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

Abstract

Introduction Notch is a family of transmembrane protein receptors whose activation requires proteolytic cleavage by γ-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 γ-secretase with small molecules may be a promising approach for breast cancer treatment. Consistent with this hypothesis, two recent papers reported that γ-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.

z-Leucinyl-Leucinyl-Norleucinal Induces Apoptosis of Human Glioblastoma Tumor–Initiating Cells by Proteasome Inhibition and Mitotic Arrest Response

“…However, we show that in our experimental context, its potency is at least one order of magnitude lower than that of LLNle. Consistently with our data, two studies published during the revision of this article show that in breast cancer cells, LLNle inhibits the proteasome, induces G 2 -M arrest, and triggers apoptosis (67), and that among LLNle, DAPT, and L685,458, only LLNle induces cell death mediated by proteasome inhibition (68).…”

γ‐Secretase inhibitor I induces apoptosis in chronic lymphocytic leukemia cells by proteasome inhibition, endoplasmic reticulum stress increase and notch down‐regulation

“…This is the first evidence that in CLL, GSI I simultaneously targets three key apoptosis regulators to induce apoptosis, even if the dual activities of GSI I on proteasome and Notch has also been demonstrated in other tumor cells. [16][17][18] These findings may have important therapeutic implications for CLL, considering the emerging interest in generating new anticancer multitarget agents, based on the current opinion that anticancer drugs, able to interfere simultaneously with multiple altered pathways, might be more effective than highly selective drugs. 4,5 Our results show that GSI I increases CLL cell apoptosis also in the presence of pro-survival signals triggered by OP9 stromal cells, suggesting the potential to reverse cytoprotection by the microenvironment.…”

“…In breast cancer cell lines 17 and in glioblastoma tumor cells, 18 the main target of GSI I is the proteasome. In precursor-B ALL cells, GSI I induces apoptosis by inhibiting Notch signaling and through Notchindependent mechanisms including, besides proteasome inhibition, the increase of reactive oxygen species production and the disruption of the AKT pro-survival pathway.…”

“…[12][13][14] It has been recently shown that GSI I (Z-Leu-Leu-Nle-CHO), a tripeptide aldehyde inhibiting chymotryptic and aspartyl protease activity, chemically similar to the proteasome inhibitor MG132 (Z-Leu-Leu-Leu-CHO), 15 induces apoptosis in breast 16,17 and glioblastoma tumor cells, 18 and in precursor-B acute lymphoblastic leukemia (ALL) cells 19 by inhibiting both c-secretase and proteasome activity or only proteasome activity. 17 The ability of GSI I to target both Notch and proteasome can render it more effective in inducing antitumor activity in those tumor cells where dysregulated survival and/ or growth are associated with alterations in both Notch signaling and proteasome activity. This might be also the case of CLL cells, given that both a constitutive Notch activation 9 and an up-regulated activity of ubiquitin-proteasome pathway 20 contribute to their apoptosis resistance.…”

“…9 Here, to define how this event contributes to GSI I-induced CLL cell apoptosis, we first examined the time-course effect of GSI I on Notch1 and Notch2 processing and Hes1 protein expression, a target of Notch2 in CLL cells. 9 We performed these experiments in CLL cells from 12 samples (2)(3)(4)(5)(6)(7)(17)(18)(19)(20)(21)(22), including patients with different characteristics. In all samples, GSI I inhibited the generation of Notch1 and Notch2 intracellular domains (IC), as well as the expression of the precursors (P) and the transmembrane/cytoplasmic portion (TM), only after 24 hr, whereas up to 8 hr, the levels of the three forms of both receptors appeared slightly increased or unchanged compared with controls (Fig.…”