Human U373 MG Astrocytoma Research Articles

Enhancement of methylene blue-induced cytotoxicity in human brain tumor cells by an iron chelator, deferoxamine

Previously, we have reported that methylene blue (MB) induces cytotoxicity in human brain tumor cells through the generation of free radicals. In this study the effect of deferoxamine (DFO), an iron chelator, on MB-induced cytotoxicity was investigated using SK-N-MC human neuroblastoma and U-373 MG human astrocytoma cells as model cellular systems. The cytotoxic effect of MB was potentiated by DFO. The potentiation effect of DFO was significantly blocked by either stoichiometric amounts of ferric ion, various antioxidants, hydroxyl radical scavengers or intracellular Ca2+ release blockers. These results suggest that hydroxyl radical DFO. These results further suggest that the combined treatment with MB and DFO may be useful for the therapeutical applications of human brain tumors.

Cyclosporin A is a substance P (tachykinin NK 1) receptor antagonist

The immunosuppressive cyclic undecapeptide, cyclosporin A, inhibited the binding of [ 125I]substance P to tachykinin NK 1 receptors expressed by human IM-9 lymphoblastoid cells, U-373 MG human astrocytoma cells and guinea pig lung parenchyma with IC 50 values of 425 ± 58, 783 ± 180, and 784 ± 163 nM respectively. The dihydro derivative of cyclosporin A (dihydro-cyclosporin A) was an equally effective inhibitor, but the O-acetylated derivative (cyclosporin A-OAc) was 3–4-fold less potent. The cyclosporin compounds also inhibited [ 125I]neurokinin A binding to human NK 2 receptors with potencies slightly less than at NK 1 sites. In contrast, they were 8–20-fold less effective inhibitors of [ 125I]MePhe 7-neurokinin B binding to guinea pig NK 3 receptors ( p < 0.001). Thus, the cyclosporin A compounds showed selectivity for NK 1 and NK 2 receptors The structure-activity pattern for the effects of cyclosporin A compounds at tachykinin receptors differs from the pattern previously described for their immunosuppressive activity. All three compounds inhibited substance P induced interleukin-6 (IL-6) secretion from U-373 MG astrocytoma cells with potencies similar to their NK 1 receptor binding affinities. In addition, cyclosporin A blocked substance P induced phosphatidylinositol (PI) turnover in U-373 MG cells without blocking the corresponding response to histamine. This novel pharmacological profile of the cyclosporin A compounds as NK 1 receptor antagonists does not appear to correlate with other known in vitro cyclosporin A functions.

Methylene blue induces cytotoxicity in human brain tumor cells.

Methylene blue (MB), a known inhibitor of guanylyl cyclase, induced cytotoxicity in SK-N-MC human neuroblastoma and U-373 MG human astrocytoma cells in a dose-dependent manner. MB did not significantly alter cellular levels of cGMP in both cells. 8-Br cGMP, a membrane-permeable analogue of cGMP, did not decrease MB-induced cytotoxicity, indicating that cGMP may not be a major target of the cytotoxic action of MB. However, hydroxyl radical scavengers or intracellular Ca2+ modulators effectively blocked the MB-induced cytotoxicity. These results suggest that hydroxyl radical and intracellular Ca2+ may have an important involvement in the cytotoxic action of MB. These results further suggest that the treatment with MB may be useful for the therapeutic applications of human brain tumors.

Intracellular Ca2+ mediates the cytotoxicity induced by bepridil and benzamil in human brain tumor cells.

The effects of bepridil and benzamil, known Na(+)-Ca2+ exchange blockers, on the growth of human brain tumor cells were evaluated using SK-N-MC human neuroblastoma and U-373 MG human astrocytoma cells as model cellular systems. These drugs induced cytotoxicity in both cells in a dose-dependent manner. Agonist (2% fetal bovine serum) alone induced a rapid increased intracellular Ca2+ concentration and then it returned to the basal level. However, the pretreatments of these drugs resulted in a more sustained high intracellular Ca2+ concentration mobilized by an agonist. Moreover, BAPTA/AM, an intracellular Ca2+ chelator, significantly blocked the cytotoxicity induced by these drugs. These results suggest that bepridil and benzamil act as effective inhibitors of in vitro growth of human brain tumor cells and that intracellular Ca2+ may be involved in the mechanism of actions of these agents.

GR138676, a novel peptidic tachykinin antagonist which is potent at NK 3 receptors

GR138676, a conformationally constrained analogue of neurokinin B, is a novel, potent NK 3 receptor antagonist. GR138676 was a competitive antagonist of neurokinin B-dependent arachidonic acid mobilization from prelabelled Chinese hamster ovary cells stably transfected with a human NK 3 receptor gene (pK B 8.3) and of contractions induced by senktide in rat portal vein (pK B 8.2). However, GR138676 was also a competitive antagonist of the increase in intracellular calcium evoked by the selective NK 1 agonist, GR73632, in the human astrocytoma U373MG cell-line (pK B 8.3). GR138676 had little activity at NK 2 receptors, inhibiting binding of the NK 2 antagonist radioligand [ 3H]-GR100679 to Chinese hamster ovary cells transfected with the human ileum NK 2 receptor with a pKi of 6.0. In summary, despite its activity at NK 1 receptors, GR138676 will be a useful tool for characterizing NK 3 receptors as well as defining the physiological and pathophysiological function of this receptor subtype.

CAMP is not involved in interleukin-1-induced interleukin-6 release from human astrocytoma cells

We have previously shown that activation of the phosphatidyl-inositol/phospholipase C pathway could induce interleukin 6 (IL-6) release from U373MG human astrocytoma cells. We also found that, although interleukin 1β (IL-1β) did not activate phosphatidylinositol turnover, it induced a robust release of IL-6. In the present study, we examined the role of adenylate cyclase/cyclic 3′,5′-adenosine monophosphate (cAMP) pathway in IL-6 release. Agents which mimicked (dibutyryl cAMP) or stimulated (isoproterenol and forskolin) cAMP formation were found to induce IL-6 release and their effects could be potentiated by 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. On the other hand, in spite of its robust action on IL-6 release, IL-1β did not stimulate cAMP formation. Other possible signal transduction mechanisms involved in IL-1β-induced IL-6 release are discussed.

Regulation of the release of interleukin-6 from human astrocytoma cells.

Recent evidence suggests that the level of interleukin-6 (IL-6) is elevated in Alzheimer's disease (AD) brains. IL-6 is produced by reactive glial cells and could potentially affect neuronal survival. Understanding the biochemical mechanism that regulates the production and release of IL-6 by astrocytic cells may help to identify potential targets for therapeutic intervention in AD. In the present study, glial fibrillary acidic protein-positive human U373MG astrocytoma cells were used as a model of reactive astrocytes. Production of IL-6 in response to drug treatment was monitored with an ELISA assay. Histamine (1-100 microM), substance P (SP; 1-100 nM), and human interleukin-1 beta (IL-1 beta; 1-30 pM) stimulated the release of IL-6 in a time- and concentration-dependent manner, with EC50 values of 4.5 microM, 8 nM, and 4.5 pM, respectively. The respective effects of histamine, SP, and IL-1 beta were effectively blocked by the histamine H1, SP, and IL-1 receptor antagonists, supporting a receptor-mediated event for these agents. Both histamine and SP enhanced the formation of inositol phosphates and increase intracellular calcium levels, suggesting that the phosphatidylinositol bisphosphate/protein kinase C pathway may be involved in the IL-6 release process. Indeed, phorbol 12-myristate 13-acetate, a protein kinase C activator, also evoked IL-6 release from the U373MG cells. On the other hand, IL-1 beta, which produces a much more robust release of IL-6 than histamine or SP, has no effect on inositol phosphate formation or intracellular calcium levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of cell growth and intracellular Ca2+ mobilization in human brain tumor cells by Ca2+ channel antagonists.

The effects of various Ca2+ channel agonists and antagonists on tumor cell growth were investigated using U-373 MG human astrocytoma and SK-N-MC human neuroblastoma cell lines. Classical Ca2+ channel antagonists, verapamil, nifedipine, and diltiazem, and inorganic Ca2+ channel antagonists, Ni2+ and Co2+, inhibited growth of these tumor cells in a dose-dependent manner. Except Ni2+, these Ca2+ channel antagonists did not induce a significant cytotoxicity, suggesting that the growth-inhibitory effects of these drugs may be the result of the influence on the proliferative signaling mechanisms of these tumor cells. In contrast, Bay K-8644, a Ca2+ channel agonist, neither enhanced the growth of tumor cells nor increased intracellular Ca2+ concentration, indicating that voltage-sensitive Ca2+ channels may not be involved in tumor cell proliferation. Moreover, growth-inhibitory concentrations of Ca2+ channel antagonists significantly blocked agonist (carbachol or serum)-induced intracellular Ca2+ mobilization, which was monitored using Fura-2 fluorescence technique. These results suggest that the inhibition of the growth of human brain tumor cells induced by Ca2+ channel antagonists may not be the result of interaction with Ca2+ channels, but may be the result of the interference with agonist-induced intracellular Ca2+ mobilization, which is an important proliferative signaling mechanism.

Differences in the interactions of Nocardia asteroides with macrophage, endothelial, and astrocytoma cell lines

An in vitro model for studying host cell interactions with Nocardia asteroides was developed. Thus, macrophage cell lines J774A.1 and P388D1, pulmonary artery endothelium cell line CPAE, rat glial tumor cell line C6, and human astrocytoma cell lines CCF-STTG1 and U-373 MG were infected with either log- or stationary-phase cells of N. asteroides GUH-2, and the host cell-nocardia interactions were determined by light microscopy and electron microscopy. Polyclonal antinocardial antibody did not enhance uptake of nocardiae by any of these cell lines; however, log-phase cells of GUH-2 infected a higher percentage of J774A.1 and P388D1 than did stationary-phase organisms. When cells infected with stationary-phase GUH-2 were incubated for 6 h, filaments developed, which indicated that nocardial growth had occurred. In J774A.1 and P388D1, only 31 to 57% of the total stationary-phase coccobacillary cells that were phagocytized formed filaments within 6 h. This indicated that there was some inhibition of growth of the phagocytized nocardiae within these macrophage cell lines; however, the nocardiae grew within the endothelial (> 87% filaments) and astrocytoma (100% filaments) cell lines. Microfilament inhibitor cytochalasin B inhibited uptake of GUH-2 by macrophages and other cell lines, except that there was no effect on uptake of nocardial cells by astrocytoma cell line U-373 MG. Scanning and transmission electron microscopy showed phagocytosis of GUH-2 by the different cell lines. In cytochalasin B-treated cells, nocardiae were shown to penetrate through the cell surface and become internalized in a manner distinct from typical phagocytosis, suggesting that filamentous forms of this organism have a phagocytosis-independent invasion factor. The extent of this cytochalasin-resistant cellular penetration by the nocardiae differed in the different cell lines.

Potentiation of anti-proliferative effect of nitroprusside by ascorbate in human brain tumor cells

The interactions of nitric oxide (NO) and ascorbate were explored on the control of growth of human brain tumor cells. Sodium nitroprusside, a NO-generating agent, inhibited the growth of SK-N-MC human neuroblastoma cells in a dose-dependent manner. The growth inhibitory effect of nitroprusside was potentiated by sodium ascorbate and inhibited by hemoglobin. Ascorbate-induced potentiation was also observed in U-373 MG human astrocytoma cells. In both tumor cell lines, this potentiation was blocked by catalase, suggesting that hydrogen peroxide may be involved in the potentiation mechanism. In astrocytoma cells, mannitol or deferoxamine also reversed ascorbate-induced potentiation, indicating involvement of hydroxyl radical. These results suggest that the combined treatment with nitroprusside and ascorbate may be a valuable therapeutic strategy for brain tumors.

