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

BMC Cancer

Eleonora Vannini³  Anna Panighini⁴  Chiara Cerri⁴  Alessia Fabbri¹  Simonetta Lisi³  Enrico Pracucci³  Nicola Benedetto²  Riccardo Vannozzi²  Carla Fiorentini¹  Matteo Caleo⁴  Mario Costa⁴ 

[1] Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy

[2] Neurochirurgia, Azienda Ospedaliero-Universitaria Pisana, Via Paradisa 2, 56100 Pisa, Italy

[3] Scuola Normale Superiore, Piazza Dei Cavalieri 7, 56100 Pisa, Italy

[4] CNR Neuroscience Institute, Via Moruzzi 1, 56124 Pisa, Italy

关键词: Temozolomide;    CNF1;    Cerebral cortex;    Mouse;    Glioma;   

Others  :  855678
DOI  :  10.1186/1471-2407-14-449

 received in 2014-01-22, accepted in 2014-06-11,  发布年份 2014

【 摘 要 】

Background

Glioblastomas 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.

Methods

We 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.

Results

In 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.

Conclusions

The activation of Rho GTPases by CNF1 represents a novel potential therapeutic strategy for the treatment of central nervous system tumors.

【 授权许可】

   
2014 Vannini et al.; licensee BioMed Central Ltd.

【 图 表 】

【 参考文献 】

• [1]Khasraw M, Lassman AB: Advances in the treatment of malignant gliomas. Curr Oncol Rep 2010, 12(1):26-33.
• [2]Stupp R, Hegi ME, Gilbert MR, Chakravarti A: Chemoradiotherapy in malignant glioma: standard of care and future directions. J Clin Oncol 2007, 25(26):4127-4136.
• [3]King GD, Curtin JF, Candolfi M, Kroeger K, Lowenstein PR, Castro MG: Gene therapy and targeted toxins for glioma. Curr Gene Ther 2005, 5(6):535-557.
• [4]Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO: Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005, 352(10):987-996.
• [5]Etienne-Manneville S, Hall A: Rho GTPases in cell biology. Nature 2002, 420(6916):629-635.
• [6]Schmidt G, Sehr P, Wilm M, Selzer J, Mann M, Aktories K: Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1. Nature 1997, 387(6634):725-729.
• [7]Flatau G, Lemichez E, Gauthier M, Chardin P, Paris S, Fiorentini C, Boquet P: Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine. Nature 1997, 387(6634):729-733.
• [8]Doye A, Mettouchi A, Bossis G, Clement R, Buisson-Touati C, Flatau G, Gagnoux L, Piechaczyk M, Boquet P, Lemichez E: CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion. Cell 2002, 111(4):553-564.
• [9]Falzano L, Fiorentini C, Boquet P, Donelli G: Interaction of Escherichia coli cytotoxic necrotizing factor type 1 (CNF1) with cultured cells. Cytotechnology 1993, 11(Suppl 1):S56-58.
• [10]Cerri C, Fabbri A, Vannini E, Spolidoro M, Costa M, Maffei L, Fiorentini C, Caleo M: Activation of Rho GTPases triggers structural remodeling and functional plasticity in the adult rat visual cortex. J Neurosci 2011, 31(42):15163-15172.
• [11]Diana G, Valentini G, Travaglione S, Falzano L, Pieri M, Zona C, Meschini S, Fabbri A, Fiorentini C: Enhancement of learning and memory after activation of cerebral Rho GTPases. Proc Natl Acad Sci U S A 2007, 104(2):636-641.
• [12]Zagzag D, Miller DC, Chiriboga L, Yee H, Newcomb EW: Green fluorescent protein immunohistochemistry as a novel experimental tool for the detection of glioma cell invasion in vivo. Brain Pathol 2003, 13(1):34-37.
• [13]Miyatake S, Martuza RL, Rabkin SD: Defective herpes simplex virus vectors expressing thymidine kinase for the treatment of malignant glioma. Cancer Gene Ther 1997, 4(4):222-228.
• [14]Falzano L, Fiorentini C, Donelli G, Michel E, Kocks C, Cossart P, Cabanie L, Oswald E, Boquet P: Induction of phagocytic behaviour in human epithelial cells by Escherichia coli cytotoxic necrotizing factor type 1. Mol Microbiol 1993, 9(6):1247-1254.
