Hironaga Kamiyama

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.

Lipophilic fraction of Panax ginseng induces neuronal differentiation of PC12 cells and promotes neuronal survival of rat cortical neurons by protein kinase C dependent manner

Panax ginseng is a traditional Chinese herb with a wide range of therapeutic benefits. Recent studies focusing on its effect on the central nervous system have revealed that ginseng has neurotrophic effects including differentiation of neurons. However, most studies involve use of the water-soluble fraction called saponin, and little is known about the effect of the lipophilic fraction. In the present study, we have shown that the lipophilic fraction of ginseng at a concentration of between 0.1 and 50 microg/ml can induce neurite outgrowth of PC12 cells in a dose-dependent manner. Nearly all cells showed morphological differentiation in response to the lipophilic fraction. This morphological differentiation of PC12 cells appeared to be similar to that of NGF. The lipophilic fraction of ginseng also induced neurite extension and promoted survival of rat cortical neurons at a concentration of between 0.025 and 1 microg/ml. These neurotrophic effects on PC12 cells and cortical neurons were not inhibited by K252b, which selectively blocks neurotrophin actions by inhibiting trk-type receptor tyrosine phosphorylation. This suggests that trks do not participate in the neurotrophic action of the lipophilic fraction. However, the effects were completely attenuated by sphingosine, polymyxin B or staurosporin, known inhibitors of protein kinase C (PKC) and calmodulin-dependent kinases. Our results suggest that the lipophilic fraction of ginseng exerts its neurotrophic effects via PKC-dependent pathways.

A rare astrocytic tumor with rhabdoid features

We report an extremely rare tumor presenting with rhabdoid features in the left temporoparietal lobe near the trigone in an 18-year-old Japanese man. This tumor mainly consisted of medium to large round cells that proliferated diffusely and incoherently with a scant extracellular matrix. These tumor cells had an eccentric nucleus and an eosinophilic cytoplasm containing inclusion bodies and bundles of intermediate filaments. The nuclei of these cells were vesicular with prominent nucleoli. This tumor had an area appearing to be diffuse astrocytoma peripherally and lacked a primitive neuroectodermal tumor component, a mesenchymal component, and epithelial differentiation. INI expression, which is not observed in atypical teratoid/ rhabdoid tumor (AT/RT), was found in this tumor. From these findings, we concluded that this tumor was not AT/RT but an astrocytic tumor with rhabdoid features. We also concluded that the tumor cells exhibiting rhabdoid features had secondarily arisen from the peripheral area presenting an appearance of diffuse astrocytoma.

In vitro and in vivo growth inhibition of human malignant astrocytoma cells by the farnesyltransferase inhibitor B1620.

Abstractp21-Ras, the protein product of the proto-oncogene Ras is overactivated in malignant astrocytomas despite the absence of mutation. It is known that p21-Ras participates in signaling events from membrane tyrosine kinase receptors and a variety of intracellular biochemical pathways to downstream targets. Signal transduction inhibition by targeting against Ras is now thought to be a promising therapeutic strategy for malignant astrocytomas. This study demonstrates that Ras pathway inactivation by a farnesyltransferase inhibitor, B1620, effectively inhibits in vitro and in vivo growth of human astrocytoma cells, although normal human astrocytes (NHA) derived from fetal brain are resistant to B1620. Anti-proliferative effect of B1620 on in vitro growth of astrocytoma cells was examined by MTT assays and soft agar colony formation assay. B1620 inhibited anchorage-dependent growth of six astrocytoma cell lines with a median effective dose (IC50) ranging from 2.0 to 20.7µM. However, growth of NHA was not significantly affected by B1620 even at the concentration of 100µM. All astrocytoma cells showed apoptotic figures after Hoechst 33258 staining, when treated for 5 days at each IC50 concentration against B1620. Anchorage-independent growth of these astrocytoma cell lines was inhibited at a much lower concentration than that of anchorage-dependent growth. Daily treatment of U87 xenograft-bearing athymic mice with B1620 at 100 or 50mgkg−1 resulted in significant inhibition of tumor growth. A histological study of the B1620-treated tumor tissue showed decreased vascularity with numerous TUNEL-positive apoptotic cells. These results suggest that the mechanism of the growth-inhibitory effect of B1620 is anti-angiogenesis, apoptosis induction and reversion of the transformed phenotype. The potential clinical use of B1620 could be expanded to malignant astrocytomas.

Effect of immunity on gene delivery into anterior horn motor neurons by live attenuated herpes simplex virus vector.

Efficient and prolonged foreign gene expression has been demonstrated in the bilateral anterior horn motor neurons of the spinal cord by intramuscular inoculation with attenuated herpes simplex virus (HSV) expressing latency associated transcript promoter-driven β-galactosidase (βH1). To examine the effect of immunity on the gene delivery, βH1 was applied in rats immunized subcutaneously or intramuscularly with the parent HF strain. Rats were immunized subcutaneously with HF strain and 28 days later when the high antibody titer was maintained, βH1 was inoculated into the right gastrocnemius muscle. Second, 35 days after inoculation with HF strain into the right gastrocnemius muscle, βH1 was inoculated at the same site. In both ways of immunization, immunity did not abolish or prevent the transgene expression in the anterior horn motor neurons, but attenuated the range and the number of the β-galactosidase-positive neurons from about 85% to 50–65% on 28 days after inoculation with βH1. However, β-galactosidase activity was observed in a wide range of the bilateral anterior horn motor neurons without significant pathological changes. These findings support the feasibility of the attenuated HSV vector in gene delivery into the central nervous system, even in the presence of immunity.

An operating microscope with higher magnification and higher resolution for cerebral aneurysm surgery: preliminary experience-technical note.

We describe a higher magnifying power operating microscope system to improve one method of high-quality microsurgical clipping for cerebral aneurysm in some cases. This higher magnification is achieved by a new lens design in the optical system, which makes the image of the object very clear at high magnifications (distinctiveness of 7 μm). This higher-resolution operating microscope system provides the surgeon with higher-magnified images (at the maximum of more than 30× magnifications as each working distance) in the operating field. The magnifications can be changed from low power (2.9×) to high power (62.0×) depending on the circumstances in a given procedure. We have used this operating microscope system on 11 patients with microsurgical clipping for cerebral aneurysms. Microsurgical treatment could be performed safely and precisely. All aneurysms were treated without any technical complications. We think that the use of this microscope would have potential benefits for microsurgical treatment for cerebral aneurysms because of better visualization.

Microvascular anastomosis at 30-50× magnifications (super-microvascular anastomosis) in neurosurgery.

Background: We report a safe and precise technique of microvascular anastomosis at higher magnifications (30 – 50 ×) in neurosurgery and evaluate our experiences to examine the utility of this method for cerebral revascularization in various situations. Methods: A retrospective review was carried out of patients who underwent microvascular anastomosis using a high-magnified operating microscope. This method was performed in 30 patients with 35 microvascular anastomoses in various situations. This microscope has two optical systems, a standard zooming system and a newly developed high magnification system. High resolution and good depth of focus are achieved by a new lens design in the optical system, which makes the image of the object very clear at higher magnifications. In this operating microscope, the combination of a 10 × eyepiece and the 200, 250, and 300-mm objective lens enables a range of final magnifications from 2.9 × to 50.4 ×. Results: This method enabled one to pay attention to performing atraumatic manipulations of small vessels and correct suturing, intima-to-intima, of vessel walls. Microvascular anastomoses were performed safely and precisely at higher magnifications. All anastomoses were patent. Conclusion: It is obvious that practical final magnifications of more than 30 × in neurosurgery would be super-magnified operative views. Microvascular anastomosis at 30 – 50 × magnifications (super-microvascular anastomosis) can help neurosurgeons to improve their skills, with good visualization, and to be safe and accurate when conducting cerebral revascularization in various situations.

Herpes simplex virus-induced, death receptor-dependent apoptosis and regression of transplanted human cancers.

Inoculation of a live attenuated herpes simplex virus (HSV) vector, bH1, into human U87MG glioblastoma cells transplanted into athymic nude mice induced complete regression of tumors. The infected cells underwent histochemically confirmed apoptosis without lymphocyte infiltration after expressing CD30, CD30 ligand (CD30L), tumor necrosis factor (TNF)‐a, TNF receptor 1 (TNF‐R1), FAS, and FAS ligand (FAS‐L) with activation of caspases 3 and 8. Induction of the transcripts of these receptors and ligands in inoculated tumors was confirmed by quantitative RT‐PCR. To examine the specificity of apoptosis in the transplanted tumor, we inoculated bH1 into transplanted human lung, breast, gastric, and colon cancer tumors, and similar tumor regression with apoptosis was observed in all tumors. We analyzed the roles of expression of CD30, CD30L, TNF‐a, TNF‐R1, FAS, and FAS‐L in the tumors, and found that HSV‐induced apoptosis was suppressed by the respective antibodies. These findings indicate that the CD30/CD30L, TNF‐a/TNF‐R1, and FAS/FAS‐L interactions resulted in apoptosis and tumor regression in immunocompromised mice. In addition to the death receptor‐dependent apoptosis induced by HSV, the expressed ligands and receptors might enhance the susceptibility of tumor cells to cell‐mediated cyto‐toxicity and augment the activation of tumor‐killing lymphocytes in immunocompetent models.

Malignant transformation of oligoastrocytoma: a case report

We report a case of oligoastrocytoma resembling dysembryoplastic neuroepithelial tumor (DNT) with malignant transformation. A 35-year-old woman presented with headache and generalized convulsion in May 2003. Magnetic resonance imaging (MRI) revealed an extensive left temporal lobe tumor. She underwent partial resection of the tumor under awake surgery, while preserving her language function. The surgical specimen showed that the majority of the tumor was composed of a glioneuronal element. However, there was also an abundant oligoastrocytoma component. Therefore, our first pathological diagnosis was oligoastrocytoma and DNT. She then underwent radiation therapy. The tumor recurred at the left temporal lobe in June 2005. She then underwent open biopsy. The pathological diagnosis was anaplastic oligoastrocytoma with a MIB-1 staining index of 79%. She received PAV (procarvazine, ACNU, and vincristine) chemotherapy, and the tumor subsided transiently. However, she died 3 years after the first operation. Although the histological findings of the first surgical specimen closely resembled those of DNT, radiologic findings and clinical course were different from those of DNT. The authors concluded that this tumor could be a malignant transformation of oligoastrocytoma mimicking DNT, and we wish to give warning that the presence of a glioneuronal component is not an absolute benign hallmark.