Takeo Anda

EFFECT OF TUMOR NECROSIS FACTOR-ALPHA ON THE PERMEABILITY OF BOVINE BRAIN MICROVESSEL ENDOTHELIAL CELL MONOLAYERS

The administration of chemotherapy to patients with tumors of the central nervous system is often blocked by the blood-brain barrier. Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that promotes vascular permeability in addition to its pro-inflammatory effects. However, no direct evidence exists as to whether TNF-alpha may increase permeability of the BBB. We evaluated the effect of TNF-alpha on the transport of cisplatin (CDDP) or high molecular weight dextran labeled with fluorescein isothiocyanate (FITC-dextran) across bovine brain microvessel endothelial cell (BMEC) monolayers that was conducted on side-by-side diffusion chambers in vitro. The permeability coefficient for the transport of CDDP across the untreated monolayer was 3.80 x 10(-5) cm sec-1 at 30 minutes. After treating the BMEC monolayer with TNF-alpha (50 U ml-1 and 500 U ml-1) for 36 hours, the PC of CDDP increased significantly to 8.94 x 10(-5), and 14.43 x 10(-5) cm sec-1 respectively (p < 0.01). TNF-alpha had no effect on the transport of FITC-dextran across the BMEC monolayers. Electron microscopy showed that the tight junctions between the BMECs persisted even after treatment with TNF-alpha, whereas they had been partially disrupted following exposure to mannitol, 1600 mOsm kg-1. TNF-alpha selectively promoted the in vitro permeability of the blood-brain barrier to CDDP without disrupting tight junctions. This system could be used as a model for experimental studies of chemotherapy. Findings suggested that the combined administration of TNF-alpha and CDDP may be clinically useful.

Expression of the constitutively activated RelA/NF-κB in human astrocytic tumors and the in vitro implication in the regulation of urokinase-type plasminogen activator, migration, and invasion

Although malignant gliomas are highly invasive tumors, a characteristic that contributes to the commonly observed therapeutic failures and local disease recurrences, the molecular events that regulate invasion in these tumors remain poorly understood. Because the transcription factor RelA/NF-κB has been shown to regulate invasion during several cellular processes, we have examined immunohistochemically expression of the constitutively activated RelA/NF-κB in tissues obtained from 49 astrocytic tumors [8 diffuse astrocytomas, 9 anaplastic astrocytomas (AAs) and 32 glioblastomas (GBMs)]. In addition, we examined the in vitro effects of antisense oligonucleotides and curcumin on the expression and activation of RelA/NF-κB, urokinase-type plasminogen activator (u-PA) expression, migration, and invasion in the T98G glioma cell line. Expression of the constitutively activated RelA/NF-κB was observed in 2 (25%) of 8 cases of diffuse astrocytomas, 5 (55.6%) of 9 cases of AAs, and 30 (93.8%) of 32 cases of GBMs. This expression was significantly correlated with the malignant potential in astrocytic tumors (P < 0.001). Moreover, antisense oligonucleotides and curcumin inhibited phorbol-12-myristate-13-acetate (PMA)-induced RelA/NF-κB expression or activation (or both), down-regulated u-PA expression, and reduced the migration and invasive potentials of T98G glioma cells. Thus, the expression of constitutively activated RelA/NF-κB is associated with malignancy potential in astrocytic tumors and may play a critical role in the regulation of u-PA expression and invasiveness in gliomas. RelA/NF-κB may therefore be an intriguing candidate for studies aimed at understanding and prevention of the invasiveness of gliomas.

Immunohistochemical expression of E-cadherin in metastatic brain tumors

The adhesion molecule E-cadherin has been shown to influence malignant transformation of tumors, including local and distant metastases. We examined the expression of E-cadherin to determine its relationship to the development of metastasis in metastatic brain tumors. Immunohistochemistry for E-cadherin and Ki-67 was carried out in 76 formalin-fixed, paraffin-embedded archival specimens of metastatic brain tumors and in 14 corresponding available primary tumors from patients who received treatment for metastatic brain tumors. The primary tumors were mainly lung cancers (51.3%), followed by gastrointestinal tumors (28.9%). E-cadherin was expressed in 62 (81.5%) of 76 cases examined. In metastatic adenocarcinomas, a consistent tendency for E-cadherin expression was noted, regardless of the degree of differentiation or the extent of spread of the disease (P=0.4). There was a direct correlation between E-cadherin expression and high MIB-1 index in all metastatic brain tumors (P=0.0007). Pairwise analysis in 14 primary tumors and the corresponding metastatic specimens revealed high E-cadherin and MIB-1 staining in metastatic brain tumors. These results provide a unique association between E-cadherin, systemic metastasis, and proliferation potential in metastatic brain tumors.

In-vivo Microdialysis Study of the Distribution of Cisplatin into Brain Tumour Tissue after Intracarotid Infusion in Rats With 9L Malignant Glioma

Simultaneous brain microdialysis in tumour and non‐tumour tissues has been used for kinetic determination of the local distribution of an anticancer agent, cisplatin, in rats.

