| Background: Medulloblastoma is the most frequent malignant brain tumor in children,occurred at the age of 3-4 and 7-8.For a long time,the MB tissue is controversial.The molecular mechanism of MB is still not perfected,so the auxiliary treatment of MB is limited to surgery and chemotherapy assisted with drugs.But the effect is not ideal,the rate that survived more than 10 years is 40%-60% and these patients have physical and mental trauma even if prognosis.So clearing mechanism of tumor and searching for more accurate therapeutic targets and effective targeted drugs are the key to cure the MB.Resveratrol is a kind of flavonoid polyphenol compounds widely found in grapes,peanuts and more than 70 plants.It has many kinds of biological function such as anti-inflammatory,anti-tumor,cardiovascular disease resistance,oxidation resistance and so on by through the blood brain barrier,and has non-toxic side effects to the body.Studies have found that resveratrol could inhibit tumor cell proliferation by anti-angiogenesis.Tumor angiogenesis plays a key role in the occurrence and invasion of the tumors,and is regulated by angiogenesis and inhibiting factors.One of them is named VEGF and it is up-regulated in gastric cancer,breast cancer,liver cancer and medulloblastoma and associated with malignant degree of tumors.CALR(Calreticulin)is a kind of common and highly conservative endoplasmic reticulum calcium binding protein,regulating various biological functions such as calcium balance,assisting in protein folding and processing,involving in antigen presenting,angiogenesis and apoptosis regulation.CALR are found in a variety of tumors such as leukemia,bladder cancer,breast cancer and liver cancer and so on.Studies have shown that CALR was significantly upregulated in neuroblastomas after the treatment of NGF and It also has been upregulated in esophageal cancer and breast cancer by proteomic technology.VDAC1(Voltage?dependent anion channel 1)is one of the main channel protein located in the outer membrane,regulating material and energy metabolism inside and outside of the mitochondria,taking part in cell apoptosis and inhibiting angiogenesis.Its abnormal expression can affect the function of mitochondrial,and it is associated with heart disease,stroke,cancer and neurodegenerative diseases.The expression of VDAC1 mRNA and protein increased significantly in gastric cancer cells after the treatment of H.pylori,and It also has been upregulated in multiple myeloma cell lines(U266)after CD45.At present,resveratrol on medulloblastoma in CALR and VDAC1 regulation has not been reported.Objective: This study was to explore the expression and the biological significance of CALR and VDAC1 in medulloblastoma cell lines: UW228-3 and DAOY which have different sensitivity to resveratrol.1)Explore the different reactivity in medulloblastoma cell lines UW228-3 and DAOY before and after treatment of resveratrol;2)Test different expression of CALR and VDAC1 in these two cell lines before and after the resveratrol;3)Compare different sensitivity to resveratrol in UW228-3 and DAOY,analyze the reasons for the different sensitivity;4)Explore the molecular mechanism of anti-tumor angiogenesis of resveratrol,look for potential drug targets for anti-tumor angiogenesis,provide theoretical basis to clinically cured medulloblastoma thoroughly.Methods: 1)Cell culture: Cultivate medulloblastoma UW228-3 and DAOY cells respectively in the low sugar DMEM and 1640 medium containing 10% fetal bovine serum;collect cells and climbs after the treatment of 100?M resveratrol for 48 h;2)Morphological detection: Observe and detect the change of morphology and the activication of cells in UW228-3 and DAOY by HE staining,MTT test and flow cytometry instrument technology before and after resveratrol;3)Proteomics: Compare the protein expression of medulloblastoma cell lines UW228-3 and DAOY before and after resveratrol using two-dimensional gel electrophoresis technology,identifit and analyse different protein by mass spectrometry,screen potential drug targets of anti-tumor angiogenesis;4)Detection of downstream: Test the expression of CALR and VDAC1 in medulloblastoma tissues by IHC and make statistical analysis;test the expression of CALR and VDAC1 in UW228-3 and DAOY by RT-PCR,ICC,IF and Western blotting methods before and after resveratrol.Results: 1)HE staining,MTT and flow cytometry showed that the number of the UW228-3 cells reduced obviously and the cells were spindler after treatment of 100?M resveratrol for 48 hours,and the number of DAOY cells and shapes did not change significantly;2)Two-dimensional electrophoresis and mass spectrometry(ms)showed that CALR and VDAC1 were upregulated in DAOY and UW228-3 after the treatment of resveratrol.CALR was 2.9 times higher after the treatment of resveratrol for 48 h in cell line DAOY,and VDAC1 is 3.6 times higher in cell line UW228-3;3)Detection of downstream IHC showed that the expression of VDAC1 and CALR were higher in relatively normal tissue compared with medulloblastoma,and the statistical results was statistically significant;RT-PCR,ICC,IF and Western blotting showed that expression of CALR and VDAC1 up-regulated after treated with resveratrol for 48 h in UW228-3 and DAOY,and the up-regulation is more obvious in UW228-3 than DAOY.Conclusion: 1)There was different sensitivity in human medulloblastoma cell lines: UW228-3 and DAOY.Resveratrol can inhibit UW228-3 cells proliferation and induce its apoptosis,but the effect is not obvious in DAOY;2)Protein expression profiles for human medulloblastoma cell lines had differences before and after resveratrol treated;3)Up-regulation of CALR and VDAC1 is obvious in UW228-3 cell line which was sensitive to resveratrol,and that is not obvious in DAOY,prompting that the expressions changes of CALR and VDAC1 may influence the sensitivity of the two cell lines,and they were likely to be one of the targets of resveratrol antitumor reaction. |
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