Proteomic Regulation Of Resveratrol In Human Glioblastoma And Medulloblastoma Cells: A Preliminary Study

BACKGROUND AND PURPOSE:glioblastoma and medulloblastoma who had high diffusion and aggression clinically were common neurological malignancies, and no effective clinical treatment, even the surgery combined with radiotherapy and chemotherapy could make a big impact on the prognosis of patients. Studies showed that drug treatment had an important means to improve the quality of life of patients. Resveratrol was a natural polyphenolic compounds with a variety of biological effects and its effect of anti-tumor was growing attention. This project aimed to study the impact of resveratrol on brain tumor cell line proteome and explored resveratrol possible mechanism of differences in the sensitivity of cell lines, looking for targets of anti-tumor or tumor resistance mechanisms might provide a basis for further research conducted in this field. Experiments include(1) the impact of protein levels: Observed the changes of proteome in different brain tumors which had differences in the sensitivity of resveratrol,with resvertrol treatment.(2) screening and identification: Selected a number of pharmaceutical proteins differentially expressed in the presence of post process, identified proteins by mass spectrometry on its property.(3) made use of immunohistochemistry and RT-PCR for the identification of proteins differentially expressed in cells before and after treatment with resveratrol for the verification.(4) Data analyzed: draw possible biological networks of tumor growth.METHODS:(1) screening and drug treatment: Chose two kinds of brain tumor cell lines(gliomas and medulloblastomas), each cell line were selected for differences in susceptibility resveratrol cell lines(glioma-sensitive : U251, less sensitive: LN18 and medulloblastoma sensitive: UW228-3, less sensitive: DAOY). Cell line U251, LN18 UW228-3 cultured in L-DMEM medium(Low-Dulbecco’s Modified Eagle Medium,L-DMEM), DAOY cultured in RPMI1640 medium(Roswell Park Memorial Institute 1640,RPMI 1640), each medium containing 10% fetal bovine serum and 1% antibiotics(penicillin and streptomycin), and drug treatment groups were with 100μM resveratrol processing 48 h, cells were observed by HE staining.(2) 2-DE separation and MS protein identification: whole cell was extracted for 2-DE, protein digestion technology changed for protein digestion and mass spectrometry(MS) was on digested samples.(3) The results of protein identification: Verified the new protein gene expression by using immunohistochemistry、immunoblotting techniques and RT-PCR technique.RESULTS:(1) morphological changes: cell line U251 and UW228-3 had a significant change in the quantity and morphology with resveratrol treatment, there were changes in the neuron-like and reducing the number of cells, and cell lines LN18 and DAOY had little changed in its shape and the number of tips: U251, UW228-3 and LN18, DAOY had different sensitivity to resveratrol.(2) proteomics experiments: 1) the overall impact of the proteome: Raised mainly, glioma cell lines, U251 experimental group, the normal group N detected 310 protein spots were detected in the control group Res 271 proteins, including computer software, a total of 170 matches, increasing rate 64.7%; in LN18 cells, the normal set of N 422 protein spots were detected in the control group Res 405 protein spots were detected, the computer software, a total of 269 matches, increasing rate 64.3%. Medulloblastoma cell lines, DAOY group, the normal group N were detected 298 points in the control group were detected Res 342 points, the software matches 234, increasing rate 42.7%; UW228-3 group, the normal group N 177 points were detected,in the control Res group detected 118 points, software paired 69, increasing rate 49.3%. 2) identification of differentially expressed proteins: protein identification results spectrum enolase, phosphoglycerate mutase, keratin, annexin, moesin, heat shock protein.(3) differences in protein verification: randomly selected a few proteins for RT-PCR validation, ENO1, PDIA1, AKR1B1 etc. showed expression levels decreased after treatment, whereas the two keratin scarcely detected in two susceptible cell lines, increased slightly in insensitive cell lines after treatment, CAP1 appeared upregulated in DAOY and U251,and downregulated in UW228-3, had no significant changes in LN18.(4) we selected protein enolase in common experimental results to WB results and immunocytochemistry were consistent, and all reducation after drug treatment, in line with the results of two-dimensional electrophoresis.CONCLUSIONS:(1) We get around four brain tumor cell lines treated with resveratrol protein expression profile, provide a reference for further research initially.(2) either sensitive or insensitive in four cell lines after resveratrol treatment, proteome have different levels of change.(3) after drug treatment, significant differences in the expression of the protein are mainly in terms of energy metabolism, cytoskeletal structure, tumor migration and inflammatory stress response, suggesting that resveratrol may have multiple biological functions.(4) the expression of these proteins which changes in the experiment are linked to form a biological network directly or indirectly, for further experimental study of the direction.