Dictamnine Inhibits WNT Pathway and EMT Progression in Prostate Cancer and Remodels the Tumor Microenvironment

TL;DR

Dictamnine, a natural compound, shows promise in treating prostate cancer by inhibiting cancer growth and changing the tumor environment.

Contribution

Dictamnine is shown to directly stabilize DKK1, inhibit Wnt/β-catenin signaling, and modulate the tumor microenvironment in prostate cancer.

Findings

Dictamnine suppresses prostate cancer malignancy by stabilizing DKK1 and inhibiting Wnt/β-catenin signaling.

It reverses epithelial–mesenchymal transition and modulates tumor microenvironment factors like VEGF-A and MMP-9.

In vivo, dictamnine inhibits tumor growth and angiogenesis, and increases macrophage infiltration in tumors.

Abstract

Prostate cancer (PCa) remains challenging to treat, particularly once it metastasizes. This study investigates the therapeutic potential of dictamnine, a natural compound, in prostate cancer. Our findings demonstrate that dictamnine exerts its anti-tumor effects by directly binding to and stabilizing the DKK1 protein. This interaction inhibits the oncogenic Wnt/β-catenin signaling pathway, thereby suppressing cancer cell proliferation and metastasis. Additionally, dictamnine inhibits tumor angiogenesis and modulates the tumor immune microenvironment. These findings unveil a novel multi-targeted mechanism of dictamnine, establishing a strong foundation for its development as a lead compound or therapeutic agent for prostate cancer treatment.

Objective: This study investigated the anti-prostate cancer mechanism of dictamnine (DIC), focusing on its potential to reverse EMT via DKK1-mediated Wnt/β-catenin inhibition and modulate the tumor microenvironment. Methods: Cell viability, proliferation, migration, and invasion were assessed using CCK-8, colony formation, EdU, wound healing, and Transwell assays. Key targets were identified via transcriptomics and bioinformatics, and validated through molecular docking, co-immunoprecipitation, and cellular thermal shift assay. Protein expression was analyzed by Western blot. Gain/loss-of-function and rescue experiments confirmed target roles. A subcutaneous xenograft model and immunohistochemistry were used for in vivo validation. Results: DIC suppresses prostate cancer malignancy in a concentration-dependent manner. The primary mechanism involves its direct binding to and stabilization of DKK1, which enhances DKK1’s interaction with LRP6. This upregulation of DKK1 inhibits the Wnt/β-catenin signaling pathway, downregulating downstream targets β-catenin/c-Myc/Cyclin D1, and reverses epithelial–mesenchymal transition (EMT) markers. Additionally, DIC modulates key tumor microenvironment factors, including VEGF-A, MMP-9, IL-11, and CXCL-12. Overexpression of DKK1 mimics the antitumor effects of DIC, while knockdown of DKK1 attenuates them. In vivo, DIC inhibits tumor growth, an effect partly mediated through the DKK1/β-catenin axis. Furthermore, DIC potently suppresses angiogenesis (reduced CD31+ staining) independently of DKK1. It also increases tumor-associated macrophage infiltration (elevated F4/80+ cells) in a DKK1-independent manner. Conclusions: DIC exerts its core antitumor effects by targeting DKK1 to inhibit Wnt/β-catenin signaling and EMT. Additionally, it independently suppresses angiogenesis and remodels the immune tumor microenvironment. This multi-level mechanism positions DIC as a promising lead compound for prostate cancer therapy.

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1. Introduction

Prostate cancer is one of the most common malignancies in men globally, with persistently high incidence and mortality rates [1,2,3,4]. Although androgen deprivation therapy (ADT), the cornerstone of treatment for advanced disease, shows initial efficacy, most patients eventually progress to castration-resistant prostate cancer (CRPC), facing limited therapeutic options and poor prognosis [5,6]. Tumor metastasis and recurrence are the primary causes of patient mortality [7,8,9], driven by two core biological mechanisms: the intrinsic epithelial–mesenchymal transition (EMT) of tumor cells and the supporting tumor microenvironment (TME) [10,11,12,13].

EMT is a critical step in which tumor cells acquire invasive and metastatic capabilities [14]. During this process, cells lose epithelial characteristics (e.g., cell adhesion) and gain mesenchymal traits (e.g., migratory ability). Among the complex regulatory pathways, the Wnt/β-catenin signaling pathway plays a pivotal role. Aberrant activation of this pathway can directly drive EMT in prostate cancer cells, thereby promoting invasion, metastasis, and therapy resistance [15].

Concurrently, tumors are embedded within a dynamic and complex tumor microenvironment (TME), which provides the “soil” for growth and metastasis. Immunologically, the prostate cancer TME is typically immunosuppressive: infiltration of immunosuppressive cells can weaken anti-tumor immunity and promote immune evasion. Regarding angiogenesis, tumor cells secrete pro-angiogenic factors like vascular endothelial growth factor (VEGF), inducing the formation of dysfunctional neovessels [16]. These vessels not only nourish the tumor but also serve as “channels” for dissemination. Aberrant angiogenesis is closely coupled with the EMT process, jointly accelerating metastasis. Therefore, strategies capable of simultaneously targeting tumor cell EMT and reshaping the immunosuppressive and abnormally vascularized TME hold promise as more effective therapeutic approaches [16].

However, current chemotherapeutic and targeted agents face challenges such as limited efficacy, significant toxicity, and the development of drug resistance in inhibiting prostate cancer metastasis [17,18]. In this context, discovering effective and low-toxicity anti-tumor compounds from traditional Chinese medicine offers a valuable resource and unique perspective for developing novel anti-metastatic drugs. Dictamnine mainly originates from the root bark of the plant Rutaceae species, and is a type of furanquinoline alkaloid with multiple biological activities [19,20]. Modern pharmacological studies have revealed its various antibacterial and anti-inflammatory activities [21,22,23]. Numerous studies have confirmed that dictamnine has extensive anti-cancer activity. The earliest research found that it could induce apoptosis of lung adenocarcinoma cells [24], and it has also been confirmed to inhibit tumors through various mechanisms in colon cancer, lung cancer, and pancreatic cancer [25,26,27]. Focusing on its intervention in the malignant process of tumors, it was found that dictamnine can clearly inhibit the EMT process and reduce the migration and invasion ability of human umbilical vein endothelial cells, suggesting its potential to regulate the tumor metastasis microenvironment [28]. However, whether dictamnine can inhibit the invasion and metastasis of prostate cancer, particularly by simultaneously regulating both EMT and angiogenesis, remains unclear, and the underlying molecular mechanisms require in-depth investigation.

Based on this background and preliminary transcriptomic data, this study aims to investigate whether and how dictamnine exerts anti-tumor effects in prostate cancer by inhibiting specific signaling pathways and influencing the tumor microenvironment. We hypothesize that dictamnine suppresses prostate cancer progression by inhibiting the Wnt/β-catenin signaling pathway and by affecting the tumor microenvironment. These two mechanisms may work synergistically to reduce tumor invasiveness and metastasis.

