Eugenol as a game-changer: overcoming osimertinib resistance in non-small cell lung cancer by inhibiting glycolysis via the tripartite motif containing 59/extracellular signal-regulated kinase pathway.

Introduction
Lung cancer is the malignant tumor with second morbidity and the first mortality in the world, with a 5-year survival rate of 19% [1]. More than 60% of lung cancer patients are locally advanced or have distant metastasis (stage III or IV) at the time of diagnosis, losing the opportunity for surgical resection [2]. Therefore, drug therapy is still the main treatment method for lung cancer, but the occurrence of drug resistance has become the main reason for treatment failure, which needs to be solved urgently. Lung cancer is histologically divided into two main types: small cell lung cancer and non-small cell lung cancer (NSCLC). NSCLC is the most common type of lung cancer, accounting for 80 to 85% [3]. Therefore, non-small cell carcinoma has become the key to lung cancer research. For patients with advanced non-small cell carcinoma lacking target genes and indications for immunotherapy, osimertinib is a targeted drug with significant effects [4]. Unfortunately, long-term drug resistance seriously affects the desired effect of osimertinib in clinical treatment, often resulting in treatment failure, poor prognosis, and high mortality [5].
Traditional Chinese medicine has obvious advantages in the treatment of advanced cancer, including lung cancer [6–10]. Clinical studies have shown that traditional Chinese medicine combined with chemotherapy in the treatment of non-small cell carcinoma is superior to chemotherapy alone in terms of effective rate and disease control rate, and also has advantages in improving patient symptoms and reducing adverse reactions [11]. In addition, traditional Chinese medicine is also one of the important means to reduce the drug resistance of non-small cell carcinoma [12]. Eugenol is a phenylpropanoid compound, which can be extracted from clove tree, Chai Gui and other plants [13]. Studies have shown that eugenol can inhibit the occurrence of NSCLC by inhibiting the activation of nuclear factor kappa-B (NF-kB)/tripartite motif containing 59 (TRIM59) [14]. In ovarian cancer, TRIM59 promotes ovarian cancer progression and upregulates glycolysis through the MKP3/extracellular signal-regulated kinase (ERK) pathway [15]. In NSCLC, glycolysis levels are upregulated in osimertinib resistance [16]. However, the effect of eugenol on the drug resistance of osimertinib in the treatment of NSCLC has not been reported yet. Therefore, in order to further explore the role of eugenol in the sensitivity of NSCLC to osimertinib, we constructed drug-resistant cell lines to reveal the results through molecular experiments. In osimertinib-resistant NSCLC, we investigated the mechanism through which eugenol modulates glycolysis by targeting the TRIM59/ERK signaling pathway. By understanding the molecular interactions between eugenol and this pathway, we sought to provide insights into its potential therapeutic role in overcoming resistance to osimertinib, offering a novel approach for improving treatment outcomes in NSCLC.

Materials and methods

Cell culture
NSCLC cell line PC9 was purchased from the Cell Bank of Chinese Academy of Sciences (SCSP-5085, Shanghai, China). Lung adenocarcinoma cell line HCC827 was ordered from the American Type Culture Collection (CRL-2868, Manassas, Virginia, USA). All cells have undergone short tandem repeat analysis and mycoplasma testing. PC9 cells and HCC827 cells were treated with osimertinib (AZD9291; S7297, Selleck, Shanghai, China) to construct drug-resistant cell lines (PC9-AR and HCC827-AR) [17]. The main method was to gradually increase the osimertinib concentration from 5 nM to 2 μM in 6 months. All cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI-1640, R8758, Sigma-Aldrich, St. Louis, Missouri, USA) medium containing 10% fetal bovine serum (C0235, Beyotime, Shanghai, China) and 1% penicillin/streptavidin (CA005-010, GenDEPOT, Barker, Texas, USA).

