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

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.


Introduction
Lung cancer, the most frequent cancer in both sexes (18.4% of the total cancer cases in 2018), has the highest mortality rate of all malignant tumors globally [1,2]. Adenocarcinomas, which comprise approximately 80% of lung cancer cases [3], are classified as non-small-cell lung cancers (NSCLCs). exhibits significant anticancer activities that inhibit the growth of prostate cancer (PC-3, DU-145 and LNCaP cells) in-vitro and in-vivo [51,52] and induce apoptosis in hT-29 human colon cancer cells [53]. In addition, many critical literatures has been reported that ViceninII has shown anti-inflammatory effect through the inhibition of TGF-β-induced protein signaling pathway as well as induced apoptosis of lung cancer H23 cell via PI3K/Akt/mTOR signaling [54,55]. However, none of the previous investigations investigated the anti-metastasis effects and its molecular mechanism of ViceninII in lung adenocarcinoma A549 and H1299 cells.
In this experiment, the structure of ViceninII was identified by UV, ESIMS, and NMR, and confirmed by comparing the spectral data following extraction from Dendrobium officinale leaves. The addition of 5 ng/mL of TGF-β1 can facilitate EMT and activate the TGF-β/Smad and PI3K/Akt/mTOR pathways in A549 and H1299 cells, so we assumed that ViceninII would reverse TGF-β1-induced EMT by inactivating these two signaling pathways. From these investigations, we present evidence that ViceninII prominently antagonizes TGF-β1-induced EMT by inactivating TGF-β/Smad and PI3K/Akt/mTOR pathways in lung adenocarcinoma A549 and H1299 cells. This is the first time that the anti-metastatic effect of ViceninII has been proven, and a reliable molecular mechanism provided. ViceninII may be a promising repressor against the metastasis of lung adenocarcinoma. Compound 1 turned fuchsia after reacting with magnesite powder and HCl, producing a purple cycle between the two liquid levels after the Molish reaction. UV spectrum absorption peaks were exhibited at 336 nm and 271 nm, suggesting the characteristics of the flavonoid glycoside skeleton. The ESI-MS results showed that the m/z of compound 1 was 593 [M−H] − in the negative ion mode. It produced four prominent fragment ions at m/z 503 (C 24 O 7 ) in the MS 2 spectra. Therefore, the molecular weight of the compound was 594. The MS spectra, MS 2 spectra, and molecular formula of the spectra of C 27 H 30 O 14 are shown in Figure 1.

Structural Identification of ViceninII
The 1 H-NMR spectrum of 1 showed a signal at δ 6.58 and the absence of H-6 and H-8, indicating that C-6 and C-8 had been substituted. The signals at δ 6.89 and δ 7.94 suggested that only a p-hydroxy group existed in ring B. The signals at δ 4.92 and δ 5.11 came from the anomeric positions of sugar. After the subtraction of 15 carbons from the flavone skeleton, another 12 carbons remained in the aglycone of 1. A comparison of the spectral data with the reference and literature data confirmed the identification of compound 1 as ViceninII [56]. The 1 H-NMR and 13
As shown in Figure 2C, TGF-β1 (5 ng/mL) treatment successfully promoted the EMT by remarkably inducing the disappearance of intercellular junctions and spindle-like appearance of A549 and H1299 cells over 48 h, instead inducing a fibroblast-like appearance with a long shape and central nucleus. These morphological changes were obviously reversed by the cotreatment of TGF-β1 with ViceninⅡ (2.5, 5, and 10 μM) for the last 24 h.
As shown in Figure 2C, TGF-β1 (5 ng/mL) treatment successfully promoted the EMT by remarkably inducing the disappearance of intercellular junctions and spindle-like appearance of A549 and H1299 cells over 48 h, instead inducing a fibroblast-like appearance with a long shape and central nucleus. These morphological changes were obviously reversed by the cotreatment of TGF-β1 with ViceninII (2.5, 5, and 10 µM) for the last 24 h.

Clonogenic Formation in Human Lung Adenocarcinoma A549 and H1299 Cells
A colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. To investigate the clonogenic potential and further anticancer activity of ViceninII (2.5, 5 and 10 µM) on its long-term efficacy against A549 and H1299 cells, a colony formation assay was performed. The results suggested that TGF-β1 treatment significantly increased colony amounts compared with the untreated cells, while significant dose-dependent decreases in the number of colonies were observed for ViceninII (10 µM) treatments in A549 and H1299 cells (Figure 3, p < 0.01).

Clonogenic Formation in Human Lung Adenocarcinoma A549 and H1299 Cells
A colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. To investigate the clonogenic potential and further anticancer activity of ViceninⅡ (2.5, 5 and 10 μM) on its long-term efficacy against A549 and H1299 cells, a colony formation assay was performed. The results suggested that TGF-β1 treatment significantly increased colony amounts compared with the untreated cells, while significant dose-dependent decreases in the number of colonies were observed for ViceninⅡ (10 μM) treatments in A549 and H1299 cells ( Figure 3, p < 0.01).  . 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.

ViceninⅡ Suppressed TGF-β1-Induced Migration and Invasion in A549 and H1299 Cells
The TGF-β1-mediated EMT of human lung cancer cells is closely related to the lung cancer metastatic characteristics, including migration and invasion. Compared with the control group, TGF-β1-treated cells showed highly enhanced migration potential in a wound-healing assay after 48 h, which was significantly suppressed by co-treatment with ViceninⅡ (2.5, 5, and 10 μM) for the last 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.

ViceninII Suppressed TGF-β1-Induced Migration and Invasion in A549 and H1299 Cells
The TGF-β1-mediated EMT of human lung cancer cells is closely related to the lung cancer metastatic characteristics, including migration and invasion. Compared with the control group, TGF-β1-treated cells showed highly enhanced migration potential in a wound-healing assay after 48 h, which was significantly suppressed by co-treatment with ViceninII (2.5, 5, and 10 µM) for the last 24 h ( Figure 4A,B, p < 0.01). In addition, TGF-β1 obviously promoted invasion, as evidenced by the increased number of migrated cells in the transwell assay, which also dramatically decreased in a dose-dependent manner when co-treated with ViceninII ( Figure 4A,B, p < 0.01). These results prove that ViceninII effectively inhibited the TGF-β1-induced migration and invasion of A549 and H1299 cells. 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.

ViceninⅡ Suppressed TGF-β1-Induced Migration and Invasion in A549 and H1299 Cells
The TGF-β1-mediated EMT of human lung cancer cells is closely related to the lung cancer metastatic characteristics, including migration and invasion. Compared with the control group, TGF-β1-treated cells showed highly enhanced migration potential in a wound-healing assay after 48 h, which was significantly suppressed by co-treatment with ViceninⅡ (2.5, 5, and 10 μM) for the last 24 h ( Figure 4A,B, p < 0.01). In addition, TGF-β1 obviously promoted invasion, as evidenced by the increased number of migrated cells in the transwell assay, which also dramatically decreased in a dose-dependent manner when co-treated with ViceninⅡ ( Figure 4A,B, p < 0.01). These results prove that ViceninⅡ effectively inhibited the TGF-β1-induced migration and invasion of A549 and 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.

ViceninII Reversed TGF-β1-Induced Expression of EMT Biomarkers in A549 and H1299 Cells
In order to find out whether ViceninII plays a critical role in TGF-β1-induced EMT in A549 and H1299 cells, we used Western blot and immunofluorescence assays to analyze the protein expression of EMT-associated biomarkers. The immunofluorescent staining results showed that TGF-β1-treatment obviously reduced the intensive green fluorescence expression of the epithelial marker E-cadherin and significantly increased the mesenchymal marker vimentin compared to control cells. These phenomena were completely reversed following co-incubation with 10 µM ViceninII ( Figure 5A). In addition, the TGF-β1-induced protein expression of N-cadherin, vimentin, MMP-2, snail, and slug was significantly inhibited after treatment with ViceninII (2.5, 5, and 10 µM), whereas the expression of E-cadherin, Claudin-1, and ZO-1, which was inhibited by TGF-β1, increased obviously in a dose-dependent manner. These results demonstrate that ViceninII could effectively reverse TGF-β1-induced alterations in the protein expression of EMT biomarkers in A549 and H1299 cells. 2.6. The Inhibitory Effect of ViceninII on TGF-β1-Induced EMT in Lung Cancer A549 and H1299 Cells through the TGF-β/Smad and PI3K/Akt/mTOR Pathways TGF-β1 has been shown to activate the canonical Smad pathway and PI3K/Akt/mTOR pathways [16,22]. To analyze the signaling pathways involved in EMT reversion induced by ViceninII in A549 and H1299 cells, we detected the protein expression of the TGF-β1-induced TGF-β/Smad and PI3K /Akt /mTOR pathways. The band intensities shown in the western blot results ( Figure 6A,B) indicate that the proteins p-Smad2, p-Smad3, p-mTOR, p-Akt and p-P70S6K were prominently up-regulated when the cells were treated with 5 ng/mL of TGF-β1, whereas total protein levels of Smad2, Smad3, mTOR, Akt, and P70S6K were essentially unchanged. However, when we added ViceninII (2.5, 5 and 10 µM) as a co-treatment, the phosphorylation of Smad2, Smad3, Akt, mTOR, and P70S6K decreased in a dose-dependent manner ( Figure 6A,B, p < 0.05). Based on the above results, we further explored the functions of the TGF-β/Smad and PI3K/Akt/mTOR pathways by using the specific pharmacological inhibitors SB431542 (SB, TGF-β/Smad inhibitor) and LY294002 (LY, PI3K/Akt inhibitor). A549 and H1299 cells were incubated with 5 ng/mL of TGF-β1 for 48 h and subsequently, co-incubated with 10 µM ViceninII, 20 µM SB431542 and 20 µM LY294002 for the last 24 h. As previously demonstrated, down-regulation of E-cadherin and ZO-1, and up-regulation of N-cadherin, vimentin, MMP-2, snail, and slug by TGF-β1 were reverted by ViceninII in A549 and H1299 cells ( Figure 6C, p < 0.05). Similar and even more effective results were observed following the LY and SB treatment, which was used as a positive control ( Figure 6C, p < 0.05). These results demonstrate, once again, that ViceninII antagonizes TGF-β1-induced EMT through inhibition of the TGF-β/Smad and PI3K/Akt/mTOR pathways in lung adenocarcinoma A549 and H1299 cells.

Discussion
The aim of this study was to analyze the anti-metastatic effects of ViceninII on TGF-β1-induced EMT in lung adenocarcinoma cell lines and to determine its possible molecular mechanism(s) of action.
Lung cancer has the highest morbidity and frequency of all cancers worldwide [1]. Metastasis of lung cancer cells to other vital organs initially happens at the early stages of the disease and leads to an overall five year survival rate of less than 15% [4,57]. Thus, the suppression of lung adenocarcinoma metastasis is vital to improve the survival of lung adenocarcinoma patients. Growing evidence suggests that the EMT is involved in the metastasis process [7], and many studies have reported that TGF-β1 contributes to the induction of EMT through the regulation of E-cadherin, N-cadherin, vimentin, snail, and MMP-2. Other representative characteristics of EMT also account for the migration and invasion of lung cancer cells [58,59]. However, Zheng et al. reported that in genetically engineered mouse models of pancreatic adenocarcinoma development, carcinoma cells could metastasize without activating EMT programs [60]. But the conclusion that EMT is not required for metastatic dissemination would require further evidence [61]. Hence, it is extremely important to develop a novel anti-metastasis agent that targets EMT-related pathways to benefit patients with metastatic lung adenocarcinoma. Due to Chinese medicine theory claims that it clears heat and nourishes the lungs, Dendrobium officinale is widely used in China to treat lung cancer as an adjunctive therapy in order to improve the life quality of patients. ViceninII, a C-glycoside flavonoid extracted from Dendrobium officinale, has been identified and isolated from various medicinal herbs and has shown multiple pharmacological activities, particularly anti-cancer activities [51][52][53], but none of the previous research has focused on the anti-metastasis effects. Therefore, we wondered whether ViceninII could inhibit the TGF-β1-mediated EMT metastasis of lung cancer cells.
In this study, an MTT assay showed that ViceninII has no toxic effect at concentrations less than 10 µM. If the does of ViceninII exceed 10 µM, it would has the killer effect on cells and we could not guarantee the anti-metastatic effect wasn't due to its cytotoxic effects. Treatment with TGF-β1 at >10 ng/mL has an obvious cytotoxic effect as anastomosed in the reports on the regulation of cell proliferation and apoptosis [62]. Meanwhile, some literature has reported the successful induction of EMT on A549 and H1299 cells with 5 ng/mL of TGF-β1 [12,16], and the results of morphology changes prove this. Therefore, we finally chose concentration levels of ViceninII (2.5, 5 and 10 µM) and TGF-β1 (5 ng/mL) to proceed with in further experiments. Furthermore, we found that ViceninII obviously inhibits TGF-β1-induced cloning efficiency, migration, and invasion in a dose-dependent manner, as evidenced by the responses of A549 and H1299 cells to the clonogenic formation, wound healing, and transwell assay.
EMT is characterized by morphological changes of cells, the downregulation of epithelial markers (E-cadherin, ZO-1 and Claudin-1), and the upregulation of mesenchymal markers (N-cadherin and vimentin) and transcription factors (snail and slug) [9]. Members of the matrix metalloproteinase (MMP) family (such as MMP-2) are required for tumor invasion and metastasis [13]. Immunofluorescence staining and western blot analyses demonstrated that ViceninII remarkably reverses the aforementioned EMT-related protein expression induced by TGF-β1. Subsequently, we investigated the mechanisms through which this occurs. The association of TGF-β/Smad and PI3K/Akt/mTOR signaling pathways with the molecular mechanisms of EMT in tumor cells is receiving increasing attention in research [63][64][65]. In the current study, TGF-β1 upregulated the phosphorylate levels of Smad2/3, AKT, and mTOR in parallel with p70S6K, but these effects were inhibited in a dose-dependent manner after the addition of ViceninII, which preliminarily revealed that ViceninII works through the TGF-β/Smad and PI3K/Akt/mTOR pathways.
LY294002, PI3K/Akt pathway inhibitor, has been shown to block PI3 kinase-dependent Akt phosphorylation and kinase activity [66]. SB431542, TGF-β/Smad pathway inhibitor, is a potent and selective ATP-competitive inhibitor of the TGF-β1 activin receptor-like kinases [67]. To further explore the relationship between the EMT and the TGF-β/Smad and Akt/mTOR/P70S6K pathways in lung adenocarcinoma, we used the LY294002 and the SB431542 to confirm that ViceninII plays a suppressive role in the EMT by regulating these pathways, as evidenced by the expression of E-cadherin, N-cadherin, ZO-1, vimentin, snail, slug, and MMP-2. The selection of inhibitor does (20 µM) was referred to the previous literature and specification [39]. The results of the group added inhibitors are similar to those found when co-treating with ViceninII. That is to say, as depicted in Figure 7, ViceninII reverses TGF-β1-mediated EMT via deactivating the TGF-β/Smad and PI3K/Akt/mTOR signaling pathways in A549 and H1299 cells. explore the relationship between the EMT and the TGF-β/Smad and Akt/mTOR/P70S6K pathways in lung adenocarcinoma, we used the LY294002 and the SB431542 to confirm that ViceninⅡ plays a suppressive role in the EMT by regulating these pathways, as evidenced by the expression of E-cadherin, N-cadherin, ZO-1, vimentin, snail, slug, and MMP-2. The selection of inhibitor does (20 μM) was referred to the previous literature and specification [39]. The results of the group added inhibitors are similar to those found when co-treating with ViceninⅡ. That is to say, as depicted in Figure 7, ViceninⅡ reverses TGF-β1-mediated EMT via deactivating the TGF-β/Smad and PI3K/Akt/mTOR signaling pathways in A549 and H1299 cells.
Previous studies have reported that isoviolanthin, a C-glycoside flavonoid with apigenin as the parent nucleus, inhibits TGF-β1-induced EMT in hepatocellular carcinoma cells via the deactivation of the TGF-β/Smad and PI3K/Akt/mTOR signaling pathways [39]. ViceninⅡ, known as apigenin-6,8-di-C-β-D-glucoside, belongs to the rare C-glycoside group of flavonoid glycosides consisting of a flavonoid moiety and two sugar units [38]. The nuclear parent of ViceninⅡ is apigenin and the sugar units, located in C-6 and C-8 respectively, are both glucose compounds. The structure of Vicenin-2 is very similar to that of isoviolanthin. Both of them have an apigenin moiety and C-8 glucose. The difference between them is that the isoviolanthin has a rhamnose unit in C-6. Thus, we speculate that the structural characteristic of the apigenin moiety and polyhydroxyl group may affect the TGF-β/Smad and PI3K/Akt/mTOR signaling pathways. Furthermore, we have identified other compounds with this structural characteristic, and the signaling pathways of those compounds will be experimentally verified in the future.
In summary, the study provides strong evidence that ViceninⅡ inhibits TGF-β1-induced EMT migration and invasion in lung adenocarcinoma A549 and H1299 cells. This is the first time these novel insights into the anti-metastatic effect of ViceninⅡ as a potential repressor have been demonstrated.  Previous studies have reported that isoviolanthin, a C-glycoside flavonoid with apigenin as the parent nucleus, inhibits TGF-β1-induced EMT in hepatocellular carcinoma cells via the deactivation of the TGF-β/Smad and PI3K/Akt/mTOR signaling pathways [39]. ViceninII, known as apigenin-6,8-di-C-β-D-glucoside, belongs to the rare C-glycoside group of flavonoid glycosides consisting of a flavonoid moiety and two sugar units [38]. The nuclear parent of ViceninII is apigenin and the sugar units, located in C-6 and C-8 respectively, are both glucose compounds. The structure of Vicenin-2 is very similar to that of isoviolanthin. Both of them have an apigenin moiety and C-8 glucose. The difference between them is that the isoviolanthin has a rhamnose unit in C-6. Thus, we speculate that the structural characteristic of the apigenin moiety and polyhydroxyl group may affect the TGF-β/Smad and PI3K/Akt/mTOR signaling pathways. Furthermore, we have identified other compounds with this structural characteristic, and the signaling pathways of those compounds will be experimentally verified in the future.
In summary, the study provides strong evidence that ViceninII inhibits TGF-β1-induced EMT migration and invasion in lung adenocarcinoma A549 and H1299 cells. This is the first time these novel insights into the anti-metastatic effect of ViceninII as a potential repressor have been demonstrated.

