Yoshifumi Tsuboi

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.

Thrombin-induced cell proliferation and platelet-derived growth factor-AB release from A172 human glioblastoma cells

Background: In a previous study, we found that thrombin induced proliferation of TM‐1 and T98G human glioma cells and that the mitogenic effect was abolished by hirudin. Objectives: We investigated thrombin’s effects on the proliferation of A172 human glioblastoma cells and the induction of growth factors. Furthermore, we examined whether or not the expression of heparin cofactor II (HCII) in A172 cells using adenovirus vector could suppress thrombin’s effects. Methods: The effect of thrombin on cell proliferation was assessed using a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide assay. The amount of growth factors in the conditioned medium was measured by enzyme‐linked immunosorbent assay. The level of platelet‐derived growth factor (PDGF)‐B mRNA was assessed by reverse transcriptase‐polymerase chain reaction analysis. Results: Thrombin‐induced proliferation of A172 cells primarily depended on the enhanced secretion of PDGF‐AB by thrombin. The action of thrombin depended on its proteolytic activity. However, thrombin‐induced PDGF‐AB secretion was not abolished by anti‐protease‐activated receptor (PAR) antibody. The PAR‐1 agonist peptide had no effect on cell growth and PDGF‐AB levels. Thrombin did not increase PDGF‐B gene expression. Expression of HCII effectively suppressed thrombin‐induced PDGF‐AB release. Conclusions: These results indicate that thrombin may play an important role in the proliferation of A172 cells by inducing PDGF‐AB secretion and that thrombin’s action is mediated by its proteolytic activity. Inhibition of thrombin’s proteolytic activity may be a new therapeutic method for gliomas.

Malignant transformation of supratentorial clear cell ependymoma

Recurrence of clear cell ependymoma is not a rare condition, but malignant transformation of clear cell ependymoma has not yet been well presented. The authors report a 44‐year‐old man who presented with progressive right hemiparesis. A brain tumor in the left frontal premotor area was removed and an initial pathological diagnosis of oligodendroglioma was made. The tumor recurred 4 months later, and reoperation of the tumor and adjuvant local radiotherapy were performed. The patient subsequently underwent surgical removal of recurrent tumors on another four occasions (6 times in total) during a period of 11 years and finally died of the original disease. Histopathological studies of all surgical and autopsy specimens were carried out. The first and second surgical specimens did not contain any ependymal rosettes or pseudorosettes, and thus a diagnosis of oligodendroglioma was made. However, the third surgical specimen showed pseudorosettes. At this time, the tumor had an ultrastructural appearance compatible with ependymoma. Thereafter, the recurrent tumors showed anaplastic features such as nuclear pleomorphisms and necrosis with pseudopallisading. The autopsy specimens resembled a feature of glioblastoma but the tumor was sharply demarcated from the surrounding parenchyma.

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.