z-Leucinyl-Leucinyl-Norleucinal Induces Apoptosis of Human Glioblastoma Tumor–Initiating Cells by Proteasome Inhibition and Mitotic Arrest Response Massimiliano Monticone, 1 Emanuela Biollo, 2 Andrea Fabiano, 4 Marina Fabbi, 3 Antonio Daga, 3 Francesco Romeo, 3 Massimo Maffei, 3 Alice Melotti, 3,4 Walter Giaretti, 3 Giorgio Corte, 3,4 and Patrizio Castagnola 3 1 Centro Biotecnologie Avanzate; 2 Dip. Chimica e Tecnologie Farmaceutiche ed Alimentare, Università di Genova; 3 Istituto Nazionale per la Ricerca sul Cancro; and 4 Dip. Oncologia Biologia e Genetica, Università di Genova, Italy Abstract γ-secretase inhibitors have been proposed as drugs able to kill cancer cells by targeting the NOTCH pathway. Here, we investigated two of such inhibitors, the Benzyloxicarbonyl-Leu-Leu-Nle-CHO (LLNle) and the N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), to assess whether they were effective in killing human glioblastoma tumor–initiating cells (GBM TIC) in vitro. We found that only LLNle was able at the micromolar range to induce the death of GBM TICs by apoptosis. To determine the cellular processes that were activated in GBM TICs by treatment with LLNle, we analyzed the amount of the NOTCH intracellular domain and the gene expression profiles following treatment with LLNle, DAPT, and DMSO (vehicle). We found that LLNIe, beside inhibiting the generation of the NOTCH intracellular domain, also induces proteasome inhibition, proteolytic stress, and mitotic arrest in these cells by repressing genes required for DNA synthesis and mitotic progression and by activating genes acting as mitotic inhibitors. DNA content flow cytometry clearly showed that cells treated with LLNle undergo arrest in the G 2 -M phases of the cell cycle. We also found that DAPT and L-685,458, another selective Notch inhibitor, were unable to kill GBM TICs, whereas lactacystin, a pure proteasome inhibitor, was effective although at a much less extent than LLNle. These data show that LLNle kills GBM TIC cells by inhibiting the proteasome activity. We suggest that LLNle, being able to target two relevant pathways for GBM TIC survival, may have a potential therapeutic value that deserves further investigation in animal models. (Mol Cancer Res 2009;7(11):1822–34) Introduction Glioblastomas (GBM) are poorly differentiated astrocytic tumors arising in the central nervous system, which despite re- cent improved treatment modalities are still characterized by very poor prognosis. Several studies have shown the existence of a subpopulation of cells within glioma tumors displaying cancer stem cells properties (1-3). The term “tumor initiating cells” (TIC) is frequently used to describe such cells (4), and we shall use it throughout this article to indicate cells with can- cer stem cell capacity. Due to the fact that TICs promote the tumor chemoresistance (5, 6), radioresistance (3, 7), and angio- genesis (5, 8), it is conceivable that finding a manner to kill them would improve GBM therapy (9, 10). The NOTCH pathway plays important roles during the cen- tral nervous system development, contributing to the mainte- nance of neural stem/progenitor cell pool (11), to promote the neural lineage entry of embryonic stem cells (12) and in the differentiation of astroglia from the adult hippocampus-derived multipotent progenitors in rat (13). NOTCH signaling activa- tion requires the proteolytic processing of this type I integral membrane protein by a two-step cleavage process catalyzed first by a metalloprotease and then by the γ-secretase complex composed of the integral membrane proteins presenilin, nicas- trin, Aph1, and Pen-2 (14-17). Increased activation of the NOTCH signaling has been reported in several tumor types (18). Recent studies showed that this pathway induces the sur- vival and/or proliferation in GBM and glioma cells (19-22), and the expression of stem cell markers in glioma cells (21, 22). According to these findings, the inhibition of this pathway leads to depletion of stem-like cells and to the block of the engraftment in embryonal brain tumors (23). Furthermore, enhanced NOTCH signaling may lead to the tumor radiore- sistance mechanisms deployed by GBM (24). Targeting the NOTCH pathway specifically in GBM TICs seems therefore a rational approach for exploring novel and hopefully more effective therapeutic strategies for the management of this malignancy. Several molecular tools are available for targeting the Notch pathway such as specific siRNAs, shRNAs, or drugs Received 5/21/09; revised 8/21/09; accepted 9/9/09; published OnlineFirst 10/27/09. Grant support: CIPE 2007-Regione Liguria (Stem Cells) and by Regione Liguria- “Accordo collaborazione scientifica tra Liguria e Piemonte, per l'anno 2008.” The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Authors' contributions: M. Monticone designed research, performed research, analyzed data, wrote the article; M. Fabbi performed research, analyzed data, wrote the article; E. Biollo, A. Fabiano, M. Maffei, A. Melotti, and A. Daga performed research; W. Giaretti and G. Corte, analyzed data; P. Castagnola designed research, performed research, analyzed data, wrote the article. Requests for reprints: Patrizio Castagnola, Istituto Nazionale per la Ricerca sul Cancro, Largo R. Benzi, 10, 16132 Genova, Italy. Phone: 39-0105737500; Fax: 39-0105737505. E-mail: patrizio.castagnola@istge.it Copyright © 2009 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-09-0225 Mol Cancer Res 2009;7(11). November 2009 1822