Effect of vitamin D on metastasis of human A549 cells

Lung adenocarcinoma is the most common of all lung cancers. It has serious consequences for human health due to its features such as not showing symptoms at an early stage, being in a rapidly progressive type, being in stage IV, which often shows metastatic features when detected it, and survival time of less than 5 months if distant organ metastases are present. Epithelial mesenchymal transition (EMT) induced by transforming growth factor-beta (TGF-β) contributes to tumor metastasis in lung adenocarcinoma. Prevention of metastasis is one of the targeted methods in cancer therapy. Studies show that adequate levels of vitamin D (VitD) may be important in the prevention of lung cancer. VitD is known to be effective in EMT regulation. The most characteristic feature in EMT is the loss of E-cadherin protein in epithelial cells. Therefore, prevention of loss of E-cadherin protein is of great importance in the prevention of tumor development and spread. The effect of VitD on the promoter methylation of the CDH1 gene encoding the E-cadherin protein is unknown in lung adenocarcinoma. The aim of the current study is to determine the effect of VitD treatments on CDH1 promoter methylation and the effective molecules in this process in human A549 cells stimulated with TGF-β. In the current study, three experimental groups were formed from A549 cells. The cells in the first group were stimulated with TGF-β (5ng/ml), the cells in the second group were treated with 100 µM VitD half an hour before the TGF-β stimulation, the cells in the third group were treated with VitD only. DNA/RNA and protein were isolated from cells collected at 24, 48 and 72 hours after substance administration. Alterations at EMT activity and the expression of molecules effective on CDH1 promoter methylation, such as Kaiso and DNA methyl transferase 3 alpha (DNMT3A) were determined by Western blot, qRT-PCR and bisulfite conversion methylation analyzes in A549 cells stimulated with TGF-β at the presence and absence of VitD. In addition, the effect of VitD in inhibiting cell migration was evaluated by wound healing assays. TGF-β treatment decreased the amount of E-cadherin, CDH1 mRNA, while it increased CDH1 promotor methylation and the expressions of Snail, Kaiso, DNMT3A, ACTA2 mRNA and alpha-smooth muscle actin (α-SMA) molecules. VitD pretreatments regressed TGF-β-induced EMT in A549s. VitD decreased the TGF-β signaling pathway activation and caused a decrease in the CDH1 promotor methylation, amount of Snail protein, which is the repressor transcription factor of the CDH1 gene, and the amounts of Kaiso and DNMT3A proteins, which methylate the CDH1 promoter. Thus, the amount of E-cadherin protein was increased by VitD pretreatment, while the amount of α-SMA was decreased in A549 cells. The current study provides in vitro evidence that the use of VitD can prevent metastasis through reduction of EMT. The data of the current study show that VitD may also be effective in preventing tumor metastasis, by reversing EMT through CDH1 silencing via DNA methylation in A549 cells. Testing of experimental data with clinical studies will determine whether the combined use of VitD with anti-carcinogenic therapeutics in lung adenocarcinoma will be beneficial.