Induction of apoptosis by a cachectic-factor in murine myotubes and inhibition by eicosapentaenoic acid.
Treatment of C(2)C(12) myotubes with a tumour-derived proteolysis-inducing factor (PIF) at concentrations between 1 and 10 nM was shown to stimulate the activity of the apoptotic initiator caspases-8 and -9 and the apoptotic effector caspases-2, -3 and -6. This increased caspase activity was attenuated in myotubes pretreated with 50 microM eicosapentaenoic acid (EPA). At least part of the increase in caspase activity may be related to the increased proteasome proteolytic activity, since a caspase-3 inhibitor completely attenuated the PIF-induced increase in 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the proteasome. However, Western blot analysis showed that PIF induced an increase in expression of the active form of caspase-3, which was also attenuated by EPA. Further Western blot analysis showed PIF increased the cytosolic content of cytochrome c, as well as expression of the pro-apoptotic protein bax but not the anti-apoptotic protein bcl-2, which were both attenuated by 50 microM EPA. Induction of apoptosis by PIF in murine myotubes was confirmed by an increase in free nucleasomes formation and increased DNA fragmentation evidenced by a nucleasomal ladder typical of apoptotic cells. This process was again inhibited by pre-incubation with EPA. These results suggest that in addition to activating the proteasome, PIF induces apoptosis in C(2)C(12) myotubes, possibly through the common intermediate arachidonic acid. Both of these processes would contribute to the loss of skeletal muscle in cancer cachexia.
Smith
H J
HJ
Pharmaceutical Sciences Research Institute, Aston University, Birmingham B4 7ET, UK.
Tisdale
M J
MJ
D000818:Animals; D017209:Apoptosis; D001798:Blood Proteins / Q000378:metabolism; D015153:Blotting, Western; D002100:Cachexia / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D002918:Chymotrypsin / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D015118:Eicosapentaenoic Acid / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D009707:Nucleosomes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011509:Proteoglycans; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein
C494827:Bax protein, mouse; D001798:Blood Proteins; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D009707:Nucleosomes; D011509:Proteoglycans; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C042057:proteolysis-inducing peptide; D045304:Cytochromes c; D015118:Eicosapentaenoic Acid; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex