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Evodiamine inhibits proliferation and induces apoptosis of nasopharyngeal carcinoma cells via the SRC/ERBB2-mediated MAPK/ERK signaling pathway

Journal of Translational Medicine volume 22, Article number: 859 (2024) Cite this article

Abstract

This study aimed to investigate the effect and potential mechanism of evodiamine (EVO) on proliferation and apoptosis of nasopharyngeal carcinoma (NPC) cells. EVO inhibited proliferation, blocked cell cycle progression, and induced apoptosis of NPC cells. There are 27 known anti-NPC targets of EVO, of which eight are core targets, namely SRC, ERBB2, STAT3, MAPK8, NOS3, CXCL8, APP, and HDAC1. Molecular docking analysis showed that the binding of EVO with its key targets (SRC, ERBB2) was good. EVO also reduced the expression of SRC and ERBB2, the key proteins p-MEK and p-ERK1/2 of the MAPK/ERK signaling pathway, and the downstream proteins PCNA and XIAP. EVO inhibited the growth of NPC xenografts in nude mice and reduced the expression levels of SRC, ERBB2, ERK1/2, p-ERK1/2, PCNA and XIAP in NPC tissue. When the MAPK/ERK signaling pathway was activated by epidermal growth factor (EGF), the expression levels of PCNA and XIAP increased, the cell proliferation index increased, and the apoptosis rate decreased in the EGF + EVO treatment group compared to treatment with EVO alone. These changes indicated that the inhibitory effect of EVO on proliferation and apoptosis of NPC cells was related to the down-regulation of SRC and ERBB2 expression, and further inhibition of the MAPK/ERK signaling pathway.

Introduction

Nasopharyngeal carcinoma (NPC) is geographically prevalent in Asian countries, and the number of cases is increasing each year. It is currently believed that the incidence of NPC is related to dietary habits, lifestyle, environmental toxins, viral infection, race, and genetics [1]. Radiotherapy, chemotherapy or combined therapy are common current treatments for NPC. Early NPC is sensitive to radiotherapy, while concurrent radiotherapy and chemotherapy are usually used in the treatment of advanced NPC. However, adverse reactions and resistance as well as other side effects limit the application of chemotherapy, which creates a major challenge for the clinical treatment of NPC [2]. Creating targeted therapies that block NPC-related signaling pathways to improve the efficacy of treatment for early and locally-advanced NPC has been a concern for researchers [3].

Evodiamine (EVO) is a type of alkaloid with a quinazolinocarboline skeleton, which is a primary active component of Evodiae Fructus. EVO has a wide range of pharmacological effects, including anti-inflammatory [4], regulation of immune function [5], improvements in Alzheimer’s disease [6], and anti-tumor effects. Studies have shown that EVO can inhibit the biological activity of a variety of malignant tumors, including colorectal cancer, pancreatic cancer, urothelial cell carcinoma, glioma, thyroid carcinoma, hepatocellular carcinoma, gastric cancer, non-small cell lung cancer [7,8,9,10,11,12,13,14,15,16,17]. EVO induces significant levels of apoptosis and inhibits the proliferation of K562 leukemia cells, via a mechanism which may involve inhibiting activation of the TRIB2/AKT signaling pathway to induce the expression of apoptosis-related proteins, and ultimately inhibit cell growth [18]. Liu et al. found that EVO inhibits the proliferation and apoptosis of glioma SHG-44 cells by promoting the expression of cleaved caspase-3 and cleaved caspase-9 [19]. Jiang et al. confirmed that EVO inhibits the progression of non-small cell lung cancer (NSCLC) by increasing the number of CD8 + T cells and inhibiting the MUC1-C/PD-L1 axis, which may be a targeted treatment strategy for patients with advanced-stage adenocarcinoma [16]. In recent years, researchers have discovered that Evodiamine (EVO) exhibits significant synergistic effects when used in combination with other compounds, further enhancing its bioactivity. For instance, one study demonstrated that EVO can significantly increase the sensitivity of freshly removed gastric cells to 5-FU [20]. Additionally, the combination of Berberine (BBR) and EVO shows synergistic effects in inhibiting P-glycoprotein (P-gp) positive colorectal cancer cells. Moreover, Berberine can mitigate the damage induced by EVO in normal colonic mucosal epithelial cells and cardiomyocytes [21].Regarding NPC, Peng et al. found that the inhibitory effect of EVO on the migration of HONE1 and CNE1 NPC cells was related to down-regulation of MMP2 expression [22], but the pharmacological action of EVO and its mechanism in NPC is not well understood.

Mitogen-activated protein kinases (MAPKs) play a crucial role in various physiological processes, particularly in cell growth and cell death [23]. As a family of protein kinase cascades, MAPKs form a highly interactive network and are significantly altered during carcinogenesis. This leads to insensitivity to growth and proliferation signals and evasion of apoptosis, which are major pathways in cancer development. Approximately one-third of cancers are directly or indirectly associated with dysregulation of the MAPK signaling pathway [24].

The mitogen-activated protein kinase (MAPK) signaling pathway plays a critical role in the development of cancer, influencing cell proliferation, differentiation, and survival. In recent years, researchers have increasingly recognized the potential of alkaloid compounds in regulating the MAPK signaling pathway, making them emerging candidates for cancer treatment. Alkaloids, such as camptothecin and berberine, have been shown to inhibit key nodes within the MAPK pathway through various mechanisms, thereby inducing cancer cell apoptosis, and inhibiting tumor growth and metastasis. For example, Camptothecin encapsulated in β-cyclodextrin nanosponges (CN-CPT) inhibits migration, invasion, and metastasis in anaplastic thyroid carcinoma (ATC), suppresses tumor angiogenesis, and phosphorylation of the Erk1/2 MAPK [25]. Meanwhile, berberine induces autophagy and inhibits cell proliferation in gastric cancer cells by modulating the ERK, JNK, and p38 MAPK pathways [26]. Studies have shown that evodiamine can inhibit cancer cell growth and metastasis by interfering with key molecules within the MAPK pathway, such as ERK, JNK, and p38 [10, 27, 28]. These findings provide new theoretical support for evodiamine as a potential anticancer therapeutic and open up new avenues for developing novel cancer treatment strategies based on the MAPK signaling pathway.

Network pharmacology integrates systems biology, pharmacology, and computer analysis technology. This is helpful for exploring the mechanism of action of traditional Chinese medicines or components in diseases, and molecular docking simulations can serve to analyze the interaction between drugs and their targets [29, 30]. Therefore, to clarify the molecular mechanism of EVO against NPC, we used network pharmacology and molecular docking technology to analyze the binding energy between key targets and EVO in NPC.

In this study, investigated the effects of EVO on the proliferation, cell cycle progression and apoptosis of highly-differentiated nasopharyngeal squamous cell carcinoma lines (CNE1, 6-10B) and poorly differentiated nasopharyngeal squamous cell carcinoma lines (CNE2, 5–8 F), as well as the growth of transplanted NPC tumor into nude mice. In addition, we also aimed to verify the role of the SRC/ERBB2-mediated MAPK/ERK signaling pathway in its inhibition of NPC.

