Factors Affect Glioma Invasion And The Role Of Direct Current Electric Field In Directional Migration Of U87 Glioma Cell~*

Background:Gliomas are the most common primary brain tumor. The feature of malignant gliomas is the invasion of tumor cells in surrounding normal brain tissue and recurrence after surgery. It is still the biggest challenge to neurosurgery. The factors affect the invasion of glioma including the intrinsic biological features of glioma, the vascularization, the microenvironments, and the interaction between the tumor and the immune system of the host. Although the high invasive features of gloma have been widely accepted, there have no effective specific anti-invasive therapies for glioma. Except the mono-antibody for EGFR, other inhibitors against intergrins, MMPs and tyrosine kinases have not shown therapeutic effects. The invasive mechanisms of malignant gliomas need to be widely studied.The glioma cells invade 3⁵ cm away form the primary tumor at the same time the formation of the tumors. Many factors, such as the hypoxia, mechanical forces, mass effects, chemotaxi and the stimulation of surgery, can"push"glioma cells separate from the tumor and invade into the surrounding brain. If there is a method can overrided these forces and"pull"the invaded cells back to the tumor for more radical treatment? These new stratages maybe improve the outcome of patients especially in the condition the functional area of brain has been affected. The tumor would be resected more radically and more functions would be kept if the invaded tumor cells"withdraw"from these areas before surgery or radiotherapy.In order to know more about the mechanisms of glioma invation and to explore new stratagies for glioma treatment, the role of Caveolin-1 (Cav1), reactive oxygen species (ROS) and the immune status of host in the growth and invasion of glioma were studied, the devices for in vitro cell biological behavor observasion under direct current electric field (DCEF) were established, and the role of DCEF in the migration of U87 glioma was observed. Moreover, the mechanisms of DCEF induced directional migration of U87 glioma were discussed.Materials and methods:(1) The expression and distribution of Cav1 were analysed by immunochemical histology and PR-PCR in 27 patients with glioma. The relationship between the expression of Cav1 and the prognosis of patients were analysed by progression free survival follow-up.(2) The down regulation of Cav1 was gained by using of methyl-β-cyclodextrin (MβCD) or siRNA in C6 rat glioma; the ability of in vitro migration and invasion of C6 gliomas were detected by Wound Healing and Transwell; and the protein level of Cav1, EGFR and Pyk2 were measured by Western-blot.(3) The down- or up-regulation of Cav1 in human U87 glioma were gained by using the CMV infection; the cell cycles were analysed by flow cytometry and the growth were measured by MTT; the ability of in vitro migration and invasion of U87 gliomas were detected by Wound Healing and Transwell.(4) Over expression manganese superoxide dismutase (MnSOD) to 3.5 folds in U87 glioma by plasmid transfection, the ROS was detected by dying with DCFH-DA or HE. N-acetyl-L-cysteine (NAC) or over expression of mCatlase were used to decrease the intracellular hydrogen peroxide; the ability of in vitro migration and invasion of U87 gliomas were detected by Wound Healing and Transwell; the levels of total as well as the phosphorylated Akt, s6-ribosomal protein, Erk1/2 and JNK were measured by Western-blot; the activation of Akt and Erk1/2 were inhibited by their specific inhibitors of LY294002 or PD98059 respectively.(5) G422 mice glioma was implanted into the brain of BALB/c mice (immunocompetent mice), nude mice (T cells-related immunodeficient mice) and complement C3 knockout mice (complement C3- related immunodeficient mice). The survival time of mice and the growth of tumors were compared. The proportion of CD68⁺ lymphocyte in the tumor tissue were analysed by flow cytometery and the TNF-αand IFN-γwere measured by ELISA. (6) The devices for realtime in vitro cell biological behavor observasion under direct current electric field (DCEF) were established.(7) Migration of U87 glioma in the DCEF was observed under realtime microscopy; the levels of total and phosphorylated 60 proteins related to the motivation of cell were analysed by antibody array; the ROS was detected by dying with DCFH-DA or HE; NAC or over expression of MnSOD and/or mCatlase were used to decrease the intracellular superoxide and/or hydrogen peroxide; the levels of total as well as the phosphorylated Akt, Erk1/2, JNK and p38 were measured by Western-blot.Results:1,The role of Cav1, ROS and immune status in the growth and invasion of glioma(1) The levels of the protein and mRNA of Cav1 were low in the normal brain and WHOâ… grade gliomas. It showed quite differents in WHOâ…¡grade gliomas but were increased significantly in WHOâ…¢andâ…£grade gliomas. The expression of Cav1 was much higher in the patients with≥10/HF Ki67 positive cells than <10 /HF. No mutations of Cav1 cDNA were found in 27 cases of glioma patients. The mean PFS of patients with low Cav1 was 28.40±9.49 m with a median PFS of 30 m, previously higher than patients of higer Cav1 with mean PFS of 9.08±7.01 m and median PFS of 7 m.(2) The abilities of in vitro migration and invasion of C6 glioma were reduced with the down regulation of Cav1 by using MβCD or siRNA. The growth of U87 gloma cells was not affected by up- or down-regulation of Cav1. In the condition of 10% FBS, the abilities of in vitro migration and invasion of U87 glioma were promoted by down-regulation of Cav1 but reduced by up-regulation of Cav1. In the contrast, in the condition of 0.1% FBS, the abilities of in vitro migration and invasion of U87 glioma were reduced by down-regulation of Cav1 but promoted by up-regulation of Cav1.