Different susceptibility to neurokinin 1 receptor antagonists of substance P and septide-induced interleukin-6 release from U373 MG human astrocytoma cell line

In a human astrocytoma cell line U373 MG, the activation of the neurokinin 1 (NK1) receptor by substance P (SP) increase, in a concentration-related manner (1 nM to 10 μM), the basal release of interleukin-6 (IL-6) as assayed by an ELISA method, in cell supernatants after 18 h of incubation. Septide, a selective NK1 receptor agonist, is equipotent to SP in inducing the IL-6 release showing similar E max (2644 ± 285 and 2830 ± 271 pg/ml) and EC 50 (15.6 ± 3.6 and 13.8 ± 3.2 nM). However, in binding assays on intact cells, septide was an about 50-fold weaker displacer of the binding of [ 3H][Sar 9,Met(O 2) 11]SP than SP ( K i's were 0.28 ± 0.1 nM and 14.2 ± 5.0 nM for SP and septide, respectively). NK2- and NK3-selective agonists (up to 1 μM) had no binding or functional effect. Highly selective non-peptide (CP96,345) or peptide (GR 82,334) NK1 receptor antagonists were more effective in antagonizing septide- (IC 50's 0.2 ± 0.06 nM and 70 ± 18 nM) than SP- (IC 50's 6.7 ± 1.3 nM and 1.95 ± 0.4 μM) induced IL-6 secretion. These data support the existence, also in human U373 MG cells, of a septide-sensitive NK1 receptor subtype(s) and/or epitope(s) blocked with high affinity by NK1 antagonist.

Evidence for the existence of ‘septide’ sensitive NK receptors in U373 MG human astrocytoma cell line

Evidence for the existence of ‘septide’ sensitive NK receptors in U373 MG human astrocytoma cell line

Calcium‐dependence of histamine‐ and carbachol‐induced inositol phosphate formation in human U373 MG astrocytoma cells: comparison with HeLa cells and brain slices

1. Histamine (1 mM) induced an accumulation of inositol monophosphate ([3H]-IP1) in the U373 MG human astrocytoma cell line which increased with time in the presence of 30 mM Li+. After a 30 min incubation period with 1 mM histamine [3H]-IP1 was the major product detected (84 +/- 1% of total [3H]-IPx) and was present at a level 11 (+/- 1) fold of basal accumulation. 2. Concentration-response curves for histamine-induced [3H]-IP1 accumulation in U373 MG cells (EC50 5.4 +/- 0.5 microM) were shifted to the right in a parallel fashion by mepyramine (slope of a Schild plot 0.99 +/- 0.08), yielding a Kd for mepyramine of 3.5 +/- 0.3 nM, consistent with the involvement of histamine H1-receptors. 3. The temelastine-sensitive binding of [3H]-mepyramine to a membrane fraction from U373 MG cells was hyperbolic and had a mean Kd of 2.5 +/- 1.0 nM. The maximum amount of temelastine-sensitive binding was 86 +/- 19 pmol g-1 membrane protein. 4. Carbachol also induced [3H]-IP1 accumulation in U373 MG cells, 2.8 (+/- 0.1) fold of basal with 1 mM carbachol, with an EC50 of 48 +/- 8 microM. Pirenzepine shifted carbachol concentration-response curves to the right (slope of Schild plot 0.89 +/- 0.07) giving a Kd for pirenzepine of 0.10 +/- 0.01 microM, suggesting that phosphoinositide hydrolysis in U373 MG cells is mediated by the M3-, rather than the M1-, muscarinic receptor subtype. 5. [3H]-IP1 accumulation induced by both 1 mM histamine and by 1 mM carbachol increased when the Ca2+ concentration of the medium was increased from 'zero' (no added Ca2+) to 0.3 mM. Histamine-stimulated [3H]-IP1 accumulation was further increased, although not so markedly, as the Ca2+ was raised to 4 mM. The same pattern was apparent with histamine-induced accumulations of [3H]-IP2 and [3H]-IP3. In contrast, [3H]-IPx accumulation in response to carbachol increased between 0.3 and 1.3 mM, but thereafter remained unchanged ([3H]-IP1) or declined ([3H]-IP2 and [3H]-IP3). 6. In HeLa cells, [3H]-IP1 accumulations induced by 1 mM histamine and 1 mM carbachol showed the same pattern of Ca2+ dependence and were independent of extracellular Ca2+ above 0.3 mM (histamine) or 1.3 mM (carbachol). The response to carbachol appeared to be mediated by an M3-muscarinic receptor (apparent Kd for pirenzepine 0.09 microM). 7. In cross-chopped slices of guinea-pig cerebral cortex and guinea-pig cerebellum, [3H]-IPI accumulation induced by 1 mM histamine in the presence of 10 mM Li+ increased as the extracellular Ca2+ was increased from 0.3 to 2.5 mM, but a further increase to 4 mM had no further effect. In contrast the response to histamine in rat cerebral cortex increased markedly between 1.3 and 4 mM Ca2+. Accumulations of [3H]-IP1 induced by carbachol in guinea-pig or rat cerebral cortical slices were not increased as extracellular Ca2+ was raised from 0.3 to 4 mM.8. Nimodipine (100 nM) and w-conotoxin (3 microM) had no significant effect on histamine-induced [3H]-IP1accumulation in rat cerebral cortical slices or in U373 MG cells. 9. We conclude that histamine-induced [3H]-IP1 accumulation in U373 MG cells does appear to have a component dependent on the extracellular Ca2+ concentration. The degree of Ca2+-dependence approaches that observed in guinea-pig cerebral cortex but is much less than in rat cerebral cortex.Whether U373 MG cells will be of use as a model system for the apparent Ca2+-entry component observed in guinea-pig or rat brain slices remains to be established.

