Yukari Yoshida

Blockage of Ca 2+ -permeable AMPA receptors suppresses migration and induces apoptosis in human glioblastoma cells

Glioblastoma multiforme is the most undifferentiated type of brain tumor, and its prognosis is extremely poor. Glioblastoma cells exhibit highly migratory and invasive behavior, which makes surgical intervention unsuccessful. Here, we showed that glioblastoma cells express Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors assembled from the GluR1 and/or GluR4 subunits, and that their conversion to Ca2+-impermeable receptors by adenovirus-mediated transfer of the GluR2 cDNA inhibited cell locomotion and induced apoptosis. In contrast, overexpression of Ca2+-permeable AMPA receptors facilitated migration and proliferation of the tumor cells. These findings indicate that Ca2+-permeable AMPA receptors have crucial roles in growth of glioblastoma. Blockage of these Ca2+-permeable receptors may be a useful therapeutic strategy for the prevention of glioblastoma invasion.

Ca2+-Permeable AMPA Receptors Regulate Growth of Human Glioblastoma via Akt Activation

Evidence has been accumulated that glioblastoma cells release and exploit glutamate for proliferation and migration by autocrine or paracrine loops through Ca2+-permeable AMPA-type glutamate receptors. Here, we show that Ca2+ signaling mediated by AMPA receptor regulates the growth and motility of glioblastoma cells via activation of Akt. Ca2+ supplied through Ca2+-permeable AMPA receptor phosphorylated Akt at Ser-473, thereby facilitating proliferation and mobility. A dominant-negative form of Akt inhibited cell proliferation and migration accelerated by overexpression of Ca2+-permeable AMPA receptor. In contrast, introduction of a constitutively active form of Akt rescued tumor cells from apoptosis induced by the conversion of Ca2+-permeable AMPA receptor to Ca2+-impermeable receptors by the delivery of GluR2 cDNA. Therefore, Akt functions as downstream effectors for Ca2+-signaling mediated by AMPA receptor in glioblastoma cells. The activation of the glutamate-AMPA receptor-Akt pathway may contribute to the high degree of anaplasia and invasive growth of human glioblastoma. This novel pathway might give an alternative therapeutic target.

Carbon Ion Radiotherapy at the Gunma University Heavy Ion Medical Center: New Facility Set-up

Carbon ion radiotherapy (C-ion RT) offers superior dose conformity in the treatment of deep-seated tumors compared with conventional X-ray therapy. In addition, carbon ion beams have a higher relative biological effectiveness compared with protons or X-ray beams. C-ion RT for the first patient at Gunma University Heavy Ion Medical Center (GHMC) was initiated in March of 2010. The major specifications of the facility were determined based on the experience of clinical treatments at the National Institute of Radiological Sciences (NIRS), with the size and cost being reduced to one-third of those at NIRS. The currently indicated sites of cancer treatment at GHMC are lung, prostate, head and neck, liver, rectum, bone and soft tissue. Between March 2010 and July 2011, a total of 177 patients were treated at GHMC although a total of 100 patients was the design specification during the period in considering the optimal machine performance. In the present article, we introduce the facility set-up of GHMC, including the facility design, treatment planning systems, and clinical preparations.

Input- and subunit-specific AMPA receptor trafficking underlying long-term potentiation at hippocampal CA3 synapses

Hippocampal CA3 pyramidal neurons receive synaptic inputs from both mossy fibres (MFs) and associational fibres (AFs). Long‐term potentiation (LTP) at these synapses differs in its induction sites and N‐methyl‐D‐aspartate receptor (NMDAR) dependence. Most evidence favours the presynaptic and postsynaptic mechanisms for induction of MF LTP and AF LTP, respectively. This implies that molecular and functional properties differ between MF and AF synapses at both presynaptic and postsynaptic sites. In this study, we focused on the difference in the postsynaptic trafficking of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors (AMPARs) between these synapses. To trace the subunit‐specific trafficking of AMPARs at each synapse, GluR1 and GluR2 subunits were introduced into CA3 pyramidal neurons in hippocampal organotypic cultures using the Sindbis viral expression system. The electrophysiologically‐tagged GluR2 AMPARs, produced by the viral‐mediated transfer of the unedited form of GluR2 (GluR2Q), were inserted into both MF and AF postsynaptic sites in a neuronal activity‐independent manner. Endogenous Ca2+‐impermeable AMPARs at these synapses were replaced with exogenous Ca2+‐permeable receptors, and Ca2+ influx via the newly expressed postsynaptic AMPARs induced NMDAR‐independent LTP at AF synapses. In contrast, no GluR1 AMPAR produced by the gene transfer was constitutively incorporated into AF postsynaptic sites, and only a small amount into MF postsynaptic sites. The synaptic trafficking of GluR1 AMPARs was triggered by the activity of Ca2+/calmodulin‐dependent kinase II or high‐frequency stimulation to induce LTP at AF synapses, but not at MF synapses. These results indicate that MF and AF postsynaptic sites possess distinct properties for AMPAR trafficking in CA3 pyramidal neurons.

