma cells in culture, using both serum or EGF as mitogens.^¹⁴,²⁹ However, these promising results were not translated to in vivo tumoral models due to the reduced reproducibility and yield of neurostatin isolation and purification methods and the ganglioside high susceptibility to hydrolysis under physiological conditions, leading to activity loss.^¹⁴ As an alternative strategy, the neurostatin oligosaccharide analogue (TS4) was synthesized, showing inhibitory activity on growth in culture of astroblasts, glioma, and neuroblastoma cells,^³⁰ and promoting, at high concentrations (millimolar range), in vivo destruction of an experimental brain glioma,^¹⁵ leaving unexplored the natural compound (neurostatin) activity.

Thus, we recently set up the conditions for obtaining neurostatin and neurostatin-related compounds by chemical semi-synthetic methods, improving the availability and stability of the compound.^¹⁶,²⁰ This method also permitted us to obtain neurostatin analogues more resistant to hydrolysis than neurostatin itself, substituting the acetyl chain by a butyryl group (O-But GD1b) and increasing in addition the antiproliferative activity by 2-fold. In the present work, we evaluate, for the first time, the in vivo activity of neurostatin over glioma cell growth, compared with the butyrylated derivative (O-But GD1b) and the un-substituted parental compound (GD1b). Our results show that neurostatin effectively inhibits tumor growth, both at the cell culture level and, experimental glioma models, its activity being substantially improved by the substitution of the acetyl group by a butyryl chain (O-But GD1b). The increased activity of the butyrylated GD1b-derivatives could be explained by a substantial increase in the compound stability to alkaline hydrolysis.^²⁰ Under biological conditions (ie, xenografts in nude mice), the compound stability would be crucial in sustaining its inhibitory activity, taking into consideration that a loss of the acetyl or butyryl chain will transform the molecule to GD1b, a ganglioside without antiproliferative activity, as observed here. However, the differences between O-Ac GD1b and O-But GD1b were not observed as evident in the intracranial glioma treatment, probably due to the participation of the immune system in the coordination of the response.

The reported mode of action and effective dose of the TS4 neurostatin oligosaccharide analogue involved the induction of necrotic cell death in intracranial gliomas,^³⁰ not fitting with the cytostatic effects reported for neurostatin itself.^¹³ Neurostatin and O-But GD1b, in the models used in the present study, caused an arrest of the cell cycle progression, with the consequent reduction in the cell proliferation, leading to increased apoptosis. The antitumoral activity of the compounds in vivo was extremely specific, in accordance with low doses of the compound used in this study, compared with other antitumoral drugs. Moreover, both O-Ac GD1b and O-But GD1b do not induce neuronal toxicity, or control astroblast division without causing cell death, making the compounds suitable for clinical treatment of brain tumors.

As observed in our results, both O-Ac GD1b and O-But GD1b seem to share the same mode of action on tumoral cells. Taking into consideration our previous results, one potential candidate for regulating neurostatin-derived compound activity could be the EGFR.^²⁷,²⁸ EGFR activation is related to tumor growth through inhibition of apoptosis, cellular proliferation, promotion of angiogenesis, and metastasis.^³¹ In the last years, the EGFR pathway has been a deeply investigated therapeutic target,^³² being the most promising strategy in the use of blocking monoclonal antibodies.^³¹,³³ The activity of the EGFR is regulated by various gangliosides, with a remarkable action of GM3.^⁶ Gangliosides can modulate ligand binding, regulate the receptor dimerization, or control receptor activation state and subcellular localization.^⁶ There is no direct evidence of the interaction of either O-Ac GD1b or O-But GD1b with the EGF–EGFR complex, but the parental compound GD1b has been proposed to regulate the EGFR activation through binding to the receptor extracellular domain.^³⁴ GD1b also inhibits dimerization of the PDGF receptor (PDGFR) and PDGF-dependent cell growth.^³⁵ Thus, the possible action of the neurostatin-related compounds on EGFR activation appears as a possible mode of action on the tumor cells, being under current investigation by our group.

The results presented in this work support the possibility that the observed inhibitory action of O-Ac GD1b and O-But GD1b on the cell cycle progression is related to regulation of the response to growth factors. The main consequence of EGFR activation in tumoral cells is the transduction of signals that promote the cell cycle progression.^³⁶ Xenografts in nude mice, treated with the modified gangliosides, overexpressed the cell cycle checkpoint inhibitors p27 and p21. These proteins are directly related to the observed downregulation of CDK–cyclin proteins (CDK6 and cyclinD1), crucial regulators of the transition from G₁ to the S phase. The main consequence was a reduction of the phosphorylation (activation) of histone H3, a marker of mitosis, and the reduction of cell proliferation indicated by reduced BrdU incorporation. These results fit with the observations in cell culture, where the activity of the compounds on the cycle profile was determined, showing an accumulation of cells in G₀–G₁ phase. Therefore, the inhibition of the C6 response to EGF could account for the inhibition of the cell cycle progression and the consequently diminished proliferation.

The compounds activity in culture correlated with that observed on xenotransplants in nude mice. However, the activity on intracranial gliomas was slightly different. Tumor growth and cell proliferation were also reduced by treatment with both O-Ac GD1b and O-But GD1b. In addition, we also observed increased immune cell infiltration (macrophages and T-cells) within the tumor. The presence of immune cells in glioma tumors has been well documented, although their activity does not fully account with tumor growth.^³⁷,³⁸ Malignant gliomas elaborate an immunosuppressive response involving the production of transforming growth factor b (TGF-b) and interleukin 10 (IL-10), down-regulating T-cell function.^³⁹ T-cell antitumoral response can only be completed with the support of an antigen presenting cell (APC)-mediated priming response,^⁴⁰ normally provided by activated microglia/macrophages. The relationship between neurostatin-related compounds and the immune cell response has been studied in models of inflammatory activation after CNS injury, showing a role in regulating IL-15-dependent responses.^²¹,⁴¹ Whether the compounds directly induce immune cell infiltration within the tumor or whether they cause a loss of immunosuppression by glioma cells remains to be established in future work.

In conclusion, we report the first in vivo evidence of the antitumoral activity of neurostatin (O-Ac GD1b) and its related ganglioside, O-But GD1b. The compounds show potent inhibition of tumor cell proliferation, through inhibition of cell cycle progression and induction of cell death. This work opens the field of the regulation of tumor cell growth by gangliosides and supports a promising future of new tumor growth inhibitors.

Funding

This work was supported by grants from the “Inocente-Inocente” and FUHNPAIIN Foundations and from the Spanish Department of Ciencia e Innovación (SAF 2006-11224).

Acknowledgements

This work was partially presented during the Eighth Congress of the European Association for Neuro-Oncology (EANO).^¹⁶ We thank Dr Alfredo Martínez Ramos for advice during C6-GFP cells transfection and generation. We also thank Jesus Díaz Tobarra for technical help.

Conflict of interest statement. None declared.

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