Daizo Yoshida

The expression of matrix metalloproteinase-2 and-9 in human gliomas of different pathological grades

Matrix metalloproteinases (MMPs) have been implicated to play a critical role in glioma invasiveness. In this study, we aimed to investigate, the expression of MMP-2 and MMP-9 in human gliomas of different degrees of malignancy, and evaluated the correlation between MMP-2 and MMP-9 expression in gliomas. The samples from 65 cases of glioma were divided into four groups according to the WHO classification: there were 16 cases of grade I, 17 cases of grade II, 20 cases of grade III, and 12 cases of grade IV. Normal brain samples served as the control group, and biopsy specimens were obtained from 8 glioma patients with a needle placed into the adjacent brain 1 cm from the margin after tumor resection. All the samples were stained with hematoxylin and eosin and immunohistochemistry. A computer-aided image-analysis system was employed to measure the integral optical density (IOD) of positive slides. No positive staining was found in the control group. The positive staining was localized in the cytoplasm of glioma cells, the extracellular matrix (ECM), the basement membrane (BM), and the endothelial cells of blood vessels. Positive staining rates increased significantly when the degree of malignancy of gliomas was elevated. The IOD value of MMP-2 and MMP-9 also indicated that the intensity of MMP-2 and MMP-9 expression was elevated significantly with the degree of malignancy of the gliomas. There was a positive correlation between MMP-2 and MMP-9 expression in gliomas. Glioma invasion and angiogenesis were particularly seen in the biopsied tissues, and MMP-9 immunostaining seemed to be much more intense and extensive than MMP-2 immunostaining in these samples. These results suggest that MMP-2 and MMP-9 staining in gliomas is localized in the cytoplasm of tumor cells, BM, and endothelial cells, and that MMP-2 and MMP-9 together play an important role in the invasiveness of gliomas, mediating the degradation of the ECM and angiogenesis. MMP-2 and MMP-9 could be molecular targets in the treatment of malignant glioma.

Quantitative analysis of copper, zinc and copper/zinc ratio in selected human brain tumors

SummarySerum copper and zinc concentrations and copper/zinc ratios have been shown to be increased in several types of human malignancies, including human brain tumors. In this study, copper and zinc levels and copper/zinc ratios were determined by atomic absorption analysis in tissue and serum from 29 primary and metastatic brain tumor patients. Metastatic carcinomas and malignant gliomas revealed significantly higher tissue copper concentrations than control tissues and meningiomas. Malignant gliomas demonstrated significantly higher tissue copper/zinc ratios. Both serum copper and copper/zinc ratio were significantly higher in the metastatic carcinoma group than control; however, serum copper levels in malignant glioma patients were not significantly different from control tissues. There were no differences both in the serum and the tissue concentrations of these trace elements in meningiomas and controls. These data suggested that copper, an important angiogenic factors, is accumulated within the malignant tissues of metastatic carcinoma and malignant glioma, but not meningiomas. These findings may have implications regarding angiogenesis in these tumors.

Copper chelation inhibits tumor angiogenesis in the experimental 9L gliosarcoma model.

To investigate the effects of copper (Cu)-depletion diet and D-penicillamine treatment (CDPT) on both tumor growth and angiogenesis, we studied Fischer-344 rats in which 9L gliosarcoma cells had been subcutaneously implanted. We focused primarily on the alteration of Cu contents and the vascular density. Compared with the normal diet group, the CDPT group showed a significant reduction of tumor weight and a decrease in Cu concentration. Furthermore, the CDPT group demonstrated smaller blood vessels with significantly lower vascular density. This decrease of tumor growth was achieved by angiosuppression. Our study indicated that CDPT selectively caused Cu chelation from the tumor tissue; the normal brain tissue did not show lower Cu concentration after the treatment. The prevention of tumor angiogenesis by this method may be very useful in cancer therapy and may help elucidate the microenvironmental mechanisms for cancer cells.

Expression of hypoxia-inducible factor 1α and vascular endothelial growth factor in pituitary adenomas

Hypoxia-inducible factor (HIF)-1α is a transcription factor in hypoxia adaptation mechanisms. In malignant tumors, HIF-1α upregulates vascular endothelial growth factor (VEGF) expression to induce tumor angiogenesis. Although VEGF and HIF-1α are expressed in pituitary adenomas, the relationships of these factors remain unclear. Therefore, we examined the expression of HIF-1α and VEGF using real-time RT-PCR and immunohistochemistry to clarify the relationship of these factors in pituitary adenomas. HIF-1α mRNA and VEGF mRNA levels in pituitary adenoma tissues from 25 operated patients were quantified using real-time RT-PCR. Some tissues were also studied by double fluorescent immunohistochemical methods. HIF-1α mRNA and protein were expressed in all pituitary adenomas examined. Their expression tended to be higher in GH-producing and lower in ACTH-producing tumors. VEGF mRNA and protein were also expressed in all pituitary adenomas. There was no significant correlation in the expression levels of HIF-1α and VEGF mRNA. The mutual expression of HIF-1α and VEGF was of no significance; in only a few cells were HIF-1α and VEGF co-localized. Our results suggest that in pituitary adenomas VEGF expression may not depend strongly on HIF-1α expression.

Suppression of cell invasion on human malignant glioma cell lines by a novel matrix-metalloproteinase inhibitor SI-27: in vitro study.

Matrix metalloproteinase (MMP) has come to be highlighted by its close relation to the cell invasion of gliomas. Suppression of MMP activity in malignant glioma cells would be meriting to local delivery of genes or chemotherapeutic agents. In this study, we employed a novel MMP inhibitor, SI-27 to investigate inhibition of cell invasiveness in human malignant glioma cell lines, U87MG, U251MG, and U373MG. We evaluated with zymogram, reverse zymogram, and cell invasion assay after exposure of SI-27 for 24 h followed by preliminary MTT assay to find non-cytotoxic dose range, 5 10 50 100 μg/ml compared with non-treatment group as the control. Common to three glioma cell lines, zymogram disclosed that expressions of MMP-2 and -9 were suppressed in a dose-dependent fashion, meanwhile those of tissue inhibitor of MMP (TIMMP) in reverse zymogram were not. The numbers of invading cells through Boyden chamber were significantly reduced in a dose-dependent manner, while those with 5 μg/ml were not diminished common to those three lines. In conclusion, dose concentration ranging 10–100 μg/ml of SI-27 inhibited MMP-2 and -9 mediated cell invasiveness in malignant glioma cell lines. This is the first report for chemotherapeutic effect of SI-27 on glioma cells.

Signalling Pathway Mediated by CXCR7, an Alternative Chemokine Receptor for Stromal-Cell Derived Factor-1α, in AtT20 Mouse Adrenocorticotrophic Hormone-Secreting Pituitary Adenoma Cells

Stromal cell‐derived factor (SDF)‐1 and its receptor, CXCR4, have been identified in both neurones and glia of many brain areas. Previous studies have mainly focused on the role of SDF‐1 and CXCR4 in modulating the hypothalamic‐pituitary axis and their possible involvement in the development of pituitary adenomas. An alternative SDF‐1 receptor, CXCR7, has recently been identified, but it has not been studied in the context of pituitary adenomas. The present study aimed to investigate the distribution and function of CXCR7 in pituitary adenomas. The expression of CXCR7, normalised to β‐actin, was assessed by tissue microarray analysis of 62 adenomas, including 23 growth hormone (GH)‐producing adenomas, 22 nonfunctioning adenomas, seven prolactin (PRL)‐producing adenomas, six adrenocorticotrophic hormone‐producing adenomas and four thyroid‐stimulating hormone‐producing adenomas. In vitro functional studies used RNA interference (RNAi) and cDNA microarray analysis to evaluate the CXCR7 signalling pathway in AtT‐20 mouse pituitary adenoma cells treated with recombinant mouse SDF‐1α and transfected with RNAi against Cxcr7 or control RNAi. In tissue microarray analysis, prominent expression of CXCR7 was observed in GH‐producing adenomas and PRL‐producing adenomas, and in macroadenomas (P < 0.05). Intracellular signalling via CXCR7 up‐regulated Bub1, Cdc29 and Ccnb1, and down‐regulated Asns, Gpt, Pycr1, Cars and Dars. The present study demonstrates that the SDF‐1α/CXCR7 signalling pathway regulates genes involved in cell cycle control, amino acid metabolism and ligase activity, which comprise targets that are distinct from those of CXCR4.

Suppression of 9L gliosarcoma growth by copper depletion with copper-deficient diet and D-penicillamine

SummaryTrace element such as Cu and Zn have important chemical and biological properties. Recently, tissue Cu and Zn concentrations have been correlated with prognosis in selected malignancies. In addition, depletion of trace metals has suppressive effects on tumor growth in experimental rat models. We measured tissue levels of Cu and Zn and investigated the inhibitory effects on tumor growth in a rat brain tumor model by the Cu-depletion. 9L gliosarcoma cells were injected subcutaneously in 24 anesthetized 5 week old male Fischer-344 rats. Control animals (n = 12) were given a normal diet throughout the experiment and hypocupremic rats (n = 12) were given a Cu-deficient diet beginning 3 weeks before and after tumor implantation, and administered 2 mg of D-Penicillamine peri os., once daily, 3 days before and after implantation. At the time of sacrifice, samples were taken to measure tumor weights. To determine tissue Cu and Zn levels, atomic absorption spectrophotometery was used. Cu, Zn and Cu/Zn ratio were significantly higher in control tumors than brain tissues. Cu levels and Cu/Zn ratio were significantly lower in hypocupremic tumors than those in control tumors. Zn levels in hypocupremic tumors were significantly higher than control tumors. Our study indicated that Cu depletion by a Cu-deficient and D-Penicillamine reduced Cu concentration and Cu/Zn ratio in a tumor model with reduction of tumor weight. A metabolic approach that restricts Cu to alter the microenvironment within the cell must become a new horizon of cancer therapy.

Anti-invasive effect of an anti-matrix metalloproteinase agent in a murine brain slice model using the serial monitoring of green fluorescent protein-labeled glioma cells.

OBJECTIVEWe aimed to analyze the anti-invasive effect of the anti-matrix metalloproteinase (anti-MMP) agent SI-27 by quantitative tracking of enhanced green fluorescent protein (EGFP)-labeled human malignant glioma cell lines in a brain slice model. METHODSPersistent expression of EGFP in human malignant glioma cell clones (U87MG, U251MG, and U373MG) was established with the use of the pEGFP-C1 vector. Tumor spheroid in 1 &mgr;l Matrigel was implanted into the caudate nucleus-putamen of a severe combined immunodeficient mouse brain slice. To allow the quantitative assessment of tumor cell invasion, the invasion area index was measured on Days 1, 3, 5, and 7 with a fluorescence stereomicroscope and an image analyzer in the presence of various concentrations of SI-27 (0, 1, 10, 50, or 100 &mgr;g/ml). RESULTSIn the control group (0 &mgr;g/ml), all glioma cell lines invaded in a fingerlike fashion and reached the contralateral hemisphere through the corpus callosum. SI-27 at concentrations of 10, 50, and 100 &mgr;g/ml significantly suppressed the invasion area index on Days 5 and 7 in a dose-dependent manner, whereas 1 &mgr;g/ml had no effect. Transmission electron microscopy and laser confocal microscopy indicated that the tumor cells had penetrated the brain slice and that the normal structural integrity of the brain was maintained until Day 7. CONCLUSIONThis model enabled unequivocal periodic tracking of individual invading tumor cells in normal brain. The significant suppression of glioma cell invasion by noncytotoxic concentrations of SI-27 indicates that anti-MMP treatment may represent an important future therapeutic strategy for malignant cerebral neoplasms.

Cavernous hemangioma of the skull in a neonate

Abstract Cavernous hemangiomas rarely occur in the calvarium and most commonly present in middle-age. Although a congenital vascular disorder can theoretically cause a diploic lesion in any age group, a calvarial cavernous hemangioma has not been reported in newborn. A 4-month-old male infant presented with a large left parietal mass that had been present since birth. Total resection was performed. Pathological examination revealed a cavernous hemangioma developing within the diploic space adjacent to prior hemorrhages. Surgery was performed in this case because of the size and persistence of the lesion. Removal of tumors of a benign nature from the calvarium can be done safely. Cavernous hemangioma of the skull in a neonate should be considered as one of the differential diagnoses in the case of suspected ossified cephalohematoma.

Transcranial Doppler Ultrasonography for Diagnosis of Cerebral Vasospasm After Aneurysmal Subarachnoid Hemorrhage: Mean Blood Flow Velocity Ratio of the Ipsilateral and Contralateral Middle Cerebral Arteries

BACKGROUND:Transcranial Doppler (TCD) is widely accepted to monitor cerebral vasospasm after subarachnoid hemorrhage (SAH); however, its predictive value remains controversial. OBJECTIVE:To investigate the predictive reliability of an increase in the mean blood flow velocity (mBFV) ratio of the ipsilateral to contralateral middle cerebral arteries (I/C mBFV) compared with the conventional absolute flow velocity. METHODS:We retrospectively investigated the clinical and radiologic data of consecutive patients with SAH admitted from July 2003 to August 2009 who underwent TCD ultrasonography. The highest mBFV value in bilateral middle cerebral arteries was recorded, while delayed cerebral ischemia (DCI) was defined as neurological deficits or computed tomographic evidence of cerebral infarction caused by vasospasm. The ipsilateral side was defined as the side with higher mBFV value when evaluating the I/C mBFV. We thus elucidated the reliability of this rate in comparison with the conventional method for predicting DCI with receiver operating characteristic (ROC) analysis. RESULTS:One hundred and forty-two patients were retrospectively analyzed with specific data from 1262 TCD studies. The ROC curve showed that the overall predictive value for DCI had an area under the curve of 0.86 (95% confidence interval: 0.76-0.96) when the I/C mBFV was used vs 0.80 (0.71-0.88) when the absolute flow velocity was used. The threshold value that best discriminated between patients with and without DCI was I/C mBFV of 1.5. CONCLUSION:In patients with SAH, the I/C mBFV demonstrated a more significant correlation to vasospasm than the absolute mean flow velocity.