Plant Material and Reagents
Leaves of Dendrobium officinale were obtained from the Xinyu Ecological Development Co., Ltd.

Extraction and Isolation
The dried Dendrobium officinale leaves (2 kg) were ground and extracted in 70% ethanol (3 × 5 L × 2 h) at room temperature. The extractions were filtered and concentrated to a residue (1183 g), which was suspended in an adequate amount of distilled water. Two volumes of petroleum ether, ethyl acetate, and n-butyl alcohol were sequentially added into the suspension for the extraction. A portion of the n-butyl alcohol extract (142 g) was chromatographed on AB-8 macroporous absorption resin (Guangzhou Wanye Chemical CO., Ltd., Guangzhou, China) and eluted with ethanol-H 2 O (0→15%→30%→50%→90%) to obtain four fractions. The 30% fraction was subjected to ODS (Tianjin Bohong Resin Technology CO., Ltd., Tianjin, China), and eluted with methanol-H 2 O (0→20%→30%→40%→50%). The 30% fraction was further purified by recrystallization to obtain compound 1.

Structural Analysis of ViceninII
The characterization of the flavonoid was done with magnesite powder and HCl. The sugar substituent was evaluated by the Molish reaction. UV, ESIMS (Thermo Finnigan LCQ FLEET, Riviera Beach, FL, USA), 1 H-NMR and a 13 C-NMR (Avance III HD 500 MHz Digital NMR spectrometer Bruker, Karlsruhe, Germany) were used to further analyze the structure.

