Cardol exposure induces cytotoxic and transcriptional changes consistent with CDK2 inhibition in Hep-G2 cells

A exposição ao cardol induz alterações citotóxicas e transcricionais condizentes com a inibição de CDK2 em células Hep-G2

Abstract

Cardol (5-[8(Z), 11(Z)-pentadecadienyl] resorcinol) is a long chain phenolic lipid with reported antitumor properties, but its molecular targets and mechanisms of growth inhibition against hepatocellular carcinoma cells remain unclear. This study aimed to elucidate its cytostatic mechanism and molecular targets in Hep-G2 hepatocellular carcinoma cells. Cytotoxicity was assessed by MTT assay, revealing an IC₅₀ value of 24.56 ± 0.31 µM, and morphological examination revealed cell rounding, shrinkage, and detachment indicative of stress induced death. Gene expression analysis by RT-qPCR after 48 h treatment with cardol at its IC₅₀ value demonstrated significant upregulation of p21 accompanied by downregulation of Cyclin E, Cyclin A, and replication licensing genes (CDC6, MCM2, PCNA), indicative of checkpoint activation and G1/S transition arrest. Molecular docking across 35 cancer-related proteins identified CDK2 (ΔG_bind = -7.84 kcal/mol; K_i ≈ 1.8 µM) and MEK1 (ΔG_bind = -7.66 kcal/mol; K_i ≈ 2 µM) as high-affinity targets, with interaction residues overlapping those of reference inhibitors roscovitine and refametinib, respectively. Inhibition of CDK2 explains the G₁/S arrest and repression of replication factors, whereas MEK1 inhibition suppressed ERK signaling and was likely associated with compensatory upregulation of STAT3, AKT, and c-MYC as part of a survival feedback loop. Consequently, these findings suggest that cardol acts as a multitarget modulator that suppresses proliferation through coordinated inhibition of CDK2-RB-E2F and MEK-ERK signaling, leading to cytostatic arrest that progresses toward cytotoxicity.

Keywords:
cardol; Hep-G2 cell; cell cycle arrest; CDK2 inhibition; molecular docking

Resumo

O cardol (5-[8(Z), 11(Z)-pentadecadienil] resorcinol) é um lipídio fenólico de cadeia longa ao qual são atribuídas propriedades antitumorais, porém seus alvos moleculares e mecanismos de inibição do crescimento contra células de carcinoma hepatocelular permanecem incertos. Este estudo teve como objetivo elucidar seu mecanismo citostático e seus alvos moleculares em células de carcinoma hepatocelular Hep-G2. A citotoxicidade foi avaliada pelo ensaio MTT, revelando um valor de CI₅₀ de 24,56 ± 0,31 µM, e o exame morfológico revelou arredondamento, retração e desprendimento celular, indicativos de morte induzida por estresse. A análise da expressão gênica por RT-qPCR, após 48 horas de tratamento com cardol na concentração equivalente à CI₅₀, demonstrou um aumento significativo na expressão de p21, acompanhado pela redução da expressão de Ciclina E, Ciclina A e de genes de licenciamento da replicação (CDC6, MCM2, PCNA), indicando ativação de ponto de verificação (checkpoint) e paralisação na transição G₁/S. Estudos de ancoramento molecular (docking) em 35 proteínas relacionadas ao câncer identificaram CDK2 (ΔG_bind = -7,84 kcal/mol; K_i ≈ 1,8 µM) e MEK1 (ΔG_bind = -7,66 kcal/mol; K_i ≈ 2 µM) como alvos de alta afinidade, com resíduos de interação sobrepostos aos dos inibidores de referência roscovitina e refametinibe, respectivamente. A inibição de CDK2 explica a paralisação em fase G₁/S e a repressão dos fatores de replicação, enquanto a inibição de MEK1 suprimiu a sinalização ERK e esteve provavelmente associada ao aumento compensatório da expressão de STAT3, AKT e c-MYC, como parte de um mecanismo de retroalimentação de sobrevivência. Consequentemente, esses achados sugerem que o cardol atua como um modulador multialvo que suprime a proliferação por meio da inibição coordenada das vias de sinalização CDK2-RB-E2F e MEK-ERK, levando a uma parada citostática que progride para citotoxicidade.