Cell administration and transfection
Eugenol (CAS: 97-53-0) with a purity of 99% was purchased from Sigma-Aldrich (St. Louis, Missouri, USA), its chemical formula was C10H12O2, its molecular weight was 164.2 g/mol, and its chemical structure was shown in Fig. 1a. PC9, PC9-AR, HCC827 and HCC827-AR cells were treated with eugenol at concentrations of 100 µM for 24 h [14]. For inhibiting the ERK pathway, 1 µM LY3214996 was used to treat PC9, PC9-AR, HCC827, and HCC827-AR cells for 24 h [4]. The constructed TRIM59 overexpression vector [pRP(Exp)-EGFP-CMV>hTRIM59(NM_173084.3)] and Vector [pRP(Exp)-EGFP-CMV>ORF_Stuffer] (Vectorbuilder, Guangzhou, China) were transfected into cells with a cell fusion degree of about 80% by Lipofectamine 3000 (L3000015, Thermo Fisher, Waltham, Massachusetts, USA) transfection reagent. After 48 h, Gene transfection efficiency was detected by quantitative reverse transcription PCR (qRT-PCR). The cells were divided into five groups: Vector, Eugenol, Eugenol + TRIM59, Eugenol + LY3214996, and Eugenol + TRIM59 + LY3214996. PC9-AR or HCC827-AR cells in the Vector, Eugenol + TRIM59, and Eugenol + TRIM59 + LY3214996 groups were transfected with the corresponding plasmids and cultured for 48 h. Then, cells in the Eugenol + TRIM59 and Eugenol + TRIM59 + LY3214996 groups were treated with 100 µM eugenol for 24 h. Subsequently, cells in the Eugenol + TRIM59 + LY3214996 group were further treated with 1 µM LY3214996 for 24 h. PC9-AR or HCC827-AR cells in the Eugenol and Eugenol + LY3214996 groups were treated with 100 µM eugenol for 24 h, followed by an additional treatment with 1 µM LY3214996 for 24 h in the Eugenol + LY3214996 group.
In addition, synthesized siTRIM59 or siNC (Genewiz Company, Shanghai, China) was transfected into PC9-AR or HCC827-AR cells with a cell fusion degree of about 80% by Lipofectamine 3000 (L3000015, Thermo Fisher, USA) transfection reagent. The PC9-AR or HCC827-AR cells were respectively divided into four groups: PC9-AR, PC9-AR + siNC, PC9-AR + Eugenol, PC9-AR + Eugenol + siTRIM59 group; HCC827-AR, HCC827-AR + siNC, HCC827-AR + Eugenol, HCC827-AR + Eugenol + siTRIM59 group. PC9-AR or HCC827-AR cells in the PC9-AR + siNC or HCC827-AR + siNC, PC9-AR + Eugenol + siTRIM59 or HCC827-AR + Eugenol + siTRIM59 groups were transfected with the siNC or siTRIM59 and cultured for 48 h. Then, cells in the PC9-AR + Eugenol, HCC827-AR + Eugenol, PC9-AR + Eugenol + siTRIM59, HCC827-AR + Eugenol + siTRIM59 groups were treated with 100 µM eugenol for 24 h.

Cell viability
To evaluate the effect of eugenol on the viability of cells that treated with 2 µM osimertinib, and the effects of different concentrations (0.6125, 0.125, 0.25, 0.5, 1, 2, and 4 µM) of osimertinib on cell viability, cell counting kit-8 (CCK-8, KTC011001, Amyjet Scientific Inc, Wuhan, China) was used to measure the viability of eugenol treated cells. 20 µl CCK-8 solution was added to PC9, PC9-AR, HCC827 and HCC827-AR cells incubated in a 96-well plate. After 1 h of incubation, absorbance was measured at 450 nm using Varioskan LUX multimode microporous plate reader (Beijing Pingliyang Trade Co., LTD, China). The Half Maximal Inhibitory Concentration (IC50) was determined using the online calculator (https://www.aatbio.com/tools/ic50-calculator).

Colony formation assay
After 48 h of cell transfection, 1000 cells of each group were added to each well of the 6-well plates and cultured in RPMI-1640 medium contaiong 10% fetal bovine serum. They were routinely placed in an incubator for culture. After 14 days, the formed colonies were stained with 0.1% crystal violet (C0121, Beyotime, Shanghai, China) for 5 min. Then, the number of colonies was recorded and counted using a camera (Nikon, Tokyo, Japan). The formula for calculating the colony formation rate is as follows: (number of clones/number of cells inoculated) × 100%.

Flow cytometry assay
Cell apoptosis was assessed according to the instructions of the apoptosis assay kit (640914, BioLegend, San Diego, California, USA). The cells to be tested were washed twice with phosphate buffer solution (C0221A, Beyotime, Shanghai, China), followed by 5 μl AnnexinV-fluorescein isothiocyanate dye and 10 μl propidium iodide dye. The cells were dyed at 4 ℃ for 30 min under dark conditions. Cell apoptosis was quantified using the NovoCyte Penteon flow cytometer (Agilent Technologies, Santa Clara, California, USA).

Transwell assay
Cell invasion ability and migration ability were detected using the transwell assay. For invasion assay, 20 μl Matrigel glue (C0372, Beyotime, China) was mixed with 100 μl RPMI-1640 serum-free medium precooled at 4 ℃, then added to transwell chamber (FTW010, Beyotime, China) and incubated at 37 ℃ for 4 h to dry into a gel. Next, humidify the chamber 2 h before starting the test. 200 μl of cell suspension was added to the upper chamber, and cell culture medium containing 10% fetal bovine serum was added to the lower chamber. Then the Transwell was cultured in the incubator for 24 h. Rinse twice with PBS and place in 4% paraformaldehyde for 30 min. After staining, the cells were observed and counted under a microscope (250×, Nikon Eclipse 80i, Japan). For migration detection, cells were placed directly in the Transwell upper chamber without Matrigel coating the Transwell upper chamber.

Glycolysis levels determination
After the cells were transfected, take an appropriate amount of 0.25% trypsin (40101ES25, YEASEN, Shanghai, China) to digest the cells in each group, inoculate the logarithmically growing cells in a 96-well plate (FCP962, Beyotime, China) at an appropriate density, set three replicate wells in each group, and place them in a 37 °C, 5% CO2 incubator (Thermo Fisher, USA). After culturing for a certain period of time, discard the supernatant. After washing the cells twice with PBS, 1% Triton-X-100 (ab286840, Abcam, Cambridge, UK) was added to the wells to act on the cells to be tested for 10 min, and the glucose uptake kit (S0554S, Beyotime, China) and lactate release detection kit (A020-1-2, Nanjing Jiancheng Bioengineering Institute, China) were used for detection. The absorbance was measured at 450 nm (glucose consumption) and 440 nm (lactate production) using a microplate reader (Bio-Rad, California, USA).

Quantitative reverse transcription PCR detection
Total RNA was extracted by RNA extraction kit (A33250, Thermo Fisher, USA), and 2100 bioanalyzer (Agilent, Santa Clara, California, USA) was used to identify the total RNA integrity of the samples. cDNA was synthesized according to the instructions of the miScriptII Reverse Transcription Kit (218160, Univ-bio, Shanghai, China), and the Reverse-transcription master mix was prepared on ice. The RNA was incubated in a PCR machine at 37 °C for 60 min and then at 95 °C for 5 min to inactivate the miScript Reverse Transcriptase Mix in the tube. The obtained cDNA was diluted by a certain fold and added to the SYBR Green PCR master mix (4344463, Thermo Fisher, USA) for quantitative detection. The reaction system was prepared, and the reaction results were detected by ABI stepone quantitative instrument (USA). The specificity of RT-PCR product detection was analyzed by the amplification curve and dissolution curve. A control group was set, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the internal reference gene. Primer sequence (Sangon Biotech, Shanghai, China) of TRIM59 is: Forward: 5’-AAGATCCTCGTGTACTGCCAT-3’; reverse: 5’- CAATGCCAGTTGGAGCAATTTC-3’. Primer sequence of GAPDH is: Forward: 5’- AGGTCGGTGTGAACGGATTTG-3’; reverse: 5’- GGGGTCGTTGATGGCAACA-3’. 2−ΔΔCt method was used to calculate the relative expression of the target gene TRIM59 mRNA [18].

Western blotting
Cells were extracted with radioimmunoprecipitation assay buffer (P0013, Beyotime, China) for total protein, and the protein concentration was determined by bicinchoninic acid (BCA; P0009, Beyotime, China) method. 30 μg/well of protein was taken for 12% SDS-PAGE (S0690, Beyotime, China), and the proteins separated by electrophoresis were transferred to polyvinylidene fluoride membrane (FFP24, Beyotime, China). Blocked with blocking solution containing 5% nonfat milk for 1 h at room temperature, and incubated with primary antibodies overnight at 4 °C. Then, horseradish peroxidase-conjugated secondary antibody was added, incubated at room temperature for 2 h, and the membrane was washed 3 times with TBST, 10 min each time. All antibodies were provided by Abcam (UK). The target bands were obtained by developing with a Bio-Rad ultra-sensitive chemiluminescence instrument (Bio-Rad, USA) and chemiluminescent (ECL) Kit (KGC4902, Keygene, Nanjing, China). ImageJ software (California, USA) was applied to quantify the protein band intensity. Table 1 lists the antibodies that were employed.

Tumor formation in nude mice
The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC), ZJCLA (Approval No. ZJCLA-IACUC-20011161). PC9/AR cells were subcutaneously injected into the abdomen of nude mice to establish an in vivo xenograft model. After modeling, the tumor growth of nude mice was observed, and the tumor volume was measured every 3 days. After the tumor volume increased to 50 mm3, drug treatment was started, 50 mg/kg, ip Eugenol was injected intraperitoneally, three times a week, until the end of the experiment. A control group was setup, and nude mice were given the same volume of normal saline every 3 days. Nude mice in the osimertinib group were treated with 5 mg/kg, i.g., osimertinib, once a day. The experiment lasted for 21 days. After the experiment, the mice were sacrificed with 150 mg/kg sodium pentobarbital, and then the tumor tissue was collected for immunohistochemical detection.

Immunohistochemistry
Immunohistochemistry was used to determine the positive rate of TRIM59 protein in different tissues. The sections were added with 3% methanol and hydrogen peroxide for 10 min to block endogenous peroxidase, washed with PBS for 5 min, and washed three times continuously. PBS was added to adjust the pH value of the whole solution to 6.0, citrate buffer was added, and slowly heated to complete the repair of defective proteins. Goat serum was added to the tissue sections for blocking for 20 min, and then the TRIM59 primary antibody was added for incubation. After washing with PBS, it was slowly heated, and secondary antibody was added to incubate for 30 min in a wet box, and PBS was added at 37 °C for washing, and diaminobenzidine (DAB) was used for color development. Take pictures under the microscope.

Statistical analysis
All measurement data were described by mean ± SD, and one-way analysis of variance (ANOVA) was used for comparison between multiple groups. All statistical analyses were implemented by Graphpad 8.0 software (California, USA) and P < 0.05 was considered statistically significant.

Results

Effects of osimertinib on viability and tripartite motif-containing 59 expression in non-drug-resistant lung cancer cells and drug-resistant lung cancer cells
In order to verify the resistance of lung cancer cells and drug-resistant cell lines to osimertinib, we treated the cells with different concentrations of osimertinib, detected the activity by CCK-8 assay, and calculated IC50. The results are shown in Figs. 1b, c, with the increasing concentration of osimertinib, the cell viability was inhibited, and it was obvious that the IC50 of the drug-resistant cell lines was significantly higher than that of normal cancer cells (P < 0.001). Western Blotting was used to detect the expression of TRIM59 in the cells of each group with different concentrations of osimertinib. Osimertinib could up-regulate the expression of TRIM59 in each group, and the expression of TRIM59 in the resistant cell group was higher than that in the nonresistant group (Figs. 1d–f, P < 0.05).

Effects of eugenol on the viability, apoptosis, proliferation, invasion, and migration of osimertinib-induced non-drug-resistant lung cancer cells and drug-resistant lung cancer cells
In order to explore the chemosensitivity of eugenol to lung cancer cells, both non-drug-resistant cancer cells and drug-resistant cancer cells were treated with 0.5 μM osimertinib for 24 h, and CCK-8 assay was used to detect cell viability. The final results showed that eugenol increased the susceptibility of resistant strains to osimertinib (Figs. 1g, h, P < 0.01). Flow cytometry was used to detect the effect of eugenol on cell apoptosis in each group, and eugenol could increase cell apoptosis in each group (Figs. 1i–l, P < 0.05). The colony formation assay was used to examine the ability of cells to form colonies, and eugenol reduced the formation of colonies in each group (Figs. 2a–d, P < 0.05). Transwell was used to detect cell migration and invasion ability, and eugenol reduced the migration and invasion ability of resistant and nonresistant cells (Figs. 2e–i, P < 0.01).

Effects of eugenol on glucose consumption, lactic acid release, glycolysis, and tripartite motif containing 59/extracellular signal-regulated kinase expression in osimertinib-induced non-drug-resistant lung cancer cells and drug-resistant lung cancer cells
In this study, in order to explore the effect of eugenol on the glycolysis level of drug-resistant and non-drug-resistant lung cancer cell lines, we detected the levels of intracellular glucose and lactate. The results showed that eugenol could reduce the glucose consumption of cells in each group (Figs. 3a, b, P < 0.01) and lactate release (Figs. 3c, d, P < 0.01), and the detection of glycolysis-related proteins found that eugenol also reduced the expression of glycolysis-related proteins (c-Myc and LDHA) (Figs. 3e–i, P < 0.05). In addition, Western blotting was used to detect the expression of TRIM59/ERK in the cells of each group, and the results showed that eugenol could inhibit the effect of osimertinib on the expression of TRIM59 and p-ERK/ERK (Figs. 3j–n, P < 0.01).

Silencing of tripartite motif containing 59 enhanced the effect of eugenol on the viability, glucose consumption, lactic acid release, glycolysis, and tripartite motif containing 59/extracellular signal-regulated kinase expression of osimertinib-induced drug-resistant lung cancer cells
In order to further explore whether eugenol can affect the drug resistance of lung cancer cells by regulating TRIM59, we transfected the siTRIM59 into the cells. The result showed that silencing of TRIM59 decreased the expression of TRIM59 in the PC9-AR and HCC827-AR cells (Figs. 4a, b, P < 0.001), it indicated that the transfection was successful. As shown in Figs. 4c–h, the result showed that eugenol decreased the viability, glucose consumption, and lactic acid release in the PC9-AR and HCC827-AR cells (P < 0.001), and silencing of TRIM59 further enhanced the effect of eugenol (Figs. 4c–h, P < 0.05). In addition, eugenol treatment inhibited the expression of c-Myc and LDHA (Figs 4i–m, P < 0.001), and silencing of TRIM59 further enhanced the effect of eugenol (Figs 4i–m, P < 0.01). The effect of eugenol on the expression of TRIM59/ERK was detected, and the result showed that eugenol treatment inhibited the expression of TRIM59 and p-ERK/ERK (Fig. 5, P < 0.01), and silencing of TRIM59 further enhanced the effect of eugenol (Fig. 5, P < 0.05).

Effects of eugenol on the viability and apoptosis of osimertinib-induced drug-resistant lung cancer cells by regulating tripartite motif containing 59/extracellular signal-regulated kinase expression
In order to further explore whether eugenol can affect the drug resistance of lung cancer cells by regulating TRIM59, we transfected the constructed TRIM59 overexpression plasmid into the cells. Firstly, the gene transfection efficiency was detected by qRT-PCR. The expression of TRIM59 in the cells transfected with the overexpression plasmid was significantly increased, which confirmed that we successfully obtained the cells transfected with TRIM59 for further experimental research (Fig. 6a, P < 0.001). Cell viability was detected by CCK-8, and the results showed that eugenol could increase the sensitivity of drug-resistant cell lines to osimertinib, while overexpression of TRIM59 reversed the effect of eugenol (Figs. 6b, c, P < 0.001). In addition, in order to explore the effect of ERK on eugenol, the ERK inhibitor LY3214996 was used to treat drug-resistant cancer cells and nonresistant cancer cells. The ERK inhibitor could reverse the effect of overexpression of TRIM59 on promoting cell viability and enhance the therapeutic effect of eugenol (Figs. 6b, c, P < 0.01). Cell apoptosis was detected by flow cytometry, and eugenol could increase cell apoptosis, TRIM59 overexpression reversed the effect of eugenol, and ERK inhibition reversed the effect of TRIM59 and enhanced the therapeutic effect of eugenol (Figs. 6d–f, P < 0.01).

