betadex and Glioma

The lack of clinical response to the alkylating agent temozolomide (TMZ) in pediatric diffuse midline/intrinsic pontine glioma (DIPG) has been associated with

Topics: Animals; Antineoplastic Agents, Alkylating; Apoferritins; beta-Cyclodextrins; Cell Line, Tumor; Drug Carriers; Glioma; Humans; Liposomes; Male; Nanoparticles; Rats, Wistar; Spheroids, Cellular; Temozolomide

In this paper, we examined arsthinol-cyclodextrin complexes, which display an anticancer activity. The association constants were 17,502±522 M(-1) for hydroxypropyl-β-cyclodextrin and 12,038±10,168 M(-1) for randomized methylated β-cyclodextrin. (1)H NMR experiments in solution also confirmed the formation of these complexes and demonstrated an insertion of the arsthinol (STB) with its dithiarsolane extremity into the wide rim of the hydroxypropyl-β-cyclodextrin cavity. Complexed arsthinol was more effective than arsenic trioxide (As2O3) and melarsoprol on the U87 MG cell line. Importantly, in the in vivo study, we observed significant antitumor activity against heterotopic xenografts after i.p. administration and did not see any signs of toxicity. This remains to be verified using an orthotopic model.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Animals; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; beta-Cyclodextrins; Brain Neoplasms; Cell Line, Tumor; Excipients; Female; Glioma; Humans; Injections, Intraperitoneal; Magnetic Resonance Spectroscopy; Melarsoprol; Mice; Mice, Nude; Oxides; Xenograft Model Antitumor Assays

Camptothecin (CPT), a plant alkaloid, is a potent anticancer drug in cell culture studies but it is clinically inactive due to rapid hydrolysis under physiological conditions. The drug exists in two forms depending on the pH value, an active lactone form at pH below 5 and an inactive carboxylate form at basic pH and this is a reversible reaction. In this study, nanoparticulate delivery systems were developed with either amphiphilic cyclodextrins, poly(lactide-co-glycolide) or poly-ɛ-caprolactone in order to maintain the active lactone form and prevent the drug from hydrolysis. All nanoparticles were prepared with nanoprecipitation technique. Mean particle sizes were 130-280nm and surface charges were negative. The encapsulation efficiency was significantly higher for amphiphilic cyclodextrin nanoparticles when compared to polymeric nanoparticles. Nanoparticle formulations based on cyclodextrins showed a controlled release profile extended up to 12 days. 6-O-Capro-β-cyclodextrin (1.44μg/60μL CPT) and concentrated 6-O-Capro-β-cyclodextrin (2.88μg/60μL CPT) nanoparticles significantly modified the growth or lethality of the 9L gliomas, since the median survival time was 26 days for the untreated group and between 27 and 33 days for amphiphilic cyclodextrin nanoparticle groups. These results indicate that, CPT-loaded amphiphilic cyclodextrin nanoparticles may provide a promising carrier system for the effective delivery of CPT in comparison to polymeric analogues.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Animals; Antineoplastic Agents, Phytogenic; beta-Cyclodextrins; Brain Neoplasms; Camptothecin; Cell Line, Tumor; Drug Carriers; Drug Compounding; Female; Glioma; Lactic Acid; Nanoparticles; Neoplasm Transplantation; Particle Size; Polyesters; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Rats, Inbred F344; Surface-Active Agents

CD44 is a cell surface adhesion molecule for hyaluronan and is implicated in tumor invasion and metastasis. Proteolytic cleavage of CD44 plays a critical role in the migration of tumor cells and is regulated by factors present in the tumor microenvironment, such as hyaluronan oligosaccharides and epidermal growth factor. However, molecular mechanisms underlying the proteolytic cleavage on membranes remain poorly understood. In this study, we demonstrated that cholesterol depletion with methyl-β-cyclodextrin, which disintegrates membrane lipid rafts, enhances CD44 shedding mediated by a disintegrin and metalloproteinase 10 (ADAM10) and that cholesterol depletion disorders CD44 localization to the lipid raft. We also evaluated the effect of long term cholesterol reduction using a statin agent and demonstrated that statin enhances CD44 shedding and suppresses tumor cell migration on a hyaluronan-coated substrate. Our results indicate that membrane lipid organization regulates CD44 shedding and propose a possible molecular mechanism by which cholesterol reduction might be effective for preventing and treating the progression of malignant tumors.

Topics: ADAM Proteins; ADAM10 Protein; Amyloid Precursor Protein Secretases; beta-Cyclodextrins; Cell Line, Tumor; Cell Movement; Cholesterol; Epidermal Growth Factor; Glioma; Humans; Hyaluronan Receptors; Hyaluronic Acid; Membrane Microdomains; Membrane Proteins; Neoplasm Metastasis; Neoplasm Proteins

Type-1 (CB1) and type-2 (CB2) cannabinoid receptors belong to the rhodopsin family of G protein-coupled receptors, and are activated by endogenous lipids termed "endocannabinoids". Recent reports have demonstrated that CB1R, unlike CB2R and other receptors and metabolic enzymes of endocannabinoids, functions in the context of lipid rafts, i.e. plasma membrane microdomains which may be important in modulating signal transduction. Here, we present novel data based on cell subfractionation, immunoprecipitation and confocal microscopy studies, that show that in C6 cells CB1R co-localizes almost entirely with caveolin-1. We also show that trafficking of CB1R in response to the raft disruptor methyl-beta-cyclodextrin (MCD) is superimposable on that of caveolin-1, and that MCD treatment increases the accessibility of CB1R to its specific antibodies. These findings may be relevant for the manifold CB1R-dependent activities of endocannabinoids, like the regulation of apoptosis and of neurodegenerative diseases.

Topics: Animals; beta-Cyclodextrins; Caveolin 1; Cell Fractionation; Cell Line, Tumor; Glioma; Immunoprecipitation; Microscopy, Confocal; Protein Transport; Rats; Receptor, Cannabinoid, CB1; Time Factors

Tenascin-C is an extracellular matrix glycoprotein, whose expression is highly restricted in normal adult tissues, but markedly up-regulated in a range of tumors, and therefore serves as a potential receptor for targeted anticancer drug or gene delivery. We describe here a liposomal carrier system in which the targeting ligand is sulfatide. Experiments with tenascin-C-expressing glioma cells demonstrated that binding of liposomes to the extracellular matrix relied essentially on the sulfatide-tenascin-C interaction. Following binding to the extracellular matrix, the sulfatide-containing liposomes were internalized via both caveolae/lipid raft- and clathrin-dependent pathways, which would ensure direct cytoplasmic release of the cargoes carried in the liposomes. Such natural lipid-guided intracellular delivery targeting at the extracellular matrix glycoproteins of tumor cells thus opens a new direction for development of more effective anticancer chemotherapeutics in future.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Antibodies; beta-Cyclodextrins; Calcitriol; Clathrin; Endocytosis; Extracellular Matrix; Glioma; Humans; Liposomes; Protein Binding; RNA, Small Interfering; Sphingosine; Sucrose; Sulfoglycosphingolipids; Tenascin; Tumor Cells, Cultured; Type C Phospholipases

Several G protein-coupled receptors function within lipid rafts plasma membrane microdomains, which may be important in limiting signal transduction. Here we show that treatment of rat C6 glioma cells with the raft disruptor methyl-beta-cyclodextrin (MCD) doubles the binding efficiency (i.e. the ratio between maximum binding and dissociation constant) of type-1 cannabinoid receptors (CB1R), which belong to the rhodopsin family of G protein-coupled receptors. In parallel, activation of CB1R by the endogenous agonist anandamide (AEA) leads to approximately 3-fold higher [35S]GTPgammaS binding in MCD-treated cells than in controls, and CB1R-dependent signaling via adenylate cyclase, and p42/p44 MAPK is almost doubled by MCD. Unlike CB1R, the other AEA-binding receptor TRPV1, the AEA synthetase NAPE-PLD, and the AEA hydrolase FAAH are not modulated by MCD, whereas the activity of the AEA membrane transporter (AMT) is reduced to approximately 50% of the controls. We also show that MCD reduces dose-dependently AEA-induced apoptosis in C6 cells but not in human CHP100 neuroblastoma cells, which mirror the endocannabinoid system of C6 cells but are devoid of CB1R. MCD reduces also cytochrome c release from mitochondria of C6 cells, and this effect is CB1R-dependent and partly mediated by activation of p42/p44 MAPK. Altogether, the present data suggest that lipid rafts control CB1R binding and signaling, and that CB1R activation underlies the protective effect of MCD against apoptosis.

Topics: Animals; Apoptosis; Arachidonic Acids; beta-Cyclodextrins; Biological Transport; Cannabinoid Receptor Modulators; Cell Line, Tumor; Cell Membrane; Cell Separation; Cholesterol; Cyclic AMP; Dose-Response Relationship, Drug; Endocannabinoids; Flow Cytometry; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hydrolysis; Kinetics; Lipid Metabolism; Lipids; Membrane Microdomains; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuroblastoma; Neurons; Polyunsaturated Alkamides; Protein Binding; Rats; Receptor, Cannabinoid, CB1; Receptors, Cannabinoid; Rhodopsin; Signal Transduction; Time Factors

We describe a simple and widely applicable method to measure cell migration in time-lapse sequences of fluorescently labeled cells in culture. Briefly, binarized cell images obtained after thresholding were cumulatively projected, and the covered areas were measured. This procedure determines the time course of the track area successively covered by the cell population. Under conditions where cell growth is negligible, a robust index of cell motility is derived from normalized plots for the displacement of cells over time. We applied this method to quantitatively examine the migration of B35 neuroblastoma cells transiently expressing GFP and to C6 glioma cells after staining with Hoechst 33258. This sensitive assay detected the influence of agents which inhibit actin polymerization (cytochalasin B) or interfere with the maintenance of cell polarity (methyl-beta-cyclodextrin) on cell migration. Thus, this assay is a versatile tool to measure quickly the migration of different cell types using different labeling strategies.

