Melittin inhibits NSCLC metastasis by targeting USP10 and promoting RNF20-mediated ubiquitination and degradation of PSMA7

Abstract

Background and aims

Non-small cell lung cancer (NSCLC) accounts for > 80% of lung cancer cases, and its mortality is predominantly attributed to its metastatic nature. Therefore, developing novel therapeutic drugs and treatment methods to improve the prognosis of patients with NSCLC is critical.

Purpose

This study examined the effect of Melittin (MEL) on NSCLC metastasis and explored the underlying regulatory mechanisms.

Methods

The effects of different doses of MEL on NSCLC cells was examined using Transwell and wound healing assays. RT-qPCR and western blotting were performed to assess the mRNA and protein expression of proteasome 20S subunit alpha 7 (PSMA7) and epithelial-mesenchymal transition-related genes such as E-cadherin and N-cadherin. A PSMA7 overexpressing lentiviral vector was constructed and transfected into HCC1833 and A549 cells to examine the inhibitory effect of MEL on NSCLC malignant progression. Proteomics analyses were performed to identify differentially expressed proteins in NSCLC cells following MEL treatment. The potential involvement of the ubiquitin degradation pathway was examined using cycloheximide and MG132. Co-immunoprecipitation (Co-IP) assays were performed to detect interactions between PSMA7, ubiquitin-specific peptidase (USP)-10, and ring finger protein (RNF)-20. An in vivo lung metastasis model was used to evaluate the effects of MEL on NSCLC metastasis.

Results

MEL treatment suppressed the malignant migration of NSCLC cells, as shown in Transwell and wound healing assays. MEL treatment also inhibited NSCLC lung metastasis in vivo. Proteomics analysis showed that MEL inhibited PSMA7 expression. Existing studies have shown that PSMA7 affects EMT process mainly by regulating the protein stability of key EMT factors. MEL downregulated EMT key factors, N-cadherin and upregulated E-cadherin, and it activated the ubiquitin degradation pathway, as shown by cycloheximide and MG132 treatments. Co-IP experiments showed that PSMA7 interacts with USP10 and RNF20. Molecular docking validated the interaction of MEL with USP10, confirming the involvement of the ubiquitin pathway in the effects of MEL on promoting the degradation of PSMA7. PSMA7 overexpression significantly reversed the inhibitory effect of MEL on NSCLC cell metastasis in vitro.

Conclusion

MEL suppresses the metastasis of NSCLC by targeting USP10 and promoting RNF20-mediated ubiquitination and degradation ofPSMA7.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12967-025-06903-7.

Introduction

Lung cancer is the leading cause of cancer-related death in the world [1]. Non-small cell lung cancer (NSCLC) accounts for > 80% of lung cancer cases, and its mortality is mainly attributable to metastasis [2, 3]. The first-line treatment for advanced NSCLC includes local surgery, radiotherapy, targeted therapy, immunotherapy, or chemotherapy/immunotherapy [4]. The treatment of NSCLC has improved considerably in the last 10 years; however, despite advances in diagnostic and treatment techniques, a large number of patients experience recurrence and metastasis leading to death [5]. Therefore, it is indispensable to explore novel therapeutic drugs and treatment methods to improve the prognosis of NSCLC.

Melittin (MEL), which is considered a traditional Chinese medicine (TCM), is the main bioactive component of the venom of honeybees (Apis mellifera), and it has been used in cancer therapy for a long time. MEL is a 26-amino acid polypeptide with various biological and pharmacological properties [6, 7]. In previous work, we showed that MEL inhibits malignant NSCLC progression by promoting CTSB-mediated hyperautophagy [8]. However, the mechanism underlying the effect of MEL on the malignant progression of NSCLC, especially recurrence and metastasis, remains to be elucidated.

In this study, we showed that MEL inhibits the malignant migration of NSCLC cells by targeting ubiquitin-specific peptidase 10 (USP10) and promoting ring finger protein 20 (RNF20)-mediated ubiquitination and degradation of proteasome 20S subunit alpha 7 (PSMA7). These findings provide insight into the role of ubiquitin in mediating the effects of therapy and suggest a potential mechanism underlying the anticancer effect of MEL, paving the way for its future clinical application.

