![]() Some studies have used GO with therapeutic purposes by inducing oxidative stress in tumor cells ( 9, 10). ![]() The Aspergillus niger glucose oxidase (GO) enzyme catalyzes the conversion of glucose to gluconic acid, generating hydrogen peroxide ( 7), which can diffuse freely through the cellular membranes generating oxidative damage ( 8). The uncoupling of the respiratory chain at complex IV level by cyanide also induces electron escape, especially through complex I and III, which could react with oxygen molecules generating reactive oxygen species, mainly superoxide radicals and hydrogen peroxide (H 2O 2 ref. Targeting the main energy source of the cell, cyanide induces a rapid ATP depletion and a drop in the mitochondrial Δ Ψ, causing DNA fragmentation and cell death ( 4, 5). Cyanide inhibits the cytochrome c oxidase of the mitochondrial respiratory chain, blocking the oxidative phosphorylation. The toxic molecule diffuses freely across cellular membranes, inducing a potent bystander effect that amplifies the therapeutic potential and counteracts the poor transduction efficiencies attained in vivo ( 3). When expressed in cells, linamarase is exported and cyanide is produced in the extracellular environment. We are here describing a system based on the linamarase plant gene ( lis) that encodes a cyanogenic β-glucosidase that hydrolyses linamarin (lin 2-hydroxy isobutyronitrile-β- d-glucopyranoside) to acetone, glucose, and cyanide ( 2). Malignant gliomas are very invasive and tumors generally recur within a year despite aggressive treatment using radical surgery, radiation, and chemotherapy ( 1) therefore, novel strategies are essential to improve the prognosis. The combined system induces autophagic cell death that can be rescued by 3-methyladenine and is characterized by the presence of double-membrane vesicles and punctate LC-3 pattern. The lethal process is caspase independent poly(ADP-ribose) polymerase 1 is not implicated and there is no apoptosis-inducing factor translocation to the nucleus. The synergic combination causes mitochondrial membrane depolarization and phosphatidylserine externalization and accelerates death by 48 hours. ![]() We show here the antitumoral effect of the lis/lin/GO therapy in a canine glioma cell line and in a xenograft glioma model in nude mice. GO produces hydrogen peroxide, inducing oxidative damage and increasing cellular stress. The combination of lis/lin with an otherwise nontoxic level of glucose oxidase (GO) enhances the therapeutic potential (IC 50 of 50 μg/mL at 48 hours). Dog glioma cells carrying the lis gene are thus sensitive to lin (IC 50 of 250 μg/mL at 48 hours) and cell death is accompanied by mitochondrial fission and ATP depletion. We present here a suicide therapy against malignant gliomas based on the transfer to tumor cells of a gene encoding a β-glucosidase, linamarase ( lis), which in the presence of the innocuous substrate linamarin (lin) produces cyanide, blocking the mitochondrial respiratory chain.
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