| Gliomas are the most frequent tumors in the central nervous system accounting for about 50% all primary brain tumors. Malignant gliomas are characterized by a high proliferation rate, marked local invasion and extensive angiogenesis. Combating glioma remains a major clinical challenge, and thus far, the existing therapeutic protocols have proven disappointing. Novel approach has been needed to resolve the task.Pigment epithelium-derived factor (PEDF) was first identified by Tombran-Tink in conditioned medium of cultured fetal human retinal pigment epithelium(RPE) cells. It is a 50 KD approximately glycoprotein and a member of the serine protease inhibitor gene family. PEDF gene is expressed in different fetal and adult tissues, including neurons, non-neurons and virtually all areas of the human brain.It was initially confirmed as a potential neurotrophic factor that induced neuritic outgrown from retinoblastoma cells and promoted the survival of cerebellar granule cells, spinal motor neurons, and developing primary hippocampal neurons exposed to neurotoxins. IN addition to its neurotrophic activity on the nervous system and retina, PEDF was recently shown to have potent anti-angiogenic activity as it specifically inhibited the migration of vessel endothelial cells in vitro and in vivo, an essential step in angiogenesis, and was more effective than the angiogenesis inhibitors: angiostatin, thrombospondin-1, and endostatin with the effective concentration of 0.4 nm. The result of these studies placed PEDF among the most potent endogenous inhibitors of angiogenesis.What the most interested us is: PEDF can completely blocks granulocyte-macrophage colony stimulation factor stimulated cell division of microglia, which is called "gliastatic" effect. The gliastatic" effect was beneficial to the therapy of central nervous system (CNS).Some researchers found that the expression of PEDF was decreased in the thoracic malignancies and oral squamous cell carcinoma; Abramson reported that the high level of PEDF in normal kidneys contrasted with the lower anti-angiogenic activity of PEDF in Wilms. The tumor sizes reduced by 60% treated with PEDF compared with the control group. The date of Doll indicated that androgen inhibited the PEDF expression of prostate, PEDF induced the endothelium cells to apoptosisand inhibited the growth of tumor by triggering endothelial apoptosis of prostate.In view of the neurotrophic, neuroprotective, anti-angiogeni, unique "gliastatic" and apoptosis-induced effect of PEDF, we considered that it will act as a potential therapeutic agent against diseases of the CNS, especially gliomas. PEDF, as a potent and ideal factor, could be used to protect brain cells and decline the growth of tumor cells as well as to inhibit the relapse and metastasis of tumor combined with surgery. But so far, there are no study about the role of PEDF on the glioma has been performed. In this paper, we obtained the biological activity recombinant PEDF (rPEDF) and explored whether gliomas angiogenesis and tumor growth could be inhibited by rPEDF in vitro and in vivo in order to provide a novel approach of developing anti-angiogenic therapies to treat gliomas. Part I. Production of active pigment epithelium-derived factor in E.coliMost of the PEDF was prepared from medium cultured by human RPE cell line that was restricted by both the low abundance and the rare availability of its source. Becerra et al. have tried to express PEDF in E.coli, however, in the form of inclusion bodies. Therefore a bacterial expression vector, pET41(a), was selected to over-express the rPEDF. Compared with the expression rates in the inclusion bodies, we found a higher rates of soluble protein using GST fusion. Approximately 90% of the rPEDF fusion protein was expressed at a high level as a soluble protein. The yield of purified GST fusion protein was 14 mg/L culture, and the final yield of purified rPEDF was 2.1 times higher than in the inclusion bodies. Most importantly, the Human umbilical vein endothelial cells (HUVECS) treated with rPEDF formed small clumps and few intact tubes were observed. In the absence of rPEDF, the HUVECS were observed to be in the characteristic tube formation of vascular endothelial cells. The active rPEDF laid a fundation for the further research of inhibitory effect on gliomas in vitro and in vivo. Part II .The impact of rPEDF on the growth and invasion of the glioma cellsThe progressive growth of glioblastoma is thought to be dependent on angiogenesis. It is believed that angiogenic homeostasis is controlled by the balance between angiogenic stimulators and angiogenic inhibitors. But so far, no study evaluating the role of PEDF on glomas has been performed. ELISA was used to evaluate whether rPEDF would affect the production of positive and negative factors of angiogenesis. Our data demonstrated that U87pedf and A172pedf cells exhibited decreased expression levels of VEGF and increased the expression of TSP-1. Bothchanges were essential and, together, were sufficient to shift the angiogenic phenotype of glioma cells to anti-angiogenic. Cell proliferation assays were performed by MTT assay. PEDF inhibited glioma cells proliferation at initial concentration (25 ng/ml), and the inhibitory rates were 54.11% and 49.76% in U87 and A172 cells, respectively. These results indicated that rPEDF blocked the proliferation of glioma cells in a dose-dependent manner.Local invasiveness is an important characteristic of gliomas and it contributes substantially to the failure of curative treatments of disease. Both the cell migratory and cell invasion assay showed that the percentage of migratory cells was found higher for U87con cells than that of U87Pedf- Similar results were obtained with A172 cells. When added the VEGF to the U87pedf and A172pedf media, the migration was blocked effectively compared with control cells(P<0.05). These results suggested that rPEDF could decrease the invasive and migratory capacity of the glioma cells even in the presence of VEGF.Part III. The research about the mechanism of rPEDF on the growth and invasion of glioma cellWe have reported the rPEDF has the potent inhibitory capacity of growth and migration on glioma cells, but the mechanistic basis for the effect of induction of rPEDF in glioma cells, are questions and remain unclear. To evaluate whether the growth inhibition of rPEDF was associated with apoptosis, we used TUNEL and flow cytometer to examine U87 and A172 cells. The apoptotic cells were higher for U87PEDF and A172PEDF than that of control cells by TUNEL. On flow-cytometric analysis, both cells showed 21.84% and 20.78% of Pl-negative/annexin V-positive population indicating the glioma cells went through the early stage of apoptosis. Apoptosis was further confirmed on the significant increase of the sub-Gl peak of DNA content. Treated with rPEDF In U87 and A172, significantly increased the distribution of cells in the Go/Gi phase, with a concomitant decrease in the S phase, suggesting Go/Gi arrest of the cells. Meanwhile, we found that rPEDF induced apoptosis was associated with the increases of p53, Bax and inhibition of Bcl-2 using real-time RT-PCR and western-blot. Apoptosis of endothelial cells in vascularized tumors would lead to a reduction of the number of vessels supporting tumor cell growth, this could explain the significantly reduced tumor growth rates observed in our experiments.Evidence that Laminin-8 and MMP-9 are localized to the glioma invasion has...
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