Mechanism Study Of Methylselenic Acid Against Human Glioma Cell Growth By Triggering Oxidative Stress Damage

Objective:To evaluate the inhibitory effect of methylseleninic acid（Me Se）against human glioma cells growth and explore the underlying molecular mechanism.Methods:Human glioma cell lines U251 and U87 were cultured in vitro,and cells were treated by 2.5-40μM Me Se for 72 h.Cells viability of U251 and U87 cells was detected by MTT method.Cells morphology of U251 cells was observed by phase contrast microscope.Cell cycle changes and apoptosis of U251 were detected by flow cytometry after PI staining.Specfic fluorescence substrates were used to detect the caspases activation.Total reactive oxygen species（ROS）and superoxide anion production were detected by DCFH-DA and DHE fluorescence probes,respectively.Western blotting was used to detect the expression of apoptosis and DNA damage related proteins.The tumor-bearing model of nude mice was established in vivo,and the body weight and tumor weight of the mice were detected.The tumor volume was detected by molybdenum imaging.Angiogenesis,cell proliferation and apoptosis in tumor tissue were detected by HE staining,immunohistochemistry and immunofluorescence.Results:MTT results showed that Me Se dose-dependently inhibited the growth of U251 and U87 cells,and U251 cells were more sensitive to Me Se.Cells morphology observation showed that the U251 cells after Me Se treatment showed significant cell shrinkage,decreased cells number,and the formation of apoptotic bodies.Flow cytometry results showed that Me Se induces apoptosis of U251 cells in a dose-dependent manner,as convinced by the increase of sub-G1 peak.Me Se treatment dose-dependently activated the expressions of caspase-3,caspase-8 and Caspase-9 in U251 cells.Western blotting results showed that Me Se dose-dependently activated the expressions of active-caspase-3,active-caspase-8 and active-caspase-9.Immunofluorescence detection found that Me Se caused significant production of ROS and superoxide anions in a time-dependent manner,which subsequently resulted in DNA damage,as demonstrated by the increased phosphorylation expression of Ser428-ATR,Ser1981-ATM,Ser15-p53 and Ser139-H₂A.However,addition of glutathione（GSH）,an inhibitor of ROS,effectively blocked ROS geenration,attenuated caspase activation and DNA damage,and finally inhibited Me Se-induced cells killing against U251 cells.Animal experiments results suggested that Me Se inhibited the glioma volume,but had no significant effect on the body weight of mice.Imagings by molybdenum target showed that Me Se effectively inhibited the tumor growth.Western blotting results showed that Me Se increased the phosphorylated expression of active-caspase-3,Ser15-p53 and ser139-H2A,indicating that Me Se induced glioma cell apoptosis and DNA damage.HE staining and Ki67 staining confirmed that Me Se effectively inhibited the proliferation of glioma cells in vivo.TUNEL staining confirmed that Me Se induced significant cells apoptosis of glioma cells in vivo.CD34 staining revealed that Me Se effectively inhibited the angiogenesis of glioma in vivo.Conclusions:Me Se treatment effectively inhibited the growth of glioma cells in vitro and in vivo by triggering ROS-dependent DNA damage,which validated that Me Se may act as a potential glioma chemotherapy agent.