Effects Of NAMPT On Epithelial-Mesenchymal Transition In A549Cells

Background:Nicotinamide phosphoribosyltransferase (NAMPT) is the key rate-limiting enzyme involved in the NAD (Nicotinamide adenine dinucleotide) salvage synthesis pathway in mammalian cells. It is found that NAMPT is related with tumorigenesis. Compared to normal cells, tumor cells demand much more material and energy, thus their demand for NAMPT is also much higher than that of normal cells. NAMPT specific inhibitor FK866can induce tumor cell death by inhibiting NAMPT activity and reducing synthesis of NAD. Clinical studies showed that the serum level of NAMPT was significantly increased in many cancer patients, and the increment of NAMPT was coincident with the degree of malignancy increased. Some researchers suggested that the level of NAMPT in serum may be thought as a biomarker of tumor malignancy degree. It was reported that NAMPT also increase the expression of VEGF and MMP2/9, consequently, to promote tumor cell migration. It suggests that NAMPT is associated with the development of tumor. There are many biological affairs are involved in the invasion and metastasis of tumor, epithelial-mesenchymal transition (EMT) is proved to be one of the most important.However, there is few study on clarifing the relation between NAMPT specific inhibitor or NAMPT and tumor invasion and metastasis. Therefore, we aim to observe the effects of FK866and extracellular NAMPT on EMT in A549cells, and try to explore the underlying molecular mechanisms involved in these process. Objectives:To observe the effects of nicotinamide phosphoribosyltranserase(NAMPT) inhibitor FK866and extracellular NAMPT (eNAMPT) on epithelial-mesenchymal transition(EMT) in A549cells, and to explore the mechanisms which are involved in the process.Method:1. To investigate the effects of NAMPT inhibitor FK866on EMT in A549cells.1) TGF-β1was used to induce EMT in A549cells, and established EMT model; To detect the expression of epithelial markers E-cadherin, mesenchymal markers vimentin and a-SMA by RT-qPCR assay after FK866treatment.2) FK866was alone administrated to A549cells for different concentrations and different duration, then RT-qPCR was performed to detect the mRNA expression of E-cadherin, vimentin and α-SMA in A549cells.3) The expression of TGF-β1was determined by RT-qPCR assay after the treatment of FK866in A549cells;4) Used NMN to pretreat A549cells, then FK866was used to induce EMT. The expression of E-cadherin, vimentin and α-SMA in A549cells was detected by RT-qPCR assay.5) Used Western blotting to detect the protein expression of ERK1/2and p-ERKl/2; To further confirm ERK pathway was involved, ERK inhibitor U0126was used as pretreatment in FK866-induced EMT in A549cells;6) Detected EMT transcriptional factor Snaill, Slug, Twist by RT-qPCR after FK866treatment in A549cells.2. To investigate the effects of extracellular NAMPT on EMT in A549cells.1) Recombinant human NAMPT proteins, wild-type NAMPT and mutant NAMPT/ H247A were used. TGF-β1was used as positive control.The epithelial markers of E-cadherin, mesenchymal markers of vimentin and α-SMA expression were detected byRT-qPCR.2) ERK inhibitor U0126was used before the treatment of eNAMPT in A549cells,then detected the epithelial markers of E-cadherin, mesenchymal markers of vimentin and α-SMA expression by RT-qPCR.3) Detected EMT transcriptional factor Snaill, Slug, Twist expression by RT-qPCR after A549cells were treated with recombinant human NAMPT protein (500ng/ml).Results:1.1) TGF-β1induced A549cell EMT in concentration-dependent and time-dependent manner; FK866failed to reverse TGF-β1-incuded EMT in A549cells;2) FK866alone caused a significant time-dependent increase in the mRNA expression of mesenchymal markers vimentin and a-SMA;3) FK8661nM significantly increased the mRNA expression of TGF-β1, while FK86610nM decreased the expression of TGF-β1in A549cells, which indicated that upregulated expression of TGF-β1might not be involved in high concentration FK866induced-EMT;4) NMN reversed FK866-induced expression of EMT-related molecular markers, upregulation of E-cadherin and downregulation of vimentin in A549cells;5) Treatment of FK866significantly increased the expression of p-ERK1/2in A549cells. ERK inhibitor U0126pretreatment significantly reversed FK866-induced upregulation of vimentin and α-SMA in A549cells.6) FK866(1nM,72h) treatment increased the expression of EMT transcriptional factors Snaill, Slug, Twist.2.1) Recombinant human NAMPT proteins (wild-type and enzymatic site mutant NAMPT/H247A) reduced the expression of epithelial markers E-cadherin and increased the expression of mesenchymal markers vimentin significantly in A549cells;2) ERK inhibitor U0126pretreatment significantly reversed eNAMPT(500ng/ml) induced-downregulation of E-cadherin and upregulation of vimentin in A549cells;3) Extracellular NAMPT significantly increased the expression of transcriptional factor Snaill, Slug and Twist in A549cells.Conclusions:1. NAMPT inhibitor FK866induce EMT in A549cells, and inhibition of NAMPT enzymatic activity, activation of ERK signaling pathway and regulation of EMT transcriptional factor may be involved in the underlying mechanism of FK866-induced EMT.2. Extracellular NAMPT (in high concentration) induce EMT via non-enzymatic action in A549cells, that is partly through activation of ERK signaling pathway. Futhermore, regulation of EMT transcriptional factor is involved in the mechanism.