Published OnlineFirst October 27, 2009; DOI: 10.1158/1541-7786.MCR-09-0225 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 Castagnola3 1Centro Biotecnologie Avanzate; 2Dip. Chimica e Tecnologie Farmaceutiche ed Alimentare, Università di Genova; 3Istituto Nazionale per la Ricerca sul Cancro; and 4Dip. Oncologia Biologia e Genetica, Università di Genova, Italy Abstract may have a potential therapeutic value that deserves γ-secretase inhibitors have been proposed as drugs further investigation in animal models. (Mol Cancer Res able to kill cancer cells by targeting the NOTCH pathway. 2009;7(11):1822–34) 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 Introduction t-butyl ester (DAPT), to assess whether they were effective Glioblastomas (GBM) are poorly differentiated astrocytic in killing human glioblastoma tumor–initiating cells tumors arising in the central nervous system, which despite re- (GBM TIC) in vitro. We found that only LLNle was able at cent improved treatment modalities are still characterized by the micromolar range to induce the death of GBM TICs very poor prognosis. Several studies have shown the existence by apoptosis. To determine the cellular processes that of a subpopulation of cells within glioma tumors displaying were activated in GBM TICs by treatment with LLNle, we cancer stem cells properties (1-3). The term “tumor initiating analyzed the amount of the NOTCH intracellular domain cells” (TIC) is frequently used to describe such cells (4), and and the gene expression profiles following treatment with we shall use it throughout this article to indicate cells with can- LLNle, DAPT, and DMSO (vehicle). We found that LLNIe, cer stem cell capacity. Due to the fact that TICs promote the beside inhibiting the generation of the NOTCH intracellular tumor chemoresistance (5, 6), radioresistance (3, 7), and angio- domain, also induces proteasome inhibition, proteolytic genesis (5, 8), it is conceivable that finding a manner to kill stress, and mitotic arrest in these cells by repressing them would improve GBM therapy (9, 10). genes required for DNA synthesis and mitotic progression The NOTCH pathway plays important roles during the cen- and by activating genes acting as mitotic inhibitors. DNA tral nervous system development, contributing to the mainte- content flow cytometry clearly showed that cells treated nance of neural stem/progenitor cell pool (11), to promote the with LLNle undergo arrest in the G2-M phases of the cell neural lineage entry of embryonic stem cells (12) and in the cycle. We also found that DAPT and L-685,458, another differentiation of astroglia from the adult hippocampus-derived selective Notch inhibitor, were unable to kill GBM TICs, multipotent progenitors in rat (13). NOTCH signaling activa- whereas lactacystin, a pure proteasome inhibitor, was tion requires the proteolytic processing of this type I integral effective although at a much less extent than LLNle. These membrane protein by a two-step cleavage process catalyzed data show that LLNle kills GBM TIC cells by inhibiting first by a metalloprotease and then by the γ-secretase complex the proteasome activity. We suggest that LLNle, being able composed of the integral membrane proteins presenilin, nicas- to target two relevant pathways for GBM TIC survival, 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 Received 5/21/09; revised 8/21/09; accepted 9/9/09; published OnlineFirst 10/27/09. the expression of stem cell markers in glioma cells (21, 22). Grant support: CIPE 2007-Regione Liguria (Stem Cells) and by Regione Liguria- According to these findings, the inhibition of this pathway “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 leads to depletion of stem-like cells and to the block of the page charges. This article must therefore be hereby marked advertisement in engraftment in embryonal brain tumors (23). Furthermore, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. enhanced NOTCH signaling may lead to the tumor radiore- Authors' contributions: M. Monticone designed research, performed research, analyzed data, wrote the article; M. Fabbi performed research, analyzed data, sistance mechanisms deployed by GBM (24). Targeting the wrote the article; E. Biollo, A. Fabiano, M. Maffei, A. Melotti, and A. Daga NOTCH pathway specifically in GBM TICs seems therefore performed research; W. Giaretti and G. Corte, analyzed data; P. Castagnola a rational approach for exploring novel and hopefully more designed research, performed research, analyzed data, wrote the article. Requests for reprints: Patrizio Castagnola, Istituto Nazionale per la Ricerca sul effective therapeutic strategies for the management of this Cancro, Largo R. Benzi, 10, 16132 Genova, Italy. Phone: 39-0105737500; Fax: malignancy. 