Ugonin J was synthesized by Dr. Chih-Chuang Liaw (National Sun Yat-sen University, Taiwan), utilizing the process described in an earlier report (Fig. 1A) [24,25]. The purity of Ugonin J was >95%. The ADAM9 (SC-135822), Akt (SC-5298), β-actin (SC-47778), β-Catenin (SC-133240), p-p85a (SC-12929), p85 (SC-1637), p65 (SC-8008), Snail (SC-271977), and Slug (SC-166476) antibodies, PI3K activator (PI3K activator; SC-3036), AKT activator (Fumonisin B1; SC-201395), p65 activator (Prostratin; SC-203422) as well as ADAM9 siRNA (SC-41408) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). E-cadherin (ab40772) and N-cadherin (ab76057) antibodies were bought from Abcam (Cambridge, MA, USA). p-Akt (4060S) and p-p65 (3033) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). ADAM9 cDNA plasmid was obtained from Origene Technologies (RC222453; Rockville, MD, USA).

The human prostate cancer cell lines PC3 (CRL-1435) and DU145 (HTB-81) were acquired by the American Type Culture Collection (Manassas, VA, USA). Cells were grown at 37°C in a humidified atmosphere containing 5% CO₂, using RPMI-1640 media (Gibco, Rockville, MD, USA) supplemented with 10% FBS. All cell lines have been authenticated using short tandem repeats (STR). Contamination by Mycoplasma was regularly examined using PCR analysis. Ugonin J was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution and diluted in culture medium to the indicated concentrations for treatment.

An MTT assay was used to examine cell viability, in accordance with our earlier publications [26]. 100 μL of culture media was used to seed PC3 and DU145 cells (5 × 10³) onto 96-well plates. Following a 24-h incubation period to promote cell adhesion, cells were incubated to varying doses of Ugonin J (0–10 μM) for a 24-h period before undergoing a cell-viability test. MultiskanTM FC microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) will be used to measure the absorbance at 570 nm with the MTT reagent (M2128; Sigma-Aldrich, St. Louis, MO, USA).

As per the manufacturer’s instructions, total RNA was extracted from PC3 cells treated with or without Ugonin J (3 μM) using TRIzol reagent (2001; Cyrusbioscience, Taipei, Taiwan). The isolated RNA samples were utilized for the subsequent library preparation. Then, libraries with different indices were multiplexed and sequenced on an Illumina HiSeq/Illumina Novaseq/MGI2000 instrument (Illumina, San Diego, CA, USA) using a 2 × 150 paired-end configuration as per the manufacturer’s guidelines. Heatmaps and volcano plots were generated to analyze differentially expressed genes using the ‘DESeq2’ (v1.26.0) in R (v3.6.3) within the Bioconductor (v3.10) package. We utilized a |log2fold change (FC)| > 1 with adjusted p value < 0.05 as the threshold for significantly differential expression [27].

Differentially expressed genes (DEGs) identified from RNA-seq analysis were submitted to the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.genome.jp/kegg/pathway.html, accessed on 01 October 2025) database to explore potential pathways and analyze biological functions.

ADAM9 levels in normal prostate tissue, primary PCa, and lymph node metastatic tissues were analyzed using the University of Alabama at Birmingham CANcer data analysis Portal (UALCAN: https://ualcan.path.uab.edu/?utm_source=chatgpt.com, accessed on 01 October 2025) dataset. Additionally, ADAM9 levels in normal, primary, and metastatic PCa tissues, stratified by cancer stage, were evaluated using the TNMplot (https://tnmplot.com/?utm_source=chatgpt.com, accessed on 01 October 2025) dataset. Gene Expression Profiling Interactive Analysis (GEPIA: https://gepia.cancer-pku.cn/?utm_source=chatgpt.com, accessed on 01 October 2025) was performed to assess ADAM9, Snail, and Slug expression levels in healthy prostate and tumor tissue samples from The Cancer Genome Atlas (TCGA).

PC3 and DU145 cells (~1 × 10⁴) were plated in 6-well dishes and incubated with Ugonin J (0, 0.3, 1, or 3 μM) for 24 h at 37°C. Using the TRIzol reagent as per the manufacturer’s guidelines, a total of 1 μg of RNA was extracted from PC and DU145 cells. An oligo-DT primer was used to convert the RNA into cDNA. The SYBR Green (A46012; Thermo Fisher Scientific, Waltham, MA, USA) was utilized to combine a 100 ng cDNA sample with specific primers, using GAPDH as the internal control. The sequences of the PCR primer (5^′-3^′) were as follows: ADAM9 forward, CTTGC TGCGA AGGAA GTACCTG and reverse, CACTC ACTGG TTTTT CCTCGGC. Thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. A StepOnePlus sequence detection system (v2.4; 4444202; Thermo Fisher Scientific, Waltham, MA, USA) was used to perform the qPCR tests in triplicate. The expression of ADAM9 mRNA was assessed via the ∆Cq comparative methods. To calculate relative expression, the comparative CT approach was employed [27,28].

RIPA lysis buffer (P0013; Beyotime Institute of Biotechnology, Shanghai, China) was used to extract total protein from PC3 and DU145 cells. In each lane, a total of 30 μg of protein was loaded, as calculated using the BCA kit (23225; Thermo Fisher Scientific, Waltham, MA, USA). Proteins were separated with resolving gels of 8% or 10%. After being separated through SDS-PAGE, proteins were transferred to Immobilon® PVDF membranes. A 5% non-fat milk solution was used to block the membrane for 1 h at room temperature. The membranes were incubated with the primary antibodies overnight at 4°C, followed by a horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG, SC-2357; 1:3000; goat anti-mouse IgG, SC-516102; 1:3000; Santa Cruz Biotechnology; Santa Cruz, CA, USA) for 1 h at room temperature. All antibodies were used at a final dilution of 1:1000. The expression of the target protein was measured using an ECL kit (WBULS0100; Millipore Sigma, St. Louis, MO, USA) and visualized with an ImageQuantTM LAS 4000 biomolecular imager (GE Healthcare, Little Chalfont, UK) [29,30].

Migration experiments were conducted using Transwell inserts (Costar, Corning Incorporated, New York, NY, USA; 8-mm pore size) in 24-well plates. Prior to the migration and invasion assay, PC3 and DU145 cells were pretreated with Ugonin J (0, 0.3, 1 or 3 μM) for 30 min. In the upper compartment, there were approximately 1 × 10⁴ cells. For an invasion experiment, the same protocol was followed, but instead, 5 × 10⁴ cells were applied to a top chamber coated with Matrigel (BD Biosciences, Bedford, MA, USA). The cells were preserved using 3.7% formaldehyde for five minutes, incubated at 37°C with 5% CO₂ for 24 h, and stained with PBS containing 0.05% crystal violet. Using cotton-tipped swabs, cells were taken off the upper side of the filters, which were then washed with PBS (0.01 M, pH 7.4) [31–33].

After seeding the PC3 and DU145 cells (5 × 10⁵ cells/well) onto 6-well plates, the cells were pre-treated with PI3K (10 μM), Akt (10 μM) or NF-κB (10 μM) activators for 30 min or transfected with ADAM9 cDNA plasmid (1 μg) or ADAM9 siRNA (10 μM) for 24 h, using Lipofectamine 2000 transfection reagent (11668019; Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s instructions, then treated with Ugonin J (3 μM) for 24 h.

After transfection of the cells with an NF-κB firefly luciferase plasmid (Stratagene; St. Louis, MO, USA) using Lipofectamine 2000 (11668019; Thermo Fisher Scientific, Waltham, MA, USA), Ugonin J was stimulated for 24 h. Luciferase activity was measured using the dual luciferase assay system (E1910, Promega; Madison, WI, USA) following the manufacturer’s instructions. To account for transfection efficiency, Firefly luciferase activity was normalized against Renilla luciferase activity. The resulting data were presented as relative luciferase activity in comparison to untreated control cells [34,35].

Statistical analyses were performed using GraphPad Prism 8.2 (GraphPad Software, San Diego, CA, USA). Data are presented as the mean ± standard deviation (SD) from at least three independent biological replicates. Statistical comparisons among more than two groups were analyzed by one-way ANOVA with Bonferroni’s post hoc test. Differences between groups were considered significant if the p-value was < 0.05.

To investigate the roles of Ugonin J on migration and invasion, we first assessed its cytotoxicity in PCa cells. Ugonin J (0–10 μM) exhibited no significant cytotoxicity in PC3 and DU145 cells (Fig. 1B,C). We then evaluated Ugonin J’s inhibitory abilities on PCa cell migration and invasion after 24 h of treatment. Ugonin J significantly suppressed migration and invasion in PC3 and DU145 cells concentration-dependently (Fig. 2A,B). To investigate the molecular mechanisms behind Ugonin J’s anti-motility effects, we conducted RNA-seq analysis on PC3 cells treated with or without Ugonin J. Gene expression changes after Ugonin J treatment were revealed by the heatmap and volcano plot (Fig. 3A,B). GO biological process analysis indicated involvement in the regulation of epithelial to mesenchymal transition and cell motility, both linked to tumor metastasis (Fig. 3C). We therefore examined EMT markers after Ugonin J stimulation. The epithelial marker E-cadherin protein expression augmented after Ugonin J stimulation, but the mesenchymal indicators N-cadherin and β-catenin showed diminished expression (Fig. 3D). The transcriptional factors Snail and Slug are major activators of EMT [36]. Ugonin J treatment markedly diminished protein levels of Snail and Slug in PCa cells (Fig. 3E). These findings demonstrate that Ugonin J inhibits EMT, migration, and invasion in PCa cells.

ADAM family proteins are critical modulators of cancer cell adhesion and metastasis [11–13]. Our RNA-seq analysis revealed that ADAM9 is the most downregulated of 13 ADAM proteins following Ugonin J treatment (Fig. 4A). TCGA analysis demonstrated markedly higher ADAM9 levels in PCa tissues compared to adjacent normal tissues (Fig. 4B). ADAM9 expression levels were also linked with tumor stage, lymph node metastasis, and distant metastasis (Fig. 4C–E). Furthermore, Kaplan–Meier survival analysis using the UALCAN database revealed that PCa patients with high ADAM9 expression and high Gleason scores exhibited poorer survival rates (p < 0.0001; Fig. 4F). We therefore investigated the role of Ugonin J on ADAM9 synthesis. Treatment of PCa cells with Ugonin J concentration-dependently inhibited ADAM9 mRNA and protein expression (Fig. 5A,B). Transfection of PCa cells with ADAM9 cDNA reversed Ugonin J-mediated inhibition of cell migration and invasion (Fig. 5C,D). In addition, ADAM9 siRNA enhanced Ugonin J-mediated inhibition of cell motility (Fig. 5C,D). ADAM9 expression levels were positively correlated with Snail and Slug expression levels in PCa patients without adjustment for confounders (Fig. 5E). Furthermore, ADAM9 cDNA transfection antagonized Ugonin J-mediated suppression of Snail and Slug expression (Fig. 5F). Thus, Ugonin J inhibits PCa EMT, migration, and invasion by suppressing ADAM9 expression.

The PI3K-Akt signaling pathway, which encompasses the PI3K, Akt, and NF-κB mechanisms, was found to be mediated by Ugonin J through KEGG enrichment pathway analysis (Fig. 6A,B). Ugonin J treatment of PCa cells resulted in the inhibition of PI3K and Akt phosphorylation (Fig. 6C). The PI3K and Akt activators countered the reduction of ADAM9 production, migration, and invasion induced by Ugonin J (Fig. 6D–F). In addition, the use of an NF-κB activator during incubation counteracted the effects caused by Ugonin J (Fig. 7A–C). Ugonin J treatment of PCa cells inhibited p65 phosphorylation and NF-κB luciferase activity, as shown in Fig. 7D,E. Ugonin J also decreased the luciferase activity of NF-κB, which was reinstated through the activation of PI3K and Akt (Fig. 7F). Thus, Ugonin J mitigates ADAM9-mediated migration and invasion via the PI3K, Akt, and NF-κB pathways.