Effects of eugenol on the proliferation, invasion and migration of osimertinib-induced drug-resistant lung cancer cells by regulating tripartite motif containing 59/extracellular signal-regulated kinase expression
The colony formation assay was used to detect the colony formation ability of each group of cells. Eugenol could reduce the colony formation, overexpression of TRIM59 reversed the effect of eugenol, and ERK inhibition reversed the effect of TRIM59 and enhanced the therapeutic effect of eugenol (Fig. 7, P < 0.001). Transwell was used to detect the migration and invasion ability of cells transfected with the TRIM59 overexpression vector. Eugenol could reduce cell migration and invasion, TRIM59 overexpression reversed the effect of eugenol, and ERK inhibition reversed the effect of TRIM59 and enhanced the therapeutic effect of eugenol (Fig. 8, P < 0.001).

Effects of eugenol on glucose consumption, lactic acid release, glycolysis, and tripartite motif containing 59/extracellular signal-regulated kinase expression in osimertinib-induced drug-resistant lung cancer cells by regulating tripartite motif containing 59/extracellular signal-regulated kinase expression
Next, a kit was used to detect the glucose uptake of cells in each group after transfection, and the results showed that eugenol could reduce glucose consumption, overexpression of TRIM59 reversed the effect of eugenol, and ERK inhibition reversed the effect of TRIM59 and enhanced the therapeutic effect of eugenol (Figs. 9a, b, P < 0.01). The detection of intracellular lactate release showed that eugenol could reduce lactate release, TRIM59 overexpression reversed the effect of eugenol, and ERK inhibition reversed the effect of TRIM59 and enhanced the therapeutic effect of eugenol (Figs. 9c, d, P < 0.001). Western blotting was used to detect the expression of glycolysis-related proteins in the cells of each treatment group. The results showed that eugenol could reduce the expression of c-Myc and LDHA, overexpression of TRIM59 reversed the effect of eugenol, and ERK inhibition reversed the effect of TRIM59 and enhanced the effect of eugenol (Figs. 9e–j, P < 0.001). Western Blotting was used to detect the expression of TRIM59 and ERK-related proteins. Eugenol reduced TRIM59 and inhibited ERK activation, TRIM59 overexpression reversed eugenol effect, and ERK inhibition reversed TRIM59 effect on ERK and enhanced eugenol therapeutic effect (Fig. 10, P < 0.001).

Effects of eugenol on tumor growth and tripartite motif containing 59/extracellular signal-regulated kinase expression in osimertinib-treated mice
In order to further study, we established a PC9/AR nude mouse model in vivo, treated the mice with eugenol on the basis of osimertinib treatment, and measured the tumor volume of the nude mice. It was found that the osimertinib treatment group had a limited effect. However, eugenol treatment significantly suppressed tumor volume (Figs. 11a, b). The expression of TRIM59 in the tumor was detected by immunohistochemical experiments, and the results showed that the expression of TRIM59 increased in the osimertinib treatment group, and eugenol inhibited the effect of osimertinib to promote the expression of TRIM59 in the tumor (Fig. 11c). Western blotting was used to detect the expression level of TRIM59/ERK in tumor tissue. The results showed that osimertinib promoted the phosphorylation of ERK, and eugenol inhibited the effect of osimertinib. The results of both for TRIM59 expression were similar to the results of immunohistochemical detection (Figs. 11d–f).

