Chemical Investigation of Diketopiperazines and N-Phenethylacetamide Isolated from Aquimarina sp. MC085 and Their Effect on TGF- β -Induced Epithelial–Mesenchymal Transition

Investigation Diketopiperazines and N-Phenethylacetamide Isolated from Aquimarina sp. MC085 and Their Effect on TGF- β -Induced Epithelial–Mesenchymal Transition. Abstract: Chemical investigations of Aquimarina sp. MC085, which suppressed TGF- β -induced epithelial–mesenchymal transition (EMT) in A549 human lung cancer cells, led to the isolation of compounds 1 – 3 . Structural characterization using spectroscopic data analyses in combination with Marfey’s analysis revealed that they were two diketopiperazines [ cyclo ( L -Pro- L -Leu) ( 1 ) and cyclo ( L -Pro- L -Ile) ( 2 )] and one N-phenethylacetamide ( 3 ). Cyclo ( L -Pro- L -Leu) ( 1 ) and N-phenethylactamide ( 3 ) inhibited the TGF- β /Smad pathway and suppressed the metastasis of A549 cells by affecting TGF- β -induced EMT. However, cyclo ( L -Pro- L -Ile) ( 2 ) downregulated mesenchymal factors via a non-Smad-mediated signaling pathway.


Introduction
Marine natural products are considered as rich sources of cytotoxins, many of which could serve as lead compounds for anticancer drug development [1]. Recently, many marine natural products have become accepted as the biosynthetic products of marine microorganisms [2]. Chemical investigations on marine microorganisms have resulted in the discovery of anticancer agents, such as bretuximab vedotin and salinosporamide A [3].
Epithelial-mesenchymal transition (EMT) is an important morphological process that differentiates epithelial cells into mesenchymal phenotype. It is characterized by the loss of polarity, cell-cell contact, and gain of mesenchymal markers. Epithelial cadherin is degraded in the plasma membrane, and desmosomes are inhibited by transcription. EMT facilitates the migration and invasion of cancer cells [4][5][6]. Therefore, EMT inhibitors are effective in cancer chemotherapy, particularly against metastasis.
As part of our efforts to discover bioactive natural products from marine microorganisms, we found that the ethyl acetate extract of marine-derived Aquimarina sp. MC085 inhibited TGF-β-induced EMT in A549 cells. The genus Aquimarina belongs to the family Flavobacteriaceae and is known for its aerobic and halophilic characteristics. Chemical investigations of this genus were rarely reported. The Aquimarina genus was reported to produce algicidal proteins against toxic cyanobacterium Microcystis aeruginosa [15]. Genome mining of Aquimarina sp. Ap349 resulted in the discovery of cuniculenes 6A and 6 B derived from polyketide synthase [16]. However, no other natural product with biological activity was reported from the bacterium in this genus. Bioactivity-guided isolation and chemical investigations of Aquimarina sp. MC085 led to the isolation of three natural products, and a spectroscopic data analysis led to the structural assignment of the isolates.

General Experimental Procedure
Optical rotation was measured using a Jasco DIP-1000 polarimeter (Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra were recorded using a 250 MHz Bruker NMR spectrometer (DMX 250) and 600 MHz Varian NMR spectrometer (VNS-600, Palo Alto, Santa Clara, CA, USA) at the Core Research Support Center for Natural Products and Medical Materials (CRCNM). Low-resolution electrospray ionization MS (LR-ESI-MS) was performed using an Agilent 6120 single-quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with a C3 column (Agilent SB-C3 Zorbax, 5 µm, 4.6 × 150 mm). Isolation of the compounds was carried out using a Waters 1525 binary high-performance liquid chromatography (HPLC) pump having a Waters 996 photodiode array (PDA) with a reversed-phase HPLC (RS Tech, Cheongju, Republic of Korea, Hector-M 5 µm C18, 250 × 4.6 mm).

Fermentation of Aquimarina sp. MC085 and Preparation of Extracts
The bacterial strain Aquimarina sp. MC085 (GenBank Accession No. MG016025) was incubated at 25 • C in a shaking incubator at 150 rpm in 5 L of SYP media. After 7 days, the broth media of strain MC085 was extracted twice with ethyl acetate and the combined extract was evaporated.

Acid Hydrolysis and C3 Marfey's Analysis of Compounds 1 and 2
Each compound (100 µg) was dissolved in 6 N HCl (400 µL) in 4 mL glass vials. The vials were incubated at 110 • C for 4 h with stirring. The resulting acid hydrolysates were dried under a stream of N 2 gas. The dried acid hydrolysates were resuspended in deionized water. After the removal of water under a N 2 gas stream, the hydrolysates were

Cell Culture and Treatment
A549 (human lung carcinoma epithelial) cells were incubated in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum and 1% penicillin-streptomycin at 37 • C in a humidified atmosphere of 5% CO 2 and 95% air. The cells were then treated with compounds 1-3, respectively, in medium for 6 h. This was followed by treatment with TGF-β (5 ng/mL) for 42 h in the presence or absence of compounds at the indicated concentrations.

Cell Viability Assay
An EZ-Cytox assay kit (DoGenBio, Seoul, Korea) was used to assess cell viability. Cells (5 × 10 4 cells/well) were seeded into a 96-well plate. After 24 h, compounds 1-3 were added to serum-free medium for 6 h before TGF-β (5 ng/mL) stimulation. EZ-Cytox reagent was added to the plate and incubated for 1 h at 37 • C. The absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) plate reader.

Western Blotting Analysis
A549 cells were treated with compounds 1-3, as previously described. After 6 h, cells were treated with 5 ng/mL of TGF-β with or without each peptide for 42 h. Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.25% deoxycholate, 1% NP-40, and 1 mM EDTA), and protein concentration was determined using BCA reagent (ThermoScientific, Rockford, IL, USA) according to the manufacturer's instructions.

