The Study On Anti-tumor Effect Of Lappaol F Targeting YAP

ObjectiveLappaol F(LAF)is a lignin component in Arctium lappa L.In present study,effects of LAF on proliferation,apoptosis,migration and invasion of tumor cells were investigated in four cancer cell lines.Further studies on the effects of LAF on expression of Yes-Associated Protein(YAP),the carcinogenic gene,and its downstream target genes were carried out to detect the regulation of LAF on YAP.This research enriches the underlying mechanisms of LAF in anti-proliferation in vitro.Methods(1)Inhibitory effects of LAF on proliferation of Hela cells,SW480 cells,PC3 cells and MDA-MB-231 cells were assessed via sulforhodamine B staining.(2)Apoptosis of cancer cells induced by different concentrations of LAF was detected using Annexin V-FITC/PI double staining.(3)Effects of LAF on migration and invasion of HeLa cells and MDA-MB-231 cells were analyzed by the real time xCELLigence RTCA DP system.(4)Effects of LAF on the expression and phosphorylation of YAP,the expression of BIRC5,Bcl-2 and C-myc(YAP target genes)and 14-3-3 O(a regulatory protein of YAP)were detected by Western-Blotting.(5)Effects of LAF on expression and localization of YAP were detected by immunofluorescence analysis,in both 14-3-3 σ normally expressed and 14-3-3 σ knockdown HeLa cells.Results(1)LAF(10-75 μmol/L)significantly inhibited the proliferation of Hela cells,SW480 cells,PC3 cells and MDA-MB-231 cells in time-and dose-dependent manners.Treatment with 50 umol/L of LAF for 48 h achieved inhibitory rates of~50%on cell proliferation.(2)LAF(25 μmol/L-75 μmol/L)induced apoptosis of Hela cells,SW480 cells,PC3 cells and MDA-MB-231 cells concentration-dependently.Treatment with 75 μmol/L of LAF for 48 hours achieved apoptotic rates of 53.6%,64.2%,81.2%and 75.8%for Hela cells,SW480 cells,PC3 cells and MDA-MB-231 cells,respectively.(3)Pretreatment with LAF(10-50μmol/L)for 48 hours significantly suppressed the migration and invasion of Hela cells and MDA-MB-231 cells.The suppression of LAF on migration and invasion of the two kinds of cancer cells exhibited strong concentration-dependence,with the suppression to MDA-MB-231 cells stronger.Compared to control cells,MDA-MB-231 cells treated with 50 μmol/L of LAF showed only 6.6%and 24.7%of the migration rate and invasion rate.(4)Hela cells,SW480 cells,PC3 cells and MDA-MB-231 cells treated with LAF(10-50μmol/L)for 24 hours,48 hours or 72 hours expressed reduced protein levels of YAP,C-myc,BIRC5 and Bcl-2.The inhibition of LAF on the expression of YAP and its target genes was dose-and time-dependent.VP(1 μmol/L),an inhibitor of YAP,also significantly reduced the expression of YAP,C-myc,BIRC5 and Bcl-2 in the four cancer cell lines.(5)Results from immunofluorescence analysis showed that LAF significantly inhibited the accumulation of YAP within the nucleus.(6)Pretreatment with LAF(10-50μmol/L)for 24,48 and 72 hours elevated the protein level of 14-3-3 σ in Hela cells,SW480 cells,PC3 cells and MDA-MB-231 cells significantly.The stimulatory effect of LAF on 14-3-3 σ gene expression showed to be concentration-and time-dependent.(7)The inhibition of 14-3-3 σ by siRNA interference resulted in elevated expression of YAP in HeLa cells.Along with the inhibition of 14-3-3 σ,the inhibition on YAP expression by LAF was significantly weaken.ConclusionIn this study,LAF significantly inhibited the proliferation,migration and invasion of human cervical cancer Hela cells,human colon cancer SW480 cells,human prostate cancer PC3 cells and human breast cancer MDA-MB-231 cells,and promoted the apoptosis of these four kinds of cancer cells.The mechanism maybe that LAF upregulated the expression of 14-3-3 σ,which retained the YAP in the cytoplasmic by binding with YAP,leading to the degradation of YAP and reduced nuclear accumulation of YAP,and therefore inhibition of YAP-mediated proliferation,migration,invasion and apoptosis.