Experimental Study Of Molecularly Targeted Therapy For Glioma In Vitro

Malignant gliomas are the most common and aggressive primary brain tumors . Despite considerable progress has been made in traditional treatment of surgery, radiotherapy, and chemotherapy , the prognosis of patients with malignant gliomas has remained dismal. Molecularly targeted therapy is a highlight of new pattern for cancer treatment, which has been a Research hot spot in recent years with its Specificity and Efficacy. In this experiment,we examined the effect of growth inhibition and apoptosis on human glioma cell line U251 with recombinant retrovirus vector expressing shRNAs targeting CDC2(CDC2 shRNA)and gefitinib ('Iressa', ZD1839), an epidermal growth factor receptor tyrosine kinase inhibitor, providing experimental evidences for application value of CDC2 and EGFR in glioma treatment as the gene for the onset and development of gliomas.Part one : Depletion of CDC2 inhibits the proliferation of human glioma cell line U251【Objective】It is known that CDC2 (cyclin-dependent kinase 1, CDK1) as an active sub-unit of the M-phase promoting factor (MPF) play a key role in the control of the G2/M transition in mitosis. There have been many evidences that CDC2 was overexpressed in many different carcinoma types including malignant gliomas. And it was highly correlated with the pathologic grades and prognosis of human gliomas. We have obtained success on the experiment of CDC2 depletion inhibiting proliferation of human glioma cells SHG44. In this part, we will investigate the effect of inhibiting CDC2 on proliferation of human glioma cell line U251, providing experimental evidence for application value of CDC2 in glioma treatment as the gene for the molecular target of different gliomas.【Methods】The recombinant plasmid P-Super-Retro-CDC2 constructed by ourselves was transfected into human glioma cell line U251. The resulting phenotypic changes of the target gene were investigated. Changes in cell morphology and quantity were observed by inverted microscopy; the expression level of CDC2 mRNA was measured using reverse transcription-polymerase chain reaction (RT-PCR) and real-time fluorescent quantitative PCR; the expression level of CDC2 protein was examined by Western blot; changes in the cell cycle and apoptosis were detected by Flow cytometry; ultramicroscopic structure of tumor cells was observed with the transmission electron microscope (TEM).【Results】After 48 h transfection of the recombinant retrovirus plasmid expressing shRNA targeting CDC2 into human glioma cell line U251, adherent tumor cells gradually became floating, proliferation and CDC2 mRNA were suppressed by more than 98% and the protein expression was down-regulated by over 92%. Meanwhile, the percentage of cells blocked at G2/M phase was high and that of apoptosis increased from 0.57% to 13.01%. The ultrastructure was obviously injured.【Conclusion】CDC2 is one of the genes for the onset and development of various gliomas., which plays an important role in the maintenance of proliferation of human gliomas. Downregulation of CDC2 could prevent the progression of the malignant phenotype of human glioma cells. And CDC2 could become a potential target on gene therapy of different human gliomas.Part two: The effect of gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, on the inhibition of the proliferation in human glioma cell line U251【Objective】To detect the effect of gefitinib ('Iressa', ZD1839), an inhibitor of epidermal growth factor receptor tyrosine kinase , on the proliferation and apoptosis in human glioma cell line U251, and to provide the experimental evidence for EGFR as a potential molecular target in glioma treatment .【Methods】After different concentration of gefitinib treatment for 48 h,MTT method was used to examined the effect of gefitinib on the cellular proliferation of U251 cells.changes in the cell cycle and apoptosis of tumor cells were detected via flow cytometry.【Results】Gefitinib could suppress the proliferation of U251 cells significantly in a dose-dependent manner. The IC50 of gefitinib on U251 cells was 18.99μM. The percentage of cells blocked at G0/G1 phase increased from 50.15% (without gefitinib treated ) to 82.66% (25.3μM with gefitinib treated ) . Meanwhile, the apoptosis of tumor cells was from 1.45%(without gefitinib treated ) rise to 40.36%(25.3μM with gefitinib treated ).【Conclusion】EGFR is one of the genes for the onset and development of gliomas. Cell proliferation coud be suppressed effectively via inhibiting the activity of EGFR, so it is worthy of the future studies as a potential molecular target .