| Objective: To investigate the activation of peroxisome proliferators activated receptor-γ(PPARγ)signaling pathway,the inhibition of metastasis with the potential mechanism in triple negative breast cancer(TNBC)by VSP-17,a novel PPARγ agonist.Methods:(1)MDA-MB-231 cells were resuspended with PBS and injected into the left and right mammary fat pads of female nude mice to establish the MDA-MB-231 xenograft model.Two weeks later,the mice were treated with VSP-17(20 mg/kg)or rosiglitazone(ROS,20 mg/kg).Then seven weeks later,the mice were dissected and RNA was extracted from there tumor in situ.At the same time,RNA was extracted from MDA-MB-231 cells treated with VSP-17(1,3,10 μM)or ROS(1 μM)for 24 hours,and RT-q PCR was used to analyze the expression of CD36,the target gene of PPARγ,in mice and cells;(2)The human breast cancer cells MDA-MB-231 were regard as the study objects with VSP-17(0.01,0.03,0.1,0.3,1,3,10,30,100 μ m)cultured for 24 h,then MTT reagent was added to detect the cell activity and select the appropriate concentration.According to the results of MTT assay,MDA-MB-231 cells were treated with VSP-17(1,3,10 μM)or ROS(1 μM)for 24 h,and expressions of the metastasis-related factors including breast cancer metastasis suppressor 1(BRMS1),E-cadherin,C-X-C motif chemokine ligand 12(CXCL-12)and metal matrix protein 9(MMP9),ORAI calcium release-activated calcium modulator 1(Orai 1),stromal interaction molecule 1(Stim1),transforming growth factor-β(TGF-β)and vascular endothelial growth factor(VEGF)were analyzed by RTq PCR;Cell migration and invasion assay were used to detect the ability of VSP-17 anti-metastasis in MDA-MB-231 cells.MDA-MB-231 xenograft model was taken as the research object,extract the RNA from its tumor and the m RNA expressions of BRMS1,E-cadherin,CXCL-12,MMP9,Orai1,STIM1,TGF-βand VEGF were detected.Hematoxylin-eosin staining(H&E staining)was used to analyze the effect of VSP-17(20 mg/kg)and ROS(20 mg/kg)on metastasis of tumor cells in mouse livers.(3)Taking human breast cancer cells MDA-MB-231 cells and MDA-MB-453 cells as research objects,and the activation effect of VSP-17(1,3,10 μm)on adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)signaling pathway in TNBC cell lines were analyzed by western blot;MDA-MB-231 cells and MDA-MB-453 cells were treated with AMPK inhibitor compound C(1 μM)or si AMPK and/or VSP-17(10 μM),and the effect of AMPK signaling pathway on VSP-17 anti-migration and invasion were detected by cell migration and invasion assay;The effect of AMPK on VSP-17anti-epithelial mesenchymal transition(EMT)were detected by western blot;After AMPK signaling pathway was inhibited,the effect of VSP-17 on the expression of E-cadherin were analyzed by RT-q PCR.PPARγ(1 μM)antagonist GW9662 or si PPARγ and/or VSP-17(10 μM)were treated with MDA-MB-231 cells and MDA-MB-453 cells,and the effect of PPARγ on the activation effect of VSP-17 on AMPK signaling pathway was analyzed by western blot;In addition,Cell migration and invasion test,western blot,RT-q PCR and immunofluorescence assay were used to detect the effect of PPARγ signaling pathway on vsp-17 antimetastasis.Results:(1)VSP-17 increased the m RNA expression of CD36 in MDA-MB-231 cells and the situ tumor of MDA-MB-231 xenografts.(2)VSP-17 increased the activity of PPARγ reporter gene.(3)VSP-17 bounded to PPARγwith a kinetic inhibition constant(Ki)of 0.27 μM.(4)VSP-17 increased the m RNA expression of E-cadherin in MDA-MB-231 xenograft mice.(5)VSP-17 inhibited the liver metastasis of MDA-MB-231 xenograft mice.(6)VSP-17 inhibited the migration and invasion of MDA-MB-231 cells.(7)VSP-17 increased the m RNA expression of E-cadherin in MDA-MB-231 cells.(8)VSP-17 increased the phosphorylation level of AMPK in MDA-MB-231 and MDAMB-453 cells.(9)Inhibition of AMPK signaling pathway reversed the effect of VSP-17anti-migration and invasion in MDA-MB-231 and MDA-MB-453 cells.(10)Inhibition of AMPK signaling pathway reversed the effect of VSP-17 antiEMT in MDA-MB-231 and MDA-MB-453 cells.(11)Inhibition of PPARγsignaling pathway reversed the effect of VSP-17 increasing AMPK phosphorylation level in MDA-MB-231 cells and MDA-MB-453 cells.(12)Inhibition of PPARγ signaling pathway reversed the effect of VSP-17 antimigration and invasion of MDA-MB-231 cells and MDA-MB-453 cells.(13)Inhibition of PPARγ signaling pathway reversed the effect of VSP-17 anti-EMT in MDA-MB-231 cells and MDA-MB-453 cells.Conclusions:(1)VSP-17 inhibited the metastasis of MDA-MB-231 xenogeneic model by up-regulating the expression of E-cadherin.(2)VSP-17 inhibited the EMT process and inhibited the metastasis of triple negative breast cancer lines via PPARγ/AMPK signaling pathway. |
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