Palmaturbine Inhibits Pancreatic Ductal Adenocarcinoma by Suppressing the JAK2/STAT3 Signaling Pathway

TL;DR

Palmaturbine, a natural compound, shows anti-tumor effects in pancreatic cancer by blocking the JAK2/STAT3 pathway, offering a potential new treatment.

Contribution

This study identifies palmaturbine as a novel JAK2/STAT3 pathway inhibitor with anti-tumor activity in pancreatic ductal adenocarcinoma.

Findings

Palmaturbine inhibits PDAC cell proliferation, migration, and invasion while inducing apoptosis.

Palmaturbine suppresses the JAK2/STAT3 signaling pathway by inhibiting phosphorylation of JAK2 and STAT3.

Palmaturbine significantly inhibits tumor growth in mouse xenograft models without causing toxicity.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with an extremely poor prognosis, and current clinical treatment options are limited. Natural products, due to their multi-target and low-toxicity characteristics, have emerged as an important direction for the development of anti-tumor drugs. Palmaturbine (Pal), an isoquinoline alkaloid derived from Coptis chinensis and Berberis species, has shown anti-inflammatory and anti-tumor potential in preliminary studies; however, its mechanism of action in PDAC remains unclear. This study systematically evaluated the anti-tumor effects and molecular mechanisms of Pal on PDAC through in vitro and in vivo experiments. In vitro, Pal significantly inhibited PDAC cell proliferation, migration, and invasion, induced G2/M phase cell cycle arrest, and promoted apoptosis. Transcriptomic sequencing and Western blot analysis revealed that Pal suppressed the JAK2/STAT3 signaling pathway by inhibiting the phosphorylation of JAK2 and STAT3. Animal experiments further indicated that Pal significantly inhibited the growth of subcutaneous xenograft tumors in nude mice without causing obvious toxicity. In summary, Pal, as a natural JAK/STAT pathway inhibitor, exhibits favorable anti-tumor activity and safety in PDAC treatment, holding potential as a novel candidate drug for PDAC.

Figures

Full text

1. Introduction

Pancreatic cancer is one of the most aggressive and lethal malignancies globally, with its incidence and mortality rates continuously rising over the past few decades [1]. It is projected to become the second leading cause of cancer-related deaths by 2030 [1]. Pancreatic ductal adenocarcinoma (PDAC) constitutes the predominant histological subtype, accounting for over 90% of all cases [2]. The clinical management of PDAC faces significant challenges due to its late presentation, lack of effective early screening methods, and high postoperative recurrence rates [3]. Consequently, systemic drug therapy (chemotherapy, targeted therapy, etc.) serves as the core strategy for prolonging survival and improving the quality of life in most PDAC patients [3,4,5].

Currently, gemcitabine-based regimens (e.g., gemcitabine monotherapy or in combination with nab-paclitaxel) and the FOLFIRINOX regimen (comprising oxaliplatin, irinotecan, leucovorin, and 5-fluorouracil) represent the first-line standard treatments for advanced PDAC [4,5]. Although these regimens have improved patient survival to some extent, their efficacy remains far from satisfactory [5]. Moreover, PDAC cells frequently exhibit inherent and acquired chemotherapy resistance, involving complex mechanisms such as increased drug efflux, enhanced DNA damage repair, inactivation of apoptosis pathways, and a highly fibrotic and hypoxic tumor microenvironment (TME) [6]. Therefore, the development of novel, efficient, and low-toxic anti-PDAC drugs, along with a deeper understanding of their mechanisms of action, has become a critical scientific issue in oncology research.

Against this backdrop, active ingredients derived from medicinal plants and natural sources have regained widespread attention. Natural products, owing to their structural diversity, multi-target effects, and relatively low toxicity, have served as a rich source for innovative drug discovery [7,8,9]. Many widely used chemotherapy drugs, such as paclitaxel and vincristine, are either directly or indirectly derived from natural products. Recently, several research teams have focused on identifying anti-pancreatic cancer compounds from traditional Chinese medicines and natural plants, achieving notable progress. For instance, curcumin inhibits cancer cell proliferation by modulating Beclin1 expression and suppressing the hypoxia-inducible factor-1α-mediated glycolytic pathway [10], while celastrol exhibits potential in inhibiting cancer cell invasion and metastasis by downregulating CXCR4 expression [11]. Notably, some isoquinoline alkaloids have demonstrated significant anti-cancer potential [12]. These studies not only validate the potential of natural products in PDAC treatment but also suggest that they may exert multi-faceted anti-tumor effects by interfering with key signaling pathways involved in tumorigenesis and progression.

