The bacterial protein toxin, cytotoxic necrotizing factor 1 (CNF1) provides long-term survival in a murine glioma model

Abstract

BackgroundGlioblastomas are largely unresponsive to all available treatments and there is therefore an urgent need for novel therapeutics. Here we have probed the antineoplastic effects of a bacterial protein toxin, the cytotoxic necrotizing factor 1 (CNF1), in the syngenic GL261 glioma cell model. CNF1 produces a long-lasting activation of Rho GTPases, with consequent blockade of cytodieresis in proliferating cells and promotion of neuron health and plasticity.MethodsWe have tested the antiproliferative effects of CNF1 on GL261 cells and human glioma cells obtained from surgical specimens. For the in vivo experiments, we injected GL261 cells into the adult mouse visual cortex, and five days later we administered either a single intracerebral dose of CNF1 or vehicle. To compare CNF1 with a canonical antitumoral drug, we infused temozolomide (TMZ) via minipumps for 1 week in an additional animal group.ResultsIn culture, CNF1 was very effective in blocking proliferation of GL261 cells, leading them to multinucleation, senescence and death within 15 days. CNF1 had a similar cytotoxic effect in primary human glioma cells. CNF1 also inhibited motility of GL261 cells in a scratch-wound migration assay. Low dose (2 nM) CNF1 and continuous TMZ infusion significantly prolonged animal survival (median survival 35 days vs. 28 days in vehicle controls). Remarkably, increasing CNF1 concentration to 80 nM resulted in a dramatic enhancement of survival with no obvious toxicity. Indeed, 57% of the CNF1-treated animals survived up to 60 days following GL261 glioma cell transplant.ConclusionsThe activation of Rho GTPases by CNF1 represents a novel potential therapeutic strategy for the treatment of central nervous system tumors.

Bacterial Toxins and Targeted Brain Therapy: New Insights from Cytotoxic Necrotizing Factor 1 (CNF1)

“…In vitro and in vivo assays showed that CNF1 triggers molecular and morphological hallmarks of senescence ( Figure 2 ), which eventually lead murine and human glioma cells to death. Specifically, CNF1-treated glioma cells show an overexpression of p21 and p16, while FoxG1 is downregulated; these markers point to an activation of the senescence process, that was also confirmed by β-galactosidase staining ( Figure 2 ) [ 62 , 63 ].…”

“…We found that CNF1 impairs motility and proliferation of both murine and human glioma cells causing their death in 15 days [ 62 , 63 ]. In vitro and in vivo assays showed that CNF1 triggers molecular and morphological hallmarks of senescence ( Figure 2 ), which eventually lead murine and human glioma cells to death.…”

“…Moreover, in vivo analysis revealed that glioma-bearing mice treated with a single, low dose of CNF1 (2 nM) showed a significantly extended survival which is comparable to that obtained with a prolonged treatment with temozolomide (TMZ; 140 µM), a widely-used chemoterapic agent. Increasing the CNF1 concentration up to 80 nM leads to a dramatic increase in the survival of glioma-bearing mice with 57% of animals surviving up to 60 days following glioma cell transplant [ 62 ]. Consistently, CNF1-treated glioma-bearing mice revealed a strongly reduced tumoral volume when compared to both TMZ- and vehicle-treated animals [ 63 , 64 ].…”

The bacterial toxin CNF1 as a tool to induce retinal degeneration reminiscent of retinitis pigmentosa

“…Specificity of CNF1 action in photoreceptors may also result from a selective vulnerability: these metabolically very active cells are likely sensitive to dysregulation of intracellular signaling, so that continuous activation of Rho GTPase signaling and changes in cytoskeletal organization may lead to their degeneration. This interpretation is supported by the cytotoxic effects of CNF1 on proliferating cells such as glioma cells 33 . In addition, mutations in proteins involved in the dynamics of actin cytoskeleton lead to severe photoreceptor phenotypes and retinal blindness, as it is the case of ciliopathies including Usher syndrome 34 .…”

Ultra-High Mass Resolution MALDI Imaging Mass Spectrometry of Proteins and Metabolites in a Mouse Model of Glioblastoma

“…To induce glioma formation, C57BL/6 mice (12–14 weeks old) received a stereotaxically guided injection of 40,000 GL261 cells into the visual cortex (2 mm lateral to the midline and in correspondence with lambda) using fine glass micropipettes (tip diameter 40 μm) 16, 43 .…”