Discussion
Common mutations in NSCLC are more common in lung adenocarcinoma, and receptor tyrosine kinases such as epidermal growth factor receptor, EML4-ALK, and c-met are known driver mutations [19,20]. These mutations promote tumor growth and metastasis by activating downstream signaling pathways [21,22]. Clinical trials have shown that osimertinib can improve progression-free survival in patients with the T790M mutation [23]. Additionally, due to its ability to cross the blood-brain barrier, osimertinib can significantly reduce brain metastases in NSCLC patients [24].
However, it is a common phenomenon for cancer cells to acquire drug resistance. In terms of enhancing the drug resistance of osimertinib, some studies have promoted the sensitivity of cancer cells to the drug by matching with traditional Chinese medicine preparations [25,26]. In this study, after treatment of drug-resistant NSCLC with eugenol, the cell proliferation, invasion, and migration abilities were significantly weakened relative to the blank control group. This indicates that the combined use of eugenol and osimertinib can improve the sensitivity of cancer cells.
The energy metabolism of tumor cells depends on glycolysis and oxidative phosphorylation, and aerobic glycolysis is an important pathway of energy metabolism in tumor cells. The imbalance of glucose and lactate metabolism in the process of glycolysis indicates that the internal mechanism is disordered. The enhanced glycolytic capacity of cancer cells under aerobic conditions is the best example of metabolic reprogramming [25,27]. Research has shown that the level of glycolysis is up-regulated in osimertinib-resistant cell lines in NSCLC [16]. Glycolysis has been implicated in osimertinib resistance in NSCLC, as alterations in this pathway can enhance cell survival and proliferative capacity despite EGFR inhibition [28]. Glucose consumption and lactate release are key indicators of glycolytic activity, and the upregulation of c-Myc and LDHA further promotes this metabolic shift [29]. C-Myc, a transcription factor, activates glycolysis-related genes, while LDHA facilitates the conversion of pyruvate to lactate [30]. In this study, eugenol could reduce glucose consumption and lactate release in drug-resistant cell lines, as well as reduce the expression of glycolysis-related proteins c-Myc and LDHA. This indicates that eugenol can inhibit the glycolytic ability of osimertinib-resistant cancer cells.
In ovarian cancer, TRIM59 promotes ovarian cancer progression by upregulating glycolysis through the MKP3/ERK signaling pathway [15]. ERK is abnormally expressed in a variety of tumor cells and is closely related to tumor growth [31–34]. Studies have shown that inhibiting this signaling pathway has potential application value in the treatment of lung cancer [35]. In the study of human fibrosarcoma cells, eugenol can inhibit the activation of the ERK signaling pathway [36], so we speculate that eugenol can increase the sensitivity of NSCLC to osimertinib, and its mechanism of action may be to inhibit TRIM59/ERK, and then inhibit glucose fermentation. The results showed that TRIM59 overexpression reversed the inhibitory effects of eugenol on cell proliferation, glycolysis, migration and invasion. At the same time, after the cells were treated with LY3214996, the effect of TRIM59 overexpression was reversed, and the therapeutic effect of eugenol was enhanced. It can be concluded that eugenol may inhibit the TRIM59 and ERK pathways when promoting the sensitivity of NSCLC cells to osimertinib.
This study has the following limitations. Firstly, it was conducted in vitro using drug-resistant lung cancer cell lines, and the results may not fully represent the complexity of osimertinib resistance in vivo or the effects of eugenol in the whole organism. Although the study identified the TRIM59/ERK pathway as a key regulator of glycolysis and resistance, it did not explore further detailed molecular mechanisms.
The study reveals that eugenol can overcome osimertinib resistance in NSCLC by inhibiting cell proliferation, migration, invasion, and promoting apoptosis. Eugenol reduces the expression of TRIM59 and ERK phosphorylation. Additionally, overexpression of TRIM59 reverses the effects of eugenol, while ERK inhibition enhances its therapeutic effect. These findings highlight the potential of eugenol as a promising adjunctive therapy, which can enhance chemotherapy efficacy by targeting the TRIM59/ERK pathway, offering a novel approach to overcoming resistance in NSCLC.

Acknowledgements

Conflicts of interest
There are no conflicts of interest.