s-trans-trans-farnesylthiosalicylic-acid and Neuroblastoma

Several studies suggest that soluble Amyloid β (Aβ) oligomer-induced aberrant neuronal cell cycle re-entry is the initial trigger for a significant part of the neuronal degeneration and loss in Alzheimer's disease (AD). In this study, we investigated the role of Ras, which is a well-known protooncoprotein, in soluble Aβ oligomer-induced aberrant neuronal cell cycle activation and subsequent cell loss using retinoic acid differentiated human SH-SY5Y neuroblastoma cells as model system. In line with previous literature, we showed that in vitro preparations of soluble Aβ42 oligomers triggered cell cycle activation but not cell proliferation. As a new finding, we showed that Farnesylthiosalicylic acid (FTS), a specific chemical Ras inhibitor, prevented soluble Aβ42 oligomer preparation-induced cell cycle activation. Moreover, we showed that the expression of dominant negative mutant H-Ras (S17N) prevented soluble Aβ42 oligomer preparation-induced cell cycle activation, confirming the specific role of Ras in this pathway. As a possible better mimic of the situation in the AD brain, we prepared soluble oligomers from Aβ42 : Aβ40 (3:7) peptide mixture and showed that this oligomer preparation similarly induced cell cycle activation which was also inhibited by the Ras inhibitor. Finally, we showed that FTS prevented soluble Aβ42 oligomer preparationinduced cell death in our retinoic acid differentiated SH-SY5Y cells. Overall, our results strongly suggest that Ras activity is required for soluble Aβ oligomer-induced aberrant neuronal cell cycle reentry and subsequent neuronal loss, which are considered important mechanisms in AD pathogenesis.

Topics: Amyloid beta-Peptides; Cell Cycle; Cell Death; Cell Differentiation; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Inhibitors; Farnesol; Gene Expression Regulation; Humans; Microtubule-Associated Proteins; Neuroblastoma; Neurons; Peptide Fragments; ras Proteins; Salicylates; Time Factors

In neuroblastoma LAN-1 cells harboring an amplified MycN gene, disruption of cooperation between Ras and MycN proteins by the Ras inhibitor farnesylthiosalicylic acid (FTS, Salirasib) reportedly arrests cell growth. Our aim was to establish whether this is a general phenomenon. We examined the effects of FTS on gene-expression profiles, growth and death of NCIH929 myeloma cells and K562 leukemia cells, which-like LAN-1 cells-exhibit Myc gene amplification and harbor active Ras. Under specified conditions, FTS reduced Ras and Myc and induced cell growth arrest and death in all Myc-amplified cell lines but not in SHEP, a neuroblastoma cell line without Myc gene amplification. Gene-expression analysis revealed a common pattern of FTS-induced endoplasmic reticulum (ER) stress, known as the unfolded protein response (UPR), in Myc-amplified cells, but not in SHEP. Thus, Ras negatively regulates ER stress in cells with amplified Myc. ER stress was also inducible by dominant-negative (DN)-Ras or shRNA to Ras isoforms, all of which induced an increase in BIP (the master regulator of ER stress) and its downstream targets Nrf2 and eIF2alpha, both regulated by active p-PERK. FTS also induced an increase in p-PERK, while small interfering RNA to PERK reduced Nrf2 and ATF4 and rescued cells from FTS-induced death. BIP and its downstream targets were also increased by inhibitors of MAPK p38 and MEK. Ras, acting through MAPK p38 and MEK, negatively regulates the ER stress cascades BIP/PERK/Nrf2 and eIF2alpha/ATF4/ATF3. These findings can explain the Ras-dependent protection of Myc-amplified cells from ER stress-associated death.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; eIF-2 Kinase; Endoplasmic Reticulum; Enzyme Inhibitors; Farnesol; Gene Amplification; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; K562 Cells; Mitogen-Activated Protein Kinase Kinases; N-Myc Proto-Oncogene Protein; Neuroblastoma; NF-E2-Related Factor 2; Nuclear Proteins; Oncogene Proteins; ras Proteins; RNA, Small Interfering; Salicylates; Transcription Factors; Unfolded Protein Response

Topics: Cell Line, Tumor; Cell Proliferation; Farnesol; Gene Expression Regulation, Neoplastic; Humans; Models, Biological; Neuroblastoma; Proto-Oncogene Proteins c-myc; ras Proteins; Salicylates; Signal Transduction

