Effect Of Mifepristone On The Activity Of Human Glioma U251 Cells And Its Mechanism

ObjectiveMifepristone(U486) is a synthetic l9-norsteroid compound. As a common clinical steroidal potent antagonist of progesterone which can effectively binding with progesterone receptor. Studies have found that mifepristone can significantly inhibit the tumor growth, and its antagonistic effect of progesterone and glucocorticoid have also been widespread concern. However, the definite anti-tumor mechanisms of mifepristone is still controversy. The progesterone and glucocorticoid synthesis are completeed in the mitochondria, cholesterol as raw materials is transported into the mitochondria through the 18 kDa translocator protein(TSPO) in mitochondrial membraneis transported into the mitochondria. Conversely, there is negative feedback for the progesterone and glucocorticoid concentration to TSPO, while the progesterone and glucocorticoid concentration increased, TSPO expression and activity decreased due to the negative feedback regulation, thereby reducing cholesterol transport and the synthesis of progesterone and glucocorticoid. TSPO is a key regulatory protein of the mitochondrial apoptosis pathway, activating the TSPO can initiate apoptosis and play a important role in the process of tumor growth inhibition. Therefore, the hypothesis of this study is: Mifepristone by inhibiting progesterone and glucocorticoid activity, reducing the progesterone and glucocorticoid negative feedback effect on TSPO, increasing TSPO expression and activity, through the mitochondrial apoptosis pathway, increasing apoptosis and inhibiting tumor growth. In this paper, we study the effect of mifepristone on human brain glioma cell line U251 in vitro. To investigate whether mifepristone on the U251 cells inhibited cell proliferation effect and observe the effect of mifepristone on apoptosis of U251 cells and the expression of TSPO, Bcl-2, Bax and Caspase-3.Primarily explore the possible mechanism of mifepristone induce the apoptosis of U251 cells and provide the basis of experiment research for new anticancer drugs development.MethodsPart I The CCK-8 microplate colorimetric detection method was applied to investigate the effect of U251 cells treated with various concentrations of mifepristone. The trypan blue exclusion assay was used to detect the mortality of U251 cells. PartⅡ Based on the results of PartI,selecting three group of different concentrations mifepristone(5, 10, 20 umol/L) for the detection of relevant indicators,including apoptosis staining and the detection of flow cytometry.Additionally, Western blotting and immumofluorescence assay were conducted to examine the expression 1evel of TSPO. Apoptosis-related protein Bcl-2, Bax and Caspase-3 were detected using western blotting assay.ResultsPart I Statistical analysis showed that the viability of U251 cells significantly decreased in 5μmol/L, 10μmol/L, 20μmol/L and 40μmol/L mifepristone groups(P<0.05), but the inhibition of cell proliferation in low concentration(2.5μmol/L) of mifepristone is not obvious(P>0.05). The results of trypan blue exclusion assay showed that U251 cells death in experimental groups were increased in experimental groups compared with the control group. But when the concentration increased to20μmol/L and 40μmol/L, there were no significant difference between the two experimental groups. PartⅡ The apoptosis rate were significantly increased in the groups of mifepristone, the effects have direct proportion with the drug concentration.Additionally, comparing with the control group, western blotting analysis and immumofluorescence demonstrated that the expression level of TSPO was significantly increased in 20 umol/L mifepristone group(P<0.05). Apoptosis-related protein Bcl-2, Bax and Caspase-3 were expressed in both control group and experimental groups, but the apoptosis rate of 20 umol/L mifepristone group was increased significantly(P<0.05).Conclusion(1)The Mifepristone inhibits the proliferation of glioma cells in an appropriate concentration range.(2) Mifepristone promotes the apoptosis of glioma U251 cells and through promoting TSPO expression and activating the mitochondrial apoptotic pathways.