endothelin-1 and Glioblastoma

Glioblastoma multiforme (GBM) is the most common and lethal type of primary brain tumor characterized by its rapid infiltration to surrounding tissues during the early stages. The fast spreading of GBM obscures the initiation of the tumor mass making the treatment outcome undesirable. Endothelin-1 is known as a secretory protein presented in various types of brain cells, which has been indicated as a factor for cancer pathology. The aim of the present study was to investigate the molecular mechanism of cell migration in GBM. We found that various malignant glioma cells expressed higher amounts of endothelin-1, ETA, and ETB receptors than nonmalignant human astrocytes. The application of endothelin-1 enhanced the migratory activity in human U251 glioma cells corresponding to increased expression of matrix metalloproteinase (MMP)-9 and MMP-13. The endothelin-1-induced cell migration was attenuated by MMP-9 and MMP-13 inhibitors and inhibitors of mitogen-activated protein (MAP) kinase and PI3 kinase/Akt. Furthermore, the elevated levels of phosphate c-Jun accumulation in the nucleus and activator protein-1 (AP-1)-DNA binding activity were also found in endothelin-1 treated glioma cells. In migration-prone sublines, cells with greater migration ability showed higher endothelin-1, ETB receptor, and MMP expressions. These results indicate that endothelin-1 activates MAP kinase and AP-1 signaling, resulting in enhanced MMP-9 and MMP-13 expressions and cell migration in GBM.

Topics: Astrocytes; Cell Line, Tumor; Cell Movement; Cell Nucleus; Central Nervous System Neoplasms; Endothelin-1; Extracellular Signal-Regulated MAP Kinases; Glioblastoma; Humans; JNK Mitogen-Activated Protein Kinases; Matrix Metalloproteinase 13; Matrix Metalloproteinase 9; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptor, Endothelin A; Receptor, Endothelin B; Transcription Factor AP-1

The invasive growth, proliferation, and transcriptional regulation of glioma C6 cells treated with endothelin-1 and PD98059, a specific inhibitor of ERK1/2 were studied. The proliferation of glioma C6 cells was assessed in different growth conditions by prior in vitro treatment with endothelin-1 followed by implantation into the brain. In vitro studies showed that PD98059 inhibited the proliferation of cultured glioma C6 cells and activated transcription factors E2F1 and Myc-Max. Endothelin-1 significantly increased the proliferation of glioma C6 cells. The model used in this study was experimental and may allow the specific features of the in vitro behavior of cultured invasive cells to be identified.

Topics: Animals; Basic-Leucine Zipper Transcription Factors; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; E2F1 Transcription Factor; Endothelin-1; Flavonoids; Glioblastoma; Mitogen-Activated Protein Kinase Kinases; Neoplasm Invasiveness; Neoplasm Transplantation; Proto-Oncogene Proteins c-myc; Rats; Rats, Wistar

The authors have monitored C6 glioma cell invasive growth, proliferation and transcriptional regulation after pretreatment with endothelin-1 and ERK1/2 specific inhibitor PD98059. To explore proliferation of C6 glioma cells in different growth conditions, they were treated in vitro with endothelin-1 and implanted into the brain. In vitro studies have indicated that PD98059 inhibited the proliferation of cultured C6 glioma cells and induced the activation of E2F1 and Myc-Max transcriptional factors. Endothelin-1 strongly increased C6 glioma cell proliferation. The model used in this study is experimental, but it may provide an insight into the specific behavior of in vitro cultured invasive cells.

Topics: Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; E2F2 Transcription Factor; Endothelin-1; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Glioblastoma; Male; Neoplasm Transplantation; Neuropeptides; Proto-Oncogene Proteins c-myc; Rats; Rats, Wistar

Endothelin-1 (ET-1) levels in the culture medium were considered to reflect the transcription of the ET-1 gene and the subsequent secretion of ET-1 from cultured cells. It has not been clarified how different ET-1 mRNA expression levels and immunoreactive (IR)-ET levels in the culture medium are in the cell culture system. We studied ET-1 mRNA expression levels and IR-ET levels in the medium of T98G glioblastoma cells treated with cytokines. T98G glioblastoma cells were cultured with cytokines (interferon-gamma 100 U/ml, tumor necrosis factor-alpha 20 ng/ml and interleukin-1beta 10 ng/ml) under normoxia or hypoxia (1% O(2)). Northern blot analysis showed that ET-1 mRNA expression levels were increased by tumor necrosis factor-alpha alone or a combination of tumor necrosis factor-alpha and interleukin-1beta, or three cytokines, and the increase was further enhanced under hypoxia. Particularly, relative expression levels of ET-1 mRNA were significantly higher under hypoxia than in normoxia in the treatment with a combination of three cytokines. IR-ET levels in the medium were increased by treatment with tumor necrosis factor-alpha, interleukin-1beta or a combination of tumor necrosis factor-alpha and interleukin-1beta, or three cytokines. In contrast to the mRNA expression levels, IR-ET levels in the medium of T98G cells treated with a combination of three cytokines were rather decreased under hypoxia compared with those in normoxia. These findings indicate that hypoxia induces ET-1 mRNA expression in the treatment of three cytokines, but IR-ET levels in the medium do not reflect this induction in T98G glioblastoma cells.

