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D-aminoacid oxidase enzymatic therapy potentiates the effects of radiotherapy in glioblastoma primary cultures

María Paz Ventero1, Victor Barberá1, Marta García1, María Fuentes Baile2, Jesús Sanz2, Luis Fernández Fornos3, Pilar Dorado Rodriguez3, Pilar García Morales1 and Miguel Saceda1.
1 Unidad de Investigación del Hospital General de Elche. Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana, Elche, Spain
2 Instituto de Biología Molecular y Celular de la Universidad Miguel Hernández de Elche, Elche, Spain
3 ERESA grupo médico


One of the main effects of radiotherapy is the generation of free radicals as a consequence of the incidence of radiation on the aqueous molecules present in the cells. The enzyme D-aminoacid oxidase (DAO) is also able to generate free radicals when converting D-aminoacids in their corresponding cetoacids. Based on this property, we tested in primary cultures from glioblastoma, the combination of radiotherapy and DAO trying to improve the results obtained with radiotherapy alone.

We have used primary cultures from patients with multiform glioblastoma. Recombinant DAO carrying the CLytA domain specific for binding to choline was immobilized to magnetic nanoparticles having a magnetite core covered with DEAE cellulose. Primary cultures were radiated at 7 and 15 Gy. After radiation the cultures were treated or not either with free DAO or with nanoparticles immobilized DAO. Cell cycle distribution was determined in all cases by flow cytometry.

Free as well as immobilized DAO dramatically potentiates the radiation effect on primary cultures from multiform glioblastoma. Treatment either with radiation alone or DAO alone induces cell accumulation in the G2+M phase of the cell cycle as well as an increase in the subG1 population associated to cell death. When both treatments are combined, most of the cells (60%-100%) appear in the subG1 phase.

The combination of radiotherapy and enzymatic therapy with DAO based on the nanotechnology, induces an increase in cell death when compared with either therapy alone.

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