Our aim was to examine whether active Ras and MycN cooperation contributes to the malignant phenotype of human neuroblastoma with amplified MycN gene, an aggressive incurable tumor.. Human neuroblastoma LAN-1 cells, in which the MycN gene is amplified, were used to examine the impact of the Ras inhibitor farnesylthiosalicylic acid on cell growth, on the levels Ras and MycN proteins, and on profiles of gene expression.. We show that LAN-1 cells express relatively large amounts of MycN and active Ras-GTP. Inhibition of active Ras by farnesylthiosalicylic acid led to attenuation of the Raf-MEK-ERK and phosphoinositide 3-kinase-Akt-glycogen synthase-3 (GSK-3) pathways, to reduction in cyclin D1, phospho-retinoblastoma, and E2F, and to increase in the cyclin-dependent kinase inhibitor p27 and in retinoblastoma-binding protein-1, an inhibitor of E2F transcriptional activity. Ras inhibition by farnesylthiosalicylic acid or by a dominant-negative Ras also led to complete disappearance of MycN protein from the nuclei of LAN-1 cells. This was a result of blocking of Akt inactivation of GSK-3, leading to GSK-3-dependent phosphorylation with consequent proteosomal degradation of MycN. Loss of active Ras and of MycN in LAN-1 cells was manifested in profiles of gene expression that could be expected from the loss of MycN transcriptional activity and of Ras signaling. These changes explain the farnesylthiosalicylic acid-induced inhibition of LAN-1 cell growth.. Active Ras is needed to block MycN degradation, promoting cooperative Ras- and MycN-dependent cell cycle progression in LAN-1 cells. Ras inhibitors are therefore likely candidates for the treatment of advanced neuroblastoma characterized by high expression of MycN.

Topics: Carrier Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Farnesol; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Green Fluorescent Proteins; Humans; Immunoblotting; In Situ Hybridization, Fluorescence; Microscopy, Confocal; Mitogen-Activated Protein Kinases; Neuroblastoma; Oligonucleotide Array Sequence Analysis; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; raf Kinases; ras Proteins; Retinoblastoma-Binding Protein 1; Salicylates; Signal Transduction; Transfection; Tumor Suppressor Proteins

A lipophilic farnesyl moiety attached to the carboxyl terminal cysteine of ras proteins structurally supports their membrane anchorage, required for ras-dependent growth-factor signaling and for transforming activity of ras oncoproteins. It has been shown that inhibition of ras farnesylation can block tumor growth in nude mice but that some ras-dependent tumors escape such blockage as a result of prenylation of ras. S-trans-transfarnesylthiosalicylic acid (FTS) is a potent ras-dislodging antagonist that does not affect ras prenylation but rather acts on the mature, membrane-bound ras and facilitates its degradation. Here we demonstrate that FTS induces reappearance of stress fibers in H-ras-transformed rat-1 cells (EJ cells) in vitro, inhibits their anchorage-independent growth in vitro, and blocks EJ-tumor growth in nude mice. The anchorage-independent growth of cells expressing ErbB2 (B104), but not that of v-raf-transformed cells, is also inhibited by FTS, suggesting specificity towards activated ras. FTS treatment (5 mg/kg i.p. daily) caused inhibition (75-80%) of tumor growth in nude mice implanted with EJ, but not in mice implanted with v-raf-transformed cells, with no evidence of systemic toxicity. Moreover, FTS treatment increased the survival rate of EJ-tumor-bearing mice from 48 to 68 days. Here we demonstrate anti-tumor potency in a synthetic, non-toxic, ras-dislodging antagonist acting independently of farnesyltransferases.

Topics: 3T3 Cells; Actins; Animals; Antineoplastic Agents; Cell Division; Cell Transformation, Neoplastic; Cytoskeleton; Farnesol; Genes, erbB-2; Genes, ras; Male; Mice; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Neoplasms, Experimental; Neuroblastoma; Oncogene Proteins v-raf; Protein Prenylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins p21(ras); Rats; Receptor, ErbB-2; Retroviridae Proteins, Oncogenic; Salicylates; Substrate Specificity; Tumor Cells, Cultured