Resveratrol Inhibits Invasion And Metastasis Of Breast Cancer By Inducing Autophagy Through SIRT3/AMPK Signal Axis

Background:Breast cancer has become the most serious cancer in the world.Although its clinical treatment has been greatly improved,breast cancer is still the malignant tumor with the highest mortality.The main obstacle to the effective treatment of breast cancer is epithelial-mesenchymal transformation(epithelial-mesenchymal transition,EMT).Therefore,effective targeted therapy is urgently needed to block the EMT process.There is evidence that resveratrol(Resv)can inhibit tumor invasion and metastasis.However,the molecular mechanism of Resv in the treatment of breast cancer remains controversial.In order to explore the molecular mechanism of Resv inhibiting invasion and metastasis of breast cancer we established cell model and transplanted tumor model to provide a new idea for the treatment of breast cancer.Objectives:Establishing a model of EMT in breast cancer cell line 4T1 induced by TGF-β1,to explore the mechanism of Resv reversing EMT process and inhibiting tumor cell invasion and migration.Establishing a model of EMT and lung metastasis of transplanted tumor in mice to explore the molecular mechanism of Resv reversing EMT and inhibiting lung metastasis.Methods:1.CCK-8 and cell colony assay were used to detect the proliferation of breast cancer 4T1 cells.2.Transmission electron microscope was used to detect the autophagy structure,and laser confocal technique was used to detect the expression of LC3β,SIRT3 and MMP-9.3.Scratch test,Transwell migration test and Matrigel invasion test were used to detect the migration and invasion ability of 4T1 cells.4.Hematoxylin-eosin staining,immunohistochemistry and Bouin staining were used to detect the growth and lung metastasis of transplanted tumor in mice.5.RT-q PCR and Western blotting were used to detect the m RNA and protein expression of autophagy and EMT related markers.Results:⒈ Compared with the blank control group,Resv effectively inhibited the proliferation of breast cancer cell line 4T1 and up-regulated the level of autophagy.The invasion and metastasis of 4T1 cells pretreated with TGF-β1 was significantly enhanced,and Resv could significantly inhibit the EMT process and invasion and metastasis of 4T1 cells pretreated with TGF-β1.2.In breast cancer 4T1 cells,Resv can significantly up-regulate the SIRT3 phosphorylation of AMPK and the expression of autophagy-related markers while inhibit the proliferation of 4T1 cells mediated by TGF-β1.When 4T1 cells were treated with autophagy inhibitor 3-MA or administrated with shRNA,the phosphorylation of AMPK and the expression of autophagy-related genes and proteins was downregulated,while the expression of MMP-9 and EMT markers was up-regulated again,indicating that Resv may induce autophagy to inhibit TGF-β1-mediated EMT process and invasion,metastasis of breast cancer cells through SIRT3/AMPK pathway.3.The model of lung metastasis of transplanted tumor in mice was established.It was found that TGF-β1 could significantly induce the growth and lung metastasis of breast cancer in mice,while Resv could significantly inhibit the tumor growth,EMT and lung metastasis induced by TGF-β1,especially at 80 mg/kg and 160 mg/kg.Resv had a significant effect on the weight loss of mice at 160 mg/kg,so 80 mg/kg Resv was selected for follow-up experiment.4.4T1 cells were treated with autophagy inhibitors 3-MA and shRNA SIRT3,and then inoculated into mice.It was found that the results of transplanted tumor in mice were consistent with the cell experiment.Resv could significantly up-regulate the SIRT3,phosphorylation of AMPK and the expression of autophagy-related markers,while inhibit the growth,EMT and lung metastasis of TGF-β1-mediated breast cancer in mice.3-MA and shRNA SIRT3 inhibited the effect of Resv,indicating that Resv may induce autophagy through SIRT3/AMPK pathway to inhibit the EMT,growth and lung metastasis of TGF-β1-mediated breast cancer in mice.Conclusions:1.Resv inhibits the proliferation,invasion and metastasis of breast cancer 4T1 cell.2.Resv inhibits TGF-β1-mediated EMT process,invasion and metastasis of breast cancer cells by autophagy induced by SIRT3/AMPK pathway.3.Resv reversed the EMT process of breast cancer transplanted tumor in mice,inhibited the growth and lung metastasis of transplanted tumor as well.4.Resv induces autophagy through SIRT3/AMPK pathway to inhibit the EMT,growth and lung metastasis of TGF-β1-mediated breast cancer in mice.