# 4-hexylIncorinol Inhibits Class I Histone Deacetylases in Human Umbilical Cord Endothelial Cells
## 4 Discussion  
In our previous study, 4 HR had wide functions such as antimicrobial effects, anti-cancer effects, proangiogenic effects, anti-inflammatory effects, increasing calcification, and reducing metabolism. These divergent effects should be related to the broad spectrum regulation of cellular events. HDACs are key enzymes in post-translational modifications  
Figure 4: Enzyme inhibition assay. The administration of 4 HR decreased HDAC enzyme activity (\({}^{*}p<0.05\) compared to the control group). Figure 3: Western blot analysis after 20 nM TSA application. (**a**) The administration of 20 nM TSA did not increase the expression of TGF-$1 prominently in HUVECs. (**b**) The administration of 4 HR increased the expression of AIF in HUVECs. Figure 2: Western blot analysis. (**a**) The administration of TSA slightly decreased the expression of HDACs in HUVECs. (**b**) The administration of TSA increased the expression of Ac-lysine in HUVECs. for the epigenetic regulation of various cellular events. In this study, we found the 4 HR's role as an HDACi and that 4 HR decreased the expression level of HDAC1, 3, 4, and 5 in HUVECs. (Figure 1a). In addition, HDAC's enzyme activity was decreased by 4 HR administration (Figure 4). With inhibition of HDAC expression and activity by 4 HR, Ac-lys is increased dose and time-dependently by 4 HR administration (Figure 1b). This inhibitory effect on HDACs was comparable to those of TSA (known HDACi) (Figure 2). To our best knowledge, this was the first study of 4 HR as novel HDACi. In our previous study, 4 HR increased the expression level of AIF in HUVECs [28; 29]. In this study, TSA increased the expression level of AIF in HUVECs, too (Figure 3b). However, increased AIF expression by TSA did not result in the increasing TGF-\(\beta\)1 expression (Figure 3a). Though IC\({}_{50}\) of TSA is \(<\)38 nM, a decreased expression level of cyclin A was observed at 10 uM TSA administration [27]. As 1-100 nM TSA was used in this study, the increasing TGF-\(\beta\)1 expression might be observed in a much higher concentration of TSA. Interestingly, 0.3 uM TSA administration increased TGF \(\beta\) type II receptor expression [31]. As the expression level of AIF was observed in 20 nM TSA application, it looked evident that expression level of TGF-\(\beta\)1 might not be directly associated with AIF expression. Sp1 is a transcription factor for TGF-\(\beta\)1 and its expression is increased by butyrate--another kind of HDACi [32]. Interestingly, 4 HR also increases Sp1 expression in SCC-9 cells [20]. Therefore, increased TGF-\(\beta\)1 expression by 4 HR administration might be associated with its HDACi activity and subsequent promotion of Sp1 expression. Every HDAC has a hydrophobic pocket as its enzymatic domain. The 4 HR has a long alkyl group and is strongly hydrophobic [14]. Therefore, 4 HR might be a decoy molecule which could block the hydrophobic enzymatic domain (Figure 5). TSA has a six carbon chain for inserting the hydrophobic pocket of HDAC (Figure 5) and its derivatives also have a similar structure [27]. The 4 HR has a six carbon alkyl group, but does not have an acetyl group at the end of alkyl chain [14]. The binding affinity of 4 HR on each HDAC might be influenced by its own structure. As 4 HR does not have acetyl group, the binding affinity to HDAC might be weaker than TSA or its derivatives. Actually, TSA showed its HDACi activity at a much lower concentration than 4 HR (Figures 1 and 2). Adding an acetyl or amine group to 4 HR might be an interesting approach for novel HDACi development. In the search for novel HDACi, TSA derivatives having carboxylic acid group have 100 nM as IC\({}_{50}\), however, TSA derivatives having acetylated lysine have 31 uM as IC\({}_{50}\)[27]. Hydroxyl-benzene group might do its role as cap (Figure 5). TSA inhibits angiogenesis by suppressing hypoxia-inducible factor-1\(\alpha\) (HIF1\(\alpha\)) [33]. However, 4 HR increases angiogenesis via the HIF1\(\alpha\)-independent pathway [34].