Masanori Kurimoto

Growth Inhibition and Radiosensitization of Cultured Glioma Cells by Nitric Oxide Generating Agents

The authors examined the effect of nitric oxide (NO) generating agents on the growth and radiosensitivity of cultured glioma cells. Three glioma, rat C6, and human T98G and U87 cell lines were treated with the NO generating agents, S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP). These agents released NO in the cell culture media and inhibited the growth of the glioma cells. Growth-inhibition was attenuated by hemoglobin, a known inhibitor of NO, suggesting it is mediated by NO. When C6 and T98G cells were irradiated in the presence of SNAP or SNP at 100 µM, radiosensitization was observed. SNAP at 100 µM exhibited a sensitizer enhancement ratio (SER) of 1.4 for C6 cells and 1.8 for T98G cells. SNP at 100 µM only radiosensitized T98G cells with a SER of 1.9. The effect of SNP on radiosensitization of C6 cells was unclear. We conclude that NO generating agents are potential growth inhibitors and radiosensitizers for malignant glioma cells. NO mediated radiosensitization of glioma cells by NO generating agents may offer a new therapeutic approach for malignant glioma.

Induction of autophagic cell death and radiosensitization by the pharmacological inhibition of nuclear factor–kappa B activation in human glioma cell lines

OBJECT The intrinsic radioresistance of certain cancer cells may be closely associated with the constitutive activation of nuclear factor-kappa B (NF-kappaB) activity, which may lead to protection from apoptosis. Recently, nonapoptotic cell death, or autophagy, has been revealed as a novel response of cancer cells to ionizing radiation. In the present study, the authors analyzed the effect of pitavastatin as a potential inhibitor of NF-kappaB activation on the radiosensitivity of A172, U87, and U251 human glioma cell lines. METHODS The pharmacological inhibition of NF-kappaB activation was achieved using pitavastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Growth and radiosensitivity assays were performed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Hoechst 33258 staining, supravital acridine orange staining, and electron microscopy were performed utilizing 3 glioma cell lines with or without pitavastatin pretreatment to identify apoptosis or autophagy after irradiation. RESULTS The growth of these 3 glioma cell lines was not significantly inhibited by pitavastatin at a concentration of up to 1 microM. Treatment with 0.1 microM of pitavastatin enhanced radiation-induced cell death in all glioma cell lines, with different sensitivity. Apoptosis did not occur in any pretreated or untreated (no pitavastatin) cell line following irradiation. Instead, autophagic cell changes were observed regardless of the radiosensitivity of the cell line. An inhibitor of autophagy, 3-methyladenine suppressed the cytotoxic effect of irradiation with pitavastatin, indicating that autophagy is a result of an antitumor mechanism. Using the most radiosensitive A172 cell line, the intracellular localization of p50, a representative subunit of NF-kappaB, was evaluated through immunoblotting and immunofluorescence studies. The NF-kappaB of A172 cells was immediately activated and translocated from the cytosol to the nucleus in response to irradiation. Pitavastatin inhibited this activation and translocation of NF-kappaB. CONCLUSIONS Autophagic cell death rather than apoptosis is a possible mechanism of radiation-induced and pitavastatin-enhanced cell damage, and radiosensitization by the pharmacological inhibition of NF-kappaB activation may be a novel therapeutic strategy for malignant gliomas.

Inhibition of cellular proliferation and induction of apoptosis by curcumin in human malignant astrocytoma cell lines

SummaryNuclear factor (NF)-κB is known to control cellular proliferation and apoptosis. In malignant astrocytoma cells, it was reported that NF-κB was activated aberrantly and promoted their proliferation. Thus, inhibition of NF-κB activity is considered to be a promising therapeutic strategy for malignant astrocytoma. Recently, curcumin, the major constituent of turmeric, was reported to inhibit NF-κB activity. In this study, we investigated inhibitory effects of curcumin on NF-κB activity and cellular proliferation, and induction of apoptosis by curcumin in human malignant astrocytoma cell lines. Alteration of NF-κB activity in NP-2 human malignant astrocytoma cell line after treatment with curcumin was examined using electrophoretic mobility shift assay. Alterations of DNA synthesis and cellular growth in five human malignant astrocytoma cell lines after treatment with curcumin were examined using [3H]thymidine incorporation assay and the trypan blue dye exclusion method, respectively. Induction of apoptosis by curcumin in NP-2 and NP-3 human malignant astrocytoma cell lines was examined by DNA-fragmentation analysis and morphological observation. We found that the NF-κB activity in NP-2 was significantly reduced by curcumin. The DNA synthesis and the cellular growth were inhibited by curcumin in dose-dependent manner in all the five malignant astrocytoma cell lines. Nuclear condensation and fragmentation, and DNA fragmentation were observed in both NP-2 and NP-3 after the treatment with curcumin. These results indicate that curcumin inhibits the cellular proliferation and induces apoptosis in human malignant astrocytoma cell lines. These results are considered to be resulted from the inhibition of NF-κB activity by curcumin.

Expression of Nedd5, A Mammalian Septin, in Human Brain Tumors

The septins are a family of cytoskeletal GTPases that play an essential role in cytokinesis in yeast and mammalian cells. Nedd5 is a mammalian septin known to associate with. actin-based structures such as the contractile ring and stress fibers. In the present study, we examined the expression of Nedd5 in a series of human brain tumor cell lines and surgical specimens by northern and western analyses. The temporal expression of Nedd5 in U373 astrocytoma cells at various timepoints throughout the cell cycle was determined by an analysis of lovastatin- and nocodazole-treated, synchronized cell populations. The intracellular localization of Nedd5 was determined by immunocytochemistry of steady state cultures and nocodazole-treated cultures enriched in M phase cells. The effects of inhibiting Nedd5 expression in human brain tumors was determined by stably transfecting U373 astrocytoma cells with an antisense-Nedd5 cDNA expression vector and by analyzing clones for Nedd5 expression by immunocytochemistry, morphological changes, cell growth and nuclear content. All human brain tumor cell lines and surgical specimens expressed Nedd5 transcript and protein. Synchronized U373 astrocytoma cells showed a relative increase in Nedd5 transcript levels from late G1 to G2M phases; and an increase in Nedd5 protein levels from S to G2M phases. Maximum expression of both transcript and protein levels was observed at the G2M phase. By immunocytochemistry, Nedd5 was concentrated at the cleavage furrow of mitotic cells. Double staining with Nedd5 and F-actin showed co-localization of Nedd5 with actin filaments except during cytokinesis. Antisense-Nedd5 expression led to an accumulation of nuclear content. These data suggest that Nedd5 is involved in the process of cytokinesis in human brain tumours. Nedd5 expression may be cell cycle-dependent with increased levels found at G2M phase. Blocking Nedd5 expression in astrocytoma cells by antisense interferes with the process of cytokinesis during cell division.

Correlation of glutamate-induced apoptosis with caspase activities in cultured rat cerebral cortical neurons