Materials

EVO (purity: 99.86%, lot.23743) was purchased from MCE (Monmouth Junction, NJ, USA); 5-fluorouracil (5-FU, lot. C10616822) was purchased from MACKLIN(Shanghai, P.R.C); Cell Counting Kit-8 (CCK8, Cat. No. C0005) was purchased from TargetMol(Boston, MA, USA); Annexin V FITC Apoptosis Detection Kit I (lot. 1026022) was purchased from BD(Franklin Lakes, New Jersey, USA). PCNA rabbit monoclonal antibody (mAb) (lot. 7), XIAP rabbit mAb (lot. 5), p-MEK rabbit mAb (lot. 18), ERK1/2 rabbit mAb (lot. 14), p-ERK1/2 rabbit mAb (lot. 17), and GAPDH mouse mAb (lot. 8) were purchased from Cell Signaling Technology(Danvers, MA, USA). SRC rabbit polyclonal antibody (pAb) (lot. 00087017) and ERBB2 rabbit pAb (lot. 00063643) were purchased from Proteintech(Wuhan, Hubei, P.R.C). IRDye® 680RD donkey anti-mouse (lot: C70419-08), IRDye® 800CW goat anti-rabbit (lot: D30110-05) antibodies were purchased from LI-COR(Lincoln, NE, USA). Universal antibody diluent (lot: 20220907) was purchased from NCM Biotech(Suzhou, Jiangsu, P.R.C).

Methods

Network pharmacology and molecular docking

The Pubchem database (https://pubchem.ncbi.nlm.nih.gov/) was used with the search term “evodiamine” to identify the chemical formula of EVO. The canonical simplified molecular-input line-entry system (SMILES) system was found and was copied to the Sea Search Server (https://sea.bkslab.org/) to obtain the target of EVO. Next, we searched for the target in NPC through Gene Cards. Then the core target gene of EVO against NPC was obtained by topological network analysis. AutoDockVina was used to dock the core target gene with EVO.

Cell counting kit-8 (CCK8) assay

CNE1, CNE2, 6-10B, and 5–8 F cells as well as NP69 nasopharyngeal epithelial cells in the logarithmic growth phase were prepared as a single cell suspension of 5 × 104 cells/mL. Cells were added to wells of a 96-well plate at 100 µL per well. After cell adherence, medium containing different concentrations of EVO was added. After 24, 36, and 48 h, NP69 cells were cultured for 24 h, and CCK8 working solution was added and incubated for 1 h. Next, the absorbance at 450 nm was detected using a microplate reader (A). Cell proliferation was calculated from the following equation:

Relative cell proliferation rate = (experimental group A - blank group A)/(control group A - blank group A) *100%.

Real-Time Cellular Analysis (RTCA) technology

CNE1, CNE2, 6-10B, and 5–8 F cells in the logarithmic growth phase were prepared as a single cell suspension with 5 × 104 cells/mL, and 100 µL was added to each well of an E-plate cell culture plate. After cell adherence, medium containing different concentrations of EVO was added and the monitoring program was set up for 48 h.

Propidium iodide (PI) staining

CNE1, CNE2, 6-10B, and 5–8 F cells in the logarithmic growth phase were plated into 6 cm petri dishes. EVO at different concentrations was added after cell adherence. Cells were collected 24 h later and were fixed with 75% alcohol. Next, PI staining solution was added and incubated at 37 °C for 30 min in darkness, and the cell cycle distribution was analyzed.

Annexin-V FITC/PI double fluorescence staining

CNE1, CNE2, 6-10B, and 5–8 F cells in the logarithmic growth phase were plated into 6 cm petri dishes and EVO at different concentrations was added after cell adherence. Cells were harvested 24 h later, and 5 µL FITC as well as 5 µL PI staining solution was added and incubated in the dark. After 15 min the staining was terminated and the apoptosis rate was detected.

Establishment of xenograft model in nude mice

After one week of adaptive feeding of BALB/c-nude mice, 5–8 F cells in logarithmic growth phase were harvested and centrifuged to remove the culture medium. The cells were resuspended with PBS, and the cell density was adjusted to 5 × 107/mL, then 100 µL of cell suspension was injected subcutaneously into subcutaneous area of the right dorsal side of nude mice. The model was successfully established with the tumor protruding from the surface of the skin with a diameter of up to 5 mm. The successful nude mice were randomly divided into three groups: control group (0.9% saline), EVO group (2.0 mg·kg− 1·day− 1) and 5-FU (8 mg·kg− 1·2 day− 1) group. The control group was intraperitoneally injected with 0.1 mL 0.9% saline once a day, the EVO group was injected intraperitoneally once a day, and the 5-FU group was injected intraperitoneally once every 2 days for 16 days. The long diameter (a) and short diameter (b) of the tumor were measured every 3 days after treatment, and the survival status of the mice was observed. After 16 days, the tumor volume was calculated and the growth curve was drawn.

Tumor volume (V) = ab2/2.

Hematoxylin-eosin staining

After fixing in 4% paraformaldehyde, tissue samples were removed from the fixative and put in an embedding mold. After dehydration, clearing, wax dipping, embedding and slicing, the slides were placed in a baking oven at 70℃ for 30 min, then dewaxed in xylene for 10 min, three times, washed in anhydrous ethanol three times for 5 min each, then in 95% ethanol, three times for 5 min each, then washed twice in 85% ethanol, 5 min each time; twice in 75% ethanol, 5 min each, then removed and washed in PBS, and stained for 5 min in hematoxylin solution. Excess staining solution was washed away with running water, then slides were placed in acid–ethanol differentiation solution (1%) for 1 min, followed by 1 min in lithium carbonate aqueous solution (1%), and dehydration was carried out by immersing in 75% ethanol for 5 min, 85% ethanol for 5 min, and 95% ethanol for 5 min. Slides were then stained with eosin for 5 min, excess staining solution was rinsed off, the slides were soaked for 5 min in anhydrous ethanol, and then cleared in xylene, sealed with neutral gum, then examined and photographed under a fluorescence microscope.

Immunohistochemical staining

After dewaxing, antigen repair was carried out by the high temperature and high pressure method. Next, blocking agent was dripped onto each slide and they were placed into a humidified chamber, sealed and incubated for 30 min at 37 °C, after which they were incubated with the primary antibody overnight in a 4 °C refrigerator. Next day they were washed three times in PBS, then enhanced solution was added, and they were incubated with the secondary antibody at 37 °C for 20 min. Color development was performed for 30 min at 37 °C, followed by re-staining, differentiation, blueing, dehydration, clearing and sealing. Images were captured with a positive fluorescence microscope.

Western blotting

NPC cells in the logarithmic growth phase were plated into 6 cm petri dishes, and EVO at different concentrations was added after cell adherence. Cells were collected 24 h later and total protein was extracted. After quantification, the sample loading system was configured at 60 µg, and samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a10% gel. The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane using a semi-dry transfer instrument, and the membrane was blocked with 5% skimmed milk at room temperature for 1 h. The primary antibody was diluted with universal antibody diluent at the following dilution ratios: SRC (1:500), ERBB2 (1:500), p-MEK (1:1000), ERK1/2 (1:1000), p-ERK1/2 (1:1000), XIAP (1:1000), PCNA (1:1000), and GAPDH (1:1000), and incubated overnight with the membrane. Next day the fluorescent secondary antibody was incubated for 2 h at a working concentration of 50 ng·mL− 1. Finally, the film was scanned using an Odyssey two-color infrared fluorescence imaging system and the band signal values were analyzed. The membrane was then returned to the incubator box and incubated overnight with GAPDH monoclonal antibody solution, and the procedure was repeated the next day.

Statistical analysis

SPSS 26.0 statistical software was used for data processing, and the experimental data followed a normal distribution. One-way analysis of variance (ANOVA) was used for comparison. If homogeneity of variance was met, multiple comparison analysis was performed by least significant difference (LSD) testing and the variance was tested by the Games Howell post-hoc test (A). The rank-sum test was used for data that did not conform to a normal distribution. P < 0.05 was regarded as a statistically-significant difference.

Results

The toxic effect of EVO on proliferation of NP69 cells

In order to investigate the toxic effect of EVO on NP69 cells, we first analyzed the effect of EVO at different concentrations on the proliferation of NP69 cells by CCK8 assay. As shown in Fig. 1, after treatment for 24 h, NP69 cell proliferation was inhibited by EVO concentrations above 4 µmol/L. However, there was no significant inhibitory effect on NP69 cell proliferation at EVO concentrations below 2 µmol/L. Therefore, for subsequent experiments we used EVO concentrations that did not exceed 2 µmol/L.

Fig. 1
figure 1

The effects of different concentrations of evodiamine (EVO) at 0.5, 1.0, 2.0, and 4.0 µM on the proliferating activity of NP69 cells. *P ＜ 0.05; **P ＜ 0.01 compared with the NP69 group

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EVO inhibits proliferation of NPC cells

The effect of EVO on the proliferation of NPC cells was observed by CCK8 assay and RTCA. Compared with the control group, EVO concentrations above 0.5 µmol/L inhibited the proliferation of NPC (CNE1, CNE2, 6-10B, and 5–8 F) cells after 24, 36, and 48 h treatment, and the cell viability decreased with the increase of drug concentration (Fig. 2A–D). The results are basically consistent with those of Peng et al. [21]. Similarly, the cell proliferation index of the EVO group with different concentrations of EVO was lower than that of the control group (Fig. 2E–H).

Fig. 2
figure 2

Changes in the proliferative activity of CNE1, CNE2, 6-10B and 5–8 F cells induced by EVO measured with CCK8 assay (A-D) and RTCA assay (E-H), *P < 0.05; **P < 0.01 compared with the control group

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EVO blocks NPC cell cycle

The cell cycle plays an important role in tumor cell proliferation, growth and damage repair. G2 phase is the stage from the completion of DNA replication to the beginning of mitosis (M phase) and G2/M phase arrest may be one of the main mechanisms of tumor cell apoptosis [31, 32].To evaluate the effect of EVO on the distribution of different cell cycle phases, PI Staining was performed. After 24 h of EVO treatment in NPC cells, the G0/G1 ratio was decreased in CNE1, CNE2, 6-10B, and 5–8 F cells while the G2/M ratio was increased compared to the control group. Furthermore, the proportion of sub-G1 phase CNE2 and 5–8 F cells was also increased (Fig. 3).

Fig. 3
figure 3

The ratio of cell cycles of CNE1, CNE2, 6-10B and 5–8 F cells induced by EVO measured with PI staining, **P < 0.01 compared with the control group

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EVO induces apoptosis of NPC cells

After 24 h of EVO treatment, the apoptosis rate of NPC cells was increased significantly. The apoptosis rates of CNE1, CNE2, 6-10B, and 5–8 F cells in the EVO 1 µmol/L group were 11.23 ± 0.97%, 16.94 ± 0.70%, 8.19 ± 0.87%, and 11.23 ± 0.97%, respectively. The apoptosis rates of CNE1, CNE2, 6-10B, and 5–8 F cells in the EVO 2 µmol/L group were 21.44 ± 0.91%, 27.0 ± 0.16%, 14.40 ± 0.55%, and 19.30 ± 2.15%, respectively (Fig. 4).

Fig. 4
figure 4

Apoptosis rates of CNE1, CNE2, 6-10B and 5–8 F cells treated with EVO and detected by Annexin-V FITC/PI double fluorescence staining. *P < 0.05; **P < 0.01 compared with the control group

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EVO inhibits SRC, ERBB2, and MAPK/ERK signaling and downstream protein expression

Twenty-seven targets of EVO against NPC have been identified (Table 1). These include eight core targets of EVO against NPC: SRC, ERBB2, STAT3, MAPK8, NOS3, CXCl8, APP, and HDAC1 (Fig. 5A). Molecular docking showed that the key targets (SRC, ERBB2) were well bound to EVO (Fig. 5B, C),and the binding energies were − 8.2 kcal/mol and − 7.8 kcal/mol, respectively. Compared with the control group, EVO treatment at 1 and 2 µmol/L decreased the protein expression of SRC and ERBB2 in CNE2 and 5–8 F cells. Similarly, EVO treatment at 1 and 2 µmol/L in CNE2 and 5–8 F cells resulted in decreased expression of p-MEK and p-ERK1/2, key proteins of the MAPK/ERK signaling pathway, as well as decreased expression of the downstream proteins PCNA and XIAP (Fig. 5D–G).

Table 1 Information regarding the 27 potential targets of EVO against nasopharyngeal carcinoma
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Fig. 5
figure 5

The key targets of EVO against nasopharyngeal carcinoma (A) and the docking of EVO with SRC and ERBB2 (B, C). Effects of EVO on SRC, ERBB2 and MAPK/ERK signaling pathways in nasopharyngeal carcinoma CNE2 (D, E) and 5–8 F (F, G) cells, *P < 0.05; **P < 0.01 compared with the control group

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EVO inhibits the growth of NPC xenografts in nude mice

A subcutaneous transplanted tumor model of NPC in nude mice was established to observe the inhibitory effect of EVO on NPC in vivo. The results showed that after 16 days of treatment, the increase in the tumor volume in the EVO and 5-FU groups was significantly lower than that in the control group (Fig. 6A, B). In addition, the tumor cells in the control group were closely arranged and strongly stained, while the tissues in the EVO and 5-FU groups showed varying degrees of necrosis, nuclear destruction and loose structure (Fig. 6C). Compared with the control group, the EVO group exhibited reduced levels of SRC, ERBB2, ERK1/2, p-ERK1/2, XIAP, and PCNA expression (Fig. 6D, E).

Fig. 6
figure 6

Effect of EVO on the growth of nasopharyngeal carcinoma xenografts in nude mice (A, B, C). The effect of EVO on the expression of SRC, ERBB2, ERK1/2, p-ERK1/2, PCNA and XIAP in tumor tissues, detected by immunohistochemical staining (D, E). Bar = 100 μm

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EVO inhibits proliferation and induces apoptosis of NPC cells via the MAPK/ERK signaling pathway

After activating the MAPK/ERK signaling pathway with EGF, the expression levels of XIAP and PCNA were increased, the cell proliferation index increased, and the apoptosis rate decreased in the combined treatment (EGF + EVO) group compared to treatment with EVO alone (Fig. 7).These results demonstrated that EGF weakened the effect of EVO on the proliferation and apoptosis of NPC cells.

Fig. 7
figure 7

The role of the MAPK/ERK signaling pathway in the inhibition of proliferation and induction of apoptosis of NPC cells by EVO. After the MAPK/ERK signaling pathway was activated by EGF, the expression of PCNA and XIAP were analyzed by western blot (A, B), cell proliferation was monitored by RTCA (C), and apoptosis was evaluated by Annexin-V FITC/PI double fluorescence staining (D, E). **P < 0.01 compared with the control group.##P < 0.01 compared with the EVO (2.0 µmol·mL− 1) group

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Discussion

In view of the limited and toxic side effects of current NPC treatments, the search for safe and effective new drugs is a hot topic in the field of NPC research. In previous studies, EVO has been shown to have a wide range of anti-cancer effects, including in gastric cancer [5, 14, 33], non-small cell lung cancer [16], hepatocellular carcer [13], thyroid cancer [12], colorectal cancer [34], pancreatic cancer [7], human urological cell carcinoma [9], and NPC [35]. While Evodiamine shows potential therapeutic benefits, its adverse effects on various body systems necessitate careful consideration and further investigation to ensure safe use.It is reported that EVO has hepatotoxicity and cardiotoxicity [36, 37]. Therefore, we first used the CCK8 assay to determine the effect of EVO treatment on NP69 nasopharyngeal epithelial cells. We found that EVO concentrations above 4 µmol/L showed obvious inhibitory effects on NP69 cells, but low concentrations of EVO had no obvious inhibitory effects on cell proliferation. This phenomenon suggests that it is important to optimize the dosage of EVO in the research process to ensure safety.

Through network pharmacological analysis, we identified the main targets of EVO against NPC, which were ERBB2, SRC, STAT3, MAPK8, NOS3, CXCl8, APP, and HDAC1. GO functional analysis showed that the effect of EVO on NPC was closely related to proliferation. Abnormal proliferation of NPC cells is one of its many malignant behaviors. CCK8 and RTCA were used to investigate the effect of EVO on NPC cell lines, and we found that the relative proliferation rate of NPC cells was decreased after treatment with different concentrations of EVO. This indicated that EVO reduces the activity of NPC cells, which is consistent with previous studies [35]. According to the changes of the RTCA curve, we concluded that EVO inhibits the proliferation of NPC cells because it causes DNA damage and inhibits DNA synthesis [38]. Break et al. showed that blocking the proliferation of NPC cells in the G2/M phase reduced their vitality and promoted apoptosis [39]. In this study, we found that after EVO treatment, the proportion of NPC cells in G2/M increased significantly, while the proportion of cells in G1 phase was reduced. Studies have shown that G2/M stage tumor cells have higher DNA separation and division activity, which leads to NPC cells being more susceptible to cell damage caused by radiation therapy, eventually leading to apoptosis [40, 41]. Furthermore, Annexin V-FITC/PI double fluorescent staining showed that EVO promoted apoptosis of NPC cells. Therefore, these data further demonstrate that EVO changes the biological behavior of NPC cells by inhibiting proliferation, blocking cell cycle progression, and inducing apoptosis.

SRC is a member of the superfamily of membrane-associated non-receptor protein tyrosine kinases, which can promote cancer by catalyzing tyrosine phosphorylation of various protein substrates. It can be activated by multiple signal transduction pathways, and the activated SRC kinase can be activated by phosphorylating tyrosine residues of corresponding target proteins, including PI3K/AKT, MAPK STAT3, and other signaling pathways [42]. ERBB2 is a primary carcinogen, which has no ligand-binding domain, but can form heterodimers with family members to bind ligands such as epidermal growth factor (EGF), thus activating downstream MAPK/ERK and PI3K/AKT signaling pathways to promote cancer cell proliferation and chemotherapy resistance [43, 44]. Studies have shown SRC-mediated ERBB2 phosphorylation promotes its oncogenic signaling by positively regulating ERBB2/ERBB3 heterocomplex formation, ERBB2-activated cells have higher metastatic potentials and increased SRC activities compared with ERBB2 low-expressing cells, and SRC activation stimulates mitochondrial ATP production and suppresses energy stress, which sustains the activation of mTORC1 and increases the translation of Ezh2 and Suz12, thereby driving ERBB2-related tumorigenesis and metastasis. In addition, combined inhibition of SRC and ERBB-2 activities reversed trastuzumab-resistance in vitro and eliminated tumors in vivo [45]. Therefore, we explored the mechanism of EVO in NPC for the first time. The molecular docking results suggested that EVO could bind well to SRC and ERBB2, which may be an important target of the drug. Moreover, our results showed that the expression levels of SRC and ERBB2 were reduced by EVO, confirming the results of network pharmacology and molecular docking analyses. The MAPK signaling pathway plays a carcinogenic role in a variety of tumors [46,47,48,49] and is also important in the development of NPC because it is closely related to the proliferation, apoptosis, migration, invasion, and chemotherapy resistance of NPC. Abnormal activation of the MAPK/ERK signaling pathway affects the continuous proliferation of NPC. A previous study has shown that drugs inhibit NPC cell proliferation via inhibition of the MAPK/ERK signaling pathway [50]. Similarly, our study showed that EVO reduced the expression of p-MEK and p-ERK1/2 proteins in NPC cells, indicating that EVO inhibits abnormal activation of the MAPK/ERK signaling pathway. In order to further confirm the regulatory role of the MAPK/ERK signaling pathway in proliferation and apoptosis of NPC cells, we activated MAPK/ERK signaling in cells prior to treatment with EVO. We found that prior activation of MAPK/ERK signaling blunted the effect of EVO to decrease the expression of p-MEK, p-ERK1/2, PCNA, and XIAP. The ability of EVO to block proliferation and apoptosis of NPC cells was also lessened with prior MAPK/ERK activation. Taken together, these findings suggest that the ability of EVO to inhibit proliferation and promote apoptosis in NPC cells may be achieved through regulation of the MAPK/ERK signaling pathway.

