The cytotoxicity of gamma-secretase inhibitor I to breast cancer cells is mediated by proteasome inhibition, not by gamma-secretase inhibition.
BACKGROUND
Notch is a family of transmembrane protein receptors whose activation requires proteolytic cleavage by gamma-secretase. Since aberrant Notch signaling can induce mammary carcinomas in transgenic mice and high expression levels of Notch receptors and ligands correlates with overall poor clinical outcomes, inhibiting gamma-secretase with small molecules may be a promising approach for breast cancer treatment. Consistent with this hypothesis, two recent papers reported that gamma-secretase inhibitor I (GSI I), Z-LLNle-CHO, is toxic to breast cancer cells both in vitro and in vivo. In this study, we compared the activity and cytotoxicity of Z-LLNle-CHO to that of two highly specific GSIs, DAPT and L-685,458 and three structurally unrelated proteasome inhibitors, MG132, lactacystin, and bortezomib in order to study the mechanism underlying the cytotoxicity of Z-LLNle-CHO in breast cancer cells.


METHODS
Three estrogen receptor (ER) positive cell lines, MCF-7, BT474, and T47D, and three ER negative cell lines, SKBR3, MDA-MB-231, and MDA-MB-468, were used in this study. Both SKBR3 and BT474 cells also overexpress HER2/neu. Cytotoxicity was measured by using an MTS cell viability/proliferation assay. Inhibition of gamma-secretase activity was measured by both immunoblotting and immunofluorescent microscopy in order to detect active Notch1 intracellular domain. Proteasome inhibition was determined by using a cell-based proteasome activity assay kit, by immunoblotting to detect accumulation of polyubiquitylated protein, and by immunofluorescent microscopy to detect redistribution of cellular ubiquitin.


RESULTS
We found that blocking gamma-secretase activity by DAPT and L-685,458 had no effect on the survival and proliferation of a panel of six breast cancer cell lines while Z-LLNle-CHO could cause cell death even at concentrations that inhibited gamma-secretase activity less efficiently. Furthermore, we observed that Z-LLNle-CHO could inhibit proteasome activity and the relative cellular sensitivity of these six breast cancer cell lines to Z-LLNle-CHO was the same as observed for three proteasome inhibitors. Finally, we found that the cell killing effect of Z-LLNle-CHO could be reversed by a chemical that restored the proteasome activity.


CONCLUSIONS
We conclude that the cytotoxicity of Z-LLNle-CHO in breast cancer cells is mediated by proteasome inhibition, not by gamma-secretase inhibition.
Han
Jianxun
J
Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta, Canada T6G 1Z2. jianxun@ualberta.ca
Ma
Ivy
I
Hendzel
Michael J
MJ
Allalunis-Turner
Joan
J
D000230:Adenocarcinoma / Q000201:enzymology / Q000473:pathology*; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors*; D001943:Breast Neoplasms / Q000201:enzymology / Q000473:pathology*; D002219:Carbamates / Q000494:pharmacology; D045744:Cell Line, Tumor / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D004151:Dipeptides / Q000494:pharmacology; D016503:Drug Delivery Systems; D004967:Estrogens; D005260:Female; D006801:Humans; D009363:Neoplasm Proteins / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009376:Neoplasms, Hormone-Dependent / Q000201:enzymology / Q000473:pathology; D009842:Oligopeptides / Q000494:pharmacology / Q000633:toxicity*; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects; D061988:Proteasome Inhibitors; D051881:Receptor, Notch1 / Q000378:metabolism; D011960:Receptors, Estrogen / Q000032:analysis
D002219:Carbamates; D004151:Dipeptides; D004967:Estrogens; C410009:L 685458; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester; C497761:NOTCH1 protein, human; D009363:Neoplasm Proteins; D009842:Oligopeptides; D061988:Proteasome Inhibitors; D051881:Receptor, Notch1; D011960:Receptors, Estrogen; C523462:benzyloxycarbonyl-leucyl-leucyl-norleucinal; D053829:Amyloid Precursor Protein Secretases; D046988:Proteasome Endopeptidase Complex