Increased cytotoxicity of ionizing radiation in combination with membrane-targeted apoptosis modulators involves downregulation of protein kinase B/Akt-mediated survival-signaling.
OBJECTIVE
The membrane-targeted apoptosis modulators erucylphosphocholine (ErPC) and erucylphosphohomocholine (ErPC3) induce apoptosis in highly apoptosis resistant malignant glioma cell lines and enhance radiation-induced cell death and eradication of clonogenic tumor cells in vitro. Aim of the present study was to elucidate molecular mechanisms of combined action.


METHODS
Induction of apoptosis was evaluated by determination of nuclear morphology (fluorescence microscopy), alteration of mitochondrial function and caspase-activation (flow cytometry, Western blot). Activity of protein kinase B (PKB/Akt) and key downstream effectors involved in apoptosis regulation was verified by Western blot analysis using activation-specific antibodies.


RESULTS
Increased cytotoxicity of the combination was linked to a more efficient activation of the intrinsic apoptosis pathway with increased damage of the mitochondria and caspase-activation. Moreover, activity of the survival kinase PKB/Akt was downregulated upon treatment with ErPC/ErPC3 alone or in combination with ionizing radiation. Inhibition of PKB/Akt was associated with decreased phosphorylation and thus activation of the pro-apoptotic Bcl-2 protein Bad as well as dephosphorylation of the transcription factor FOXO3A (FKHRL1) that may be responsible for the observed increased expression of the pro-apoptotic Bcl-2 protein Bim.


CONCLUSIONS
Our data suggest a role for inhibition of PKB/Akt-mediated anti-apoptotic signaling in increased efficacy of the combination.
Handrick
René
R
Department of Radiation Oncology, Experimental Radiation Oncology, University of Tübingen, Tübingen, Germany.
Rübel
Amelie
A
Faltin
Heidrun
H
Eibl
Hansjörg
H
Belka
Claus
C
Jendrossek
Verena
V
D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D001254:Astrocytoma / Q000188:drug therapy / Q000201:enzymology / Q000532:radiotherapy*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D003131:Combined Modality Therapy; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation; D004883:Erucic Acids / Q000494:pharmacology*; D005909:Glioblastoma / Q000188:drug therapy / Q000201:enzymology / Q000532:radiotherapy*; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D004883:Erucic Acids; C518520:erucylphosphohomocholine ErPC3; D010767:Phosphorylcholine; C075010:erucylphosphocholine; D051057:Proto-Oncogene Proteins c-akt; D020169:Caspases