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 → 3GalN
Acβ1 → 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
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.