In cultured rat cortical neurons lactate dehydrogenase (LDH) activity in the medium, a cell-death marker, increased gradually after exposure to glutamate (100 microM to 1 mM) for 60 min and reached a plateau at 24 to 30 h. Neuronal death was mainly apoptotic as suggested by typical electron microscopic findings, fluorescent double staining with membrane-permeating and nonpermeating chromatin dyes, nick end labeling, and assessment of DNA fragmentation by agarose gel electrophoresis. After 1 mM glutamate exposure, a rise of interleukin-1beta converting enzyme (ICE)-like protease activity in neurons was parallel to cysteine protease p32 (CPP32)-like protease activity and declined before CPP32-like protease activity reached the peak (at 6 h). LDH activity in the medium of glutamate-exposed neurons was decreased by specific ICE and/or CPP32 inhibitors, acetyl-L-tyrosyl-L-valyl-L-alanyl-L-aspart-1-al (Ac-YVAD-CHO) and acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspart-1-al (Ac-DEVD-CHO), respectively, in a dose-dependent manner. Fluorescent double staining of nuclei also demonstrated that at 100 microM each inhibitor prevented neuronal apoptosis and that this effect was additive. Among agonists corresponding to various glutamate receptor subtypes, N-methyl-D-aspartate (NMDA) and kainate induced apoptosis in cortical neuronal cultures while alpha-amino-3-hydroxy-5-methylisoxazole-4-propinate (AMPA) did not. The metabotropic glutamate receptor agonist, 1-aminocyclopentane-1S, 3R-dicarboxylate (ACPD) prevented apoptosis. Interestingly, apoptosis at 24 h after agonist or antagonist exposure correlated closely with caspase activity 6 h after exposure.

Tissue factor and cancer procoagulant expressed by glioma cells participate in their thrombin-mediated proliferation.

The relationship between coagulation cascade activation and glioma cell proliferation was examined. The human glioma cell lines T98G, TM-1 and normal human astrocyte cell strain (NHA) were examined. Using anti-tissue factor (TF) antibody, immunocytochemical detection of TF antigen was obtained in both cell lines and cell strain. TF antigen in cell lysates was also measured by enzyme linked immunosorbent assay (ELISA). In a one-stage clotting assay, T98G, TM-1 and NHA revealed procoagulant activity (PCA) in normal human plasma and factor VII deficient plasma. PCA in normal human plasma was significantly inhibited by both inhibitory anti-TF antibody and cysteine protease inhibitor HgCl2. This result indicates that T98G, TM-1 and NHA cells express not only TF but also cancer procoagulant (CP) at the same time.In a cell proliferation assay, thrombin induced proliferation in T98G and TM-1 cells in a dose-dependent fashion and in NHA cell in a bell-shaped fashion. This mitogenic stimulant was inhibited by the specific thrombin inhibitor hirudin. The combinations of coagulation factors II, V, and X with or without factor VII induced proliferation in T98G, TM-1, and NHA cells. The maximal mitogenic stimulatory effects were larger in glioma cells than in NHA. These mitogenic stimulatory effects were also inhibited by hirudin. Each coagulation factor on its own or in any other combination of coagulation factors had no proliferative effect. Thus, these mitogenic stimulatory effects were considered to be the effect of thrombin.In conclusion, T98G and TM-1 human glioma cells express two different types of procoagulants TF and CP. In the presence of coagulation factors, these glioma cells can generate thrombin and this thrombin generation is capable of inducing glioma cell proliferation in vitro.

Cerebrospinal Fluid Tissue Factor and Thrombin-Antithrombin III Complex as Indicators of Tissue Injury After Subarachnoid Hemorrhage

BACKGROUND AND PURPOSE No marker that reflects and predicts brain injury due to subarachnoid hemorrhage (SAH) and cerebral vasospasm has been reported. We hypothesized that membrane-bound tissue factor (mTF) and thrombin-antithrombin III complex (TAT) in the cerebrospinal fluid (CSF) of patients with SAH become markers indicating brain injury. To evaluate the hypothesis, we correlated levels of mTF and TAT in the CSF of patients with SAH with clinical severity, the degree of SAH, and outcome. METHODS We assayed CSF mTF, TAT and myelin basic protein (MBP) in patients with SAH at intervals that included days 0 to 4 and days 5 to 9 after ictus. Classification of clinical severity of disease on admission was based on Hunt and Hess grade, degree of SAH on CT on Fishers grading, and outcome 3 months after SAH on the Glasgow Outcome Scale. RESULTS In the interval from days 0 to 4, mTF and TAT correlated with Hunt and Hess and Fisher grades, and occurrence of cerebral infarction due to vasospasm. Only mTF correlated significantly in this period with outcome. TAT, mTF, and MBP all correlated significantly with each other. From days 5 to 9, only mTF correlated with cerebral infarction, infarction volume, MBP levels, and outcome. CONCLUSIONS Both mTF and TAT reflected brain injury from SAH and predicted vasospasm, though mTF was more sensitive and a better predictor of outcome. Unlike mTF, TAT did not correlate with vasospasm during the interval when it most commonly occurs, which raised doubt about thrombin activation as a cause.

