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Paeonol inhibits the Glycolysis in oral squamous cell carcinoma though suppressing NAT10-mediated ac4C modification

BMC Cancer volume 25, Article number: 629 (2025) Cite this article

Abstract

Background

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral and maxillofacial regions. Paeonol, derived from Moutan Cortex, has diverse pharmacological effects including anti-inflammatory and anticancer activities. The N-acetyltransferase 10 (NAT10)-mediated N4-acetylcytidine (ac4C) modification is a newly discovered RNA epigenetic mechanism. This study aimed to investigate the role of paeonol in OSCC and its underlying mechanisms of action.

Methods

Cell viability and migration were assessed using a cell counting kit-8 and transwell migration assays. Glycolysis-related indices were detected using commercial kits. The interaction between NAT10 and hexokinase 2 (HK2) was examined using RNA immunoprecipitation and dual-luciferase reporter assays. A tumor-bearing mouse model was established.

Results

The results showed that paeonol treatment decreased the viability, migration, and glycolysis of OSCC cells. Moreover, paeonol treatment inhibited NAT10-mediated ac4C modifications in OSCC cells. In addition, NAT10 overexpression upregulates glycolysis and cell migration in OSCC cells. Moreover, NAT10 upregulated ac4C levels of HK2 in OSCC cells. Animal studies have revealed that paeonol treatment decreases OSCC tumor growth.

Conclusion

This study revealed that paeonol inhibited glycolysis and cell migration in OSCC by suppressing the NAT10-mediated ac4C modification of HK2.

Peer Review reports

Introduction

Oral squamous cell carcinoma (OSCC) represents a significant global health burden with substantial morbidity and mortality, accounting for approximately 90% of all malignant neoplasms in the head and neck [1]. The complexity of OSCC pathogenesis involves a combination of genetic predisposition and environmental exposure [1]. Chronic irritation and inflammation from poorly fitting dentures, chronic infections such as human papillomavirus (HPV), tobacco use, alcohol consumption, betel nut chewing, and poor oral hygiene can contribute to disease onset [2, 3]. Moreover, genetic alterations, including mutations in tumor suppressor genes and oncogenes, play a critical role in disease progression [4]. Despite advances in early detection and multimodal therapies, including surgical resection, radiation therapy, chemotherapy, and targeted treatments [3], the five-year survival rate of OSCC has not significantly improved over the past few decades. In addition, these treatments often have considerable side effects that affect patients’ quality of life. Given the limitations of current therapeutic options and the need for less toxic alternatives, exploring natural compounds derived from traditional Chinese medicine (TCM) is a promising strategy.

Tumor cells often undergo significant metabolic changes to support rapid growth and proliferation. One of the key adaptations they make is to enhance glycolysis, this shift towards increased glycolysis, despite the availability of oxygen, is referred to as the “Warburg effect” [5]. Increased glycolytic activity in tumor cells leads to the production of lactate, even in the presence of oxygen, which can contribute to an acidic tumor microenvironment [6]. This acidic environment can promote tumor progression, invasion, and metastasis as well as help the tumor evade the immune response. Hexokinase 2 (HK2) is a pivotal enzyme in the glycolytic pathway that plays a critical role in the initial step of glycolysis by catalyzing the phosphorylation of glucose to glucose-6-phosphate [7]. HK2 is often upregulated in cancer cells, which supports its rapid proliferation and survival [8].

Moutan Cortex, a well-regarded component in TCM, has been utilized for centuries because of its numerous therapeutic properties [9]. It is particularly noted for its ability to dissipate heat and cool blood, a concept in TCM that refers to its therapeutic effects in reducing inflammation, alleviating fever, and improving blood circulation [10]. In TCM, “heat” and “blood heat” are pathological conditions often associated with inflammatory diseases, fever, and certain bleeding disorders [11]. By dissipating heat and cooling blood, Moutan Cortex helps restore balance within the body, making it a valuable component in TCM formulations. One of the key active compounds found in Moutan Cortex is mpeony phenol, commonly known as paeonol [12]. This naturally occurring compound possesses unique structural features that contribute to its broad-spectrum biological effects, including anti-inflammatory, antipyretic, and analgesic properties [12]. In recent years, paeonol has garnered increasing attention for its potential role in cancer therapy owing to its antitumor activity. Studies have indicated that paeonol exerts its antitumor effects through multiple mechanisms, such as regulating apoptosis and anti-inflammation, and modulating the expression of various genes involved in tumor progression [13,14,15]. A previous study found that paeonol exhibited protective effects against 7,12-dimethylbenz(a)anthracene-induced oral carcinogenesis [14]. However, the specific mechanism of action of paeonol in OSCC remains unclear.

N4-acetylcytidine (ac4C) is a unique and significant post-transcriptional modification that plays a crucial role in regulating RNA function and stability [16]. This modification involves the addition of an acetyl group to the nitrogen atom at the fourth position of the cytidine base in RNA molecules. The presence of ac4C has been shown to influences various aspects of RNA biology, including mRNA stability, translation efficiency, and overall dynamics of gene expression [16]. N-acetyltransferase 10 (NAT10) is the primary catalyst responsible for the addition of an acetyl group to cytosine, which facilitates the formation of ac4C [17]. This enzyme plays a pivotal role in the dynamic regulation of RNA modifications. Research has shown that NAT10 exhibits abnormal activity under various pathological conditions, particularly in the context of cancer [17, 18]. However, research on NAT10-mediated ac4C modifications in OSCC is limited.

Given this background, the aim of this study was to investigate the effect of paeonol on glycolysis in OSCC and its potential mechanism of action, which may provide a potential therapeutic strategy for OSCC.

Methods and materials

Study design

This study is a preclinical experimental investigation combining in vitro and in vivo approaches to evaluate the therapeutic potential and mechanisms of paeonol in OSCC. In vitro experiments were performed using OSCC cell lines, while in vivo experiments utilized a xenograft mouse model to validate the findings.

Materials

Human oral keratinocytes (HOK, #YS1199C, WHELAB Biotech Co., LTD, Shanghai, China), HOK medium (#M1004A, WHELAB), SCC9 cells, (#QS-H241, Keycell Biotech Co. Ltd., Wuhan, China) and HSC3 (#QS-H267, Keycell), HSC3 cells (#QS-H267, Keycell), SCC9 medium (SCC9, Keycell), HSC3 medium (#QS-H267A, Keycell), Opti-MEM® medium (#31985062, Thermo Fisher Scientific, Waltham, MA, USA), paeonol (#H35803, purity: 99%, Sigma-Aldric, St. Louis, MO, USA), Lipofectamine 3000 (#L3000001; Thermo Fisher), CCK8 kit (#PF00004; Proteintech Biotech Co. Ltd., Wuhan, China), glucose assay kit (#ab136955; Abcam, Cambridge, MA, USA), lactate assay kit (#ab65331; Abcam), BCA kit (#20200ES76; Yeason Biotech Co. Ltd., Shanghai, China), Seahorse Glycolytic Stress Test kit (#103020-100; Agilent Technologies, Santa Clara, CA, USA), Seahorse Mitochondrial Stress Test kit (#103015-100; Agilent), oligomycin (#495455, Sigma), FCCP (#C2920, Sigma), rotenone (#557368, Sigma), antimycin A (#77332, Sigma), glucose (#49163, Sigma), 2-deoxyglucose (#D8375, Sigma), methanol (#322415; Merck Millipore, Billerica, MA, USA), crystal violet (#32675; Merck), TRIzol (#15596026CN, Thermo Fisher), TBST (#60145ES76, Yeason), enhanced chemiluminescence kit (#RM00021; ABclonal Biotech Co. Ltd., Wuhan, China), Hifair® III Reverse Transcriptase kit (#14601ES03; Yeason), Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix kit (#11184ES03; Yeason), primers (Genescript Biotech Co. Ltd., Nanjing, China), RNA fragmentation reagent (#AM8740; Thermo Fisher), protein A/G (#88802; Thermo Fisher), RIP lysis buffer (#89900; Thermo Fisher), pGL3 vector (Promega, Madison, WI, USA), DNA purification kit (Qiagen, Duesseldorf, Germany), nude mice (Vital River, Beijing, China), pentobarbital sodium (#P3761; Sigma).

Antibodies: ac4C (#A18806; 1/2000; ABclonal Biotech Co. Ltd., Wuhan, China), HRP-conjugated goat anti-rabbit IgG secondary antibody (#AS014; 1/10,000 for dot blot, 1/200 for immunohistochemical; ABclonal), NAT10 (#ab194297; 1/80; Abcam), Ki67 (#ab16667; 1/200; Abcam), NAT10 (#ab194297; 1/500; Abcam), HK2 (#ab209847; 1/500; Abcam).

Cell culture and drug treatment

Human oral keratinocytes were maintained in a specialized medium. SCC9 and HSC3 cells were cultivated in their respective commercial media. All the cell cultures were maintained under standard conditions at 37 °C in a 5% CO2 atmosphere. To determine the optimal concentration of paeonol, HOK, SCC9, and HSC3 cells were exposed to varying concentrations (0, 100, 200, and 400 µg/ml) of paeonol for 48 h.

Cell transfection

For transfection experiments, SCC9 and HSC3 cells were seeded in 6-well plates at a density of 5 × 10^5 cells per well one day prior to transfection to ensure that they reached approximately 80% confluency. Transfections were performed using Lipofectamine 3000 according to the manufacturer’s protocol. Briefly, vector control and NAT10 overexpression plasmids were separately diluted in Opti-MEM® medium. Lipofectamine 3000 reagent and PLUS Reagent were mixed in Opti-MEM® medium according to the guidelines provided. After 5 min of incubation, the diluted plasmids were combined with the reagent mixture and incubated for 15 min at room temperature to allow complex formation. The transfection complex was added dropwise to the cells. Following a 4-hour incubation period at 37 °C in a 5% CO2 atmosphere, the medium was replaced with fresh complete growth medium. The cells were further incubated for 48 h post-transfection to achieve optimal expression levels.

Cell counting kit-8 (CCK-8) assay

To perform the cell viability assay, HOK, SCC9, and HSC3 cells were plated in 96-well plates at a concentration of 1 × 10^3 cells per well in 100 µL of complete medium and incubated at 37 °C for 24 h. Subsequently, each well received 10 µL of CCK8 reagent was added. The plates were then returned to the incubator at 37 °C for 2 h. Optical density readings were taken at 450 nm using a microplate reader (Thermo Fisher Scientific) to quantify cell proliferation.

Glucose consumption and lactate production measurement

To evaluate glucose consumption and lactate production in SCC9 and HSC3 cells, biochemical assays were performed using commercial kits. Cells were seeded in 24-well plates at a density of 2 × 10^5 cells per well and incubated overnight to achieve approximately 90% confluence. Glucose consumption was assessed using a glucose assay kit, which measured the amount of glucose remaining in the supernatant. Lactate production was quantified using a lactate kit that detects the level of lactate produced during glycolysis. The standards and samples were prepared according to the manufacturer’s instructions. The absorbance at the specified wavelength was recorded using a spectrophotometer. Glucose consumption and lactate production were normalized to the protein content of the cells. The protein content was determined using a BCA kit.

Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) detection

To investigate the cellular metabolic profiles of SCC9 and HSC3 cells, we measured the ECAR and OCR using the Seahorse XFe24 Extracellular Flux Analyzer (Agilent). Prior to the experiment, cells were seeded at a density of 1 × 10^5 cells per well in a Seahorse XF cell culture microplate and allowed to attach overnight. On the day of the assay, the Seahorse XFe24 instrument was calibrated according to the manufacturer’s instructions to ensure accurate measurement. The cell culture medium was then replaced with Seahorse XF assay medium prewarmed to 37 °C and supplemented with glucose, glutamine, and pyruvate. The Seahorse Glycolytic Stress Test kit and Seahorse Mitochondrial Stress Test kit were used to assess glycolytic and mitochondrial activities, respectively. For mitochondrial function analysis, cells were sequentially treated with oligomycin (1 µM), FCCP (0.3 µM), and a mixture of rotenone and antimycin A (Rote/AA; 0.1 µM). To evaluate glycolysis, cells were exposed to glucose (10 mM), oligomycin (0.5 µM), and 2-deoxyglucose (2-DG; 50 mM). After metabolic assessments, the cells were lysed, and protein concentrations were determined using a BCA kit. ECAR and OCR values were normalized to the protein content to account for differences in cell numbers and metabolic status among the wells. Data were analyzed using Seahorse Wave software, and results were expressed as pmol/min for OCR and mpH/min for ECAR and normalized to protein content.

Cell migration measurement

Cell migration was assessed using a transwell migration assay. SCC9 and HSC3 cells were suspended in serum-free medium at 5 × 10^4 cells/mL and added to transwell inserts with 8.0 μm pores placed in 24-well plates with 10% FBS in the lower chamber. The plates were incubated for 24 h at 37 °C in an atmosphere of 5% CO2. Non-migrated cells were removed and migrated cells were fixed with methanol and stained with 0.1% crystal violet. Cells were counted in five random fields per insert at 200 × magnification.

Ac4C Dot blot assay

To determine the total levels of ac4C in SCC9 and HSC3 cells, a dot blot assay was performed. Cells were harvested, and RNA was extracted using TRIzol reagent according to the manufacturer’s instructions. The RNA concentration was measured using a Nanodrop spectrophotometer, and equal amounts (1 µg) were spotted onto a nylon membrane and allowed to air-dry. The membrane was then UV-crosslinked to immobilize RNA. The membrane was blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST) for 1 h at room temperature. Following blocking, the membrane was incubated overnight at 4°C with a primary antibody targeting ac4C. After washing three times with TBST, the membrane was incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Finally, the signals were detected using an enhanced chemiluminescence (ECL) kit and imaged using a chemiluminescence detection system. The experiment was repeated thrice to ensure reproducibility.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from SCC9 and HSC3 cells using the TRIzol reagent. Hifair® III Reverse Transcriptase kit was used for reverse transcription to generate the cDNA. The Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix was used for qPCR amplification, following the recommended reaction conditions: one cycle of 95 °C for 2 min, 40 cycles of 95 °C for 10 s and 60 °C for 30 s, and a melt curve stage. The used primers are listed below: NAT10, forward, 5′-ATAGCAGCCACAAACATTCGC-3′ and reverse, 5′-ACACACATGCCGAAGGTATTG-3′; glucose transporter type 1 (GLUT1), forward, 5′-TCTGGCATCAACGCTGTCTTC-3′ and reverse, 5′-CGATACCGGAGCCAATGGT-3′; lactate dehydrogenase B (LDHB), forward, 5′-TGGTATGGCGTGTGCTATCAG-3′ and reverse, 5′-TTGGCGGTCACAGAATAATCTTT-3′; HK2, forward, 5′-GAGCCACCACTCACCCTACT-3′ and reverse, 5′-CCAGGCATTCGGCAATGTG-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward, 5′-TGTGGGCATCAATGGATTTGG-3′ and reverse, 5′-ACACCATGTATTCCGGGTCAAT-3′.

Molecular docking

Molecular docking was used to analyze the mode of action of paeonol and the target protein NAT10. The molecular structure of paeonol was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The NAT10 protein structure was obtained from the RCSB Protein Data Bank (https://www.rcsb.org/). Molecular docking was performed using the Glide module in Schrodinger Maestro software. Finally, the mode of action of the compound and target protein was analyzed, and the interaction between the compound and protein residue was determined.

Surface plasmon resonance (SPR) assay

The binding assays based on SPR technology were performed in a Biacore 8 K instrument (GE Healthcare) at 25 °C using PBST (PBS, pH 7.4, containing 0.005% Tween-20) as the running buffer. The protein sample was dissolved in coupling buffer (20 µg/mL in 10 mM sodium acetate [pH 5.0]) and then immobilized onto a CM5 chip that had been equilibrated with PBST overnight. Paeonol (1.25 µM) was serially diluted and then injected at a flow rate of 30 µL/min for 90 s (contact phase), followed by 90 s (dissociation phase). The binding data were collected using Biacore 8 K evaluation software (GE Healthcare).

Methylated-RNA immunoprecipitation (Me-RIP)

To quantify the ac4C modification in GLUT1, LDHB, and HK2 transcripts within SCC9 and HSC3 cells, a Me-RIP assay was employed. Total RNA was extracted from cells. RNA samples were fragmented into fragments 100 nucleotides long using the RNA fragmentation reagent. Fragmented mRNAs (400 ng) was incubated with an ac4C antibody overnight at 4°C. Magnetic beads conjugated with protein A/G were added to the RNA-antibody complex and incubated for 2 h at room temperature to facilitate the binding and precipitation of ac4C-modified RNA fragments. The beads were subsequently washed three times with low- and high-salt buffer solutions to remove the unbound material. The ac4C-modified RNA was eluted from the beads using elution buffer and analyzed by RT-qPCR.

RNA immunoprecipitation (RIP) assay

To validate the interaction between NAT10 and HK2 in SCC9 and HSC3 cells, RIP assay was conducted. Cells were harvested and lysed in RIP lysis buffer supplemented with RNase inhibitors. Lysates were cleared by centrifugation, and supernatants were collected. For the RIP reaction, 50 µL of magnetic beads conjugated with protein A/G was incubated with an anti-NAT10 antibody or an isotype-specific IgG control overnight at 4 °C. The antibody-bound beads were then added to the cell lysates and incubated for 4 h at 4 °C to allow the formation of NAT10-containing RNA-protein complexes. After incubation, the beads were washed four times with RIP wash buffer to remove non-specifically bound materials. The immune complexes were eluted from the beads using an elution buffer. Finally, the level of HK2 expression was measured by qPCR.

Prediction of ac4C sites of HK2

The bioinformatics analysis tool: prediction of ac4C sites in mRNA (PACES http://rnanut.net/paces/) was used to predict the ac4C sites of HK2.

Dual-luciferase reporter assay

To further elucidate the regulatory mechanism of NAT10 on HK2, a dual-luciferase reporter assay was performed. Plasmids were constructed using GenScript and included wild-type HK2 (WT) and mutant variants (mut1 [1#], mut2 [2#], and mut3 [3#]). Wild-type HK2 sequences containing potential ac4C sites were amplified and inserted into a pGL3 vector. The corresponding mutant sequences were cloned into the same vector. SCC9 and HSC3 cells were co-transfected with these WT or mutant plasmids alongside either empty or NAT10 overexpression vectors using the transfection reagent. Luciferase activity was measured 48 h post-transfection using a Dual-Luciferase Reporter Assay System (Promega).

Chromatin immunoprecipitation (ChIP)-qPCR

To verify the effect of NAT10 overexpression on the promoter activity of HK2 in SCC9 and HSC3 cells, a ChIP assay was performed. Cells were transfected with either an empty vector or NAT10 overexpression vector. At 48 h post-transfection, the cells were crosslinked with 1% formaldehyde for 10 min at room temperature to stabilize the protein-DNA interactions. The cross-linking reaction was quenched with glycine at a final concentration of 0.125 M for 5 min. The cells were then washed twice with ice-cold PBS, harvested, and lysed in lysis buffer containing protease inhibitors. The chromatin was sheared by sonication to obtain DNA fragments ranging from 200 to 1000 bp. After centrifugation, the chromatin was precleared with protein A/G magnetic beads and incubated overnight at 4 °C with an antibody specific to NAT10 or with an isotype-matched IgG control. The following day, the immunocomplexes were captured using protein A/G magnetic beads and washed sequentially with low-salt, high-salt, LiCl, and TE buffers. The bound DNA was eluted from the beads with elution buffer and reverse-crosslinked overnight at 65 °C. The DNA was purified using a DNA purification kit to remove proteins. Finally, the obtained DNA was subjected to RT-qPCR. Fold enrichment was calculated after normalization to 1% input.

Animal study

Twelve nude mice were randomly divided into two groups, each comprising six animals: a control group and a paeonol-treated group. Subcutaneous injections of 1 × 10^5 SCC9 cells suspended in 0.1 mL were administered to all mice to establish a tumor-bearing model. Beginning 24 h after SCC9 cell injection, the paeonol group received intraperitoneal injections of paeonol every other day for a total of six administrations over 12 days, with observations continuing for 28 days. Tumor volumes were measured weekly using a Vernier caliper and calculated using the following formula: volume (mm³) = (length × width²)/2. After the fourth volume measurement, the mice were anesthetized with pentobarbital sodium (60 mg/kg) and euthanized by cervical dislocation. The tumors were excised and weighed for further analysis. This study was approved by the Ethics Committee of MDKN Biotechnology Co., Lt (Approval number: MDKN-2024-178). All animal experiments were performed in accordance with the ARRIVE guidelines. All methods were performed in accordance with relevant guidelines and regulations.

Immunohistochemical (IHC)

IHC analysis was performed to evaluate the expression levels of NAT10 and HK2 in tumor tissues from mice. Tumor tissue Sect. (4 μm thick) were cut from paraffin-embedded blocks and mounted on glass slides. The sections were deparaffinized with xylene and rehydrated using graded ethanol solutions. Antigen retrieval was performed by heating the slides in citrate buffer (pH 6.0) in a microwave oven for 20 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min, and non-specific binding was minimized by incubating sections with 5% normal goat serum for 30 min. The sections were then incubated overnight at 4 °C with primary antibodies in a humidified chamber. After washing with PBS, the sections were incubated with the secondary antibody for 1 h at room temperature, followed by treatment with a streptavidin-peroxidase complex for 30 min. Color development was achieved using diaminobenzidine as the chromogen, and the slides were counterstained with hematoxylin. Finally, images were captured using a light microscope (Thermo Fisher).

Statistical analysis

SPSS software (version 21.0) was used to analyze the data. Data are expressed as means ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for comparisons between groups. Statistical analyses were performed using the GraphPad Prism software (v8.0.1, GraphPad Software Inc., San Diego, CA, USA). p < 0.05 indicates that the difference is statistically significant.

Results

Paeonol treatment decreased the viability of SCC9 and HSC3 cells

The chemical structure of paeonol is shown in Fig. 1A. We subsequently treated OSCC cells (SCC9 and HSC3) and normal human oral keratinocytes (HOK) cells with varying concentrations of paeonol. The results indicated that treatment with 100, 200, and 400 µg/mL paeonol significantly decreased the viability of SCC9 and HSC3 cells, with a more pronounced inhibitory effect observed at higher concentrations. Conversely, in HOK cells, 400 µg/mL paeonol led to a reduction in cell viability, suggesting potential toxicity at this level. Therefore, further experiments were conducted using 200 µg/mL paeonol (Fig. 1B).

Fig. 1
figure 1

Paeonol treatment decreased the viability of SCC9 and HSC3 cells. A, The chemical structure of paeonol; B, The cell viability of HOK, SCC9, and HSC3 cells treated with different concentrations (0, 100, 200, and 400 µg/mL) of paeonol was detected by CCK-8 (n = 3). (*p < 0.05, ***p < 0.001)

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Paeonol treatment inhibited Glycolysis and cell migration in OSCC

In comparison with the control group, treatment with paeonol resulted in a reduction in glucose consumption, lactate production, and ECAR in SCC9 and HSC3 cells. Additionally, paeonol treatment led to an increase in OCR in these cells relative to that in the control group (Figs. 2A-F). Moreover, paeonol treatment significantly decreased the migration of SCC9 and HSC3 cells compared to that in the control group (Fig. 2G). These findings indicated that paeonol treatment effectively inhibited glycolysis and cell migration in OSCC.

Fig. 2
figure 2

Paeonol treatment inhibited glycolysis and cell migration in OSCC. A, Glucose consumption and B, lactate production of SCC9 and HSC3 cells in the control and paeonol groups (n = 3); The OCR in C, SCC9 and D, HSC3 cells (n = 3); The ECAR in E, SCC9 and F, HSC3 cells (n = 3); G, Transwell migration assay was performed to analyze cell migration in the control and paeonol groups. (***p < 0.001)

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Paeonol treatment regulated NAT10-mediated ac4C modification in OSCC

Paeonol treatment led to decreased relative ac4C levels and NAT10 mRNA expression in SCC9 and HSC3 cells. Additionally, molecular docking analysis showed that paeonol and NAT10 could be bonded by hydrogen and hydrophobic bonds (Fig. 3C-E). To further validate this interaction, SPR experiments were performed, which confirmed that paeonol effectively binds to NAT10 (Fig. 3F). These results indicate that paeonol treatment regulates NAT10-mediated ac4C modifications in OSCC.

Fig. 3
figure 3

Paeonol treatment regulated NAT10-mediated ac4C modification in OSCC. A, Dot blot assay was performed to assess the ac4C level in each group (n = 3); B, RT-qPCR was performed to analyze the mRNA level of NAT10 in the control and paeonol groups (n = 3); C-E, Molecular docking of NAT10 and paeonol; F, The binding sensorgram (for the interactions between paeonol and NAT10). (***p < 0.001)

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Overexpression of NAT10 upregulated the Glycolysis and cell migration in OSCC

To further explore the role of NAT10 in glycolysis and cell migration in OSCC, an NAT10 overexpression vector was transfected into SCC9 and HSC3 cells. The results showed that the mRNA levels of NAT10 were upregulated after NAT10 overexpression (Fig. 4A). Moreover, compared with the vector group, NAT10 overexpression increased cell viability, glucose consumption, lactate production, and ECAR, whereas OCR was decreased in the NAT10 overexpression group relative to the vector group in SCC9 and HSC3 cells (Figs. 4B-H). Transwell migration assay results indicated that NAT10 overexpression increased cell migration in SCC9 and HSC3 cells compared to that in the vector group (Fig. 4I).

Fig. 4
figure 4

Overexpression of NAT10 upregulated the glycolysis and cell migration in OSCC. A, After NAT10 overexpression, the mRNA level of NAT10 in SCC9 and HSC3 cells was analyzed by RT-qPCR (n = 3); B, Cell viability in each group was detected by CCK-8 (n = 3); C, Glucose consumption and D, lactate production of SCC9 and HSC3 cells in each group (n = 3); The OCR in E, SCC9 and F, HSC3 cells (n = 3); The ECAR in G, SCC9 and H, HSC3 cells (n = 3); I, Transwell migration assay was performed to analyze cell migration in each group (n = 3). (***p < 0.001 vs. the vector or the control group; ###p < 0.001 vs. the paeonol + vector group)

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NAT10 upregulated the ac4C level of HK2 in SCC9 and HSC3 cells

To investigate the downstream targets of NAT10, we evaluated the ac4C levels of several important glycolytic enzymes, GLUT1, LDHB, and HK2, using a MeRIP assay. The findings indicated that the overexpression of NAT10 led to an increase in the relative ac4C level of HK2, whereas the ac4C levels of GLUT1 and LDHB remained unaffected in SCC9 and HSC3 cells (Fig. 5A). Additionally, RIP analysis revealed that NAT10 interacted with the mRNA of HK2 in SCC9 and HSC3 cells (Fig. 5B). The potential ac4C modification sites on HK2 mRNA (sites 1153–1167, 1777–1791, and 2260–2274) were predicted using the PACES tool (Figs. 5C). Dual-luciferase reporter assays demonstrated that NAT10 specifically bound to HK2 at site 1# (1153–1167), as opposed to site 2# (1777–1791) or site 3# (2260–2274) in SCC9 and HSC3 cells (Figs. 5D-F). Furthermore, CHIP analysis indicated that the overexpression of NAT10 enhanced the activity of the HK2 promoter in SCC9 and HSC3 cells (Fig. 5G). Moreover, RT-qPCR results suggested that NAT10 overexpression resulted in an increase in the relative mRNA levels of HK2 in SCC9 and HSC3 cells (Fig. 5H).

Fig. 5
figure 5

NAT10 upregulated the ac4C level of HK2 in SCC9 and HSC3 cells. A, The ac4C levels of GLUT1, LDHB, and HK2 after NAT10 overexpression in SCC9 and HSC3 cells was detected using MeRIP-qPCR assay (n = 3); B, RIP assay was conducted to examine the interaction between NAT10 and HK2 in SCC9 and HSC3 cells (n = 3); C, The three ac4C sites of HK2 were predicted by the bioinformatics analysis tool: prediction of ac4C sites in mRNA; Dual-luciferase reporter assay was performed to evaluate the binding of NAT10 and HK2 in SCC9 and HSC3 cells at sites D, 1#, E, 2#, and F, 3# (n = 3); G, CHIP-qPCR was performed to analyze the HK2 promoter activity in SCC9 and HSC3 after NAT10 overexpression (n = 3); H, RT-qPCR was used to assess the HK2 mRNA level in SCC9 and HSC3 after NAT10 overexpression (n = 3). (*p < 0.05, ***p < 0.001)

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Paeonol treatment decreased tumor growth in OSCC

Finally, an in vivo OSCC tumor-bearing mouse model was established. The results showed that paeonol treatment decreased tumor size, weight, and volume compared to the control group (Figs. 6A-C). Moreover, the IHC results suggested that paeonol treatment downregulated the expression of Ki67, NAT10, and HK2 in tumor tissues (Fig. 6D). These results suggest that paeonol treatment decreases OSCC tumor growth.

Fig. 6
figure 6

Paeonol treatment decreased tumor growth in OSCC. The A, tumor size, B, volume, and C, weight in the control and paeonol groups (n = 6); D, IHC was performed to detect the Ki67, NAT10, and HK2 expression in the control and paeonol groups (n = 6). (***p < 0.001)

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Discussion

TCMs has gained recognition for its diverse applications in cancer treatment, offering potential therapeutic benefits with fewer side effects. Paeonol, a major constituent of Moutan Cortex, has shown promising anticancer effects in various malignancies through its multiple properties such as anti-inflammation and oxidation resistance [13, 14]. Despite its potential, investigations of paeonol in OSCC remain limited. In this study, paeonol at a concentration of 200 µg/mL was selected for subsequent experiments because this concentration significantly reduced the cell viability of OSCC and did not affect the viability of HOK cells. Similarly, paeonol inhibits proliferation, migration, and invasion of ovarian cancer cells at a concentration of 200 µg/mL [19]. The treatment concentration of paeonol varied greatly among different tumor cells. For example, 20–100 µg/mL paeonol treatment reduces the viability and colony formation ability of non-small cell lung cancer cells [20]. Nevertheless, in hepatocellular carcinoma, 100–500 µg/mL paeonol treatment inhibits cell viability, invasion, and migration [21].

Aberrant glycolysis is a critical mechanism in OSCC [22, 23]. Further investigation revealed that paeonol treatment inhibited glycolysis in OSCC cells. However, the effect of paeonol on OSCC glycolysis has not been previously reported. However, paeonol has been shown to inhibit glycolysis in other types of cancers such as gastric and lung cancers [24, 25]. Additionally, other TCM components have been demonstrated to be involved in suppressing OSCC by modulating glycolysis. For instance, Lin et al. demonstrated that nobiletin, a polymethoxylated flavone found in citrus, inhibited cell growth by restraining aerobic glycolysis in OSCC [26]. In addition, Li et al. [27] reported that Tanshinone IIA suppressed OSCC by reducing signaling-mediated aerobic glycolysis.

RNA epigenetic modifications such as N6-methyladenosine (m6A) and 5-methylcytosine (m5C) have been established as crucial mechanisms in the development of OSCC [28, 29]. However, another RNA modification, ac4C, has been studied less extensively in OSCC. In this study, we found that paeonol inhibited glycolysis and cell migration by suppressing NAT10-mediated ac4C modifications in OSCC. The regulatory role of paeonol in NAT10-mediated ac4C modification has not yet been explored. Consistent with our findings, NAT10 is significantly upregulated in OSCC and laryngeal squamous cell carcinoma (LSCC), and its knockdown inhibits cell proliferation, migration, and invasion [30, 31]. Additionally, NAT10 is involved in the malignant progression of other cancers, such as gastric and triple-negative breast cancers, by promoting glycolysis addiction [32, 33]. Interestingly, Mei et al. [34] reported that NAT10-mediated ac4C acetylation drives m6A modification, regulates glycolysis, and promotes osteosarcoma progression. However, the role of TCM components in disease progression via NAT10-mediated ac4C modification remains unclear. Only one study found that the Mijiao formula, a traditional herbal remedy, regulates osteoporosis via NAT10-mediated ac4C modification [35].

HK2 is a pivotal enzyme that plays a critical role in glycolysis and facilitates the first committed step of glucose metabolism [7]. A previous review suggested that targeting HK2 is a viable strategy for cancer therapy [36]. In this study, we found, for the first time, that HK2 is the downstream target of NAT10-mediated ac4C modification in OSCC. Similarly, pan-cancer analysis revealed that HK2 is highly expressed in most tumors and is related to the progression of some tumors [37]. The correlation between HK2 and ac4C modifications has not been previously reported. However, previous studies have shown that HK2 expression and activity are related to m6A modifications. Ye et al. [38] indicated that downregulation of obesity-associated protein (FTO) and alkylation repair homolog protein (ALKBH)5 enhances glycolysis in colorectal cancer by regulating insulin-like growth factor-binding protein (IGF2BP)2-mediated m6A methylation of HK2 mRNA. In addition, methyltransferase-like (METTL)3 enhances HK2 stability through YTH domain family (YTHDF)1-mediated m6A modification, thereby promoting the Warburg effect in cervical cancer [39].

In summary, this study demonstrated that paeonol inhibited glycolysis and migration in OSCC by suppressing the NAT10-mediated ac4C modification of HK2, which may offer a potential therapeutic strategy for OSCC. If validated in clinical studies in the future, paeonol could offer a novel treatment option for OSCC patients, particularly those who are resistant to conventional therapies. Furthermore, the potential for combining paeonol with existing treatments, such as chemotherapy or radiotherapy, might enhance therapeutic efficacy and improve patient outcomes. However, there are limitations to this study. For example, the tumor volume in this study was assessed using caliper measurements, and radiological imaging techniques, such as Computed Tomography (CT) or Magnetic Resonance Imaging (MRI), were not employed. Future studies could consider incorporating radiological measurements to further enhance the accuracy of tumor volume assessment. Besides, while our preclinical findings demonstrated the potential of paeonol as a therapeutic agent for OSCC, its applicability in humans requires further investigation. Currently, no clinical studies have explored the use of paeonol for OSCC treatment. Future research should focus on pharmacokinetic studies, toxicity assessments, and clinical trials to evaluate the safety and efficacy of paeonol in human patients. Additionally, exploring the combination of paeonol with existing therapies may enhance its clinical relevance and therapeutic outcomes. Furthermore, to further validate the findings of this study, future research should include additional in vivo models such as patient-derived xenograft (PDX) or genetically engineered mouse models (GEMMs), deeper mechanistic investigations using omics technologies, and combination therapy studies with standard treatments. Moreover, biomarker identification could help guide its clinical application.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Funding

The work was supported by Open Project of Shaanxi Key Laboratory of Chinese Medicine Fundamentals and New Drugs Research under grant number KF2201; Xi’an Science and Technology Bureau under grant number 23YXYJ0042.

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  1. Department of Pharmacy, Xi’an Hospital of Traditional Chinese Medicine, No.69, Fengcheng 8th Road, Weiyang District, Xi’an, 710021, China

    Kang Yang, Baosen Yue, Huan Tian, Xiao Yang & Weiying Zhang

  2. Xi’an Beilin Zhongqingyun Dental Clinic, Xi’an, 710032, China

    Lu Wang

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Contributions

All authors participated in the design and interpretation of the studies, analysis of the data, and review of the manuscript. K Y drafted the work and revised it critically for important intellectual content; B Y, H T, L W, and X Y were responsible for the acquisition, analysis, and interpretation of data; W Z made substantial contributions to the conception or design of the work. All authors have read and approved the final manuscript.

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Correspondence to Weiying Zhang.

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Yang, K., Yue, B., Tian, H. et al. Paeonol inhibits the Glycolysis in oral squamous cell carcinoma though suppressing NAT10-mediated ac4C modification. BMC Cancer 25, 629 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12885-025-14000-7

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