## 4 Discussion  
EMT is induced by several signaling pathways, including transforming growth factor-\(\beta\)1 (TGF-\(\beta\)1), hypoxia, notch, and hedgehog [19]. In our study, EMT of human lung cancer A549 cells was induced by TGF- \(\beta\). EMT is characterized by the loss of epithelial markers, including vimentin, N-cadherin, fibronectin, and \(\alpha\)-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.  
Compound **1** is known to have diverse bioactivities, including antibacterial [22], antifungal [23], antifouling [24], anti-biofilm activities [25], quorum sensing modulation [26; 27], inhibition of dengue virus replication [28], and induction of mitochondria-mediated apoptosis and tumor growth suppression [29]. Compound **2** was reported to have antifungal [30] and quorum sensing inhibitory activities [26]. Compound **3** was reported to inhibit chymotrypsin A [31], increase the lifespan of _Caenorhabditis elegans_ by SIR-2.1 induction [32], and reverse the resistance of doxorubicin-resistant leukemia P388 cells [33]. However, the EMT-related bioactivities of compounds **1**-**3** were not previously investigated. Compounds **1** and **3** downregulated MMP-2 through the inhibition of TGF-\(\beta\)-mediated phosphorylation of Smad2/3 in A549 cells. Compound **3** also reversed TGF-\(\beta\)-induced EMT markers, N-cadherin, vimentin, and Snail. Although compound **2** exhibited activities through a non-Smad-mediated signaling pathway, it attenuated TGF-\(\beta\)-dependent induction of mesenchymal markers, such as vimentin and Snail. In addition, it modulated the expression level of MMP-2 compared to that in the untreated controls in the A549 cell line. High MMP expression is associated with tumor invasion and metastasis. The present study showed that the increased expression of MMP-2 by TGF-\(\beta\) treatment was inhibited by compounds **1**-**3** (Figure 5). Smad2/3 activated by TGF-\(\beta\) induction translocates to the nucleus, and \(\beta\)-catenin might form a complex with active Smad2/3. The complex may interact with various transcription factors and regulate EMT-related genes. _Cyclo_(1-Pro-L-Leu) (**1**) and N-phenethyllactamase (**3**) inhibited TGF-\(\beta\)/Smad pathways and suppressed metastasis of A549 cells by affecting TGF-\(\beta\)-induced EMT. However, _cyclo_(1-Pro-L-Ile) (**2**) downregulated mesenchymal factors via a non-Smad-mediated signaling pathway. The chemical investigation of _Aquimarina_ sp. MC085 led to the identification of two diketopiperazines [_cyclo_(1-Pro-L-Leu) (**1**) and _cyclo_(L-Pro-L-Ile) (**2**)] and one N-phenethylacetamide (**3**), which inhibit TGF-\(\beta\)-mediated EMT.  
Figure 4: Comparison of the effects of compounds **1**-**3** (50 \(\upmu\)M) on matrix metalloproteinase-2 (MMP-2) activity in A549 human lung carcinoma cells, tested using gelatin zymography. The cell supernatants were harvested and concentrated by centrifugation using Amicon Ultra-4 centrifugal filter devices. Clear bands represent gelatin-cleaved areas.