Topics: Cell Hypoxia; Cell Line, Tumor; Culture Media, Conditioned; Cytokines; Endothelin-1; Glioblastoma; Humans; Radioimmunoassay; RNA, Messenger; Up-Regulation

Endothelin-1 (ET-1) is mitogenic and/or antiapoptotic in human cancers, and antagonists to ET-1 receptors are under evaluation for cancer treatment. Inhibition of ET-1 activation by the endothelin-converting enzymes 1(a)(-)(d) (ECE-1(a)(-)(d); EC 3.4.24.71) represents another approach to block the ET-1 effect in cancer. To evaluate this potential, we synthesized and characterized a series of low nanomolar nonpeptidic thiol-containing ECE-1 inhibitors, and evaluated their effect, as well as the effect of inhibitors for the related metalloproteases neprilysin (NEP; EC 3.4.24.11) and angiotensin-converting enzyme (ACE; EC 3.4.15.1), on human glioblastoma cell growth. Only ECE-1 inhibitors inhibited DNA synthesis by human glioblastoma cells. Exogenous addition of ET-1 or bigET-1 to glioblastoma cells did not counterbalance the growth inhibition elicited by ECE-1 inhibitors, suggesting that ECE-1 inhibitors block the proliferation of human glioblastoma cells most likely via a mechanism not involving extracellular production of ET-1. This class of molecules may thus represent novel therapeutic agents for the potential treatment of human cancer.

Topics: Antineoplastic Agents; Aspartic Acid Endopeptidases; Carbamates; Cell Line, Tumor; Cell Proliferation; Central Nervous System Neoplasms; Drug Screening Assays, Antitumor; Endothelin-1; Endothelin-Converting Enzymes; Glioblastoma; Humans; Hydrazines; Metalloendopeptidases; Proline; Pyrimidines; Pyrrolidines; Structure-Activity Relationship; Sulfhydryl Compounds

Glioblastomas multiforme (GBMs) are hypervascular tumors characterized by endothelial cell (EC) proliferation. There is increasing evidence that ECs that infiltrate systemic tumors are different from normal blood vessel cells; whether this difference is seen in the central nervous system between GBM and normal brain tissue is not known. The goal of this investigation was to characterize and compare the functional and phenotypic properties of GBM-associated ECs and normal brain ECs.. Human ECs were isolated from fresh tissue specimens, purified using flow cytometry, and characterized by immunostaining. Proliferation was measured by determining bromodeoxyuridine incorporation and Ki-67 staining, and by performing the monotetrazolium assay. The migration rate of the cells was determined using the modified Boyden chamber technique. Apoptosis was evaluated by performing the TUNEL assay, cell death enzyme-linked immunosorbent assay (ELISA), and annexin V staining. Growth factor production was analyzed using the ELISA technique. The brain tumor ECs differed from normal brain ECs morphologically and by their expression and distribution of specific markers (that is, vascular endothelial cadherin [VE-cadherin] and CD31). Functional differences between the two cell populations were also evident. The brain tumor ECs proliferated more slowly and underwent less apoptosis than normal brain ECs; however, the tumor ECs migrated faster than the normal ECs. The normal ECs were sensitive to growth factors such as vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1), whereas the tumor ECs were not. In addition, the brain tumor ECs constitutively produced higher levels of ET-1 and VEGF, compared with the normal ECs.. The data demonstrated that ECs derived from normal brain and from GBMs have significant phenotypic and functional distinctions. Further characterization of brain tumor ECs is essential for efficient antiangiogenic treatment of gliomas.

