The Study Of Effects Of Celecoxib Combination With Temozolemide On Glioma U251Cells And The Possible Machanisms

ObjectiveTo detect the expression of COX-2in glioma U251cells; observe anti-tumor effects of celecoxib alone and in combination with Temozolomide on glioma U251cells in vitro; explore the possible mechanisms,which provides experimental evidence for COX-2-targeted therapy of glioma.Methods1、Detecting the expression of COX-2in glioma U251cellsWe cultured glioma U251cells in vitro in the appropriate conditions,the inmmunohistochemical and Western Blot were used for detecting the expression of COX-2in glioma U251cells.2、The study of anti-tumor effects of celecoxib alone and in combination with Temozolomide on glioma U251cells in vitro(1) There were four groups in this expriment depending on the intervening factors:①control group；②celecoxib group (only used celecoxib)③Temozolomide (only usde Temozolomide)④ヽombination group (Celecoxib combinated with Temozolomide)(2) We cultured glioma U251cells in vitro,when the density was80percent,according to the above grouping we intervened the cells using drugs,and after72hours detcted U251cells from the following aspects respectively:①we used microscope to observe the morphology of cells in every group;②deceted the proliferation of cells in every group by MTT assay;③detected the apoptosis rate of cells in every group in early stage and later period by Annexin V-FITC/PI on flow cytometry;﹐bserved apoptosis morphology of cells in every group by Hoechst33258on fluorescence microscope.(3) We cultured glioma U251cells in vitro,when the density was80percent,making a scratch model, according to grouping we intervened the cells by drugs,after24hours observed the migration ablity of cells in every group on microscope.3、The study of possible mechanism for the effects of celecoxib alone and in combination with Temozolomide on glioma U251cellsWe cultured glioma U251cells in vitro,when the density was80percent, according to grouping we intervened the cells by drugs,after72hours we detected the expression of Bax and Bcl-2of cells in every group.Results1. The expression of COX-2in glioma U251cells(1) The expression of COX-2in gioma U251cells was detected by inmmunohistochemical method:COX-2was in cytoplasm and nucleus,we could see apparent brown grains in cytoplasm and nucleus of glioma U251cells,the high expression of COX-2was in glioma U251cells.(2) The expression of COX-2was detected in gioma U251cells by Western Bolt:we could see that the high expression of COX-2was in glioma U251cells by Western Bolt.2、The study of anti-tumor effects of celecoxib alone and in combination with Temozolomide on glioma U251cells in vitro (1) The morphology of glioma U251cells intervened by drugs was observed on microscope:in control group,the numbers of cells was more than anyother group,the growth state was fine,the form of cells was spindle,the transmittance and refraction was better and the speed of proliferation was faster than anyother group. The volume> transmittance and refraction cells of Celecoxib group and Temozolomide group reduced,Tomozolomide group was more notable than Celecoxib group,the volume of combination group was smaller and refraction was weaker than anyother group,there was point flake debris surrounding cells,a lot of cells were disinegration and floating,the medium was turbid.(2) The proliferation of glioma U251cells intervened by drugs was detected by MTT assay.By MTT assay we could see that the OD of control goup was0.79±0.03, the OD of celecoxib group was0.68±0.03,the OD of Temozolomide goup was0.61±0.04, the OD of combination goup was0.45±0.13;the OD and proliferation rate of control group were higher than anyother anyother group (P<0.01), the OD and proliferation rate of combination group were lower than Clelecoxib group and Temozolomide group (P<0.01)(3) The apotosis rate of glioma U251cells was deteced by Annexin V-FITC/PI on flow cytometry.The apotosis rate detected by flow cytometry of cells in each group was4.20%±0.33%、19.12%±0.91%、24.76%±0.87%、42.98%±0.56%. the apotosis rate of control group was lower than anyother anyother group (P<0.01), the apotosis rate of combination group was higher than Clelecoxib group and Temozolomide group(P<0.01).We compared the apotosis rate of U251at early stage and later period, the apotosis rate of control group at early stage and later period was lower than anyother anyother group(P<0.01), the apotosis rate of combination group at early stage and later period was higher than Clelecoxib group and Temozolomide group (P<0.01) (4) The apoptosis morphology of glioma U251cells intervened by drugs was observed by Hoechst33258on fluorescence microscope.Cells was colored by Hoechst33258staining and analyzed by fluorescence microscopy. DNA was uniform distribution and no shrinkage in normal cells. Some large understained nuclei could be observed in view. However, DNA accumulated and different degrees of shrinkage formed in apoptotic cells. Hyperchromatic density of nuclei could be observed in some cells.Cells of control group were little apotosis, the number of apotosis cells in Celecoxib group and Temozolomide increased,combination group was more significant.(5) The migration abilitiy of glioma U251cells intervened by drugs was observed by migration test.The cell growth condition at scratches was observed microscope. Comparing the dates of migration at the same space at different times, width variation indicated abilities of migration and the wider of the width, the stronger of the migration ability. The results showed that after cultured24hours,the migration abilitiy of cells treated by drugs decreased, combination group was more significant.3> The study of possible mechanism for the effects of celecoxib alone and in combination with Temozolomide on glioma U251cellsThe expression of Bax and Bcl-2in glioma U251cells was detected by Western Blot.The value of A (gray value of sample/gray value of internal reference) represented the expression level of protein.By Western Blot we saw that the value of A of Bax in each group was0.88±0.45、0.96±0.43、1.17±0.39、1.54±0.47, the expression level of Bax in Celecoxib group was higher than that in control group (p<0.05); the expression level of Bax in Temozolomide group and combination group were higher than that in control group (P<0.01); the expression level of Bax in combination group was higher than that in Celecoxib group and Temozolomide group (P<0.01); The value of A of Bcl-2in each group was:0.55±0.0.47、0.47±0.31、0.43±0.22、0.37±0.0.22, the expression level of Bcl-2in control group was higher than that in anyother group (P<0.01), the expression level of Bcl-2in combination group was lower than that in Celecoxib group (P<0.01) and Temozolomide group (P<0.05).Conclusions1.The expression of COX-2in glioma U251cells was at a high level;2.The proliferation and migration of glioma U251cells treated by Celecoxib combinating with Temozolomide could be inhibited,and the apoptisis could be promoted;3.The possible mechanism of promoting apoptisis may be that the expression of Bax increased and the expresssion of Bcl-2decreased.