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Downregulation of Apaf-1 and caspase-3 by RNA interference in human glioma cells: Consequences for erucylphosphocholine-induced apoptosis
Authors:W.?Kugler  author-information"  >  author-information__contact u-icon-before"  >  mailto:wkugler@med.uni-goettingen.d"   title="  wkugler@med.uni-goettingen.d"   itemprop="  email"   data-track="  click"   data-track-action="  Email author"   data-track-label="  "  >Email author,F.?Buchholz,F.?K?hler,H.?Eibl,M.?Lakomek,B.?Erdlenbruch
Affiliation:(1) Abteilung Pädiatrie I, Zentrum Kinderheilkunde und Jugendmedizin, Universitätsklinikum Göttingen, Robert-Koch-Str. 40, D-37099 Göttingen, Germany;(2) Max-Planck-Institut für biophysikalische Chemie, Am Faβberg, D-37077, Göttingen
Abstract:Erucylphosphocholine (ErPC) exerts strong anticancer activity in vivo and in vitroand induces apoptosis even in chemoresistant glioma cell lines. We investigated the contribution of Apaf-1 and caspase-3 to the apoptotic response to ErPC using RNA interference (RNAi) in human glioblastoma cells. We could demonstrate that human glioma cell lines are susceptible to RNAi. Apaf-1 and caspase-3 are amenable to specific small interfering RNA (siRNA)-induced degradation resulting in a reduction of protein levels to 8–33% (Apaf-1) and to 30–50% (caspase-3). Transfection of siRNA directed to Apaf-1 and caspase-3 specifically reduced caspase-3 processing induced by ErPC treatment and yielded a reduction in cells that undergo ErPC-induced apoptosis to 17–33% (Apaf-1) and to 38–50% (caspase-3). The caspase-3 siRNA experiments were corroborated in caspase-3-deficient and -reconstituted MCF-7 breast cancer cells. Survival assays and morphological observations revealed that caspase-3 reconstitution significantly sensitized MCF-7 cells to ErPC. Exploring the caspase cascade responsible for ErPC-induced apoptosis MCF-7 cells provided evidence that caspase-3 is required for the activation of caspases-2, -6 and -8 and also participates in a feedback amplification loop. Our results provide evidence that Apaf-1 and caspase-3 are major determinants of ErPC-induced apoptosis and the possible use of ErPC in a clinical setting is discussed.
Keywords:Apaf-1  apoptosis  caspase-3  erucylphosphocholine  glioma  siRNA
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