Anticancer activities of cyclohexenone derivatives

We designed 21 ethyl 3,5-diphenyl-2-cyclohexenone-6-carboxylate derivatives to identify compounds exhibiting anticancer activity. To measure the inhibitory effects of the compounds on cancer cell growth, a long-term survival clonogenic assay was performed. Since compounds containing a cyclohexenone moiety inhibit the enzyme acetylcholinesterase, an in vitro acetylcholinesterase assay was performed for all 21 cyclohexenone derivatives. To examine the effect of the derivative that exhibited the best cancer cell growth inhibition on the induction of apoptosis by demonstrating the activation of caspases and apoptosis regulatory proteins, immunoblotting and immunofluorescence microscopic analyses were performed. The binding mode between the cyclohexenone derivatives and acetylcholinesterase was elucidated at the molecular level using in silico docking. Druggability was evaluated based on ligand efficiency.

Of the multiple methods that can be used to screen anticancer compounds, we opted for a clonogenic long-term survival assay, given its ability to distinguish small differences in the antitumor activity caused by compounds with similar structures [18]. To verify the biological activity of the cyclohexenone derivatives, we determined the activation of the apoptosis regulatory protein caspases [19], through immunoblotting and immunofluorescence microscopy in HCT116 colon cancer cells. Compounds containing a cyclohexenone moiety have been reported to inhibit acetylcholinesterase (AChE) [20]. In this study, we predicted that ethyl 3,5-diphenyl-2-cyclohexenone-6-carboxylate derivative binds to AChE using an in silico docking experiments. The druggability of the cyclohexenone derivatives was evaluated based on ligand efficiency. Our results suggest that ethyl 3,5-diphenyl-2-cyclohexenone-6-carboxylate derivatives inhibit AChE activity and trigger apoptosis in HCT116 colon cancer cells.

Materials and methods
Twenty-one derivatives of ethyl 3,5-diphenyl-2-cyclohexenone-6-carboxylate (listed in Table 1) were prepared according to the previous report [21]. A clonogenic longterm survival assay was performed in HCT116 human colon cancer cells, as previously described [22]. HCT116 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (HyClone, Logan, UT, USA). The cells were seeded into 24-well tissue culture plates (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) at 3 × 10 3 cells per well. After attachment, the cells were exposed to different concentrations of the derivatives (0, 5, 10, 20, and 40 μM) for 7 days, followed by fixation in 6% (w/v) glutaraldehyde and staining with 0.1% (w/v) crystal violet [23]. For all of these analyses, previously reported methods were followed [25].
An in vitro acetylcholinesterase enzyme activity assay was performed using an acetylcholinesterase assay kit (fluorometric-green; ab138872; Abcam, Cambridge, UK). Thiocholine produced from the hydrolysis of acetylthiocholine by AChE can be quantified using thiol green indicator. The intensity of thiol green indicator was measured on an FS-2 fluorescence spectrophotometer (Scinco, Seoul, Korea) fitted with a xenon lamp and bandwidth-adjustable filters for the excitation and emission wavelengths. Samples of all 21 derivatives were dissolved in dimethylsulfoxide and adjusted to 1, 5, 10, 50, and 80 μM concentrations. Donepezil (used as a reference drug) solutions were prepared at 0.001, 0.01, 0.1, 1, 5, and 10 μM concentrations. The excitation and emission wavelengths were 490 and 520 nm, respectively. IC 50 values were determined using Sigma-Plot, following the manufacturer's protocol.
Immunofluorescence staining was conducted as described previously [26]. HCT116 cells were cultured on coverslips and treated with derivative 21 for 12 h. After fixing the cells with 4% paraformaldehyde and permeabilizing them with 0.1% (v/v) Triton X-100, the cells were incubated with primary antibodies against α/β-tubulin and cleaved caspase-7 for 2 h at 25 ℃. It was probed with Alexa Fluor 488-(for α/β-tubulin; green signal) and Alexa Fluor 555-conjugated (for cleaved caspase-7; red signal) secondary antibodies for 30 min at 25 ℃. Nuclear DNA was counterstained with 1 μg/mL Hoechst 33,258 for 10 min (blue signal). Fluorescence was examined under an EVOS FL fluorescence microscope (Advanced Microscopy Group; Bothell, WA, USA).
As a three-dimensional (3D) structure of AChE, 4ey7. pdb deposited in the protein data bank (PDB) was selected [27] for the in silico docking experiments conducted on a Linux PC using the Sybyl program (Tripos,  St. Louis, MO, USA). The experimental procedures followed have been described previously in detail [28].

Results and discussion
The 21 cyclohexenone derivatives used in this study contained 3 substituents, including 3,5-diphenyl rings and ethyl 6-carboxylate (Fig. 2). The 3,5-diphenyl group was substituted with 1-naphthalene or 2-naphthalene. The clonogenic assay shows that most of the derivatives inhibited the clonogenic ability of HCT116 cells at concentrations of 20-40 μM (Fig. 3). The 1-naphthalenyl substituents exhibited better inhibitory activity on the clonogenicity of HCT116 cells than the 2-naphthalenyl substituents, and the number and position of the substituents attached to the 3-phenyl ring, including the hydroxy and methoxy groups, did not affect the clonogenicity of HCT116 cells (Table 1). These data suggest that cyclohexenone derivatives used in this study exhibit anticancer properties (Fig. 4).
AChE is a serine hydrolase found at cholinergic synapses and neuromuscular junctions. It plays a crucial role in terminating neuronal transmission by hydrolyzing acetylcholine into acetic acid and choline [30]. In addition to the classical function of acetylcholine hydrolysis, AChE also exerts non-cholinergic functions such as the promotion of apoptosis in HeLa cells [31] and fibroblasts [32]. However, several studies reported that acetylcholine induces the proliferation of gastric cancer cells [33] and stimulates lung cancer growth [34,35]. The enzymatic activity of AChE is enhanced in meningiomas, gliomas, and vestibular nerve schwannomas [36][37][38][39]. Thus, AChE functions differently in different tissue types. In human colon cancer cells, the stimulation of the muscarinic cholinergic M3 receptor induced cellular proliferation [40] and stimulated migration and invasion [41,42]. In addition, acetylcholine is synthesized and mediates the autocrine stimulation of colon cancer cell proliferation [43]. These findings suggest that AChE inhibitors may be useful chemotherapeutic agents against colon cancer.
Caspases are cysteine-dependent proteases that mediate the apoptosis triggered by various anticancer agents [19]. Caspases are activated via proteolytic cleavage during the initiation (caspase-9) and execution stages (caspase-3 and -7) of apoptosis [19]. To investigate whether the inhibition of AChE induces apoptosis, we treated HCT116 cells with 50 μM derivative 21, which showed the highest AChE inhibitory activity, and examined the activation of caspases through immunoblotting. Upon Fig. 3 Effect of 21 cyclohexenone derivatives on the clonogenicity of HCT116 cells treatment with derivative 21, the proteolytic fragments of the initiator caspase (caspase-9) and executioner caspases (caspase-3 and -7) increased in a time-dependent manner (Fig. 5a). Poly(ADP-ribose) polymerase (PARP) is a substrate protein of caspase-3 and -7 [29]. The extent of the proteolytic cleavage of PARP was also increased as a function of time, following treatment with derivative 21 (Fig. 5a). Immunofluorescence microscopy also showed high levels of cleaved caspase-7 upon treatment with derivative 20 (Fig. 5b). Notably, fragmented nuclei, a characteristic of apoptotic cells, appeared after treatment with derivative 21. These data suggest that derivative 21 triggers apoptosis via the activation of the caspase cascade in HCT116 colon cancer cells.
