The Multidrug Resistances And Mechanism Research Of Brain Tumor Stem Cells From Human Neuroepithelial Tumors

OBJECTIVE The purpose of the first part to isolate the CD133 positive(CD133~+, brain tumor stem cells, BTSC) and CD133 negative(CD133~-, differentiated tumor cells, TC) from human neuroepithelial tumor by magnetic activated cells sorting (MACS) based on CD133 expression, further to determine the percentage of CD133~+ cells and study their proliferating and monoclonal formation characteristics. The main aim of the second part is to explore the BTSC characteristic marker Nestin expression and clinical significance in human neuroepithelial tumors and the relationship between Nestin as well as CD133. The next two parts are to analyze the expression of ABC superfamily transporters and MGMT between the CD133~+ and CD133~-cells, exploring the correlation and significances among them in the multidrug resistances of BTSC from neuroepithelial tumor. Further to study the feasibility to reversing the P-gp,MRP1 chemoresistance by verapamil.METHODS In the first part, samples from 35 gliomas and 8 ependymomas were obtained from patients undergoing microsurgical tumor resection. The BTSC were isolated based on CD133 marker by MACS and determined the percentage of CD133~+ cells from the tumors, thus CD133~+ cells were cultured and mechanically proliferated and passaged in serum-free medium or complete culture medium. The monoclonal formation experiments were performed to determine CD133~+ cells proliferating capacity and these cells were labeled by BrdU to observe the brain tumor neurospheres (BTS) formations processes. The correlation between pathological grade and CD133~+ cells monoclonal formation frequencies proportion in the tumors was also analyzed. The next part was to explore the expression of Nestin protein and activity in human neuroepithelial tumors by immunocytochemistry and RT-PCR respectively and its clinical significance, thus suggested the Nestin representing the BTSC contents in the tumors. The final two parts were to analyze and compare the multidrug resistances proteins P-gp,MRP1,MGTM gene expression between CD133~+ and CD133~-cells by RT-PCR, then P-gp,MRP1 protein expression in tumor tissue level by immunocytochemistry and in cell level by immunofluorescence.RESULTS CD133~+ cells and CD133~-cells could be isolated successfully by MACS. CD133~+ cells percentage and Nestin expression were positive correlation to the pathological grades. CD133~+ cells generated free-floating neurosphere-like BTS and abnormal proliferating capacity in the serum-free medium in vitro. 3 cases from glioblastoma stem cells could form BTS in the complete medium and culturede for 1-3 passage. There is nearly no expression of Nestin protein in low pathological grade neuroepithelial tumors and metastatic tumors contrasted to distinctly expression in anaplastic nuclei and cytoplasm for high grade tumors. The tumor vascular endothelial cells expressed Nestin in all of the tumors and no relationship to the pathological grade. The expressional range of P-gp and MRP1 protein was 18%-67%, 23%-73% in CD133~+ cells and 12%-38%, 18%-44% in glioma respectively. CD133~+ cells expressed high level of MDR1 MRP1 mRNA as well as higher level of MGMT mRNA than TC, the protein activities were totally increased to 16.1,19.6,34.0 times respectively compared with TC and have positive correlation with pathological grades of tumors. MDR1 MRP1 MGMT genes were not expressed in all the tumors and positive expressional rates were 76.7%,86.7%,73.3% respectively in all. MDR1 MRP1 and MGMT protein activity expression have obvious correlation in malignant tumors.CONCLUSIONS 1.Only a small proportion of cells in the neuroepithelial tumors are CD133~+ cells with the ability to self-renew and abnormal proliferation, which could be isolated by MACS. BTSC have more powerfully proliferated ability than NSC. 2. The "sternness gene" Nestin expression and monoclonal formation frequencies were positive correlation with the pathological grades and both important methods in evaluating the malignant grades of the neuroepithelial tumors. The Nestin expression was achieved in the genetic level, which was considered as a marker for proliferative endothelium during rapid growth and an independently valuable factor in determining malignant process and prognosis. However, the Nestin expression in all of the tumor vascular endothelial cells was no relationship to the pathological grade. The upregulation of Nestin protein and activity expression in glioma maybe an important mechanism in the process of tumor malignant transformation. 3. For the first time this study provided evidence that CD133~+ cells display strong capacity on tumor's resistances to chemotherapy. There was marked correlation among the MDR1 MRP1 MGMT activity. This multidrug resistances was probably contributed by the CD 133~+ cell with higher expression of P-gp MRP1 MGMT. BTSC were the root of multidrug resistances and key therapeutic target for the neuroepithelial tumors. This study provides a new vision of the development for drug strategy therapeutically.