![]() ![]() ![]() 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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