Proliferation Inhibition Effect Of Lutein On Human Breast Cancer MCF-7 Cell Via Inhibiting AP-1 Activation And Its Mechanism

BackgroundBreast cancer is a kind of common malignant tumorin women,which caused by the interaction of many factors and multiple genes.Its morbidity and mortality increased year by year.But the exact etiology and pathogenesis are not fully determinated.Currently,the treatments for breast cancer are gradually evolving.Nowadays,it is the synthesis of various treatment methods,combined with surgery,chemotherapy,radiotherapy,and target therapy.However,it can cause indelible pains to the patients.Therefore,it is of great significance to find natural compounds,with efficient and low toxicity effects,for the prevention and treatment of breast cancer.Lutein,which is abundant in the green plants such as flowers,fruits and vegetables,especially in marigold,is a kind of natural compounds with antioxidant activity.Lutein could be ingested and delivered to the tissues through the circulatory system.Epidemiological studies have shown that lutein plays a significant role in the physical health,such as protection of vision,prevention of cataracts,delay of atherosclerosis,and prevention of cancer and so on.ObjectiveThe aim of this experiment is to detect the level of activator protein 1(AP-1)and related factors of signal pathway in human breast cancer MCF-7 cell which interfered with lutein,and to research the effects and mechanism of lutein on the proliferation of MCF-7 cell.And the result may provide a theoretical basis for the linical research of chemoprevention on breast cancer,and lutein plays an critical role as a natural anticancer compound with low side effect.Methods1.Treated MCF-7 cell with different concentrations of lutein(0,5,10,20,40,80?g/ml)and cultured for 12,24 and 48 h respectively,then detected the cell inhibition rate though MTT assay,and analysis the results.2.Flow cytometry was used to detect the levels of reactive oxygen species(ROS)in the control group and the experimental groups those were treated with different concentrations of lutein(0,5,10,20,40,80?g/ml)after 24 h.3.RT-qPCR was used to detect the Nrf2,HO-1 mRNA expression in control group and experimental groups.4.Western blot was used to detect the Nrf2 and HO-1 protein expression in the control group and the experimental groups.5.Set experimental groups on the basis of the results of MTT method,namely the control group,3-MA group,lutein group,3-MA+ lutein group for the subsequent experiments.Flow cytometry was used to examine the cell cycle and apoptosis in control group and experimental groups.6.RT-qPCR was used to detect PI3 K,AP-1,Cox-2 and CyclinD1 mRNA expression in control group and experimental groups.7.Western blot was used to detect PI3 K,AP-1,Cox-2 and CyclinD1 protein expression in the control group and the experimental groups.Results1.The result of MTT showed that lutein can inhibit the proliferation of human breast cancer MCF-7 cell.And the inhibition was in a dose and time dependent manner.2.Flow cytometry results showed: compared with the control group,the level of ROS had obvious difference,levels of ROS of experiment groups were lower than control group,while the lutein was high,the group level of ROS was lower than that of control group,the differences were statistically significant(P<0.05).3.RT-qPCR results showed: compared with the control group,Nrf2,HO-1 mRNA expression in experiments groups were increased,4.Western blot results showed: compared with the control group,the protein expression of HO-1 in experiment groups increased.Nrf2 protein expression were up-regulated in nucleus.5.Annexin V-FITC/PI apoptosis assay showed: cultured 24 h later,compared with the control group,3-MA group,lutein(40?g/ml)group and 3-MA+lutein(40?g/ml)group apoptosis rate were significantly different(P<0.05),the percentage of apoptotic cells of 3-MA+lutein(40?g/ml)group were significantly greater than lutein(40?g/ml)group and 3-MA group(P<0.05),and lutein(40?g/ml)group compared with 3-MA group,there was no significant difference between the two groups.6.Flow cytometry results showed: compared with the control group,the cell of experiment groups in G0/G1 increased with the control group.Especially,the 3-MA +Lutein group blocked in G1?7.RT-qPCR results showed: compared with the control group,PI3K?AP-1?Cox-2?CyclinD1 mRNA expressionin 3-MA group were decreased,and in lutein(40?g/ml)group and 3-MA+lutein(40?g/ml)group,they were significantly up-regulated.However,compared with the control group,PI3K?AP-1?Cox-2?CyclinD1 mRNA expressionin 3-MA group were up-regulated,and in lutein(40?g/ml)group and 3-MA+ lutein(40?g/ml)group,they were decreased significantly(P<0.05).8.Western blot results showed: compared with the control group,the protein expression of PI3K?AP-1?Cox-2?CyclinD1 in 3-MA group were decreased.Compared with the control group,in lutein(40?g/ml)group and 3-MA+ lutein(40?g/ml)group,PI3K?AP-1?Cox-2?CyclinD1 protein expression were significantly increased(P<0.05).Conclusion1.Lutein can inhibit the proliferation of human breast cancer cell MCF-7,and the inhibitory effect was in a time and dose dependent manner.2.Lutein can induce the expression and nuclear translocation of Nrf2,and reduce the ROS level.3.The proliferation inhibition effect of Lutein on human breast cancer cell MCF-7 might be probably mediated by up-regulating the expression of Nrf2 and HO-1,decreasing the level of ROS,inhibiting the expression of the PI3 K,whereby down-regulating the expression of AP-1 and its corresponging downstream target gene.