Suppression of Global Protein Synthesis and Hepatocellular Carcinoma Cell Growth by Benzimidazoisoquinoline, 4,11-Dichloro-BBQ.

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor best known for mediating biological responses to a wide range of xenobiotics, such as dioxins and polycyclic aromatic hydrocarbons. Recently, AhR has emerged as an important player in cancer biology, with the potential for therapeutic applications through targeted modulation of its activity in specific cancer types. In this study, we report that 4,11-dichloro-BBQ (DiCl-BBQ), a benzimidazoisoquinoline, exhibits AhR-mediated antiproliferative activity in HepG2 hepatocellular carcinoma cells. DiCl-BBQ was found to decrease cell growth at nanomolar concentrations, and this antiproliferative effect persisted even after the compound’s removal. Using inducible shRNA expression system, we demonstrated that the inhibitory effect of DiCl-BBQ was significantly reduced following AhR knockdown. Flow cytometric analysis revealed that DiCl-BBQ halted cell division and induced G1 cell cycle arrest in an AhR-dependent manner. Proteomic profiling identified the top four enriched pathways following DiCl-BBQ exposure: metabolism of RNA, translation, ribonucleoprotein complex biogenesis, and carboxylic acid metabolic processes. Notably, DiCl-BBQ caused a dramatic downregulation of translation-associated proteins, with this response diminished in AhR-depleted cells. Consistently, global protein synthesis was significantly repressed in DiCl-BBQ-treated cells. Together, these results indicate that DiCl-BBQ effectively inhibits HepG2 cells growth by inducing G1 cell cycle arrest and downregulating the protein translation machinery in an AhR-dependent manner.

1. Introduction

Hepatocellular carcinoma (HCC) accounts for 90% of primary liver cancers, with a 5-year survival rate of less than 20% [1]. HCC is notably resistant to radiotherapy and chemotherapy. Currently, six tyrosine kinase inhibitors are approved for HCC therapy. However, mutations in target receptors and downstream pathways, along with off-target toxicity from these inhibitors, continue to limit the treatment efficacy and outcomes[2, 3]. These challenges underscore the need to explore novel therapeutic targets for effective intervention.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that regulates the expression of a diverse set of genes by binding to the xenobiotic response element (XRE) in association with its heterodimerization partner, the AhR nuclear translocator (ARNT) protein [4]. First reported in 1974 as a “sensor” for foreign chemicals that mediates the biological responses to various environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), AhR has since attracted significant interest in the field of toxicology [5]. In recent decades, its physiological roles have expanded to include numerous biological functions, such as angiogenesis, drug metabolism, cell motility, and immunity, beyond its traditional function in toxicology [6]. It has also become increasingly evident that AhR activation plays a role in cancer biology.

The role of AhR in cancer is complex and multifaceted, as evidenced by notable discrepancies in its reported pro-tumorigenic and anti-tumorigenic activities across studies. Elevated AhR activity has been linked to increased tumorigenesis by enhancing tumor-intrinsic malignancy, such as proliferation and migration, while suppressing anti-tumor immunity [7, 8]. However, AhR can also exert tumor-suppressive effects, inhibiting both tumor formation and metastasis, depending on the tumor type and the stage of development [9, 10]. For example, in invasive ductal carcinoma breast cancer, AhR levels were found to be inversely correlated with histological tumor grade [11]. In early-stage lung cancer, AhR was reported to suppress metastasis, with low AhR expression associated with reduced overall patient survival [12]. In mouse model, loss of AhR in intestinal epithelial cells promoted colon tumorigenesis [13, 14]. In a separate study, feeding mice natural AhR ligands, such as indole derivatives, reduced intestinal tumor development; however, this protective effect was absent in AhR-deficient mice [15]. A previous study from our group also demonstrated that AhR functions as a tumor suppressor in a genetically engineered mouse model. In these p53-deficient models, the loss of AhR expression led to a broader range of tumors, significantly larger thymic lymphomas, and reduced survival rates compared to p53-deficient mice with AhR expression [16]. Using an unbiased genome-wide knockout screen, a recent study identified AhR as a regulator of proliferation and tumorigenesis in p53-knockout cells [17]. These findings suggest that AhR functions as a suppressor of carcinogenesis by repressing growth factor signaling and interacting with the p53 pathways.

The “Janus-faced” nature of AhR in cancer likely stems from the complexity of its activation and functions, which depend on the cell type, the specific ligand, and the tumor microenvironment. Given its diverse roles, tailoring AhR tumor suppressive activity in specific cancer types could provide a promising strategy for improving treatment outcomes. Since no single AhR ligand is universally effective for cancer therapy, our laboratory screens small molecules that either activate tumor-suppressive signaling or induce cancer cell death through AhR in various cancer cell types. Through these efforts, we have identified and characterized numerous drugs and compounds as AhR ligands with potential for cancer treatment. For instance, we showed that flutamide, an antiandrogen used in prostate cancer treatment, induced growth arrest in hepatocellular carcinoma cells by promoting TGF-β1 induction [18]. Similarly, Semaxanib (SU5416), a VEGF receptor and angiogenesis inhibitor, was found to suppress hepatoma cell growth through upregulation of the cell cycle inhibitor p21^cip1/waf1 [19]. Lastly, CGS-15943, an adenosine receptor antagonist, was shown to trigger cell death in breast cancer and hepatoma cells via the apoptosis regulators FasL and BAX [20].

In this study, we report that 4,11-dichloro-BBQ (DiCl-BBQ) functions as an antiproliferative agent in HepG2 hepatocellular carcinoma cells. DiCl-BBQ belongs to a newly identified class of benzimidazoisoquinolines that act as high-affinity, rapidly metabolized AhR ligands [21]. The IC50 of DiCl-BBQ, determined by its ability to competitively inhibit the binding of the radiolabeled AhR ligand 3-methylcholanthrene (3-MC), was estimated to be 5.2 nM, comparable to TCDD’s IC50 of 4.7 nM. Notably, its analog, 11-Cl-BBQ, has been shown to induce AhR-dependent growth inhibition in triple-negative breast cancer cells without affecting the growth of fibroblasts and normal human mammary epithelial cells [22]. In addition, 11-Cl-BBQ decreased cell proliferation in both small and non-small lung cancer cell lines by activating p53 and upregulating the cell cycle inhibitor p27^Kip1 [23]. Building on these findings, we demonstrate here that DiCl-BBQ effectively inhibits HepG2 cell growth by downregulating the protein translation machinery and inducing G1 cell cycle arrest in an AhR-dependent manner.

2. Materials and Methods

2.1. Cell culture

HepG2 and Huh-7 hepatocellular carcinoma cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Avantor, Radnor Township, PA, USA) and 1% penicillin-streptomycin (Corning, Corning, NY, USA). The cultures were maintained at 37°C in a humidified incubator with 5% CO₂.

2.2. Cell proliferation and viability assay

HepG2 cells were seeded and allowed to grow overnight before treatment with DiCl-BBQ (4,11-dichloro-7H-benzimidazo[2,1-a]benzo[de]isoquinolin-7-one) (ChemBridge, San Deigo, CA, USA). Cell viability was assessed by quantifying ATP levels in metabolically active cells. Specifically, CellTiter-Glo reagent (Promega, Madison, WI, USA) was added directly to cultured cells, inducing lysis and generating a luminescent signal proportional to ATP content. Luminescence was measured using a microplate luminometer (Berthold, Bad Wildbad, Germany), normalized to the vehicle control, and expressed as a percentage. Alternatively, cell viability was determined by manual counting with a hemocytometer, using trypan blue exclusion to differentiate live cells.

2.3. Inducible AhR knockdown in HepG2 cells

The inducible AhR knockdown cell line was generated as previously described [24]. Briefly, HepG2 cells were transduced with lentiviral particles containing shRNA targeting AhR (sense, 5′-CGGGCTCTTTCAAGATAGTAAA-3′) under the control of a tetracycline-inducible promoter. Successfully transduced cells were selected in medium containing 3 μg/ml puromycin (VWR, Radnor, PA, USA). These cells were cultured as described above, except they were incubated with 2 μg/mL doxycycline (RPI, Mount Prospect, IL, USA) for 3 days before experiments involving AhR knockdown. shRNA expression was verified by RFP fluorescence under a microscope, and reduced AhR protein levels were confirmed by Western blot.

2.4. Western blot

Cell lysates were prepared in RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 8, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate). Protein concentrations were measured using the BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were mixed 1:1 with Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) containing β-mercaptoethanol, denatured at 100°C for 5 minutes, and subjected to SDS-PAGE. Separated proteins were transferred to a PVDF membrane, followed by blocking with 5% nonfat dry milk in TBST buffer (20 mM Tris, 150 mM NaCl, and 0.1% Tween 20) and overnight incubation at 4°C with primary antibodies. The following primary antibodies were used: anti-AhR (#SC-5579, Santa Cruz Biotechnologies), anti-GAPDH (#SC-365062, Santa Cruz Biotechnologies), and anti-puromycin (#PMY-2A4, DSHB). The membrane was then incubated for 1 hour at room temperature with the horseradish peroxidase (HRP)-conjugated secondary antibodies. Blots were visualized using the SuperSignal West Pico chemiluminescent substrate (Pierce, Waltham, MA, USA) and imaged on a bioimaging system (Synoptics LTD, Cambridge, UK).

2.5. Colony formation assay

HepG2 cells were treated as indicated, then trypsinized and re-seeded into 6-well plates at a density of 300 cells per well. After 11 days of incubation, colonies were fixed and stained with 0.2% methylene blue in 50% methanol, followed by manual counting for quantification.

2.6. Fluorescent dye-based cell proliferation assay

HepG2 cells were gently rinsed with phosphate-buffered saline (PBS) before being incubated with 2 μM CM-DiI, a carbocyanine membrane probe (Invitrogen, Waltham, MA, USA), at 37°C for 5 minutes followed by 4°C for 15 minutes. After labeling, cells were rinsed twice with PBS and treated as indicated in fresh medium. Following treatments, cells were trypsinized, rinsed once with PBS, and analyzed by flow cytometry using the CytoFlex system (Beckman Coulter, Brea, CA, USA). As the fluorescence dye transfers from mother to daughter cells during division, the progressive decrease in fluorescence intensity allows for monitoring of cell cycle progression [25].

Cell proliferation rate was also determined by the incorporation of 5’-bromo-2’-deoxyuridine (BrdU). HepG2 cells were incubated with 10 μM BrdU (MilliporeSigma, Burlington, MA, USA) for 30 minutes at 37°C, then trypsinized and rinsed with PBS. Following fixation in cold 60% ethanol, cells were treated with 2 M HCl to denature DNA and expose incorporated BrdU, then neutralized with 0.1 M sodium borate (pH 8.5). Next, cells were incubated with a FITC-conjugated anti-BrdU antibody (#11–5071-42, Thermo Fisher Scientific, Waltham, MA, USA) for 30 minutes at room temperature. After rinsing with 0.1% (w/v) bovine serum albumin in PBS, BrdU positivity, indicating actively proliferating cells, was assessed by flow cytometry [26].

2.7. Cell cycle analysis with propidium iodide staining

HepG2 cells were trypsinized, washed with PBS, and fixed in cold 70% (v/v) ethanol. The cells were then incubated in PBS containing 0.1% (v/v) Triton X-100 (Fisher Scientific, Waltham, MA, USA), 500 μg/ml RNase A (MilliporeSigma, Burlington, MA, USA), and 20 μg/ml propidium iodide (MilliporeSigma, Burlington, MA, USA) for 45 minutes at room temperature. Flow cytometry was used to determine the percentage of cells in each cell cycle phase.

2.8. Caspase-3/7 assay

Caspase-3 and −7 activities were measured using the Caspase-Glo 3/7 assay kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. The reagent was added directly to cultured cells, inducing lysis and caspase-mediated substrate cleavage, which generated a luminescent signal proportional to caspase activity. Luminescence was measured using a microplate luminometer, and results were expressed as fold change relative to the control.

2.9. Proteomic sample preparation

HepG2 cells, with or without AhR knockdown, were seeded at a density of 1 × 10⁶ cells per 100 mm dish and treated with 3 μM DiCl-BBQ or 0.1% (v/v) dimethyl sulfoxide (DMSO). After 5 days of incubation, cells were trypsinized, washed twice with PBS, and collected in 50 mM ammonium bicarbonate containing 8 M urea and protease inhibitors (MilliporeSigma, Burlington, MA, USA). Cells were sonicated, and debris was removed by centrifugation at 15,000 × g for 15 minutes at 4°C. Protein concentration was measured using the Bradford assay and normalized to ensure equal loading. Proteins were reduced with 5 mM dithiothreitol at 56°C for 30 minutes and alkylated with 10 mM iodoacetamide in the dark at room temperature for 1 hour. Proteins were precipitated by overnight incubation with ice-cold isopropanol at −20°C. After centrifugation at 15,000 × g for 15 minutes at 4°C, protein pellets were resuspended in 50 mM ammonium bicarbonate containing 0.1% (w/v) RapiGest (Waters Corporation, Milford, MA, USA), an MS-compatible surfactant. Proteolytic digestion was performed using MS-grade trypsin (Promega, Madison, WI, USA) at a 1:50 (w/w) ratio for 8 hours at 37°C.

2.10. LC-MS/MS Analysis

Label-free shotgun proteomic analysis was performed using an Orbitrap Fusion Lumos mass spectrometer coupled with a Waters nanoACQUITY UPLC system. Each sample was injected in triplicate. For each injection, 0.5 μg of peptide was loaded onto a trapping column (180 μm × 20 mm, 5 μm particle size) for online desalting with 3% (v/v) acetonitrile and 0.1% (v/v) formic acid at a flow rate of 5 μL/min for 3 minutes. Peptide separation was performed on a Waters nanoACQUITY UPLC BEH130 C18 analytical column (100 μm × 100 mm, 1.7 μm particle size) using a linear gradient from 3% (v/v) to 30% (v/v) mobile phase B (acetonitrile with 0.1% (v/v) formic acid) over 102 minutes at a flow rate of 500 nL/min.

Peptides were ionized via nano-electrospray using a 2000 V potential applied to the emitter, and the ion transfer tube temperature set to 300°C. The mass spectrometer operated in data-dependent acquisition mode using XCalibur software with “Top Speed” acquisition. High-resolution MS1 spectra of precursor ions were acquired in the Orbitrap mass analyzer over an m/z range of 350–1500, with a resolving power of 120,000 at m/z 200. The automatic gain control target for MS1 was set to 4 × 10⁵, with a maximum injection time of 50 ms. Precursor ions exceeding an intensity threshold of 5 × 10³ and carrying a charge state of 2 or higher were selected for MS/MS fragmentation. Ions were isolated using a quadrupole with a 1.6 Da isolation window and fragmented via collision-induced dissociation with a normalized collision energy of 30%. MS/MS spectra of fragment ions were recorded in the linear ion trap over an m/z range of 100–2000. The automatic gain control target for MS/MS scans was set to 1 × 10⁴, with a maximum injection time of 35 ms to ensure rapid and efficient fragment ion detection. Dynamic exclusion was enabled for 30 seconds to prevent repeated selection of the same ions, while the system operated in Top Speed mode to maximize the number of MS/MS scans per cycle.

2.11. Data Processing

Raw files were processed using Proteome Discoverer (v3.1) with the Sequest HT search engine. MS1 spectra were searched against the UniProtKB/Swiss-Prot human protein database, supplemented with a contaminant database. The precursor mass range was set to 350 – 5000 Da, with a mass tolerance of 10 ppm for MS1 spectra in the Orbitrap and 0.6 Da for MS/MS spectra in the ion trap. Up to two missed cleavages were allowed. Dynamic modifications included methionine oxidation, methionine loss at the N-terminus, N-terminal acetylation, and phosphorylation on serine, threonine, and tyrosine residues. Carbamidomethylation of cysteine was set as a static modification. Peptide spectral matches were filtered using a false discovery rate of 1%, calculated with a target-decoy approach. Only high-confidence peptides that met the false discovery rate threshold were included for further analysis.

2.12. Bioinformatic analysis

Proteins detected in fewer than 25% of all replicates were excluded from the analysis. For the remaining proteins, missing values were replaced using the k-nearest neighbor method. Data processing was performed using the MetaboAnalyst 6.0 web tool [27], applying sum normalization, log transformation, and z-score scaling. Sum normalization was used because equal protein concentrations were injected for all samples, while log transformation helped manage small values. Z-score scaling, calculated as z = (x_i − x)/S, adjusted protein levels to a standard deviation of 1. Here, x_i represents the individual peak area for a particular protein in each replicate, x is the mean peak area of that protein across all replicates, and 𝑆 is the standard deviation of the peak area for that protein across all samples. This scaling enables data analysis based on correlations rather than covariances, as achieved through centering [28].

Differentially expressed proteins were identified using a standard t-test followed by multiple testing correction. Principal component analysis showed that PC1 and PC2 accounted for 44.6% and 14.7% of the total variation, respectively, across all samples. Functional enrichment analysis of the differentially expressed proteins was conducted using Metascape (2024 update) [29], where clusters with an enrichment score ≥ 1.3 (corresponding to a -log10 p-value ≤ 0.05) were considered as significant. The heatmap of translation-related proteins, identified from the Reactome pathway (R-HSA-72766), was generated using the “pheatmap” package in RStudio (Kolde, 2019; RStudio Team, 2020).

2.13. RNA isolation and quantitative PCR

Total RNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA concentration was measured with a NanoDrop spectrophotometer, and 1 μg of RNA was used to synthesize complementary DNA with random hexamer primers and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). For quantitative PCR, cDNA, along with forward and reverse primers, was combined with FastStart Universal SYBR Green Master (MilliporeSigma, Burlington, MA, USA). Reactions were performed under standard thermal cycling conditions using the 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The primers used for gene expression analysis were as follows: p21^Cip (forward 5′-CCTGGTGATGTCCGACCTG-3′; reverse 5′-CCATGAGCGCATCGCAATC-3′), p27^Kip1 (forward 5′-TCAAACGTGAGAGTGTCTAACG-3′; reverse 5′-CCGGGCCGAAGAGATTTCTG-3′), and GAPDH (forward 5′-AGGTCGGTGTGAACGGATTTG-3′, reverse 5′-TGTAGACCATGTAGTTGAGGTCA-3′).

2.14. Generation of CRISPR-Cas9 knockout cell line

The HepG2 cell line with CRISPR knockout of the AhR gene was generated as previously described [30]. Briefly, HepG2 cells at 80–90% confluence were transfected with plasmid DNA using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Transfected cells were cultured in medium containing 2 μg/mL puromycin for one week to enable selection. Individual colonies were isolated using a cloning cylinder, and reduced AhR expression was confirmed by Western blot. The AhR-targeting vector containing the guide RNA sequence (5′-TTGCTGCTCTACAGTTATCC-3′) was obtained from GenScript (Piscataway, NJ, USA), while the control vector was a gift from Dr. Feng Zhang (Addgene Plasmid #52961).

2.15. Surface sensing of translation (SUnSET) assay

The SUnSET assay detects changes in protein synthesis by recognizing puromycin-labeled peptides [31]. Following DiCl-BBQ treatment, HepG2 cells were incubated with 10 μg/mL puromycin for 1 hour. Puromycin-labeled peptides were detected using an anti-puromycin antibody (#PMY-2A4, DSHB), followed by either an HRP-conjugated secondary antibody (for Western blot) or an Alexa Fluor 647-conjugated secondary antibody (for flow cytometry).

2.16. Statistical analysis

Comparisons among multiple groups were performed using one-way ANOVA followed by a Bonferroni post hoc test for pairwise comparisons. Differences between two groups were analyzed using an unpaired t-test. All statistical analyses were conducted using GraphPad Prism 10.

3. Results

3.1. DiCl-BBQ reduced HepG2 cell growth in an AhR-dependent manner

In our preliminary test, benzimidazoisoquinolines, including BBQ, 10-Cl-BBQ, and DiCl-BBQ, were evaluated for their antiproliferative effects on HepG2 cells. Among them, DiCl-BBQ exhibited the strongest effect and was therefore selected for further investigation. As shown in Figures 1A and 1B, the 5-day incubation with 500 nM DiCl-BBQ led to a substantial 15-fold reduction in cell number compared to the vehicle. The decrease in cell viability was both time- and concentration-dependent (Figure 1C). At the lowest concentration (1 nM), cell viability remained unaffected on Day 1 but decreased by 25% by Day 5. At the highest concentration (10 μM), viability declined by 30% on Day 1 and reached an 80% reduction by Day 5. The IC₅₀ of DiCl-BBQ for HepG2 cell viability was estimated to be 50 nM following a 5-day incubation (Figure 1D). A concentration of 3 μM was selected for subsequent studies to investigate its mechanism of action.

Figure 1. DiCl-BBQ decreased HepG2 cell growth and viability.

(A-B) HepG2 cells were seeded and incubated with vehicle control (0.1% DMSO) or 500 nM DiCl-BBQ for 5 days. (A) Representative images of cells under each treatment were captured using a phase contrast microscopy (scale bar = 100 μm). (B) The cell numbers were estimated with a hemocytometer. (C-D) Cell viability was monitored using an ATP-based assay. HepG2 cells were incubated with various concentrations of DiCl-BBQ ranging from 1 nM to 10 μM for up to 5 days. Cell viability was calculated and normalized to vehicle control (set to 100%), and the IC50 was determined using nonlinear regression in GraphPad. Figures show mean ± SD (n=3), with statistical significance indicated (****p < 0.0001).

To determine whether AhR is essential for the antiproliferative effect of DiCl-BBQ, we generated a stably transfected HepG2 cell line expressing a tetracycline-inducible shRNA targeting AhR. In these cells, incubation of doxycycline (Dox), a tetracycline analog, induces shRNA-mediated AhR silencing. As shown in Figure 2A, DiCl-BBQ had a minimal effect on Dox-treated cells compared to untreated cells. Quantitatively, in the (−) Dox group, DiCl-BBQ reduced cell number by 8-fold relative to the vehicle. However, in the (+) Dox group, this inhibitory effect was largely reversed (Figure 2B). In these (+) Dox cells, AhR protein levels were reduced, as confirmed by Western blot (Figure 2C). Consistent with the role of AhR in mediating DiCl-BBQ’s effects, another hepatocellular carcinoma cell line, Huh-7, exhibited reduced sensitivity to DiCl-BBQ (Figure 2D), as it expresses lower AhR protein levels than HepG2 cells (Figure 2E).

Figure 2. AhR knockdown attenuated the effect of DiCl-BBQ on HepG2 cell growth.

(A-C) HepG2 cells stably transfected with Dox-inducible shRNA targeting AhR were plated in the presence or absence of Dox and treated with 0.1% DMSO or 3 μM DiCl-BBQ for 5 days. (A) Representative images of HepG2 cells under each condition were captured using a phase contrast microscopy (scale bar = 100 μm). (B) Cell numbers were estimated with a hemocytometer. (C) Western blot analysis of AhR protein expression in cells cultured with or without Dox. GAPDH was used as a loading control. (D) Cell viability of HepG2 and Huh-7 cells treated with indicated concentrations of DiCl-BBQ for 5 days. Viability was normalized to the untreated control (set to 100%). (E) Western blot analysis of AhR protein expression in HepG2 and Huh-7 cells. GAPDH was used as a loading control. Figures are presented as mean ± SD (n=3), with significance levels indicated (ns, not significant; *p < 0.05; **p < 0.01; ****p < 0.0001). Western blot experiments were repeated twice to confirm the results.

3.2. The antiproliferative effect of DiCl-BBQ persisted after its removal

One of the challenges in cancer therapy is that most tumors in remission or under therapeutic control may rebound upon treatment discontinuation [32]. To investigate the reversibility of DiCl-BBQ’s antiproliferative effects, HepG2 cells were incubated with or without DiCl-BBQ for 5 days, then transferred to fresh new medium for up to 11 days. Colony formation assay was used to monitor cell growth. As shown in Figure 3A, in the (−) Dox group, cells previously exposed to DiCl-BBQ failed to form colonies during the 11-day recovery period. In contrast, despite prior DiCl-BBQ exposure, cells with Dox-induced AhR knockdown formed a considerable number of colonies, roughly 40% of those formed by the Dox-treated control. Similarly, in the viability assay, cell growth did not recover following a 5-day DiCl-BBQ treatment, resulting in a 75% viability reduction compared to control cells after 3 days. Conversely, in cells with reduced AhR expression, viability remained at approximately 80% of the control during the same treatment-free period (Figure 3B). These findings collectively indicate that DiCl-BBQ exerts a prolonged inhibitory effect on HepG2 cell growth in an AhR-dependent manner, even after its removal.

Figure 3. Prolonged antiproliferative effect of DiCl-BBQ on HepG2 cell growth after its removal.

HepG2 cells, with or without AhR knockdown by Dox, were treated with 0.1% DMSO or 3 μM DiCl-BBQ for 5 days. Following the treatments, HepG2 cells were trypsinized and re-seeded in fresh medium: (A) for 11 days before colony counting, or (B) monitored for viability over 3 days. Viability was calculated and normalized to cells treated with 0.1% DMSO (set to 100%). Both figures display mean ± SD (n=3), with significance levels indicated (**p < 0.01; ****p < 0.0001).

3.3. AhR-dependent suppression of cell division by DiCl-BBQ

The antiproliferative effect of DiCl-BBQ is primarily mediated through the inhibition of cell division. This was demonstrated using the cell-tracking dye CM-DiI, which integrates into the cell membrane and is transferred to daughter cells, causing fluorescence intensity to diminish with each cell division. As shown in Figure 4A, cells treated with 500 nM DiCl-BBQ retained strong fluorescence intensity, indicating a substantial population of non-dividing cells. In contrast, the addition of the AhR antagonist CH223191 significantly reduced this effect, as fluorescence intensity decreased by about 40% in the combination group compared to the DiCl-BBQ-treated group (Figure 4B). Similarly, in the BrdU assay, where BrdU incorporation marks proliferating cells, the BrdU-positive cell population was reduced by 75% upon DiCl-BBQ treatment (5.4% vs. 20.8% in the vehicle-treated group) (Figure 4C). However, in the presence of Dox (i.e., with reduced AhR expression), the percentage of BrdU-positive cells in the DiCl-BBQ group (13.4%) was comparable to that of the control (14.9%). These results confirm that DiCl-BBQ effectively halts cell division, while suppression of AhR expression significantly reduces its antiproliferative effect.

Figure 4. DiCl-BBQ inhibited cell division in an AhR-dependent manner.

(A) HepG2 cells were first labeled with CM-DiI and treated as indicated. After 1 day, the cells were switched to fresh medium and allowed to grow for 3 days. On Day 5, the cells were harvested through trypsinization and subjected to analysis by flow cytometry. Vehicle: 0.1% DMSO. DiCl-BBQ: 500 nM. CH223191: 3 μM. (B) The mean fluorescence intensity (MFI) from CM-DiI was calculated for cells in each group. The figure shows mean ± SD (n = 3), with significance levels indicated (**p < 0.01; ****p < 0.0001). (C) HepG2 cells, with or without AhR knockdown by Dox, were treated with 0.1% DMSO or 500 nM DiCl-BBQ for 4 days. After BrdU labeling, cells were incubated with an anti-BrdU fluorescent antibody and analyzed by flow cytometry. Groups without BrdU labeling served as negative controls. Both CM-Dil and BrdU assays were performed independently three times, and representative results from each experiment are shown.

3.4. DiCl-BBQ induced G1 Cell Cycle Arrest

The observed suppression of cell division was associated with G1 cell cycle arrest, as indicated by propidium iodide staining, a fluorescent dye that intercalates with cellular DNA. Figures 5A and 5B show that following DiCl-BBQ treatment, the proportion of cells in the S/G2 phase was drastically reduced, with 90% of cells accumulating in the G1 phase. However, in Dox-induced AhR knockdown cells, G1-phase accumulation increased only marginally to 70% after DiCl-BBQ treatment, a level comparable to that of vehicle-treated cells (60%).

Figure 5. Induction of G1 cell cycle arrest by DiCl-BBQ.

(A-B) Dox-inducible AhR knockdown cells were treated with vehicle or 3 μM DiCl-BBQ for 1 day, then switched to fresh medium and cultured for an additional 2 days. On Day 4, cells were collected, stained with propidium iodide, and analyzed by flow cytometry. (A) Representative cell cycle distribution from one experiment; the experiment was repeated once more with comparable results. (B) Percentage of cells in different cell cycle phases was calculated for each treatment group. (C) After a 3-day treatment with 3 μM DiCl-BBQ, cells were harvested for caspase-3/7 activity measurement. Data are presented as mean ± SD (n=2) (ns, not significant).

The reduction in cell growth could also be attributed to apoptosis. To assess this possibility, a key apoptotic marker, caspase-3/7 activity was examined. As shown in Figure 5C, DiCl-BBQ exposure resulted in a slight but statistically insignificant increase in caspase-3/7 activity, suggesting that DiCl-BBQ primarily induces cell cycle arrest rather than apoptosis.The slight increase in caspase-3/7 activity is likely due to a subset of cells undergoing apoptosis as a consequence of prolonged G1 phase arrest [33].

3.5. Proteomic analysis revealed downregulation of translation-related proteins by DiCl-BBQ

To investigate the mechanisms underlying cell cycle arrest and growth inhibition, cells with or without Dox-induced AhR knockdown were treated with either vehicle or DiCl-BBQ for 5 days, followed by proteomic analysis. Principle component analysis (PCA) of proteomic profiles across experimental groups revealed treatment-induced shifts and changes associated with AhR knockdown (Figure 6A). The separation along the PC1 dimension was primarily driven by DiCl-BBQ treatment, with wild-type cells shifting more toward the negative side, whereas AhR knockdown cells exhibited a less pronounced shift.

Figure 6. Proteomic profiling of DiCl-BBQ-induced changes in wild-type and AhR knockdown HepG2 cells.

(A) Principal component analysis (PCA) displays the proteomic profiles of wild-type and Dox-induced AhR knockdown cells, either untreated or treated with DiCl-BBQ (3 μM for 5 days) (n = 3). The plot shows the separation of experimental groups based on their global proteomic profiles, with PC1 accounting for 44.6% of the variance and PC2 accounting for 14.7%. (B) Heatmap depicting DiCl-BBQ-induced changes in the levels of proteins associated with AhR target genes and p53 pathway-related genes. Colors represent relative protein abundance calculated using z-score normalization. (C) The mRNA-levels of p21^Cip1 and p27^Kip1 were measured by qPCR in wild-type HepG2 cells treated with 3 μM DiCl-BBQ for 4 days. Data are presented as mean ± SD (n = 3), with significant differences from the untreated control indicated (*p < 0.05; ****p < 0.0001).

In wild-type cells treated with DiCl-BBQ, proteins corresponding to AhR target genes (CYP1A1, CYP19A1, CYP3A5, ALDH3A1, and NQO1) were upregulated, whereas their expression was reduced in AhR knockdown groups. In both vehicle-treated controls, these proteins were minimally expressed (Figure 6B). Similar to its analog 11-Cl-BBQ, which exerts antiproliferative effects through the p53 pathway [22, 23], DiCl-BBQ also upregulated proteins associated with this pathway, including TP53BP1, TP53I3, TP53RK, p21^Cip1, and p27^Kip1 in wild-type cells. The upregulation of p21^Cip1 and p27^Kip1 RNA levels was further confirmed by qPCR (Figure 6C).

Consistent with the PCA plot, DiCl-BBQ treatment significantly altered the levels of 2,215 proteins in wild-type cells compared to the vehicle (adjusted p-value < 0.05). In AhR knockdown cells, however, the number decreased to 1,008. Pathway enrichment analysis revealed that both cell types shared the same top four enriched pathways following DiCl-BBQ exposure: metabolism of RNA, translation, ribonucleoprotein complex biogenesis, and carboxylic acid metabolic processes. Notably, translation-related enrichment (Reactome: R-HSA-72766) was markedly reduced in knockdown cells. In wild-type cells, 136 of 292 translation-related proteins exhibited significant changes, whereas only 51 out of 292 were altered in AhR knockdown cells (Figure 7A).

Figure 7. Functional enrichment analysis shows downregulation of translation-related proteins by DiCl-BBQ.

(A) The figure illustrates the top four enriched pathways following DiCl-BBQ treatment in both wild-type and AhR knockdown cells. The X-axis displays the percentage of pathway-related proteins that are differentially expressed after DiCl-BBQ treatment. The Y-axis represents the total number of proteins associated with each functional pathway. (B) Cluster heatmap of 262 proteins associated with translation, identified from Reactome (R-HSA-72766). Colors indicate relative protein abundance, with clustering based on protein expression across various experimental conditions.

A total of 262 translation-related proteins were identified in our proteomic analysis, with their relative abundance illustrated in Figure 7B. DiCl-BBQ treatment resulted in a pronounced downregulation of more than half of these proteins in wild-type cells, an effect that was markedly attenuated in AhR knockdown cells. Specifically, 98 translation-related proteins, including aminoacyl-tRNA synthetases (ARSs), translation initiation factors (EIFs), and ribosomal proteins (MRPLs and RPs), exhibited at least a 2-fold downregulation in wild-type cells, compared to only 26 in knockdown cells. In contrast, the number of upregulated proteins remained modest in both groups, with 37 in wild-type cells and 24 in knockdown cells (Table 1).

Table 1. Significantly altered proteins associated with translation.

The table lists differentially expressed proteins from the indicated comparisons (q < 0.05). Symbols indicate the direction of change: ↑ for upregulation and ↓ for downregulation.

Wild type: DiCl-BBQ vs. Vehicle
37 proteins ↑	CARS2, DARS2, EEF2, EIF3M, EIF5B, FARSB, GFM2, GSPT1, MRPL4, MRPL55, MRPS33, MRPS6, PPA2, RPL11, RPL17, RPL18, RPL18A, RPL21, RPL28, RPL29, RPL3, RPL7, RPS2, RPS24, RPS27, RPS27A, SARS1, SEC61A1, SEC61B, SPCS1, SRP68, SRP9, SRPRB, SSR2, TARS2, TRAM1, YARS1
98 proteins ↓	AARS1, AIMP2, DARS1, DDOST, EEF1A1, EEF1B2, EEF1G, EIF2B4, EIF2B5, EIF2S1, EIF3B, EIF3C, EIF3D, EIF3E, EIF3F, EIF3H, EIF3J, EIF3K, EIF4A1, EIF4A2, EIF4B, EIF4EBP1, EIF4G1, EPRS1, ERAL1, GADD45GIP1, GFM1, HARS2, LARS1, LARS2, MRPL1, MRPL16, MRPL19, MRPL20, MRPL24, MRPL27, MRPL28, MRPL3, MRPL37, MRPL40, MRPL41, MRPL43, MRPL44, MRPL53, MRPS10, MRPS14, MRPS17, MRPS18A, MRPS22, MRPS23, MRPS27, MRPS28, MRPS31, MRPS34, MRPS35, MRPS5, MRPS7, MRPS9, MRRF, NARS1, NARS2, PABPC1, RARS1, RPL13A, RPL23, RPL23A, RPL31, RPL34, RPL39, RPL4, RPL7A, RPL8, RPN1, RPN2, RPS10, RPS14, RPS15, RPS15A, RPS19, RPS20, RPS21, RPS25, RPS26, RPS28, RPS29, RPS3, RPS4X, RPS9, SEC11A, SEC11C, SEC61G, SRP54, SRP72, SSR1, TRMT112, TUFM, WARS1, YARS2
AhR Knockdown: DiCl-BBQ vs. Vehicle
24 proteins ↑	ERAL1, ETF1, GFM1, IARS2, LARS1, MRPL50, MRPS14, MRPS28, PTCD3, RPL15, RPL18, RPL23A, RPL8, RPS11, RPS13, RPS14, RPS2, RPS28, RPS29, RPS3A, SPCS3, SRP9, SSR1, TRMT112
26 proteins ↓	EIF3F, EIF3G, EIF3L, EIF4EBP1, FARSB, FAU, GFM2, GSPT1, GSPT2, MRPL53, MRPL55, MRPL58, MRPL9, MRPS10, MRPS5, RARS2, RPL13, RPL7, RPN1, RPN2, RPS25, RPS9, SEC11A, SSR4, TSFM, YARS1
Vehicle: Wild type vs. AhR knockdown
6 proteins ↑	IARS1, MRPL19, MRPS28, MRPS7, RPLP1, RPS4X
4 proteins ↓	EEF1E1, RPL5, RPS17, RPS25

3.6. DiCl-BBQ suppressed global protein synthesis

To evaluate the effect of DiCl-BBQ on translation, we performed the SUnSET assay using HepG2 cells with CRISPR-mediated AhR knockout. These cells exhibited resistance to the growth-inhibitory effects of DiCl-BBQ, similar to the Dox-inducible AhR knockdown cells (Figures 8A and 8B).

Figure 8. Repression of global protein synthesis by DiCl-BBQ.

(A) Western blot analysis of AhR protein levels in individual clones of cells transfected with CRISPR vectors, with or without the guide RNA (gRNA). Clone #6, showing successful AhR reduction, was selected for subsequent experiments, with clone #2 used as a mock control. The experiment was repeated to confirm the results. (B) Cell numbers were estimated using a hemocytometer, and the results are presented as mean ± SD (n = 3), with significant differences indicated (*p < 0.05; **p < 0.01). (C) Detection of puromycin-labeled peptides by Western blot in mock and AhR-KO cells. (D) Puromycin incorporation in mock and AhR-KO cells was normalized to the GAPDH loading control and expressed as a percentage relative to untreated cells. (E) Immunofluorescent staining and flow cytometric analysis of puromycin-labeled peptides in mock and AhR-KO cells. In all panels, cells were treated with either vehicle (0.1% DMSO) or 3 μM DiCl-BBQ for 5 days. Both Western blot and flow cytometric analysis of the SUnSET assay were performed independently twice, with representative results from each experiment shown.

The SUnSET assay measures protein synthesis by detecting puromycin-labeled peptides incorporated into newly synthesized proteins. Puromycin incorporation, quantified using an anti-puromycin antibody, provides a direct readout of translation activity. As shown in Figures 8C and 8D, DiCl-BBQ reduced puromycin incorporation by 50% in mock cells but had minimal effect in AhR-KO cells. Flow cytometry data further supported this finding, showing a reduction in puromycin-positive cells in DiCl-BBQ-treated mock cells, as indicated by a leftward shift in the treatment population (red peak). However, in AhR-KO cells, no difference was observed, as the vehicle (green) and treated (red) populations remained overlapping (Figures 8E and 8F). These results align with the proteomics data, demonstrating that DiCl-BBQ inhibits protein translation, thereby contributing to cell cycle arrest and growth inhibition.

4. Discussion

Given the context-dependent roles of AhR in tumor promotion or suppression, ligands that modulate AhR activity in a tumor-specific manner and inhibit tumor growth represent a potential therapeutic strategy. Selective AhR modulators are compounds that elicit specific AhR-dependent responses without triggering the toxicity associated with traditional AhR ligands, such as TCDD [34, 35]. For example, 6-methyl-1,3,8-trichlorodibenzo-p-dioxin (MCDF) inhibits the growth and metastasis of estrogen receptor-negative breast cancer cells in mouse models while minimally activating canonical AhR target genes like CYP1A1 [36, 37]. Similarly, compounds such CGS-15943, 11-Cl-BBQ, and BBQ (Analog 523), identified in our laboratory as “select modulators of AhR-regulated transcription” (SMAhRTs), induce apoptosis or suppress the growth of various cancer cell types, including liver, breast, and lung cancers, via mechanisms distinct from the traditional TCDD-AhR signaling pathway [20, 22, 23, 38]. In this study, we further report that DiCl-BBQ mediates AhR-dependent antiproliferative activity in HepG2 hepatocellular carcinoma cells.

DiCl-BBQ exerted a strong AhR-dependent growth-inhibitory effect in HepG2 cells, as evidenced by an increase in non-dividing, G1-arrested cells, along with reduced cell growth and viability. Proteomic profiling revealed downregulation of translation-related proteins, which was further confirmed by the SUnSET assay showing a corresponding reduction in global protein synthesis.

Protein synthesis is fundamental to cell growth and proliferation [39]. Each phase of the cell cycle follows a tightly regulated sequence of events that controls protein synthesis and degradation, ensuring that production of essential proteins for cell division [40]. However, in permanently growth-arrested or senescent cells, global protein synthesis declines, along with inhibited ribosome biogenesis and reduced expression of translation factors [41, 42]. RNA metabolism also plays a critical role in cellular senescence, as all types of RNA—ribosomal, transfer, and messenger RNAs—undergo processing and maturation. Disruption in the metabolism of these RNA species have been linked to senescence [43]. Our findings align with these hallmarks of irreversible growth arrest. DiCl-BBQ treatment downregulated aminoacyl-tRNA synthetases (ARSs), translation initiation factors (EIFs), and ribosomal proteins (MRPLs and RPs), as shown in Table 1. Beyond translation, enrichment analysis identified RNA metabolism and ribonucleoprotein complex biogenesis as significantly affected pathways following treatment (Figure 7A). Additionally, the fourth functional group, carboxylic acid metabolic process, may be relevant to senescence, as irreversible cell cycle arrest is often accompanied by metabolic shifts, including alterations in the TCA cycle and its associated pathways [44, 45]. Together, these results suggest that DiCl-BBQ induces a permanent growth arrest, reflected by the observed reduction in global protein synthesis and downregulation of translation machinery.

Recent studies from our laboratory have characterized the antiproliferative effect of the analog 11-Cl-BBQ in breast and lung cancer cells, showing increased SA-β-gal activity, a biomarker of cellular senescence or sustained growth arrest, through activation of the tumor suppressor protein p53 [22, 23]. In line with this, another study from our group identified AhR as a tumor suppressor in p53-deficient mice [16]. p53 is a well-studied transcription factor that suppresses cell growth and reduces protein synthesis by regulating key components of the translation machinery, including ribosome biogenesis, eukaryotic initiation factors, and the assembly of translation initiation complexes [46, 47]. Thus, it is likely that DiCl-BBQ exerts its growth-inhibitory effect through a mechanism involving p53 pathways, as supported by the upregulation of pathway-associated proteins in the proteomic analysis and the increased transcription of cell cycle regulators p21^Cip1 and p27^Kip1 (Figures 6B and 6C).

In addition to p53, AhR may also have a role in protein synthesis. Figures 8C and 8E reveal a marked reduction in the protein synthesis rate in AhR-KO cells compared to mock controls. This observation aligns with previous studies, demonstrating that AhR regulates protein synthesis by activating the transcription of genes essential for ribosome biogenesis and translation, such as OGFOD1 and NOLC1 [48], or by modulating the expression and ubiquitination of elongation factor 2 kinase [49]. Our proteomic data further identified four additional proteins (EEF1E1, RPL5, RPS17, and RPS25) that were significantly downregulated in AhR-knockdown cells (Table 1), suggesting their potential involvement in AhR-mediated regulation of protein synthesis.

Finally, the sustained antiproliferative effect of DiCl-BBQ on cell growth, even after its removal (Figure 3), suggests potential epigenetic involvement. Our proteomics analysis supports this notion, as Figure 9 demonstrates changes in proteins associated with epigenetic modifications, including SUMOylation, histone modifications, and DNA methylation. Recent studies have recognized AhR as an epigenetic regulator that influences chromatin structure by interacting with transcriptional co-activators or repressors, as well as mediating histone- or DNA-modifying enzymes [6]. Given that epigenetic alteration is a hallmark of senescence [50] and that AhR and its ligands are linked to aging [51–53], it is likely that DiCl-BBQ acts through AhR and induces changes in the epigenome, leading to a permanent cell growth arrest. Future studies could further investigate the role of AhR activation in mediating these epigenetics effects. In conclusion, our study identifies DiCl-BBQ as a compound that suppresses HepG2 cell growth via AhR, supporting its further development as a potential therapeutic for hepatocellular carcinomas with high AhR expression.

Figure 9. DiCl-BBQ-induced changes of proteins involved in epigenetic regulation.

The heatmap demonstrates alterations in protein levels associated with SUMOylation, histone modification, and DNA methylation following DiCl-BBQ treatment. Colors indicate relative protein abundance, calculated using z-score normalization.

Abbreviations:

AhR

Aryl hydrocarbon receptor

DiCl-BBQ

4,11-dichloro-7H-benzimidazo[2,1-a]benzo[de]isoquinolin-7-one

HCC

Hepatocellular carcinoma

TCDD

2,3,7,8-tetrachlorodibenzo-p-dioxin

PBS

Phosphate-buffered saline

BrdU

5-bromo-2’-deoxyuridine

DMSO

Dimethyl sulfoxide

MS

Mass spectrometry

PCA

Principal component analysis

Dox

Doxycycline

SUnSET

Surface sensing of translation

SMAhRT

Select modulator of AhR-regulated transcription