Farnesylthiosalicylic acid induces caspase activation and apoptosis in glioblastoma cells.

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.

5-Hydroxytryptamine2 (5-HT2) structure-function relationships of the nitro and amino phenylpiperazines on intact human platelets.

Nitro and amino phenylpiperazines were synthesized to study the agonist and antagonist activities of the phenylpiperazines at the human platelet 5-hydroxytryptamine2 (5-HT2) receptor. Amplification of ADP-induced aggregation and binding competition experiments with [3H]ketanserin were used to evaluate receptor interactions in this system. All the monosubstituted phenylpiperazines were antagonists despite the wide variation in electronic and hydrophobic properties. The parent compound, unsubstituted phenylpiperazine (PP), had the lowest affinity for the [3H]ketanserin binding site. The intensely electron-withdrawing NO2 substituent increased binding affinity at all ring positions and this activity correlated with antagonist potency in platelet aggregation studies (rank order: 4-NO2-PP greater than 3-NO2-PP greater than 2-NO2-PP). NH2 substitution decreased binding affinity at the 4- and 2-positions compared with the analogous NO2 substituted compounds; however, evaluation of NH2-PP antagonist potency in aggregation studies was complicated due to slow association with the receptor. To compare the activities of the phenylpiperazines at other 5-HT sites, binding competition experiments were performed using [3H]5-HT in bovine brain membranes. The rank order of the affinities for the NO2 substituted compounds was distinctly different from that determined with platelets, reflecting the heterogeneous composition of 5-HT receptor subtypes in the brain membrane preparations. The platelet aggregation experiments demonstrated that marked alteration of the electronic and hydrophobic properties of phenylpiperazine by ring substitution did not impart 5-HT2 agonist activity. By contrast, 5-HT2 antagonist activity appeared to be enhanced markedly by electron-withdrawing resonance effects which decreased the electron density at the 1'-piperazine nitrogen. This enhancement appeared to be specific for the 5-HT2 receptor subtype.

Modulatory effects of aluminum, calcium, lithium, magnesium, and zinc ions on [3H]MK-801 binding in human cerebral cortex.

The independent and combined effects of Ca2+, Mg2+, Zn2+, Al3+ and Li+ on [3H]MK-801 binding in human cerebral cortical membranes were studied to further characterize the modulatory effects of metal ions on the N-methyl-D-aspartate (NMDA) receptor-ionophore. Glycine, in the presence of glutamate, significantly intensified the Mg2+ inhibition of [3H]MK-801 binding whereas it masked the Ca2+ enhancement and slightly diminished the Zn2+ inhibition. Both Ca2+ and Mg2+ reduced the Zn2+ inhibitory potency. Aluminum demonstrated a potent, relatively glycine-insensitive inhibition of [3H]MK-801 binding as an amorphous Al(OH)3 polymer rather than as the free ion. Cationic modulation of the NMDA receptor-ionophore appears to be regulated at multiple sites which have significant allosteric interactions.

Elimination of ascorbic acid-induced membrane lipid peroxidation and serotonin receptor loss by Trolox-C, a water soluble analogue of vitamin E.

Ascorbic acid is commonly used as an antioxidant to prevent the decomposition of ligands in neurotransmitter receptor studies, but may alter biological membranes by initiating lipid peroxidation in the presence of physiologic metal ions. The aim of the present study was to characterize the effect of ascorbic acid-induced lipid peroxidation on an applicable membrane receptor and to examine an appropriate antioxidant system. Ascorbic acid generated significant lipid peroxidation (5.5 to 45 fold increase in malonaldehyde levels) in three diverse tissues having different membrane properties: bovine brain, mouse teratoma, and rat kidney. In membranes from bovine cerebral cortex, ascorbate-induced lipid peroxidation was associated with a 26% decrease in [3H]-serotonin receptor binding (Bmax = 159 +/- 11 from control of 216 +/- 10 fmol/mg protein), with no significant change in KD. Trolox-C, a water soluble analogue of vitamin E, completely blocked the ascorbate-induced loss of serotonin receptor binding in brain membranes, and the combination of Trolox-C and ascorbate prevented [3H]-serotonin decomposition in solution. Trolox-C also prevented ascorbate-induced lipid peroxidation in brain, teratoma, and kidney membranes. Lipid peroxidation may be a significant factor in the ascorbate-induced alteration of brain membranes as reflected by reduced binding to serotonin receptors. The combination of Trolox-C (200 microM) and ascorbic acid (1.0 mM) maintains a protective environment for oxygen sensitive neurotransmitters while blocking the deleterious effects of ascorbic acid on lipid membranes.

Pertussis toxin activates platelets through an interaction with platelet glycoprotein Ib.

Platelets present a unique model to study the B-oligomer effects of pertussis toxin because they become activated in response to the B oligomer but are not susceptible to ADP-ribosylation by the holotoxin. In these studies, the B oligomer of pertussis toxin caused concentration-dependent platelet activation, as determined by increases in intracellular calcium concentration, dense granule secretion, and platelet aggregation. Stirring was required for pertussis toxin to increase intracellular calcium. A monoclonal antibody against platelet glycoprotein Ib abolished increases in intracellular calcium concentration and increased the latency and reduced the slope of the aggregation response elicited by the B oligomer. Pertussis toxin also evoked [14C]serotonin release from platelets, and this effect was inhibited, though not eliminated, by an antibody against platelet glycoprotein Ib. Binding of pertussis toxin to glycoprotein Ib was observed after nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that the B oligomer of pertussis toxin induces platelet activation mediated, at least in part, by an interaction with platelet glycoprotein Ib.

Plasmin modulates the thrombin-evoked calcium response in C6 glioma cells.

Extracellular proteinases may be selectively targeted to cell surfaces by specific receptors or binding sites. In previous studies, we have characterized cellular binding sites for plasminogen and plasmin on rat C6 glioma cells. In this investigation, we studied the response of C6 cells to alpha-thrombin and plasmin by measuring the rapid kinetics of free intracellular Ca2+ concentrations ([Ca2+]i). Thrombin produced a strong, concentration-dependent rise in [Ca2+]i with an onset within 3 s and peak levels achieved in less than 10 s. A similar response was also evoked by an SFLLRN-containing thrombin-agonist peptide. C6 cells did not respond to plasmin (25 nM-1.5 microM). By contrast, pretreatment of C6 cells with 100 nM plasmin significantly inhibited the [Ca2+]i response to thrombin and the thrombin-agonist peptide. The peak [Ca2+]i response to thrombin, in cells pretreated with plasmin, was reduced by approx. 50%. The effect of plasmin on the cellular response to thrombin was selective, as pretreatment of the cells with plasmin did not affect the [Ca2+]i response to platelet-activating factor. Di-isopropylphosphorylplasmin and plasminogen did not inhibit the cellular response to thrombin, indicating that plasmin activity is required and that occupancy of cellular plasmin(ogen)-binding sites alone is insufficient. These studies demonstrate that plasmin does not directly induce a response in C6 cells, but may affect cellular function by specifically modulating the thrombin response.

Phorbol 12-myristate 13-acetate induces protein kinase ceta-specific proliferative response in astrocytic tumor cells.

Protein kinase C (PKC) activation has been implicated in cellular proliferation in neoplastic astrocytes. The roles for specific PKC isozymes in regulating this glial response, however, are not well understood. The aim of this study was to characterize the expression of PKC isozymes and the role of PKC-eta expression in regulating cellular proliferation in two well characterized astrocytic tumor cell lines (U-1242 MG and U-251 MG) with different properties of growth in cell culture. Both cell lines expressed an array of conventional (alpha, betaI, betaII, and gamma) and novel (theta and epsilon) PKC isozymes that can be activated by phorbol myristate acetate (PMA). Another novel PKC isozyme, PKC-eta, was only expressed by U-251 MG cells. In contrast, PKC-delta was readily detected in U-1242 MG cells but was present only at low levels in U-251 MG cells. PMA (100 nm) treatment for 24 h increased cell proliferation by over 2-fold in the U-251 MG cells, whereas it decreased the mitogenic response in the U-1242 MG cells by over 90%. When PKC-eta was stably transfected into U-1242 MG cells, PMA increased cell proliferation by 2.2-fold, similar to the response of U-251 MG cells. The cell proliferation induced by PMA in both the U-251 MG and U-1242-PKC-eta cells was blocked by the PKC inhibitor bisindolylmaleimide (0.5 micrometer) and the MEK inhibitor, PD 98059 (50 micrometer). Transient transfection of wild type U-251 with PKC-eta antisense oligonucleotide (1 micrometer) also blocked the PMA-induced increase in [(3)H]thymidine incorporation. The data demonstrate that two glioblastoma lines, with functionally distinct proliferative responses to PMA, express different novel PKC isozymes and that the differential expression of PKC-eta plays a determining role in the different proliferative capacity.

Epidermal growth factor differentially regulates low density lipoprotein receptor-related protein gene expression in neoplastic and fetal human astrocytes.

Low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytotic receptor that may modify the biological activity of reactive astrocytes in neuroplasticity and neurodegeneration and of malignant astrocytes in brain invasion. In this study, the regulation of LRP by epidermal growth factor receptor (EGFR) ligands in both cultured human fetal astrocytes and astrocytic tumor cell lines (U-251 MG and U-1242 MG) was investigated. All astrocytic cell types expressed LRP, as determined by the binding of activated alpha2-macroglobulin (alpha2M*) on intact cells and by Western and Northern blot analyses of cell extracts. Primary cultured astrocytes expressed the highest levels of alpha2M*-binding capacity (Bmax = 30 fmol/mg protein). This was twofold higher than for the U-1242 MG astrocytoma cells (Bmax = 15 fmol/mg protein) and fourfold greater than for the glioblastoma U-251 MG cells (7.0 fmol/mg protein). Receptor affinity (K(D)) ranged from 0.25 to 0.6 nM in all the astroglial cell types. Functional LRP at the surface was down-regulated by EGF, compared with controls, as indicated by a reduction of both Bmax and LRP-mediated endocytosis by approximately 50% and 60%, respectively. In comparison, EGF treatment of primary astrocytes did not down-regulate LRP expression or LRP-mediated endocytosis. Treatment of the tumor cells with EGF or TGFalpha (25 ng/ml) significantly down-regulated total cellular LRP. Receptor-associated protein (RAP) mRNA expression was not affected by EGF in either tumor cells or primary astrocytes. The reduction of LRP in the tumor cells resulted from a specific decrease in LRP mRNA transcription, as determined by Northern blot and nuclear run-on experiments. These data suggest that EGF mediates a functional down-regulation of LRP endocytotic activity in astrocytic tumor cells and that LRP expression is differentially regulated in neoplastic and non-neoplastic astrocytes.