br Acknowledgments The authors thank Claudia Wittrock for

Acknowledgments The authors thank Claudia Wittrock for her excellent technical assistance. The work was supported by grants of the Deutsche Forschungsgemeinschaft (SFB974 TP B05) and of the Anton-Betz-Stiftung to N.K. A.M. is an associate member of the iBrain graduate school at the University of Düsseldorf.
Introduction Photodynamic therapy (PDT) involves selective uptake of a photosensitizer (PS) by tumor cells and irradiation of tumor with appropriate light wavelength to activate the PS, which generates reactive oxygen species (ROS) and leads to cytotoxicity (Robertson et al., 2009). Over decades of improvement on PS and light source (radiator), PDT has been applied clinically in several diseases, including certain dermatosis and some sorts of malignant tumors located in lung, ovary, brain, and so on (Bredell et al., 2010, Song et al., 2005, Zhao and He, 2010). We have investigated the effects of PDT on intracranially implanted VX2 tumors in rabbits (Xiao et al., 2009) and C6 glioma in rats (data published in Chinese), and found that PDT was useful in prolonging the survival duration of tumor-burdened animals. Glioma cells release glutamate during growth, which produces a high concentration of extracellular glutamate microenvironment, and causes excitotoxicity to the surrounding neurons by Ca2+ influx through N-methyl-d-aspartate receptor (NMDAR) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic PR619 glutamate receptor (AMPAR). But glioma cells itself escape the excitotoxicity under physiological conditions. Furthermore, the proliferation, migration and invasion of glioma cells require this specific microenvironment of high concentration of extracellular glutamate (Lyons et al., 2007, Roesler et al., 2010). However, the role of glutamate and its receptors in the photodynamic therapy on glioma cells is still unclear. Thus, the purpose of this study is to prospectively evaluate the influence of PDT on C6 glioma cells in vitro and the role of glutamate and its receptors AMPAR during PDT. This study indicates that PDT may kill C6 glioma cells partly through glutamate and its receptors AMPAR, which induces Ca2+ influx and then cells apoptosis. These results allow us to further understand the effects and molecular mechanism of PDT on glioma.
Results
Discussion The identification of glutamate as a key factor in microenvironment which promotes tumor growth and invasion has profound implications for current glioma biology study (Lyons et al., 2007, Roesler et al., 2010, Vanhoutte and Hermans, 2008). Glioma cells regulate the neurotransmitter glutamate as well as a variety of cytokines and growth factors (Sontheimer, 2003, Sontheimer, 2008). Here we observed changes of glutamate release after PDT and applied interventions that targeted glutamate receptors during PDT. Our data showed that the concentration of extracellular glutamate was significantly increased after PDT in the culture medium. We also observed increased expression of AMPAR subunits, which indicated AMPA receptors might increase on the membrane of C6 glioma cells. Moreover, CNQX, an AMPAR antagonist, decreased [Ca2+]i and apoptosis of C6 glioma cells. These results indicate that PDT may lead to death of C6 glioma cells partly through glutamate and its receptors AMPA, which induces Ca2+ influx and then cells apoptosis. We found that the extracellular glutamate in groups treated with PDT was three times higher than that in the groups without PDT at 1h after PDT. In physiological conditions, glutamate is transferred by the glutamate/cystine antiporter (Xc− system), which exchanges one molecule of intracellular glutamate with one molecule of extracellular cystine to counteract oxidative stress, including ROS arising from the increased metabolic rate of the tumor. In general, the high concentration of extracellular glutamate results from reduced glutamate uptake and/or increased glutamate release by glioma cells (de Groot et al., 2005, Sontheimer, 2008). PDT, which generates ROS to kill cells, also enhances the function of Xc− system. Thus, extracellular glutamate increased significantly. However, other specific mechanism of increased extracellular glutamate during PDT is still unknown which needs to be investigated.