In our study ERK and JNK inhibitors

In our study, ERK and JNK inhibitors effectively inhibited the migration of glioma dacomitinib while the effect of the p38 inhibitor was not as significant. Hence, we further evaluated the effect of downregulation of uPAR and cathepsin B on the expression of ERK and JNK. Reduction in the expression levels of p-ERK with pUC and pUC+10Gy and the inhibition of migration of the cells with ERK inhibitor indicated that the activation of ERK is necessary for cell migration (Lind et al., 2006; Nguyen et al., 1999). shRNA treatment and the inhibitor treatment with respect to the activation of JNK and migration of glioma cells seemed to be contradictory. These phenomena prompted us to further concentrate on the involvement of JNK in the migration of glioma cells. Based on the stimuli, compartment-specific localization and activation of JNK have been demonstrated in several earlier reports (Bjorkblom et al., 2008; Coffey et al., 2000). In our study, pUC treatment alone and in combination with radiation induced the activation and translocation of JNK into the nucleus of non-GICs and GICs. Even though a minor pool of nuclear activity existed in the irradiated cells, the majority of p-JNK remained in their cytoplasmic compartments. This means that the pUC-induced translocation of p-JNK into the nucleus might impair its ability to influence the migration of cells. Overexpressing uPAR and cathepsin B by transfecting the cells with FLU and FLC induced an increase in the accumulation of p-JNK in the cytoplasm of the cells. Overexpression of uPAR and cathepsin B did not show any effect on the nuclear pools of p-JNK. Also an increase in the migration of the glioma non-GICs and GICs was observed after FLU and FLC treatments. Taken together, these results further provide evidence that uPAR and cathepsin B-mediated activation of cytoplasmic JNK is required for the migration of the cells whereas the nuclear pool of active JNK cannot impact migration. Previously, it was reported that the activation of cytoplasmic pool of JNK was required for the migration of NRK cells (Rosse et al., 2009) and dendritic cells (Bjorkblom et al., 2005). The dynamic assembly and disassembly of focal adhesions play central roles in cell migration. Adapter proteins such as Paxillin, Vinculin, α-Actinin and Talin are very important for the formation of these focal adhesion complexes at the leading edge of the migrating cell (Huttenlocher and Horwitz, 2011; Vicente-Manzanares et al., 2009). In our present study, the non-GICs and GICs treated with pUC and pUC+10Gy showed a significant decrease in the expression of the adhesion machinery molecules p-Paxillin, Vinculin, α-Actinin, Talin, Integrin αvβ3 and Integrin β1. Along with various well-known transcription factors and apoptosis-related proteins that are substrates for JNK, several cytoskeleton-associated proteins and signaling molecules as well as adaptor proteins have recently been identified (Bogoyevitch and Kobe, 2006; Huang et al., 2004b). Radiation, FLU and FLC treatments increased the expression of the above mentioned adhesion molecules, which were inhibited by treating the cells with the JNK inhibitor. This result further confirms that uPAR and cathepsin B regulate the migration and adhesion of glioma cells through the activation of cytoplasmic JNK. Localized activation of JNK at the leading edge of migrating NRK cells (Rosse et al., 2009) and the localization of JNK to the actin dense membrane ruffles of the migrating fibroblast cells (Amagasaki et al., 2006) were observed earlier. In our present study, p-JNK interacted with p-Paxillin at the leading edge of the migrating glioma cells. Along with p-Paxillin, p-JNK directly interacted with Vinculin as well as α-Actinin. The direct interaction between these molecules was inhibited by treating the cells with pUC alone or in combination with radiation. This further provides evidence for uPAR and cathepsin B-mediated regulation of p-JNK and its interaction with the focal adhesion molecules required for glioma cell migration. Inhibition of co-localization of p-JNK and p-Paxillin was also evident in the in vivo sections treated with pUC alone or in combination with radiation and led to the regression of tumor growth.