γ-secretase inhibitor I inhibits neuroblastoma cells, with NOTCH and the proteasome among its targets

As high-risk neuroblastoma (NB) has a poor prognosis, new therapeutic modalities are needed. We therefore investigated the susceptibility of NB cells to γ-secretase inhibitor I (GSI-I). NOTCH signaling activity, the cellular effects of GSI-I and its mechanisms of cytotoxicity were evaluated in NB cells in vitro and in vivo. The results show that NOTCH signaling is relevant for human NB cells. Of the GSIs screened in vitro GSI-I was the most effective inhibitor of NB cells. Both MYCN-amplified and non-amplified NB cells were susceptible to GSI-I. Among the targets of GSI-I in NB cells were NOTCH and the proteasome. GSI-I caused G2/M arrest that was enhanced by acute activation of MYCN and led to mitotic dysfunction. GSI-I also induced proapoptotic NOXA. Survival of mice bearing an MYCN non-amplified orthotopic patient-derived NB xenograft was significantly prolonged by systemic GSI-I, associated with mitotic catastrophe and reduced angiogenesis, and without evidence of intestinal toxicity. In conclusion, the activity of GSI-I on multiple targets in NB cells and the lack of gastrointestinal toxicity in mice are advantageous and merit further investigations of GSI-I in NB.

2 repeat (STR) profiles. For STR profiling of cell lines the GenePrint 10 System (Promega, Mannheim, Germany) was used according to the manufacturer's recommendation. For STR profiling of short-term cultures forensic STR loci were determined by the Institute of Forensics, Ulm University.
CD11a, CD19 and CD20 flow cytometry U-NB1 and U-NB2 cells were stained for the lymphocyte markers CD11a, CD19 and CD20. 2x10 5 cells per sample were used for staining. PBMCs isolated from buffy coat served as positive staining controls. CD11a was detected using anti-human Growth medium with 1 µM GSI-I was replaced twice a week until colony formation was observed. Colonies were stained with 1 mg/ml MTT (Sigma).

Clonogenic growth assay
A single cell suspension of NB cells in clonal density, i.e. 1000 cells/well was seeded in 6-well plates. Growth medium with 1 µM GSI-I was added 24h after seeding.

Generation and transfection of pcDNA3-dnMAML-GFP
The dnMAML-GFP cDNA was isolated by BglII/ClaI digestion of the retroviral vector

PAS-staining
Periodic acid-Schiff (PAS) staining was performed according to standard protocols.
Briefly, formalin-fixed, paraffin-embedded tissue specimens were deparaffinized and rehydrated. Periodic acid (Carl Roth, Karlsruhe, Germany) was added for 5 min and slides were incubated at room temperature. After washing in distilled water Schiff's reagent (Carl Roth) was added for 15 min at room temperature. Subsequently, specimens were washed in running tap water for 5 min and nuclei were counterstained with Meyer's hematoxylin (Sigma-Aldrich) for 5-10 sec. Finally, samples were dehydrated and mounted in non-aqueous mounting medium (Merck, Darmstadt, Germany).