Effects Of Resveratrol On GRP78、ATP5B And VEGF In Human Medulloblastoma Cells:Potential Biological Implications

Background:Medulloblastomas are the most common malignant brain tumors in children and occur under 10 years of age. Medulloblastomas arise in the fourth ventricle of the cerebellum. They dependent on angiogenesis to maintain oxygen and nutrient supply. Tumor vessels are distributed unevenly, disorganized and lack of basement membrane. They have high permeability, thus contributing to the growth of tumor invasion and providing a way for the transfer and spread of the tumor. Currently, multimode treatment including surgery, chemotherapy and radiotherapy has daily matured. However, to achieve radical treatment with a high degree of invasion intracranial tumor is still very difficult. While large doses of radiotherapy and chemotherapy have some toxic side effects, such as cerebellar abnormalities, bone marrow suppression, liver function abnormalities and change in blood, and it has delayed and cumulated. Therefore, It has important clinical significance to explore an effective, reliable and non-toxic side effects’ anti-angiogenic drugs for the prevention and treatment of medulloblastomas. Resveratrol is a polyphenol compound that widely found in more than 70 kinds of plants such as grapes, peanuts, knotweed and so on. With a variety of biological activity, no significant side effects on the body, and strong fat-soluble, low molecular weight, easily through the blood brain barrier, it is thought of a potential drug for the prevention and treatment of cancer. Previous studies showed that resveratrol could inhibit tumor angiogenesis through a variety of channels, thereby inhibit tumor development. Vascular endothelial growth factor (VEGF) is known as a key angiogenic factor, which is over-expressed in a variety of tumors, such as lung cancer, breast cancer, glioblastoma, medulloblastoma and so on. It is closely related to the degree of tumor malignancy. Glucose-regulated protein 78 is also known as immunoglobulin heavy chain binding protein, which is a molecular chaperon located in the endoplasmic. It is expressed in many tumors such as lung cancer, stomach cancer, prostate cancer, etc. It is involved in tumor metastasis and angiogenesis. ATP5B gene encodes ATP synthase β subunit. It is not only located in the mitochondria but also in the surface of tumor cells. It plays a role in tumor angiogenesis. Currently, the regulation effect of resveratrol on ATP5B and GRP78 in human medulloblastoma cells has not been reported.Objective:This study aims to explore the effects and biological significance of resveratrol on GRP78、ATP5B and VEGF in human medulloblastoma cells.1) Explore the reaction difference in human medulloblastoma cells (UW228-3, DAOY) before and after resveratrol treated; 2) Detect the expression of GRP78 and ATP5B in human medulloblastoma cells before and after resveratrol treated; 3) Explore the molecular mechanism of resveratrol anti-angiogenic and analyze the molecular basis of resveratrol’s sensitivity differences of anti-medulloblastoma, so as to provide the basis for a solid theoretical foundation for revealing the anti-angiogenic drug targets and individualized treatment of medulloblastoma.Methods:Human medulloblastoma cell line UW228-3 and DAOY were cultured in DMEM and RPM1640 culture medium containing 10% FBS, then collected after 100μM resveratrol treated for 48 hours. Study the sensitivity difference of medulloblastomas by HE staining, MTT and flow cytometry. Compare and identify the expression difference of proteins before and after resveratrol treated in two cell lines by 2D-PAGE and MS, and select potential anti-angiogenic drug targets. Detect the expression of GRP78 and ATP5B in human medulloblastoma tissue arrays through IHC. RT-PCR, ICC and Western-blotting were used to detect the expression changes of GRP78, ATP5B and VEGF.Results:1) HE staining, MTT and flow cytometry showed that medulloblastoma cell lines UW228-3 cell morphology changed and the number of cells reduced significantly, while DAOY cells had no significant change; 2) 2D-PAGE and MS found two potential anti-angiogenic drug targets:GRP78 and ATP5B, thus GRP78 increased 2.1 fold and ATP5B increased 3.0 fold after resveratrol treated in DAOY cells; 3) IHC staining found that the expression of GRP78 and ATP5B exist differences between medulloblastoma and normal tissues of human brains; 4) RT-PCR, ICC and Western-blotting results showed that the expression of GRP78 and ATP5B up-regulated and the expression of VEGF down-regulated after resveratrol treated for 48 hours. While in sensitive cells UW228-3, the expression of VEGF down-regulated significantly.Conclusions:1) There was anti-tumor effect difference of resveratrol in two human medulloblastoma cell lines, relative to DAOY, UW228-3 cells were more sensitive to a certain concentration of resveratrol.2) The expression of proteins existed difference before and after resveratrol treated.3) Resveratrol could induce the expression of GRP78 and ATP5B, and down-regulate the expression of VEGF in medulloblastoma cells.4) The expression trend of GRP78 and ATP5B was similar in resveratrol sensitive/less sensitive cells. While the expression of VEGF had more significant reduction in sensitive cells, suggesting that GRP78 and ATP5B might not be important mechanism for regulating tumor angiogenesis, while VEGF might be one of the important resveratrol anti-angiogenic targets.