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized 125I‐endothelin‐1 (125I‐ET‐1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High‐affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (KD = 4.7 ± 1.0 × 10−10 and 1.6 ± 0.3 × 10−10M, respectively; Bmax = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ‐123, a selective antagonist for the ETA receptor, potently competed for 125I‐ET‐1 binding to sections of the microvessels with IC50 values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10−6M BQ‐123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET‐3 showed two components (IC50 = 5.7 ± 2.5 × 10−10 and 1.4 ± 0.2 × 10−6M for tumor microvessels, 1.8 ± 0.6 × 10−10 and 1.1 ± 0.3 × 10−6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high‐affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ETA subtype.

A selective endothelin ETA antagonist, BQ-123, inhibits125I-endothelin-1 (125I-ET-1) binding to human meningiomas and antagonizes ET-1-induced proliferation of meningioma cells

Summary1. We studied the effects of BQ-123, a selective ETA receptor antagonist, on125I-endothelin-1 (125I-ET-1) binding to cell surface receptors in surgically excised human meningiomas and on ET-1-induced DNA synthesis in cultured human meningioma cellsin vitro, using a quantitative receptor autoradiographic technique with radioluminography and3H-thymidine incorporation, respectively.2. All of the human meningiomas expressed high-affinity binding sites for125I-ET-1, regardless of differences in histological subtypes (Kd=2.6±0.2 nM,Bmax=374±93 fmol/mg; mean ± SE;n=9).3. BQ-123 competed for125I-ET-1 binding to sections of meningiomas with IC50s of 3.2±0.9×10−7M, and 10−4M BQ-123 displaced 80% of the binding.4. ET-1 significantly stimulated DNA synthesis in cultured human meningioma cells, up to 170% of the basal level in the presence of 10−9M ET-1. BQ-123 inhibited ET-1 (10−9M)-induced DNA synthesis in meningioma cells, in a dose-dependent manner, and 10−5M BQ-123 reduced it to 120% of the basal level.5. The number of meningioma cells determined after 4 days in culture was dose dependently increased in the presence of ET-1 (10−9 and 10−7M). The growth rate of meningioma cells, incubated with 10−9M ET-1, was reduced by 50% in the presence of 10−7M BQ-123.6. Our data suggest that (a) human meningioma cells express a large number of ETA endothelin receptors, with a small proportion of non-ETA receptors linked to proliferation of the cells, and (b) ET receptor antagonists, including BQ-123, might prove to be effective treatment for patients with meningioma.

Endothelin receptor in microvessels isolated from human meningiomas: Quantification with radioluminography

Summary1. We characterized specific125I-endothelin-1 (125I-ET-1) binding sites in microvessels isolated from human meningiomas, using anin vitro quantitative receptor autoradiographic technique coupled to a radioluminographic imaging plate system.2. This newly developed and highly sensitive method revealed high-affinity ET receptors present in pellet sections of the microvessels from all the meningiomas studied, regardless of histological subtypes (dissociation constant, 1.2 ± 0.3 nM; maximum binding capacity, 185 ± 56 fmol/mg; means ± SE for nine tumors).3. In five cases of meningiomas, ET-3 competed for125I-ET-1 binding to microvessels from those tumors with a low affinity [50% inhibiting concentration (IC50) of 1.6 ± 0.4 × 10−6M], and a selective ETB receptor agonist, sarafotoxin S6c, up to 10−6M, did not displace ET binding from the sections.4. In the sections of microvessels from four other tumors, biphasic competition curves were obtained in the case of incubation in the presence of increasing concentrations of ET-3, with an IC50 of 1.1 ± 0.2 × 10−9M for the high-affinity component and 1.6 ± 0.3 × 10−6M for the low-affinity component, respectively. In addition, S6c competed for ET binding to those sections (IC50=2.3 ± 0.2 × 10−10M) and 10−6M S6c displaced 30% of the control, corresponding to the high-affinity component of competition curves obtained in the presence of ET-3.5. Our results suggest that (a) capillaries in human meningiomas express a large number of high-affinity ETA (non-ETB) receptors with a small proportion of ETB receptors, and (b) ET may have a role in neovascularization, tumor blood flow, and/or function of the blood-tumor barrier in meningioma tissues by interacting with specific receptors present on the surface of the endothelium.

Factors affecting rapid growth of unruptured cerebral aneurysms during the acute stage of subarachnoid hemorrhage.

Abstract Background and purpose: Several unruptured cerebral aneurysms have been reported to grow and rupture. To determine which factors affect the growth of these aneurysms during the acute stage of subarachnoid hemorrhage (SAH), a retrospective review was performed. Methods: Between January 2000 and January 2003, 130 patients with angiographically proven ruptured cerebral aneurysms were treated at our institution. Of these patients, 32 also had simultaneous unruptured aneurysms, and the total number of the unruptured aneurysms was 40, including two neck remnants which had remained since the past clipping. Seventeen patients had 17 unruptured aneurysms and two neck remnants. The unruptured aneurysms were not treated during the acute stage of SAH but had received a complete short term follow-up. Results: The rapid growth of one unruptured aneurysm and two neck remnants was confirmed by a second angiogram performed on average40 days after the first angiogram. Several candidate factors responsible for the growth of aneurysm were selected, and the results of a statistical analysis indicate that a systolic blood pressure above 200 mmHg during the acute stage of SAH and vasospasm, confirmed by transcranial Doppler ultrasound (TCD) or neurological examination, and neck remnants, are risk factors that affect the growth. Conclusions: Short term follow-up angiography is thus important for patients with untreated unruptured cerebral aneurysms after the acute stage of SAH.