Jun-ichiro Hamada

The strategy for enhancing temozolomide against malignant glioma

A combined therapy of the alkylating agent temozolomide (TMZ) and radiotherapy is standard treatment, and it improves the survival of patients with newly diagnosed glioblastoma (GBM). The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) removes the most cytotoxic lesions generated by TMZ, O6-methylguanine, establishing MGMT as one of the most important DNA repair mechanisms of TMZ-induced DNA damage. Thus, the expression of MGMT, its activity, and its promoter methylation status are associated with the response of GBM to TMZ, confirming that MGMT promotes clinical resistance to TMZ. Previous studies have shown that a variety of drugs such as interferon-β (IFN-β), levetiracetam (LEV), resveratrol, and valproic acid (VAP) increased the sensitivity of TMZ through MGMT-dependent or MGMT-independent mechanisms. In this review, we describe drugs and promising molecules that influence the responsiveness of GBM to TMZ and discuss their putative mechanism of action. In MGMT-positive GBMs, drugs that modulate MGMT activity could enhance the therapeutic activity of TMZ. Thus, administration of these drugs as an adjunct to TMZ chemotherapy may have clinical applications in patients with malignant gliomas to improve the outcome.

Receptor Tyrosine Kinases: Principles and Functions in Glioma Invasion

Protein tyrosine kinases are enzymes that are capable of adding a phosphate group to specific tyrosines on target proteins. A receptor tyrosine kinase (RTK) is a tyrosine kinase located at the cellular membrane and is activated by binding of a ligand via its extracellular domain. Protein phosphorylation by kinases is an important mechanism for communicating signals within a cell and regulating cellular activity; furthermore, this mechanism functions as an on or off switch in many cellular functions. Ninety unique tyrosine kinase genes, including 58 RTKs, were identified in the human genome; the products of these genes regulate cellular proliferation, survival, differentiation, function, and motility. Tyrosine kinases play a critical role in the development and progression of many types of cancer, in addition to their roles as key regulators of normal cellular processes. Recent studies have revealed that RTKs such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), c-Met, Tie, Axl, discoidin domain receptor 1 (DDR1), and erythropoietin-producing human hepatocellular carcinoma (Eph) play a major role in glioma invasion. Herein, we summarize recent advances in understanding the role of RTKs in glioma pathobiology, especially the invasive phenotype, and present the perspective that RTKs are a potential target of glioma therapy.

Force-detecting gripper and force feedback system for neurosurgery applications

AbstractPurpose For the application of less invasive robotic neurosurgery to the resection of deep-seated tumors, a prototype system of a force-detecting gripper with a flexible micromanipulator and force feedback to the operating unit will be developed. n Methods Gripping force applied on the gripper is detected by strain gauges attached to the gripper clip. The signal is transmitted to the amplifier by wires running through the inner tube of the manipulator. Proportional force is applied on the finger lever of the operating unit by the surgeon using a bilateral control program. A pulling force experienced by the gripper is also detected at the gripper clip. The signal for the pulling force is transmitted in a manner identical to that mentioned previously, and the proportional torque is applied on the touching roller of the finger lever of the operating unit. The surgeon can feel the gripping force as the resistance of the operating force of the finger and can feel the pulling force as the friction at the finger surface.n Results A basic operation test showed that both the gripping force and pulling force were clearly detected in the gripping of soft material and that the operator could feel the gripping force and pulling force at the finger lever of the operating unit. n Conclusions A prototype of the force feedback in the microgripping manipulator system has been developed. The system will be useful for removing deep-seated brain tumors in future master–slave-type robotic neurosurgery.

Combination therapy using Notch and Akt inhibitors is effective for suppressing invasion but not proliferation in glioma cells

Molecular targeted therapy can potentially provide more effective treatment for patients with high-grade gliomas. Notch and Akt are notable target molecules as they play important roles in a variety of cellular processes, such as regeneration, differentiation, proliferation, migration, and invasion. Here, we assessed the therapeutic possibility of inhibiting Notch and Akt in gliomas using the clinically available, selective small molecule inhibitors MRK003 and MK-2206. We evaluated their efficacy individually and as a combination therapy in U251 and U87 glioma cell lines. We confirmed that MK-2206 effectively inhibits Akt phosphorylation in a dose-dependent manner, whereas MRK003 inhibits Notch signaling and Akt phosphorylation. Both MRK003 and MK-2206 significantly inhibited cell growth, migration, and invasion in a dose-dependent manner. Akt dephosphorylation was enhanced by combination therapy with MRK003 and MK-2206. However, the effect of combination treatment did not exceed that of MK-2206 monotherapy in proliferation assay. Inhibition of invasion, further enhanced by combination therapy, correlated with increased Akt inactivation. In summary, combination therapy with MRK003 and MK-2206 may be effective for inhibiting invasion but not proliferation.

Characterizing invading glioma cells based on IDH1-R132H and Ki-67 immunofluorescence

AbstractnGlioma, the most common primary brain tumor, is characterized by proliferative-invasive growth. However, the detailed biological characteristics of invading glioma cells remain to be elucidated. A monoclonal antibody (clone HMab-1) that specifically and sensitively recognizes the isocitrate dehydrogenase-1 (IDH1) protein carrying the R132H mutation can identify invading glioma cells by immunostaining. To investigate the degree of invasion in gliomas of distinct grades and the proliferative capacity of the invading cells, immunofluorescent staining was conducted using antibodies against IDH1-R132H and Ki-67 on 11 surgical and autopsy specimens of the tumor core and the invading area. Higher numbers of IDH1-R132H-positive cells in the invading area correlated with a higher tumor grade. Double staining for IDH1-R132H and Ki-67 demonstrated that most invading cells that expressed IDH1-R132H were not stained by the Ki-67 antibody, and the ratio of Ki-67-positive cells among IDH1-R132H-positive cells was significantly lower in the invasion area than in the tumor core in all grades of glioma. These data suggest that higher grade gliomas have a greater invasive potential and that invading cells possess low proliferative capacity.

Association between carotid plaque composition assessed by multidetector computed tomography and cerebral embolism after carotid stenting

IntroductionWe aimed to assess the relationship between atherosclerotic carotid plaque composition analyzed using multidetector computed tomography (MDCT) and the appearance of new ischemic lesions detected by diffusion-weighted images (DWI) after carotid artery stenting (CAS).MethodsWe quantitatively and qualitatively analyzed plaque characteristics in carotid arteries using MDCT before CAS in 19 patients. Carotid plaques were expediently subdivided into four components with Hounsfield unit (HU) values of <0, 0–60, 60–130, and >600. The incidence of distal embolism was evaluated with DWI. Pearsons correlation analyses were used to assess the association between plaque composition and the incidence of cerebral embolization.ResultsFifteen patients (79%) demonstrated new DWI lesions after CAS. High-signal DWIs were noted as follows: one in six patients, 2u2009~u20095 in five patients, 6u2009~u200910 in two patients, and >10 in two patients. The mean volumes of the plaque components for HUu2009<u20090, 0–60, 60–130, and >600 were 5.4, 200, 260, and 59xa0mm3, respectively. There was a strong correlation between the number of high-signal DWI lesions in the ipsilateral side and the plaque volume of HUu2009<u20090 (ru2009=u20090.927; Pu2009<u20090.0001). There was a moderate correlation between the number of high-signal DWI lesions and the plaque volume of HU 0–60 (ru2009=u20090.568; Pu2009=u20090.0099) and the sum total of HUu2009<u20090 and HU 0–60 (ru2009=u20090.609; Pu2009=u20090.0047).ConclusionsQuantitative and qualitative tissue characterization of carotid plaques using MDCT might be a useful predictor for silent ischemic lesions after CAS.

Force detecting gripper and flexible micro manipulator for neurosurgery

In order to realize a less invasive robotic neurosurgery for the deeply seated tumor, a force detecting gripper with a flexible micro manipulator has been developed. Gripping force applied on the gripper is detected by strain gages fit on the gripper clip. Signal is conducted to the amplifier by the cables through the inner pipe of the manipulator. In order to approach to the deeply seated tumor through a narrow hole, a micro manipulator which can flex at the end part to face the gripper for the target and can rotate the closing direction of the gripper at the end of the manipulator has been developed. Some operation test showed that the developed manipulator can approach flexibly to the target, and the taking out force of a target on the soft material was detected clearly.

Molecular analysis of a recurrent glioblastoma treated with bevacizumab.

We treated a case of recurrent glioblastoma (GBM) with bevacizumab and assessed its effect biologically. A 55-year-old man with a left frontal lobe GBM was experiencing recurrence 7xa0months postoperation. We administered bevacizumab concomitant with temozolomide (TMZ). Follow-up magnetic resonance imaging (MRI) showed dramatic but temporal tumor reduction; however, the patient died of re-recurrent disease 6xa0months after beginning bevacizumab. We obtained an autopsy and analyzed the detailed molecular change. In the autopsy specimen, the quantity of microvessels was significantly reduced. Vascular endothelial growth factor receptor (VEGFR) 1 and VEGFR2 were downregulated, most likely due to a negative feedback mechanism by blocking of VEGF signaling. Matrix metalloproteinase (MMP)-2 and membrane-type 1 MMP were upregulated, resulting in the higher activation of MMP-2 in the autopsy specimen. MIB-1 staining index and phosphorylation levels of p44/42-mitogen-activated protein kinase did not change, whereas phosphorylated protein kinase B (Akt) was decreased in the autopsy specimen, suggesting compensation and/or amplification of other proliferative signaling pathways such as suppression of apoptosis signaling. Consequently, bevacizumab might inhibit the VEGF autocrine loop, which then causes a change in molecular expression related not only to enhancement of tumor invasion but also maintenance of tumor proliferation.

Recurrent anaplastic meningioma treated by sunitinib based on results from quantitative proteomics

Anaplastic meningiomas are aggressive tumours that account for 1% to 3% of all meningiomas [1]. They tend to recur even after complete surgical resection and subsequent radiation therapy. The prognosis for anaplastic meningiomas is poor, with a median survival of less than 2 years [2]. However, none of the chemotherapeutic drugs have shown any convincing effect on anaplastic meningiomas [3]. Currently, novel therapeutic drugs that act on growth factor receptors are being manufactured and tested for anaplastic meningiomas [3]. In this report, we present the case of a patient with intractable anaplastic meningioma that recurred repeatedly in a short period. Our proteomics analysis showed that platelet-derived growth factor receptor (PDGFR) b was highly expressed in the tumour. Immunohistochemistry confirmed expression and activation of PDGFRb in the surgical specimen. According to our results, sunitinib (Sutent, Pfizer Inc., New York , NY, USA), a rationally designed small-molecule inhibitor that blocks PDGFRb signalling, was administered [4]. To the best of our knowledge, this is the first case where a tailored chemotherapy has been administered for anaplastic meningioma on the basis of proteomics results. A 62-year-old woman was admitted to our hospital (Kanazawa University Hospital) for the treatment of sphenoid ridge meningioma. The detailed clinical course was reported by Kawahara et al. as the extremely aggressive anaplastic meningioma [5]. Briefly, she was diagnosed with anaplastic meningioma and underwent surgery three times and radiotherapy twice for the repeated recurrences. However, the tumour showed local recurrence and cerebrospinal fluid dissemination 4 months after the first surgery. We analysed the tumour samples resected during the first surgery with quantitative target absolute proteomics, as described previously [6]. The research protocols for the present study were approved by the ethical committees of the Graduate School of Pharmaceutical Sciences, Tohoku University and Graduate School of Medical Science, Kanazawa University Hospital. The plasma membrane fractions of the brain samples were processed using differential centrifugation with a sucrose density gradient and were digested by trypsin followed by reduction and alkylation. The tryptic digests were acidified with formic acid for analysis with the high-performance liquid chromatography (HPLC) system (Agilent1200 system, Agilent, Santa Clara, CA, USA), which was connected to an electrospray ionization-triple quadrupole mass spectrometer (API5000, ABSCIEX, Foster City, CA, USA). HPLC was performed with C18 capillary columns (ZORBAX SBC18 0.5 mm inner diameter 150 mm, 5 mm particles; Agilent). The mass spectrometer was set up to run a multichannel reaction monitoring for 11 target molecules that have available molecular-targeted drugs (Table 1). Linear gradients of 1–50% acetonitrile in 0.1% formic acid were applied to elute the peptides at a flow rate of 50 ml/min for 50 min. The protein expression levels were determined as described previously [7]. Peptide sequences and selected ions for quantification of each membrane protein are shown in Table S1. As shown in Table 1, PDGFRb, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), CD33 and CD37 were detected in the plasma membrane fraction of the tumour tissues, and PDGFRb exhibited the greatest protein expression levels. Moreover, PDGFRb protein level in this case was higher than the levels obtained from glioblastomas (mean SD, 0.270 0.731 fmol/mg protein: n = 21), whereas PDGFRb is undetectable in normal brain (n = 1). Immunohistochemistry was performed to validate the proteomics data. Briefly, paraffin-embedded tissue blocks were sectioned (6-mm thick) on to slides and then deparaffinized. Sections were immunostained using the ABC Elite Kit (Funakoshi, Tokyo, Japan) with anti-PDGFRb monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA) and phosphorylated PDGFRb (Tyr857) polyclonal antibody (Santa Cruz Biotech., Santa Cruz, CA, USA). Sections were quenched with 3% hydrogen peroxide in methanol for 20 min, microwaved for 15 min in citrate buffer, blocked by incubation in 0.3% H2O2 solution in methanol for 20 min, and blocked for 20 min with diluted

Identification of the Inflow Zone of Unruptured Cerebral Aneurysms: Comparison of 4D Flow MRI and 3D TOF MRA Data

BACKGROUND AND PURPOSE: The hemodynamics of the inflow zone of cerebral aneurysms may be a key factor in coil compaction and recanalization after endovascular coil embolization. We performed 4D flow MR imaging in conjunction with 3D TOF MRA and compared their ability to identify the inflow zone of unruptured cerebral aneurysms. MATERIALS AND METHODS: This series comprised 50 unruptured saccular cerebral aneurysms in 44 patients. Transluminal color-coded 3D MRA images were created by selecting the signal-intensity ranges on 3D TOF MRA images that corresponded with both the luminal margin and the putative inflow. RESULTS: 4D flow MR imaging demonstrated the inflow zone and yielded inflow velocity profiles for all 50 aneurysms. In 18 of 24 lateral-projection aneurysms (75%), the inflow zone was located distally on the aneurysmal neck. The maximum inflow velocity ranged from 285 to 922 mm/s. On 4D flow MR imaging and transluminal color-coded 3D MRA studies, the inflow zone of 32 aneurysms (64%) was at a similar location. In 91% of aneurysms whose neck section plane angle was <30° with respect to the imaging section direction on 3D TOF MRA, depiction of the inflow zone was similar on transluminal color-coded 3D MRA and 4D flow MR images. CONCLUSIONS: 4D flow MR imaging can demonstrate the inflow zone and provide inflow velocity profiles. In aneurysms whose angle of the neck-section plane is obtuse vis-a-vis the imaging section on 3D TOF MRA scans, transluminal color-coded 3D MRA may depict the inflow zone reliably.