Nonhomologous End-Joining Repair Plays a More Important Role than Homologous Recombination Repair in Defining Radiosensitivity after Exposure to High-LET Radiation

DNA double-strand breaks (DSBs) induced by ionizing radiation pose a major threat to cell survival. The cell can respond to the presence of DSBs through two major repair pathways: homologous recombination (HR) and nonhomologous end joining (NHEJ). Higher levels of cell death are induced by high-linear energy transfer (LET) radiation when compared to low-LET radiation, even at the same physical doses, due to less effective and efficient DNA repair. To clarify whether high-LET radiation inhibits all repair pathways or specifically one repair pathway, studies were designed to examine the effects of radiation with different LET values on DNA DSB repair and radiosensitivity. Embryonic fibroblasts bearing repair gene (NHEJ-related Lig4 and/or HR-related Rad54) knockouts (KO) were used and their responses were compared to wild-type cells. The cells were exposed to X rays, spread-out Bragg peak (SOBP) carbon ion beams as well as with carbon, iron, neon and argon ions. Cell survival was measured with colony-forming assays. The sensitization enhancement ratio (SER) values were calculated using the 10% survival dose of wild-type cells and repair-deficient cells. Cellular radiosensitivity was listed in descending order: double-KO cells > Lig4-KO cells > Rad54-KO cells > wild-type cells. Although Rad54-KO cells had an almost constant SER value, Lig4-KO cells showed a high-SER value when compared to Rad54-KO cells, even with increasing LET values. These results suggest that with carbon-ion therapy, targeting NHEJ repair yields higher radiosensitivity than targeting homologous recombination repair.

Reduction of nitric oxide level enhances the radiosensitivity of hypoxic non-small cell lung cancer

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (E‐TKI) resistance has emerged as an important clinical issue. To overcome this resistance, researchers have examined different modalities, either for use as a monotherapy or in combination with E‐TKI therapy. In the present study, we investigated whether a decrease in nitric oxide (NO) levels affects the radiosensitization of non‐small cell lung cancer (NSCLC) cell lines. A549 and H3255 NSCLC cells were examined. They were subjected to hypoxic conditions and monotherapy, or combined therapy using radiation and NG‐monomethyl‐l‐arginine, monoacetate (LNMMA). Reductions in nitric oxide levels enhanced the radiosensitivity of both cell lines and significantly reduced the expression of both hypoxia‐inducible factor‐1α (HIF‐1α) and EGFR in H3255 cells compared to A549 cells. Since NO is significantly associated with cell metabolism, we measured the levels of pyruvate dehydrogenase kinase‐1 (PDK‐1), reactive oxygen species, and oxygen and observed that the expression of PDK‐1 was significantly reduced. This reduction was seen simultaneously after the silencing of HIF‐1α; however, not following LNMMA treatment. The oxygen concentration was significantly increased in the treated cells, and their viability decreased in parallel. Reactive oxygen species were decreased after LNMMA and radiation treatment. Adding EGFR–TKI to cells with reduced NO levels further suppressed cell viability when combined with radiation. This study suggests that a reduction in the NO level might substantially overcome the radioresistance of mutant NSCLC cells. (Cancer Sci 2011; 102: 2150–2156)

Serum‐dependence of AMPA receptor‐mediated proliferation in glioma cells

Glutamate may cause Ca2+ entry through Ca2+‐permeable glutamate receptors, which in turn stimulates the anti‐apoptotic signaling cascade in glioma cells. It was found that a human glioma cell line, U‐87 MG, expressed subunits of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate acid‐type glutamate receptors (AMPAR). Ca2+ entry through AMPAR was detected in approximately 40% of U‐87 MG cells. AMPAR agonists facilitated cell proliferation in low‐serum medium containing 0.5% fetal calf serum (FCS). Unexpectedly, cell proliferation by the activation of AMPAR was not detected in serum‐rich medium containing 10% FCS. Overexpression of Ca2+‐permeable AMPAR facilitated proliferation of U‐87 MG cells in the low‐serum condition, whereas it had again no effect in the serum‐rich condition. Cell proliferation of U‐87 MG cells is likely to be under the regulation of both growth factors contained in the serum and Ca2+ entry through AMPAR, and that the latter regulation becomes evident only when serum factors are deprived of culture medium.

