nutlin-3a and Glioblastoma

Glioblastoma multiforme is the most aggressive brain tumor, due to its high invasiveness and genetic heterogeneity. Moreover, the blood-brain barrier prevents many drugs from reaching a therapeutic concentration at the tumor site, and most of the chemotherapeutics lack in specificity toward cancer cells, accumulating in both healthy and diseased tissues, with severe side effects. Here, we present in vitro investigations on lipid-based nanovectors encapsulating a drug, nutlin-3a, and superparamagnetic iron oxide nanoparticles, to combine the proapoptotic action of the drug and the hyperthermia mediated by superparamagnetic iron oxide nanoparticles stimulated with an alternating magnetic field. The nanovectors are functionalized with the peptide angiopep-2 to induce receptor-mediated transcytosis through the blood-brain barrier and to target a receptor overexpressed by glioma cells. The glioblastoma multiforme targeting efficiency and the blood-brain barrier crossing abilities were tested through in vitro fluidic models, where different human cell lines were placed to mimic the tumor microenvironment. These nanovectors successfully cross the blood-brain barrier model, maintaining their targeting abilities for glioblastoma multiforme with minimal interaction with healthy cells. Moreover, we showed that nanovector-assisted hyperthermia induces a lysosomal membrane permeabilization that not only initiates a caspase-dependent apoptotic pathway, but also enhances the anticancer efficacy of the drug.

Topics: Blood-Brain Barrier; Cell Line, Tumor; Ferric Compounds; Glioblastoma; Humans; Imidazoles; Lysosomes; Magnetic Iron Oxide Nanoparticles; Nanoparticles; Peptides; Piperazines

Glioblastoma multiforme is one of the deadliest forms of cancer, and current treatments are limited to palliative cares. The present study proposes a nanotechnology-based solution able to improve both drug efficacy and its delivery efficiency.. Nutlin-3a and superparamagnetic nanoparticles were encapsulated in solid lipid nanoparticles, and the obtained nanovectors (nutlin-loaded magnetic solid lipid nanoparticle [Nut-Mag-SLNs]) were characterized by analyzing both their physicochemical properties and their effects on U-87 MG glioblastoma cells.. Nut-Mag-SLNs showed good colloidal stability, the ability to cross an in vitro blood-brain barrier model, and a superior pro-apoptotic activity toward glioblastoma cells with respect to the free drug.. Nut-Mag-SLNs represent a promising multifunctional nanoplatform for the treatment of glioblastoma multiforme.

Topics: Antineoplastic Agents; Biological Transport; Blood-Brain Barrier; Cell Line, Tumor; Cell Survival; Drug Carriers; Drug Liberation; Glioblastoma; Humans; Imidazoles; Kinetics; Lipids; Magnetite Nanoparticles; Particle Size; Piperazines; Surface Properties

Glioblastoma (GB) is the most common and aggressive malignant tumor of the central nervous system. Despite current intensive treatment regimens, consisting of surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide (TMZ) chemotherapy, the prognosis of patients with GB remains extremely poor. Considering that alterations of the p53 tumor suppressor pathway have a key role in both GB development and resistance to TMZ treatment, the re‑activation of p53 could be an effective therapeutic approach against GB. In this study, we challenged p53 wild‑type and mutant GB cell lines with RITA, a molecule originally identified for its ability to restore p53 functions, although it was subsequently shown to act also through p53‑independent mechanisms. We examined the effects of RITA on GB cell viability, through MTS and clonogenic assays, and analyzed cell death through cytoflourimetric analyses. In all the tested GB cell lines, RITA significantly reduced the cell proliferative and clonogenic potential and induced cell accumulation in the S and/or G2/M cell cycle phases and massive p53‑dependent apoptosis. Moreover, RITA was more effective than the well‑known p53 re‑activating molecule, nutlin‑3, and did not affect the viability of normal astrocytes. In addition, RITA decreased survivin expression and induced DNA damage, two mechanisms that likely contribute to its anti‑tumor effects. Furthermore, RITA synergized with TMZ and was able to decrease the expression of MGMT, which is a crucial player in TMZ resistance. Thus, although further studies are warranted to clarify the exact mechanisms of action of RITA, the data of this study suggest the potential of such an approach for GB therapy, which may also help to overcome resistance to TMZ.