2. Materials and Methods

2.1. Cell Culture

PC3 cells (purchased from Procell, Wuhan, China) were cultured in Ham’s F-12K medium (BasalMedia, Shanghai, China) supplemented with 10% fetal bovine serum (FBS) (ABW, Shanghai, China). DU145 and 22Rv1 cells (purchased from iCell Bioscience, Shanghai, China) were cultured in MEM medium (BasalMedia, Shanghai, China) and RPMI-1640 medium (BasalMedia, Shanghai, China), respectively, both supplemented with 10% FBS. HUVECs (purchased from iCell Bioscience, Shanghai, China) were cultured in Endothelial Cell Medium (ScienCell, Carlsbad, CA, USA) supplemented with 5% FBS and Endothelial Cell Growth (ScienCell, Carlsbad, CA, USA) Supplement. All culture media were supplemented with 1% penicillin-streptomycin (BasalMedia, Shanghai, China) to prevent contamination. Cells were maintained in a humidified incubator at 37 °C with 5% CO_2_. For genetic manipulation, DKK1-knockdown and DKK1-overexpressing cell lines were established using lentiviral particles constructed by GenePharma (Shanghai, China). For knockdown, cells were infected with lentivirus expressing one of two independent shRNA sequences targeting human DKK1 (a non-targeting shRNA served as the control). For overexpression, the full-length human DKK1 cDNA was cloned into a plasmid vector (empty vector as control). After infection, stable polyclonal populations were selected with 2 μg/mL puromycin (BasalMedia, Shanghai, China) for 7–10 days. Successful modulation of DKK1 expression was confirmed by Western blotting. All experiments were performed using cells between passages 5 and 15 after thawing (or within 10 passages after puromycin selection for the engineered lines).

2.2. CCK-8 Assay

Cells in the logarithmic growth phase were seeded in 96-well plates at a density of 3000 cells/well in medium containing 10% FBS and cultured at 37 °C with 5% CO_2_. After treatment, the medium was replaced with fresh medium containing 10% CCK-8 reagent (Servicebio, Wuhan, China) and incubated for 1 h. Absorbance at 450 nm was measured using a microplate reader.

2.3. Colony Formation Assay

Cells in the logarithmic growth phase were seeded in 6-well plates at 1000 cells/well in medium containing 10% FBS and cultured for 14 days at 37 °C with 5% CO_2_. Colonies were fixed with 4% paraformaldehyde (Servicebio, Wuhan, China), stained with 0.1% crystal violet (Servicebio, Wuhan, China), and photographed.

2.4. EdU Assay

Cells in the logarithmic growth phase were seeded in 24-well plates at 50,000 cells/well in medium containing 10% FBS and cultured for 12 h. Cell proliferation was assessed using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime Shanghai, China) according to the manufacturer’s instructions. Following EdU addition, cells were incubated for 2–3 h, and then processed for imaging.

2.5. Wound Healing Assay

Cells in the logarithmic growth phase were seeded in 6-well plates and cultured until reaching >90% confluency. A straight scratch was made using a 200 µL pipette tip. After washing with PBS, cells were cultured in medium containing 2% FBS. Images were captured at 0 and 24 h to record cell migration.

2.6. Transwell Invasion Assay

After serum starvation for 12 h, 30,000 cells/well were seeded into the upper chamber of a Matrigel-coated (Corning, Corning, NY, USA) Transwell insert containing 200 µL serum-free medium. The lower chamber contained 500 µL medium with 10% FBS. After 24 h, cells on the upper surface were removed. Cells that invaded through the membrane to the lower surface were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and photographed.

2.7. Western Blot Analysis

Cells were lysed on ice for 10 min using RIPA (Solarbio, Beijing, China) lysis buffer containing 1% protease inhibitor cocktail (Solarbio, Beijing, China). Lysates were scraped, centrifuged, and protein concentration was determined using the BCA method. Proteins were denatured by heating with 5× SDS-PAGE loading buffer (Solarbio, Beijing, China) at 95 °C. 20 µg of protein per lane was separated by SDS-PAGE (Solarbio, Beijing, China) and transferred to PVDF membranes (Yeasen, Shanghai, China). After blocking with 5% non-fat milk, membranes were incubated with primary and secondary antibodies. Protein bands were visualized using chemiluminescence reagents (Yeasen, Shanghai, China) and analyzed with ImageJ software (Fiji v5.0.3).

2.8. Co-Immunoprecipitation (Co-IP)

Cells were washed with ice-cold PBS and lysed in non-denaturing lysis buffer (Solarbio, Beijing, China) containing protease inhibitors at 4 °C for 30 min. Lysates were centrifuged, and the supernatant was collected as total protein. A portion was saved as Input control. Total protein was incubated overnight at 4 °C with rotation with a specific primary antibody. Subsequently, Protein A/G magnetic beads (BEAVER, Suzhou, China), pre-equilibrated with lysis buffer, were added and incubation continued for 2 h. The bead complexes were washed five times with pre-cooled wash buffer. Bound proteins were eluted by boiling with 1× SDS loading buffer at 95 °C for 10 min. Eluted proteins and Input controls were analyzed by SDS-PAGE and Western blotting using antibodies specific for the interacting proteins. Normal IgG served as a negative control.

2.9. Cellular Thermal Shift Assay (CETSA)

Cells were resuspended in PBS and aliquoted into PCR tubes. The cell suspensions were subjected to gradient heating in a thermal cycler to induce protein denaturation. After heating, samples underwent freeze–thaw cycles in liquid nitrogen for complete lysis, followed by high-speed centrifugation at 4 °C for 20 min. The supernatant containing thermostable proteins was collected for Western blot analysis of the target protein.

2.10. Animal Experiments

Four- to five-week-old male BALB/c nude mice were acclimatized for one week under specific pathogen-free (SPF) conditions. PC3 cells in the logarithmic growth phase were trypsinized, resuspended in pre-cooled PBS to form a single-cell suspension, and 5 × 10^6^ cells were subcutaneously injected. Animal status was monitored daily. Euthanasia and termination of the experiment were implemented immediately upon reaching any of the following criteria: tumor volume exceeding 1000 mm^3^, tumor ulceration or infection, or body weight loss exceeding 20%.

2.11. Processing Tools

Original image processing and quantitative analysis were performed using ImageJ (Fiji v5.0.3). Statistical analysis and chart generation were carried out using GraphPad Prism 10.1.2. Molecular docking simulation and interaction analysis were conducted using AutoDockTools-1.5.6. The final layout was done using Adobe Photoshop 2024. The relevant image materials are from “Home for Researchers.”

3. Results

3.1. Dictamnine Inhibits the Malignant Biological Behavior of Prostate Cancer

CCK-8 assay results (Figure 1A) showed that DIC significantly inhibited PCa cell viability in a concentration- and time-dependent manner. The half-maximal inhibitory concentration (IC50) was calculated for subsequent experiments: 227.3 µM for DU145, 232.6 µM for PC-3, and 228.0 µM for 22Rv1. Colony formation (Figure 1B) and EdU assays (Figure 1C) further confirmed that treatment with DIC at the IC50 concentration significantly inhibited the long-term clonogenic capacity and short-term DNA replication activity of PCa cells. Moreover, wound healing (Figure 1D) and Transwell invasion assays (Figure 1E) demonstrated that DIC treatment effectively slowed PCa cell migration and significantly impaired their transmembrane invasive ability. These results collectively indicate that DIC effectively inhibits the malignant biological behaviors of PCa cells in vitro.

3.2. Transcriptomic Profiling Identifies DKK1 as a Candidate Target of Dictamnine

To elucidate the mechanism through which DIC reduces migration, we performed transcriptome sequencing analysis on PC3 cells before and after DIC treatment (Detailed sequencing methods and quality control information can be found in Supplementary File S3). RNA-seq analysis of PC3 cells treated with or without DIC revealed that DIC induced extensive gene expression reprogramming (Figure 2A). Intersecting the differentially expressed genes with prostate cancer-related genes from the GeneCards and TTD databases identified five potential key targets: PDGFRA, VDR, BRD2, DKK1, and TLR2 (Figure 2B). These genes were significantly differentially expressed (|log_2_FC| > 1, padj < 0.05) in DIC-treated cells.

KEGG pathway analysis of the DEGs revealed that the most significantly enriched pathway was “Cytokine-cytokine receptor interaction” (hsa04060, padj = 0.0203) (Figure 2D), which contained 27 genes, including multiple chemokines (e.g., CXCL12, CCL25, CCL28) and interleukins. Notably, chemokines are a specific subfamily of cytokines primarily responsible for chemotaxis. Consistently, GO molecular function analysis showed significant enrichment of terms related to “chemokine activity” (GO:0008009, padj = 0.022) and “chemokine receptor binding” (GO:0042379, padj = 0.022) (Figure 2C,F). These findings point to a potential mechanism whereby DIC may modulate the tumor microenvironment by affecting chemokine and cytokine networks, a possibility supported by previous studies demonstrating the importance of the CXCL12/CXCR4 axis in tumor microenvironment regulation [29].

In addition to the cytokine-related pathways, GO biological process analysis (Figure 2C,E) also showed enrichment of terms including “cell surface receptor signaling pathway” (GO:0007166, padj = 0.099) and “cell adhesion” (GO:0007155, padj = 0.144), with the latter containing several cadherin family genes. And the enrichment of the “Wnt signaling pathway” itself did not reach statistical significance in either GO (GO:0016055, padj = 0.099) or KEGG (hsa04310, padj = 0.315) analyses, we noted that among the five candidate targets identified from the intersection analysis, DKK1—a gene with significant upregulation (|log_2_FC| > 1, padj < 0.05)—is a well-established secreted antagonist of the canonical Wnt/β-catenin pathway [30,31]. Given the critical role of Wnt/β-catenin signaling in prostate cancer progression and EMT regulation [15,32,33], we hypothesized that DIC might exert its anti-migratory effects, at least in part, through DKK1-mediated modulation of this pathway. To test this hypothesis, we focused subsequent validation on DKK1 and its functional involvement in Wnt signaling and EMT.

3.3. Dictamnine Directly Targets DKK1 to Inhibit Wnt Signaling and EMT, and Modulates the Tumor Microenvironment

Molecular docking was employed to simulate the interaction between DIC and the DKK1 protein. The results showed that DIC could dock with high affinity into multiple pockets of DKK1 (Figure 3A), suggesting stable binding potential.

Cellular Thermal Shift Assay (CETSA) demonstrated that DIC treatment caused a significant rightward shift in the thermal stability curve of the DKK1 protein (Figure 3B), directly proving that DIC binds to and stabilizes DKK1 within cells. Co-immunoprecipitation (Co-IP) experiments further revealed that DIC treatment enhanced the binding between DKK1 and the Wnt co-receptor LRP6 (Figure 4A). This indicates that DIC stabilizes DKK1 and promotes its interaction with LRP6, leading to effective blockade of Wnt signaling at the protein level.

Western blot analysis confirmed (Figure 4B) that DIC treatment significantly upregulated DKK1 protein levels, while downregulating active β-catenin and its downstream targets c-Myc and Cyclin D1. Consistent with this, the EMT process was reversed: the epithelial marker E-cadherin increased, and the mesenchymal marker Vimentin decreased. These results demonstrate that DIC stabilizes DKK1, an event associated with both the inhibition of Wnt/β-catenin signaling and the blockade of EMT.

In addition to its effects on the Wnt/EMT axis, DIC also influenced factors related to the tumor microenvironment. Western blot analysis showed decreased expression of pro-angiogenic VEGF-A and matrix metalloproteinase MMP-9, both of which are associated with invasion and metastasis (Figure 4B). Interestingly, the chemokine CXCL12 was up-regulated, while the pro-inflammatory cytokine IL-11 was down-regulated (Figure 4B). These protein-level changes are consistent with the significant enrichment of the “Cytokine-cytokine receptor interaction”(hsa04060, padj = 0.0203) pathway observed in our transcriptomic analysis, confirming that DIC modulates the chemokine/cytokine network at both the transcriptional and translational levels. These findings indicate that DIC targets both the Wnt Signaling, EMT and the tumor immune microenvironment, together contributing to its multi-faceted anti-tumor activity.

3.4. Overexpression of DKK1 Mimics the Anti-Tumor Effects of Dictamnine

DKK1 overexpression significantly inhibited PCa cell proliferation (EdU assay, Figure 5A), migration (wound healing assay, Figure 5B), and invasion (Transwell assay, Figure 5C), mimicking the effects of DIC treatment. At the molecular level, DKK1 overexpression similarly led to downregulation of β-catenin, c-Myc, Cyclin D1, upregulation of E-cadherin, and downregulation of Vimentin (Figure 5D). This directly demonstrates that upregulating DKK1 alone is sufficient to inhibit the Wnt pathway, reverse EMT, and suppress the malignant phenotype of PCa cells.

3.5. The Upregulation of DKK1 by Dictamnine Exhibits Anti-Cancer Effects

A stable DKK1-knockdown PCa cell line was successfully established (Figure 6A). Functional rescue assays showed that DKK1 knockdown itself promoted PCa cell proliferation (Figure 6B) and invasion (Figure 6C). In this context, DIC treatment significantly rescued the pro-proliferative and pro-invasive phenotypes induced by DKK1 knockdown.

Similarly, the promotion of cell migration by DKK1 knockdown was also markedly rescued by DIC treatment (Figure 7A). At the molecular level, Western blot results (Figure 7B) further supported this regulatory relationship. In control cells, DKK1 knockdown effectively upregulated β-catenin and its downstream target Cyclin D1, while downregulating the epithelial marker E-cadherin and upregulating the mesenchymal marker Vimentin. In DKK1-knockdown cells, DIC treatment still partially restored the regulation of these key proteins. These rescue experiments provide strong evidence from both positive and negative aspects that DIC’s inhibition of the Wnt/β-catenin pathway and its anti-EMT effects primarily depend on its upregulation of DKK1.

3.6. In Vivo Verification of the Anti-Cancer Effect of Dictamnine

The results (Figure 8A,B) show that knockdown of DKK1 in tumor cells significantly promoted the growth and tumor weight of transplanted tumors, while DIC treatment could reverse this tumor-promoting effect.

The immunohistochemical results (Figure 8C) indicate that knockdown of DKK1 upregulated the expression of the Wnt pathway nuclear effector β-catenin and the proliferation marker Ki67, while DIC treatment significantly reduced the expression of both.

In terms of angiogenesis, knockdown of DKK1 increased the expression of the microvascular density marker CD31 by activating the Wnt pathway, and DIC treatment not only completely reversed this phenomenon, but its inhibitory effect was even lower than that of the control group, suggesting that DIC may also have other anti-angiogenic pathways.

In addition, knockdown of DKK1 had no significant effect on the infiltration of macrophage marker F4/80, but DIC treatment could increase the F4/80 positive signal and change the factors secreted by tumor cells, such as CXCL-12 and IL-11, indicating that DIC may promote the recruitment of macrophages by regulating the remodeling of the tumor-derived factor network and the immune microenvironment.

4. Discussion

This study, integrating multi-omics analysis, molecular docking, functional validation, and animal models, identifies DKK1 stabilization as the central event through which dictamnine exerts its anti-cancer effects, linking the inhibition of Wnt/β-catenin signaling to the concurrent blockade of EMT. DIC directly binds to and stabilizes the DKK1 protein, enhancing its interaction with the Wnt co-receptor LRP6. This leads to inhibition of the Wnt pathway core effector β-catenin and its downstream targets c-Myc and Cyclin D1, alongside the upregulation of E-cadherin and downregulation of Vimentin—outcomes of DKK1-mediated signaling. A rigorous chain of evidence demonstrates that targeting the DKK1-Wnt axis is the cornerstone of DIC’s direct anti-cancer action. Notably, the pivotal role of DKK1 as a tumor suppressor through inhibiting the Wnt/β-catenin pathway finds support in other cancer contexts, such as in hepatocellular carcinoma where upregulation of DKK1 contributes to reduced malignancy [34]. This convergence of evidence across cancer types underscores the fundamental importance of the DKK1-Wnt axis as a therapeutic target.

Secondly, an important finding of this study is that DIC’s remodeling effect on the tumor microenvironment exhibits a complex characteristic of being both DKK1-dependent and -independent, which may be key to its potent tumor suppression. Notably, the role of DKK1 in prostate cancer is increasingly recognized as context-dependent. While our findings demonstrate that DIC-mediated DKK1 stabilization inhibits Wnt/β-catenin signaling and EMT—consistent with its tumor-suppressive functions reported in certain contexts [35]—emerging evidence also implicates DKK1 in promoting immune evasion and modulating the bone metastatic niche [36,37]. For instance, DKK1 has been shown to contribute to an immunosuppressive tumor microenvironment by altering natural killer cell activity [37], and to influence the complex balance between osteoblastic and osteolytic lesions during bone metastasis [36,38]. This dualistic nature of DKK1 underscores the importance of the broader microenvironmental context in determining its net functional outcome. In this light, DIC’s ability to independently modulate chemokine networks—such as upregulating CXCL-12 and downregulating IL-11—may represent a critical complementary mechanism. Through its direct effects on tumor cell-derived chemokines and cytokines, DIC could potentially coordinate with DKK1 stabilization to rewire the TME in a manner that overcomes the pro-tumorigenic aspects of DKK1 signaling, while preserving its anti-oncogenic effects on cancer cells themselves. In addition to indirectly inhibiting VEGF-A via the DKK1/Wnt axis, DIC likely possesses one or more direct anti-angiogenic pathways independent of this axis. This “dual-insurance” mechanism allows DIC to powerfully suppress the tumor’s vascular support system even under the adverse conditions of DKK1 deficiency and Wnt pathway activation. Mechanistically, this could involve the inhibition of other pro-angiogenic pathways. For instance, dictamnine has been shown to downregulate hypoxia-induced HIF-1α protein synthesis by inhibiting the mTOR/p70S6K and MAPK pathways in other cancer models [26], which represents a potent VEGF-independent anti-angiogenic mechanism. This suggests that DIC’s pleiotropic action may simultaneously target multiple angiogenesis-supporting hubs. Furthermore, DKK1 knockdown had no effect on macrophage infiltration (F4/80+), but DIC treatment significantly increased the number of F4/80+ macrophages within tumors (Figure 8C). This phenomenon aligns with earlier molecular findings: DIC treatment significantly altered the cytokine secretion profile of tumor cells, particularly downregulating IL-11 and upregulating CXCL-12 (Figure 4). CXCL-12 is a known key chemokine for immune cell recruitment, and its altered expression may directly drive the recruitment of immune cells like macrophages to the tumor site. This indicates that DIC can actively reverse the immunosuppressive state by directly modulating the tumor cell’s own “secretome,” thereby changing the immune factor network within the TME—a process independent of its regulation of the DKK1/Wnt pathway. Intriguingly, dictamnine’s capacity to modulate macrophages is corroborated by research in colorectal cancer, where it was found to impede the M2 polarization of macrophages, as evidenced by inhibited expression of CD163, TGF-β, and Arg-1 [25]. This prompts a critical future question: are the macrophages recruited by DIC in our prostate cancer model polarized towards an anti-tumor (M1) or pro-tumor (M2) phenotype? The answer will determine whether this recruitment ultimately benefits or hinders therapy. Specifically, if DIC promotes M1 polarization, it could act as a strategic immune-priming agent to help convert the characteristically immunosuppressive, “cold” tumor microenvironment of prostate cancer [39] into a more immunogenic state, thereby potentially creating a foundation for enhanced efficacy of subsequent immunotherapies.

This dual-action characteristic of DIC—“targeting the core pathway” and “remodeling the microenvironment”—holds promise for overcoming the limitations of single-target therapies, such as the ease of developing resistance. Particularly, its ability to independently recruit macrophages and potentially polarize them towards an anti-tumor phenotype provides a potential natural drug candidate for combination immunotherapy aimed at converting “cold” tumors into “hot” tumors. This is especially relevant for prostate cancer, which is characteristically an immunologically “cold” tumor with a poor response to single-agent immune checkpoint inhibitors like PD-1/PD-L1 blockers [34]. It is noteworthy that dictamnine’s anticancer mechanisms appear to be context-dependent. While it acts primarily via the DKK1-Wnt axis in our prostate cancer model, studies in pancreatic and lung cancers highlight its inhibition of the PI3K/AKT [27] and c-Met/PI3K-AKT/mTOR pathways [26], respectively. This multi-target potential, while a strength, also necessitates a careful evaluation of its safety profile for future translation.

However, the promising multi-faceted efficacy of dictamnine must be balanced against its documented safety concerns. Several studies have reported that dictamnine can cause acute liver injury in mice, primarily through the metabolic activation of its furan ring to a reactive epoxy intermediate, leading to oxidative stress and protein adduction [40,41]. This potential hepatotoxicity is a significant consideration for its clinical development. To mitigate this risk and enhance tumor-specific delivery, future formulations could leverage advanced drug delivery strategies. For example, encapsulating dictamnine in nanoparticles (e.g., PLGA nanocarriers) has been shown to enhance its dermal penetration and efficacy in a dermatitis model while potentially modulating release [42]. More innovatively, engineered exosomes or plant-derived nanoparticles displaying targeting ligands could be employed to specifically deliver dictamnine to prostate cancer cells, minimizing systemic exposure and off-target toxicity in the liver, a strategy proven effective for other drugs like doxorubicin [43] and andrographolide [44].

Notably, the DKK1/Wnt/β-catenin/EMT signaling axis elucidated in this study intersects with known resistance biology in oncogenic KRAS contexts. Wnt/β-catenin signaling and EMT programs are frequently implicated in adaptive resistance pathways across cancers. Particularly relevant is the established role of RHO/RAC1–PAK signaling axis activation in mediating acquired resistance to KRASG12C inhibitors. Studies have shown that KRASG12C inhibitor treatment can induce the expression and activation of RHO family GTPases, driving YAP nuclear translocation via an ROCK-dependent mechanism to promote resistance [45]. Furthermore, in sotorasib-resistant cells, PAK kinases are activated and re-activate the MAPK pathway by phosphorylating MEK and c-Raf, while concurrently forming a positive feedback loop with the PI3K/AKT pathway to sustain the resistant phenotype [46]. These findings highlight targeting alternative or parallel pathways, such as Wnt/EMT or RHO/PAK, as a viable strategy to overcome targeted therapy resistance. The multi-target potential of dictamnine—encompassing inhibition of Wnt/β-catenin, PI3K/AKT/mTOR, and c-Met pathways—positions it as a candidate worthy of further investigation for potentially preventing or reversing treatment resistance driven by these adaptive signaling networks.

Based on the findings of this study, future work could focus on elucidating the direct targets through which DIC mediates anti-angiogenesis and immune remodeling, dissecting the specific phenotypes and functions of the macrophages it regulates, and exploring its synergistic potential with existing therapies to promote the development of dictamnine-based combination treatment strategies for prostate cancer. Given that dictamnine can inhibit c-Met [26] and that Marsdenia tenacissima extract (which modulates DKK1) can overcome gefitinib resistance in lung cancer by affecting Wnt/β-catenin and ferroptosis [47], exploring DIC’s synergy with androgen receptor-targeted therapies or chemotherapies for prostate cancer is a highly logical next step. Furthermore, integrating mechanisms from other models—such as its ability to induce ferroptosis [25] or inhibit EMT via HIF-1α/Slug [28]—into the prostate cancer research paradigm may reveal additional layers of its antitumor action.

5. Conclusions

Dictamnine directly binds to and stabilizes the DKK1 protein, thereby inhibiting the Wnt/β-catenin pathway and reversing EMT to suppress prostate cancer malignancy. Furthermore, it remodels the tumor microenvironment by inhibiting angiogenesis and improving the immune landscape, highlighting its potential as a multi-target therapeutic agent.