Palavras-chave:
cardol; célula Hep-G2; parada do ciclo celular; inibição de CDK2; ancoramento molecular

1. Introduction

Hepatocellular carcinoma (HCC) is the most prevalent primary liver malignancy, accounting for over 90% of cases worldwide (Kinsey and Lee, 2024). Over 500 genes contribute to its progression, typically through the activation of multiple signaling pathways, including receptor tyrosine kinases (RTKs), RAS/RAF/MEK/ERK, PI3K/AKT/mTOR, and JAK/STAT (Shiragannavar et al., 2023; Zhou et al., 2024). Conventional treatments for HCC include surgical resection, liver transplantation, and systemic chemotherapy. However, these options are often limited by late-stage diagnosis, drug resistance, and severe adverse effects (Zhu and Sun, 2019). Molecular targeted agents such as sorafenib and lenvatinib offer modest efficacy and are frequently associated with systemic toxicity and acquired resistance (Shaaban et al., 2014). The exploration of alternative therapeutic strategies from natural sources has become increasingly imperative. Propolis, a resinous substance produced by bees from plant exudates, has been widely recognized for its diverse pharmacological activities (El-Didamony et al., 2024). Propolis from stingless bees (Tetragonula spp.) found in tropical regions contains a unique chemical profile rich in phenolics, flavonoids and alkylresorcinols, many of which show potent cytotoxicity against cancer cell lines. (Forma and Bryś, 2021). Phenolic and flavonoid compounds such as chlorogenic acid and gallic acid inhibit Hep-G2 proliferation by inactivating ERK. Similarly, apigenin from Apis mellifera suppresses HCC cell growth by inducing G1 arrest in Hep-G2 cells through activation of the p38/MAPK-p21 pathway and regulation of cyclin D1/CDK4 (Li et al., 2020).

In this study, we investigate the molecular mechanisms by which cardol (5-pentadecylresorcinol) exerts cytotoxic effects on Hep-G2 cells. While cardol has been identified in cashew (Anacardium occidentale), pistachio (Pistacia vera), and mango (Mangifera indica) (Makwana et al., 2022), its specific antitumor mechanisms against hepatocellular carcinoma remain underexplored. We aim to bridge the gap between natural product chemistry and molecular oncology by integrating in vitro assays and in silico docking analysis. In this experiment, cardol compound extracted from Tetragonula laeviceps propolis in previous studies (Meemongkolkiat et al., 2024) was investigated for cytotoxicity and morphological effects in Hep-G2 cells. Subsequently, molecular docking screened cardol against thirty-five proteins associated with HCC to identify potential targets. Finally, RT-qPCR was utilized to quantify the expression of genes associated with cell cycle progression, replication, and survival. This study highlights the therapeutic potential of stingless bee derived compounds and supports the development of anticancer agents with improved specificity and lower toxicity for hepatocellular carcinoma.

2. Material and Methods

2.1. Cell culture

The liver cancer (Hep-G2, ATCC^®️ HB-8065^TM) cell line was used for testing for the in vitro antiproliferative activity. This cancer cell line was culted in complete media (Dulbecco’s Modified Eagle Medium, DMEM medium (Gibco) containing 10% (v/v) fetal bovine serum (FBS; Gibco), L-glutamine (2 μM) (Invitrogen), and an antibiotic mixture of penicillin (100 U/mL) and streptomycin (100 μg/mL) (Invitrogen). Cell line was kindly donated by Assistant Prof. Cherdsak Boonyong (Pharmacology and Toxicology Unit, Faculty of Science, Rangsit University) and was incubated at 37 ^oC in a humidified air atmosphere containing 5% (v/v) CO₂.

2.2. MTT assay of cell viability and proliferation

Cardol (C15:2 (5-[8(Z), 11(Z)-pentadecadienyl]resorcinol)) was purified fromTetragonula laevicepspropolis (Bankha, Ratchaburi) following previously reported procedures (Figure 1) (Meemongkolkiat et al., 2024). Hep-G2 cells were seeded at 2 x 10⁴ cells/well in 96-well plates and cultured overnight. Cells were treated with cardol (10 to 160 μM) or roscovitine (Catalog. 557360, Sigma-Aldrich, St. Louis, MO)) for 48 h. Following incubation, the medium was replaced with 100 μL of 0.1% MTT solution in PBS and incubated for 30 min at 37°C. The resulting formazan was solubilized in 100 μL of DMSO and incubated in the dark for 30 min at room temperature. Absorbance was measured at 560 nm. Cell viability was expressed as a percentage relative to the control, and IC₅₀ values were determined from dose-response curves (Sophonnithiprasert et al., 2025).

Figure 1
Cardol C15:2 (5-[8(Z), 11(Z)-pentadecadienyl]resorcinol) structure.

2.3. Cell imaging

The Hep-G2 (2 x 10⁴ cells) was cultured in 100 μL medium in each well of 96-well plates and incubated for overnight. Then, they were treated with 0.5% (v/v) DMSO as control to with various concentration of cardol or roscovitine. The cell morphology of the Hep-G2 cell line after treatment was observed and photographed after 48 h of incubation using a Motic AE31 microscope (Universal City, TX, USA) at 200 × magnification.

2.4. Compound structure preparation for molecular docking

The cardol (15:2) files were obtained from the PubChem ligand structure database (www.pubchem.ncbi.nlm.nih.gov). The Online Simplified Molecular Input Line Entry System (SMILES) Structure and Structure File Generator (https://cactus.nci.nih.gov/translate/) was used to translate the ligands from 3D SDF files to Protein Data Bank (PDB) format. The AutoDock Auxiliary Tool (ADT) version 4.1 was used to add all polar hydrogen atoms, written in a Partial Charge (Q) and Atom Type (T) for PDBQT file format (Rattanaburee et al., 2023).

2.5. Molecular docking

Potential target proteins were predicted based on 2D and 3D similarity measures using SwissTargetPrediction (http://www.swisstargetprediction.ch). Then, thirty five proteins associated with hepatocarcinoma cells were selected for investigation (Supplementary Material Table 1). The 3D protein structures were obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB). The criteria for selecting accession number of each protein were the protein that particularly have kinase structures, including the catalytic domain, generally consist of two lobes (an N-terminal and a C-terminal lobe) connected by a hinge region predicted by UniProt (https://www.uniprot.org/) (Pundir et al., 2017). The Autodock (ADT) software was used to separate water molecules and all co-crystallized ligands. Every co-crystallized ligand was eliminated. All polar hydrogen atoms were included in the Protein Data Bank (PDB) file format to simulate hydrogen bond interactions. Binding affinities and interactions were evaluated using Autodock4 version 4.2 (Rattanaburee et al., 2020). Docking was performed in a 126 (x) x 126 (y) x 126 (z) Å grid box using the Lamarckian Genetic Algorithm (LGA) with 50 runs and a population size of 200. The docking score was reported as the lowest predicted binding energy (ΔG_bind) in kcal/mol, with conformations clustered at a root mean square deviation (RMSD) tolerance of 3.0 Å (Morris et al., 2009). The docking protocol was validated by re-docking the reference inhibitor into its respective protein target, comparing the docked pose and binding site to the original crystal structure. All 3D visualizations were performed using the Visual Molecular Dynamics (VMD) package, while 2D interaction maps and docked positions were analyzed using BIOVIA Discovery Studio 2025 (Haque et al., 2022).

2.6. Change in gene expression

Changes in the transcription levels of selected genes from three signaling pathways implicated in hepatocellular carcinoma were investigated. The first group included genes involved in cell cycle progression (Pellarin et al., 2025), particularly in G₁/S transition and S phase (Cyclin D, Cyclin E, Cyclin A, p21, p27). The second group comprised DNA replication related genes (CDC6, MCM2, and PCNA) (Oehlmann et al., 2004). The last group included genes involved in the mitogen signaling pathway, which is involved in proliferation, survival, and differentiation (STAT3, AKT, c-MYC) (Luo et al., 2024). The Hep-G2 cells were seeded at 2 x 10⁵ cells per well into 6-well plates, cultured overnight, and treated with (i) cardol in DMSO at IC₅₀ value and half of IC₅₀ value (ii) roscovitine, and (iii) 0.5% (v/v) DMSO only as a control. After 48 h of incubation at 37 ^oC with 5% (v/v) CO₂, total RNA was extracted from the treated cells using TRIzol^TM reagent (Invitrogen, Carlsbad, CA, USA). RNA quantity was spectrophotometrically evaluated using the A₂₆₀/A₂₈₀ nm ratio (Nanodrop^TM 2000C spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA). One microgram of total RNA was reverse-transcribed using iScript^TM Reverse Transcription Supermix for RT-qPCR (Bio-Rad laboratories, Inc., MA, USA). RT-qPCR was performed on a CFX96 Connect^TM system with CFX Maestro^TM software (Bio-Rad Laboratories, Inc., MA, USA) using Luna^® universal qPCR master mix (New England Biolabs, Inc, MA, USA). Each 10 μL reaction contained 50 - 100 ng of total cDNA, 5 μL of 2x Luna^®Universal qPCR Master Mix, and 0.25 μL of each forward and reverse primer (10 μM) (Supplementary Material Table 2). Primer specificity was verified by performing a melting curve analysis Supplementary Material Figure 1). Relative expression levels were normalized to GAPDH as an internal control. Each assay was performed in triplicate using a thermal cycling program of 95 ^oC for 60 s, followed by 40 cycles of 95 ^oC for 15 s and 60 ^oC for 30 s. Expression levels were quantified using the 2^-ΔΔCT method (Sophonnithiprasert et al., 2025).

2.7. Statistic analysis

All data are presented as the mean ± standard deviation (SD). Statistical analysis of the data was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test for the significance of differences between the means. Data was considered statistically significant at the P < 0.05 levels.

3. Results

3.1. Cytotoxicity of cardol and roscovitine against Hep-G2 cells

To determine the cytotoxic IC₅₀ values of Cardol and roscovitine for direct comparison, Hep-G2 cells were cultured on a small scale (2 x 10⁴ cells in 100 µL of medium in each well of a 96-well plate) for 48h. According to the cell viability of the treated Hep-G2 cells, both cardol and roscovitine showed clearly inhibited cell growth in a dose-dependent manner (Figure 2). For the cardol compound, the average IC₅₀ value was 24.56 ± 1.09 µM. The average concentration around 0 - 10 µM did not affect cell proliferation/survival, resulting in more than 90% cell survival. But after the concentration increased from 20 to 26 µM, the cell viability was sharply decreased from approximately 81.88% at 20 µM to 36.49% at 26 µM, indicating dose effects the cell growth (Figure 2A). For roscovitine, the inhibitor for Hep-G2, the average IC₅₀ value was 11.77 ± 0.95 µM. The average concentration around 0 - 5 µM did not affect cell proliferation/survival, resulting in more than 90% cell survival. After the concentration increased from 10, 15 and 20 µM, the cell viability was gradually decreased from approximately 68.51%, 29.03% and 14.09%, respectively (Figure 2B). Although both compounds showed the level of cytotoxicity to the Hep-G2 cells, the toxicity of cardol was about 2-fold less than that of roscovitine.

Figure 2
Cytotoxicity of (A) Cardol and (B) Roscovitine on Hep-G2 cells, the cell viability (%) was estimated by the MTT assay after 48 h exposure. Data are shown as the mean ± SD, derived from three independent repeats.

3.2. Morphological characteristics of Hep-G2 cells grown on cardol and roscovitine

Under the microscope (200× magnification), clear differences were seen in Hep-G2 cell cultures treated with the test compound (Cardol, IC₅₀ = 24.56 µM) and the positive control inhibitor, Roscovitine (IC₅₀ = 11.23 µM) compared with untreated cells. In the control cell (Figure 3A), cells cultured on plate were clustered and form aggregates. The cells were densely packed with intact intercellular junctions and a granular cytoplasm. When treated with the test compound (Cardol, IC₅₀ = 24.56 µM), cells showed obvious morphological changes (Figure 3B). Colonies were smaller and disrupted, with many cells becoming rounded, shrunken, and detaching from the surface. Many cells lost adhesion and floated in the medium, suggesting cytotoxic or apoptotic effects. Overall, cell density was reduced, consistent with approximately 50% viability suppression. Positive control inhibitor, Roscovitine (11.23 µM, IC₅₀), also resulted in substantial morphological changes (Figure 3C). Colonies became sparse and irregular, with fewer viable cells remaining adherent. Taken together, these findings show that both the new compound and Roscovitine strongly suppress Hep-G2 cell growth, with the test compound mainly causing cell rounding and detachment.

Figure 3
Morphological changes in Hep-G2 cells after treatment with compound and positive control inhibitor. Representative phase-contrast images of Hep-G2 cells are shown with 200X magnification. (A) Control cells, (B) Cells treated with Cardol (24.56 µM, IC₅₀) and (C) Cells treated with the positive control inhibitor Roscovitine (11.23 µM, IC₅₀). Scale bar = 200 µm.

3.3. Molecular docking results

3.3.1. Target screening

Molecular docking was conducted against thirty-five cancer associated proteins. Two targets exhibited more favorable predicted affinities for cardol than their reference inhibitors: cyclin-dependent kinase 2 (CDK2), and mitogen-activated protein kinase kinase 1 (MEK1).

Predicted binding energies were -7.84 kcal/mol for CDK2 (Roscovitine, -6.71 kcal/mol, K_i = 1.78 µM) and -7.66 kcal/mol for MEK1 (Refametinib, -5.32 kcal/mol, corresponding to a predicted K_i = 2 µM) (Supplementary Material Table 1). The affinity value (kcal/mol) represented the binding capacity of cardol, where the lower the binding capacity, the more stable the ligand receptor binding. Based on these values, CDK2 and MEK1 were selected for further structural interaction analysis.

3.3.2. Binding interactions of cardol with CDK2

CDK2 binding interactions are summarized in Table 1 and shown in Figure 4. Cardol occupied the same ATP-binding pocket as roscovitine, sharing interactions with seven residues: IIle10, Glu12, Val18, Ala31, Leu83, Gln131, and Leu134 (Figure 4A, 4B). In top scoring pose, IIle10 and Glu12 formed hydrogen bonds with cardol hydroxyl groups, while Leu134, Val18, and Ala31 contributed alkyl or π-alkyl hydrophobic contacts. Cardol also engaged Lys33 (alkyl interaction) and Phe80 (π-alkyl stacking), involving the glycine-rich loop, hinge, and hydrophobic sub-pocket. Overall, the overlap of these key residues with those identified for roscovitine confirms that cardol occupied the same ATP-competitive site within CDK2 (Figure 4B).

Table 1
Interacting amino acids of CDK2, and MEK1 with Cardol and inhibitors.

CDK2		MEK1
Cardol	Roscovitine (Inhibitor)	Cardol	Refametinib (Inhibitor)
Ile10	Ile10		Gly77
Glu12	Glu12		Lys97
Val18	Val18	Leu115
Ala31	Ala31	Leu118
Lys33		Cys121
Val64		Phe129	x
Phe80		Ile141	Ile141
Phe82		Met143
Leu83	Leu83		Asp190
	His84		Lys192
	Asp86	Cys207
	Lys89	Asp208	Asp208
Gln131	Gln131	Phe209
Leu134	Leu134	Val211
Ala144		Ser212
		Leu215	Leu215

Figure 4
Ligand interaction with CDK2 and MEK1 structure. Interaction of the CDK2 and its important binding domain with roscovitine (inhibitor) (A) and active compound (cardol) (B). Interaction of the MEK1 and its important binding domain with refametinib (inhibitor) (C) and active compound (cardol) (D).

3.3.3. Binding interactions of cardol with MEK1

Residue specific interactions are presented in Table 1 and Figure 4. Refametinib, a clinically established MEK1 allosteric inhibitor was used for residue comparison. Refametinib interacted with Ile141, Asp208, and Leu215, which characterize the MEK1 allosteric pocket (Figure 4C). Cardol bound within the same cavity and shared these three key residues. Additional contacts were observed with Leu115, Leu118, Met143, and Ser212. Leu115, Leu118, and Met143 formed π-alkyl interactions with the hydrophobic chain, while Ser212 formed a hydrogen bond with a cardol hydroxyl group (Figure 4D). All these residues shape the hydrophobic lining, outer pocket surface, and activation segment region. The hydrogen bond to Ser212 and multiple hydrophobic interactions helped stabilize the ligand. The predicted binding energy (−7.66 kcal/mol) corresponded to an estimated K_i of ~2 µM, indicating a stable interaction under the docking conditions employed.

3.3.4. Impact of cardol and roscovitine on Hep-G2 cell cycle and signaling genes

To evaluate the effect of Cardol on transcriptional regulation, Hep-G2 cells were treated for 48 h with Cardol at half of its IC₅₀ (12.28 µM) and IC₅₀ (24.56 µM). Roscovitine was used as a positive control at half of its IC₅₀ (5.89 µM) and IC₅₀ (11.77 µM). The mRNA expression levels of eleven genes were analyzed by qRT-PCR relative toGAPDH, including cell-cycle regulatory genes (Cyclin D, Cyclin E, Cyclin A, p21 and p27; Figure 5A), DNA-replication genes (CDC6, MCM2,andPCNA; Figure 5B) and upstream signaling genes (STAT3, AKT and c-MYC; Figure 5C). At half IC₅₀ of Cardol, most genes exhibited modest downregulation with significant decreases in MCM2 (0.36-fold, p < 0.05) and STAT3 (0.56-fold, p < 0.05). At the IC₅₀ concentration, several S-phase-related genes were suppressed. Significant reductions were observed inCyclin A(0.18-fold,p<0.05),PCNA(0.31-fold,p<0.05), andMCM2(0.43-fold,p<0.05). AlthoughCyclin E(0.56-fold) andCDC6(0.49-fold) did not reach statistical significance, both exhibited downward trends. Early G₁-phase regulators Cyclin D (0.92-fold), p21 (1.05-fold), and p27 (0.62-fold) were unchanged. For the upstream signaling group,STAT3(1.18-fold), and AKT(1.73-fold) remained statistically unchanged with slightly in upregulation trend, whilec-MYCwas significantly upregulated (1.63-fold,p< 0.05).

Figure 5
Change in transcript expression levels of selected cell-cycle regulatory genes (Cyclin D, Cyclin E, Cyclin A, p21, p27) (A), DNA-replication genes (CDC6, MCM2,andPCNA) (B) and upstream signaling genes (STAT3, AKT and c-MYC) (C). Hep-G2 cells were cultured with 0.5% (v/v) DMSO (control), cardol (12.28 and 24.56 µM), or Roscovitine (5.89 and 11.77 µM) for 48 h. Data are presented as mean ± SD from three independent replicates. Significant differences between control and treated cells are indicated as p < 0.05.

Roscovitine produced a broadly similar transcriptional profile. At half IC₅₀ (5.89 µM), only c-MYC was significantly elevated (1.65-fold, p < 0.05). At the IC₅₀ concentration (11.77 µM), Roscovitine caused suppression of replication related genes and activation of certain upstream regulators. Cyclin A (0.38-fold, p < 0.05) and MCM2 (0.58-fold) were decreased, while Cyclin E (1.58-fold) and CDC6 (0.77-fold) displayed minor, non-significant fluctuations. Conversely, strong upregulation was observed in STAT3 (1.82-fold, p < 0.05) and c-MYC (1.87-fold, p < 0.05), accompanied by moderate increases in AKT (1.95-fold).

Cardol and Roscovitine exhibited a congruent inhibitory trend on Cyclin A, PCNA, and MCM2, together with limited influence on Cyclin D and p21. This similarity supports the notion that both agents affect the transcriptional program downstream of CDK2. The consistent downregulation of replication licensing factors indicates that Cardol likely exerts a CDK2-inhibitory effect on transcriptional control of the cell cycle machinery in Hep-G2 cells (Figure 6).

Figure 6
Proposed schematic representation of the molecular mechanism of cardol in Hep-G2 cells. Black arrows indicate the forward direction of signaling. Black arrows with a cross denote inhibitory signaling within a forward pathway. Black lines ending with a bar (cardol) represent direct inhibitory interactions predicted from molecular modeling results, including CDK2 and MEK1 inhibition. Green arrows denote increased gene expression, whereas red arrows indicate decreased gene expression.

4. Discussion

Cardol and cardanol have been linked to propolis originating from Southeast Asia, particularly from IndonesianTrigona incisaand ThaiApis mellifera, where it has been identified among the major alkylphenolic constituents (Kustiawan et al., 2015; Teerasripreecha et al., 2012). It is also found in edible plants of the Anacardiaceae family. Cardol enriched fractions derived from these natural sources have demonstrated pronounced antiproliferative effects in various carcinoma cell lines, accompanied by characteristic cell-death morphology and cell-cycle arrest (Kustiawan et al., 2017). In this study, cardol showed dose-dependent cytotoxicity in Hep-G2 cells, accompanied by stress related morphology such as rounding and shrinkage, consistent with growth arrest and cell death (Figure 3). These findings congruent with previous reports showing that cardol/cardanol rich fractions reduce viability and induce cytotoxic morphological changes in colon, gastric, lung, and breast cancer cells. Clinical evidence shows that roscovitine, despite being a classical CDK inhibitor, has limited potency and tolerability (Le Tourneau et al., 2010). These limitations underscore the need for more stable and selective CDK2 inhibitors. Despite a slightly higher IC₅₀, cardol exhibited stronger CDK2 binding (-7.84 kcal/mol), suggesting an alternative multitarget modulator for liver cancer therapy.

Computational docking was then used to nominate protein level mechanisms. Two targets, CDK2 and MEK1 were prioritized because predicted free energies were favorable relative to reference inhibitors, and, notably for MEK1, the estimated K_i was in the low micromolar range. In the present docking study of cardol with CDK2, cardol occupied the ATP binding site in a pose that closely resembled that of roscovitine (Hamed et al., 2024). Its hydroxyl groups formed hydrogen bonds with Ile10 and Glu12 near the hinge, while the long alkyl chain extended into the hydrophobic sub-pocket, creating van der Waals interactions with Val18, Ala31, Leu83, Gln131, and Leu134 (Figure 4B). Additional π-alkyl stacking between the phenolic ring of cardol and the gatekeeper residue Phe80 (Vulpetti et al., 2005), alongside contacts with Lys33 near the triphosphate binding region, further stabilized the complex (Shimazaki and Tachikawa, 2022). These combined interactions suggest that cardol bridges both the hinge and the glycine-loop regions, effectively mimicking the anchoring features of ATP competitive inhibitors but reaching deeper into the hydrophobic cavity (Li et al., 2015; Mandour et al., 2022). This extended fit likely enhances binding stability and supports a mechanism in which CDK2 activity is suppressed, leading to inhibition of the G₁/S transition. For MEK1, cardol was found to occupy the same allosteric pocket adjacent to the activation segment, overlapping closely with the binding mode of refametinib (Suryavanshi et al., 2024). The hydroxyl group of cardol formed a stabilizing hydrogen bond with Ser212 within the activation loop, while its hydrophobic chain engaged residues Leu115, Leu118, Ile141, Met143, Asp208, and Leu215 through a combination of van der Waals and π-alkyl interactions (Figure 4D) (McDermott and Qin, 2015). These contacts collectively anchored the ligand within the hydrophobic core that maintains MEK1 in its inactive state (AlZahrani et al., 2022). The similarity in interacting residues particularly Asp208 and Leu215, supports a comparable mechanism to known allosteric inhibitors (Iverson et al., 2009). By stabilizing the inactive conformation and restricting movement of the activation loop, cardol likely functions as a non-ATP competitive modulator that suppresses MEK1 catalytic activation.

Linking these docking-based mechanisms to transcriptional changes provides a consistent biological narrative. These target level findings help rationalize the gene expression pattern determined by qRT-PCR. Inhibition at CDK2 would be expected to blunt G₁/S machinery by reducing Rb hyperphosphorylation (Gerosa et al., 2024), thereby reinforcing RB-E2F repression of S-phase genes (e.g., CCNA2, MCMs, PCNA, CDC6) and promoting G₁ arrest (Cook et al., 2002). From the RT-qPCR results, the up-regulation of p21 (CDKN1A)and p27 (CDKN1B)was accompanied by marked reductions ofCyclin E (CCNE1)andCyclin A (CCNA2), whileCyclin D (CCND1)remained unchanged. The mild induction ofp21andp27indicates activation of checkpoint control that inhibits Cyclin E/A-CDK2 complexes, maintaining RB in its hypophosphorylated form and preventing E2F-dependent transcription (Morris et al., 2000; Moser et al., 2018). Downregulation ofCyclin EandCyclin Afurther limits the G₁/S transition, whereas the stable level ofCyclin Dsuggests that upstream mitogenic signaling was not completely abolished. This pattern is characteristic of selective CDK2 inhibition (Figure 6) (Kim et al., 2022).

The downregulation ofCDC6,MCM2, andPCNAsupports a second layer of proliferation blockade. Their suppression reflects sustained RB activity and transcriptional silencing of replication genes (Figure 6) (Wong et al., 2011). The combined downregulation signifies failure of replication licensing and reinforces the notion of a dual blockade at both G₁/S transition and initiation of DNA replication (Nevis et al., 2009). The unchanged expression ofCyclin Dtogether with the moderate upregulation ofSTAT3,AKT, andc-MYCsuggests the activation of feedback survival pathways. Inhibition of MEK1 often interferes with ERK-mediated feedback. When this restraint is lost, receptors can reactivate upstream signals that stimulate both the PI3K-AKT and JAK-STAT3 cascades (Figure 6) (Lee et al., 2014; Turke et al., 2012). Activated STAT3 can increase the transcription ofc-MYCandCyclin D, explaining whyCyclin Dexpression remained relatively stable whilec-MYCwas elevated despite a likely reduction in ERK signaling (Bowman et al., 2001). Together, these events indicate a coordinated feedback response that sustains pro-survival signaling without restoring full proliferative drive. However, these compensatory activations appear insufficient to overcome the dominant cell cycle inhibition imposed by CDK2 inactivation and replication gene suppression. Thus, the elevated c-MYC level may intensify replication stress in cells already arrested by CDK2 inhibition, further contributing to apoptotic progression during sustained exposure (Peripolli et al., 2024).

Overall, the pattern represents a balance between checkpoint activation and survival signaling. CDK2 inhibition enforces G₁ arrest and replication licensing failure, while MEK1 inhibition triggers feedback loops that activate STAT3 and AKT. This coordinated response explains the cytostatic to cytotoxic transition observed and supports the view that cardol acts as a multitarget modulator that disrupts both the CDK2-RB-E2F and MEK-ERK-STAT3-AKT pathways, suppressing cell proliferation. Although our docking and gene expression results suggest that cardol disrupts cell cycle and survival signaling in Hep-G2 cells, we acknowledge that transcriptional changes do not always correspond linearly to functional protein levels. Experimental validation using proteomics or western blotting will be necessary to confirm these downstream execution steps. Nevertheless, this work provides a molecular starting point for understanding how bioactive phenolic lipids from Tetragonula laeviceps propolis inhibit cancer cell growth.

Supplementary Material

Supplementary material accompanies this paper.

Supplementary Table 1

Potential targets of Cardol

Supplementary Table 2

Primer sequences of studied genes

Supplementary Table 3

cytotoxic results by MTT assay

Supplementary Table 4

q-PCR raw data of eleven genes

Supplementary Figure 1

Melt curve of eleven cancer related genes and housekeeping gene (GAPDH) for qRT-PCR

This material is available as part of the online article from https://doi.org/10.1590/1519-6984.305353

Acknowledgements

This research funding was supported by the RSU Research Institute of Rangsit University, Thailand, for the academic year 2024 (Grant No. 64/2024). The authors would like to acknowledge the Cell Culture Laboratory, Building 4, Room 603, Faculty of Science, Rangsit University, for providing the facilities for cell culture and MTT assay experiments.

Data availability statement

The dataset supporting the findings of this study are available within the article and its Supplementary Materials

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Edited by

• Editor:
  Marcelo A.M. Esquisatto

Publication Dates

• Publication in this collection
  21 Aug 2026
• Date of issue
  2026

History

• Received
  08 Feb 2026
• Accepted
  03 July 2026