Flavone Regulates NO And ROS Production And Effects DLC1/RhoA Pathway In Breast Cancer Cells

Rho family GTPases are key regulators of actin cytoskeleton and act as on-off molecular switch, cycling between active GTP-bound form and inactive GDP-bound form. RhoA is one of the most studied and characterized members of Rho family GTPases. Tumor suppressor protein DLC1(Deleted in Liver Cancer 1) is a RhoGAP(RhoGTPase-activating protein) for RhoA, promotes active RhoA-GTP returning to inactive RhoA-GDP. DLC1 is frequently lost or down-regulated in multiple kinds of human cancers.NO(nitric oxide) and ROS(reactive oxygen species) act as signal molecules and regulate a series of cellular signal transduction pathways. NO is synthesized from by nitric oxide synthase(NOS) and regulates a lot of cellular responses by S-nitrosylation. It has been reported that NO affects activity of RhoA. ROS directly regulates RhoA activity in fibroblasts, and this process is not dependent on GEF or GAP. ROS regulates p190Rho-GAP and Cdc42 GAP. But, ROS-induced regulations on DLC1 are still not clear.Flavone(2-phenyl-4H-1-benzopyran-4-one) is the core structure of flavonoids,and inhibits proliferation of human colon and breast cancer cells. Flavone regulates intracellular NO in murine macrophages and rat hepatocyte, effects ROS production in zebrafish embryos, human hepatoma HepG2 cells and human colon cancer HT29 cells, and increases DLC1 mRNA expression in breast cancer cells, but the mechanisms are not clear.This study investigated flavone-induced regulations on DLC1/RhoA pathway,regulations on NO and ROS-related cellular processes and related mechanisms in MCF-7 and MDA-MB-231 breast cancer cells, and further explored whether flavone-induced regulations of DLC1/RhoA pathway is mediated by NO or ROS.The results of MTT and Hoechst/PI nuclear staining indicated flavone inhibited cell proliferation and induced apoptosis with a concentration- and time-dependentmanner in MCF-7 and MDA-MB-231 breast cancer cells. Flavone induced no effect on cell necrosis. Our results showed that flavone up-regulated DLC1 protein expressions in breast cancer cells. Proteasome inhibitor MG132 treatment increased protein levels of DLC1 and protein synthesis inhibitor cycloheximide(CHX) caused DLC1 degradation rapidly, which suggested DLC1 is unstable and degraded through proteasome pathway. Flavone suppressed the DLC1 degradation process in the presence of CHX in both MCF-7 and MDA-MB-231 cells, which indicated flavone increased DLC1 protein stability.This study found flavone inhibits NO generation by does-dependent suppressing NOS enzymatic activity. The decreases of NO production were detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. We further investigated flavone-induced regulation on protein S-nitrosylation. Protein S-nitrosylation is a reversible post-translational modi?cation which occurs on protein cysteine residues,by attaching a NO moiety with a reactive cysteine thiol group to form an S-nitrosothiol(SNO). This study detected protein S-nitrosylation based on the“Biotin-switch” method. All S-nitrosylated proteins were labeled with maleimide-biotin, and then purified by streptavidin magnetic beads, and detected by western blot. Results showed that flavone down-regulated protein S-nitrosylation in breast cancer cells.This study further explored whether flavone-induced inhibition on cell proliferation and up-regulation of DLC1 were mediated by NO. NO donor SNP was added to restore the NO levels. SNP reversed flavone-induced suppression on cell proliferation, which indicated flavone inhibits proliferation by decreasing NO in breast cancer cells. SNP caused no effect on DLC1 protein expressions in both MCF-7and MDA-MB-231 cells, which indicated that flavone-induced up-regulation of DLC1 expressions was not mediated by NO.This study investigated flavone-induced effect on ROS levels in MCF-7 and MDA-MB-231 breast cancer cells. Flavone decreased ROS production by enhancing SOD activity. The decrease of ROS levels were detected by fluorescencespectrophotometer and fluorescence microscopy. Flavone up-regulated DLC1 expression, and then inhibited RhoA activity and cell migration in breast cancer cells.This study further explored whether flavone-induced regulation on DLC1/RhoA pathway is mediated by ROS. Hydrogen peroxide was added to restore the ROS levels.The functions of ROS are related to its concentration. ROS in physiological level act as signal molecules and regulates a series of cellular signal transduction pathways; but cause cell damage and death in excessive level. The aim of present study is investigating regulations induced by ROS within the physiological range on DLC1/RhoA pathway, 40μM hydrogen peroxide restored ROS level approximately to the control groups. So, 40μM hydrogen peroxide was used to restore flavone-induced decrease of ROS in the subsequent experiments.The results showed that, after treated by 150 and 200μM flavone for 24 and 72 h,DLC1 protein expression in both MCF-7 and MDA-MB-231 cells were up-regulated.Hydrogen peroxide inhibited the flavone-induced up-regulation of DLC1 expression,which indicated flavone increased DLC1 expression by decreasing ROS. Pull-down assay showed that after treated by 150 and 200μM flavone for 24 h, RhoA activity in both MCF-7 and MDA-MB-231 cells were down-regulated, and hydrogen peroxide reversed flavone-induced inhibition on RhoA activity. Flavone-induced inhibitions on cell migration were reversed by hydrogen peroxide. These results demonstrated flavone regulated DLC1/RhoA pathway by decreasing ROS production.In conclusion, this study demonstrated flavone inhibited cell prolifetation and induced apoptosis in MCF-7 and MDA-MB-231 breast cancer cells. Flavone increased DLC1 protein expressions and enhanced DLC1 protein stability. Flavone decreased NO generation by inhibiting NOS activity, and then down-regulated protein S-nitrosylation in breast cancer cells. Flavone inhibited cell proliferation by decreasing NO level. Results showed that flavone-induced up-regulations of DLC1 expression did not mediated by NO. Flavone decreased ROS production by increasing SOD activity. Flavone inhibited cell migration by regulating DLC1/RhoA pathway,and after the ROS levels were restored by hydrogen peroxide, flavone-induced up-regulation of DLC1, down-regulation of RhoA activity and inhibition of cellmigration were all reversed, which indicated flavone regulated DLC1/RhoA pathway by decreasing ROS production.This study demonstrated flavone regulated NOS/NO/S-nitrosylation cellular processes and inhibited cell proliferation by decreasing NO, and flavone inhibited cell migration through DLC1/RhoA pathway by decreasing ROS production in breast cancer cells.