Topics: Apoptosis; Brain; Brain Neoplasms; Cell Movement; Cell Proliferation; Endothelial Cells; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Glioblastoma; Humans; In Situ Nick-End Labeling; Neovascularization, Pathologic; Phenotype; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

Endothelin-1 (ET-1), a vasoactive and mitogenic peptide mainly produced by vascular endothelial cells, may be involved in the progression of several human tumors. Here, we present an immunohistochemical analysis of the expression pattern of ET-1 receptor subtypes (ET(A)-R and ET(B)-R) and a functional study of their potential role in human oligodendrogliomas and oligoastrocytomas. By comparison, we assessed the corresponding expression patterns of glioblastomas. Interestingly, a nuclear localization of ET-1 receptor subtypes (associated or not with a cytoplasmic labeling) was constantly observed in tumor cells from all three glioma types. Moreover, we noted a distinct receptor distribution in the different gliomas: a nuclear expression of ET(B)-R by tumor cells was found to be restricted to oligodendrogliomas and oligoastrocytomas, while a nuclear expression of ET(A)-R was only detected in tumor cells from some glioblastomas. Using primary cultures of oligodendroglial tumor cells, we confirmed the selective expression of nuclear ET(B)-R, together with a plasma membrane expression, and further demonstrated that this receptor was functionally coupled to intracellular signaling pathways known to be involved in cell survival and/or proliferation: extracellular signal-regulated kinase and focal adhesion kinase activation, actin cytoskeleton reorganization. In addition, impairment of ET(B)-R activation in these cells by in vitro treatment with an ET(B)-R-specific antagonist induced cell death. These data point to ET-1 as a possible survival factor for oligodendrogliomas via ET(B)-R activation and suggest that ET(B)-R-specific antagonists might constitute a potential therapeutic alternative for oligodendrogliomas.

Topics: Actin Cytoskeleton; Antihypertensive Agents; Astrocytoma; Brain Neoplasms; Cell Membrane; Cell Nucleus; Cell Survival; Cytoplasm; Endothelin B Receptor Antagonists; Endothelin-1; Extracellular Signal-Regulated MAP Kinases; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Glioblastoma; Humans; Immunohistochemistry; Oligodendroglioma; Oligopeptides; Piperidines; Protein-Tyrosine Kinases; Receptor, Endothelin A; Receptor, Endothelin B; Tumor Cells, Cultured

There is accumulating evidence showing that glial cells and gliomas secrete some neuropeptides and vasoactive peptides, such as adrenomedullin and endothelin-1. We have previously shown that expression of these two peptides is induced by inflammatory cytokines in T98G human glioblastoma cells. Glucocorticoids are frequently used for the treatment of inflammatory diseases and glioblastomas. We therefore studied effects of dexamethasone on expression of adrenomedullin and endothelin-1 in T98G human glioblastoma cells. Dexamethasone dose-dependently increased adrenomedullin mRNA levels and immunoreactive-adrenomedullin levels in the medium in T98G cells, whereas it decreased immunoreactive-endothelin levels in the medium. A combination of three cytokines, interferon-gamma (100 U/ml), tumor necrosis factor-alpha (20 ng/ml) and interleukin-1beta (10 ng/ml) induced expression of adrenomedullin and endothelin-1 in T98G cells. Dexamethasone (10(-8) mol/l) suppressed increases in expression of both adrenomedullin and endothelin-1 induced by these three cytokines. Thus, dexamethasone alone increased adrenomedullin expression whereas it suppressed the cytokine-induced expression of adrenomedullin in T98G cells. These findings raised the possibility that effects of dexamethasone on brain inflammation and glioblastomas may be partly mediated or modulated by its effects on expression of adrenomedullin and endothelin-1.

Topics: Adrenomedullin; Blotting, Northern; Cell Line, Tumor; Chromatography, High Pressure Liquid; Cycloheximide; Cytokines; Dactinomycin; Dexamethasone; Dose-Response Relationship, Drug; Endothelin-1; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Interferon-gamma; Interleukin-1; Nucleic Acid Synthesis Inhibitors; Peptides; Protein Synthesis Inhibitors; Radioimmunoassay; RNA, Messenger; Tumor Necrosis Factor-alpha