In summary, we found that EVO binds to SRC and ERBB2 to inhibit their expression, thereby reducing activity of the MAPK/ERK signaling pathway, and ultimately inhibiting the proliferation and inducing apoptosis of NPC cells (Fig. 8). However, the road to anti-tumor drug development is long and tortuous. While these models provide valuable insights, human physiology can differ significantly, and direct extrapolation may not be entirely accurate. Future studies should include clinical trials to confirm these effects in humans. And comprehensivedose-response studies are necessary to identify the precise therapeutic window and potential toxic levels of EVO.

Fig. 8
figure 8

Mechanism of EVO inhibiting the biological activity of NPC

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Data availability

All the original data of this experiment can be obtained from the corresponding author if necessary.

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Acknowledgements

We would like to thank the members of Hunan Provincial Key Lab for the Prevention and Treatment of Ophthalmology and Otolaryngology Diseases with Traditional Chinese Medicine, Hunan University of Chinese Medicine for providing valuable advises to this study.

Funding

This work was supported in part by the National Sciences Foundation of China (Grant No.82305329), the National Natural Science Foundation of Hunan Province (Grant No.2023JJ30449, 2024JJ6343, 2023JJ40500),the Project of Hunan Provincial Department of Education (23A0298,23B0361), Chinese Academy of Engineering Academician Liang Liu’s Workstation of Hunan University of Chinese Medicine (22YS001), First-class Discipline Construction Project of Hunan University of Chinese Medicine (22JBZ011),Hunan Provincial Health High-Level Talent Scientific Research Project (R2023111).

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Author notes

  1. Jie Liu and Lan He contributed equally to this work.

Authors and Affiliations

  1. Hunan University of Chinese Medicine, Changsha, 410208, China

    Jie Liu, Wenqing Zhang, Yinggang Tang, Jingying Fan & Yingchun He

  2. The First Affiliated Hospital of Hunan University of Chinese Medicine, Changsha, 410007, China

    Lan He

  3. Hunan Provincial Engineering and Technological Research Center for Prevention and Treatment of Ophthalmology and Otolaryngology Diseases with Chinese Medicine and Protecting Visual Function, Hunan University of Chinese Medicine, Changsha, 410208, China

    Jingying Fan & Yingchun He

  4. Hunan Provincial Key Lab for the Prevention and Treatment of Ophthalmology and Otolaryngology Diseases with Traditional Chinese Medicine, Hunan University of Chinese Medicine, Changsha, 410208, China

    Yingchun He

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  1. Jie Liu
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  2. Lan He
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  3. Wenqing Zhang
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  4. Yinggang Tang
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Contributions

J L and L H preformed the main experiments and original draft; WQ Z finished the statistical analysis; YG T and JY F consulted relevant literature; J L revised the manuscript; YC H designed the topic and guided the experiment.

Corresponding author

Correspondence to Yingchun He.

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Ethical approval

This study was approved by the Ethics Committee of Hunan University of Chinese Medicine.The ethics code is LL2022092801.

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Liu, J., He, L., Zhang, W. et al. Evodiamine inhibits proliferation and induces apoptosis of nasopharyngeal carcinoma cells via the SRC/ERBB2-mediated MAPK/ERK signaling pathway. J Transl Med 22, 859 (2024). https://doi.org/10.1186/s12967-024-05656-z

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Journal of Translational Medicine

ISSN: 1479-5876

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