Bergamottin demonstrates preclinical effectiveness against pancreatic cancer by modulating PPAR-γ to induce apoptosis

Introduction

Pancreatic cancer (PC) is one of the most fatal malignancies globally, characterized by high malignancy, rapid progression, and poor prognosis (1). Despite significant advances in diagnostic techniques and multimodal therapeutic strategies, the survival outcome remains unsatisfactory, with a 5-year survival rate of merely 12% (2). Clinically, gemcitabine is widely used for the treatment of PC, either as a monotherapy or in combination with other agents; however, its therapeutic efficacy is limited by the lack of specific targeted drugs. Notably, emerging research indicates that PC may become the second leading cause of cancer-related deaths worldwide by 2030, thereby imposing a substantial burden on global mortality (3,4). Given its inherent resistance to traditional therapies, the discovery and development of novel, effective therapeutic agents are therefore urgently required (5).

Against this backdrop, natural compounds—with their extensive structural diversity (including coumarins and other classes)—serve as a rich resource for pharmaceutical research and development (6). Among these natural compounds, bergamottin, a substance isolated from bergamot oil, belongs to the furanocoumarin family of organic compounds and is primarily present in grapefruit juice (at a concentration of approximately 25 µM) and lime juice (at a concentration of approximately 100 µM) (7,8). Previous studies have demonstrated that bergamottin exhibits diverse biological activities (9), and due to its broad-spectrum bioactivity and favorable biosafety profile, it has attracted growing attention from researchers as a potential anti-cancer agent, with its anti-cancer effects having been investigated in several cancer models (10). However, current studies mainly focus on describing the biological effects of bergamottin, such as its ability to induce apoptosis in various cancer cell lines (8,9), while its precise molecular targets and underlying mechanisms remain unclear. More importantly, research on the anti-cancer effects of bergamottin in PC is relatively scarce and limited, creating a critical research gap.

To address this research gap, herein we aim to further elucidate the role of bergamottin—a citrus-derived compound isolated from bergamot orange—in the progression of PC. The primary objective of this study is to investigate the biological effects of bergamottin on the development and progression of PC, explore the involved intracellular mechanisms, and identify its potential molecular targets. To achieve this goal, we employed a series of analytical techniques to evaluate the effects of bergamottin on multiple biological processes, including proliferation, invasion, migration, and apoptosis, in both PC cells and a xenograft mouse model, thereby enabling more accurate and comprehensive data analysis. Furthermore, RNA sequencing (RNA-seq) analysis was used to identify potential molecular targets of bergamottin, which suggested that bergamottin may target peroxisome proliferator-activated receptors (PPARs); this finding was subsequently verified through drug synergistic intervention experiments. Collectively, our results provide additional evidence supporting the potential therapeutic value of bergamottin in the treatment of PC, thus bridging the aforementioned research gap. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0837/rc).

Methods

Drugs and antibodies

Bergamottin (CAS No. 7380-40-7, formula: C₂₁H₂₂O₄, purity: 99.80%) and GW9662 (CAS No. 22978-25-2, formula: C₁₃H₉ClN₂O₃, purity: 99.79%) was purchased from MedChemExpress (Shanghai, China) and prepared as a 10 mM stock solution in dimethyl sulfoxide (DMSO). The Annexin V-FITC Apoptosis Detection Kit was obtained from BestBio Company (Shanghai, China). Ki67 primary antibody (catalog No. ab279653) was supplied by Abcam (Cambridge, UK). CoraLite488-conjugated Affinipure secondary antibody (catalog No. SA00013-2) was supplied by Proteintech (Wuhan, China). All reagents and antibodies were stored according to the manufacturers’ instructions to ensure their activity and stability.

Cells and cell culture

Two human PC cell lines, PANC-1 and PATU8988, were acquired from the American Type Culture Collection (ATCC, Manassas, USA). All cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, GENOM, Hangzhou, China) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, USA) to prevent bacterial contamination. The cells were cultured in a humidified incubator with 5% CO₂ at 37 ℃ and passaged regularly when they reached 80–90% confluence. Cells in the logarithmic growth phase were used for all subsequent experiments to ensure consistency in cell viability and responsiveness. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Cell Counting Kit-8 (CCK-8)

Cells were seeded into 96-well plates at a density of 5×10³/well and cultured for 24 h under routine conditions. Gradient concentrations of bergamottin (0, 3.12, 6.25, 12.5, 25, 50 µM) were administered to cells with three biological replicates per group, at a volume of 100 µL per well. Plates were incubated for another 24 h at 37 ℃ with 5% CO₂ and saturated humidity. After drug intervention, the original medium was discarded and refreshed. Subsequently, 100 µL CCK-8 working solution consisting of serum-free DMEM and CCK-8 reagent at a volume ratio of 9:1 was supplemented to each well. Samples were incubated in the dark at 37 ℃ for 2 h. The absorbance at 450 nm was finally detected to calculate relative cell viability.

RTCA

PANC-1 and PATU8988 cells were seeded into the wells of an E16-plate (ACEA Biosciences, San Diego, USA) at a density of 1×10⁴ cells per well. After cell adhesion, the cells were treated with different concentrations of bergamottin (0, 20, 40 µM), with three replicate wells set for each concentration. The RTCA system (Roche, Penzberg, Germany) was used to automatically monitor and measure the cell proliferation rate in real time over the experimental period. The cell index (CI), an indicator of cell viability and proliferation, was automatically calculated using the RTCA software package v1.2. Normalization was performed at each time point to eliminate background interference and ensure the accuracy of the results.

Immunofluorescence staining

Cells were seeded in polystyrene 6-well plates and cultured until they reached 60–70% confluence. The cells were then treated with bergamottin at concentrations of 0, 20, and 40 µM for 24 hours. After treatment, the cells were rinsed three times with phosphate-buffered saline (PBS) to remove residual medium and drug. Subsequently, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, Missouri, USA) at room temperature for 20 minutes, followed by permeabilization with 0.1% Triton X-100 (Sigma-Aldrich, Missouri, USA) for 10 minutes to enhance antibody penetration. Non-specific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 30 minutes. The cells were then incubated with the Ki67 primary antibody (1:400 dilution) overnight at 4 ℃. After three washes with PBS, the cells were incubated with Cora Lite 488-conjugated Affinipure secondary antibody (1:1,000 dilution) at room temperature for 1 hour in the dark. The nuclei were stained with DAPI for 5 minutes, and excess dye was removed by washing with PBS. Images were captured using a fluorescence microscope (Olympus, Tokyo, Japan), and the percentage of Ki67-positive cells was calculated using the formula: (number of Ki67-positive cells)/(number of DAPI-positive cells) × 100%. Three random fields were selected per well for quantification to ensure statistical reliability.

Colony formation assay

PANC-1 and PATU8988 cells in the logarithmic growth phase were seeded into 6-well plates at a density of 1,000–2,000 cells per well, with three replicate wells for each group. The cells were treated with bergamottin (0, 20, 40 µM) for 24 hours, after which the drug-containing medium was replaced with fresh complete medium. The cells were then incubated for 14 days under standard culture conditions (37 ℃, 5% CO₂) to allow colony formation. After incubation, visible colonies were washed twice with PBS, fixed with 4% formaldehyde for 15 minutes, and stained with 0.1% crystal violet solution for 20 minutes. Excess crystal violet was gently washed away with running water, and the plates were air-dried at room temperature. Colonies with a diameter exceeding 0.1 mm or containing more than 50 cells were counted manually. This assay was performed in triplicate to evaluate the effect of bergamottin on cell proliferation and clonogenic capacity.

Wound healing (migration) assay

Cells in the exponential growth phase were seeded into 6-well plates at a density of 5×10⁵ cells per well and cultured at 37 ℃ for 48 hours until they formed a confluent monolayer. A sterile 200 µL pipette tip was used to scratch a straight, uniform linear gap in the cell monolayer. The detached cells were washed away with PBS, and the remaining cells were cultured in medium containing different concentrations of bergamottin (0, 20, 40 µM). Images of the scratch area were captured using an inverted microscope (Olympus, Tokyo, Japan) at 0 and 24 hours after scratching. The extent of cell migration was quantified using ImageJ software (National Institutes of Health, Bethesda, USA), and cell motility was represented by the change in scratch length, calculated as: scratch length at 0 hours − scratch length at 24 hours. Each group was set with three replicates, and the experiment was repeated three times independently.

Transwell invasion assay

The invasiveness of PC cells was evaluated using a Transwell chamber (8 µm pore size) coated with Matrigel (Corning, New York, USA). Briefly, 1×10⁵ cells in 200 µL of serum-free medium containing different concentrations of bergamottin (0, 20, 40 µM) were added to the upper chamber of the Transwell insert. The lower chamber was filled with 600 µL of complete medium containing 10% FBS as a chemoattractant. The cells were incubated at 37 ℃ with 5% CO₂ for 24 hours. After incubation, the cells that had not migrated through the membrane were gently removed from the upper surface of the membrane using a cotton swab. The cells that had migrated to the bottom surface of the membrane were fixed with 4% formaldehyde for 20–30 minutes and stained with 0.1% crystal violet for 15 minutes. Three random visual fields per well were observed under an optical microscope (Olympus, Tokyo, Japan), and the number of invading cells was counted. The degree of cell invasion was quantified as the average number of invading cells per microscopic field. Each experiment was performed in triplicate.

Flow cytometry analysis for apoptosis

Cell apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit (BestBio, Shanghai, China) strictly following the manufacturer’s instructions. Cells were seeded into 6-well plates and treated with bergamottin (0, 20, 40 µM) for 24 hours. After treatment, the cells were harvested by trypsinization [without ethylenediaminetetraacetic acid (EDTA)] and centrifuged at 1,000 rpm for 5 minutes. The cell pellet was washed twice with cold PBS and resuspended in 500 µL of binding buffer. Then, 5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI) were added to the cell suspension, which was incubated at room temperature in the dark for 15 minutes and 5 minutes, respectively. All experimental steps were performed in the dark to avoid fluorescence quenching. Flow cytometry was performed using a FACS C6 instrument (BD Biosciences, San Jose, USA), and the acquired data were analyzed using FlowJo 7.6 software (FlowJo LLC, Oregon, USA). The apoptotic rate was calculated as the percentage of Annexin V-FITC-positive and/or PI-positive cells. Each group was set with three replicates, and the experiment was repeated three times.

RNA-seq and pathway enrichment analysis

Total RNA was extracted from PANC-1 cells in the control group (0 µM bergamottin) and the bergamottin-treated group (20 µM) using TRIzol reagent (Thermo Fisher Scientific, California, USA) according to the manufacturer’s instructions. RNA purity and integrity were verified using a Nanodrop spectrophotometer and agarose gel electrophoresis, respectively. RNA-seq analysis was performed by Lianchuan Biological (Hangzhou, China). The raw sequencing data were processed using Cutadapt software to remove adapter sequences and low-quality reads. The clean reads were mapped to the human reference genome (hg38) using HISAT2 software. The expression levels of genes were estimated using StringTie and Ballgown software. Differentially expressed genes (DEGs) were screened using DESeq2 software with the criteria of fold change (FC) >2 or <0.5 and P value <0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to annotate the biological functions and signaling pathways of the DEGs (11). The RNA-seq datasets generated in this study are available from the corresponding author upon reasonable request (no public database deposition was performed, and no restrictions on data availability exist).

Xenograft mouse models

Six-week-old male BALB/C nude mice (weight: 18–22 g) were purchased from SiPeiFu Biotechnology (Beijing, China) and housed under specific pathogen-free (SPF) conditions with a 12-hour light/dark cycle, constant temperature (22–25 ℃), and constant humidity (50–60%). All animal experiments were performed under a project license (ethical approval code: YXSW2204103831-2) granted by the Animal Ethics Committee of Hangzhou Yanqu Information Technology Co., Ltd., in compliance with the national guidelines for the care and use of laboratory animals. A protocol was prepared before the study without registration. Xenograft mouse models were established by subcutaneous injection of 1×10⁷ PANC-1 cells (suspended in 100 µL PBS) into the axillary region of each nude mouse. Two weeks after tumor inoculation, when the tumor volume reached approximately 100 mm³, the mice were randomly divided into two groups (n=6 mice per group): the control group (intraperitoneal injection of PBS) and the bergamottin-treated group (intraperitoneal injection of bergamottin at 25 mg/kg·d). The injections were administered once daily for 14 consecutive days. After the treatment period, the mice were sacrificed by cervical dislocation, and the tumors were dissected and weighed. The tumor volume was calculated using the formula: volume = (length × width²)/2, where length was defined as the longest diameter of the tumor. All tumor tissues were stored at −80 ℃ for subsequent experiments.

Histopathological analysis

Tumor tissues dissected from nude mice were fixed in 10% neutral buffered formalin for 24 hours, dehydrated through a graded ethanol series, and embedded in paraffin. Paraffin-embedded tissues were sectioned into 4 µm-thick slices using a microtome. The sections were deparaffinized in xylene and rehydrated through a graded ethanol series, then stained with hematoxylin and eosin (HE) stain (Yuanye Biotechnology, Shanghai, China) according to standard protocols. The stained sections were dehydrated, cleared in xylene, and mounted with neutral balsam. Images were captured using a DFC420C digital camera system (Leica Microsystems, Wetzlar, Germany). Two independent professional pathologists evaluated the histopathological changes of the tumor tissues in a blinded manner to avoid bias.

Molecular docking

Molecular docking was performed using the SYBYL v2.1.1 Surflex-Dock Suite with default parameters. The 3D molecular structure of bergamottin was downloaded from the PubChem database (12), and the 3D crystal structure of PPAR-γ was retrieved from the Protein Data Bank (PDB) (13). The structure of bergamottin (in SDF format) was converted to MOL2 format using OpenBabel v2.4.1 software. The PPAR-γ protein was prepared using SYBYL software, including energy minimization and removal of water molecules and heteroatoms, to generate the SFXC file format. The ligand (bergamottin in MOL2 format) was docked to the active site of PPAR-γ, and the docking results were analyzed to evaluate the binding affinity and interaction mode between bergamottin and PPAR-γ.

Statistical analysis

All statistical analyses were performed using GraphPad Prism v8.1 software (GraphPad Software, San Diego, USA). All experiments were conducted in triplicate, and the data are presented as the mean ± standard error (SE). The Student’s t-test was applied for comparisons between two independent groups. For comparisons among three or more experimental groups including the control, 20 and 40 µM groups, one-way analysis of variance (ANOVA) combined with Tukey’s post-hoc test was adopted. Statistical significance was defined as P<0.05.

Results

Bergamottin inhibits cell proliferation in human PC cells

The effect of bergamottin on cell proliferation was evaluated using real-time cell analysis (RTCA), colony formation assay, and Ki67 immunofluorescence staining. The viability of PANC-1 and PATU8988 cells was assessed via CCK-8 assay following treatment with varying concentrations of bergamottin (0 to 50 µM). The respective half-maximal inhibitory concentration (IC₅₀) values of bergamottin against PANC-1 and PATU8988 were determined to be 15.11 and 24.39 µM. Hence, we chose 20 µM (near IC₅₀) and 40 µM to establish a proper concentration gradient to observe its dose-dependent effects. RTCA results revealed a dose-dependent reduction in the proliferation rate of PANC-1 and PATU8988 cells following treatment with 20 and 40 µM bergamottin (Figure 1A). Ki67, a well-recognized marker of cell proliferation and tumor malignant potential, showed markedly decreased expression in bergamottin-treated groups compared to the control (Figure 1B,1C). Consistently, colony formation assays demonstrated that bergamottin significantly suppressed the clonogenic capacity of both PANC-1 and PATU8988 cells (Figure 1D). Collectively, these results indicate that bergamottin dose-dependently inhibits the growth of PC cells, supporting its potential as an anti-proliferative agent for PC.

Figure 1 Bergamottin inhibits the proliferation of PANC-1 and PATU8988 cells in a dose‑dependent manner. (A) Real‑time cell growth curves of PANC-1 and PATU8988 cells treated with 0 µM (red line), 20 µM (green line), and 40 µM (dark green line) bergamottin, monitored over 80 hours. (B) Representative immunofluorescence images of PANC-1 and PATU8988 cells, showing the proliferation marker Ki67 (green fluorescence) and nuclear counterstain DAPI (blue fluorescence) under control and bergamottin treatment (20, 40 µM). (C) Quantification of Ki67‑positive cells in PANC-1 and PATU8988 cells, demonstrating a dose‑dependent reduction in Ki67 expression following bergamottin treatment. (D) Colony formation assay of PANC-1 and PATU8988 cells treated with 0, 20, and 40 µM bergamottin (stained with crystal violet after 14 days of culture) and corresponding statistical analysis of colony numbers, confirming the dose‑dependent inhibition of cell proliferation. Data are presented as mean ± SD of three independent biological replicates (N=3). *, P<0.05; **, P<0.01; ***, P<0.001 versus the control group. DAPI, 4',6-diamidino-2-phenylindole; SD, standard deviation.

Bergamottin inhibits the invasion and migration of PC cells

Cancer cell invasion and migration are critical drivers of tumor malignancy and recurrence. Transwell invasion assays showed that untreated PANC-1 and PATU8988 cells exhibited strong invasive capacity, which was notably attenuated by bergamottin in a concentration-dependent manner (Figure 2A,2B). Wound-healing assays further confirmed that bergamottin treatment dose-dependently impaired the migratory ability of both cell lines (Figure 2C,2D). These findings indicate that bergamottin exerts significant inhibitory effects on the invasive and migratory phenotypes of PC cells.

Figure 2 Bergamottin inhibits the invasion and migration of PANC-1 and PATU8988 cells in a dose-dependent manner. (A) Representative transwell invasion images (stained with 0.1% crystal violet) of PANC-1 and PATU8988 cells treated with 0, 20, and 40 µM bergamottin for 24 h (scale bar: 100 µm), demonstrating dose-dependent suppression of cell invasion. (B) Quantitative analysis of the number of invasive cells in transwell assays for PANC-1 and PATU8988 cells. (C) Representative wound healing images of PANC-1 and PATU8988 cells at 0 and 24 h post-treatment with 20, 40 µM bergamottin, or control medium (scale bar: 200 µm). (D) Quantitative analysis of cell mobility from wound healing assays, confirming dose-dependent inhibition of pancreatic cancer cell migration. Data are presented as mean ± SD of three independent biological replicates (N=3). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 versus the control group. SD, standard deviation.

Bergamottin induces apoptosis of PC cells and suppresses PC progression in vivo

Dysregulation of apoptosis is a key hallmark of cancer. Flow cytometry analysis using Annexin V-FITC/propidium iodide staining revealed that treatment with 20 and 40 µM bergamottin for 24 h significantly increased the apoptosis rate of PANC-1 and PATU8988 cells in a dose-dependent manner (Figure 3A). Following treatment with 20 µM bergamottin, both cell lines showed marked TUNEL-positive signals that co-localized with DAPI-stained nuclei (Figure S1). These results align with our flow cytometry data, providing additional evidence that bergamottin induces apoptosis in PC cells. To validate the in vivo anti-tumor efficacy of bergamottin, a PANC-1 xenograft tumor model was established in nude mice (Figure 3B). Tumors from bergamottin-treated mice were significantly smaller than those from control mice, and histological analysis revealed apoptotic cavities in the bergamottin group (Figure 3C). Furthermore, tumor volume and weight were markedly reduced in bergamottin-treated mice compared to controls (Figure 3D-3F). These data demonstrate that bergamottin promotes apoptosis of PC cells both in vitro and in vivo, thereby effectively suppressing tumor progression.

Figure 3 Bergamottin induces apoptosis in pancreatic cancer cells and suppresses tumor growth in vivo. (A) Flow cytometric analysis of apoptosis in PANC-1 and PATU8988 cells treated with 0, 20, and 40 µM bergamottin, demonstrating a dose‑dependent increase in apoptotic cell rate. (B) Representative image of nude mice bearing PANC-1 xenografts, with the red circle indicating the tumor in the control (CON) group and the green circle indicating the tumor in the bergamottin‑treated (BGM) group. (C) HE staining of tumor tissues (scale bar: 50 µm), showing morphological alterations indicative of inhibited tumor cell growth in the BGM group. The black arrows indicate apoptotic cavities. (D) Representative images of excised tumors from CON and BGM groups. (E,F) Quantitative analysis of tumor volume (E) and tumor weight (F) in nude mice, confirming significant inhibition of tumor growth in the BGM group. Data are presented as mean ± SD of three independent biological replicates (N=3). *, P<0.05; ***, P<0.001; ****, P<0.0001 versus the control group. HE, hematoxylin and eosin; SD, standard deviation.

Bergamottin activates the PPAR signaling pathway in human PC

To elucidate the molecular mechanism underlying the anti-tumor effects of bergamottin, RNA-seq was performed on PANC-1 cells from the control and bergamottin-treated groups. Principal component analysis (PCA) confirmed clear separation between the two groups, indicating distinct gene expression profiles (Figure 4A). Using a threshold of |log₂FC| ≥1 and P<0.05, we identified 317 upregulated and 111 downregulated DEGs, which were visualized in a volcano plot (Figure 4B). A heatmap of the top 100 DEGs (sorted by P value) further illustrated the distinct gene expression patterns between the two groups (Figure 4C).

Figure 4 Transcriptomic profiling of bergamottin-treated pancreatic cancer cells by RNA-seq. (A) PCA of gene expression profiles across all samples. (B) Volcano plot of DEGs, red dots represent significantly up-regulated genes, blue dots represent significantly down-regulated genes, and gray dots represent genes with no significant expression difference. (C) Hierarchical clustering analysis of the top 100 DEGs ranked by the lowest P value; genes involved in the PPAR signaling pathway are highlighted in red. (D) KEGG pathway enrichment analysis of DEGs, with the PPAR signaling pathway identified as a key enriched pathway. Data are presented as mean ± SD of three independent biological replicates (N=3). DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; PCA, principal component analysis; SD, standard deviation.

GO enrichment analysis showed that DEGs were primarily involved in biological processes, including “positive regulation of angiogenesis”, “positive regulation of neutrophil chemotaxis”, “PERK-mediated unfolded protein response”, and “regulation of autophagy” (Figure S2A,S2B). KEGG pathway enrichment analysis revealed significant enrichment of cancer- and immune-related pathways, including the “p53 signaling pathway” and “IL-17 signaling pathway”. Notably, the “PPAR signaling pathway” emerged as the top enriched pathway, containing seven DEGs, suggesting it is a key molecular target of bergamottin (Figure 4D).

Inhibition of PPAR-γ abolishes bergamottin-mediated anti-tumor effects via binding to its pocket

It is well established that PPAR-γ activation promotes cell death in cancer (14). To determine whether PPAR-γ mediates the anti-PC effects of bergamottin, we used the specific PPAR-γ inhibitor GW9662. Wound-healing assays demonstrated that pretreatment with GW9662 effectively reversed the inhibitory effect of bergamottin on PANC-1 cell migration (Figure 5A,5B). Similarly, colony formation assays showed that GW9662 diminished the anti-proliferative effect of bergamottin (Figure 5C,5D), confirming the involvement of PPAR-γ in bergamottin’s anti-tumor activity. Notably, emerging evidence has demonstrated that PPAR-γ mainly functions as a tumor suppressor in PC, and its activation inhibits proliferation, migration, and promotes apoptosis of PC cells.

Figure 5 Inhibition of PPAR reverses the bergamottin-mediated anti-tumor effects, and PPAR directly binds bergamottin. (A) Representative wound healing images of PANC-1 cells treated with bergamottin (BGM, 20 µM) alone or in combination with the PPAR inhibitor GW9662 (10 µM), monitored at 0 and 24 h (scale bar: 200 µm). (B) Quantitative analysis of cell mobility. (C) Representative colony formation images (stained with 0.1% crystal violet) of PANC-1 cells under the same treatment conditions as in (A). (D) Quantitative analysis of colony number. (E) Molecular docking model of PPAR (green) and bergamottin, generated using SYBYL software (total score: 7.4160); the surface structure of the PPAR active site is highlighted in blue. (F) Overlaid view of the binding mode between bergamottin (cyan) and the PPAR ligand-binding pocket, with hydrogen bonds depicted as red dashed lines and key interacting residues labeled. Data are presented as mean ± SD of three independent biological replicates (N=3). *, P<0.05; **, P<0.01 versus the indicated group. BGM, bergamottin-treated group; GW9662, PPAR inhibitor-treated group; SD, standard deviation.

Bergamottin (5-geranoxypsoralen, CAS: 7380-40-7) is a lipophilic furanocoumarin with extremely low aqueous solubility. The calculated water solubility is 1.04×10⁻² mg/L (LogS =−4.51), with a LogP of 5.30–5.61, indicating high lipophilicity (15). It is soluble in organic solvents such as DMSO (30–50 mg/mL) and ethanol (~10 mg/mL), but practically insoluble in water. Bergamottin is light-, heat-, and pH-sensitive. The solid powder is stored at 2–8 ℃, protected from light; DMSO stock solutions are stable for 6 months at −80 ℃ or 1 month at −20 ℃ under light protection. Preliminary pharmacokinetic studies in humans show that following oral administration of 6 and 12 mg bergamottin, the Cmax values are 2.1 and 5.9 ng/mL, and Tmax values are 0.8 and 1.1 h, respectively (16). It is rapidly absorbed but exhibits low oral bioavailability, is primarily metabolized by CYP3A4 to 6',7'-dihydroxybergamottin (DHB), and has a short elimination half-life (17). Intraperitoneal administration of bergamottin at 50 mg/kg exerts prominent anti-hepatocellular carcinoma effects in nude mice, and combined treatment markedly potentiates the anticancer efficacy of lenvatinib in vivo (18).

To further clarify the direct interaction between bergamottin and PPAR-γ, molecular docking simulations (SYBYL-X 2.1.1) were performed using the PPAR-γ crystal structure (PDB ID: 4EMA) that revealed a stable binding interaction with a total score of 7.4160 (Figure 5E). Bergamottin formed hydrogen bonds with residue S342 (bond length <5 Å) and interacted with 11 residues (Y473, H449, F363, I341, L333, M329, I326, H323, R288, R289, C285) within the ligand-binding pocket, inducing conformational changes that remodel cofactor binding sites for gene activation (Figure 5F). These findings demonstrate that bergamottin functions as a direct PPAR-γ agonist rather than indirectly regulating PPAR-γ expression, thereby activating PPAR-γ signaling to induce apoptosis and suppress PC progression.

Discussion

Despite advances in early detection and therapeutic strategies, PC remains one of the most lethal malignancies, characterized by high invasiveness, early metastasis, and frequent diagnosis at advanced stages (19). Surgical resection is the only curative option, yet it is feasible in only ~20% of patients, and recurrence rates remain high even after complete resection (20). Chemotherapy (e.g., FOLFIRINOX, S-1, nab-paclitaxel plus gemcitabine) is the mainstay of treatment for unresectable or recurrent disease (21,22), but long-term survival outcomes remain unsatisfactory, highlighting the urgent need for novel therapeutic agents and molecular targets (23,24).

Natural products represent a rich source of anti-cancer compounds (25). Bergamottin, a furanocoumarin derived from bergamot oil, has been reported to exhibit antibacterial, cytochrome P450-modulating, and anti-proliferative activities in various cancers, including neuroblastoma (26,27), liver cancer and gastric cancer (9), colon cancer (27), and lung cancer (28). It also exhibits antibacterial activity against a variety of pathogens and improves the bioavailability of compounds such as anticancer drugs by interacting with certain subtypes of the cytochrome P450 enzyme (9). Zsidó et al. conducted cell viability and proliferation tests using NIH3T3 (mouse embryonic fibroblasts) and HeLa (human cervical cancer cells) to analyze the cytotoxicity of bergamottin, and found that bergamottin had no significant toxicity to fibroblasts, suggesting that bergamottin has low toxic side effects on normal cells (29). We performed HE staining on major organs including the heart, liver, spleen, lung, and kidney after treatment with 200 mg/kg bergamottin, and found no obvious pathological abnormalities or structural damage, preliminarily confirming its good safety in the body (Figure S3). RTCA, Ki67 staining, colony formation detection, and transwell invasion demonstrated that 20 and 40 µM concentrations of bergapten could significantly inhibit the proliferation and invasion of PANC-1 and PATU8988 cells. Flow cytometry showed that bergamottin could promote apoptosis of PC cells, and 25 mg/kg bergamottin treatment could significantly inhibit tumor growth in nude mice. Our research provides comprehensive in vitro and in vivo evidence indicating that bergamottin has high safety and inhibits the proliferation, invasion, and migration of PC cells in a dose-dependent manner, while inducing cell apoptosis and thereby inhibiting tumor development.

PPARs are nuclear transcription factors that regulate cellular differentiation, metabolism, and tumorigenesis (30). Among the three subtypes (PPAR-α, PPAR-β, PPAR-γ), PPAR-γ is a key regulator of lipid and glucose metabolism, inflammation, and cell differentiation, and its dysregulation—typically downregulation—has been implicated in multiple cancers (31-33). Activation of PPAR-γ has been shown to inhibit cell cycle progression and suppress the malignant behavior of PC cells (34), and PPAR-γ agonists (e.g., troglitazone) have demonstrated anti-tumor effects in preclinical models (35). Koga et al. further found that PPAR-γ ligands enhance the cytotoxic effects of the first-line anticancer drug gemcitabine on human PC cells (36). Our RNA-seq analysis revealed that the PPAR signaling pathway was the most significantly enriched pathway following bergamottin treatment. Molecular docking further confirmed that bergamottin binds stably to the ligand-binding pocket of PPAR-γ, forming hydrogen bonds with S342 and interacting with key residues to induce conformational changes and activate PPAR-γ signaling, similar to other compounds such as medium-chain fatty acids (37).

Analysis of DEGs within the PPAR signaling pathway identified three upregulated genes (PCK2, SLC27A1, PLIN2) and two downregulated genes (SCD, OLR1). Dysregulation of these genes is closely linked to cancer progression: upregulation of PCK2 and SLC27A1 is associated with enhanced survival of colorectal and ovarian cancer cells (38,39), while overexpression of PLIN2 significantly slows the progression of clear cell renal cell carcinoma (40). Conversely, downregulation of SCD and OLR1 inhibits PC growth (41,42). Our data suggest that bergamottin modulates the expression of these cancer-related genes via PPAR-γ activation, thereby exerting its anti-tumor effects. Flow cytometry analysis confirmed that bergamottin induces apoptosis in PC cells. To verify the role of PPAR-γ in this process, we used the PPAR-γ inhibitor GW9662, which reversed the inhibitory effects of bergamottin on PANC-1 cell migration and proliferation. These results further confirm that PPAR-γ activation is the dominant and critical mechanism mediating the anti-tumor activity of bergamottin. Although we cannot completely exclude other apoptosis-related pathways, the PPAR-γ-independent apoptotic mechanism has been largely excluded based on the significant reversal effect of GW9662.

In addition to the PPAR signaling pathway, our RNA-seq data also revealed significant enrichment of the p53 signaling pathway and IL-17 signaling pathway, implying potential multitarget mechanisms of bergamottin against PC. The p53 pathway is a well-established tumor suppressor axis that governs cell cycle arrest, apoptosis, and DNA damage repair, and its dysfunction is closely associated with the malignant progression and therapeutic resistance of PC (43). Similarly, the IL-17 signaling pathway participates in chronic inflammation, immune microenvironment remodeling, and tumor invasion, which are closely linked to PC metastasis and poor prognosis (44). Although the current study focuses on PPAR-γ as the core target, we cannot rule out the possibility that bergamottin simultaneously modulates p53 and IL-17 pathways to enhance its anti-PC effects. These observations support the multi-target property of natural products and provide new directions for further exploring the comprehensive mechanisms of bergamottin in PC suppression.

Although we have confirmed that bergamottin exerts regulatory effects on the PPAR-γ signaling pathway at the cellular level, this study still has some limitations. Notably, RNA-seq analysis was only conducted in PANC-1 cells. Further transcriptomic investigation in additional PC cell lines is therefore necessary to fully verify and elucidate the detailed molecular mechanisms. Additionally, given the inherent chemoresistance of PC (1,5), future studies should explore the synergistic effects of bergamottin in combination with first-line chemotherapies (e.g., gemcitabine) or other targeted agents (e.g., COX-2 inhibitors) to improve therapeutic outcomes and delay the development of drug resistance. Furthermore, given the prominent anti-PC activity of bergamottin and its clear mechanism of inducing apoptosis and regulating the PPAR-γ signaling pathway, bergamottin also exhibits great potential for combination therapy with conventional clinical chemotherapeutic agents. Synergistic anti-tumor effects may be achieved when bergamottin is combined with first-line drugs such as gemcitabine, which could help reduce the dosage of chemotherapeutic drugs, alleviate chemotherapy-related toxic side effects, and delay the occurrence of drug resistance. Therefore, the efficacy and safety of bergamottin in combination therapy will be included in our subsequent experimental design and preclinical systematic evaluation, so as to further improve and perfect the translational research basis of bergamottin for PC treatment.

Nevertheless, the present study has certain limitations that warrant consideration. All data were obtained from two established PC cell lines and a subcutaneous xenograft mouse model, which may not fully recapitulate the complex tumor microenvironment, genetic heterogeneity, or clinical characteristics of human PC. Therefore, it is still too preliminary to conclude that bergamottin can be directly developed as a therapeutic drug at the current stage. Further validation using clinically relevant models, including patient-derived primary PC cells, patient-derived organoids (PDOs), or orthotopic tumor models, is urgently needed in future studies to confirm the anti-tumor efficacy, biosafety, and translational potential of bergamottin before clinical application.

Conclusions

In conclusion, this study demonstrates that bergamottin significantly inhibits the malignant biological behavior of PC cells and promotes tumor cell apoptosis both in vitro and in vivo. Mechanistically, bergamottin acts as a PPAR-γ agonist by binding to its ligand-binding pocket, thereby activating PPAR-γ signaling and inducing apoptosis. These findings demonstrate that bergamottin exerts favorable preclinical anti-tumor effects against PC, supporting its potential value for further translational study.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0837/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0837/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0837/prf

Funding: This work was supported by the Guizhou Provincial Basic Research Program (Natural Science) (No. QianKeHe basics-MS [2026] 349), the Taizhou Science and Technology Project (No. 25ywb202), the Science and Technology Fund Project of Guizhou Provincial Health Commission (No. gzwkj2026-044), the Guizhou Province Traditional Chinese Medicine and Ethnic Medicine Science and Technology Research Project (No. QZYY-2026-213), and the Talent Project of Guizhou Provincial People’s Hospital (No. [2024]-28).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0837/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All animal experiments were performed under a project license (ethical approval code: YXSW2204103831-2) granted by the Animal Ethics Committee of Hangzhou Yanqu Information Technology Co., Ltd., in compliance with the national guidelines for the care and use of laboratory animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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