Acute pathologic features with angiographic correlates of the nearly or completely occuluded lesions of the cervical internal carotid artery

BACKGROUND The true pathologic process of nearly or completely occluded lesions of the cervical internal carotid artery (ICA) has not been studied sufficiently. This information is important in determining the critical indications for endarterectomy. METHODS Acute pathologic features of these advanced occlusive lesions of the ICA were studied in 40 patients who underwent emergency carotid endarterectomy. Gross morphologic and histopathologic features of these occlusive lesions were examined, and the relationship between the clinical information and the pathologic characteristics was investigated. RESULTS Thirty-seven lesions had histologic features of advanced atherosclerosis complicated by fresh intraplaque hemorrhages with or without transintimal extension. Thinwalled neovessels were thought to be an important etiologic factor in producing intraplaque hemorrhage. The remaining three lesions without these changes had strangulated embolic material at the occluded portion. A good correlation between these pathologic features and angiographic findings was found. CONCLUSION The presented results clearly indicate that intraplaque hemorrhage is the most important factor in producing and determining the acute pathologic features of symptomatic and advanced atheromatous occlusive ICA lesions.

Lower incidence of symptomatic vasospasm after subarachnoid hemorrhage owing to ruptured vertebrobasilar aneurysms.

OBJECTIVE: In this study, we evaluated the difference in incidence of symptomatic vasospasm between ruptured aneurysms in the anterior and posterior circulation using multiple logistic regression analysis. METHODS: A total of 145 consecutive patients who underwent surgery for aneurysms within 72 hours after subarachnoid hemorrhage (SAH) were studied. RESULTS: The ruptured aneurysm was in the anterior circulation in 128 patients (88.3%) and in the posterior circulation in 17 patients (11.7%). Forty patients (27.6%) had symptomatic vasospasm and 105 patients (72.4%) did not. Univariate and multivariate analyses were performed to assess relationships among various variables and the occurrence of symptomatic vasospasm after SAH. Finally, Grade III to V (Hunt and Hess grade) and Group 3 (Fisher’s classification) on admission were found to be independently positively associated with the occurrence of symptomatic vasospasm while ruptured vertebrobasilar aneurysm were negatively associated. CONCLUSION: Although a poor clinical grade and a severe SAH classification on admission such as Hunt and Hess grade and Fisher’s classification are established powerful predictors of symptomatic vasospasm, ruptured vertebrobasilar aneurysm are for the first time reported to be a predictor of symptomatic vasospasm based on results of a recent reliable statistical analysis.

Intracranial Germinoma Associated with Down’s Syndrome

It is well known that Down’s syndrome is sometimes associated with leukemia. However, there have been only a few case reports of a relationship between Down’s syndrome and brain tumors. We report 2 cases with histological diagnoses of germinoma. The 1st case was a 10-year-old boy with Down’s syndrome complaining of seizure and left hemiparesis. Computed tomographic (CT) scan and magnetic resonance imaging (MRI) showed a mass lesion in the right basal ganglia and thalamus. Histological examination indicated two cell pattern germinomas. The 2nd case was a 20-year-old man with Down’s syndrome complaining of headache and vomiting. CT scan and MRI showed a pineal region tumor with marked hydrocephalus. Surgical specimens showed typical germinoma. Only 13 cases of brain tumors associated with Down’s syndrome have been reported. A higher incidence of germ cell tumors seems to be related to chromosomal abnormalities.