Study On The Effect And Molecular Mechanism Of Metastases Inhibition By Arenobufagin In Prostate Cancer Cells

Background and Objective: Epithelial-mesenchymal transition(EMT)plays an important role in prostate cancer(PCa)metastasis.Thus,development of EMT inhibitors may be a feasible treatment for metastatic PCa.Arenobufagin,isolated from toad venom by our group,is a leading compound of antineoplastic drugs and can inhibit the proliferation of various solid tumors.In this study,we try to investigate whether arenobufagin inhibits prostate cancer PC3 cells EMT and metastases and reveal the molecular mechanism.Methods:(1)MTT assay was used to determine the non-toxic concentration of arenobufagin in PC3 cells;(2)PC3 cells were stained with rhodamine-phalloidin to visualize the actin cytoskeleton;(3)Western blotting was applied to detect the expression of EMT markers in PC3 cells;(4)Wound healing and Transwell assays were carried out to investigate the effect of arenobufagin on migration and invasion of PC3 cells;(5)Immunofluorescence assay was performed to determine the effect of arenobufagin on the nuclear translocation of ?-catenin;(6)qPCR was used to analyze the mRNA level of ?-catenin/TCF/LEF-1 complex related target genes;(7)Dual-luciferase reporter gene assay was applied to detect the transcriptional activity of ?-catenin/TCF/LEF-1 complex;(8)?-catenin overexpression plasmid and siRNA were transiently transfected into PC3 cells respectively to investigate the role of ?-catenin in arenobufagin-mediated EMT inhibiton;(9)Subcutaneous xenograft nude mouse model and lung metastasis model of PC3 cells were established to examine the inhibitory effect of arenobufagin on PC3 cells EMT and metastases in vivo.Immunohistochemistry was conducted to determine the expression of EMT markers in xenograft tumors.Hematoxylin-eosin staining of lung tissues were performed to distinguish tumorous metastatic foci and normal tissues and other visceral tissues were stained with hematoxylin-eosin for pathological examination.Results: The result of MTT assay showed arenobufagin inhibited 10% of PC3 cell growth at the dose of 8 nM,namely the non-toxic concentration.Rhodamine-phalloidin staining assay demonstrated the cytoskeleton of PC3 cells treated with arenobufagin transformed from a mesenchymal-like shape(spindle)to an epithelial-like shape(spherical).Western blotting results showed arenobufagin reduced the expression of mesenchymal markers(ZEB1,N-cadherin,Vimentin,Snail,Slug and Twist1)and elevated the expression of epithelial markers(E-cadherin,ZO1 and Claudin1).Wound healing and Transwell assays displayed arenobufagin attenuated the migration and invasion of PC3 cells.The above results indicated arenobufagin inhibits PC3 cells EMT.In addition,we found arenobufagin down-regulated the mRNA and protein expression of ?-catenin in PC3 cells.Immunofluorescence assay showed the nuclear translocation of ?-catenin was reduced after arenobufagin treatment.The dual-luciferase reporter gene assay demonstrated the transcriptional activity of ?-catenin/TCF/LEF-1 complex was weakened by arenobufagin.The result of qPCR revealed ?-catenin/TCF/LEF-1 complex target genes,which are related to tumor invasion and metastasis,were down-regulated after arenobufagin treatment.Overexpression of ?-catenin in PC3 cells antagonized the EMT inhibition activity of arenobufagin,while knockdown of ?-catenin enhanced the inhibitory effect of arenobufagin on EMT,indicating that ?-catenin is involved in the EMT inhibition effect of arenobufagin in PC3 cells.In addition,arenobufagin restrained EMT of xenograft tumors from PC3 cells-xenografted nude mice,as demonstrated by decreased mesenchymal markers expression and increased epithelial markers expression and reduced the tumor metastatic foci in lung.And no abnormal pathological changes were observed in the visceral organs of arenobufagin-treated nude mice.Conclusion: This study first reported that arenobufagin inhibits PC3 cell EMT by down-regulating ?-catenin,thereby suppressing PCa cells metastases.It also provides new evidence for the development of arenobufagin as a treatment for metastatic prostate cancer.