Yoshifumi Mizobuchi

Akt2 and Akt3 play a pivotal role in malignant gliomas

Akt, one of the major downstream effectors of phosphatidylinositol 3-kinase, is hyper-expressed and activated in a variety of cancers including glioblastoma. However, the expression profiles of the Akt isoforms Akt1/PKBalpha, Akt2/PKBbeta, and Akt3/PKBgamma and their functional roles in malignant glioma are not well understood. Therefore, we examined the protein and mRNA expression patterns of Akt isoforms in tissues from human astrocytomas, glioblastomas, and non-neoplastic regions. We also explored the biological role of each Akt isoform in malignant glioma cells using RNA interference-mediated knock-down and the over-expression of plasmid DNA of each isoform. The expression of Akt1 protein and mRNA was similar in glioma and normal control tissues. Although the protein and mRNA level of Akt2 increased with the pathological grade of malignancy, the expression of Akt3 mRNA and protein decreased as the malignancy grade increased. In U87MG, T98G, and TGB cells, the down-regulation of Akt2 or Akt3 by RNA interference reduced the expression of the phosphorylated form of Bad, resulting in the induction of caspase-dependent apoptosis. Akt1 knock-down did not affect cell growth or survival. We first demonstrate that the over-expression of Akt2 or Akt3 down-regulated the expression of the other protein and that endogenous Akt3 protein showed high kinase activity in U87MG cells. Our data suggest that Akt2 and Akt3 play an important role in the viability of human malignant glioma cells. Targeting Akt2 and Akt3 may hold promise for the treatment of patients with gliomas.

REIC/Dkk-3 induces cell death in human malignant glioma

The progression of glioma to more malignant phenotypes results from the stepwise accumulation of genetic alterations and the consequent disruption of the apoptotic pathway and augmentation of survival signaling. REIC/Dkk-3, a member of the human Dickkopf (Dkk) family, plays a role as a suppressor of the growth of several human cancers; however, to date it has not been identified in brain tumors. We compared the gene and protein expression of REIC/Dkk-3 in human malignant glioma and normal brain tissues using quantitative real-time PCR, Western blotting, and immunohistochemistry. We also performed small interfering REIC/Dkk-3 (siREIC/Dkk-3) knockdown and REIC/Dkk-3 overexpression experiments to examine the role of REIC/Dkk-3 in human malignant glioma cells in vitro. In brain tissue from patients with malignant glioma, the gene and protein expression of REIC/Dkk-3 was lower than in normal brain tissue and was related to the malignancy grade. In the primary glioblastoma cell line, REIC/Dkk-3 transfection led to apoptosis owing to the activation of phosphorylated JUN, caspase-9, and caspase-3 and the reduction of beta-catenin; in REIC/Dkk-3 knockdown experiments, cell growth was augmented. Our results suggest that REIC/Dkk-3 regulates the growth and survival of these cells in a caspase-dependent and -independent way via modification of the Wnt signaling pathway. Our work is the first documentation that the gene and protein expression of REIC/Dkk-3 is down-regulated in human malignant glioma. Our demonstration of the mechanisms underlying REIC/Dkk-3-induced cell death indicates that REIC/Dkk-3 plays a pivotal role in the biology of human malignant glioma and suggests that REIC/Dkk-3 is a promising candidate for molecular target therapy.

Clinical results of boron neutron capture therapy (BNCT) for glioblastoma

The purpose of this study was to evaluate the clinical outcome of BSH-based intra-operative BNCT (IO-BNCT) and BSH and BPA-based non-operative BNCT (NO-BNCT). We have treated 23 glioblastoma patients with BNCT without any additional chemotherapy since 1998. The median survival time (MST) of BNCT was 19.5 months, and 2-year, 3-year and 5-year survival rates were 26.1%, 17.4% and 5.8%, respectively. This clinical result of BNCT in patients with GBM is superior to that of single treatment of conventional radiotherapy compared with historical data of conventional treatment.

Up-regulation of endogenous PML induced by a combination of interferon-beta and temozolomide enhances p73/YAP-mediated apoptosis in glioblastoma

Interferon-beta (IFN-β) is reported to augment anti-tumor effects by temozolomide in glioblastoma via down-regulation of MGMT. Promyelocytic leukemia (PML), a gene induced by IFN-β, is a tumor suppressor. Here, we report for the first time that in combination therapy, an IFN-β-induced increase in endogenous PML contributes to anti-tumor effects in p53 wild- and mutant glioma cells in a xenograft mice model. The increased PML promoted the accumulation of p73, a structural and functional homolog of p53, to fuse the coactivator Yes-associated-protein in the PML nuclear bodies. The adjuvant therapy targeted at PML may be a promising therapeutic strategy for glioblastoma.

Clinical results of BNCT for malignant brain tumors in children

It is very difficult to treat the patients with malignant brain tumor in children, especially under 3 years, because the conventional irradiation cannot be applied due to the damage of normal brain tissue. However, boron neutron capture therapy (BNCT) has tumor selectivity such that it can make damage only in tumor cells. We evaluated the clinical results and courses in patients with malignant glioma under 15 years. Among 183 patients with brain tumors treated by our group using BSH-based intra-operative BNCT, 23 patients were under 15 years. They included 4 patients under 3 years. There were 3 glioblastomas (GBM), 6 anaplastic astrocytomas(AAS), 7 primitive neuroectodermal tumors (PNET), 6 pontine gliomas and 1 anaplastic ependymoma. All GBM and PNET patients died due to CSF and/or CNS dissemination without local tumor regrowth. All pontine glioma patients died due to regrowth of the tumor. Four of 6 anaplastic astrocytoma and 1 anaplastic ependymoma patients alive without tumor recurrence. BNCT can be applied to malignant brain tumors in children, especially under 3 years instead of conventional radiation. Although it can achieve the local control in the primary site, it cannot prevent CSF dissemination in patients with glioblastoma.

Histopathological findings in autopsied glioblastoma patients treated by mixed neutron beam BNCT

Since 1998, we have introduced a mixed epithermal- and thermal neutron beam for boron neutron capture therapy (BNCT) to improve the neutron beam distribution. Sixteen patients with malignant glioma (glioblastoma, n = 14; anaplastic ependymoma, n = 1; PNET, n = 1) were treated by BNCT in Japan. Of these, 9 died; 3 due to cerebrospinal fluid (CSF) dissemination, 1 each of tumor invasion, meningitis, pneumonia, and unknown causes, and 2 patients died of local recurrence or radiation necrosis. The current postmortem study is comprised of 3 patients with glioblastoma who were treated with BNCT employing an epithermal neutron beam and sodium borocaptate (BSH: Na2B12H11SH). None of the patients manifested local regrowth at the primary site. However, in 2 patients there was CSF dissemination; tumor cells were recognized throughout the subarachnoid space. In the other patient, tumor cells had massively invaded the ipsilateral- and contralateral hemisphere and brain stem from the bottom of the tumor cavity via the corpus callosum and cerebral peduncle. Our findings indicate that BNCT can achieve local control of glioblastoma at the primary site. However, to further improve the clinical outcome after BNCT, steps must be taken to prevent CSF dissemination.

Current Topics in Sports-related Head Injuries: A Review

We review the current topic in sports-related head injuries including acute subdural hematoma (ASDH), concussion, and chronic traumatic encephalopathy (CTE). Sports-related ASDH is a leading cause of death and severe morbidity in popular contact sports like American football in the USA and judo in Japan. It is thought that rotational acceleration is most likely to produce not only cerebral concussion but also ASDH due to the rupture of a parasagittal bridging vein, depending on the severity of the rotational acceleration injury. Repeated sports head injuries increase the risk for future concussion, cerebral swelling, ASDH or CTE. To avoid fatal consequences or CTE resulting from repeated concussions, an understanding of the criteria for a safe post-concussion return to play (RTP) is essential. Once diagnosed with a concussion, the athlete must not be allowed to RTP the same day and should not resume play before the concussion symptoms have completely resolved. If brain damage has been confirmed or a subdural hematoma is present, the athlete should not be allowed to participate in any contact sports. As much remains unknown regarding the pathogenesis and pathophysiology of sports-related concussion, ASDH, and CTE, basic and clinical studies are necessary to elucidate the crucial issues in sports-related head injuries.

Blocking of the interaction between Wnt proteins and their co- receptors contributes to the anti-tumor effects of adenovirus- mediated DKK3 in glioblastoma

The effect of the third member of the Dickkopf family (DKK3) in the Wnt pathway in glioblastoma remains unclear. We first demonstrated the non-specific interaction of Wnt3a and Wnt5a with the receptors LRP6 and ROR2 and the up-regulation of the Wnt pathway in glioblastoma cells. We used an adenovirus vector and found that an increase in DKK3 protein attenuated the expression of Wnt3a, Wnt5a and LRP6, but not of ROR2, and their interaction, thereby affecting both canonical- and non-canonical Wnt downstream cascades. This produced anti-tumor effects in GBM xenograft models. The suppression of Wnt pathways upstream by DKK3 may have promise for the treatment of glioblastoma.

Long-survivors of glioblatoma treated with boron neutron capture therapy (BNCT)

The purpose of this study was to compare the radiation dose between long-survivors and non-long-survivors in patients with glioblatoma (GBM) treated with boron neutron capture therapy (BNCT). Among 23 GBM patients treated with BNCT, there were five patients who survived more than three years after diagnosis. The physical and weighted dose of the minimum gross tumor volume (GTV) of long-survivors was much higher than that of non-long survivors with significant statistical differences.

Generalized dystonia in a patient with a novel mutation in the GLUD1 gene

Hyperinsulinism-hyperammonemia syndrome (HHS; MIM 606762) is a form of congenital hyperinsulinism caused by mutations in the glutamate dehydrogenase 1 gene (GLUD1). This gene encodes glutamate dehydrogenase (GDH), which catalyzes the oxidation of glutamate to ammonia. Mutations in GLUD1 cause GDH hyperactivity by lowering the enzyme’s sensitivity to guanosine-50-triphosphate (GTP), an allosteric inhibitor of GDH, resulting in persistent hyperammonemia and hyperinsulinism. Although GLUD1-related HHS is frequently associated with epilepsy and mental retardation, generalized dystonia has only been reported in 1 case. Here, we confirm this association and describe a fulminant clinical progression of dystonia to dystonic storm, as well as significant improvement with bilateral globus pallidus internus deep brain stimulation (GPi-DBS). The female patient grew normally until 6 months of age, when she had an episode of generalized tonic–clonic seizures that responded poorly to antiepileptic drugs. Laboratory tests at age 33 months revealed hypoglycemia and hyperinsulinemia followed by persistent hyperammonemia. She also had moderate mental retardation, although her EEG and brain MRI appeared normal. A diagnosis of HHS was made, and we began treatment with diazoxide, which controlled her blood glucose levels and stopped the seizures. At age 11, she developed stereotyped repetitive neck rotation and upper arm contraction. The dystonic movements spread to whole areas of the body within a few months and were refractory to pharmacotherapy that included trihexyphenidyl, risperidone, clonazepam, baclofen, and L-3,4-dihydroxyphenylalanine. The patient eventually developed a life-threatening dystonic storm at age 16, and bilateral GPi-DBS was performed. GPi-DBS had a striking impact on her symptoms; her preoperative Burke-Fahn-Marsden Dystonia Rating Scale score of 112 decreased to 11.5 after GPi-DBS (Video). This improvement was essentially stable on examination 1 year after surgery. The genetic study was approved by the ethics committee, and participants provided written informed consent. We analyzed GLUD1 gene exons by PCR sequencing and confirmed the presence of a heterozygous sequence variation in exon 7: c.943C>T (Fig. 1A). Basal GDH activity was 35 nmol/min/mg of protein (control, 29), and allosteric inhibition by GTP (IC50) was 1819 nM (control, 135). These results are consistent with HHS caused by GLUD1 gene mutations. The patient underwent magnetic resonance spectroscopy (H-MRS) at age 14 (Fig. 1B–D), according to a previously reported method. Coding exons of the TOR1A and THAP1 genes did not contain mutations. Bahi-Buisson et al previously described generalized dystonia with prominent cranial-cervical features in a patient affected with the major GTP-binding site mutation. The mutation in the presented case lies in the same region. Further parallels include the age of dystonia onset, repeated hypoglycemic seizures, and moderate learning disability. Although epilepsy is reported frequently with the major mutation in the GTP-binding site, clonazepam may have masked the patient’s EEG aberrations. This case further supports that mutations in the GLUD1 gene could be a potential cause of dystonia and also suggests that GLUD1-related dystonia occurs through unique GLUD1 gene mutation– induced disruption of metabolic pathways. GLUD1 mutations can induce GDH overactivity that chronically depletes glutamate, which serves as the precursor of GABA. Loss of GABAergic inhibition is critical in dystonia development. Indeed, an MRS study showed that this patient had reduced levels of GABA and glutamate in the basal ganglia. Moreover, our patient showed decreased GABA levels in CSF and plasma (96 pmol/mL [normal controls, 239 6 76] and 74 pmol/mL [normal controls, 120–210], respectively). These data suggest that HHS-associated dystonia may be caused by altered GABAergic neurotransmission from GDH overactivity following direct mutation of the major GTP-binding site.