The pathogenesis of PDAC involves a complex network of signaling cascades. While mutations in KRAS (>90%), TP53, SMAD4, and CDKN2A are considered driver events, directly targeting these mutations has proven pharmacologically challenging [13]. Alternatively, the JAK/STAT3 pathway, which is often hyperactivated by upstream inflammatory cytokines (e.g., IL-6) and oncogenic KRAS signaling, serves as a critical hub for tumor growth and stromal interactions [14,15]. Therefore, targeting the JAK2/STAT3 axis represents a viable therapeutic strategy to disrupt this oncogenic network.

Specifically, the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, particularly the JAK2/STAT3 axis, plays a central role in PDAC initiation, progression, metastasis, and immune evasion [16]. Under normal physiological conditions, the JAK/STAT pathway is tightly regulated by cytokines and participates in cell proliferation, differentiation, apoptosis, and immune responses. However, in PDAC, this pathway is often persistently and abnormally activated due to excessive cytokine secretion (e.g., IL-6) or receptor mutations. Activated JAK2 phosphorylates STAT3 at specific tyrosine residues (e.g., Tyr705), leading to STAT3 dimerization and nuclear translocation, where it acts as a transcription factor to directly bind DNA and initiate the transcription of downstream target genes, including those promoting cell cycle progression (e.g., Cyclin D1), anti-apoptosis (e.g., Bcl-2, Bcl-xL, Mcl-1), and angiogenesis (e.g., VEGF) [17]. Thus, sustained STAT3 activation provides PDAC cells with robust proliferative drive, apoptosis resistance, and microenvironmental adaptation advantages. Numerous preclinical studies have shown that inhibiting the JAK2/STAT3 signaling pathway effectively suppresses PDAC growth and enhances its sensitivity to chemotherapy [18,19,20]. Nevertheless, no specific JAK2/STAT3 inhibitors have been approved for clinical use in PDAC, highlighting the significant clinical value of developing novel, safe, and effective inhibitors targeting this pathway.

Pal is an isoquinoline alkaloid isolated from Coptis chinensis and Berberis species, sharing structural similarities with berberine [18]. Early pharmacological studies have indicated that Pal possesses various biological activities, including antibacterial, anti-inflammatory, and cardiovascular protective effects [21,22]. More recently, its anti-tumor potential has been gradually unveiled, with studies demonstrating its inhibitory effects on lung cancer cells through mechanisms involving cell cycle arrest and apoptosis [21,22]. However, little is known about its role in PDAC, the most aggressive and treatment-refractory malignancy. Whether Pal can inhibit PDAC cell proliferation, migration, and invasion, and whether its action is related to the modulation of the critical signaling pathway, as well as its anti-tumor efficacy and safety in vivo, remain unanswered questions that warrant systematic investigation.

In this study, we aimed to determine the anti-PDAC efficacy of Palmaturbine and elucidate its mechanism of action. We hypothesized that Palmaturbine exerts its anti-tumor effects by directly targeting the hyperactivated JAK2/STAT3 signaling axis, thereby disrupting the cell cycle and inducing apoptosis in pancreatic cancer cells. To test this, we systematically evaluated the phenotypic changes in PDAC cells, validated the molecular targets using transcriptomics and docking analysis, and assessed the therapeutic potential in a xenograft mouse model.

2. Results

2.1. Palmaturbine Specifically Inhibits the Viability of PDAC Cells in a Dose- and Time-Dependent Manner

The molecular structure of Pal is presented in Figure 1A. To evaluate the effects of Pal on PDAC cells, we first observed morphologically that Pal exerts inhibitory effects on PDAC cells: Cells treated with Pal exhibited typical cytotoxic responses, including cell rounding, shrinkage, reduced adherence, and increased refractivity. Subsequently, we assessed its cytotoxicity against four different PDAC cell lines (ASPC-1, BxPC-3, CFPAC-1, and SW1990) (Figure 1B). The results of the CCK-8 assay demonstrated that, over a treatment period of 24 to 72 h, Pal exhibited significant growth-inhibitory effects on all tested PDAC cell lines, showing a clear dose- and time-dependent pattern (Figure 1C). The half-maximal inhibitory concentration (IC50) values for each cell line at 24, 48, and 72 h were calculated and are summarized in Supplementary Table S1. Collectively, these findings indicate that Pal is cytotoxic to PDAC cells, suggesting its potential as a candidate chemotherapy agent for PDAC.

2.2. Palmaturbine Effectively Inhibits the Proliferative Capacity of PDAC Cells

The infinite proliferative potential of tumor cells is a core characteristic. We employed the EdU assay and the colony formation assay to separately evaluate the impact of Pal on the proliferative capacity of PDAC cells. The EdU incorporation assay was used to assess the effect of Pal on cellular DNA synthesis. As shown in Figure 2A,B, after 24 h of treatment with 20 μM and 40 μM Pal, the EdU-positive rates in CFPAC-1 and SW1990 cells were significantly reduced in a dose-dependent manner. At the protein level, Western blot analysis revealed that Pal treatment significantly downregulated the expression of two proliferation marker proteins, Ki-67 and PCNA (Figure 2C). To evaluate the impact of Pal on the long-term proliferative and self-renewal capacity of cells, we conducted a colony formation assay. The results, as shown in Figure 2D,E, demonstrated that compared to the control group, the number and size of cell colonies formed in the Pal-treated groups were significantly reduced, and this inhibitory effect was enhanced with increasing drug concentration. Combining the results of the EdU, Western blot, and colony formation assays indicates that Pal can suppress proliferative potential at multiple levels, from inhibiting DNA synthesis to weakening long-term colony formation.

2.3. Palmaturbine Inhibits the Migration and Invasion Processes of PDAC Cells

Metastasis is the primary cause of the poor prognosis in PDAC patients. We evaluated the effects of Pal on the migration and invasion capabilities of PDAC. The wound healing assay demonstrated that Pal treatment significantly slowed down the healing rate of scratches in CFPAC-1 and SW1990 cells (Figure 3A,B). The Transwell migration assay further corroborated this phenomenon, with the number of cells migrating through the membrane significantly decreasing with increasing Pal concentrations (Figure 3C,D). More importantly, in the invasion assay using Transwell chambers pre-coated with Matrigel to simulate the extracellular matrix, Pal also exhibited potent inhibitory effects, significantly reducing the ability of cells to penetrate the Matrigel (Figure 3E,F). Epithelial–mesenchymal transition (EMT) is a key process that drives tumor cells to acquire migration and invasion capabilities. We examined the protein expression of EMT-related markers. As shown in Figure 3G, after Pal treatment, the expression level of the epithelial marker E-cadherin was upregulated, while the expression of mesenchymal markers N-cadherin, Vimentin, and Snail was significantly downregulated. This series of molecular changes provides a mechanistic explanation for Pal’s inhibitory effects on cell migration and invasion: by reversing or inhibiting the EMT process, it maintains cells in a more epithelial-like phenotype, thereby reducing their motility and invasiveness.

2.4. Palmaturbine Induces G2/M Cell Cycle Arrest and Activates the Intrinsic Apoptotic Pathway in PDAC Cells

Next, we examined whether Pal affects the cell cycle of PDAC. Flow cytometric cell cycle analysis revealed that Pal treatment led to a significant increase in the proportion of CFPAC-1 and SW1990 cells in the G2/M phase (Figure 4A,B), suggesting that the drug arrests cells in the G2/M phase. Western blot analysis of cell cycle regulatory proteins corroborated this result: the expression levels of key proteins promoting the G1/S transition, Cyclin D1 and Cyclin E1, as well as the S phase marker protein Cyclin A2, all decreased. Conversely, the expression of Cyclin B1, a key protein driving the G2/M phase progression, was upregulated (Figure 4E). This disarray in the expression of cyclins is the direct cause of cell cycle arrest.

Subsequently, we analyzed cell apoptosis. Annexin V-FITC/PI double-staining flow cytometry demonstrated that Pal dose-dependently induced apoptosis in PDAC cells (Figure 4C,D). At the molecular level, Western blot results showed that Pal treatment downregulated the anti-apoptotic protein Bcl-2 (Figure 4F). The levels of Caspase-3, a key executor of the apoptotic pathway, were significantly reduced, and its substrate PARP-1 was cleaved. These results clearly indicate that Pal induces cell apoptosis by regulating apoptotic proteins.

2.5. Transcriptomic Analysis Reveals JAK/STAT Signaling Pathway as the Core Target of Palmaturbine

To comprehensively investigate the mechanism of action of Pal, we conducted RNA-seq analysis on cells treated with Pal and untreated cells. PDAC cells were treated with 40 μM Palmaturbine for 24 h prior to RNA-seq analysis. The volcano plot of differentially expressed genes (DEGs) revealed significant alterations in the expression of a substantial number of genes (Figure 5A). Both KEGG pathway enrichment analysis and gene set enrichment analysis (GSEA) consistently indicated that the JAK/STAT signaling pathway was one of the most prominently downregulated pathways affected by Pal (Figure 5B,C).

This bioinformatic prediction was substantiated at the molecular level. Quantitative RCR analysis confirmed changes in gene expression related to the JAK/STAT signaling pathway after Palmaturbine treatment (Figure 5D). Western blot analysis demonstrated that Pal dose-dependently suppressed the phosphorylation levels of JAK2 and STAT3 (p-JAK2, p-STAT3), without affecting their total protein amounts (Figure 5E). Functional rescue experiments revealed that when cells were co-treated with the JAK/STAT pathway agonist Colivelin (50 μg/mL) and Pal, the inhibitory effect of Pal on cell viability and its pro-apoptotic effect were partially reversed by Colivelin (Figure 5F–H). These data provide compelling evidence that inhibiting the activation of the JAK2/STAT3 signaling pathway is the core molecular mechanism underlying the anti-PDAC action of Pal.

To further validate the selectivity of Palmaturbine and explore its impact on upstream signaling, we examined the expression of the upstream receptor IL-6R and other STAT family members (STAT1 and STAT5) by Western blotting. As shown in Supplementary Figure S1A, Palmaturbine treatment resulted in a dose-dependent decrease in IL-6R expression, suggesting that the drug may interfere with the pathway at the receptor level. In contrast, the total protein levels of STAT1 and STAT5 remained largely unchanged following treatment. These findings indicate that Palmaturbine selectively targets the IL-6R/JAK2/STAT3 axis in PDAC cells without broadly affecting other STAT family members.

2.6. Molecular Docking Predicts Direct Interaction Between Palmaturbine and JAK2/STAT3

To further elucidate whether Palmaturbine directly targets the JAK2/STAT3 signaling pathway, we performed molecular docking analysis. The simulation results revealed that Palmaturbine could fit favorably into the ATP-binding pocket of JAK2 with a binding energy of −7.4 kcal/mol (Supplementary Figure S1B). Hydrogen bonds were predicted to form between Palmaturbine and amino acid residues of JAK2. Similarly, Palmaturbine exhibited a strong binding affinity (−5.9 kcal/mol) for the SH2 domain of STAT3, potentially blocking its dimerization, stabilized by hydrogen bonding with residues (Supplementary Figure S1B). These computational data suggest that Palmaturbine may exert its inhibitory effect by directly binding to key functional domains of JAK2 and STAT3.

2.7. Palmaturbine Effectively Inhibits Tumor Growth In Vivo and Demonstrates Favorable Safety Profile

We evaluated the in vivo efficacy of Pal in a nude mouse model with subcutaneously transplanted CFPAC-1 tumors (Figure 6A). After 21 days of treatment, both the low-dose (5 mg/kg) and high-dose (10 mg/kg) Pal groups exhibited significant inhibition of tumor growth compared to the solvent control group. This was manifested by a notably flattened tumor volume growth curve (Figure 6B,C) and a significant reduction in the final tumor weight (Figure 6D,E). Throughout the experimental period, there was no significant decrease in the body weight of mice in the Pal treatment groups compared to the control group (Figure 6F), indicating low systemic toxicity of Pal. Although a slight transient weight loss was observed, it remained within the ethical limits (<20%), indicating a manageable safety profile.

Immunohistochemical analysis revealed a significant decrease in the expression of the proliferation marker Ki-67 and an increase in the expression of the apoptotic protein Bax in the tumor tissues (Figure 6G). Western blot analysis showed a marked reduction in the expression of phosphorylated STAT3 and JAK2 (Figure 6H,I). These in vivo results are highly consistent with the in vitro findings, indicating that Pal effectively inhibits the progression of PDAC in vivo by inhibiting the JAK/STAT pathway, suppressing proliferation, and inducing apoptosis, while demonstrating acceptable safety.

3. Discussion

Pancreatic ductal adenocarcinoma, known as the “king of cancers” due to its extreme treatment resistance and high mortality rate, poses a significant challenge in oncology [23]. Current standard therapies have limited efficacy, underscoring the urgent need for the development of novel therapeutic strategies. Unlike previous reports focusing on lung or breast cancer, this study is the first to systematically reveal the promising anti-tumor activity of Pal specifically in the context of PDAC—a malignancy characterized by a distinct, dense stromal microenvironment and aggressive biology. We elucidated in depth its molecular mechanism of action, which involves inhibiting the JAK2-STAT3 signaling pathway. This finding provides a promising candidate molecule for the pharmacological treatment of PDAC.

Our research has revealed that the anti-tumor effects of Pal are multifaceted. Firstly, it weakens both short-term and long-term cell proliferation capabilities by inhibiting DNA synthesis and the expression of key proliferation-related proteins. Secondly, it significantly suppresses cell migration and invasion, an effect closely related to its ability to reverse the epithelial–mesenchymal transition (EMT) phenotype. EMT is a critical early step in tumor metastasis. By upregulating E-cadherin and downregulating N-cadherin, Vimentin, and Snail, Pal “locks” cells in a more benign, less migratory epithelial state, offering a new approach to intervene in PDAC invasion and metastasis. Thirdly, Pal interferes with the cell cycle regulatory network, arresting cells in the G2/M phase [24]. The G2/M checkpoint is crucial as it ensures DNA damage repair is completed before mitosis proceeds. The accumulation of Cyclin B1 and the disruption of other cyclins caused by Pal suggest that it may activate this checkpoint by causing DNA damage or interfering with mitotic spindle function, leading to cell cycle arrest. Fourthly, and most importantly, Pal effectively activates the intrinsic apoptotic pathway in cells. It downregulates Bcl-2, upregulates Bax, and subsequently activates the Caspase cascade, ultimately resulting in programmed cell death. Importantly, our results distinguish the effect of Palmaturbine as cytotoxic rather than merely cytostatic. While cell cycle arrest at the G2/M phase contributes to growth inhibition, the significant increase in Annexin V-positive cells (Figure 4D) and the cleavage of executioner Caspase-3 and PARP (Figure 4F) provide definitive evidence that Palmaturbine actively induces programmed cell death. This cytotoxic characteristic is crucial for eliminating PDAC cells and preventing tumor recurrence.

The core contribution of this study lies in the use of transcriptomics combined with molecular validation to identify the JAK-STAT signaling pathway as the key target of Palmaturbine. Although our RNA-seq analysis indicated the modulation of multiple signaling pathways, we prioritized the JAK2/STAT3 axis as the central mechanistic driver for several reasons. Firstly, it emerged as the top-ranked downregulated pathway in both KEGG and GSEA analyses. Secondly, functional rescue experiments using the agonist Colivelin demonstrated that restoring JAK/STAT signaling significantly reversed Pal-induced apoptosis and growth inhibition, confirming its causative role. We interpret alterations in other signaling cascades as likely being secondary downstream effects or compensatory responses that are non-essential to the primary anti-tumor mechanism of Palmaturbine in this context. The continuous activation of the JAK-STAT pathway, particularly STAT3, plays a central role in the initiation, progression, immune evasion, and treatment resistance of PDAC. Activated STAT3, acting as a transcription factor, can directly upregulate the expression of a series of pro-cancer genes, such as Cyclin D1, Bcl-2, Bcl-xL, and Mcl-1 (which inhibit apoptosis). Our data show that Palmaturbine specifically inhibits the phosphorylation of JAK2 and STAT3 without affecting their total protein levels, indicating that it intervenes in the activation step of this pathway rather than protein synthesis. The resulting downregulation of downstream target genes, Cyclin D1 and Bcl-2, perfectly explains the observed G1/S phase transition block and the breakdown of apoptotic resistance. Functional rescue experiments further establish a causal relationship between Palmaturbine and the JAK-STAT pathway. Unlike previous reports suggesting that Palmaturbine acts through the PI3K/AKT pathway, this study reveals a new, more universally applicable axis of action in PDAC [18]. While previous studies in lung cancer suggested Pal might act via cell cycle regulation independent of STAT3, our findings in PDAC indicate that the JAK2/STAT3 axis is the dominant driver, likely due to the high prevalence of IL-6/STAT3 hyperactivation in the pancreatic tumor microenvironment. Given that KRAS mutation is the signature driver event in >90% of PDAC cases, our study utilized CFPAC-1 and SW1990 cell lines, both of which harbor KRAS mutations, to mimic the clinical scenario. Our findings suggest that Palmaturbine effectively suppresses the proliferation of these KRAS-mutant cells, likely by intercepting the downstream JAK2/STAT3 signaling, which is often amplified by oncogenic RAS. The broad oncogenic nature of the JAK-STAT pathway implies that drugs targeting this pathway may have broad-spectrum anti-cancer potential, and Palmaturbine, as a naturally derived inhibitor, has multi-target properties that may help overcome the drug resistance that often arises with single-target drugs.

Our study further elucidates the specificity of Palmaturbine’s action. The observation that Palmaturbine downregulates IL-6R expression while sparing STAT1 and STAT5 (Supplementary Figure S1A) provides compelling evidence of its selectivity. This suggests that Palmaturbine not only inhibits JAK2/STAT3 phosphorylation but may also disrupt the autocrine or paracrine IL-6 signaling loop by targeting the receptor itself, thereby distinguishing it from broad-spectrum kinase inhibitors that often cause off-target toxicity. Furthermore, our molecular docking analysis (Supplementary Figure S1B) supports a direct interaction, showing that Palmaturbine fits favorably into the ATP-binding pocket of JAK2, suggesting a dual mechanism of action involving both receptor downregulation and kinase inhibition.

Of course, this study also has some limitations. Firstly, the direct binding sites of Palmaturbine to JAK2 or STAT3 proteins need to be further confirmed using techniques such as surface plasmon resonance (SPR) or cellular thermal shift assay (CETSA). Secondly, it is worth noting that the relationship between the effective in vitro concentration (20–40 μM) and the in vivo dosage (5–10 mg/kg) involves complex pharmacokinetics that were not explicitly determined in this study. While 10 mg/kg effectively inhibited tumor growth, the actual drug concentration reaching the tumor microenvironment depends on absorption, distribution, and metabolism rates. The dosage was selected based on our preliminary tolerance tests and previous studies on similar alkaloids. Future pharmacokinetic studies are required to bridge the gap between in vitro potency and in vivo efficacy. Additionally, while we observed dose-dependent effects within the tested range, the precise therapeutic window and the upper limit of dose-dependency before reaching toxicity saturation require further investigation. Regarding safety, although we observed a manageable profile with no lethal toxicity or significant weight loss in mice, we acknowledge that body weight alone provides an incomplete measure of systemic safety. To fully rule out potential off-target effects, comprehensive toxicological evaluations, including hematological, biochemical, and histopathological analyses of major organs, are required in future studies. Thirdly, this study primarily focused on tumor cells themselves, while the JAK-STAT pathway plays a crucial role in the tumor microenvironment (TME), particularly in regulating the function of immune cells such as myeloid-derived suppressor cells and tumor-associated macrophages. Whether Palmaturbine can reshape the immunosuppressive microenvironment of PDAC and thus synergize with immune checkpoint inhibitors is an attractive future research direction.

4. Materials and Methods

4.1. Cell Lines and Reagents

Human PDAC cell lines ASPC-1, BxPC-3, CFPAC-1, and SW1990 were obtained from the China Center for Type Culture Collection (CCTCC). All cells were cultured in RPMI-1640 medium (Gibco, New York, NY, USA) supplemented with 10% Fetal Bovine Serum (FBS, Gibco) and 1% penicillin-streptomycin double antibiotic solution (Sigma-Aldrich, Carlsbad, CA, USA) in a humidified incubator at 37 °C with 5% CO_2_. Palmaturbine (purity ≥ 98%, HPLC analysis) was purchased from Chengdu Herbpurify Biotechnology Co., Ltd. (Chengdu, China). It was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) to prepare a 10 mM stock solution, which was aliquoted and stored at −20 °C in the dark.

4.2. Cell Viability Assay

Cell viability was assessed using the CCK-8 kit (Beyotime, Shanghai, China). Cells in the logarithmic growth phase were seeded into 96-well plates at a density of 3 × 10^3^ cells per well, with three replicate wells per group. After 24 h of incubation, the medium was replaced with fresh medium containing different concentrations of Palmaturbine (0, 2.5, 5, 10, 15, 20, 30, 40 μM), and the cells were treated for 24, 48, and 72 h. At the designated time points, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for an additional hour. The absorbance at 450 nm was measured using a full-wavelength multifunctional microplate reader (Bio-Rad, Hercules, CA, USA). Cell viability was calculated by subtracting the blank well value (containing no cells) and normalizing to the control group. The working concentrations of Palmaturbine (5, 10, 20, 40 μM) were selected based on the IC50 values determined from preliminary CCK-8 cytotoxicity assays.

4.3. EdU Incorporation Assay

Cell proliferation was evaluated using the BeyoClick™ EdU Cell Proliferation Assay Kit (Beyotime). Cells (1.7 × 10^4^ cells per well) were seeded into 24-well plates and cultured until they reached approximately 60% confluence. They were then treated with different concentrations of Palmaturbine for 24 h. Subsequently, the cells were incubated with EdU working solution for 4 h, fixed, and labeled with click reaction reagent (containing Azide-488) according to the manufacturer’s instructions. The cell nuclei were counterstained with Hoechst 33342. At least three non-overlapping fields were randomly selected and imaged under a microscope. The percentages of EdU-positive cells (green fluorescence) and total cells (blue fluorescence) were counted using ImageJ software (version 1.8.0), and the EdU-positive cell ratio was calculated.

4.4. Colony Formation Assay

CFPAC-1 and SW1990 cells in the logarithmic growth phase were collected and seeded into 6-well plates at a low density (1 × 10^3^ cells per well), with three replicate wells per group. After 24 h of incubation to allow cell attachment, the medium was replaced with fresh medium containing different concentrations of Palmaturbine, and the cells were treated for 24 h. Subsequently, the drug-containing medium was carefully aspirated, and fresh complete medium was added. The cells were cultured with the medium changed every 3 days. After 7–10 days, the cells were fixed and stained with crystal violet. Colonies containing more than 50 cells were manually counted. The drug was dissolved in DMSO (<0.1%). Data are presented as the absolute number of colonies per well.

4.5. Wound Healing Assay and Transwell Migration/Invasion Assays

Wound healing assay: Cells were seeded into 6-well plates at a high density and cultured until they reached 90% confluence. A straight scratch was made in the monolayer using a sterile 200 μL pipette tip. The cells were gently washed three times with PBS to remove detached cell debris and then cultured in serum-free medium containing different concentrations of Palmaturbine. Images were taken at the same position under an inverted microscope at 0 and 24 h post-scratch. The scratch area was measured using ImageJ software, and the relative healing rate was calculated.

Transwell Assay: Both migration and invasion assays were performed using 8.0 μm pore size Transwell chambers. For the invasion assay, the upper chamber was pre-coated with diluted Matrigel basement membrane matrix. Cells (1.5 × 10^5^ cells per well) in the logarithmic growth phase were collected, resuspended in serum-free medium, and adjusted to a density of 2 × 10^5^ cells/mL. A 200 μL cell suspension was added to the upper chamber, and 600 μL of complete medium containing 20% FBS was added to the lower chamber as a chemoattractant. The cells on the lower surface were fixed with 4% paraformaldehyde, stained with 1% crystal violet for 10 min, and imaged under an inverted microscope. The number of cells that migrated/invaded through the membrane was counted in at least three randomly selected fields.

4.6. Flow Cytometric Analysis of Cell Cycle and Apoptosis

Cell Cycle: Cells (1.0 × 10^5^ cells per well) were treated with Pal for 24 h, collected, washed twice with pre-cooled PBS, and fixed overnight at 4 °C with 70% ethanol. After centrifugation, the ethanol was removed, and the cells were washed with PBS. They were then resuspended in staining buffer containing RNase A and propidium iodide (PI) and incubated at 37 °C for 30 min in the dark. The cell cycle distribution was analyzed using a flow cytometer(Beckman, Brea, CA, USA), and the data were processed with FlowJo software (version 10.8.1, FlowJo LLC, Ashland, OR, USA)to determine the proportion of cells in each phase.

Apoptosis: Cell (1.0 × 10^5^ cells per well) apoptosis was detected using the Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime). Treated cells were collected, washed with pre-cooled PBS, and resuspended in 1× binding buffer. Annexin V-FITC and PI staining solutions were added sequentially, and the cells were gently mixed and incubated at room temperature for 15 min in the dark. Immediately after incubation, 1× binding buffer was added, and the samples were analyzed by flow cytometry within 1 h. The cells were divided into four quadrants: Annexin V−/PI− (live cells), Annexin V+/PI− (early apoptosis), Annexin V+/PI+ (late apoptosis), and Annexin V−/PI+ (necrotic cells). The total apoptosis rate was the sum of early and late apoptosis rates.

4.7. RNA Sequencing and Bioinformatics Analysis

CFPAC-1 cells (1.0 × 10^6^ cells per well) treated with Palmaturbine for 24 h and DMSO-treated controls were used for RNA sequencing. Total RNA was extracted using TRIzol reagent, and RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Santa Clara, CA, USA) and Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA). Qualified samples were sent to Shanghai Majorbio Co., Ltd. (Shanghai, China) for library construction using a strand-specific library protocol and sequenced on the Illumina NovaSeq 6000 platform (San Diego, CA, USA). Differential expression analysis was performed using the DESeq2 R package (version 1.42.0), with a screening criterion of |log_2_ fold change (Fold Change)| > 1. KEGG pathway enrichment analysis and gene set enrichment analysis (GSEA) were conducted to identify significantly regulated signaling pathways by Pal.

4.8. Molecular Docking

To predict the binding mode and affinity between Palmaturbine and its potential targets, molecular docking simulations were performed using AutoDock Vina software (version 1.1.2). The crystal structures of JAK2 and STAT3 were retrieved from the RCSB Protein Data Bank. The 3D structure of Palmaturbine was constructed and energy-minimized using Chem3D (version 22.0) software. Before docking, water molecules were removed, and polar hydrogen atoms and Kollman charges were added to the protein structures. A grid box was defined to cover the ATP-binding pocket of JAK2 and the SH2 domain of STAT3. The docking results were analyzed and visualized using PyMOL (version 2.3.4) to identify key interacting residues and hydrogen bonds.

4.9. Real-Time Quantitative PCR

Total RNA was extracted from cells (1.0 × 10^5^ cells per well) using the TRIzol method, and its concentration was determined using a NanoDrop. One microgram of total RNA was reverse transcribed into cDNA using the PrimeScript RT Master Mix reagent kit (Takara, Tokyo, Japan) according to the manufacturer’s instructions. Quantitative PCR (qPCR) reactions were performed using SYBR Premix Ex Taq II on a QuantStudio 5 (ABI, Carlsbad, CA, USA) real-time fluorescence quantitative PCR system. Relative gene expression levels were calculated using the 2^−ΔΔCt^ method. All primer sequences were synthesized by Sangon Biotech (Shanghai, China).

4.10. Western Blotting

After collecting the treated cells (1.0 × 10^5^ cells per well), lyse them on ice for 30 min using pre-chilled RIPA lysis buffer. Centrifuge at 12,000 rpm for 15 min at 4 °C and collect the supernatant containing total protein. Determine the protein concentration using the BCA method. Take equal amounts of protein (30 μg) for SDS-PAGE electrophoresis separation, followed by transfer to a PVDF membrane using the wet transfer method. After blocking with 5% skim milk at room temperature for 1 h, incubate the membrane with diluted primary antibodies overnight at 4 °C. The next day, wash the membrane three times with TBST for 10 min each time, then incubate with the corresponding horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h. Finally, expose and develop the membrane using an enhanced chemiluminescence (ECL) substrate on a chemiluminescence imaging system. Analyze the band gray values using ImageJ software.

4.11. Animal Experiments

To ensure the generalizability of the results and minimize potential sex-based bias, three male and three female BALB/c nude mice (5 weeks old) were selected for each experimental group (Total n = 6 per group). The mice were acclimated in an SPF-grade environment. The sample size of six was determined based on a power analysis to ensure sufficient statistical power (>0.8 at α = 0.05) to detect significant differences in tumor volume, while strictly adhering to the 3R principle (Replacement, Reduction, and Refinement) for ethical animal use. All animal experiments strictly adhered to the guidelines of the Experimental Animal Ethics Committee of Zhejiang Chinese Medical University and received approval (Approval No.: SYXK (Zhejiang) 2018-0012). CFPAC-1 cells in the logarithmic growth phase were collected and resuspended in PBS to a concentration of 1 × 10^7^ cells/mL. Each mouse was subcutaneously injected with 100 μL of the cell suspension (containing 1 × 10^6^ cells) into the right flank. Daily observations were conducted when the tumor volume reached approximately 100 mm^3^, the tumor-bearing mice were randomly divided into three groups (six mice per group): ① Solvent control group; ② Low-dose Palmaturbine group (5 mg/kg); ③ High-dose Palmaturbine group (10 mg/kg). The drugs were dissolved in a vehicle mixture containing 10% DMSO and 90% Saline, and administered via intraperitoneal injection every three days for 21 days. During this period, the long diameter (L) and short diameter (W) of the tumors were measured every three days using vernier calipers, and the tumor volume was calculated using the formula V = 0.5 × L × W^2^, while the body weight of the mice was also recorded. At the end of the experiment, the mice were euthanized by cervical dislocation, and the tumor tissues were completely dissected and weighed.

4.12. Immunohistochemistry and H&E Staining

Tumor tissues obtained from the animal experiments were fixed in 4% paraformaldehyde, routinely dehydrated, cleared, embedded in paraffin, and sectioned into 4 μm-thick slices. For immunohistochemistry: After dewaxing and hydration, the sections were subjected to heat-induced antigen retrieval using sodium citrate buffer. Subsequently, endogenous peroxidase activity was blocked with 3% H_2_O_2_, and non-specific sites were blocked with 5% BSA. Primary antibodies were added and incubated overnight at 4 °C, followed by the addition of HRP-labeled secondary antibodies and incubation at room temperature for 30 min the next day. Finally, the sections were developed using a DAB color development kit, counterstained with hematoxylin for nuclear staining, dehydrated, mounted, and observed and photographed under a microscope.

4.13. Statistical Analysis

All quantitative data are presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical analyses were performed using GraphPad Prism software (Version 8.0, GraphPad Software, Inc., San Diego, CA, USA). The normality of the data distribution was verified using the Shapiro–Wilk test. For comparisons between two groups, Student’s t-test (two-tailed) was used for normally distributed data, while the Mann–Whitney U test was used for non-normally distributed data. For comparisons among multiple groups, One-way Analysis of Variance (ANOVA) followed by Tukey’s post hoc test was employed. * Indicates p < 0.05, and ** indicates p < 0.01.

5. Conclusions

In conclusion, this study systematically elucidates the role and mechanism of the natural compound Pal in inhibiting the proliferation, migration, invasion, and inducing apoptosis of pancreatic ductal adenocarcinoma in vitro and in vivo by targeting and inhibiting the JAK2/STAT3 signaling pathway. These findings not only provide solid preclinical evidence for the development of Pal as an anti-PDAC candidate drug but also open up new avenues for research on JAK-STAT pathway inhibitors based on natural products. Future work should focus on precise target identification, pharmacokinetic optimization, and exploration of combination therapies with existing treatments (such as chemotherapy and immunotherapy), with the aim of advancing Pal towards clinical translation and benefiting PDAC patients as soon as possible.