Hironobu Harada

Accuracy of diffusion tensor magnetic resonance imaging-based tractography for surgery of gliomas near the pyramidal tract: a significant correlation between subcortical electrical stimulation and postoperative tractography.

BACKGROUND: Diffusion tensor (DT) imaging-based fiber tracking is a noninvasive magnetic resonance technique that can delineate the course of white matter fibers. OBJECTIVE: To evaluate the accuracy and usefulness of this DT imaging-based fiber tracking for surgery in patients with gliomas near the pyramidal tract (PT). METHODS: Subjects comprised 32 patients with gliomas near the PT. DT imaging-based fiber tracks of the PT were generated before and within 3 days after surgery in all patients. A tractography-integrated navigation system was used during the operation. Cortical and subcortical motor-evoked potentials (MEPs) were also monitored during resection to maximize the preservation of motor function. The threshold intensity for subcortical MEPs was examined by searching the stimulus points and changing the stimulus intensity. Minimum distance between the resection border and the illustrated PT was measured on postoperative tractography. RESULTS: In all subjects, DT imaging-based tractography of the PT was successfully performed, preoperatively demonstrating the relationship between tumors and the PT. With the use of the tractography-integrated navigation system and intraoperative MEPs, motor function was preserved postoperatively in all patients. A significant correlation was seen between threshold intensity for subcortical MEPs and the distance between the resection border and PT on postoperative DT imaging. CONCLUSION: DT imaging-based fiber tracking is a reliable and accurate method for mapping the course of subcortical PTs. Fiber tracking and intraoperative MEPs were useful for preserving motor function in patients with gliomas near the PT.

Introduction of wild-type p53 enhances thrombospondin-1 expression in human glioma cells

Malignant gliomas are distinguished from low-grade gliomas by their intense angiogenesis. In gliomas, p53 is the most frequently altered gene and is involved in the early phase of glioma development. In contrast, homozygous p16 gene deletion is more common in high-grade gliomas. In order to understand the mechanism by which gliomas become more angiogenic during the malignant transformation, we examined the relationship between thrombospondin-1, a negative regulator in angiogenesis, and these tumor suppressor genes in malignant gliomas. Human glioma cell line U-251 MG, which has mutated p53 and deleted p16, was transduced with recombinant replication-defective adenovirus vectors containing the cDNA of wild-type p53, p16, and p21. Only the induction of wild-type p53 enhanced expression of thrombospondin-1 mRNA and the protein in U-251 MG cells. Furthermore, thrombospondin-1 that was secreted in the culture medium was significantly increased (3.8-fold) as compared with that of the viral control 36 h after infection with Ad5CMV-p53. In the presence of wild-type p53 plasmid DNA, the promoter activity was increased 7.4-fold as compared with an empty expression vector control. These studies may suggest that mutation of p53 gene endows gliomas with an angiogenic phenotype by reducing thrombospondin-1 production as well as enhancing the angiogenesis inducers in the early phase of malignant progression.

Endothelial nitric oxide synthase expression in tumor vasculature is correlated with malignancy in human supratentorial astrocytic tumors

OBJECTIVE Endothelial nitric oxide synthase (eNOS) may play an important role in the regulation of tumor blood flow and vascular permeability. However, there have been no reports describing alterations of eNOS expression in relation to malignant progression in human astrocytic tumors. We immunohistochemically studied the relationship between eNOS expression in tumor vasculature and malignancy in supratentorial astrocytic tumors. METHODS Tissue samples were obtained from 12 patients with low-grade astrocytomas, 10 with anaplastic astrocytomas, and 17 with glioblastomas. Normal brain tissue samples were obtained from four patients with other brain diseases. Immunohistochemical staining was performed using the avidin-biotin complex method, with polyclonal anti-eNOS antibody, and the levels of eNOS expression in endothelial cells were evaluated as slight, moderate, or intense on the basis of eNOS immunoreactivity. The proliferative potential was assessed as the MIB-1 staining index for tumor cells. RESULTS The expression of eNOS was slight in all specimens of normal brain tissue, slight in 7 and moderate in 5 specimens of low-grade astrocytoma, slight in 2, moderate in 6, and intense in 2 specimens of anaplastic astrocytoma, and moderate in 5 and intense in 12 specimens of glioblastoma. The MIB-1 staining index (mean+/-standard deviation) was 0.2+/-0.2% for normal specimens, 1.8+/-0.6% for low-grade astrocytomas, 9.6+/-6.9% for anaplastic astrocytomas, and 18.5+/-7.7% for glioblastomas. The MIB-1 staining indices for slight, moderate, and intense eNOS expression were 2.0+/-2.3%, 10.8+/-9.8%, and 16.9+/-7.7%, respectively. CONCLUSION Expression of eNOS in tumor vessels was significantly correlated with histological grade and proliferative potential. These findings suggest that astrocytic tumor vessels possess higher activity for nitric oxide production than do normal vessels.

miR340 suppresses the stem-like cell function of glioma-initiating cells by targeting tissue plasminogen activator.

Glioma-initiating cells (GIC) have stem-like cell properties thought to be sufficient for recurrence, progression, and drug resistance in glioblastomas. In the present study, we defined miRNA (miR)-340 as a differentially expressed miRNA in human GICs that inhibit GIC-mediated tumorigenesis. Furthermore, we defined tissue plasminogen activator (PLAT) as a critical direct target of miR340 for inhibition. Among miRNAs screened, we found that miR340 expression was decreased in all human GICs and in human glioblastoma tissues, compared with human neural stem cells and normal brain tissues. miR340 overexpression in GICs suppressed their proliferative, invasive, and migratory properties in vitro, triggering cell senescence in vitro and inhibiting GIC-induced tumorigenesis in mouse brains. shRNA-mediated silencing of PLAT in GICs phenocopied the effects of miR340 overexpression in vitro and in vivo, suggesting a potential role for tissue factor in stem-like cell function. Taken together, our results identified miR340 as a tumor suppressor that functions in GIC to enforce PLAT blockade and ablate their stem-like functions.

Oct-3/4 promotes migration and invasion of glioblastoma cells

As a result of increased glioblastoma migration and invasion into normal brain parenchyma, treatment of local tumor recurrence following initial treatment in glioblastoma patients remains challenging. Recent studies have demonstrated increased Oct‐3/4 expression, a self‐renewal regulator in stem cells, in glioblastomas. However, little is known regarding the influence of Oct‐3/4 in glioblastoma cell invasiveness. The present study established Oct‐3/4‐overexpressing glioblastoma cells, which were prepared from human glioblastoma patients, to assess migration, invasion, and mRNA expression profiles of integrins and matrix metalloproteinases (MMPs). Compared with control cells, Oct‐3/4 expressing‐glioblastoma cells exhibited increased migration and invasion in wound healing and Matrigel invasion assays. Oct‐3/4 overexpression resulted in upregulated FAK and c‐Src expression, which mediate integrin signals. Vinculin accumulated along the leading edges of Oct‐3/4 expressing‐glioblastoma cells and associated with membrane ruffles during cell migration. Oct‐3/4 expressing‐cells exhibited increased MMP‐13 mRNA expression and MMP‐13 knockdown by shRNA suppressed cell invasion into Matrigel and organotypic brain slices. These results suggested that Oct‐3/4 enhanced degradation of surrounding extracellular matrix by increasing MMP‐13 expression and altering integrin signaling. Therefore, Oct‐3/4 might contribute to tumor promoting activity in glioblastomas. J. Cell. Biochem. 113: 508–517, 2012.

Downregulation of laminin α4 chain expression inhibits glioma invasion in vitro and in vivo

The laminin family is a structural constituent of the extracellular matrix that plays an essential role in promoting the motility of infiltrative tumor cells. We investigated the role of laminin α4 chain, a subset of laminin‐8, ‐9 and ‐14, in the motile and invasive activities of human glioma cells. All malignant glioma cell lines examined expressed more mRNA for the laminin α4 and β1 chains than for the β2 chain, indicating that these cells predominantly express the laminin‐8 isoform. Introducing an antisense oligonucleotide for laminin α4 chain (AS‐Ln‐α4) into the glioma cells resulted in downregulation of laminin α4 expression. AS‐Ln‐α4 also significantly suppressed glioma cell adhesion and migration. Furthermore, invasiveness was significantly reduced in cells transfected with AS‐Ln‐α4 compared to those transfected with the sense oligonucleotide (S‐Ln‐α4). Indeed, when glioma spheroids were implanted into rat brain slices, AS‐Ln‐α4‐transfected cells failed to invade surrounding normal brain tissues. In addition, intracerebral injection of glioma cells transfected with AS‐Ln‐α4 into nude mice resulted in the formation of a noninvasive tumor, whereas injection of cells transfected with S‐Ln‐α4 resulted in diffuse invasion of brain tissue. These results suggest that mainly laminin‐8 is essential for the invasive activity of human glioma cells; thus, a novel therapeutic strategy could target this molecule to treat patients with malignant glioma.

Downregulation of laminin alpha4 chain expression inhibits glioma invasion in vitro and in vivo.

The laminin family is a structural constituent of the extracellular matrix that plays an essential role in promoting the motility of infiltrative tumor cells. We investigated the role of laminin α4 chain, a subset of laminin‐8, ‐9 and ‐14, in the motile and invasive activities of human glioma cells. All malignant glioma cell lines examined expressed more mRNA for the laminin α4 and β1 chains than for the β2 chain, indicating that these cells predominantly express the laminin‐8 isoform. Introducing an antisense oligonucleotide for laminin α4 chain (AS‐Ln‐α4) into the glioma cells resulted in downregulation of laminin α4 expression. AS‐Ln‐α4 also significantly suppressed glioma cell adhesion and migration. Furthermore, invasiveness was significantly reduced in cells transfected with AS‐Ln‐α4 compared to those transfected with the sense oligonucleotide (S‐Ln‐α4). Indeed, when glioma spheroids were implanted into rat brain slices, AS‐Ln‐α4‐transfected cells failed to invade surrounding normal brain tissues. In addition, intracerebral injection of glioma cells transfected with AS‐Ln‐α4 into nude mice resulted in the formation of a noninvasive tumor, whereas injection of cells transfected with S‐Ln‐α4 resulted in diffuse invasion of brain tissue. These results suggest that mainly laminin‐8 is essential for the invasive activity of human glioma cells; thus, a novel therapeutic strategy could target this molecule to treat patients with malignant glioma.

Oct-3/4 promotes tumor angiogenesis through VEGF production in glioblastoma

Accumulating evidence shows that the expression level of Oct-3/4, a self-renewal regulator in stem cells, is positively correlated with the progression of various solid tumors. However, little is known regarding the influence of Oct-3/4 in the tumor angiogenesis of glioblastomas. In the present study, we subcutaneously transplanted Oct-3/4-overexpressing human glioblastoma U251 (U251/EGFP-Oct-3/4) cells into the right thighs of nude mice to evaluate the roles of Oct-3/4 in the tumor angiogenesis. Both tumor size and the number of large vessels growing in the tumor were markedly increased. In an in vitro model of angiogenesis, the conditioned media from U251/EGFP-Oct-3/4 cells significantly accelerated capillary-like tube formation compared with that of U251/EGFP cells. In comparison with U251/EGFP cells, U251/EGFP-Oct-3/4 cells had markedly elevated the expression of vascular endothelial growth factor mRNA under the control of hypoxia-inducible factor (HIF) 1α. In U251/EGFP-Oct-3/4 cells, enhanced protein expression and nuclear translocation of HIF1α were observed. Furthermore, we demonstrated that the involvement of AKT, an oncogenic signaling molecule, in the Oct-3/4 induced upregulation of HIF1α protein. Our findings suggest that Oct-3/4-expressing glioblastoma cells have the ability to adapt to low-oxygen environments within tumor masses by promoting tumor angiogenesis through AKT-HIF1 pathway.

Utility of three-dimensional computed tomography for anatomical assistance in endoscopic endonasal transsphenoidal surgery

Endoscopic endonasal transsphenoidal surgery (ETSS) has been widely applied to pituitary adenomas. However, anatomical orientation is difficult when structures of the sphenoidal sinus are complicated. This study investigated the usefulness of three-dimensional computed tomography (3D-CT) modeling in planning surgical procedures for ETSS and providing anatomical guidance during surgery. CT data from 99 consecutive patients with pituitary adenoma treated between January 2008 and March 2014 were used to reconstruct 3D-CT models. Based on these images, the architecture of sphenoidal sinus, particularly structures surrounding the sellar floor, was visualized for preoperative simulation of surgical procedures. These 3D-CT images were also compared to surgical views during ETSS to evaluate applicability of the images. These models clearly demonstrated the morphology of the nasal cavity and structures of the sphenoidal sinus, including bony prominences of the internal carotid arteries (ICAs) and optic canals by successively eliminating sphenoidal structures. The 3D-CT images permitted determination of the maximum marginal line of the opening of the sellar floor by presenting vital structures such as ICAs and optic canals. With this 3D-CT model, the surgeon could access the sella more easily, open the floor widely enough for each individual patient, and resect the tumor maximally without complications. Preoperative 3D-CT models distinctly visualized the optic canals, bilateral ICAs, and complicated structures of sphenoidal septa. The 3D-CT images were useful for preoperative planning and as a road map during endoscopic surgery for pituitary adenoma, facilitating maximum tumor resection without complications.

Oct-3/4 modulates the drug-resistant phenotype of glioblastoma cells through expression of ATP binding cassette transporter G2

BACKGROUND Drug resistance is a major obstacle for the efficacy of chemotherapeutic treatment of tumors. Oct-3/4, a self-renewal regulator in stem cells, is expressed in various kinds of solid tumors including glioblastoma. Although Oct-3/4 expression has been implicated in the malignancy and prognosis of glioblastomas, little is known of its involvement in drug resistances of glioblastoma. METHODS The involvement of Oct-3/4 in drug resistance of glioblastoma cells was assessed by lactate dehydrogenase assay, efflux assay of an anticancer drug, poly ADP-ribose polymerase cleavage, and in vivo xenograft experiments. Involvement of a drug efflux pump ATP binding cassette transporter G2 in Oct-3/4-induced drug resistance was evaluated by quantitative PCR analysis and knockdown by shRNA. RESULTS Oct-3/4 decreased the susceptibility to chemotherapeutic drugs by enhancing excretion of drugs through a drug efflux pump gene, ATP binding cassette transporter G2. Moreover, the expression of Oct-3/4 was well correlated to ATP binding cassette transporter G2 expression in clinical GB tissues. CONCLUSION Oct-3/4 elevated the ATP binding cassette transporter G2 expression, leading to acquisition of a drug-resistant phenotype by glioblastoma cells. GENERAL SIGNIFICANCE If the drug-resistance of glioblastoma cells could be suppressed, it should be a highly ameliorative treatment for glioblastoma patients. Therefore, signaling pathways from Oct-3/4 to ATP binding cassette transporter G2 should be intensively elucidated to develop new therapeutic interventions for better efficacy of anti-cancer drugs.