Carbon-ion beam irradiation kills X-ray-resistant p53-null cancer cells by inducing mitotic catastrophe.

Background and Purpose To understand the mechanisms involved in the strong killing effect of carbon-ion beam irradiation on cancer cells with TP53 tumor suppressor gene deficiencies. Materials and Methods DNA damage responses after carbon-ion beam or X-ray irradiation in isogenic HCT116 colorectal cancer cell lines with and without TP53 (p53+/+ and p53-/-, respectively) were analyzed as follows: cell survival by clonogenic assay, cell death modes by morphologic observation of DAPI-stained nuclei, DNA double-strand breaks (DSBs) by immunostaining of phosphorylated H2AX (γH2AX), and cell cycle by flow cytometry and immunostaining of Ser10-phosphorylated histone H3. Results The p53-/- cells were more resistant than the p53+/+ cells to X-ray irradiation, while the sensitivities of the p53+/+ and p53-/- cells to carbon-ion beam irradiation were comparable. X-ray and carbon-ion beam irradiations predominantly induced apoptosis of the p53+/+ cells but not the p53-/- cells. In the p53-/- cells, carbon-ion beam irradiation, but not X-ray irradiation, markedly induced mitotic catastrophe that was associated with premature mitotic entry with harboring long-retained DSBs at 24 h post-irradiation. Conclusions Efficient induction of mitotic catastrophe in apoptosis-resistant p53-deficient cells implies a strong cancer cell-killing effect of carbon-ion beam irradiation that is independent of the p53 status, suggesting its biological advantage over X-ray treatment.

Effects of Charged Particles on Human Tumor Cells

The use of charged particle therapy in cancer treatment is growing rapidly, in large part because the exquisite dose localization of charged particles allows for higher radiation doses to be given to tumor tissue while normal tissues are exposed to lower doses and decreased volumes of normal tissues are irradiated. In addition, charged particles heavier than protons have substantial potential clinical advantages because of their additional biological effects, including greater cell killing effectiveness, decreased radiation resistance of hypoxic cells in tumors, and reduced cell cycle dependence of radiation response. These biological advantages depend on many factors, such as endpoint, cell or tissue type, dose, dose rate or fractionation, charged particle type and energy, and oxygen concentration. This review summarizes the unique biological advantages of charged particle therapy and highlights recent research and areas of particular research needs, such as quantification of relative biological effectiveness (RBE) for various tumor types and radiation qualities, role of genetic background of tumor cells in determining response to charged particles, sensitivity of cancer stem-like cells to charged particles, role of charged particles in tumors with hypoxic fractions, and importance of fractionation, including use of hypofractionation, with charged particles.

The radiobiological effectiveness of carbon-ion beams on growing neurons

Purpose: Recently carbon-ion beams have been reported to be remarkably effective for controlling various cancers with less toxicity and are thought to be a promising modality for cancer treatment. However, the biological effect of carbon-ion beams arising on normal neuron remains unknown. Therefore, this study was undertaken to investigate the effect of carbon-ion beams on neurons by using both morphological and functional assays. Materials and methods: Dorsal root ganglia (DRG) and sympathetic ganglion chains (SYMP) were isolated from day-8 and day-16 chick embryos and cultured for 20 h. Cultured neurons were exposed to carbon-ion beams and X-rays. Morphological changes, apoptosis and cell viability were evaluated with the Growth Cone Collapse (GCC), Terminal deoxynucleotidyl Transferase (TdT)-mediated deoxyUridine TriPhosphate (dUTP) nick End Labeling [TUNEL] assay and 4-[3-(4-iodophenyl)- 2-(4-nitrophenyl)- 2H-5-tetrazolio]- 1,3-benzenedisulfonate [WST-1] assays, respectively. Results: Irradiation caused GCC and neurite destruction on a time- and irradiation dose-dependent manner. Changes in morphological characteristics were similar following either irradiation. Morphological and functional assays showed that day-8 neurons were more radiosensitive than day-16 neurons, whereas, radiosensitivity of DRG was comparable to that of SYMP. The dose-response fitting curve utilising both GCC and TUNEL labeling index showed higher relative biological effectiveness (RBE) values were associated with lower lethal dose (LD) values, while lower RBE was associated with higher LD values. Conclusion: Exposure to high-linear energy transfer (LET) irradiation is up to 3.2 more efficient to induce GCC and apoptosis, in early developed neuronal cells, than low-LET irradiation. GCC is a reliable method to assess the radiobiological response of neurons.