The cytotoxicity of gamma-secretase inhibitor I to breast cancer cells is mediated by proteasome inhibition, not by gamma-secretase inhibition.

Abstract

INTRODUCTION:

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.

• Authors: Jianxun Han, Ivy Ma, Michael J Hendzel, Joan Allalunis-Turner
• Journal: Breast cancer research : BCR (Breast Cancer Res) Vol. 11 Issue 4 Pg. R57 ( 2009) ISSN: 1465-542X [Electronic] England
• PMID: 19660128 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Carbamates
• Dipeptides
• Estrogens
• L 685458
• N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester
• NOTCH1 protein, human
• Neoplasm Proteins
• Oligopeptides
• Proteasome Inhibitors
• Receptor, Notch1
• Receptors, Estrogen
• benzyloxycarbonyl-leucyl-leucyl-norleucinal
• Amyloid Precursor Protein Secretases
• Proteasome Endopeptidase Complex

Topics

• Adenocarcinoma (enzymology, pathology)
• Amyloid Precursor Protein Secretases (antagonists & inhibitors)
• Breast Neoplasms (enzymology, pathology)
• Carbamates (pharmacology)
• Cell Line, Tumor (drug effects, enzymology, pathology)
• Dipeptides (pharmacology)
• Drug Delivery Systems
• Estrogens
• Female
• Humans
• Neoplasm Proteins (analysis, antagonists & inhibitors, metabolism)
• Neoplasms, Hormone-Dependent (enzymology, pathology)
• Oligopeptides (pharmacology, toxicity)
• Proteasome Endopeptidase Complex (drug effects)
• Proteasome Inhibitors
• Receptor, Notch1 (metabolism)
• Receptors, Estrogen (analysis)