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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
Author(s) -
Andersson Yvonne,
Juell Siri,
Fodstad Øystein
Publication year - 2004
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/ijc.20371
Subject(s) - lactacystin , pseudomonas exotoxin , apoptosis , proteasome inhibitor , programmed cell death , biology , cathepsin b , cancer research , downregulation and upregulation , microbiology and biotechnology , caspase 8 , cathepsin , caspase , biochemistry , cytotoxicity , in vitro , gene , enzyme
Abstract 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‐X L . 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 (ΔΨ mito) decreased concurrently with caspase activation, showing the involvement of ΔΨ 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. © 2004 Wiley‐Liss, Inc.