To elucidate the binding mode of AChE and cyclohexenone derivatives, we conducted in silico docking experiments using derivative 21. Multiple 3D structures of AChE are available in the PDB. Even the PDB structures 1f8u.pdb and 4bdt.pdb contained the highest number of residues, with resolutions of 2.90 Å and 3.10 Å, respectively [46,47]; they did not contain donepezil as the ligand, which was used as a reference compound in the current in vitro AChE enzyme assay. Therefore, 4ey7.pdb containing donepezil as its ligand and with a resolution of 2.35 Å was selected for in silico docking [27]. Although human AChE consists of 614 amino acids, 4ey7.pdb contains 542 amino acid residues (Gly33-Thr574), with homodimer polypeptides A and B. Since chain A contained more unmodeled residues than chain B, the latter was chosen for docking purposes. The apo-protein of chain B, prepared using the Sybyl program, was subjected to energy minimization. The root mean square deviation between the crystallographic structure and energy minimized apo-protein was 0.22 Å. Residues in the binding site were determined via LigPlot analysis [48]: Try103, Trp117, Gly152, Tyr155, Glu233, Ser234, Trp317, Ser324, Phe326, Tyr368, Phe369, Tyr372, and His478. The 3D structure of derivative 21 was determined based on the 3D structure of 5-pentafluorophenyl-3-phenyl-2-cyclohexenone available at PubChem. The Sybyl program provides a flexible docking method. To confirm the accuracy of the docking procedures, the donepezil ligand of 4ey7.pdb that was docked onto the apo-protein of AChE chain B was observed to dock well (Additional file 1: Fig. S1). The Sybyl program generated 30 ligand-protein complexes owing to the 30 repeated iterations. The binding energy obtained from the docking results ranged between 13.55 and − 10.62 kcal/ mol. Likewise, in silico docking for derivative 21 revealed the binding energy to range from − 11.96 to − 10.91 kcal/mol. The derivative 21 ligands generated by 30 iteraton were docked into the apo-AChE protein well (Additional file 1: Fig. S2). The complex with the lowest binding energy showed the best docking pose (Additional file 1: Fig. S3) and was thus subjected to analysis using LigPlot (Additional file 1: Fig. S4). Nine residues, including Trp286, Leu289, Glu292, Val294, Arg296, Phe297, Phe338, Tyr341, and Gly342, showed hydrophobic interactions, and two residues, Ser293 and Phe295, formed hydrogen bonds (H-bonds). H-bonds were observed between the amino proton of the peptide bond of Ser293 and the ketone oxygen of carboxylic acid (3.11 Å) and between the amino proton The molecular weights of the 21 cyclohexenone derivatives ranged between 386 and 476 Da (Table 1). While 18 derivative compounds were novel, derivatives 1, 10, and 20, though not published, were registered with the Chemical Abstract Service (https ://www.cas.org/), vide registration numbers, 1632161-14-8, 1194722-11-6, and 52220-43-6, respectively. Lead optimization, necessary to obtain novel and active compounds, resulted in increased molecular weights, with a subsequent enhancement in hydrophobicity as well [49]. Hydrophobicity can be predicted by the logP values. In this study, the logP values that were calculated using the Sybyl/MOLCAD module, ranged from 4.10 to 4.48 (Table 1). Because highly , and whole-cell lysates were subjected to immunoblotting using antibodies specific for the cleaved caspases. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to ensure the same amount of protein loading. b HCT116 cells cultured on coverslips were treated with 50 μM derivative 21 for 12 h. The cells were fixed and incubated with antibodies against α/β-tubulin and cleaved caspase-7 for 2 h, followed by incubation with AlexaFluor 488-(green signal) and AlexaFluor 555-conjugated (red signal) secondary antibodies for 30 min. Nuclear DNA was stained with 0.1 μg/mL Hoechst 33,258 for 10 min (blue signal). Arrows indicate the apoptotic nuclear fragments. Fluorescence-positive cells were examined under an EVOS FL ® fluorescence microscope. Dotted boxes are enlarged on the right. Size bars, 50 μm lipophilic compounds have low solubility, in vivo experiments, including the preclinical phase, are challenging to perform. Therefore, this requires an additional delivery system.
Ligand efficiency (LE) is the ratio of the ligand affinity for the target molecules to the number of heavy atoms except for hydrogen atoms [49], which has been used to rank hit compounds for drug development. LE can be obtained from the equation as follows: where ΔG and HA denote the Gibbs free energy change associated with the binding of the ligand-protein complex and the number of non-hydrogen atoms, respectively [50]. The LE value for the derivative 21apo-AChE complex obtained using this equation was 0.36 (ΔG = − 11.88 kcal/mol, HA = 33), which falls into the criteria of LE values for drug-like compounds [50]. Therefore, the cyclohexenone derivatives obtained in this research can be considered potent chemotherapeutic agents. However, the study has some limitations. The limited number of tested derivatives in this study may not provide sufficient evidence for the chemotherapeutic potential. Further in vivo studies are needed to confirm the correlation between the cyclohexenone LE = − �G/HA derivative-induced AChE inhibitory activity and anticancer property.