• [15]Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C: Clonogenic assay of cells in vitro. Nat Protoc 2006, 1(5):2315-2319.
• [16]Liang CC, Park AY, Guan JL: In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2007, 2(2):329-333.
• [17]Liau LM, Lallone RL, Seitz RS, Buznikov A, Gregg JP, Kornblum HI, Nelson SF, Bronstein JM: Identification of a human glioma-associated growth factor gene, granulin, using differential immuno-absorption. Cancer Res 2000, 60(5):1353-1360.
• [18]Day BW, Stringer BW, Al-Ejeh F, Ting MJ, Wilson J, Ensbey KS, Jamieson PR, Bruce ZC, Lim YC, Offenhauser C, Charmsaz S, Cooper LT, Ellacott JK, Harding A, Leveque L, Inglis P, Allan S, Walker DG, Lackmann M, Osborne G, Khanna KK, Reynolds BA, Lickliter JD, Boyd AW: EphA3 maintains tumorigenicity and is a therapeutic target in glioblastoma multiforme. Cancer Cell 2013, 23(2):238-248.
• [19]Restani L, Cerri C, Pietrasanta M, Gianfranceschi L, Maffei L, Caleo M: Functional masking of deprived eye responses by callosal input during ocular dominance plasticity. Neuron 2009, 64(5):707-718.
• [20]Lodovichi C, Berardi N, Pizzorusso T, Maffei L: Effects of neurotrophins on cortical plasticity: same or different? J Neurosci 2000, 20(6):2155-2165.
• [21]Li YM, Hall WA: Targeted toxins in brain tumor therapy. Toxins (Basel) 2010, 2(11):2645-2662.
• [22]Fabbri A, Travaglione S, Fiorentini C: Escherichia coli cytotoxic necrotizing factor 1 (CNF1): toxin biology, in vivo applications and therapeutic potential. Toxins (Basel) 2010, 2(2):283-296.
• [23]Aktories K: Bacterial protein toxins that modify host regulatory GTPases. Nat Rev Microbiol 2011, 9(7):487-498.
• [24]Fiorentini C, Fabbri A, Flatau G, Donelli G, Matarrese P, Lemichez E, Falzano L, Boquet P: Escherichia coli cytotoxic necrotizing factor 1 (CNF1), a toxin that activates the Rho GTPase. J Biol Chem 1997, 272(31):19532-19537.
• [25]Caprioli A, Falbo V, Roda LG, Ruggeri FM, Zona C: Partial purification and characterization of an escherichia coli toxic factor that induces morphological cell alterations. Infect Immun 1983, 39(3):1300-1306.
• [26]Caprioli A, Donelli G, Falbo V, Possenti R, Roda LG, Roscetti G, Ruggeri FM: A cell division-active protein from E coli. Biochem Biophys Res Commun 1984, 118(2):587-593.
• [27]Magana-Maldonado R, Manoutcharian K, Hernandez-Pedro NY, Rangel-Lopez E, Perez-Dela Cruz V, Rodriguez-Balderas C, Sotelo J, Pineda B: Concomitant treatment with pertussis toxin plus temozolomide increases the survival of rats bearing intracerebral RG2 glioma. J Cancer Res Clin Oncol 2014, 140(2):291-301.
• [28]Chicoine MR, Won EK, Zahner MC: Intratumoral injection of lipopolysaccharide causes regression of subcutaneously implanted mouse glioblastoma multiforme. Neurosurgery 2001, 48(3):607-614.
• [29]Salhia B, Rutten F, Nakada M, Beaudry C, Berens M, Kwan A, Rutka JT: Inhibition of Rho-kinase affects astrocytoma morphology, motility, and invasion through activation of Rac1. Cancer Res 2005, 65(19):8792-8800.
• [30]De Filippis B, Fabbri A, Simone D, Canese R, Ricceri L, Malchiodi-Albedi F, Laviola G, Fiorentini C: Modulation of RhoGTPases improves the behavioral phenotype and reverses astrocytic deficits in a mouse model of Rett syndrome. Neuropsychopharmacology 2012, 37(5):1152-1163.
• [31]Travaglione S, Fabbri A, Fiorentini C: The Rho-activating CNF1 toxin from pathogenic E. coli: a risk factor for human cancer development? Infect Agent Cancer 2008, 3:4. BioMed Central Full Text
• [32]Ruban A, Berkutzki T, Cooper I, Mohar B, Teichberg VI: Blood glutamate scavengers prolong the survival of rats and mice with brain-implanted gliomas. Invest New Drugs 2012, 30(6):2226-2235.