Materials and methods

Ethics statement

BALB/c nude mice were obtained from Shanghai Jihui experimental animal feeding Co., LTD (Shanghai, China). Mice had a mean age of 4–6 weeks and weighed 15–20 g. Experiments were approved by the Ethics Committee of Shanghai University of Medicine and Health Sciences of Shanghai, China (approval no.: 2023-GZR-18–340,406,198,707,142,817). Each nude mouse was anesthetized with 30 mg/kg of pentobarbital sodium, and the animals' suffering was minimized throughout the experiments.

Cell culture

The NSCLC HCC1833 and A549 cell lines used in this study were donated by the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM (Shanghai Basal Media Technologies Co., Ltd Shanghai, China) supplemented with penicillin–streptomycin (100 ×) (Gibco, USA) and 10% FBS (bio-explorer, USA). A549 and HCC1833 cells were treated with various concentrations of MEL in normal saline for 24 h before experiments.

Quantitative reverse transcription PCR (RT-qPCR) analysis

Total RNA was extracted from tumor cells or tissues using a commercial kit according to the manufacturer’s instructions (Vazyme Biotech Co., Ltd, Nanjing, China). The purity and concentration of RNA were assessed by measuring absorbance using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The conditions used for RT-qPCR were as follows: 37 ℃ for 15 min × 3 (reverse transcription reaction) and 85 ℃ for 5 s (deactivation of reverse transcriptase).

Thermal cycling conditions for RT-qPCR were 95.0 ℃ for 30 s (hold stage), 95.0 ℃ for 10 s and 60.0 ℃ for 30 s (PCR stage for 40 cycles), and 95.0 ℃ for 15 s and 60.0 ℃ for 60 s (melt curve stage).

RNA was reverse transcribed into cDNA using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific). RT-qPCR was performed using the AB 7300 Real-Time System (Applied Biosystems, Foster City, CA, USA) with primer pairs specific for PSMA7, β-actin (Shanghai Shenggong Biological Engineering Co., LTD, Shanghai, China), and TaqMan Universal PCR Master Mix (Thermo Fisher Scientific). Gene expression was quantified using the 2^−ΔΔCt method. The primers used were as follows: PSMA7, (forward) 5′-GTGCTTTGGATGACAACGTCTGC-3′ and (reverse) 5′-TAGCGGGTGATGTACTCCACAG-3′; β-actin, (forward) 5′-CACCATTGGCAATGAGCGGTTC-3′ and (reverse) 5′-AGGTCTTTGCGGATGTCCACGT-3′.

Western blot analysis

Total protein was extracted from NSCLC cells using RIPA lysis buffer. Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked for 1 h using dry skim milk (Yili Milk Company, Inner Mongolia, China), and then incubated in the following primary antibodies: monoclonal anti-PSMA7 (1:5000 dilution; 67,817–1-Ig; Proteintech, China), monoclonal anti-E-cadherin (1:2000 dilution; 60,335–1-Ig; Proteintech, China), monoclonal anti-N-cadherin (1:5000 dilution; 66,219–1-Ig; Proteintech, China), monoclonal anti-USP10 (1:5000 dilution; 67,917–1-Ig; Proteintech, China), monoclonal anti RNF20 (1:1000 dilution; F1483; Selleck, USA) and β-actin antibodies (1:20,000 dilution; 66,009–1-1 g; Proteintech, China).

Immunoreactive bands were visualized using enhanced chemiluminescence (Thermo Fisher Scientific). Pairwise comparisons were performed using the Least Significant Difference (LSD) t-test. In cases involving comparisons across multiple groups, one-way ANOVA was used for datasets adhering to normal distribution. ImageJ was used for image analysis.

Co-immunoprecipitation (Co-IP) assays

Protein was extracted from cells using the lysis buffer in the Co-IP kit (Biolinkedin, China), and protein concentration was determined using a BCA kit (Yeason, China). A/G magnetic beads were washed in IP buffer before use. Lysates were incubated with the corresponding IP antibody or IgG control at 4 °C overnight and then incubated with magnetic beads. Immunoprecipitates were analyzed by western blotting or subjected to mass spectrometry for analysis of ubiquitination.

Transwell assays

Transwell culture chambers (Guangzhou Jet Bio-Filtration Co., Ltd., China) were used for migration experiments. The concentrations of cells treated with MEL refer to our previous studies [8]. Approximately 30,000 cells were seeded into each chamber. After 24 h, cells were fixed with 4% paraformaldehyde, washed twice with pre-cooled PBS, and dyed with crystal violet solution. Pre-cooled PBS was used for cleaning and air drying, and the results were analyzed using Image J.

Scratch wound assays

For scratch wound assays,A549 and HCC1833 cells (3 × 10⁵) were cultured in DMEM in six-well plates. The concentrations of cells treated with MEL refer to our previous studies [8]. When the cells reached a confluency of 90–95%, a “scratch” was created using a pipette tip. After two washes in PBS to remove detached cells, the wells were treated with MEL (0, 1, 2, 2.5, and 5 μg/mL) for 48 h. Wound images were obtained using an inverted microscope.

Lentiviral transfection

A549 and HCC1833 cells were seeded at a density of 2 × 10⁵ cells per well in six-well plates.

After starvation for 6 h, 20 μl PGMLV-CMV-MCS-ZsGreen1-T2A-Puro as carrier of PSMA7 lentiviral fluid or no-load liquid was added to each well. After screening with 2 μg/mL puromycin for 3 days, cells were examined under a fluorescence microscope, and transfection efficiency was assessed by western blotting and RT-qPCR.

Proteomic data collection and analyses

HCC1833 cells treated with or without MEL (2 μg/mL) were seeded in 10-cm dishes. Cells were washed with PBS and lysed with RIPA buffer containing 1 mM PMSF. Protein concentration was assessed using the BCA protein assay kit (Pierce, IL, USA). Cleared lysates were maintained at -80 °C. Samples containing 250 μg protein were treated with 50 mM IAA, followed by 1.5 mL prechilled 100% acetone to clean sediment, and centrifuged at 14,000 g three times. Samples were air-dried for 2–3 min, diluted with 500 μL NH₄HCO₃ and peptides were desalted using SPE C18 cartridges (Thermo Fisher Scientific). Experiments were performed using three replicates to verify the accuracy of protein identification, reliability, and reproducibility.

Molecular docking analysis

The structure of USP10 was predicted using AlphaFold. Molecular docking was performed using AutoDock Vina. The structure of MEL3D was obtained using Chem3D, and the MM2 module in Chem3D was used for energy minimization. The search parameters for molecular docking were 60 Å × 60 Å × 60 Å grid box, and the box size included the protein pocket. The grid spacing was set to 0.375 Å, and the results were analyzed using Pymol.

Molecular dynamics simulation

MD simulation was used GROMACS 2024 on super computing platform. The complex were solvated in a cubic box using SPC water model. The topology of protein generated by GROMOS96 53a6 force field and ligand topology by PRODRG server. In order to remove steric clashes, systems were subjected to steepest energy minimization to give the maximum force below 1000 kJ/mol/nm. The Particle Mesh Ewald method was used to calculated long-range electrostatic forces. The neighbor list was determined to the Grid method. After energy minimization, the position restraint simulation of 5000 steps (2 fs each steep) was implemented under NVT (the constant Number of particles, Volume and Temperature) and NPT (the constant Number of particles, Pressure and Temperature) condition. In the end, 300 ns MD simulation was conducted on each ensemble. The root mean square deviation (RMSD) and relative root mean square fluctuation (ΔRMSF), which are an important index for evaluating the protein structure, were calculated using GROMACS rmsd and rmsf tools.

Binding free-energy calculation

The binding free energies for WT and mutated α-L-rhamnosidase complexes were calculated with g_mmpbsa software in GROMACS5.1.4 based on the trajectory approach using MD trajectory for each complex. The binding free energy was figured out using following equations:

The complex binding free-energy of MM/PBSA is defined as

In a solution, free energy of the molecules can be expressed as

Molecular mechanics potential energy, E_MM, includes the energy of both bonded and non-bonded. It is calculated based on this equation,

where, E_bond E_angle and E_dihedral are bonded interactions consisting of bond, angle, dihedral. The non-boned interactions include both van der Waals (E_vdw) and Coulomb charge effect (E_coulomb)

The solvability of MM/PBSA is consisted of polar (G_polar) and apolar (G_apolar). G_polar is calculated by solving the Poisson-Boltzmann (PB) equation; G_apolar is estimated according to the surface area of experience method, in this, γ is a coefficient related to surface tension of the solvent, A is solvent accessible surface area (SASA) and b is fitting parameter.

To make a long story short, the MM/PBSA calculation formula is following,

Pulmonary metastasis assays

Stably-transfected luciferase-labeled HCC1833 (Luc-HCC1833) cells were used to evaluate metastasis. Nude mice were injected with Luc-HCC1833 transfected with or without lentivirus packaged PSMA7 overexpression vector (Lv-PSMA7), through the tail vein. Lung metastases were confirmed 4 weeks after injection using a bioluminescence imaging system. The concentrations of mice treated with MEL refer to our previous studies [8]. MEL dissolved in normal saline (2.5 mg/kg) was injected every 2 days, via the tail vein, and the Ctrl shRNA and sh-PSMA7 groups were injected with normal saline. Intact lung tissues were stained with H&E.

Statistical analysis

Results are presented as the mean ± SD. GraphPad Prism (GraphPad, La Jolla, CA, USA) was used for comparisons between subgroups. A P-value ≤ 0.05 was considered statistically significant.

Results

MEL suppresses NSCLC cell migration in vitro

Transwell migration assays showed that MEL treatment for 24 h suppressed the migration of A549 (Fig. 1A and C) and HCC1833 (Fig. 1B and D) cells in a dose-dependent manner. MEL showed the strongest inhibitory effect at 5 μg/mL in A549 cells and 2 μg/mL in HCC1833 cells.

Fig. 1.

Melittin (MEL) inhibits the migration of non-small cell lung cancer (NSCLC) cells in vitro. A–D Transwell assay for migration detection showed the migration ability in both A549 (A and C) and HCC1833 (B and D) cells treated with different concentrations of MEL (0, 1 and 2 μg/mL for HCC1833; 0, 2.5 and 5 μg/mL for A549) for 24 h. E–H Wound healing experiments showed the migration ability of A549 (E and G) and HCC1833 (F and H) cells treated with different concentrations of MEL (0, 1 and 2 μg/mL for HCC1833; 0, 2.5 and 5 μg/mL for A549) for 24 h. Data are expressed as the mean ± SD. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001

The results of wound healing experiments were consistent with those of migration assays. The largest wound width was observed in A549 cells treated with 5 μg/mL MEL (Fig. 1E andG) for 24 and 48 h and in HCC1833 cells (Fig. 1F and H) treated with 2 μg/mL MEL for 24 and 48 h.

MEL inhibits NSCLC pulmonary metastasis in vivo

A model of pulmonary metastasis in nude mice was constructed using Luc-HCC1833 cells, and the pulmonary metastasis in nude mice was detected using live imaging (Fig. 2A). The final fluorescence data showed that lung fluorescence intensity was significantly lower in mice treated with MEL (dissolved in normal saline to 2.5 mg/kg) than in the control group (without MEL treatment). This suggests that MEL can significantly inhibit the malignant metastasis of NSCLC in a nude mice model (Fig. 2B). HE staining data showed that the number of tumor lesions was significantly lower in the MEL treated group than in the control group, confirming that MEL inhibits NSCLC pulmonary metastasis in vivo (Fig. 2C–F).

Fig. 2.

MEL inhibits the migration of NSCLC cells in vivo. A and B Live image detection showing luc-H1833 cell pulmonary metastasis. C and D Representative images of lung tissues. E and F The numbers of metastatic foci in lung tissues were calculated according to the H&E staining (F). Data are expressed as the mean ± SD. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001

MEL inhibits the metastasis of NSCLC cells by downregulating PSMA7

The results of proteomics analyses showed that MEL significantly downregulated PSMA7 in HCC1833 cells. PSMA7 is an α subunit of the 20S proteasome core complex (Fig. 3A). The PSMA7 gene is located on chromosome 20 and is involved in different processes including the regulation of tumor metastasis.

Fig. 3.

MEL inhibits the metastasis of NSCLC cells by downregulating PSMA7. A Clustered heat map of HCC1833 cells post MEL treatment compared with control cells. D and E Western blot analysis of the PSMA7 and EMT-related protein E-cadherin and N-cadherin levels in A549 (B and C) and HCC1833 cells treated with or without MEL (2 μg/mL for HCC1833 and 5 μg/mL for A549). F and G RT-qPCR assay shows the mRNA level of PSMA7. H–K Western blot analysis of PSMA7 and EMT-related protein E-cadherin and N-cadherin levels. L–O Western blot analysis of PSMA7, E-cadherin and N-cadherin in Ctrl shRNA group, Ctrl shRNA + MEL group, shPSMA7 group and shPSMA7 + MEL group. Data are expressed as the mean ± SD. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001

Western blot analysis showed that MEL treatment for 24 h significantly downregulated PSMA7 protein levels in A549 (Fig. 3B and C) and HCC1833 (Fig. 3D and E) cells in a concentration-dependent manner. MEL significantly increased the levels of E-cadherin (E-Ca) and significantly decreased the levels of N-cadherin (N-Ca), suggesting that MEL suppressed epithelial–mesenchymal transition (EMT)-mediated migration of NSCLC.

PSMA7 overexpression reverses the inhibitory effect of MEL on NSCLC metastasis

To further examine the regulatory effect of MEL on the metastasis of NSCLC cells, A549 and HCC1833 cells were transfected with PSMA7-overexpressing vector (Lv-PSMA7) or control vector (Lv-Ctrl). RT-qPCR and western blot analysis confirmed the overexpression of PSMA7 in HCC1833 and A549 cells transfected with Lv-PSMA7 (Fig. 3F–K). Transfection with Lv-PSMA7 upregulated E-cadherin and downregulated N-cadherin compared with the Lv-Ctrl group.

Because the target bands of the PSMA7 overexpression group were too obvious, the PSMA7 imaging in the no-load group was affected. However, the focus of this section was to compare the banding trend of the pre-overexpressed cells and post-overexpressed cells treated with MEL respectively, so as to confirm whether the influence degree of MEL on key factors of PSMA7 and EMT would be changed depending on the overexpression of PSMA7 or not. Treatment with MEL for 24 h and Western Blot detection showed that the protein levels of PSMA7 and N-cadherin were significantly higher and E-cadherin levels were significantly lower in the Lv-PSMA7 + MEL than in the Lv-Ctrl + MEL group (Fig. 3L–O). These results indicate that PSMA7 overexpression reversed the effects of MEL on EMT in NSCLC cells, and demonstrated the importance of regulating the metastasis of NSCLC cells by downregulating PSMA7.

Transwell experiment (Fig. 4A–D) showed that the number of cells that penetrated into the lower culture chamber were significantly higher in the Lv-PSMA7 group than in the Lv-Ctrl group, and significantly higher in the Lv-PSMA7 + MEL group than in the Lv-Ctrl + MEL group. PSMA7 overexpression reversed the inhibitory effects of MEL on NSCLC cell migration.

Fig. 4.

Overexpression of PSMA7 reversed the inhibitory effect of MEL on malignant metastasis of NSCLC cells in vitro. A–D Transwell assays in different groups (Ctrl shRNA group, Ctrl shRNA + MEL group, shPSMA7 group and shPSMA7 + MEL group) of A549 (A and B) and HCC1833 (C and D) cells with or without MEL (2 μg/mL for HCC1833 and 5 μg/mL for A549) treatment for 24 h. E–H Wound healing assays (Ctrl shRNA group, Ctrl shRNA + MEL group, shPSMA7 group and shPSMA7 + MEL group) in A549 (E and F) and HCC1833 (G and H) cells treated with or without MEL (2 μg/mL for HCC1833 and 5 μg/mL for A549) for 24 h. Data are expressed as the mean ± SD. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001

Wound healing experiments (Fig. 4E–H) confirmed that overexpression of PSMA7 (Lv-PSMA7 group) promoted the migration of HCC1833 and A549 cells. The wound width was greater in the Lv-PSMA7 + MEL group than in the Lv-Ctrl + MEL group, and the inhibitory effects of MEL on NSCLC cell migration were significantly reversed.

MEL treatment promotes RNF20-mediated ubiquitination and degradation of PSMA7 by targeting USP10

HCC1833 and A549 cell lines were treated with cycloheximide (40 μM, a protein synthesis inhibitor) and MG132 (10 μM, a proteasome inhibitor). Western blot analysis showed that PSMA7 synthesis was significantly decreased in A549 and HCC1833 cells treated with cycloheximide for 4 and 8 h, and PSMA7 protein levels were significantly decreased after combination treatment with MEL (5 μg/mL for A549 and 2 μg/mL for HCC1833) compared with cells treated with MEL alone (Fig. 5A and B). MG132 treatment for 4 and 8 h significantly increased the levels of PSMA7 in A549 and HCC1833 cells, and PSMA7 protein levels were higher in the group treated with MG132 than in that treated with MEL alone (Fig. 5C and D). This suggests that MEL downregulates PSMA7 by promoting its ubiquitin-mediated degradation.

Fig. 5.

MEL promotes RNF20-mediated-ubiquitination and degradation of PSMA7 by targeting USP10. A–D WB experiment data showed that two cell lines were treated with cycloheximide (40 μM), a protein synthesis inhibitor (A and B), and MG132 (10 μM), a protein degradation inhibitor (C and D), for 0, 4, and 8 h to determine the protein level of PSMA7. E Schematic diagram of molecular docking data. MEL binds to the USP10 active site through seven hydrogen bonds to form a complex. WB and Co-IP data showed the protein level of β-actin, PSMA7, USP10 and RNF20 and the protein interaction between them (F–K). Data are expressed as the mean ± SD. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001

The deubiquitinase USP10 and the E3 ubiquitin ligase RNF20 were screened in cells treated with MEL. Co-IP samples were detected using mass spectrometry, and molecular docking experiments confirmed that MEL binds to USP10 in the form of hydrogen bonds at Glu551, Ile555, Asn541, Glu537, Gln632, Lys687, and Lys693. The docking score was -9.412 kcal/mol (Fig. 5E), and the results of multiple molecular docking experiments of different forms and molecular dynamics simulation experiments have all confirmed our conjecture (Fig. S1 A-D).

This suggests that MEL can bind to USP10 to block the binding of USP10 and PSMA7, thereby increasing the interaction between PSMA7 and RNF20. PSMA7 is degraded by ubiquitination, which inhibits the migration of NSCLC.

Co-IP experiments showed that MEL suppressed the binding of USP10 to PSMA7 compared with the control, whereas it increased the binding of PSMA7 to RNF20 (Fig. 5F–H). The results of the Co-IP and INPUT groups (Fig. 5I–K) confirmed the hypothesis that MEL inhibits the migration of NSCLC by promoting RNF20-mediated ubiquitination and degradation of PSMA7 by targeting USP10.

PSMA7 overexpression reverses the inhibitory effects of MEL on the metastasis of NSCLC cells in vivo

A pulmonary metastasis nude mouse model was constructed using Luc-HCC1833 cells transfected with or without Lv-PSMA7, and the pulmonary metastasis was detected by live imaging (Fig. 6A). The final fluorescence data showed that the lung fluorescence intensity was significantly higher in the Lv-PSMA7 group than in the Lv-Ctrl group, whereas the fluorescence in the Lv-PSMA7 + MEL group showed a similar trend as that in the Lv-Ctrl + MEL group, suggesting the inhibitory effect of MEL on NSCLC metastasis was reversed by overexpression of PSMA7 (Fig. 6B). HE staining data indicated that the number of lung tumor lesions was significantly higher in the Lv-PSMA7 group than in the Lv-Ctrl group and significantly higher in the Lv-PSMA7 + MEL group than in the Lv-Ctrl + MEL group (Fig. 6C–F).

Fig. 6.

Overexpression of PSMA7 significantly reversed the inhibitory effect of MEL on NSCLC metastasis in vivo. A and B Live image detection showing luc-H1833 cells and luc-HCC1833 cells overexpressing PSMA7 in pulmonary metastasis. C–F Representative images of lung tissues and the numbers of metastatic foci in lung tissues were calculated according to H&E staining. Data are expressed as the mean ± SD. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001

Discussion

Recurrence and metastasis are key factors leading to poor prognosis [9, 10], and it is challenging for clinicians to predict prognosis and select the type of surgery and postoperative adjuvant chemotherapy [11]. Standard treatments such as surgery, radiation, chemotherapy, targeted therapy, and immunotherapy have improved the prognosis of patients with lung cancer. Immunotherapy in particular has improved patient outcomes in lung cancer [12]. However, these treatments have some limitations, which has shifted the focus towards TCM.