Cell Culture and Drug Treatment
Human lung adenocarcinoma cell line A549 and H1299 cells were gifted from Dr. Biaoyan Du, Professor, Guangzhou University of Chinese Medicine, Guangzhou, China. The A549 and H1299 cells were cultured in high-glucose Dulbecco's Modified Eagle's Medium (H-DMEM), supplemented with 10% fetal bovine serum (FBS), 0.5% streptomycin, and penicillin, at 37 • C in a humidified atmosphere of 5% CO 2 . All media components were obtained from Gibco (Grand Island, NY, USA). The concentration of TGF-β1 (PeproTech, Rocky Hill, NJ, USA) used to induce EMT was chosen by the MTT assay and morphology observation, based on what some literature has reported previously [58]. In order to guarantee the final DMSO content was less than 0.1%, the cell culture solution of ViceninII was prepared by first dissolving freeze-dried powder with DMSO (Sigma, St. Louis, MO) at a concentration of 10 mM, and then diluting it with H-DMEM medium to different concentrations (2.5, 5, and 10 µM).

Wound Healing Migration Assay
A549 and H1299 cells (3 × 10 5 cells/well) were seeded in 6-well plates for 24 h and a clear cell-free line was manually created by scratching the confluent monolayers with a 100 µL pipette tip in each well. The wounded monolayers were washed three times with PBS and photographed using an inverted microscope (Olympus, Hamburg, Germany) as the picture for 0 h. Then, the cells were co-treated with TGF-β1 (5 ng/mL) for 48 h in combination with ViceninII (2.5, 5 and 10 µM) for the last 24 h. TCell migration was captured as images at 48 h. The migration area was counted by Image-Pro Plus 6.0 software (Bethesda, MD, USA).

Transwell Invasion Assay
A549 and H1299 cells (2 × 10 4 cells/well) were plated in Matrigel-coated top chambers (8 µm pore size, Corning, New York, NY, USA) with 200 µL of serum-free H-DMEM containing ViceninII (2.5, 5 and 10 µM) for 24 h. Meanwhile, 800 µL of H-DMEM (10% FBS) medium with TGF-β1 (5 ng/mL) was used as a chemoattractant in the lower chambers. After incubation for 24 h, the non-migrated cells were removed from the upper surface of the chamber, and the invaded cells were fixed with 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet for 15 min. Then, the invading cells were photographed and counted by Image-Pro Plus 6.0 software (Bethesda, MD, USA).

Immunofluorescence Assay
A549 and H1299 cells (2 × 10 4 cells/well) were seeded in glass slides on 12-well plates and incubated for 24 h. The cells were treated with TGF-β1 (5 ng/mL) for 48 h and ViceninII (10 µM) for the last 24 h. The cell slides were removed from the medium and fixed in 4% paraformaldehyde at room temperature for 15 min, blocked with 5% bovine serum albumin (BSA) and 0.5% Triton X-100 for 2 h at 37 • C, and then incubated overnight at 4 • C with anti-vimentin and anti-ecadherin antibodies (Cell Signaling Technology, Danvers, MA, USA, 1:100). After this, the cell slides were incubated with secondary fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG antibody (Cell Signaling Technology, 1:1000) for 1 h and counterstained with 4 ,6-diamidino-2-phenylindole (DAPI) (Beyotime, Guangzhou, China) for 20 min at room temperature. The cells were viewed and photographed under laser confocal microscopy 880 with Airyscan (Zeiss, Jena, Germany).

Statistical Analysis
Data are shown as the mean ± standard deviation (SD) and the results from each group were obtained using triplicate samples independently. The MS data were analyzed with the Finnigan Xcalibur 2.0 advanced chromatography workstation (Thermo Quest Corporation, San Jose, CA, USA). A one-way ANOVA was used to determine the significant differences among the groups. A probability value of p < 0.05 was considered to be statistically significant. All statistical analyses were conducted with SPSS 20.0 (SPSS Inc., Chicago, IL, USA) and Prism 6.0 software (GraphPad Software Inc., La Jolla, CA, USA).

Conclusions
ViceninII will be one of the most important components responsible for the anti-metastasis effect of Dendrobium officinale. For the first time, we demonstrate that ViceninII targets the TGF-β/Smad and PI3K/Akt/mTOR pathways to inhibit TGF-β1-induced EMT phenotypes in lung adenocarcinoma A549 and H1299 cells. Furthermore, these results provide evidence that ViceninII could be a promising repressor against the metastasis of lung adenocarcinoma by affecting TGF-β1-induced EMT.

Conflicts of Interest:
The authors declare no conflict of interest.