WIN 64821, a novel neurokinin antagonist produced by an Aspergillus sp. II. Biological activity.

WIN 64821, a secondary metabolite produced by Aspergillus sp. (ATCC 74177) was found to inhibit radiolabeled substance P (SP) binding in a variety of tissues, including those of human origin. This compound inhibited, in a competitive manner, the binding of SP with Ki values ranging from 0.24 microM in human astrocytoma U-373 MG cells to 7.89 microM in rat submaxillary membranes. Additionally, WIN 64821 was found to inhibit 125I-NKA binding to the NK2 receptor in human tissue at a concentration equivalent to its NK1 activity (0.26 microM). The inhibitory activity of WIN 64821 against an NK3 selective ligand, 3H-senktide, was found to be much weaker (Ki = 15.2 microM). WIN 64821 was also evaluated in NK1 functional assays and was found to be a competitive antagonist of SP-induced contractility in the guinea pig ileum (pA2 = 6.6) as well as an inhibitor of SP-induced 45Ca2+ efflux from human astrocytoma U-373 MG cells (IC50 = 0.6 microM). In a rat vas deferens model, WIN 64821 inhibited eledoisin-induced contractility with an IC50 of 3.4 microM indicating functional antagonism at the NK2 receptor. The data presented in this study provide biochemical, pharmacological and functional evidence supporting WIN 64821 as a competitive neurokinin antagonist.

Differential effects of methionine enkephalin on the growth of brain tumor cells.

The effect of methionine enkephalin on the growth of human brain tumor cells was investigated. The results show that this endogenous opioid has dual effects on tumor cell growth. This peptide exerted an inhibitory effect in SK-N-MC human neuroblastoma cell line; in contrast, in U-373 MG human astrocytoma cell line, the peptide showed a stimulatory effect. Treatment with naltrexone, an opioid receptor antagonist, also resulted in a similar alteration of tumor cell growth implicating that its action may be unrelated to opioid receptor blockade. These results suggest that in these tumor cell lines endogenous opioid systems may be involved in cell proliferation. Furthermore, these tumor cell lines may be useful model systems for the study of the signal transduction mechanisms of endogenous opioids.

