Lutein Inhibits Human Breast Cancer Cell Proliferation Via Nrf2/GSK-3? Signaling Pathway

Background Lutein(LUT),a natural phytochemical,is structurally an oxygenated carotenoid.Lutein has a special structure of multiple conjugated double bonds,which has anti-oxidative stress activity and various biological functions,including anti-inflammatory and anti-tumor effects.At present,breast cancer is still a worldwide public health dilemma and one of the most common malignant tumors that seriously threaten women's health and life.In the past two decades,breast cancer-related research has led us to make great progress in understanding of the development and mechanism of breast cancer,further improving the treatment.Reactive oxygen species(ROS)is associated with the development of many tumors,and an increase in the content of ROS has been detected in various tumors.Moreover,it has been shown that reactive oxygen species,as information molecules,are involved in a variety of complex signal transduction pathways and cross talk,and thus are associated with many biological behavior changes such as tumor cell proliferation and apoptosis.Tumor cells express high levels of antioxidant proteins to detoxify elevated ROS levels,establish a redox balance.The redox balance of tumor cells was significantly changed compared with the redox balance of normal cells,indicating that the intervention of reactive oxygen species may be an important mechanism for tumor intervention.Nuclear factor erythrocyte 2 related factor 2 (Nrf2),an important transcription factor,has been identified as a major factor in cellular antioxidant defense systems in many human cancers.The glycogen synthase kinase 3?(GSK-3?),a serine/threonine kinase,is a complex signaling molecule.GSK-3? is involved in the regulation of cellular oxidative stress by interacting with Nrf2,mediating changes in a variety of cellular biological behaviors.Under ROS-stimulated conditions,Nrf2 mediates the production of a series of antioxidant defense proteins and type II heterologous metabolic enzymes.Classical Keap-1 dependent and non-canonical NF-?B and GSK-3?-dependent mechanisms regulate Nrf2-mediated defense responses,regulate Nrf2 expression and expression of downstream defense proteins such as HO-1,NQO-1 and so on.Objective The purpose of this study was to investigate the inhibitory effect of lutein on the proliferation of human breast cancer cells MCF-7 and MDA-MB-231.And based on the mechanism of reactive oxygen species(ROS),Revealing the effects of lutein on the expression and activity of the most important antioxidant defense transcription factor Nrf2 through the NF-?B pathway and the GSK-3?/Fyn pathway,and the molecular mechanisms of changes in the expression levels of Nrf2 downstream signaling antioxidant enzymes or proteins.Provide a theoretical basis for the chemical intervention of lutein for breast cancer.Methods 1 Human breast cancer cells MCF-7 and MDA-MB-231 cells were treated with lutein at a concentration of 5,10,20,40,80 ?M for 12,24,and 48 h,respectively.The 0 ?M group was used as a blank control group.The MTT assay was used to evaluate the effect of lutein on cell proliferation.IC50 was calculated by the percentage of cells surviving at each concentration.2 Flow cytometry was used to measure the apoptotic rate of MCF-7 and MDA-MB-231 breast cancer cells treated with lutein(10,20,40 ?M);the apoptosis of lutein-treated cells was analyzed by Western blot.The change in the expression level of protein caspase-3.3 After treatment of MDA-MB-231 and MCF-7 cells with lutein(10,20,40 ?M)and hydrogen peroxide(positive control),the level of reactive oxygen species in the cells was measured using flow cytometry DCFH-DA.4.MDA-MB-231 and MCF-7 were exposed to different doses of lutein(10,20,40 ?M)and H2O2,and then subjected to nuclear separation experiments to collect proteins.5.After lutein treatment,the protein level of Keap-1 in MDA-MB-231 and MCF-7 cells was detected by western blot,and the levels of Nrf2 protein in cytoplasm and nucleus were detected,respectively.6.After lutein treatment,m RNA and protein expression levels of HO-1,NQO-1 in MDA-MB-231 and MCF-7 cells were detected by western blot and RT-q PCR,respectively.7 The expression of NF-?B and GSK-3?/fyn pathway-related proteins in MDA-MB-231 and MCF-7 cells treated with lutein was analyzed by Western blot.The changes of Nrf2 were detected by RT-q PCR at m RNA level.Results 1.The MTT data showed that lutein treatment of human breast cancer MDA-MB-231 and MCF-7 cells was significantly inhibited in a dose-dependent manner.2.Flow cytometry results showed that the apoptotic rate of lutein treatment of human breast cancer MDA-MB-231 and MCF-7 cells increased with increasing lutein concentration compared with the untreated group.Western blot results showed that the expression of caspase-3 increased with the increase of lutein.3.In lutein treatment of human breast cancer MDA-MB-231 and MCF-7 cells,ROS levels were decreased,whereas ROS levels were elevated during hydrogen peroxide treatment.4.Lutein significantly down-regulated the expression of Nrf2 in the cytoplasm and nucleus of MDA-MB-231 and MCF-7 cells,increased the expression level of Keap-1,and decreased the expression levels of HO-1 and NQO-1 at protein and m RNA levels.These phenomena are reversed during hydrogen peroxide treatment.5.Western blot results showed that lutein inhibited the phosphorylation of Ikk? and IKB? in the NF-?B pathway,thereby inhibiting the nuclear translocation of P65,and decreasing the expression of Nrf2 at the m RNA level,but was reversed by hydrogen peroxide.In the GSK-3? pathway,lutein inhibits phosphorylation of Gsk-3B and increases Fyn nuclear translocation,but hydrogen peroxide enhances phosphorylation of Gsk-3B and inhibits nuclear translocation of Fyn.Conclusion 1.Lutein significantly inhibits lutein treatment of human breast cancer MDA-MB-231 and MCF-7 cells proliferation.2.The mechanism of lutein inhibiting the proliferation of human breast cancer cells may be achieved by reducing the ROS level of breast cancer cells and thereby down-regulating the expression of Nrf2 mediated by the NF-?B signaling pathway,and reducing the nuclear localization of Nrf2 through the GSK-3?/Fyn pathway,further reducing the m RNA and protein expression levels of the antioxidant defense genes HO-1 and NQO-1.