Gelatin Zymography
Cell supernatants of serum-free cultures were concentrated using Amicon Ultra-4 Centrifugal Filter Devices (Millipore, Billerica, MA, USA). Matrix Metalloproteinase-2 (MMP-2) activity was determined using a Zymogram-PAGE System (Komabiotech, Seoul, Korea) according to the manufacturer's instructions. In brief, after electrophoresis, the gel was washed for 30 min to remove the SDS and then incubated overnight at 37 • C with a developing buffer. Then, the gel was stained with Coomassie Blue R-250 and distained with methanol and acetic acid. The gelatinolytic activities were then visualized as a white band and the gels were scanned.

Statistical Analysis
Results are expressed as mean ± standard deviations (SD). The statistical significance between test groups was determined using a one-way ANOVA multiple comparison test with Turkey's post-test using GraPhad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA). p < 0.05 was considered statistically significant. These data indicated that the molecular formula of compound 1 was C 11 H 18 N 2 O 2 . The 2D NMR data analysis led to the planar structure of 1 as a cyclic dipeptide, cyclo(Pro-Leu), as shown in Figure 1. The absolute configuration of 1 was determined to be cyclo(L-Pro-L-Leu) using C3 Marfey's method ( Figure S8) [17]. showed structural features similar to those of compound 1. However, compound 2 showed a distinctive methyl triplet and a methyl doublet, whereas compound 1 showed two methyl doublets. Based on a careful comparison of spectroscopic data, including 1 H and 13 C NMR data, compound 2 was identified as cyclo(Pro-Ile). In addition, C3 Marfey's analyses configured both constituting amino acids as L ( Figure S9) [17].
Compound 3 was obtained as a white powder. Its molecular formula was assigned as C 10 H 13 NO based on 1D NMR spectra and the presence of a protonated ion at m/z 164.2 [M + H] + in the ESI-MS spectrum. The 1 H and 13 C NMR spectra of compound 3 in CDCl 3 showed five proton signals in the ranges of δ H 7.15-7.35, and resonances for four sp2 methine carbons at δ C 138.97, 128.80, 128.69, and 126.57, corresponding to a mono-substituted phenyl ring. Moreover, signals of two methylene units (δ C 40.78, δ H 2.80; δ C 35.68, δ H 3.49) and an acetyl unit (δ C 170.35; δ C 23.30, δ H 1.92) were observed. By comparing the spectroscopic data with the values provided in the literature, the structure of compound 3 was confirmed to be N-phenethylacetamide [18]. Compounds 1-3 were isolated from microorganisms of the genus Aquimarina for the first time.

The Cell Viability Effect on Comparison of Compounds 1-3 on TGF-β-Induced EMT of A549 Cells
First, we investigated the cytotoxic effects of compounds 1-3 with or without TGF-β on A549 cells using an EZ-Cytox assay. As presented in Figure 2, the cells were treated with various concentrations of compounds 1-3 (100, 50, 25, and 12.5 µM) for 24 h. The cell viability analysis showed that none of the components affected cell viability at concentrations below 50 µM; however, compound 2 showed a significant decrease cell viability at 100 µM concentration. Therefore, further assays were conducted at a concentration of 50 µM.

Comparison of the Protein Expression Profiles of A549 Cells to Determine the Effect of the Treatment with Compounds 1-3 (50 µM), via the Smad2/3 Signaling Pathway, on the TGF-β-Mediated EMT of the Cells
To observe the expression levels of proteins associated with canonical Smad2/3, A549 cells were treated with 50 µM of the test compounds, followed by a TGF-β treatment. Phosphorylation of Smad2/3 was measured using western blotting. On comparison with the control group, the results showed that TGF-β induced the phosphorylation of Smad2/3. Phosphorylated Smad2/3 was inhibited by compounds 1 and 3 ( Figure 3A). We also examined the effect of compounds 1-3 on TGF-β-mediated expression of EMTrelated markers in A549 cells. N-Phenethylacetamide (3) dramatically downregulated mesenchymal phenotypic markers (N-cadherin, vimentin, and Snail) induced by TGFβ (p = 0.0014). The levels of vimentin and Snail were reduced by compound 2 in the TGF-β-treated A549 cells. However, β-catenin levels were not changed in the compounds 1-3-treated cells exposed or not exposed to TGF-β ( Figure 3B).

Comparison of the Effects of the Compounds 1-3 (50 µM) on the Activity of MMP-2 in A549 Human Lung Carcinoma Cells, Tested Using Gelatin Zymography
MMP-2 is the main proteolytic collagenase among MMPs and plays a major role in the invasion and metastasis of cancer cells. Therefore, we investigated the effects of compounds 1-3 on MMP-2 activity. The 62-KDa form was identified as MMP-2. MMP-2 expression was increased by TGF-β stimulation. All cells treated with compounds 1-3 showed decreased MMP-2 activity compared to cells subjected to TGF-β treatment alone (Figure 4).

Discussion
EMT is induced by several signaling pathways, including transforming growth factor-β1 (TGF-β1), hypoxia, notch, and hedgehog [19]. In our study, EMT of human lung cancer A549 cells was induced by TGF-β. EMT is characterized by the loss of epithelial markers, including vimentin, N-cadherin, fibronectin, and α-smooth muscle actin, and an increase in mesenchymal markers [20]. Therefore, we analyzed the protein expression of mesenchymal markers by immunoblotting. MMP-2 is a proteolytic enzyme that induces EMT by breaking down the extracellular matrix of malignant cells [21]. In this study, MMP-2 activity was evaluated using gelatin zymography.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.

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