(3) The elevation of hydrogen peroxide (H2O2) level and the enhancement of glioma migration/invasion by over expression of MnSOD were demonstrated. Over-expression of MnSOD significantly increased activation of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinases (PI3Ks), including Akts, s6-ribosomal protein, Erks and JNKs. Over-expression of MnSOD was also associated with elevations of matrix metalloproteinases-1 (MMP-1) and MMP-9 protein. The promotion of migration/invasion, activation of PI3Ks, MAPKs and up-regulation of MMPs were inhibited by general reactive oxygen species scavenger N-acetyl-l-cysteine (NAC), over-expressing of the H2O2-detoxifying enzyme mitochondrial catalase (mCAT) and specific inhibitors of LY294002 for Akt or PD98059 for Erk.(4) G422 mice glioma was implanted into the brain of BALB/c mice (immunocompetent mice), nude mice (T cells-related immunodeficient mice) and complement C3 knockout mice (complement C3- related immunodeficient mice). The growh of tumor was slowest in BALB/c mice, followed by nude mice and fastest in complement C3 knockout mice. the survival time of the host was longest (44.3±6.0 d), the tumor growh was faster in nude mice and the survival time was shorter (24.8±5.2 d), the tumor growh was fastest in complement C3 knockout mice and the survival time was the shortest one (18.6±5.8 d). Althoug the invasive CD₆₈⁺ lymphocyte in the tumors were similar in the three kinds of mice, the concentrations of TNF-αin nude mice (28.11±4.86μmol/L) and complement C3 knockout mice (22.87±6.36μmol/L) were much lower than that in BALB/c mice (230.21±39.17μmol/L). The concentrations of IFN-γwas highest in BALB/c mice (180.76±29.19μmol/L), followed by which in nude mice (113.46±23.76μmol/L) and lowest in complement C3 knockout mice (16.84±4.45μmol/L).2,The role of DCEF in the directional migration of U87 glioma cells(1) The devices established for this experiment including the glass clips, wells, salt bridges et al. could meet the needs for realtime in vitro cell biological behavor observasion under direct current electric field.(2) In the absence of an applied direct current electric field, glioma cells migrated with equal probability in all directions, with an average cosineθof 0.16±0.02. In contrast, a 200mV/cm DCEF significantly directed the migration of glioma cells toward the cathode, and the average cosineθwas 0.72±0.12. The arrangement of U87 gloma cells was changed by DCEF.(3) The Antibody array for detection of the levels of total and phosphorylated 60 proteins relating to the motivation of cell showed that the phosphorylation of 54 proteins were increased after the using of electrical field with a strength of 200mV/cm for 30 min, especially the proteins related to the PI3K and MAPK pathways.(4) Electrical field with a strength of 200mV/cm significantly increased DCFH and HE fluorescence in 15 minutes, indicative of increased hydrogen peroxide and superoxide generation. The higher electric field induced increasing of DCFH and HE fluorescence. Pre-treating cells with NAC significantly inhibited or abolished both of the hydrogen peroxide and superoxide generation detected by DCFH or HE respectively. The directional cathode migration of U87 glioma cells were also abolished by the application of NAC without obvious influence of the speed of cell migration. The electric field induced generation of hydrogen peroxide was decreased by over expression of Catalase and the generation of superoxide was totally abolished by the over expression of MnSOD. However, the directional migration of U87 glioma was totally abolished by over expression of MnSOD but not by Catalase.(5) The DCEF (200mV/cm) significantly increased overall activation of p-Akt, p-Erk 1/2, p-JNK and p-p38 in 30 min. The higher electric field strength also caused more phosphorylation of these proteins. The presence of an antioxidant NAC or over expression of MnSOD could almost completely erase the activation of Akt, Erk1/2, JNK and p38. Although the activation of JNK and p38 was inhibited by over expression of Catalase, but the activation of Akt and Erk1/2 was not affected.(6) The DCEF (200mV/cm) could induce the polarization of intracellular p-Erk 1/2 and Cav1 to the cathode, but had no affect on the distribution of total Erk1/2.Conclusions:In the present work, the role of Cav1, ROS and the immune status of host in the growth and invasion of glioma were studied, the devices for in vitro cell biological behavor observasion under DCEF were established, the role and the mechanisms of DCEF induced directional migration of U87 glioma were discussed.(1) The expression of Cav1 in glioma is related to the prognosis of the patients, but the role of Cav1 in the in vitro migration and invasion of glioma cells is affected by the culture environment of the cells.(2) The H2O2 would contribute to the MnSOD-promoted migration/invasion in glioma cells through activating of Akts and Erks.(3) The immune status of host could affect the growth of G422 glioma because of the different ability for TNF-αand IFN-γreleasing.(4) The devices established for this experiment could meet the needs for realtime in vitro cell biological behavor observasion under direct current electric field. (5) DCEF could induce the directional migration of U87 glioma to the cathode and the production of hydrogen peroxide and superoxide. The superoxide play critical role in DCEF induced directional migration of glioma cell through regulation of the activation of Akt and Erk1/2.This study provided novel insights into the understanding the role of Cav1, ROS, DCEF and immune status in the growth and invasion of the glioma. The ability of DCEF to induce the directional migration of glioma cells was clearly demonstrated and the mechanisms were discussed. The presented study offered further theoretical basis for setting DCEF as a potential new utilization for glioma therapy to"pull"the invaded cells back to the tumor for more radical treatment.