In this study, we found that MEL modulates the malignant progression of NSCLC. In vitro and in vivo experiments confirmed that MEL inhibited migration and metastasis in a dose-dependent manner as reported previously [13]. MEL treatment inhibits malignant progression in colorectal cancer, NSCLC, and other tumors [14–18]. MEL inhibits EMT and metastasis in human gastric cancer AGS cells [19]. Consistent with previous results, we found that MEL inhibited the expression of the EMT-related protein N-cadherin.

Proteomics analysis confirmed that MEL inhibited PSMA7 expression. PSMA7 is the alpha subunit of the proteasome 20S core complex, which functions in ubiquitin–proteasome-mediated protein degradation [20]. PSMA7 is expressed at high levels in breast cancer and esophageal cancer [20–23]. PSMA7 downregulation reverses tumor malignant progression. Silencing of PSMA7 causes cell cycle arrest at the G0/G1 phase leading to apoptosis in cervical cancer cells [24]. PSMA7 contributes to tumorigenesis by activating the NF-κB and MAPK signaling pathways [25, 26].

EMT ‌(Epithelial mesenchymal transition) refers to the process of Epithelial to mesenchymal cells, it gives cell metastasis and invasion ability. Previous studies have shown that the relationship between PSMA7 and EMT mainly affects the process indirectly through the degradation of EMT-related regulatory factors by proteasome. The abnormal expression of PSMA7 may affect the degradation efficiency of these factors, and then regulate the EMT process and enhance the migration and invasion ability of tumor cells.

In this study, we used proteasome inhibition to demonstrate that MEL activates the ubiquitin–proteasome degradation pathway. Molecular docking and Co-IP detection verified that MEL interacts with USP10 and promotes RNF20-mediated ubiquitination and degradation of PSMA7. PSMA7 overexpression reversed the inhibitory effects of MEL on NSCLC malignant progression. The deubiquitinase UCHL1 maintains protein homeostasis via the PSMA7-APEH-proteasome axis in high-grade serous ovarian carcinoma [20]. In this study, MEL downregulated PSMA7 expression at the protein level by removing the protective effect of USP10 and activating the RNF20-mediated ubiquitination and degradation of PSMA7. Therefore, by affecting the level of PSMA7, the key factor (E-cadherin / N-cadherin) of EMT is affected, which leads to the decrease of EMT level and the decrease of metastasis ability of NSCLC cells.

Taken together, the present results suggest that MEL inhibits the migration of NSCLC cells by targeting USP10 and promoting RNF20-mediated ubiquitination and degradation of PSMA7 (Fig. 7). These findings provide insight into the role of ubiquitination in tumor therapy and suggest a potential mechanism underlying the anticancer effect of MEL, paving the way for its future clinical application.

Fig. 7.

The graphical abstract shows that MEL inhibits the migration of NSCLC cells by targeting USP10 and promotes RNF20-mediated ubiquitination and degradation of PSMA7

Abbreviations

CHX

Cycloheximide

Co-IP

Co-immunoprecipitation

EMT

Epithelial mesenchymal transition

MEL

Melittin

NSCLC

Non-small cell lung cancer

PSMA7

Proteasome Subunit Alpha 7

RNF20

Ring finger protein 20

RT-qPCR

Quantitative reverse transcription PCR

TCM

Traditional Chinese medicine

USP10

Ubiquitin-specific peptidase 10

WB

Western Blot

Author contributions

MJ, FX, GH and CL designed and performed the experiments, analyzed the data, and drafted the manuscript; YW designed and performed the experiments and analyzed the data; XL, CZ, MQ, ZZ, JX and WY performed the experiments and analyzed the data. This investigation was conducted in compliance with current ethical standards. All authors actively participated in the study and approved submission of this article for publication.

Funding

This research was funded by the Shanghai 2024 “Science and Technology Innovation Action Plan” Natural Science Fund Project (24ZR1429400), National Natural Science Foundation of China (No. 52103171, 82127807), Shanghai Municipal Health Commission Medical New Technology research and Transformation Seed Program Project PlanAssignment (2024ZZ2056), National Key Research and Development Program of China (2020YFA0909000) and Shanghai Key Laboratory of Molecular Imaging (18DZ2260400).

Data availability

All data are published with the consent of all authors and may be provided as required.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All the authors have read and approved the final manuscript for publication.

Competing interests

None declared.