s-trans-trans-farnesylthiosalicylic-acid and Glioblastoma

We aimed to develop lipid-polyethylene glycol (PEG)-polymer hybrid nanoparticles, which have high affinity to tumour tissue with active ingredient, a new generation antineoplastic drug, farnesylthiosalicylic acid (FTA) for treatment of glioblastoma.. Farnesylthiosalicylic acid-loaded poly(lactic-co-glycolic acid)-1,2 distearoyl-glycerol-3-phospho-ethanolamine-N [methoxy (PEG)-2000] ammonium salt (PLGA-DSPE-PEG) with or without 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) hybrid nanoparticles has been prepared and evaluated for in-vitro characterization. Cytotoxicity of FTA-loaded nanoparticles along with its efficacy on rat glioma-2 (RG2) cells was also evaluated both in vitro (in comparison with non-malignant cell line, L929) and in vivo.. Scanning electron microscopy studies showed that all formulations prepared had smooth surface and spherical in shape. FTA and FTA-loaded nanoparticles have cytotoxic activity against RG2 glioma cell lines in cell culture studies, which further increases with addition of DOTAP. Magnetic resonance imaging and histopathologic evaluation on RG2 tumour cells in rat glioma model (49 female Wistar rats, 250-300 g) comparing intravenous and intratumoral injections of the drug have been performed and FTA-loaded nanoparticles reduced tumour size significantly in in-vivo studies, with higher efficiency of intratumoral administration than intravenous route.. Farnesylthiosalicylic acid-loaded PLGA-DSPE-PEG-DOTAP hybrid nanoparticles are proven to be effective against glioblastoma in both in-vitro and in-vivo experiments.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Farnesol; Female; Glioblastoma; Lipids; Nanoparticles; Polyethylene Glycols; Polymers; Rats; Rats, Wistar; Salicylates; Treatment Outcome; Tumor Burden

New drug delivery systems are highly needed in research and clinical area to effectively treat gliomas by reaching a high antineoplastic drug concentration at the target site without damaging healthy tissues. Intranasal (IN) administration, an alternative route for non-invasive drug delivery to the brain, bypasses the blood-brain-barrier (BBB) and eliminates systemic side effects. This study evaluated the antitumor efficacy of farnesylthiosalicylic acid (FTA) loaded (lipid-cationic) lipid-PEG-PLGA hybrid nanoparticles (HNPs) after IN application in rats. FTA loaded HNPs were prepared, characterized and evaluated for cytotoxicity. Rat glioma 2 (RG2) cells were implanted unilaterally into the right striatum of female Wistar rats. 10days later, glioma bearing rats received either no treatment, or 5 repeated doses of 500μM freshly prepared FTA loaded HNPs via IN or intravenous (IV) application. Pre-treatment and post-treatment tumor sizes were determined with MRI. After a treatment period of 5days, IN applied FTA loaded HNPs achieved a significant decrease of 55.7% in tumor area, equal to IV applied FTA loaded HNPs. Herewith, we showed the potential utility of IN application of FTA loaded HNPs as a non-invasive approach in glioblastoma treatment.

Topics: Administration, Intranasal; Animals; Antineoplastic Agents; Blood-Brain Barrier; Brain Neoplasms; Drug Carriers; Drug Delivery Systems; Farnesol; Female; Glioblastoma; Lipids; Magnetic Resonance Imaging; Nanoparticles; Polyesters; Polyethylene Glycols; Rats; Rats, Wistar; Salicylates; Treatment Outcome

The three oncogenes, ErbB receptors, Ras proteins and nucleolin may contribute to malignant transformation. Previously, we demonstrated that nucleolin could bind both Ras protein and ErbB receptors. We also showed that the crosstalk between the three proteins facilitates anchorage independent growth and tumor growth in nude mice, and that inhibition of this interaction in prostate and colon cancer cells reduces tumorigenicity. In the present study, we show that treatment with Ras and nucleolin inhibitors reduces the oncogenic effect induced by ErbB1 receptor in U87-MG cells. This combined treatment enhances cell death, reduces cell proliferation and cell migration. Moreover, we demonstrate a pivotal role of nucleolin in ErbB1 activation by its ligand. Nucleolin inhibitor prevents EGF-induced receptor activation and its downstream signaling followed by reduced proliferation. Furthermore, inhibition of Ras by Salirasib (FTS), mainly reduces cell viability and motility. The combined treatment, which targets both Ras and nucleolin, additively reduces tumorigenicity both in vitro and in vivo. These results suggest that targeting both nucleolin and Ras may represent an additional opportunity for inhibiting cancers, including glioblastoma, that are driven by these oncogenes.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Aptamers, Nucleotide; Brain Neoplasms; Cell Death; Cell Line, Tumor; Cell Movement; Cell Proliferation; ErbB Receptors; Farnesol; Glioblastoma; Humans; Mice, Nude; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; Phosphorylation; ras Proteins; Receptor Cross-Talk; RNA-Binding Proteins; Salicylates; Signal Transduction; Time Factors; Tumor Burden

Primary glioblastomas (GBMs) commonly overexpress the oncogene epidermal growth factor receptor (EGFR), which leads to increased Ras activity. FTA, a novel Ras inhibitor, produced both time- and dose-dependent caspase-mediated apoptosis in GBM cell lines. EGFR-mediated increase in 3H-thymidine uptake was inhibited by FTA. FACS analysis was performed to determine the percent of apoptotic cells. The sub-Go population of GBM cells was increased from 4.5 to 13.8% (control) to over 45-53.6% in FTA-treated cells within 24 h. Furthermore, FTA also increased the activities of both caspase-3 and -9, and PARP cleavage. Treatment of GBMs with FTA before or after EGF addition to the cultures blocked phosphorylation of Akt and mitogen-activated protein kinases (MAPK). FTA also significantly reduced the amount of EGF-induced Ras-GTP as reflected by a decrease in the level of Ras bound to Raf-RBD-GST. This study demonstrates that inhibition of Ras methylation may provide a therapeutic target for the treatment of GBMs overexpressing EGFR.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Farnesol; Glioblastoma; Humans; Mitogen-Activated Protein Kinases; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; ras Proteins; Salicylates; Signal Transduction; Time Factors

Active Ras contributes to the malignant phenotype of glioblastoma multiforme. Recent studies showed that the Ras inhibitor farnesyl thiosalicylic acid downregulates the transcription factor hypoxia-inducible factor-1alpha, causing shutdown of glycolysis in U87 glioblastoma cells. Farnesyl thiosalicylic acid also inhibited the growth of U87 cells. The way in which Ras inhibition affects U87 cell proliferation was not clear. Here we applied a computational method in which gene expression profile clustering is combined with promoter sequence analysis to obtain global dissection of the transcriptional response to farnesyl thiosalicylic acid in U87 cells. The analysis revealed a prominent Ras-dependent cell-cycle arrest response, in which a major component is highly enriched for the binding-site signature of the transcription factor E2F1. Electrophoretic mobility shift assays together with E2F-luciferase reporter assays showed that E2F1 was inactivated by the Ras inhibitor. Inhibition of Ras by farnesyl thiosalicylic acid promoted proteasomal degradation of cyclin D1, with a concomitant decrease in phosphorylated retinoblastoma protein accompanied by downregulation of E2F1 and decreased expression of key E2F1-regulated genes critical for cell-cycle progression. U87 cell growth arrest induced by farnesyl thiosalicylic acid was overridden by constitutive expression of E2F1. Thus, downregulation of E2F1 and of hypoxia-inducible factor-1alpha represents 2 distinct arms of the antioncogenic effect of Ras inhibitors in glioblastoma.

Topics: Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin D1; Dose-Response Relationship, Drug; E2F1 Transcription Factor; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Farnesol; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioblastoma; Green Fluorescent Proteins; Humans; Immunoblotting; Immunohistochemistry; Luciferases; Phosphorylation; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; ras Proteins; Recombinant Fusion Proteins; Retinoblastoma Protein; Salicylates

The Ras inhibitor farnesylthiosalicylic acid (FTS) has been shown to induce apoptosis in glioblastoma multiforme, but its mechanism of action was unknown. We show that FTS or dominant-negative Ras, by deregulating extracellular signal-regulated kinase and Akt signaling, decreases survivin gene transcripts in U87 glioblastoma multiforme, leading to disappearance of survivin protein and cell death. FTS affected both Ras-controlled regulators of survivin transcription and Ras-regulated survival signals. Thus, Ras inhibition by FTS resulted in release of the survivin "brake" on apoptosis and in activation of the mitochondrial apoptotic pathway: dephosphorylation of Bad, activation of Bax, release of cytochrome c, and caspase activation. FTS-induced apoptosis of U87 cells was strongly attenuated by forced expression of survivin or by caspase inhibitors. These results show that resistance to apoptosis in glioblastoma multiforme can be abolished by a single Ras inhibitor, which targets both survivin, a critical inhibitor of apoptosis, and the intrinsic mitochondrial apoptotic machinery.

Topics: Apoptosis; Caspases; Cell Line, Tumor; Farnesol; Gene Expression; Glioblastoma; Humans; Inhibitor of Apoptosis Proteins; Microtubule-Associated Proteins; Neoplasm Proteins; ras Proteins; RNA, Messenger; Salicylates; Survivin; Transfection

Active Ras and phosphatidylinositol-3-kinase-dependent pathways contribute to the malignant phenotype of glioblastoma multiformes (GBM). Here we show that the Ras inhibitor trans-farnesylthiosalicylic acid (FTS) exhibits profound antioncogenic effects in U87 GBM cells. FTS inhibited active Ras and attenuated Ras signaling to extracellular signal-regulated kinase, phosphatidylinositol-3-kinase, and Akt. Concomitantly, hypoxia-inducible factor 1alpha (HIF-1alpha) disappeared, expression of key glycolysis pathway enzymes and of other HIF-1alpha-regulated genes (including vascular endothelial growth factor and the Glut-1 glucose transporter) was down-regulated, and glycolysis was halted. This led to a dramatic reduction in ATP, resulting in a severe energy crisis. In addition, the expression of E2F-regulated genes was down-regulated in the FTS-treated cells. Consequently, U87 cell growth was arrested and the cells died. These results show that FTS is a potent down-regulator of HIF-1alpha and might therefore block invasiveness, survival, and angiogenesis in GBM.

Topics: Cell Death; Cell Growth Processes; Cell Line, Tumor; Down-Regulation; Farnesol; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioblastoma; Glycolysis; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Phosphatidylinositol 3-Kinases; ras Proteins; Salicylates; Transcription Factors