Evidence has accumulated showing that vasoactive peptides, such as endothelin-1, adrenomedullin and urotensin-II, are expressed in various kinds of tumour cells. In the present study, the expression of endothelin-1 and endothelin receptors was studied in eight human tumour cell lines: T98G (glioblastoma), IMR-32 and NB69 (neuroblastoma), BeWo (choriocarcinoma), SW-13 (adrenocortical carcinoma), DLD-1 (colonic carcinoma), HeLa (cervical carcinoma) and VMRC-RCW (renal carcinoma). Reverse transcriptase-PCR showed expression of endothelin-1 mRNA in seven out of the eight cell lines, the exception being BeWo cells. ET(A) receptor mRNA was expressed in T98G, IMR-32 and NB69 cells, but weakly in the other cells. ET(B) receptor mRNA was expressed in IMR-32, NB69 and BeWo cells, but only weakly in T98G and HeLa cells. Immunoreactive endothelin was detected in the culture media of six out of the eight cell lines, but not in that of IMR-32 or BeWo cells. Treatment of T98G cells with an anti-endothelin-1 antibody or an anti-adrenomedullin antibody for 24 h decreased cell numbers to approx. 84% and 90% of control respectively. Treatment with the ET(A) receptor antagonist BQ-610 (1 microM) significantly decreased cell number to about 90% of control, whereas the ET(B) receptor antagonist BQ-788 had no significant effect. On the other hand, exogenously added endothelin-1, adrenomedullin or urotensin-II (0.1 microM) had no significant effects on cell number. These results suggest that endothelin-1 acts as a paracrine or autocrine growth stimulator in tumours. The effect of endothelin-1 on tumour growth appears to be mediated by the ET(A) receptor.

Topics: Adrenal Cortex Neoplasms; Adrenomedullin; Antibodies, Monoclonal; Cell Division; Choriocarcinoma; Colonic Neoplasms; Endothelin Receptor Antagonists; Endothelin-1; Glioblastoma; Growth Substances; HeLa Cells; Humans; Kidney Neoplasms; Neuroblastoma; Oligopeptides; Peptides; Piperidines; Receptor, Endothelin A; Receptor, Endothelin B; RNA, Messenger; Tumor Cells, Cultured; Urotensins; Vasodilator Agents

Production and secretion of endothelin-1 (ET-1) by a human glioblastoma cell line, T98G, were studied by radioimmunoassay and Northern blot analysis. Immunoreactive ET was detected in the culture medium of T98G (17.6 +/- 0.6 fmol/10(5) cells/24 h, mean +/- SEM, n = 5). Reverse-phase high-performance liquid chromatography (HPLC) of immunoreactive ET in the culture medium extract showed a single peak eluting in the position of ET-1. Northern blot analysis showed expression of ET-1 mRNA in T98G cells. Treatment with interferon-gamma decreased the expression of ET-1. Treatment with TNFalpha or interleukin-1beta (IL-1beta) increased the expression of ET-1. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) showed expression of endothelin-A- and -B- (ET(A) and ET(B)) receptor mRNAs in T98G glioblastoma cells. These findings indicate that glioblastoma cells produce and secrete ET-1, and express ET receptor mRNAs. ET-1 secreted by glioblastoma cells may act locally on tumor cells, possibly as a growth modulator.

Topics: Endothelin-1; Glioblastoma; Humans; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Endothelin-1 (ET-1) is a powerful mitogenic and/or anti-apoptotic peptide produced by many cancer cells. To evaluate the potential role of the endothelin system in glioblastoma we first determined the cellular distribution of the mRNA and proteins of the components of the endothelin system, preproendothelin-1 (PPET-1), endothelin-converting enzyme-1 (ECE-1), and ET(A) and ET(B) receptors in human glioblastoma tissue and glioblastoma cell lines. PPET-1, ECE-1, and ET(A) receptor were highly expressed in glioblastoma vessels and in some scattered glioblastoma areas whereas ET(B) receptor was mainly found in cancer cells. This suggests that glioblastoma vessels constitute an important source of ET-1 that acts on cancer cells via the ET(B) receptor. Four human glioblastoma cell lines expressed mRNA for all of the components of the ET-1 pathway. Bosentan, a mixed ET(A) and ET(B) receptor antagonist, induced apoptosis in these cell lines in a dose-dependent manner. Apoptosis was potentiated by Fas Ligand (APO-1L, CD95L), a pro-apoptotic peptide, only in LNZ308 cells, corresponding to the known functional Fas expression in these cell lines. LNZ308 cells also expressed the long and short forms of the cellular FLICE/caspase-8 inhibitory protein (FLIP). Bosentan and a protein kinase C inhibitor down-regulated short FLIP in these cells. ET-1 induced transient phosphorylation of extracellular signal-regulated kinase but did not induce long-term thymidine incorporation in LNZ308 glioblastoma cells. These results suggest that, in glioblastoma cells, ET-1, mainly acting via the ET(B) receptor, is a survival/antiapoptotic factor produced by tumor vasculature, but not a proliferation factor, involving protein kinase C and extracellular signal-regulated kinase pathways, and stabilization of the short form of FLIP.

Topics: Base Sequence; Brain Neoplasms; DNA Primers; Endothelin-1; Fas Ligand Protein; fas Receptor; Glioblastoma; Humans; Immunohistochemistry; Membrane Glycoproteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured