Effect And Mechanism Of Apigenin On Triple-negative Breast Cancer

Objective:Triple negative breast cancer(TNBC)is one of the most malignant types of breast cancer.In previous experiments,we screened TNBC inhibitors in natural compound libraries and identified apigenin as a potential inhibitor.The purpose of this study was to investigate the inhibitory effect of apigenin on TNBC cells and its relative mechanism.It provides more evidence for celery research and more detection options for TNBC treatment.Methods:(1)Effect of apigenin on the proliferation of TNBC cells:The TNBC cells used in this experiment were MDA-MB-231 and MDA-MB-436.Effects of apigenin(0,2,4,8,16,32 and 64 ?M)on 24 hours-,48 hours-,and 72 hours-cell viability were measured by SRB assay and the IC50 values of apigenin were calculated.Effects of apigenin on proliferation of the two TNBC cell lines were examined by cloning formation assay.In this experiment,cells were treated with apigenin(0,5,10,20,40 ?M)for 72 hours,then incubated in normal medium for 10 days.After dyeing,photographs and clone counts were taken.(2)Effect of apigenin on the migration of TNBC cells:The effect of apigenin on the migration ability of two TNBC cell lines was examined by wound healing assay.After scratching uniform width,TNBC cells in 6-well plates were treated with 0,10,20 ?M apigenin in serum-free medium.At different time points(0,12,24h),photographs at the same position of each well were taken and analyzed.In addition,Transwell assay was used to verify the effect of apigenin on cell migration.TNBC cell lines were pretreated with apigenin for 24 hours,then added into chamber transwell.Fixed staining was performed 24 hours later,and photographs were taken under a microscope to count the number of cells.(3)Effect of apigenin on(breast cancer stem cells,BCSCs)properties of TNBC cells:Flow cytometry was used to detect the CD44+/CD24-cell subpopulation.The control group was treated with 0.1%DMSO,while the drug group was treated with apigenin(0,10,20 ?M)for 72 hours.Anti-CD24-PE and anti-CD44-FITC were used for staining of BCSCs.The percentage of CD44+/CD24-subpopulation was analyzed by flow cytometry.In addition,effect of apigenin on mammospheres formation ability of TNBC cells was also examined.TNBC cells were treated with 0,10 or 20 ?M apigenin for 72h and then plated into ultra-low attachment plates.Ten days later,images were taken and mammosphere number were counted.(4)Effect of apigenin on Hippo signaling pathway:TNBC cells were treated with 0.1%DMSO(Control),5,10 or 20 ?M of apigenin for 72 hours.The mRNA expression of downstream genes CTGF and CYR61 was detected by RT-qPCR.The levels of Hippo signaling pathway-related proteins(YAP,TAZ,CTGF,CYR61 and GAPDH)were detected by Western blotting.Co-Immunoprecipitation assay was employed to detect the effect of apigenin on YAP/TAZ-TEADS interaction.(5)Detection of anti-tumor ability of apigenin in vivo:MDA-MB-231 was pretreated with 0.1%DMSO and 20 ?M of apigenin respecrtively for 48 hours,and then different amount of the DMSO or apigenin-pretreated cells(1×106,1×105,or 1×104)were injected subcutaneously into nude mice(3 mice each treatment).The growth of nude mice was observed every two days.The formation efficiency,volume and weight of tumors were recorded once a week.At the end of the experiment,the weight and volume of the exfoliated tumors were recorded and photographed.Results:(1)Apigenin inhibited the proliferation of TNBC cells:The SRB protein assay showed that apigenin was basically non-toxic to normal human breast cancer cells but inhibited the proliferation of TNBC cells.The inhibition was in a dose-and time-dependent manner.When apigenin acted for 72 hours,the IC50 values of TNBC cell lines MDA-MB-436 and MDA-MB-231 were about 30 and 33 ?M.Apigenin also significantly reduced the colony formation rates of two TNBC cell lines.(2)Apigenin inhibited TNBC cell migration;Results from wound-healing assays showed that apigenin at 10 and 20?M,significantly reduced the migration gap closure of MDA-MB-231 and MDA-MB-436.In Transwell assay,apigenin reduced the number of migrating cells in a dose-dependent manner.Statistical analysis of chamber transwell migrating cell showed that apigenin could significantly reduce the migration of MDA-MB-231 and MDA-MB-436 cells.(3)Apigenin inhibited the BCSCs properties of TNBC cells:Results from flow cytometry analysis showed that apigenin reduced the percentage of CD44+/CD24-cancer stem cell subpopulation in MDA-MB-231 and MDA-MB-436 cells.In mammosphere formation assay,apigenin significantly reduced the number of mammospheres formed by the two cell lines,suggesting that apigenin could reduce the self-renewal capability of TNBC cells.(4)Apigenin inhibited the expression of TNBC cell-related genes and protein levels by hippo pathway.The results of RT-qPCR and Western blotting showed that apigenin did not significantly change the protein levels of YAP and TAZ in Hippo signaling pathway.However,apigenin significantly reduced the levels of mRNA and protein(CTGF,CYR61)in a dose-dependent manner.In addition,Apigenin disrupted the interaction of YAP/TAZ-TEADs in MDA-MB-231 and MDA-MB-436 cells.(5)Apigenin inhibited the tumor-initiating properties of TNBC cells:in nude mice,pretreated with apigenin inhibited the tumorigenic potential of MDA-MB-231 cells.After subcutaneously jection with 1×106,10×105,1×104 0.1%DMSO-pretreated cells(Control),the xenograft formation rate in nude mice were 100%,100%,and 75%respectively.However,after injection with same amount of apigenin-pretreated cells,tumor formation efficiency in the nude mice decreased to 50%,20%,and 0,respectively.Compared to control group,the tumor in apigenin group grew slowly,and the tumor volume and weight peeled off at the last experiment were significantly decreased.Conclusion:In this study,apigenin inhibited the proliferation,migration and breast cancer stem cell properties of TNBC cells through inhibiting the activity of the downstream effector factor YAP/TAZ of Hippo pathway thus reversing the malignant phenotype of TNBC,showing to be a potential drug candidate for the treatment of patients with high YAP/TAZ activity in TNBC.