Calcitriol Inhibits Migration And Invasion Through Suppressing Epithelial-Mesenchymal Transition In Renal Cell Carcinoma Cells

Background and Purpose: In recent years,many studies have shown that low vitamin D status is positively correlated with not only the risk of renal cell carcinoma(RCC)but also the progression of renal cell carcinoma.We designed this study to investigate the association between vitamin D status and renal cell carcinoma and to explore the effects of calcitriol on epithelial-mesenchymal transition,migration and invasion in renal cell carcinoma cells.Materials and Methods: This study is divided into two parts.(1)Population study: this study recruited 40 newly diagnosed renal cell carcinoma patients and 80 age and gender matched controls.Serum vitamin D status and levels of inflammatory cytokines and adhesion molecules in patients and controls were measured to investigate the correlation.(2)To investigate the mechanism by which low vitamin D status affects RCC progression,we investigated the effect of calcitriol,an active vitamin D3,on epithelial-mesenchymal transformation(EMT)of renal cell carcinoma using ACHN,786-O,and CAKI-2 renal cell cancer cell lines.We treated renal cell cancer cells with two models: Model 1,ACHN cells,786-O cells,and CAKI-2 cells,three renal cell cancer cell lines,were pretreated with active vitamin D3(200 nmol/L)for 12 hours,then treated with LPS(2.0 mg /m L)or TGF-?1(10 ng /m L).All cells were collected 24 hours after treatment with LPS or TGF-?1.Model 2,ACHN and CAKI-2 cells,two renal cell cancer cells,were incubated for 12 hours with only active vitamin D3(200 nmol/L),and then cells were collected.The effects of active vitamin D3 on migration and invasion of renal cell cancer cells were evaluated.The effects of active vitamin D3 on TGF-?-dependent or-independent EMT were assessed.Results: Firstly,we investigated the correlation between vitamin D status and serum c-reactive protein and adhesion molecules in renal cell carcinoma patients.Results showed that serum 25(OH)D level was lower in RCC patients than in controls.By contrast,serum levels of CRP,an inflammatory molecule,and ICAM,LAMA4 and Ep CAM,three adhesion molecules,were higher in RCC patients than in controls.All RCC patients were divided into two groups: H-Vit D(>20 ng/ml)or L-Vit D(<20 ng/ml).Interestingly,the levels of serum CRP and all adhesion molecules were higher in RCC patients with L-Vit D than those with H-Vit D.Nuclear vitamin D receptor(VDR)was downregulated and nuclear factor kappa B(NF-?B)was activated in cancerous tissues.The in vitro experiments found that Vit D3 suppressed NF-?B activation and adhesion molecules in RCC cells.Moreover,Vit D3 suppressed NF-?B through reinforcing physical interaction between VDR and NF-?B p65 subunit in RCC cells.Next,we investigated the effects of active vitamin D3 on the migration and invasion of renal cancer cells.Results showed that calcitriol inhibited migration and invasion not only in TGF-?1-stimulated but also TGF-?1-unstimulated RCC cells.Moreover,calcitriol suppressed E-cadherin downregulation and vimentin upregulation not only in TGF-?1-stimulated but also in TGF-?1-unstimulated ACHN and CAKI-2 cells.Calcitriol attenuated LPS-induced upregulation of MMP-2,MMP-7,MMP-9,MMP-26 and u-PA in ACHN cells.In addition,calcitriol blocked TGF-?1-induced nuclear translocation of ZEB1,Snail and Twist1 in ACHN and CAKI-2 cells.Mechanistically,calcitriol suppressed EMT through different signaling pathways:(1)calcitriol suppressed Smad2/3 phosphorylation through reinforcing physical interaction between vitamin D receptor(VDR)and Smad3 in TGF-?1-stimulated RCC cells;(2)calcitriol inhibited STAT3 activation in LPS-stimulated RCC cells;(3)calcitriol inhibited ?-catenin/TCF-4 activation through promoting integration of VDR with ?-catenin in TGF-?1-unstimulated RCC cells.Conclusion: Low vitamin D status is associated with upregulation of inflammatory cytokines and adhesion molecules in serum and tumor tissues of renal cell carcinoma patients.Active vitamin D3 can inhibit migration and invasion of renal cell cancer cells partially by inhibiting smad2/3,STAT3 and ?-catenin mediated EMT.