The AsPC-1 (passage 11) and MIA PaCa-2 (passage 13) pancreatic cancer cell lines were obtained from the Korea Cell Line Bank (KCLB). KCLB routinely authenticates cell lines via short tandem repeat (STR) profiling to confirm their identity and screen for contamination. All cell lines were cultured under standard conditions and used at the specified passage numbers to ensure experimental consistency. Cultivation of AsPC-1 cells was conducted using Roswell Park Memorial Institute (RPMI) medium (Hyclone). Concurrently, MIA PaCa-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/High (Hyclone). Both media were enriched with a supplement comprising 10% fetal bovine serum (FBS; Hyclone), and 1% Penicillin-Streptomycin (Gibco BRL). The cell lines were incubated at a temperature of 37°C in a humidified environment, with a 5% CO₂ atmosphere to ensure optimal growth conditions.

The assessment of cell viability for both AsPC-1 and MIA PaCa-2 cell lines was performed utilizing the Ez-cytox Cell Viability Assay Kit (Itsbio) according to the manufacturer's instructions. All experiments were performed in triplicate (n=3) to ensure reproducibility and statistical significance.

Lysis of AsPC-1and MIA PaCa-2 cells, as well as mouse tissue samples, was carried out using the EzRIPA Lysis kit (ATTO Corporation). Protein concentration was determined using the Bradford assay reagent (Bio-Rad). For protein visualization, Western blot analysis was performed using primary antibodies at a 1:1,000 dilution, obtained from Cell Signaling Technology. This was followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies at a 1:2,000 dilution, sourced from Vector Laboratories. Detection of specific immune complexes was achieved using the Western Blotting Plus Chemiluminescence Reagent (Millipore). The primary antibodies used targeted hPARP (Cell Signaling Technology, #9542), MCL-1 (Cell Signaling Technology, #5453), cleaved-caspase-3 (Cell Signaling Technology, #9664), TGF-βR1 (Abcam, ab31013), p-SMAD2/3 (Cell Signaling Technology, #8828s), SMAD2/3 (Cell Signaling Technology, #5678s), human E-cadherin (Abcam, ab76055), Vimentin (Abcam, ab92547), Snail, (Cell Signaling Technology, #3879), PUMA (Cell Signaling Technology, #98672), and β-actin (Sigma-Aldrich, A5541). All experiments were performed in triplicate (n=3) to ensure reproducibility and statistical significance.

AsPC-1and MIA PaCa-2 were seeded into 8-well plate (SPL Life Science) at the concentration of 8,000 cells/well. After cultured overnight, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min. Then the cells were blocked with incubation buffer (Normal Hores serum in PBS) for 1 h at room temperature. Following that, the cells were also stained with above antibodies and incubated at 4°C protected from light. After 1 h incubation, the cells were washed twice and stained with 100 ng/ml DAPI for 10 min at room temperature. The immunocytochemistry staining utilized antibodies against E-cadherin (Santa Cruze Biotechnology, sc-8426), Vimentin (Santa Cruze Biotechnology, sc-373717), Snail (Genetex, GTX125918) and GAPDH (Santa Cruze Biotechnology, sc-365062). The final observation of the samples was conducted using a fluorescence imaging system (EVOS U5000; Invitrogen; Thermo Fisher Scientific, Inc.). All experiments were performed in triplicate (n=3) to ensure reproducibility and statistical significance.

For the immunohistochemical analysis, tissue sections that were formalin-fixed and paraffin-embedded underwent deparaffinization, followed by a rehydration process using a series of ethanol solutions. Epitope retrieval was then carried out in accordance with standard methodologies. The immunohistochemical staining utilized antibodies against BIM (Cell Signaling Technology, #2933s), Mcl-1 (Santa Cruze Biotechnology, sc-377487), E-cadherin (Santa Cruze Biotechnology, sc-8426), and Snail (Genetex, GTX125918). Post-staining, the samples were analyzed for antibody expression using a laser-scanning microscope (Eclipse TE300; Nikon).

The detection of apoptosis in pancreatic cancer cells was carried out using TUNEL analysis, employing the In Situ Apoptosis Detection Kit (Takara Bio Inc.), with the protocol adhering to the manufacturer's instructions. In summary, the procedure involved incubating sample slides with 50 µl of TUNEL reaction mixture and TdT labeling reaction mix for a duration of 1 h at 37°C, ensuring the environment was dark. Following the incubation, the slides were washed three times using phosphate-buffered saline (PBS). The final observation of the samples was conducted using a fluorescence imaging system (EVOS U5000; Invitrogen; Thermo Fisher Scientific, Inc.).

The AsPC-1 and MIA PaCa-2 cell lines were cultured in 6-well plates until they reached full confluence. Subsequently, the growth medium was substituted with serum-free media, and the cells underwent a 72-h incubation period. Post-incubation, the cell monolayers were mechanically disrupted to create a wound, followed by the application of a test agent (rupatadine). An exact region of the wound was imaged using phase-contrast microscopy prior to the administration of these agents. This same region was imaged again after a 72-h period. The area of the wound was quantitatively assessed using image analysis techniques, both initially and after the 72-h interval. The rate of wound closure was calculated based on the change in wound area over time, using the formula: [(initial wound area-final wound area)/initial wound area] ×100, to express this change as a percentage. All experiments were performed in triplicate (n=3) to ensure reproducibility and statistical significance.

Our animal study design involved five-week-old male BALB/c nude mice (Orient Bio). Our animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Korea (approval No. CUMC-2022-0014-01) and were conducted in accordance with institutional and national guidelines for the care and use of laboratory animals. For the xenograft pancreatic cancer model, 1×10⁶ AsPC-1 cells were subcutaneously injected into the mice. In the evaluation of rupatadine's in vivo efficacy, mice were grouped randomly, with each group containing five mice. After two weeks post-implantation of the tumor model, the mice were treated with rupatadine (3 mg/kg) administered intraperitoneally, three times a week for three weeks. Twenty-one days after initial treatment, the animals were euthanized, and the tumors were surgically removed for subsequent analysis. Animals were euthanized using compressed carbon dioxide (CO₂) gas at a displacement rate of 30–70% of the chamber volume per minute for 2–3 min. Loss of consciousness was confirmed by observing lack of response and faded eye color, and death was verified by cessation of respiration, cardiac arrest, and pupil dilation. Throughout the experiment, animal health was monitored daily, and humane endpoints-such as weight loss >20%, severe lethargy, or ulceration-were predefined and strictly observed.

Regarding the establishment and monitoring of tumor formation before initiating Rupatadine treatment, tumors were allowed to reach a predetermined size, specifically a volume of 100 mm³, confirmed through visual inspection before the commencement of drug administration. The endpoint for the study was matched based on the time-point rather than the stage, ensuring that all groups were assessed over the same duration post-treatment initiation.

Mice received an intravenous dose of 10 mg/kg rupatadine. Plasma samples were collected at intervals from 5 to 360 min and processed by mixing with four volumes of acetonitrile, followed by centrifugation. Tissue samples at 5 and 60 min were prepared similarly. Rupatadine concentrations were measured using high-performance liquid chromatography (HPLC) with a Waters Arc system, a C18 column, and a mobile phase of methanol and 0.3 M sodium acetate (pH 4.4) at a 20:80 ratio, with a flow rate of 1 ml/min. The retention time for rupatadine was 5 min.

Statistical analysis was performed using SPSS 11.0 (SPSS Inc.), with data presented as mean ± standard deviation (SD). Normality was assessed using the Shapiro-Wilk test. The Kruskal-Wallis test with Dunn's post hoc test was used for multiple-group comparisons, while the Mann-Whitney U test and Student's t-test were applied for nonparametric and normally distributed two-group comparisons, respectively. Statistical significance was set at P<0.05.

Rupatadine is a chemical compound characterized by a cationic amphiphilic structure, consisting of a hydrophobic ring system and a side chain with a cationic amine group (Fig. 1A). To further evaluate the dose-dependent effects of rupatadine, dose-response curves were generated for AsPC-1 and MIA-PaCa-2 pancreatic cancer cells, as well as human pancreatic stellate cells (HPSC) (Fig. 1B). The IC50 values for AsPC-1 cells were 23.5 µM (24 h) and 24.9 µM (48 h), while for MIA-PaCa-2 cells, the IC50 values were 30.9 µM (24 h) and 35.1 µM (48 h). In the MIA-PACA cell line, rupatadine displayed lower cell viability compared to treatments with irinotecan (3.125–200 ng/ml), 5-fluorouracil (1–100 µM), and paclitaxel (1–10 nM) at both 24 and 48 h. This suggests that rupatadine may possess superior anticancer effects relative to these well-established chemotherapeutic agents (Fig. S1). In contrast, rupatadine exhibited significantly lower cytotoxicity in human pancreatic stellate cells (HPSC), with IC50 values of 66.8 µM at 24 h and 132.0 µM at 48 h, indicating a selectivity for pancreatic cancer cells over normal pancreatic cells. Data are presented as mean ± SD from three independent experiments. This data suggests that rupatadine preferentially inhibits pancreatic cancer cells while having a reduced effect on normal pancreatic cells.

Following the treatment with rupatadine, a concentration-dependent (0–25 µM) increase in the pro-apoptotic markers, PARP and cleaved caspase-3, and a decrease in the anti-apoptotic marker, Mcl-1, were observed in AsPC-1 pancreatic cancer cells, as evidenced by Western blot analysis (P<0.05) (Fig. 1C). A similar trend was noted in MIA PaCa-2 cells, demonstrating the compound's consistent apoptotic induction across different cell lines (P<0.05) (Fig. 1D). Data are presented as mean ± SD from three independent experiments. Additionally, Western blot analysis was employed to assess the impact of rupatadine on TGF-β signaling and EMT markers. The results indicate that Rupatadine significantly suppresses the expression of TGFβRI and diminishes the phosphorylated to total SMAD2/3 ratio in a dose-dependent manner in AsPC-1 cells (P<0.05) (Fig. 2A), implicating the inhibition of the TGFβ1 signaling pathway at the receptor level. The analysis further revealed that Rupatadine upregulated the epithelial marker E-cadherin while downregulating mesenchymal markers N-cadherin, Vimentin, and Snail, highlighting its role in thwarting EMT (P<0.05). Parallel results in MIA PaCa-2 cells reinforced rupatadine's efficacy in targeting both the TGFβ signaling pathway and EMT (Fig. 2B).

The impact of rupatadine on the migration of pancreatic cancer cells was evaluated using a wound healing assay. After creating a scratch in a confluent monolayer of AsPC-1 cells, the closure of the wound was monitored in response to increasing concentrations of rupatadine (0.01–25 µM). Increased concentrations of rupatadine, specifically at 25 µM, were correlated with a significant expansion of the scratch area, illustrating a dose-dependent suppression of cell migration in AsPC-1 cells (Fig. 3A). An analogous effect was recorded in MIA PaCa-2 cells at the 25 µM concentration of rupatadine (Fig. 3B). Data are presented as mean ± SD from three independent experiments.

The influence of rupatadine on EMT in AsPC-1 pancreatic cancer cells was investigated through immunofluorescence analysis of EMT markers following treatment with rupatadine. Increasing the concentration of rupatadine (0.1–10 µM) resulted in a concentration-dependent increase in the immunofluorescence of the epithelial marker E-cadherin, and a decrease in the immunofluorescence of mesenchymal markers Snail and Vimentin (P<0.05) (Fig. 4A). Similar effects were observed in MIA PaCa-2 pancreatic cancer cells, with rupatadine treatment leading to an increased immunofluorescence in E-cadherin and a decreased immunofluorescence in Snail and Vimentin (P<0.05) (Fig. 4B). Data are presented as mean ± SD from three independent experiments.

The in vivo xenograft mouse model of pancreatic cancer was established by direct injection of AsPC-1 cells (1×10⁶ cells) into the pancreas of BALB/c nude mice after laparotomy under anesthesia. Two weeks later, the mice were treated with rupatadine (3 mg/kg) administered intraperitoneally, three times a week for three weeks. On 21 days after initial treatment, the animals were euthanized, and the tumors were surgically removed for subsequent analysis. Fig. 5A presents a representative illustration depicting the difference in tumor size between the control group and the rupatadine-treated group on 21 days after initial treatment. Tumors in the control group weighed between 0.4 and 0.6 g, whereas those treated with Rupatadine weighed between 0.2 and 0.3 g. To assess the effects of rupatadine on tumor progression over time, tumor volume was measured at multiple time points following treatment initiation. The rupatadine-treated group exhibited a significantly slower tumor growth rate compared to the control group (Fig. 5B top). After 21 days of treatment, the average tumor volume in the control group continued to increase, whereas the tumor volume in the rupatadine-treated group showed a marked reduction (Fig. 5B top). In contrast, no significant differences in body weight were observed between the two groups throughout the experimental period, indicating that rupatadine treatment did not cause notable systemic toxicity (Fig. 5B bottom). Consistently, tumor weight analysis performed on day 21 after the initial treatment revealed a significant reduction in the rupatadine-treated group compared to the control group (P<0.05), further supporting its tumor-suppressive effects (Fig. 5C). Data are presented as mean ± SD from three independent experiments.

Subsequently, a TUNEL assay was conducted to quantify apoptosis in the excised tumors from the rupatadine-treated groups. The results of the TUNEL assay revealed a significantly higher apoptosis index in the tumors treated with rupatadine compared to those in the control groups (P<0.05) (Fig. 5D). Western blot analysis was utilized to assess the differential expression of markers associated with EMT and apoptosis in the excised tumor tissues (Fig. 5E). In the group treated with rupatadine, a notable upregulation of the epithelial marker E-cadherin was observed, in contrast to the control group. Conversely, the mesenchymal marker vimentin was significantly downregulated (P<0.05) in the rupatadine-treated tumors. Furthermore, the rupatadine-treated tumors demonstrated a significant elevation in the levels of pro-apoptotic markers, PARP and PUMA, while the expression of the anti-apoptotic marker Mcl-1 was markedly reduced (P<0.05). Data are presented as mean ± SD from three independent experiments.

Tumor tissues derived from a mouse xenograft model of pancreatic cancer underwent Hematoxylin and Eosin (H&E) staining for comparative histological examination. In the rupatadine-treated group, a notable reduction in tumor cell density was observed (Fig. 6A). This was followed by immunohistochemical staining of the excised tumor tissues from each experimental group. Analysis of the stained tissues revealed that, in the rupatadine-treated group, there was a significant increase in the percentage of immunoreactive areas for a pro-apoptotic marker, BIM, compared to the control group. Conversely, the expression of Mcl-1, an anti-apoptotic marker, was significantly decreased (P<0.05) (Fig. 6B top). Furthermore, the rupatadine-treated group exhibited a significant increase in the percentage of immunoreactive areas for the epithelial marker E-cadherin, alongside a significant decrease in Snail, a mesenchymal marker (P<0.05) (Fig. 6B bottom). Overall, these findings suggest that rupatadine treatment can attenuate the EMT process and enhance apoptosis in pancreatic cancer tissues within this mouse xenograft model.

To further investigate the in vivo pharmacokinetics of rupatadine, we analyzed its plasma concentration and tissue distribution following intravenous injection (10 mg/kg) in mice. Plasma concentration-time profiling demonstrated a rapid decline in rupatadine levels, with detectable concentrations at 5, 15, 30, and 60 min, but no detectable levels after 180 min, indicating fast clearance from circulation (Fig. 6C, left). Tissue distribution analysis revealed that rupatadine primarily accumulated in the liver and kidneys within 5 min post-injection but was largely undetectable in these tissues by 60 min, suggesting rapid metabolism and excretion (Fig. 6C, right). These findings provide essential information regarding rupatadine's pharmacokinetic profile and tissue clearance dynamics, which are critical for understanding its potential as an anticancer agent.