39-0105737505. E-mail: [email protected] Copyright © 2009 American Association for Cancer Research. Several molecular tools are available for targeting the doi:10.1158/1541-7786.MCR-09-0225 Notch pathway such as specific siRNAs, shRNAs, or drugs 1822 Mol Cancer Res 2009;7(11). November 2009 Downloaded from mcr.aacrjournals.org on September 28, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst October 27, 2009; DOI: 10.1158/1541-7786.MCR-09-0225 LLNle Kills Human Glioblastoma Cells 1823 such as γ-secretase inhibitors. The latter are small cell per- 24 hours of treatment with DAPT or LLNle, TICs did not show meant molecules able to inhibit the γ-secretase by distinct me- major changes in the amount of NICD in the samples when chanisms (25). Many researchers are carefully studying these compared with the control sample. At 48 hours, instead, a dra- molecules because they can be potentially effective in cancer matic decrease of the signal corresponding to the NICD in the and in the Alzheimer disease (18, 26). Recently, the results of LLNle-treated TICs with respect to the DMSO and DAPT- a phase I trial and of phase II trials (in Alzheimer disease) treated cells was observed (Fig. 2). No detectable protein deg- with the γ-secretase inhibitor LY450139 have been published radation was observed by Ponceau Red staining performed (27-29). before challenging with the primary antibody (Fig. 2). Similar We presently investigated, by evaluating by flow cytometry results were obtained with TICs from PT2 and PT3 (see Supple- and gene expression profiling, two drugs, N-[N-(3,5-difluoro- mentary Data S1 and S2, respectively). phenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and Benzyloxicarbonyl-Leu-Leu-Nle-CHO (LLNle), known LLNle Induces Apoptosis in GBM TICs to be active as γ-secretase inhibitors, for their ability to inter- To investigate the killing mechanism underling the activity fere in vitro specifically with survival properties of GBM TICs of LLNle in GBM TICs, we took advantage of a multicolor la- previously obtained in our laboratory (30). Here, we clearly beling system able to detect apoptosis and necrosis at the same show that LLNle is effective in killing these cells by inhibiting, time. This system utilizes the use of allophycocyanin-conjugat- in addition to the NOTCH, also the ubiquitin-proteasome path- ed Annexin V, C12-resazurine, and Sytoxgreen to discriminate way. These data suggest that preclinical studies should definite- metabolically active cells, early apoptotic, late apoptotic, and ly be carried on to evaluate whether LLNle is able to necrotic cells. The nonfluorescent C12-resazurine is reduced significantly improve the survival of immunodeficient mouse to orange-fluorescent resorufin by live cells; the far-red fluores- xenotransplanted with human GBM TICs. cent allophycocyanin-Annexin V binds to exposed phosphati- dylserine on the membrane of apoptotic cells; and Sytoxgreen, Results being cell impermeant, is able to bind to the DNA of cells with The γ-Secretase Inhibitor LLNleu Kills GBM TICs in compromised membranes such as late apoptotic and necrotic Culture cells. These cell subpopulations can be discriminated by flow To determine whether the γ-secretase inhibitors LLNleu and cytometry. After 48 hours of treatment with LLNle, PT1 GBM DAPT have any effect on GBM TICs in vitro, cells obtained TICs showed an increase in the far-red and green-labeled cell from patient (PT)1 (PT1) were cultured with medium alone subpopulation (late apoptotic) and in the far-red and orange-la- or supplemented with different amount of DMSO (vehicle), beled cell subpopulations (early apoptotic) when compared LLNleu, and DAPT. After 48 hours, the cell viability was de- with the DMSO treatment (R3 and R7, respectively, in Fig. termined by using the MTT compound. PT1 GBM TICs treated 3). It should be pointed that most cells in the region R1, after with LLNle displayed a dose-dependent reduction of cell via- 48 hours of treatment with LLNle, are still resorufine positive bility compared with untreated cells (Fig. 1). Cells treated with and hence metabolically active (Fig. 3). The flow cytometric DMSO or with DAPT displayed a reduction of viability ≥50% results obtained after 48 hours of exposure of TICs to DAPT of the untreated controls only at the higher end of the concen- were very similar to those showed by the TICs exposed to trations tested, corresponding to 3.6% and 270 μmol/L, respec- DMSO (data not shown). Similar results were obtained also tively (Fig.