Identifying the Structural Components Responsible for the Antiproliferative Properties of Hydroxychavicol

Abstract

Betel leaves are widely used as herbal medicine in Asia due to their antimicrobial properties. These properties have been attributed to the phenolic compound eugenol and its derivative, hydroxychavicol. Hydroxychavicol has also been shown to inhibit cancer cell proliferation. The main objective of this study was to investigate which structural components of hydroxychavicol are responsible for the antiproliferative property of this compound. Jurkat-E6 cells (JE6) were treated with increasing concentrations (5, 15, and 45 µM) of hydroxychavicol and structural variants of it for 48 h. The results of this study demonstrate that the catechol structure in hydroxychavicol is the structural component that exhibits the highest antiproliferative effect. More specifically, the data show that the six-carbon ring must be aromatic with the two hydroxyl groups attached in an ortho position. Furthermore, this study establishes that the oxygen in the hydroxyl groups has a vital role in the antiproliferative properties of catechol and hydroxychavicol.

1. Introduction

The leaf of Piper betel has been used as a herbal remedy in Asian countries for generations due to its various benefits. Its usage encompasses cultural, spiritual, and medical matters. Research has shown that this herb has antioxidative, anti-inflammatory, antibacterial, and anticancer properties [1,2,3,4,5,6,7].

Studies have shown that these properties are linked to eugenol and hydroxychavicol [7,8,9,10]. Eugenol and its derivative, hydroxychavicol, are phenolic compounds commonly found in and extracted from essential oils from spices such as cloves and cinnamon [3,11,12,13]. A comparative study of both compounds showed that hydroxychavicol possesses more potent antimicrobial properties when tested against various species of oral and biofilm-forming bacteria [13,14,15].

Other studies have shown that eugenol and hydroxychavicol have anticarcinogenic properties as well. Both compounds appear to be exerting their antiproliferative effect through the PI3K/AKT/mTOR pathway in multiple cancer cell lines, including colon, breast, lung, and melanoma, by causing the downregulation of cell cycle-related transcription factors [16], the inhibition of oncogenes [17], and inducing apoptosis and autophagy [6,18,19,20,21,22,23,24,25,26].

As shown in Figure 1, the structures of eugenol and hydroxychavicol both consist of an aromatic benzene ring with an attached propenyl (allyl) side chain. Additional substituents on the benzene ring give rise to the difference in the structures of the two compounds. Eugenol possesses a hydroxyl and a methyl ether moiety, whereas hydroxychavicol has two free hydroxyl groups attached to the benzene ring (a catechol structure).

Cell-based studies testing the antiproliferative effect of hydroxyflavones and trihydroxyflavones have shown that compounds with catechol-like structures have stronger anticancer properties. Almost all compounds with a catechol-like structure demonstrated this property. They have been shown to more effectively (requiring lower concentrations) suppress the proliferation of all the treated cancer cell lines [27,28].

The main objective of the present study was to investigate which structural elements—the aromatic ring, the hydroxyl groups and their positions, or the propenyl chain of the hydroxychavicol molecule—hold the key to the antiproliferative function of this compound. To help us achieve this goal, we used fourteen different compounds sharing various structural characteristics with hydroxychavicol. This study may lead to the discovery of more potent anticancer compounds.

2. Materials and Methods

2.1. Cell Culture

Jurkat, Clone E6 cells (JE6) were grown and subcultured regularly in complete growth media, RPMI 1640 with L-Glutamine and 25 mM HEPES (ThermoFisher Scientific, Grand Island, NY, USA) supplemented with 10% by volume of fetal bovine serum (ThermoFisher Scientific, Grand Island, NY, USA). All cells were grown under standard conditions (37 °C in a humidified 5% CO₂ incubator).

2.2. Chemical Solution Preparation

A 10 mM stock solution in DMSO was prepared for each analyte. This solution was then sterilized by filtration through a 0.22 µm syringe disc filter. The only analyte used in an aqueous solution was mercaptophenol. Studies involving mercaptophenol were compared against water. For all other compounds, an appropriate concentration of DMSO was used as a control.

2.3. Treatment of Cells

Cell concentrations were determined using a Vi-Cell XR Automated Cell Viability Analyzer (Beckman Coulter, Miami, FL, USA). Individual wells of a 12-well plate were seeded with 120,000 cells in 1 mL of complete growth media. After a 24 h incubation period, the wells were treated with 5 μM, 15 μM, and 45 μM of analyte and DMSO (at 0.45%, which corresponds to the DMSO content in the treatment with the highest compound concentration) or water as solvent control. The plates were then incubated for 48 h under standard conditions. After the incubation period, the cells were counted.

2.4. Cell Counting and Viability Determination

JE6 cells were triturated to form a single-cell suspension. The cell viability and count in each well were determined using a Vi-Cell Automated Cell Viability Analyzer.

3. Results

To investigate which structural components of hydroxychavicol exhibit an antiproliferative effect, a select number of structural variants of the said compound were used. The antiproliferative effect of the analytes was compared to that of hydroxychavicol by treating Jurkat, Clone E6 (JE6) cells with increasing concentrations (5 µM, 15 µM, and 45 µM) of the analytes. Following the treatments, the total number of cells in each well was determined and used to calculate the fold increase in the number of cells by comparing it to the number of the initially plated cells. The fold increase observed in the control samples treated with solvent alone (DMSO or water) was set at one and used as a reference for the results obtained from the studied compounds.

The initial study compared the antiproliferative effect of hydroxychavicol and its closest derivatives, eugenol and methyl eugenol (Figure 2). The highest concentration-dependent suppression of cell growth was observed at a 45 µM concentration of hydroxychavicol (63%) compared to the DMSO control, followed by eugenol (53%) and methyl eugenol (39%). It appears that the addition of each hydroxyl group adds to the antiproliferative effect of the compound (t-test p value <0.01 between hydroxychavicol and methyl eugenol). The data clearly show that the reason for the stronger antiproliferative effect of hydroxychavicol compared to that of eugenol is that there are two adjacent hydroxyl groups in the molecule of hydroxychavicol.

To understand if the propenyl chain of hydroxychavicol contributes to the antiproliferative effect of the compound, the JE6 cells were treated with catechol. Figure 3 shows that catechol exerts more potent antiproliferative properties than hydroxychavicol, which becomes apparent at a 45 µM concentration (t-test p-value = 0.059 at 45 µM). This observation demonstrates that the propenyl chain is the reason why hydroxychavicol has a lower antiproliferative effect than catechol.

The next objective of this study was to investigate whether the benzene ring contributes to the antiproliferative effect of hydroxychavicol. To assess this, a sample containing a mixture of cis and trans isomers of cyclohexane-1,2-diol was compared against the activity of catechol. As shown in Figure 4, cyclohexane-1,2-diol had no antiproliferative effect on the JE6 cells. (p-value < 0.00001 at 45 µM), indicating that the two hydroxyl groups must be attached to a benzene ring if the compound is to have any antiproliferative effect.

Next, the proximity, as well as the number of hydroxyl groups on the benzene ring, were investigated (Figure 5). To test the effect of the position of the hydroxyl groups, the JE6 cells were treated with catechol and resorcinol. Catechol’s hydroxyl groups are ortho to one another, whereas resorcinol’s hydroxyl groups are meta (Figure 5). Changing the orientation of the two hydroxyl groups was critical. Resorcinol had no observed effect on the proliferation of the cells (p-value < 0.05 at 45 µM compared to catechol). The number of hydroxyl groups attached to the benzene ring also proved to be important for hindering cell growth. Pyrogallol has an additional hydroxyl group, while phenol lacks one hydroxyl group compared to catechol (Figure 5). Adding a third or removing one of the hydroxyl groups from the catechol molecule also had a detrimental effect on the antiproliferative function of the compound (p-value < 0.01 at 45 µM compared to catechol). It should be noted that pyrogallol had a significantly lower inhibitory effect on cell proliferation than phenol.

