The data reported in this manuscript demonstrate the therapeutic potential of a bacterial protein toxin, CNF1, in blocking proliferation of glioma cells and prolonging the survival of glioma-bearing mice. At present, there are several evidences on the use of targeted toxins to treat cancer . However, the clinical efficacy of toxins has mainly been observed in hematological malignancies, but not in solid tumors, including GBM. The toxin used in this report, CNF1, is produced by certain pathogenic strains of E. Coli and consists of a N-terminal binding domain that interacts with membrane receptors on target cells, a middle translocation domain to enter the cytosol and a C-terminal catalytic domain [22, 23]. The catalytic moiety of CNF1 activates members of the Rho GTPase family (Rho, Rac and Cdc42) by conversion (deamidation) of a single glutamine residue into glutamic acid. This aminoacid change impairs GTP hydrolysis thus locking the Rho family proteins in their GTP-bound, active state [22, 23]. Depletion of activated Rho GTPases is then accomplished via ubiquitin-mediated proteasomal degradation .
Since Rho GTPases regulate the dynamics of the actin cytoskeleton, their activation by CNF1 triggers a rapid reorganization of F-actin . In particular, proliferating cells exposed to CNF1 acquire a multinucleated phenotype, due to the inhibition of cytokinesis despite ongoing nuclear division [22, 25, 26]. We have observed CNF1-induced multinucleation in either GL261 glioma cells or early passage cell lines from primary GBM specimens. We have also shown that these multinucleated cells degenerate within about 15 days in vitro.
These data raise the still open question of the identification of the pathway(s) by which CNF1 causes cell degeneration. Experiments with β-Galactosidase, Annexin V and Propidium Iodide labelling allow us to conclude that senescence and then necrosis account for CNF1-induced GL261 cell death. This is consistent with the senescent-like phenotype (cell enlargement, flattening, increase in size of nuclei and nucleoli) assumed by cells treated with CNF1 (see Figure 4A, inset). At the moment, we cannot exclude the possibility that the autophagy pathway contributes to the CNF1-induced phenotype. A recent paper indicates the activation of the autophagy pathway following the treatment of glioma cells with pertussis toxin and TMZ . Future studies are needed to clarify the possible role of autophagy in the antitumoral action of CNF1.
Since CNF1 is derived from E. coli, it might be argued that contamination by bacterial products (such as LPS) could contribute to the observed antineoplastic effects via the activation of immune responses in the brain . However, this hypothesis is very unlikely, as the amount of LPS is our CNF1 preparation was found to be extremely low, in a range unable to activate macrophages (see Methods).
In GBM and in experimental glioma models, the activation of Rho GTPases (such as Rac1) has been linked to increased cell invasion , pointing to these molecules as key therapeutic targets. In this scenario, the dramatic effect of CNF1 on the actin cytoskeleton reorganization renders cells virtually immobile, and this might substantially enhance the anti-tumoral properties of the toxin. Indeed, we have demonstrated, in the wound migration assay, that CNF1 dramatically decreased the motility of GL261 cells, suggesting a further potential therapeutic feature of this toxin for its possible application in the treatment of glioma tumors. This aspect is important in the context of glioma treatment, because glioma cells tend to diffuse profusely into adjacent healthy tissue. It is well established that CNF1 causes assembly of F-actin in prominent stress fibers and extreme flattening of the cell body [6, 24]. Since cells in motion need actin dynamics to attach and detach from the extracellular matrix, actin polymerization by CNF1 effectively renders cells stationary.
Furthermore, it is worth noting that the other key aspect of the action of CNF1 is on neuron plasticity and health . This cooperates with the antitumoral effect (reduced proliferation and motility) and may lead to better preservation of neuronal function in the brain areas surrounding the tumor. Experiments to address CNF1-mediated functional sparing in glioma models are currently ongoing in our laboratory.
Importantly, a key feature of CNF1 is the rapidity of its action. We found that exposure to CNF1 for 1 hr was sufficient to halt proliferation of most of the treated cells (Figure 2). Since CNF1 is an enzyme, entry of a few toxin molecules inside a cell can lead to the modification of several Rho GTPase targets, providing a dramatic amplifying effect. Thus, even a short CNF1 exposure produced nearly maximal effects on cell proliferation. This may be important for glioma therapy, as one problem of local therapies is the rapid washout of therapeutic molecules infused in the tumor area. Moreover, the effects of CNF1 appear to persist for weeks following one single administration of the toxin [10, 11, 30], likely due to persistent catalytic activity of the toxin inside cells. This further strengthens the potential of CNF1 and could avoid the need for repeated drug administration.
The potential involvement of CNF1 in cell transformation is still controversial . Several studies in vitro and in vivo, including the present results, demonstrate the anti-proliferative and cytotoxic effect of CNF1 in cancer cell lines [22, 25, 26]. Furthermore, cell transformation and tumor formation have never been observed after a single administration of CNF1 in the rodent brain (e.g. [10, 11, 30]).
In order to evaluate the effectivness of CNF1, we compared its action to that of a current standard chemotherapic drug, namely TMZ. Even if TMZ in experimental animal models is typically given orally, here we have chosen to administer it via an intracranial route to allow direct comparison with CNF1, which does not cross the blood–brain barrier. We found that TMZ, administered for one week via minipumps, was effective in prolonging animal survival but had a quite narrow therapeutic range, with concentrations of 20 μM being ineffective and concentrations > 200 μM being toxic for the animals. The limited efficacy of TMZ chemotherapy is in line with current clinical and experimental experience [4, 32] and can be attributed to both inherent and acquired tumor drug resistance. In contrast with conventional chemotherapy, CNF1 had greater efficacy and showed no obvious side effects with increasing doses. Remarkably, more than half of the animals treated with 80 nM CNF1 survived for at least 60 days following glioma cell inoculation. One important aspect of CNF1 is its long-lasting action, as one single intracerebral administration leads to Rho GTPase activation for at least 10–28 days [10, 11, 30].