Topics: Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Drug Synergism; Furans; Glioblastoma; Humans; Imidazoles; Mutation; Piperazines; Protein Binding; Proto-Oncogene Proteins c-mdm2; Temozolomide; Tumor Suppressor Protein p53

OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein-protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein-protein interactions.

Topics: Animals; Antineoplastic Agents, Alkylating; Brain Neoplasms; Combined Modality Therapy; Disease Models, Animal; Glioblastoma; Humans; Imidazoles; Piperazines; Proto-Oncogene Proteins c-mdm2; Temozolomide; Xenograft Model Antitumor Assays

Integrins play a role in the resistance of advanced cancers to radiotherapy and chemotherapy. In this study, we show that high expression of the α5 integrin subunit compromises temozolomide-induced tumor suppressor p53 activity in human glioblastoma cells. We found that depletion of the α5 integrin subunit increased p53 activity and temozolomide sensitivity. However, when cells were treated with the p53 activator nutlin-3a, the protective effect of α5 integrin on p53 activation and cell survival was lost. In a functional p53 background, nutlin-3a downregulated the α5 integrin subunit, thereby increasing the cytotoxic effect of temozolomide. Clinically, α5β1 integrin expression was associated with a more aggressive phenotype in brain tumors, and high α5 integrin gene expression was associated with decreased survival of patients with high-grade glioma. Taken together, our findings indicate that negative cross-talk between α5β1 integrin and p53 supports glioma resistance to temozolomide, providing preclinical proof-of-concept that α5β1 integrin represents a therapeutic target for high-grade brain tumors. Direct activation of p53 may remain a therapeutic option in the subset of patients with high-grade gliomas that express both functional p53 and a high level of α5β1 integrin.

Topics: Animals; Antineoplastic Agents, Alkylating; Brain Neoplasms; Cell Line, Tumor; Dacarbazine; Drug Resistance, Neoplasm; Glioblastoma; Humans; Imidazoles; Integrin alpha5beta1; Mice; Piperazines; Temozolomide; Treatment Outcome; Tumor Suppressor Protein p53

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cellular Senescence; Gene Silencing; Glioblastoma; Humans; Imidazoles; Mutation; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Overcoming the resistance of glioblastoma cells against temozolomide, the first-line chemotherapeutic agent of choice for newly diagnosed glioblastoma, is a major therapeutic challenge in the management of this deadly brain tumor. The gene encoding O(6) -methylguanine DNA methyltransferase (MGMT), which removes the methyl group attached by temozolomide, is often silenced by promoter methylation in glioblastoma but is nevertheless expressed in a significant fraction of cases and is therefore regarded as one of the most clinically relevant mechanisms of resistance against temozolomide. However, to date, signaling pathways regulating MGMT in MGMT-expressing glioblastoma cells have been poorly delineated. Here in this study, we provide lines of evidence that the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK)-murine double minute 2 (MDM2)-p53 pathway plays a critical role in the regulation of MGMT expression, using stem-like glioblastoma cells directly derived from patient tumor samples and maintained in the absence of serum, which not only possess stem-like properties but are also known to phenocopy the characteristics of the original tumors from which they are derived. We show that, in stem-like glioblastoma cells, MEK inhibition reduced MDM2 expression and that inhibition of either MEK or MDM2 resulted in p53 activation accompanied by p53-dependent downregulation of MGMT expression. MEK inhibition rendered otherwise resistant stem-like glioblastoma cells sensitive to temozolomide, and combination of MEK inhibitor and temozolomide treatments effectively deprived stem-like glioblastoma cells of their tumorigenic potential. Our findings suggest that targeting of the MEK-ERK-MDM2-p53 pathway in combination with temozolomide could be a novel and promising therapeutic strategy in the treatment of glioblastoma.

Topics: Aminoacetonitrile; Animals; Dacarbazine; DNA Modification Methylases; DNA Repair; DNA Repair Enzymes; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Imidazoles; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Piperazines; Proto-Oncogene Proteins c-mdm2; RNA Interference; Temozolomide; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins