Paeonol represses A549 cell glycolytic reprogramming and proliferation by decreasing m6A modification of Acyl-CoA dehydrogenase

Lixin Zhang; Lihua Wu; Xiangrui Zhu; Jian Mei; Yingli Chen

doi:10.4103/cjop.CJOP-D-22-00166

ORIGINAL ARTICLE

Year : 2023 | Volume : 66 | Issue : 4 | Page : 248-256

Paeonol represses A549 cell glycolytic reprogramming and proliferation by decreasing m6A modification of Acyl-CoA dehydrogenase

Lixin Zhang¹, Lihua Wu², Xiangrui Zhu³, Jian Mei³, Yingli Chen¹
¹ Central Laboratory of Harbin Medical University, Daqing, China; Department of Immunology, College of Medical Laboratory Science and Technology, Harbin Medical University, Daqing, China
² Department of Geriatrics, Daqing Oilfield General Hospital, Daqing, China
³ Department of Immunology, College of Medical Laboratory Science and Technology, Harbin Medical University, Daqing, China

Date of Submission	18-Jan-2023
Date of Decision	09-Apr-2023
Date of Acceptance	17-Apr-2023
Date of Web Publication	06-Jul-2023

Correspondence Address:
Dr. Yingli Chen
College of Medical Laboratory Science and Technology, Harbin Medical University, Xinyang Road, Daqing, Heilongjiang 163319
China

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cjop.CJOP-D-22-00166

Abstract

Aberrant glycolytic reprogramming is involved in lung cancer progression by promoting the proliferation of non-small cell lung cancer cells. Paeonol, as a traditional Chinese medicine, plays a critical role in multiple cancer cell proliferation and inflammation. Acyl-CoA dehydrogenase (ACADM) is involved in the development of metabolic diseases. N6-methyladenosine (m6A) modification is important for the regulation of messenger RNA stability, splicing, and translation. Here, we investigated whether paeonol regulates the proliferation and glycolytic reprogramming via ACADM with m6A modification in A549 cells (human non-small cell lung cancer cells). Cell counting kit 8, 5-Bromo-2-deoxyuridine, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, flow cytometry analysis, western blotting and seahorse XFe24 extracellular flux analyzer assays showed that paeonol had a significant inhibitory effect against A549 cell proliferation and glycolysis. Mechanistically, ACADM was a functional target of paeonol. We also showed that the m6A reader YTH domain containing 1 plays an important role in m6A-modified ACADM expression, which is negatively regulated by paeonol, and is involved in A549 cell proliferation and glycolytic reprogramming. These results indicated the central function of paeonol in regulating A549 cell glycolytic reprogramming and proliferation via m6A modification of ACADM.

Keywords: Acyl-CoA dehydrogenase, glycolytic reprogramming, N6-methyladenosine, paeonol, proliferation

How to cite this article:
Zhang L, Wu L, Zhu X, Mei J, Chen Y. Paeonol represses A549 cell glycolytic reprogramming and proliferation by decreasing m6A modification of Acyl-CoA dehydrogenase. Chin J Physiol 2023;66:248-56

How to cite this URL:
Zhang L, Wu L, Zhu X, Mei J, Chen Y. Paeonol represses A549 cell glycolytic reprogramming and proliferation by decreasing m6A modification of Acyl-CoA dehydrogenase. Chin J Physiol [serial online] 2023 [cited 2023 Dec 4];66:248-56. Available from: https://www.cjphysiology.org/text.asp?2023/66/4/248/380730

Lixin Zhang and Lihua Wu contributed equally to this work.

Introduction

Paeonol (2-hydroxy-4-methoxyacetophenone) belongs to the traditional Chinese medicine Ranunculaceae, and was first isolated from the root bark of Paeonia moutan.^[1],[2] It is functionally diverse and involves a wide range of pathophysiological processes such as anticonvulsant, neuroprotection, oxidative stress, antitumor and cytoprotective autophagy.^[3],[4],[5] It has been reported that paeonol plays a vital protective role in various cancers, including ovarian cancer, bladder cancer, breast cancer and colorectal cancer. For example, paeonol inhibits proliferation and induces apoptosis of bladder cancer cells in vitro and in vivo by regulating the phosphorylation expression of phosphatidylinositol 3-kinase and protein kinase B (PKB/AKT).^[6] Paeonol may activate CXCL4/CXCR3-B-mediated growth inhibitory signals to increase BTB and CNC homology 1 and decrease nuclear factor E2-related factor 2 expressions and exert anticancer and apoptotic effects in breast cancer.^[7] Notably, a recent study also illustrated that the proliferation and motility of A549 cells can be suppressed by paeonol by disrupting signal transducer and activator of transcription 3 (STAT3)/nuclear factor kappa B (NF-κB) subunit signaling.^[8] Nonetheless, whether paeonol participates in the regulation of A549 cell (human non-small cell lung cancer cell) proliferation through other signaling pathways remains to be elucidated.

