Curcumin Induced Apoptosis Of U87 Cells By ROS

Curcumin is a kind of natural dietary polyphenols isolated from turmeric rhizome,which possess many pharmacological activities,such as anticancer,anti-inflammatory,antioxidant,antibacterial,antiviral,antidiabetic and promoting wound healing.Numerous studies have shown that curcumin inhibit the growth of multiple types of cancer cells,including lung,breast,cervical,liver,pancreatic and colon cancer act as an antioxidant.but there israrely research on human glioma.Several animal experimental and clinical studies have also proved the anticancer effect of curcumin.Recent studies have revealed that curcumin may exertedanti-tumor activity by increasing cell ROS level,or oxidative stress.In this paper,human glioma cell line U87 was used as a cell model to test and evaluate the effects of curcumin on cell proliferation,ROS level,oxidative stress index and apoptosis.Aimed to investigate whether the anticancer effect of curcumin is related to its pro-oxidation propetty,and to provide data and reference for the clinical application of curcumin and the development of functional food.The results are as follows:1.The effect of curcumin on the proliferation of U87 cells was tested by CCK-8assay.The results showed that curcumin significantly inhibited the proliferation of U87 cells.In the range of 5-100μM,the proliferation rate of U87 cells decreased with the increase of curcumin concentration,and the effect is in a concentration-dependent manner,The IC₅₀of regression analysis was 68.32μM.the effect of curcumin causing cell death was detected by trypan blue exclusion assay.The results showed that the mortality of U87 cells increased with the increase of curcumin concentration after treatment for 24 h.2.The morphological changes of U87 cells treated with curcumin were observed under optical microscope.It is showed that curcumin could induce U87 cells contraction and roundness of,and the higher concentration of curcumin was used,the morphological change was more obvious When combined treatment with antioxidant5m M GSH,the cells recovered to a morphology similar to control group,suggesting that the morphological changes induced by curcumin may be related to oxidation.3.DCFH-DA fluorescence probe and electrochemical method were used to detect the ROS content of U87 cells.Results showed that the intracellular ROS of U87 cells increased after curcumin treatment,and the ROS level decreased after GSH combination.Enzyme-linked immunosorbent assay（Elisa）was used to detect the activity of NADPH oxidase in U87 cells.It is showed that the activity of NADPH oxidase in U87 cells treated with 5-40μM curcumin for 24 h was higher than control cells.When NADPH oxidase Nox1/Nox4 inhibitor GKT137831 was added,the activity of NADPH oxidase decreased,which indicated that curcumin induced the increase of intracellular ROS may be derived from the NADPH oxidase pathway.4.Cell total antioxidant capacity（T-AOC）,Malondialdehyde（MDA）,Superoxide dismutase（SOD）and Glutathione（GSH）measure showed,after treatment with 5-40μM curcumin,intracellular T-AOC was significantly decreased in U87 cells.With the increase of curcumin concentration,T-AOC decreased gradually,intracellular MDA level increased significantly in a dose-dependent manner,and intracellular GSH level decreased significantly in a concentration-dependent pattern.These results suggest that curcumin causes oxidative stress in U87 cells and the stress increased with curcumin concentration rise.The level of intracellular SOD also increased with the concentration of curcumin,which indicated that the SOD expression increased under oxidative stress,and curcumin could promote the oxidative stress.5.The apoptosis of U87 cells was detected by flow cytometry after Annexin V/PI double staining and fluorescence microscope when AO/EB fluorescence staining,which showed that U87 cells apoptosis occurred after 24 h treatment with 5-40μM curcumin,and the total apoptosis rate increased significantly in a in concentration-dependent fashion.The combination of with antioxidant GSH could attenuate the apoptosis induced by 10μM and 40μM curcumin,which indicated that the apoptosis was caused by oxidation.6.Western blot was used to detect the apoptosis-related protein signaling pathway.It was found that curcumin treatment significantly decreased the expression of NF-κB/p65 protein and that was increased by 5m M GSH,suggested that the increase of ROS induced by curcumin can inhibit the expression of NF-κB/p65protein.The results showed that curcumin could increase the expression of pro-apoptotic protein Bax and decrease the expression of anti-apoptotic protein Bcl-2,thatmeans,increase the value of Bax/Bcl-2.It was also found that curcumin induced apoptosis may be caspase-dependent.Because curcumin could down-regulate the expression of pro Caspase-3,indicating curcumin increased its activated form Caspase-3,hence induced apoptosis.It was also found that curcumin-induced cell death might be related to the apoptosis-related protein Caspase-1.The results of western blot when adding antioxidant GSH also showed that curcumin induced cell death by promoting oxidation.In summary,all the results showed that curcumin promoted ROS production by increasing the activity of NADPH oxidase in human glioma U87 cells.It caused the change of intracellular redox index,decrease of cell T-AOC and GSH,increase of MDA,tand the change of SOD activity,that indicatedcurcumin treatment lead to U87cells suffer oxidative stress.ROS production,triggering cellular signaling pathways,down-regulated the expression of NF-κB/p65,the differential expression of Bax and Bcl-2,and mitochondrial endogenous apoptosis pathway was activated,and apoptosis was initiated by apoptosis executive molecule Caspase-3.