Downregulation of Apaf-1 and caspase-3 by RNA interference in human glioma cells: consequences for erucylphosphocholine-induced apoptosis.
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.
Kugler
W
W
Abteilung Pädiatrie I, Zentrum Kinderheilkunde und Jugendmedizin, Universitätsklinikum Göttingen, Robert-Koch-Strasse 40, D-37099 Göttingen, Germany. wkugler@med.uni-goettingen.de
Buchholz
F
F
Köhler
F
F
Eibl
H
H
Lakomek
M
M
Erdlenbruch
B
B
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D053477:Apoptotic Protease-Activating Factor 1; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D001943:Breast Neoplasms / Q000503:physiopathology; D053148:Caspase 3; D020169:Caspases / Q000096:biosynthesis*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015536:Down-Regulation / Q000502:physiology*; D005909:Glioblastoma / Q000503:physiopathology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology; D034622:RNA Interference
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D051023:BH3 Interacting Domain Death Agonist Protein; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D010767:Phosphorylcholine; C075010:erucylphosphocholine; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases