Kukoamine B Inhibits EMT in Lung Adenocarcinoma Cells by Regulating Intracellular PD-L1-Mediated p65 Nuclear Translocation

TL;DR

This study shows how Kukoamine B, a natural compound, can stop lung cancer cells from spreading by blocking a specific protein interaction.

Contribution

The study reveals a new role for intracellular PD-L1 in lung cancer progression and introduces Kukoamine B as a potential treatment.

Findings

Intracellular PD-L1 promotes lung cancer progression by interacting with p65 and causing EMT.

Kukoamine B inhibits PD-L1/p65 interaction, preventing p65 nuclear translocation and EMT.

Blocking this pathway with Kukoamine B reduces cancer cell proliferation and migration.

Abstract

This study uncovers a novel, non-immune function of intracellular PD-L1 in driving LUAD progression and identifies a promising therapeutic strategy. We demonstrate that intracellular PD-L1 in LUAD cells directly interacts with phosphorylated p65 (p-p65), promoting p65 nuclear translocation and thereby inducing epithelial–mesenchymal transition (EMT) to enhance LUAD progression. Furthermore, we establish that the natural compound Kukoamine B (KuB) acts as a potent inhibitor of this pathway by competitively disrupting the PD-L1/p65 interaction. This blockade prevents p65 nuclear translocation and effectively suppresses EMT, proliferation, and migration in LUAD cells. Our findings reveal a key intracellular mechanism of PD-L1 in LUAD metastasis and highlight how KuB may serve as a tumor-intrinsic PD-L1-targeting therapeutic.

Cortex Lycii Radicis, a medicinal plant, has been reported to inhibit epithelial–mesenchymal transition (EMT) and exhibit anti-lung cancer properties. Our previous study identified its major compound, Kukoamine B (KuB), as an inhibitor of membrane PD-1/PD-L1 interaction, thereby restoring T-cell function. However, the effect of KuB on EMT and the underlying mechanism thereof remain unknown. Herein, we show that PD-L1 overexpression enhances the proliferation, migration, and EMT of LUAD cells, upregulating N-cadherin and Vimentin, while downregulating E-cadherin. Mechanistically, PD-L1 directly binds phosphorylated p65 (p-p65) and facilitates p65 nuclear translocation, an interaction confirmed by molecular simulations. We found that KuB disrupts the PD-L1/p65 complex, impedes p65 nuclear translocation, and suppresses EMT, proliferation, and migration in LUAD cells. These inhibitory effects were reversed by PD-L1 overexpression. We therefore conclude that KuB suppresses EMT in LUAD by targeting intracellular PD-L1, blocking PD-L1–p65 interaction and nuclear translocation of p65.

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1. Introduction

Based on the latest data from the National Cancer Center, lung cancer is expected to remain the top cause of cancer-related deaths in 2024 [1]. Of the different lung cancer subtypes, LUAD is the most frequently diagnosed, making up about 40% of all cases [2]. Despite progress in treatment approaches, the outlook for LUAD patients remains unfavorable, with current therapeutic options failing to yield consistently favorable outcomes [3]. Therefore, gaining deeper insights into the molecular pathways underlying LUAD and developing novel targeted therapies are essential to improving patient survival.

Programmed Cell Death-Ligand 1 (PD-L1), a type I transmembrane protein, plays a crucial role in immune evasion, predominantly expressed on the surface of tumor cells [4]. Recent research has revealed that PD-L1 is not limited to the cell membrane but also localizes to various subcellular compartments, including the cytoplasm (cPD-L1) and the nucleus (nPD-L1) [5]. This diverse localization enables PD-L1 to participate in multiple cellular functions, such as promoting tumor cell proliferation, modulating the immune response, regulating the DNA damage response, and controlling gene expression [6,7].

Notably, PD-L1 may interact with intracellular proteins to regulate cytoskeletal reorganization and intercellular junction stability [8]. This interaction facilitates the acquisition of a mesenchymal phenotype by tumor cells, thereby enhancing their metastatic potential. Furthermore, high PD-L1 expression can influence cellular adhesion, migration, and invasion by modulating intracellular signaling pathways (e.g., PI3K/AKT or MAPK), which promotes the maintenance and progression of epithelial–mesenchymal transition (EMT) [9]. However, the exact mechanisms through which PD-L1 activates endogenous signaling pathways within tumor cells remain largely unclear. Gaining a comprehensive understanding of the subcellular localization and regulation of PD-L1 expression in cancer cells could pave the way for the development of more precise and effective therapeutic strategies.

Kukoamine B, a bioactive compound isolated from the traditional Chinese medicinal herb Cortex Lycii Radicis [10], has demonstrated a range of pharmacological activities, including immunomodulatory, anti-hypertensive, anti-inflammatory, and neuroprotective effects [11]. Previous research by our group demonstrated that Cortex Lycii Radicis extracts inhibit EMT in lung cancer cells, thus exerting significant anti-metastatic effects [12]. Further studies revealed that both Cortex Lycii Radicis extracts [13] and Kukoamine B (manuscript under review), its major bioactive component, directly bind to PD-L1, thereby blocking PD-1–PD-L1 interaction and inhibiting LUAD progression. Despite these promising findings, the precise role and mechanisms of action of Kukoamine B in inhibiting lung cancer metastasis remain insufficiently understood.

This study seeks to elucidate the role of intracellular PD-L1 in LUAD and examine how Kukoamine B affects intracellular PD-L1 and related cellular processes. The results may provide new insights into targeting PD-L1-mediated immunosuppression as a potential therapeutic strategy for LUAD.

2. Materials and Methods

2.1. Cell Lines

All cell lines used in the experiments were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, including the human bronchial epithelial cell line Beas-2b (Catalog Number: GNHu 27) and two LUAD cell lines, A549 (Catalog Number: TCHu 150) and H1299 (Catalog Number: TCHu 160).

2.2. Reagents

The cell culture media (Gibco, C11995500BT; C11875500BT) were supplemented with Fetal Bovine Serum (FBS, Excell, FSP500), and all cell cultures were maintained at 37 °C in an incubator with 5% CO_2_. Every experiment included three biological replicates for consistency. Kukoamine B was sourced from TargetMol (Batch number: 151262) and dissolved in DMSO as a stock solution. BAY11-7082 was sourced from MCE (Catalog Number: HY-13453) and dissolved in DMSO as a stock solution. In this study, the antibodies utilized were as follows: Normal Rabbit IgG (2729S) and p65 (8242S) rabbit monoclonal antibodies from CST; PD-L1/CD274 (28076-1-AP), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), Vimentin (10366-1-AP), Lamin B1 (12987-1-AP), GAPDH (10494-1-AP), and HRP Mouse Anti-Rabbit IgG Light Chain Specific (SA00001-7L) from Proteintech; Phospho-p65 (AF2006) from Affinity; and secondary antibody HRP Goat Anti-Rabbit IgG from Abclonal (AS014).

2.3. Molecular Dynamics Simulations

Three-dimensional structures of CD274 (PDB ID: 7XYQ) and p65 (PDB ID: 1NFI) were retrieved from the RCSB PDB database (https://www.rcsb.org/, accessed on 5 April 2025). Docking simulation was performed using ZDOCK (https://zdock.wenglab.org/, accessed on 5 April 2025) to predict potential interaction sites, and the top five docked structures were saved for further analysis [14]. The docked complexes were subjected to MM/GBSA calculations on HawkDock (https://zdock.wenglab.org/, accessed on 14 April 2025) in order to evaluate the binding free energy [15]. The complex with the best binding free energy was selected for molecular dynamics calculations using GROMACS v2020.6 [16]. An AMBER99SB-ILDN force field was applied, while the SPC/E water model was used for solvation. Periodic boundary conditions (PBCs) were applied to simulate an infinite system. Energy minimization was performed to resolve steric hindrances before the production MD run. A production simulation of 160 nanoseconds (ns) was conducted under NPT ensemble conditions, with the temperature set to 300 K using the V-rescale thermostat and with pressure maintained at 1 bar via the Parrinello–Rahman barostat. Visualization of the protein structures and simulation results was performed using PyMOL v2.4.0 (https://www.pymol.org/, accessed on 15 April 2025). MM/GBSA free energy calculations were conducted using gmx_MMPBSA v1.6.2 (https://valdes-tresanco-ms.github.io/gmx_MMPBSA/dev/getting-started/ [17], accessed on 20 April 2025).

2.4. Western Blot

Cells were subjected to lysis in RIPA buffer (FD008, FuDe Biotech, Hangzhou, China) and spun at 12,000 rpm for 10 min at 4 °C. A cytoplasmic and nuclear protein extraction kit (BB-3112, BestBio, Shanghai, China) was used to extract nuclear proteins. We measured the protein concentration using a BCA protein assay kit (KGB2101-5000, KeyGen Biotech, Nanjing, China) and measured the absorbance at 562 nm. Proteins were denatured in SDS-PAGE loading buffer, heated to 100 °C for 5 min, and stored at −20 °C for future use. The samples were run on SDS-PAGE and transferred to PVDF membranes (03010040001, Roche, Basel, Switzerland). Membranes were blocked with 5% milk in TBST and incubated with primary antibodies overnight at 4 °C. Secondary antibody incubation was carried out for 1 h at room temperature after washing with TBST. Protein bands were visualized using an ECL chemiluminescence detection kit (P10300, NCM Biotech, Suzhou, China); images were acquired with a chemiluminescence imaging system (Tanon 5200, Tanon, Shanghai, China). Triplicate experiments were conducted for each condition, and the intensities of the bands were quantified with ImageJ software v1.51.

2.5. Cell Proliferation Assay

Cells were plated at a concentration of 5000 per well in 96-well plates. Following a 24 h incubation period, cell viability was assessed using the CCK8 assay kit (FD3788, FuDe Biotech, Hangzhou, China) according to the manufacturer’s instructions. Absorbance readings were taken at 450 nm on a microplate reader at specified time points: 0.5 h, 1 h, 2 h, and 4 h.

2.6. Clonogenic Assay

One thousand cells were seeded per well in 6-well plates. After adhering, the cells were exposed to Kukoamine B at concentrations of 5 µmol/L and 10 µmol/L for 24 h. After a 14-day incubation period, cell colonies were observed under a microscope. We next washed the cells with PBS; the cells were fixed for a duration of 20 min using 4% paraformaldehyde, subsequently stained with 1% crystal violet for 30 min, and then photographed after allowing them to air-dry.

2.7. Scratch Assay

The 6-well plates were inoculated with cells at a concentration of 6 × 10^4^ cells per well and maintained overnight at 37 °C in an environment supplemented with 5% CO_2_. Once cell confluence had reached over 80%, a vertical scratch was made with a sterile 200 L pipette tip. The wells were rinsed with PBS to clear any debris, and the cells were then incubated in serum-free medium containing 5 µmol/L and 10 µmol/L Kukoamine B. Wound healing was tracked over time and visualized with an inverted bright-field microscope.

2.8. Lentiviral Transduction

Lentiviral vectors were sourced from Shanghai GeneChem (Genechem, Shanghai, China). The A549 and H1299 cell lines were dispensed into 96-well plates with a cell density of 0.4 × 10^4^ per well, cell confluence was evaluated within the range of 30% to 50%, and lentiviral transduction was performed according to the manufacturer’s instructions. After incubating the cells overnight at 37 °C in an atmosphere containing 5% CO_2_, the cultures were subsequently refreshed with fresh complete medium. Transduced cells were selected using puromycin (P8230, Solarbio, Beijing, China) and subsequently expanded for further experimentation.

2.9. Immunofluorescence

Cells (5 × 10^4^) were plated on glass coverslips within confocal dishes. Once the cells attached, they were rinsed with PBS, fixed with 4% paraformaldehyde for a duration of 10 min, and subsequently permeabilized using 0.2% Triton X-100 for 15 min at room temperature. Blocking was then performed using 5% BSA for a period of 30 min, followed by overnight incubation at 4 °C with primary antibodies in a humidified chamber. After three PBS washes, conducted for a period of 1 h at 37 °C in the absence of light, the cells were subjected to incubation with secondary antibodies that were fluorescently tagged. Finally, the cells underwent washing followed by counterstaining with DAPI. A confocal fluorescence microscope (Zeiss LSM 800, Zeiss, Oberkochen, Baden-Württemberg, Germany) was utilized for visualization at a 630× magnification.

2.10. Co-Immunoprecipitation

Cells were seeded at a density of 4 × 10^6^ cells per dish and treated with varying concentrations of Kukoamine B for 24 h. Following the treatment, the cells were lysed with lysis buffer (P0013, Beyotime Biotech, Shanghai, China). Subsequently, the lysates were centrifuged at 14,000× g for 10 min at 4 °C. Protein concentrations were then measured using a BCA assay kit. A+G magnetic beads (P2108, Beyotime Biotech, Shanghai, China) were washed with TBS and incubated with the respective antibodies for 1 h. Overnight incubation of the protein lysates with the antibody/magnetic bead complexes was conducted at 4 °C. After multiple washes, the proteins were analyzed by means of Western blotting after eluting the proteins at 95 °C for 5 min.

2.11. Statistical Analysis

All data are expressed as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 8.2.1 and SPSS 26.0. A one-way ANOVA test was conducted, considering p values of 0.05 or less as statistically significant. Significance levels are denoted by * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.

3. Results

3.1. Altered Distribution of PD-L1 During LUAD Progression

High PD-L1 expression is frequently linked to poor clinical outcomes in LUAD [18]. By leveraging information sourced from the Human Protein Atlas (HPA) database (https://www.proteinatlas.org/, accessed on 12 May 2025), we observed that PD-L1 levels in both the cytoplasm and nucleus of LUAD tissues were markedly elevated compared to adjacent non-cancerous tissues (Figure 1A). The online Kaplan–Meier Plotter tool (https://www.kmplot.com/analysis/, accessed on 29 January 2025) was utilized to assess the prognostic impact of PD-L1 expression in lung adenocarcinoma. Survival analysis demonstrated that patients with high PD-L1 expression had a significantly lower survival probability compared to those in the low-expression group (Figure 1B). This was further validated through Western blot analysis, which demonstrated increased PD-L1 expression in both the cytoplasmic and nuclear fractions of A549 and H1299 compared to Beas-2B (Figure 1C,D). These findings indicate that PD-L1 expression undergoes significant changes in LUAD cell models, suggesting that elevated levels of cytoplasmic PD-L1 (cPD-L1) and nuclear PD-L1 (nPD-L1) may be associated with its malignant phenotype. To gain deeper insights into the role of PD-L1 in LUAD, we established stable LUAD cell lines with either PD-L1 overexpression or silencing (Figure 1E), and we confirmed that PD-L1 overexpression led to a marked elevation in intracellular PD-L1 levels (Figure 1F).

3.2. Intracellular PD-L1 Mediates Proliferation, Migration, and EMT in LUAD Cells

We established a stable PD-L1-overexpressing cell line via plasmid transfection, which markedly increased total PD-L1 levels, including both the intracellular and membrane-bound forms. Although the canonical function of PD-L1 requires membrane localization to engage PD-1, our findings do not preclude potential contributions from intracellular PD-L1 through non-canonical pathways. Consequently, the phenotypic effects observed upon PD-L1 overexpression or knockdown are likely attributable to its intracellular pool rather than the membrane-bound form. We performed a CCK8 assay to assess the effect of intracellular PD-L1 on LUAD cell proliferation, and our findings indicate that PD-L1 silencing notably decreased cell growth, while PD-L1 overexpression led to a notable increase in proliferation (Figure 2A). These findings are supported by clonogenic assays, which exhibited a similar trend in cell proliferation (Figure 2B,C). To gain deeper insights into the function of intracellular PD-L1 in cell migration, we employed a scratch assay. The results demonstrate that PD-L1 silencing inhibited cell migration, whereas overexpression of PD-L1 enhanced the cells’ migratory abilities (Figure 2D,E). Additionally, our Western blot analysis disclosed decreased E-cadherin levels and concurrent increases in N-cadherin and Vimentin levels in LUAD cells that overexpressed PD-L1 (Figure 2F,G). In summary, our findings imply that intracellular PD-L1 stimulates cell proliferation, enhances migratory capabilities, and facilitates the process of EMT in LUAD cells.

3.3. Intracellular PD-L1 Promotes p65 Nuclear Translocation by Targeting p65

We then investigated the mechanism through which intracellular PD-L1 drives EMT in LUAD. Given the central role of NF-κB in regulating EMT, which, in its inactive state, sequesters p65 in the cytoplasm, while, upon activation, p65 is released, phosphorylated [19], and translocates to the nucleus to initiate EMT-related gene transcription [20]. Prompted by this theory and the observed positive correlation between PD-L1 (CD274) and p65 (RELA) in the TIMER database (p < 0.001) (Figure S1), we subsequently focused on the activated form of p65 to investigate the potential interaction between PD-L1 and p65. Co-immunoprecipitation experiments demonstrated the presence of both PD-L1 and phosphorylated p65 (p-p65) proteins in the input samples, whereas these proteins were not detected in the IgG control group of the immunoprecipitation (IP) samples. Importantly, both PD-L1 and p-p65 were detected in the anti-PD-L1 immunoprecipitated group, suggesting a direct association between PD-L1 and p-p65, which was further strengthened by PD-L1 overexpression (Figure 3A,B). Western blot analysis revealed that nPD-L1 and nuclear phosphorylated p65 (np-p65) levels were significantly upregulated in cells overexpressing PD-L1 (Figure 3C,D). In addition, immunofluorescence staining confirmed elevated levels of both nPD-L1 and np-p65 in cells with PD-L1 overexpression (Figure 3E), indicating that intracellular PD-L1 promotes the nuclear translocation of p-p65 via its interaction with p65.

To determine whether PD-L1-driven EMT depends on the NF-κB canonical pathway, we performed a rescue experiment in H1299 cells using the NF-κB inhibitor (BAY11-7082). Western blot analysis showed that BAY11-7082 treatment reduced the expression level of P65 protein, while PD-L1 expression showed no significant decrease; it also reduced the expression levels of N-cadherin and Vimentin, and upregulated the expression of E-cadherin. (Figure S2). Under effective p65 inhibition, PD-L1 overexpression still significantly enhanced the EMT process, suggesting that the intracellular PD-L1-driven EMT in lung adenocarcinoma cells is independent of p65.

3.4. Molecular Docking and Molecular Dynamics Simulations Reveal Strong Binding of PD-L1 and p65

To explore the physical interaction between PD-L1 and p65, we conducted molecular docking followed by molecular dynamics (MD) simulations. ZDOCK docking analysis produced five potential binding structures, and the fifth-ranked model was found to have the most favorable binding energy, as assessed by means of MM/GBSA methodology, yielding an energy score of −57.56 kcal/mol. These docking results suggest that PD-L1 and p65 are capable of forming multiple stable binding conformations (Figure 4A–E). The fifth-ranked structure was selected for further MD simulation analysis. RMSD data from 60 ns to 160 ns indicated convergence, implying that the PD-L1–p65 complex exhibits substantial flexibility over this time frame (Figure 4F). B-factor analysis revealed strong rigidity in the protein regions responsible for recognition and binding, while the solvent-exposed N-terminal domain of p65 showed higher flexibility, further confirming the stability of the PD-L1–p65 binding interface (Figure 4G). Structural snapshots, which were taken at 40 ns, 80 ns, 120 ns, and 160 ns, demonstrated that the observed flexibility in the complex was mainly due to contributions from intradomain random coils (Figure 4H). Throughout the 160 ns simulation, an average of 9.059 hydrogen bonds were consistently formed between PD-L1 and p65, underscoring the crucial role of these hydrogen bonds in maintaining the stability of the complex (Figure 4I).

LigPlot analysis of the PD-L1 (chain A) and p65 (chain B) complex revealed several key interactions that contribute to the stability of the binding interface, including prominent hydrogen bonds and hydrophobic contacts, visualized in both 2D and 3D structural representations (Figure 5A–C). MM/GBSA analysis of the PD-L1–p65 protein complex indicated that electrostatic forces, van der Waals interactions, and hydrophobic contacts positively influence the binding affinity, while polar solvent interactions were found to have a destabilizing effect (Figure 5D). Several crucial residues were identified as major contributors to the binding free energy of the PD-L1–p65 complex. On the PD-L1 chain (Chain A), residues such as GLU164, ASP215, TYR123, and LEU214 emerged as significant contributors. On the p65 chain (Chain B), the residues ARG201, PHE309, and ILE312 were identified as critical to binding (Figure 5E). Notably, GLU164 and TYR123 on PD-L1 were highlighted for their stable contributions to binding energy (Figure 5F). Free energy landscape analysis revealed that the PD-L1–p65 complex possesses multiple potential energy wells, indicative of various conformational states (Figure 5G). However, one interaction region between PD-L1 and p65 displayed a singular deep potential well, indicating a particularly stable conformation in this domain (Figure 5H). Interestingly, this interaction region overlaps significantly with the binding residues observed between PD-L1 and Kukoamine B, suggesting a potential similarity in their binding mechanisms (Figure 5I). Our results reveal that the binding interaction between PD-L1 and p65 is potentiated by the involvement of random coil and beta-sheet conformations within their structures, leading to a complex with significant binding energy, which is indicative of a stable and robust interaction.

3.5. Kukoamine B Inhibits p65 Nuclear Translocation by Disrupting the Interaction Between Intracellular PD-L1 and p65

To explore the effect of Kukoamine B on intracellular PD-L1 in LUAD cells, we examined its impact on the association between PD-L1 and p65 (the chosen drug concentrations were established in prior, unpublished work by our group). Co-immunoprecipitation assays detected the existence of both PD-L1 and p65 in the input samples, while no proteins were found in the IgG control group, thereby ruling out non-specific binding. PD-L1 and p65 were detected in the anti-PD-L1 immunoprecipitated samples, confirming a direct interaction between the two proteins, which was progressively weakened with increasing concentrations of Kukoamine B (Figure 6A,B). Additionally, Western blot analysis demonstrated that Kukoamine B decreased the levels of nPD-L1 and np65 (Figure 6C,D), indicating that Kukoamine B inhibits p65 nuclear translocation by disrupting its interaction with PD-L1.

3.6. Kukoamine B Inhibits Proliferation, Migration, and EMT in LUAD Cells Through Inhibiting Intracellular PD-L1

Recognizing the role of intracellular PD-L1 in promoting proliferation, migration, and EMT in LUAD cells, we subsequently evaluated the impact of Kukoamine B on these cellular processes. Clonogenic assays revealed that 24 h treatment with 5 µmol/L and 10 µmol/L Kukoamine B significantly decreased the proliferative capacity of A549 and H1299 cells (Figure 7A,B). Additionally, scratch assays revealed that Kukoamine B treatment impaired cell migration, as indicated by the reduced wound healing areas observed at 24, 48, and 72 h compared to the control groups (Figure 7C,D). Western blot analysis of EMT-associated proteins demonstrated that Kukoamine B treatment markedly decreased N-cadherin and Vimentin levels, while increasing E-cadherin expression in both H1299 and A549 cells (Figure 7E,F). These results imply that Kukoamine B suppresses proliferation, migration, and EMT in LUAD cells. To explore whether this inhibition is mediated by intracellular PD-L1, we investigated the impact of Kukoamine B on H1299 and A549 cells with overexpressed PD-L1. The inhibitory effects exerted by Kukoamine B on N-cadherin, Vimentin, and E-cadherin expression were reversed due to PD-L1 overexpression (Figure 7G,H). Based on these results, Kukoamine B inhibits proliferation, migration, and EMT in LUAD cells by targeting PD-L1 and disrupting PD-L1–p65 interactions.

4. Discussion

Immunotherapy is considered a conventional treatment for LUAD [21,22]. Although immune checkpoint inhibitors have had a transformative impact, most solid tumors develop primary or acquired resistance [22,23]. Research on PD-L1, a common immune checkpoint, has largely focused on its extracellular functions, particularly its effects upon binding to its ligand [24], while its intracellular signaling and functions remain poorly understood. Recent discoveries indicate that intracellular PD-L1 can transduce cell-intrinsic signals independently of PD-1 [8,25]. In fact, different subcellular localization patterns of PD-L1 may lead to diverse effects. It is evident that these intracellular PD-L1 signals regulate tumor progression in ways independent of its immune checkpoint function. Drugs that block intracellular PD-L1 action could potentially enhance anti-tumor responses. Therefore, targeting intracellular PD-L1 represents a promising cancer immunotherapy strategy worthy of further investigation.

In this study, using computational simulation data, we observed a significant increase in PD-L1 levels within the tumor tissues of LUAD patients relative to adjacent non-cancerous tissues. Based on Western blot analysis, the cytoplasmic and nuclear fractions of LUAD cells A549 and H1299 showed elevated PD-L1 expression, compared with the non-cancerous Beas-2b bronchial epithelial cells. These results indicate that increased expression of intracellular PD-L1 (cPD-L1) and nuclear PD-L1 (nPD-L1) is significantly associated with the malignant phenotype of LUAD. It is hypothesized that intracellular PD-L1 may modulate intracellular signaling pathways or translocate to the nucleus to influence gene transcription [26]. PD-L1 seems to contribute to tumor malignancy through mechanisms unrelated to immune checkpoints, such as regulating DNA repair, autophagy, and enhancing cell migration [5]. The results of our study suggest that PD-L1 overexpression promotes proliferation, migration, and EMT in LUAD cells, possibly by influencing nuclear gene expression.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a ubiquitously expressed transcription factor that undergoes phosphorylation in the cytoplasm prior to its translocation into the nucleus [27]. NF-κB is essential in controlling key processes such as inflammation, tumor progression, and metastasis [28]. p65 is the core functional subunit of the NF-κB pathway; its active form is phosphorylated before entering the nucleus to initiate transcription. Immunoprecipitation assays from our study confirmed that PD-L1 binds to phosphorylated p65 (p-p65), with PD-L1 overexpression promoting the nuclear translocation of p65. Furthermore, rescue experiments demonstrated that under conditions where p65 was effectively inhibited, PD-L1 overexpression still enhanced the EMT process in lung adenocarcinoma cells, suggesting another new function of intracellular PD-L1: PD-L1 may serve as a direct binding partner of p65, facilitating its nuclear entry through a non-canonical pathway to drive EMT. Subsequently, molecular docking and dynamics simulations revealed multiple potential binding interactions between PD-L1 and p65, with the interaction region showing significant overlap with the residues observed in the binding between PD-L1 and Kukoamine B, suggesting a potentially similar binding mechanism. This finding prompted us to further investigate the mechanistic action of Kukoamine B.

Kukoamine B, a bioactive compound derived from the traditional Chinese medicinal herb Cortex Lycii Radicis, has shown potential in modulating immune responses, lowering blood pressure, reducing inflammation, and providing neuroprotection [29]. Our previous research demonstrated that Kukoamine B serves as a potent inhibitor of PD-L1, displaying notable anti-tumor activity. Through co-immunoprecipitation and Western blot analyses, we discovered that Kukoamine B competitively disrupts the interplay between PD-L1 and p65, thereby inhibiting the translocation of p65 to the nucleus. Moreover, our results from clonogenic assays, scratch assays, and Western blotting confirm that Kukoamine B significantly reverses the proliferative, migratory, and EMT characteristics of LUAD cells. The results emphasize the critical role of intracellular PD-L1 in LUAD progression, while also suggesting that Kukoamine B mediates the reversal of EMT by targeting PD-L1. Nevertheless, additional research is necessary to thoroughly elucidate the precise mechanisms underlying these phenomena.

It is important to note some limitations of our study. First, the research was limited to in vitro experiments, without in vivo studies to explore the role of intracellular PD-L1 in LUAD progression. Second, additional control experiments, such as treating PD-L1-knockout cells with KuB, or conducting other rescue experiments to verify p65 activity, would more rigorously establish the role of intracellular PD-L1 in LUAD and strengthen evidence for KuB’s PD-L1-targeting effect. We plan to conduct these in vitro and in vivo experiments in future work in order to more comprehensively validate the results and hypotheses of this study.

5. Conclusions

This research reveals a previously unrecognized mechanism in LUAD, whereby intracellular PD-L1 interacts with p65, promoting its nuclear entry through a non-canonical mechanism, thereby facilitating the EMT process. In addition, in LUAD cells, Kukoamine B suppresses EMT and blocks p65’s translocation to the nucleus by interfering with PD-L1 and p65 interactions. The results suggest that Kukoamine B may serve as a tumor-intrinsic PD-L1-targeting strategy, providing a foundation for further research into its mechanism of action in LUAD.