n-(n-(3-5-difluorophenacetyl)alanyl)phenylglycine-tert-butyl-ester and Glioblastoma

Brain cancer stem-like cells (bCSC) are cancer cells with neural stem cell (NSC)-like properties found in the devastating brain tumor glioblastoma multiforme (GBM). bCSC are proposed a central role in tumor initiation, progression, treatment resistance and relapse and as such present a promising target in GBM research. The Notch signaling pathway is often deregulated in GBM and we have previously characterized GBM-derived bCSC cultures based on their expression of the Notch-1 receptor and found that it could be used as predictive marker for the effect of Notch inhibition. The aim of the present project was therefore to further elucidate the significance of Notch pathway activity for the tumorigenic properties of GBM-derived bCSC.. Human-derived GBM xenograft cells previously established as NSC-like neurosphere cultures were used. Notch inhibition was accomplished by exposing the cells to the gamma-secretase inhibitor DAPT prior to gene expression analysis and intracranial injection into immunocompromised mice.. By analyzing the expression of several Notch pathway components, we found that the cultures indeed displayed different Notch pathway signatures. However, when DAPT-treated neurosphere cells were injected into the brain of immunocompromised mice, no increase in survival was obtained regardless of Notch pathway signature and Notch inhibition. We did however observe a decrease in the expression of the stem cell marker Nestin, an increase in the proliferative marker Ki-67 and an increased number of abnormal vessels in tumors formed from DAPT-treated, high Notch-1 expressing cultures, when compared with the control.. Based on the presented results we propose that Notch inhibition partly induces differentiation of bCSC, and selects for a cell type that more strongly induces angiogenesis if the treatment is not sustained. However, this more differentiated cell type might prove to be more sensitive to conventional therapies.

Topics: Animals; Brain Neoplasms; Cell Proliferation; Cell Survival; Dipeptides; Female; Gene Expression; Glioblastoma; Heterografts; Humans; Mice, SCID; Neoplasm Transplantation; Neoplastic Stem Cells; Receptors, Notch; Signal Transduction

Trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) is a soluble epoxide hydrolase inhibitor that suppresses glioblastoma cell growth in vitro. The aim of this study was to examine whether the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) could sensitize glioma cells to t-AUCB-induced apoptosis.. Both U251 and U87 human glioblastoma cell lines were tested. Cell growth was assessed using the cell counting kit-8. Cell apoptosis was detected with caspase-3 activity assay kits and flow cytometry. The protein levels in the p38 MAPK/MAPKAPK2/Hsp27 pathway in the cells were analyzed using Western blots.. Pretreatment with DAPT (2 μmol/L) substantially potentiated the growth inhibition caused by t-AUCB (200 μmol/L) in U251 and U87 cells. Furthermore, pretreatment with DAPT markedly increased t-AUCB-induced apoptosis of U251 and U87 cells. T-AUCB alone did not significant affect caspase-3 activity in the cells, but t-AUCB plus DAPT pretreatment caused significant increase of caspase-3 activity. Furthermore, pretreatment with DAPT completely blocked t-AUCB-induced phosphorylation of p38 MAPK, MAPKAPK2 and Hsp27 in the cells.. The γ-secretase inhibitor DAPT sensitizes t-AUCB-induced apoptosis of human glioblastoma cells in vitro via blocking the p38 MAPK/MAPKAPK2/Hsp27 pathway, suggesting that the combination of t-AUCB and DAPT may be a potentially effective strategy for the treatment of glioblastoma.

Topics: Amyloid Precursor Protein Secretases; Apoptosis; Benzoates; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Dipeptides; Glioblastoma; HSP27 Heat-Shock Proteins; Humans; Intracellular Signaling Peptides and Proteins; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Signal Transduction; Urea

The objective of the current study was to investigate the regulation of VEGF signaling and tumor angiogenesis by gamma-secretase inhibitor DAPT in glioblastoma. Effects of DAPT on VEGFR1, VEGFR2, endothelial cell proliferation and vessel function were evaluated using mouse microvascular endothelial H5V cell line and U87MG xenograft mouse models. We found that DAPT efficiently inhibited Notch signaling, increased VEGFR2 expression, but decreased VEGFR1 expression. DAPT treatment enhanced endothelial cell proliferation when used combined with VEGF, but exerted no effect if used alone. In U87MG xenograft mouse models, DAPT treatment increased tumor vessel density but compromised vessel function, as evidenced by poor perfusion and aggravated hypoxia. Therefore, DAPT treatment results in an uncoupling of tumor vessel density from vessel function and suppresses glioblastoma growth; disturbance of angiogenesis with DAPT presents a novel therapeutic approach for glioblastoma.

Topics: Amyloid Precursor Protein Secretases; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Dipeptides; Disease Models, Animal; Endothelial Cells; Female; Glioblastoma; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2

Malignant gliomas are treated with a combination of surgery, radiation, and temozolomide (TMZ), but these therapies ultimately fail due to tumor recurrence. In glioma cultures, TMZ treatment significantly decreases neurosphere formation; however, a small percentage of cells survive and repopulate the culture. A promising target for glioma therapy is the Notch signaling pathway. Notch activity is upregulated in many gliomas and can be suppressed using gamma-secretase inhibitors (GSI). Using a neurosphere recovery assay and xenograft experiments, we analyzed if the addition of GSIs with TMZ treatment could inhibit repopulation and tumor recurrence. We show that TMZ + GSI treatment decreased neurosphere formation and inhibited neurosphere recovery. This enhancement of TMZ treatment occurred through inhibition of the Notch pathway and depended on the sequence of drug administration. In addition, ex vivo TMZ + GSI treatment of glioma xenografts in immunocompromised mice extended tumor latency and survival, and in vivo TMZ + GSI treatment blocked tumor progression in 50% of mice with preexisting tumors. These data show the importance of the Notch pathway in chemoprotection and repopulation of TMZ-treated gliomas. The addition of GSIs to current treatments is a promising approach to decrease brain tumor recurrence.

Topics: Amyloid Precursor Protein Secretases; Animals; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Cell Line, Tumor; Dacarbazine; Dipeptides; Glioblastoma; Humans; Mice; Neoplasm Recurrence, Local; Receptors, Notch; RNA, Messenger; Signal Transduction; Spheroids, Cellular; Temozolomide; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Gamma-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.

Topics: Amyloid Precursor Protein Secretases; Apoptosis; Blotting, Western; Cell Cycle; Cell Survival; Dipeptides; Enzyme Activation; Flow Cytometry; Gene Expression Profiling; Glioblastoma; HSP70 Heat-Shock Proteins; Humans; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Receptors, Notch; Ubiquitin