Effect And Mechanisms Of BMP4Regulate On The Chemotherapy Sensitivity Of Human Drug-resistance Glioma Cells In Vitro

Glioma was the most common malignant tumors of the central nervous system. Treatment methods had developed by a single surgical treatment to surgery combined with radiation and chemotherapy. However, the prognosis of patients has not been significantly improved, because of their biological characteristics of invasive and high recurrence rate. Studies have shown that the patient’s survival time could benefit from the new chemotherapy drugs, but limited impact on extends survival time. Tumor resistance is the main reason for the failure of chemotherapy, especially tumor multidrug resistance (MDR), which a common way of tumor resistance, was the prior problem must be solved in current chemotherapy. BMP is a very important member of the TGF-β family subfamily, which have a similar structure of the highly conserved functional protein. In recent years, Studies found that BMP plays a key role not only in bone development and regeneration and repair, and also plays an important role in mesoderm induction and differentiation, hematopoietic tissue formation and development, nervous system development and repair, the occurrence, development and prognosis.of a variety of malignant tumors. Now less research has taken on the relationship of BMP4and glioma chemosensitivity. The subject of the project was explored the effect and mechanisms of BMP4regulate on the chemotherapy sensitivity of human drug-resistance glioma cells in vitro. We establish glioma multidrug resistant cell line, investigate the effects of BMP4on the glioma drug resistance and the expression of the related drug resistance genes, and to explore the possible mechanism of BMP4regulate on glioma drug resistance.PART1Establishment and characterization of the glioma drug-resistance cell line U251/TMZObjective:Establish the drug-resistance human glioma cell line induced by temozolomide(TMZ), which has significant value for the study of molecular mechanism of drug-resistance and find out the strategy to reverse gliomas drug-resistance. And view the characterization of the drug-resistance cell line and verify the important role of related genes in the drug resistance mechanism.Methods:Chemotherapeutic resistance in U251human glioma cells was initiated by the stepwise revulsion with TMZ(0,2,4,6,8,10,12,14,16,18,20μg/ml). The cell morphology of U251and U251/TMZ was observed under optical microscope. The drug resistance was detected by MTT (to express with IC50). Cell cycle was tested by flow cytometry. Cell doubling time was calculated through the cell count. The expression of MDRl、BCL-2、MRP5and LRP1was observed by RT-PCR.Results:We established the glioma drug-resistance cell line U251/TMZ successfully, Optical microscopy showed that the volume of drug-resistance cells was increased, pleomorphic, and irregular shaped nucleus obviously increased, intracytoplasmic granular material increased; doubling time of U251/TMZ was(14.64±1.98) h, the doubling time of the parental cell line U251was(10.26±1.03) h, which was no significant differences between the two groups; MTT method to detect the drug resistance times keep on TMZ was6.13folds; flow cytometry showed the cell cycle of U251/TMZ cell line was significantly reduced than the U251parental cell lines in the period of G0/G1and S, but increased in the period of G2/M. Detection of the expressions of MDR1, BCL-2, MRP5, LRP1resistance gene in U251/TMZ raised significantly.Conclusions:A stable drug-resistant cell line U251/TMZ induced by TMZ was established successfully in8-month culture, meanwhile, increasing the concentration of TMZ stepwisely was the important strategy. The expression of MDR1, BCL-2, MRP5, and LRP1were significantly increased, may be related to the mechanism of drug resistance formation. Part2Study on relationship between BMP4and human glioma chemotherapy resistanceObjective:Bone morphogenetic protein4(BMP4) was a multifunctional factor closely related to the development of human growth and development of a variety of malignant tumors, But the relationship with tumor resistance to chemotherapy was unclear. We discuss on the relationship between BMP4with MnSOD and BCL-2, which associated with the gliomas drug resistance, and explore the role of BMP4play in the mechanism of drug resistance of the gliomas cells.Methods:The tissue microarry (TMA) of human brain astrocytoma was constructed and analyzed for the expresion of BMP4, BCL-2and MnSOD by using SP immunohistochemistry. Temozolomide(TMZ)resistant cell line was constructed and the expression of BMP4was detected. BMP4plasmid was transfected into the TMZ resistant cells, and then detected the chemotherapy sensitivity.Results:The positive expression rates of BMP4decrease with higher grade astrocytoma, and has significant difference during each grade(P<0.05). The expression of BMP4was negatively correlated with MnSOD(r=-0.3835, P<0.05) and negatively with BCL-2(r=-0.2391, P<0.05). TMZ resistant cell line was constructed and the expression of BMP4was decreased compared to U251cells, the resistance was abolished when plasmid of BMP4was transfected into the TMZ resistant cells, thus cell inhibition rate increased significantly under the action of TMZ.Conclusions:The expression of BMP4was significantly related to drug-resistance gene MnSOD and BCL-2in human glioma tissues. Transfection of BMP4into the resistance cells could significantly improve the cell inhibition rate by TMZ, showed that it could regulate the chemosensitivity of gliomas. Part3The mechanisms of BMP4regulate on the chemotherapy sensitivity of human drug-resistance glioma cellsObjective:The mechanism of BMP4regulation on chemosensitivity of glioma drug-resistance cell is remain unclear. To study on the effect of BMP4regulate on the chemotherapy sensitivity of U251drug-resistance cell line, analysis the expression changes of drug-resistant gene MDR1, BCL-2and TopoⅡ and detect the activation of the signaling pathway after BMP4transfected into gliomas drug-resistance cell line, and to explore the possible mechanisms of BMP4and related signaling pathways in regulating chemotherapy sensitivity of U251drug-resistance cell line.Methods:BMP4plasmid was transfected into U251drug-resistance cells to establish the stable expression cell line, and then under the effect of TMZ, the cell activity was detected by MTT, expression change of MDR1, BCL-2and TopoⅡ were detected by Western-blotting in each group cells. Phosphorylation level of Smad1/5/8and p38were detected after BMP4transfected into cells drug-resistance cells. Blockade of the BMP/Smad and p38MAPK signal pathway, the cell activity was detected by MTT, expression change of MDRl, BCL-2, Topo Ⅱ were detected by Western-blotting in each group cells.Results:The drug-resistance cell activity was decreased significantly after the BMP4plasmid transfected into the cell lines, while under the effect of different concentration TMZ, that showed BMP4could enhanced the chemosensitivity of U251/TMZ drug resistance cells. and the expression of BCL-2decreased while Topo Ⅱ increased significantly in transfected group, and there was a significant difference compared with blank transfected group, meanwhile the reduce of MDRl expression was no statistically significant difference. After BMP4plasmid was transfected into U251/TMZ cell line, detected the level of Smadl/5/8and p38phosphorylation, the results showed that Smad and p38MAPK signal pathway were activated. By inhibiting BMP/Smad signal pathway in cell lines, the cell growth inhibition rate declined obviously, the expression of BCL-2was increased and Topo Ⅱ decreased significantly in transfected group, and there was a significant difference compared with untransfected group(P<0.05), while the reduce of MDRl protein expression was no statistically significant difference(P>0.05). By inhibiting p38MAPK signal pathway in cell lines, the cell growth inhibition rate declined obviously, the expression of BCL-2and MDRl were increased while Topo Ⅱ decreased significantly in transfected group, and there were a significant difference compared with untransfected group(P<0.05).Conclusions:BMP4regulate on the chemosensitivity of the glioma drug-resistance cell, and its mechanism may be related to change the expression of BCL-2and topoⅡ protein. BMP4could activaed the Smad and p38MAPK signaling pathway in glioma drug-resistance cell, and through which it regulate on the expression of BCL-2and Topo Ⅱ to reverse the chemosensitivity of the glioma drug-resistance cell.