Having established that the catechol structure is vital for antiproliferative activity, the proximity of the hydroxyl groups to the benzene was subsequently investigated. Salicyl and phthalyl alcohol were chosen because their hydroxyl groups are spaced from the benzene ring by one or two methylene units (Figure 6). Even though phthalyl alcohol appears to have a slightly stronger antiproliferative effect than salicyl alcohol, both compounds had a much lower impact on the relative cell growth compared to that of catechol (p-values < 0.05 (phthalyl) and =0.06 (salicyl) at 45 µM compared to catechol). These observations confirm that the oxygens of the two hydroxyl groups must be adjacent and directly bound to the aromatic ring.

The role of the heteroatoms in the analyte structure was also investigated by using chlorine and sulfur analogs of catechol (Figure 7). The effect on cell proliferation of 2-mercaptophenol, containing a thiol functional group instead of a hydroxy group, was almost nonexistent (p-value = 0.02 at 45 µM compared to catechol). This observation showed that the oxygens of the hydroxyl groups are essential for the antiproliferative effect of catechol and hydroxychavicol. Additional studies were conducted with compounds containing chlorine substitutions of one or both of the hydroxyl groups. When one of the hydroxyl groups was substituted (2-chlorophenol), no major difference in the activity was observed. However, when both hydroxyl groups were replaced with chlorine atoms (dichlorobenzene) or when the chlorines were attached to a non-aromatic cyclohexane ring (1,2-dichlorohexane), the antiproliferative functionality of the molecule was almost completely lost (p-value < 0.00001 at 45 µM for 1,2-dichlorohexane compared to catechol; the data from dichlorobenzene were not statistically significant). These observations further supported the essential role of the hydroxyl groups (and specifically the essential role of the oxygen atoms) in the catechol structure of hydroxychavicol.

Lastly, the antiproliferative effect of nordihydroguaiaretic acid (NDGA), a structural dimer of hydroxychavicol, was examined (Figure 8). As anticipated, NDGA performed similarly to hydroxychavicol at lower concentrations and significantly better than hydroxychavicol at higher concentrations (Figure 8). These results indicate a proportional influence of the catechol structure on an analyte’s antiproliferative activity (p-value = 0.02 at 45 µM NDGA compared to hydroxychavicol).

4. Discussion

Through conducting the present study, we aimed to identify the structural components of the hydroxychavicol molecule that contribute to its antiproliferative effect. To achieve this, a series of structural variants of hydroxychavicol were investigated, and their antiproliferative properties were compared to those of hydroxychavicol itself. Our findings provide valuable insights into the key structural features responsible for the observed antiproliferative activity.

The structure of hydroxychavicol contains catechol, which is known to have antiproliferative properties [3,4,6,7,8,13,27,28]. Our study demonstrates that the presence of the catechol-like structure is key for the antiproliferative activity of hydroxychavicol and that the attachment of an aliphatic chain to the catechol structure dampens that activity (Figure 3). The presented data show conclusively that the aromatic ring is essential (Figure 4) and also demonstrate that the two hydroxyl groups must be directly attached to the ring and adjacent to one another (Figure 5 and Figure 6). Furthermore, this work establishes that the oxygen of the hydroxyl groups plays a critical role, which cannot be achieved by atoms with similar valence electron configuration, such as sulfur (Figure 7). It appears that the unique size and electronegativity of oxygen are critical for giving catechol, and analogous structures, antiproliferative capabilities.

Understanding the structural features of catechol and hydroxychavicol provides valuable information for the rational design of novel compounds with enhanced antiproliferative activity for potential therapeutic applications. Further studies are required to elucidate the underlying mechanisms of the observed properties and explore the potential clinical applications of these compounds.