In vitro antitumor activity of cromakalim in human brain tumor cells.

The effect of cromakalim, a K+ channel opener, on the growth of human brain tumor cells was investigated. Cromakalim inhibited the growth of SK-N-MC human neuroblastoma and U-373 MG human astrocytoma cell lines in a dose-dependent manner. This effect of cromakalim was significantly blocked by the co-treatment with sulfonylureas (glibenclamide or tolbutamide) which are known as specific blockers for ATP-sensitive K+ channels. In addition, cromakalim significantly inhibited agonist-induced intracellular Ca2+ mobilization in the astrocytoma cells. This inhibition induced by cromakalim was also reversed by pretreatment with glibenclamide. These results suggest that the antitumor activity of cromakalim may be due to the activation of ATP-sensitive K+ channels leading to the inhibition of the intracellular Ca2+ signalling mechanism.

Inhibition of human brain tumor cell growth by a receptor-operated Ca 2+ channel blocker

SK&F 96365, a reported receptor-operated Ca 2+ channel blocker, inhibited the growth of U-373 MG human astrocytoma and SK-N-MC human neuroblastoma cell lines in a dose-dependent manner. Carbachol and serum which act as growth factors for these cells induced a rapid, transient increase of intracellular Ca 2+ concentration without a sustained increase. SK&F 96365 also exerted a significant inhibition of carbachol or serum-induced intracellular Ca 2+ mobilization. These results suggest that SK&F 96365 is a potent inhibitor of brain tumor cell growth and that its effect may be mediated by the inhibition of agonist-induced intracellular Ca 2+ mobilization.

Tachykinin‐Stimulated Inositol Phospholipid Hydrolysis and Taurine Release from Human Astrocytoma Cells

The activation of NK1 receptors on U373 MG human astrocytoma cells by substance P (SP) and related tachykinins was accompanied by an increase in taurine release and an accumulation of inositol phosphates. Both of these effects could be inhibited by spantide, a SP receptor antagonist. The relative potency of tachykinins in stimulating 3H-inositol phosphate accumulation correlated very well with their effects in stimulating the release of [3H]-taurine and inhibition 125I-Bolton-Hunter reagent-conjugated SP binding. The effect on [3H]taurine release was mimicked by a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA). The inactive phorbol ester analogue 4-alpha-phorbol 12,13-didecanoate, however, was without effect. Both SP- and PMA-induced releases of [3H]-taurine were markedly inhibited by staurosporine, a potent PKC inhibitor. Pretreatment of U373 MG cells with 10 microM PMA for 19 h to down-regulate PKC activity also markedly inhibited both SP- and PMA-induced releases of [3H]-taurine. Treatment of cells with 100 nM SP induced a time-dependent translocation of PKC from the cytosolic fraction to the membrane fraction. These findings are consistent with the hypothesis that an activation of NK1 receptors on U373 MG cells results in the release of inositol phosphates and activation of PKC, which in turn may regulate the release of taurine.

Functional characterization of neurokinin-1 receptors on human U373MG astrocytoma cells.

The neurokinin-1 (NK-1, substance P) receptor belongs to the class of seven transmembrane domain (7-TM) receptors that interact with cellular effector systems via guanine nucleotide binding regulatory proteins (G-proteins). In this study, coupling mechanisms of functional NK-1 receptors endogenously expressed in a human astrocytoma cell line (U373MG) were analyzed. Stimulation with substance P (SP) resulted in 1) a rapid increase in inositol 1,4,5-trisphosphate (IP3) synthesis; 2) a rise in cytosolic free calcium concentration ([Ca2+]i); 3) induction of immediate early gene transcription as monitored by c-fos and c-jun expression; and 4) a significant increase in de novo DNA synthesis. Thus, the functional responses induced by stimulation of NK-1 receptors on U373MG strongly correlate with those observed after treatment of primary astrocytes with SP and make U373MG cells a useful in vitro model system for the analysis of NK-1 receptor function on astrocytes in vivo.