Salinomycin Suppresses TGF-?1-induced Epithelial-tomesenchymal Transition In MCF-7 Human Breast Cancer Cells And It's Mechanism

Research background: Breast cancer is the most common malignant tumor in global female.Invasion and metastasis is the basic feature of malignant tumor,especially in the advanced neoplasm.Research shows that breast cancer metastasis is the major cause of death from breast cancer.The induction of epithelial-to-mesenchymal transition(EMT)is involved in most malignant tumor,thus making cancer cells obtain the characteristic of stem cells,enhancing the ability of movement,invasion and metastasis of tumor cells.EMT indicates that epithelial cells translate into mesenchymal cells via specified program.EMT plays a vital role in embryonic development,chronic inflammation,tissue reconstruction,spread of cancer cells and various fibrotic diseases.The main traits of EMT include cell adhesion molecule(E-cadherin)present low expression and mesenchymal marker(Vimentin)present high expression.When cancer cells happened EMT,On one hand,epithelial cell loses the connection with basement membrane as well as cell polarity,On the other hand,cells obtains higher ability of invasion and metastasis,anti-apoptosis,degrade extracellular matrix.Multiple signaling pathways involved in the process of EMT regulation.These pathways include TGF-?/Smad signaling,Wnt/?-catenin signaling,Ras-MAPK signaling,PI3K/AKT signaling,Src/Rho kinase and so on.These pathways interact with each other to induce the process of EMT by breaking down intercellular connection and changing the polarity of cells.The transcription factors involved in EMT include Snail/Slug,Twist,NF-?B and so on.Snail and Slug belong to the superfamily of zincfinger protein Snail,they can competitive bind the promoter region of E-cadherin in order to suppress the expression of E-cadherin.Twist belongs to alkaline spiral-ring-spiral protein family,by means of promotes the expression of Snail to induce the process of EMT.NF-?B binds to the prompter region of Vimentin,advancing the content of Twist and Vimentin.So these EMT related transcription factors have synergistic effect between each other.Salinomycin(Sal),a potassium ionophore antibiotic,traditional effectively used as an anticoccidial drug for increasing the weight of treated poultry.It was a big surprise when Gupta and his colleagues revealed that Sal selectively killed human breast cancer stem cells.Recently,we reviewed the druggability of Sal,no promising document has been found to demonstrate whether Sal plays a role in migratory and invasive properties as well as EMT process of human breast cancer cells.In our experiments,we intended to explore the effect of Sal on TGF-?1-induced migratory and invasion of MCF-7 cells as well as EMT-related phenotypic and genetic changes and the possible mechanism.This study is the first to report that Sal suppresses the TGF-?1-induced EMT in MCF-7human breast cancer cells by down regulating Smad and non-Smad signaling pathways.Our experiments provide theoretical basis for the clinical application of Sal,offering a new treatment for control of breast cancer recurrence and metastasis.Methods:(1)Cells were divided into four groups: Control(-TGF-?1/-Sal),TGF-?1alone(+TGF-?1/-Sal),TGF-?1 and Sal(+TGF-?1/+0.5?M Sal)or(+TGF-?1/+1.0?M Sal).(2)Wound healing assay measured the move ability of MCF-7 cells.Cells were scraped and allowed to migrate to sub-confluence in serum-free medium for 48 hours.The cell migration images were photographed at 0,24 and 48 hours following scraping.We examined the effect of Sal on TGF-?1-induced invasion by Boyden chamber assay.The system was incubated for 24 hours or 48 hours and then photographed.(3)We detected the protein content of MCF-7 cells by Western blot.Detection index include: matrix metalloproteinases—MMP-2,epithelial maker—E-cadherin,mesenchymal maker—Vimentin,key molecule of TGF-?/Smad signaling—p-Smad2/3,key molecule of Wnt/?-catenin signaling—?-catenin,key molecule of MAPKs—p-p38 MAPK,EMT related transcription factor—Snail1.Meanwhile,adopting gelatin zymography assay to detect MMP-2 level of MCF-7 cells,immunocytochemistry to observe the expression of E-cadherin,Vimentin and ?-catenin in MCF-7 cells,q RT-PCR technique to compare the Snail1's m RNA content of different groups.(4)Immunohistochemistry method to analysis the expression of E-cadherin and?-catenin in 55 cases of human breast cancer specimens.We calculated the correlation of them by statistics.Results:(1)According to the results of scratch and transwell assay,TGF-?1 endowed MCF-7 cells higher migration and invasion ability.Sal effectively inhibited the migration distance and invasion numbers.Western and gelatin zymography assay detected the level of MMP-2,results showed that TGF-?1 improved the level of MMP-2,Sal lower TGF-?1-induced high level of MMP-2.(2)The detection of EMT marker revealed that TGF-?1decreased the protein level of E-cadherin,elevated the protein level of Vimentin while Sal reversed the effect of TGF-?1.(3)Compared with the blank treated group,TGF-?1 group presented higher expression of P-Smad2/3,Snail1,?-catenin and p-p38 MAPK.Compared with the TGF-?1 treated group,TGF-?1+Sal(0.5?M/1.0?M)groups lowered expression of corresponding protein.(4)Immunohistochemical of 55 cases of human breast cancer tissue displayed that relative to non-metastatic breast cancer,lymph node metastatic breast cancer expressed less E-cadherin and more ?-catenin.Statistical software calculated the correlation coefficient of E-cadherin and ?-catenin(r=-0.769,P=0.033).Conclusion: Sal reversed TGF-?1 induced invasion and metastasis accompanied with down-regulation of MMP-2 by experiments on human breast cancer cell line MCF-7.Sal was able to inhibit TGF-?1-induced EMT phenotypic transition and the activation of key signaling molecules involved in Smad(p-Smad2/3,Snail1)and non-Smad(?-catenin,p-p38 MAPK)signals that cooperatively regulate the induction of EMT.Importantly,in a series of breast cancer specimens,we found strong correlation among E-cadherin expression,?-catenin expression,and the lymph node metastatic potential of breast cancer.Our research suggests that Sal promised to be a chemotherapeutic drug by suppressing the metastasis of breast cancer.