Downregulation of the antiapoptotic MCL-1 protein and apoptosis in MA-11 breast cancer cells induced by an anti-epidermal growth factor receptor-Pseudomonas exotoxin a immunotoxin.
Pseudomonas exotoxin (PE)-containing immunotoxins (ITs) act by arresting protein synthesis and promoting apoptosis, but the mechanisms of the induced apoptosis and the relationship to protein synthesis inhibition is not well elucidated. We studied these effects in MA-11 human breast cancer cells treated with 425.3PE, an unmodified PE covalently linked to the 425.3 antibody, which targets the EGF receptor. This IT induced efficient inhibition of protein synthesis with simultaneous induction of apoptosis. Thus, treatment of cells with 10 ng/ml of IT for 5 hr caused 85% inhibition of protein synthesis in parallel with caspase-3, -8 and -9 activation and PARP inactivation. Even after 72 hr of IT treatment, preincubation with the broad-spectrum caspase inhibitor z-VAD-FMK caused a significant increase in cell survival without affecting IT-induced protein synthesis inhibition. Interestingly, a combination of z-VAD-FMK and the cathepsin B/L inhibitor z-FA-FMK prevented completely IT-induced cell death in MA-11 cells after 24 hr, indicating that cathepsin activation may be important for optimal induction of IT-induced cell death. IT treatment caused after 2.5 hr a significant decrease in the level of the antiapoptotic protein Mcl-1 but not of Bcl-2 and Bcl-XL. Furthermore, Mcl-1 expression was not sensitive to caspase inhibitors but was totally prevented by the lactacystin proteasome inhibitor, suggesting that IT-induced apoptosis may be triggered by a reduction in the Mcl-1 level. Mitochondrial membrane potential (DeltaPsi mito) decreased concurrently with caspase activation, showing the involvement of DeltaPsi mito as a regulator of IT-induced apoptosis. Our results demonstrate that 425.3PE-mediated cell death involves simultaneous induction of apoptosis and protein synthesis inhibition in MA-11 cells, thus contributing to an understanding of the mechanisms involved in IT-induced apoptosis.
Andersson
Yvonne
Y
Department of Tumor Biology and Institute for Cancer Research, Norwegian Radium Hospital, Oslo, Norway. y.g.andersson@klinmed.uio.no
Juell
Siri
S
Fodstad
Øystein
Ø
D036002:ADP Ribose Transferases / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D017209:Apoptosis / Q000187:drug effects*; D001427:Bacterial Toxins / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D020169:Caspases / Q000378:metabolism; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism; D015536:Down-Regulation; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000276:immunology; D005098:Exotoxins / Q000494:pharmacology*; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D014407:Tumor Cells, Cultured; D037521:Virulence Factors / Q000494:pharmacology*; D051020:bcl-X Protein; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D020169:Caspases; D002403:Cathepsins; D004791:Enzyme Inhibitors; D066246:ErbB Receptors; D005098:Exotoxins; D000922:Immunotoxins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D011065:Poly(ADP-ribose) Polymerases; D011500:Protein Synthesis Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D037521:Virulence Factors; D051020:bcl-X Protein; D000097668:Pseudomonas aeruginosa Exotoxin A; C494811:BCL2L1 protein, human