Topics: Animals; beta-Cyclodextrins; Cell Line, Tumor; Cell Movement; Cytochalasin B; Glioma; Green Fluorescent Proteins; Microscopy, Fluorescence; Neuroblastoma; Neuroglia; Neurons; Rats

Hyaluronan (HA) is a component of the brain extracellular matrix environment that is synthesized and secreted by glioma cells. The primary cell surface receptor for HA is CD44, a membrane glycoprotein that is functionally regulated by a membrane type 1 matrix metalloproteinase (MT1-MMP). Both CD44 and MT1-MMP are partially located in Triton X-100-insoluble domains, but no functional link has yet been established between them. In the present study, we studied the regulation of HA cell surface binding in U-87 glioma cells. We show that an MMP-dependent mechanism regulates the intrinsic cell surface binding of HA as ilomastat, a broad MMP inhibitor, increased HA binding to glioma cells. HA binding was also rapidly and specifically up-regulated by 3-fold by type I collagen in U-87 cells, which also induced a significant morphological reorganization associated with the activation of a latent form of MMP-2 through a MT1-MMP-mediated mechanism. Interestingly, caveolae depletion with a cell surface cholesterol-depleting agent beta-cyclodextrin triggered an additional increase (9-fold) in the binding of HA, in synergy with type I collagen. On the other hand, HA cell surface binding was diminished by the MEK inhibitor PD98059 and by the overexpression of a recombinant, wild type MT1-MMP, whereas its cytoplasmic-deleted form had no effect. Taken together, our results suggest that MT1-MMP regulates, through its cytoplasmic domain, the cell surface functions of CD44 in a collagen-rich pericellular environment. Additionally, we describe a new molecular mechanism regulating the invasive potential of glioma cells involving a MT1-MMP/CD44/caveolin interaction, which could represent a potential target for anti-cancer therapies.

Topics: beta-Cyclodextrins; Caveolae; Cell Line, Tumor; Cell Membrane; Cholesterol; Collagen Type I; Coloring Agents; Cyclodextrins; Detergents; DNA, Complementary; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Flavonoids; Flow Cytometry; Fluorescein-5-isothiocyanate; Glioma; Humans; Hyaluronan Receptors; Hyaluronic Acid; Hydroxamic Acids; Immunoblotting; Indoles; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Models, Biological; Octoxynol; Plasmids; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA; Temperature; Transfection; Up-Regulation

Cannabinoids induce the expression of the cyclooxygenase-2 (COX-2) isoenzyme in H4 human neuroglioma cells via a pathway independent of cannabinoid- or vanilloid receptor activation. The underlying mechanism was recently shown to involve increased synthesis of ceramide, which in turn leads to activation of p38 and p42/44 mitogen-activated protein kinases (MAPKs). The present study investigates a possible contribution of membrane lipid rafts to cannabinoid-induced COX-2 expression. To address this issue, we tested the influence of methyl-beta-cyclodextrin (MCD), a membrane cholesterol depletor, on COX-2 expression by the endocannabinoid analogue R(+)-methanandamide (R(+)-MA). Incubation of H4 cells with MCD was associated with a loss of lipid raft integrity and a substantial inhibition of R(+)-MA-induced COX-2 expression and subsequent formation of prostaglandin E2. Moreover, MCD was shown to suppress signal transduction steps upstream to COX-2 induction by R(+)-MA. Accordingly, the cholesterol depletor suppressed R(+)-MA-induced formation of ceramide as well as phosphorylation of p38 and p42/44 MAPKs. Together, our results suggest that R(+)-MA induces COX-2 expression in human neuroglioma cells via a pathway linked to lipid raft microdomains.

Topics: Arachidonic Acids; beta-Cyclodextrins; Blotting, Western; Brain Neoplasms; Cell Line; Cell Line, Tumor; Ceramides; Cholesterol; Cyclooxygenase 2; Dinoprostone; Dose-Response Relationship, Drug; Glioma; Humans; Isoenzymes; MAP Kinase Signaling System; Membrane Microdomains; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction