Structure Identification of ViceninII Extracted from Dendrobium officinale and the Reversal of TGF-β1-Induced Epithelial⁻Mesenchymal Transition in Lung Adenocarcinoma Cells through TGF-β/Smad and PI3K/Akt/mTOR Signaling Pathways

Abstract

ViceninII is a naturally flavonoid glycoside extracted from Dendrobium officinale, a precious Chinese traditional herb, has been proven to be valuable for cancer treatment. Transforming growth factor-β1 (TGF-β1), promotes the induction of epithelial⁻mesenchymal transition (EMT), a process involved in the metastasis of cells that leads to enhanced migration and invasion. However, there is no previously evidence that ViceninII has an inhibitory effect on cancer metastasis, specifically on the TGF-β1-induced EMT process in lung adenocarcinoma cells. In this experiment, we used UV, ESIMS, and NMR to identify the structure of ViceninII.A549 and H1299 cells were treated with TGF-β1 in the absence and presence of ViceninII, and subsequent migration and invasion were measured by wound-healing and transwell assays. The protein localization and expressions were detected by immunofluorescence and Western blotting. The results indicated that TGF-β1 induced spindle-shaped changes, increased migration and invasion, and upregulated or downregulated the relative expression of EMT biomarkers. Meanwhile, these alterations were significantly inhibited when co-treated with ViceninII and inhibitors LY294002 and SB431542. In conclusion, ViceninII inhibited TGF-β1-induced EMT via the deactivation of TGF-β/Smad and PI3K/Akt/mTOR signaling pathways.This is the first time that the anti-metastatic effects of ViceninII have been demonstrated, and their molecular mechanisms provided.

Keywords: A549; Dendrobium officinale; EMT; H1299 cells; PI3K/Akt/mTOR; TGF-β/Smad; ViceninII; lung adenocarcinoma; structural identification.

Figure 1

Structure and MS/MS spectra of ViceninII in negative ion mode.

Figure 2

Effects of ViceninII and transforming growth factor (TGF)-β1 on the viability and morphology changes of lung adenocarcinoma A549 and H1299 cells. The cell viability of A549 and H1299 cells treated with (A) ViceninII (1.25, 2.5, 5, 10, 20, 40 and 80 μM), (B) TGF-β1 (0.625, 1.25, 2.5, 5, 10, 20 and 40 ng/mL) for 24 h and 48 h was determined by MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazoliumbromide] assay. Cells were treated with TGF-β1 (5 ng/mL) for 48 h and co-treated with ViceninII for the last 24 h, and the cell morphology was observed (C). Magnification ×200.

Figure 3

Effects of ViceninII on colony formation in human lung A549 and H1299 cells. After being co-incubated with 5 ng/mL of TGF-β1 and ViceninII (2.5, 5 and 10 μM) for 14 days, the clonogenic potential of each group was analyzed by colony formation assay. Representative graphs and a histogram (mean ± SD) are shown, n = 3. ** p < 0.01 compared with the control group; ^## p < 0.01 compared with the TGF-β1 group.

Figure 4

ViceninII inhibited TGF-β1-induced migration and invasion of (A) A549 and (B) H1299 cells. Cells were treated with TGF-β1 (5 ng/mL) for 48 h and co-treated with ViceninII (2.5, 5 and 10 µM) for the last 24 h. The migration and invasion capacities were measured by the wound healing and transwell assays. Typical graphs and a histogram (mean ± SD) are shown, n = 3. Magnification is ×100. ** p < 0.01 compared with the control group; ^# p < 0.05, ^## p < 0.01 compared with the TGF-β1-treated group.

Figure 5

Effect of ViceninII on the expression of TGF-β1-induced EMT biomarkers in A549 and H1299 cells. (A) After treatment with TGF-β1 (5 ng/mL) for 48 h and co-treatment with 10 µM ViceninII for the last 24 h, immunofluorescence staining was performed to detect E-cadherin and vimentin expression in A549 and H1299 cells by confocal microscopy. Green fluorescence indicates E-cadherin and vimentin positive expression, and blue fluorescence indicates 4′,6-diamidino-2- phenylindole (DAPI)-labeled nuclei. (B) Cells were treated with TGF-β1 (5 ng/mL) for 48 h and co-treated with ViceninII (2.5, 5 and 10 µM) for the last 24 h, and the protein expression of EMT-associated biomarkers was determined by western blot analysis. Scale bars: 20 μm. Typical graphs and a histogram (mean ± SD) are shown, n = 3. ** p < 0.01 compared with the control group; ^## p < 0.01 compared with the TGF-β1-treated group.

Figure 6

Effects of ViceninII on the TGF-β/Smad and PI3K/Akt/mTOR pathways in A549 and H1299 cells. (A) Cells were treated with 5 ng/mL of TGF-β1 for 48 h, during which the indicated concentrations of ViceninII were added for the last 24 h. The expression of TGF-β/Smad related pathway proteins, including p-Smad2, Smad2, p-Smad3, and Smad3, was examined by western blot analysis. (B) The expression of PI3K/Akt/mTOR related pathway proteins, such as p-Akt, Akt, p-mTOR, mTOR, p-P70S6K and P70S6K, was detected by western blot analysis. It was reconfirmed that ViceninII suppresses TGF-β1-induced EMT in A549 and H1299 cells through regulation of the TGF-β/Smad and PI3K/Akt/mTOR pathways. (C) Cells were pre-treated with 5 ng/mL TGF-β1 for 48 h and then treated with 10 μM ViceninII, 20 μM SB431542 (SB, TGF-β/Smad inhibitor) or 20 µM LY294002 (LY, PI3K/Akt inhibitor) for the last 24 h. Then, using western blot, the protein expression of E-cadherin, ZO-1, N-cadherin, MMP-2, vimentin, snail, and slug was analyzed. β-actin was used as a loading control. Typical graphs and a histogram (mean ± SD) are shown, n = 3. ** p < 0.01 compared with the control group; ^## p < 0.01 compared with the TGF-β1-treated group.

Figure 7

Schematic representation of the effect of ViceninII on TGF-β1-induced EMT in lung adenocarcinoma A549 and H1299 cells. ViceninII suppressed the PI3K/Akt/mTOR and TGF-β/Smad pathways and finally inhibited migration and invasion of lung adenocarcinoma cells by affecting TGF-β1-induced EMT. “→” indicates promotion; “⊥” indicates inhibition.