Luteolin Inhibits Breast Cancer Development And Progression In Vitro And In Vivo By Suppressing Notch Signaling And Regulating MiRNAs

Objective:The purpose of this experiment is to study the effect of Luteolinon thetumorigenicity and Notch-1 signaling pathway in human breast cancer cells,and to study its possible mechanism,which may provide a clinical guide for its treatment of human breast cancer.Methods:Cell growth/survival assays and cell cycle analyses were performed to evaluate cell survivalin vitro.Migration tests were carried out to analyze cell viability and aggressiveness.Tube formation assays were applied to identify the ability of Luteolin in angiogenesis with or without the presentation of breast cancer cells.Notch-1 intracellular domain,Hes-1,Hey-1,Hey-2,Cyclin D1,VEGF,MMPs and serum VEGF were detected by western blotting analyses and ELISA assays.Notch-1,Hes-1,Hey-1,Hey-2.VEGF,and MMPs mRNAs were analyzed by real-time reverse transcription-polymerase chain reaction.The activity of MMP-2 and MMP-9 was evaluated by gelatin zymography analyses.MiRNAs were analyzed using miRNAs expression assays.After transfected with Notch-1 siRNA and cDNA,miR-34a and miR-224 mimics in MDA-MB-231 cells separately,the above assays were also carried out to examine the potential changes of tumor cells.Xenograft models were applied for evaluating the treatment potency of Luteolin in breast cancer.Results:Luteolin inhibited breast cancer cell survivalLuteolin suppressed the growth of both MDA-MB-231 and MCF-7 cells,with time-effect relationship and dose-effect relationship.Tumor cells treated with 100?mol/L Luteolin showed most significant inhibition effect when compared to those treated with lower doses or with controlled DMSO.Also,both MDA-MB-231 and MCF-7 cells were blocked in S phases after treating with Luteolin,demonstrating that this drug can remarkably alter tumor cell survival.Luteolin decreased breast cancer cell migrationHorizontal migration tests revealed that scratches in cells treated with Luteolin remained virtually unchanged after 24 hours,whereas control group cells(DMSO)almost healed from the damage.Similar results were also found in vertical migration tests,which larger number of cells migrated to the lower chamber in control group than those in experimental groups.The vertical migration tests also revealed dose-effect relationship,with cells treated by 100?mol/L Luteolin showed smallest migration numbers.Reduced tube formation of HUVECs induced by declined VEGF expression in Luteolin-treated breast cancer cellsVisible tube formation of HUVECs was introduced after it was co-cultured with MDA-MB-231,MCF-7 cells,or treated with VEGF.However,this tube formation effect was reversed if Luteolin was added in MDA-MB-231 or MCF-7 cells,respectively.In order to clarify whether Luteolin would cause tube formation directly without the presentation of breast cancer cells,50?mol/L Luteolin with VEGF were applied for HUVECs incubation,and no significant change was detected 8 hours afterward when compared to HUVECs cultured with DMSO andVEGF.This result suggested that the tube formation could only be achieved with the employment of breast cancer cells.We also detected decreased VEGF level in MDA-MB-231 and MCF-7 cells treated with 50?mol/L Luteolin compared to those treated with DMSO,illustrating that reduced tube formation was induced by declined VEGF expression in Lutolin-treated breast cancer cells.Luteolin reduced Notch-1,Hes-1,Hey,VEGF,Cyclin D1 and MMPs expression in breast cancer cellsBoth MDA-MB-231 and MCF-7 cells treated with 50?mol/L Luteolin for 48 hours presented decreasing expression of Notch-1 mRNA,as well as its downstream genes Hes-1,Hey-1 and Hey-2,indicating that Luteolin significantly inactivated transcription effect in Notch pathway.Similar results were also detected in western blotting that Notch-1,Hes-1 and Cyclin D1 protein levels were lower in cells treated with Luteolin,suggesting that Luteolin may also alter protein expression in Notch signaling.RT-PCR and western blotting revealed remarkable decline of VEGF,MMP-2,MMP-9 mRNA(P<0.05)and VEGF,MMP-2,MMP-9 protein in cancer cells,indicating that Luteolin plays an important role in both tumor cell growth and matrix invasion(Figure 3A and 3B).The secretion of VEGF in media/supernatant was also lower in cells cultured with Luteolin,presenting dose-effect relationships in ELISA assays.Luteolin helped to decrease the activities of MMP-2 and MMP-9 proteins,as indicated in Gelatin zymography,which Luteolin-treated cells presented lower gray scale(Figure 3C).MiRNAs expression in Luteolin-treated breast cancer cellsThe expression of miR-181a,miR-139-5p,miR-224 and miR-246 in both MDA-MB-231 and MCF-7 cells were significantly enhanced after treated with Luteolin,while the level of miR-155 was decreased.miR-34aexpression only increased in Luteolin-treated MDA-MB-231 cells.The above results suggested that Luteolin distinctively altered miRNA levels in breast cancer cells.Effect of Notch-1 silence and overexpression on breast cancer cell survival,invasion and angiogenesisAfter transfected with Notch-1 siRNA,the expression of Notch-1,Hes-1,Cyclin D1,VEGF,MMP-2 and MMP-9 proteins decreased in MDA-MB-231 cells.Cancer cell survival,migration ability and tube formation of HUVECs were also inhibited with the down-regulation of Notch-1 introduced by siRNA transfection.Moreover,the suppress effects were increased when 25|imol/L Luteolin was added to Notch-l-silenced MDA-MB-231 cells.Western blotting also presented similar results that Notch-1 silence together with Luteolin could more effectively inactivate proteins in Notch signaling.Notch-1 down-regulation triggered by siRNA promoted Luteolin-induced VEGF,MMP-2 and MMP-9 inactivation.Effect of Notch-1 silence on miRNAs level in breast cancer cellsAfter transfected with Notch-1 siRNA,MDA-MB-231 cells presented increased expression of miR-34a,miR-181a,miR-139-5p,miR-224 and miR-246,while showed decreased level of miR-155(Figure 4B).The above results were similar to MDA-MB-231 cells treated with Luteolin.miR-34a and miR-224 regulated Notch signaling and inhibited breast cancer cell survival,migration and angiogenesismiR-34a and miR-224 mimics were successfully transfected into MDA-MB-231 cellsand high level of miRNA expression were detected compared to blank control.Transfected miRNA presented inhibiting effects on Notch-1,VEGF,MMP-2 and MMP-9 proteinsexpression and reduced the activity of MMP-2 and MMP-9 in MDA-MB-231 cells.Cancer cell viability,invasion/migration ability,and tube formation of HUVECs were also significantly suppressed after miR-34a or miR-224 mimics transfected into MDA-MB-231 cells.Luteolin inhibited the growth of breast cancer xenografts in vivoSubcutaneous breast cancer mouse model was successfully established and all animals were randomly assigned in 4 groups,treated with PBS,Paclitaxel,and different doses of Luteolin,respectively.Our results revealed that high dose Luteolin effectively inhibit tumor growth in vivo when compared to both blank control and standard therapy groups,given that significant tumor weight and volume reduction were detected in the former.High dose(40mg/kg/d)and low dose(20mg/kg/d)Luteolin groups presented a RTV inhibition rate of 28.31%,40.50%,respectively,and a tumor growth inhibition rate of 54.28%,40.38%,respectively,which all statistically different from those of the blank control group.And besides,none of the mice in Luteolin groups suffered from toxic side effects,such as weight loss or activity reduction,demonstrating that Luteoin can significantly change the growth profile of breast cancer in xenograft model,with no negative effects on animals' daily activity.Conclusions:Luteolin inhibits Notch signaling by regulating miRNAs relating to tumor development and progression.However,the effect of miRNAs on Notch pathway could be either Luteolin-dependent or Luteolin-independent.Also,Notch-1 alteration may inversely change miRNAs levels.Our data demonstrate the critical status of Luteolin,miRNAs and Notch pathway in breast cancer development and prognosis,suggesting any components within the network could be potential target for future treatment and drug delivery.