ABSTRACT
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.
Subject(s)
Apoptosis/drug effects , Dipeptides/pharmacology , Glioblastoma/drug therapy , Oligopeptides/pharmacology , Proteasome Inhibitors , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Apoptosis/genetics , Blotting, Western , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Survival/drug effects , Enzyme Activation/genetics , Flow Cytometry , Gene Expression Profiling , Glioblastoma/enzymology , Glioblastoma/genetics , Glioblastoma/pathology , HSP70 Heat-Shock Proteins/biosynthesis , HSP70 Heat-Shock Proteins/metabolism , Humans , Proteasome Endopeptidase Complex/metabolism , Receptors, Notch/antagonists & inhibitors , Ubiquitin/antagonists & inhibitors , Ubiquitin/metabolismABSTRACT
BACKGROUND: KRAS and BRAF mutations appear of relevance in the genesis and progression of several solid tumor types but the co-occurrence and interaction of these mutations have not yet been fully elucidated. Using a microsatellite stable (MSS) colorectal cancer (CRC) cell line (Colo741) having mutated BRAF and KRASWT, we also aimed to investigate the KRAS-BRAF interaction. Gene expression profiles for control KRASWT, KRAS G12V and KRAS G12D transfected cells were obtained after cell clone selection and RT-PCR screening. Extensive qPCR was performed to confirm microarray data. RESULTS: We found that the KRAS G12V state deregulated several genes associated to cell cycle, apoptosis and nitrogen metabolism. These findings indicated a reduced survival and proliferation with respect to the KRASWT state. The KRAS G12D state was, instead, characterized by several other distinct functional changes as for example those related to chromatin organization and cell-cell adhesion without affecting apoptosis related genes. CONCLUSION: These data predict that the G12D mutation may be more likely selected in a BRAF mutated context. At the same time, the presence of the KRAS G12V mutation in the cells escaping apoptosis and inducing angiogenesis via IL8 may confer a more aggressive phenotype. The present results get along with the observations that CRCs with G12V are associated with a worse prognosis with respect to the WT and G12D states and may help identifying novel CRC pathways and biomarkers of clinical relevance.