Glycolytic reprogramming, known as the “Warburg effect,” is the leading factor in the proliferation of cancer cells.^[9],[10] The main metabolic process of cancer cells is the transformation of glucose into lactic acid in the presence of oxygen.^[11] During the accumulation of lactic acid, the extracellular matrix becomes erratic under an acidic environment, which accelerates the proliferation of cancer cells.^[12] Accumulated evidence reveals that glycolysis is regulated by a variety of oncogenes and signaling pathways, which unveils the key role of glycolysis in the proliferation of A549 cells. For example, mutation of the Kirsten rat sarcoma oncogene increases A549 cell proliferation via induction of enhanced glycolytic reprogramming.^[13] Glycolysis promotes cisplatin resistance in lung adenocarcinoma A549 cells via the AKT-mTOR-c-Myc signaling pathway.^[14] However, the antiproliferative activity of paeonol in A549 cells, as well as its effect on glycolytic reprogramming has not been investigated.

Acyl-CoA dehydrogenase (ACADM) is the key protein responsible for the breakdown of fatty acids in the mitochondria. Abnormal expression of ACADM has been found in cardiovascular, metabolic and liver diseases, and suppression of ACADM promoted hepatocellular carcinoma progression.^{[15],[16],[17]} Most studies focus on the role of ACADM in fatty acid metabolism. However, to date, the role of ACADM in glycolytic reprogramming, especially in lung cancer has not been thoroughly studied.

N6-methyladenosine (m6A), a common epitranscriptomic modification, is mainly dependent on the regulation of m6A “writer” proteins (METTL3, METTL14), “eraser” proteins (FTO, ALKBH5) and “reader” proteins (YTHDC1-3, YTHDF1-3).^[18],[19] Studies have demonstrated that aberrant m6A modification of messenger RNA (mRNA) plays an important role in the development of multiple diseases, including lung cancer. For example, the m6A demethylase alkB homolog 5 (ALKBH5) promotes lung cancer cell proliferation by destabilizing the insulin like growth factor 2 mRNA binding proteins target genes cyclin dependent kinase inhibitor 1A or TIMP metallopeptidase inhibitor 3.^[20] Although the roles of m6A modification in lung cancer have been elucidated, whether paeonol is involved in regulating the m6A modification of ACADM in A549 cells remains poorly understood.

In this study, we hypothesized that paeonol could inhibit proliferation in A549 cells by ACADM-mediated glycolytic reprogramming, which provides a guide for further studies on targeted therapy for lung cancer.

Materials and Methods

Cell culture

The A549 cells used in the experiment were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). The cells were cultured in RPMI-1640 (RPMI-1640, Grand Island, NY, USA) containing 10% fetal bovine serum (Clark, USA) and 1% penicillin streptomycin (Beyotime Biotechnology, Shanghai, China) at 37°C, 5% CO₂, and 100% relative humidity. A549 cells were treated with paeonol from Sigma (Sigma, USA), and a Dimethyl sulfoxide vehicle was used to increase the paeonol solubility.

Small interfering RNAs construction and transfection

Small interfering RNAs (siRNAs) were designed and synthesized by GenePharma (Shanghai, China) and negative control siRNA (siNC) was used as an NC. The sequences were as follows:

Negative control (NC): sense, 5′-UUCUCCGAACGUGU CACGUTT-3′, antisense, 5′-ACGUGACACGUUCGG AGAATT-3′.

Homo-ACADM-1: sense, 5′-GCAGGGUCCUGAGAA GUAUTT-3′, antisense, 5′-AUACUUCUCAGGACC UGCTT-3′.

Homo-ACADM-2: sense, 5′-CCAUUGAUGUGUGC UUAUUTT-3′, antisense, 5′-AAUAAGCACACAUCAA UGGTT-3′.

Homo-ACADM-3: sense, 5′-GCUCUGAUGUAGCU GGUAUTT-3′, antisense, 5′-AUACCAGCUACAUCAG AGCTT-3′.

Homo-YTHDC1-1: sense, 5′-GGAGAAAGAUGGAG AACUUTT-3′, antisense, 5′-AAGUUCUCCAUCUUUC UCCTT-3′.

Homo-YTHDC1-2: sense, 5′-GCUCUGCAUCAGA GUCAUATT-3′, antisense, 5′-UAUGACUCUGAUGCA GAGCTT-3′.

Homo-YTHDC1-3: sense, 5′-GCAAGGAGUGUU AUCUUAATT-3′, antisense, 5′-UUAAGAUAACACUCC UUGCTT-3′.

Transfections in A549 cells were performed using X-tremeGene siRNA transfection reagents following the manufacturer's instructions. A549 cells were transfected with 2 μg siRNAs. The transfection reagents were removed after 4–6 h, and the cells were switched to 5% serum-containing medium and cultured for another 24 h.

RNA immunoprecipitation and Me-RIP

The RNA immunoprecipitation and Me-RIP assays were performed by using an RIP kit (Bes5101, BersinBio, Guangzhou, China) following the manufacturer's instructions. In brief, 1 × 10⁷ A549 cells were lysed with RIP lysis buffer. After removing DNA, 20 μL of protein A/G bead-conjugated anti-YTHDC1 antibodies or anti-m6A antibody (4 μg, A-1801, Epigentek Group Inc., Farmingdale, NY, USA) was used. After extracting the RNA from IP and input samples, the total RNA was converted to cDNA and subjected to quantitative reverse transcription polymerase chain reaction (PCR) assays.

Cell counting kit 8 assay

A549 cells were separately seeded in 96-well plates (5000 cells/well), and then treated with different concentrations of paeonol. After 24 h of incubation at 37°C, 10 μL of Cell counting kit 8 (CCK8) reagent (C0037, Beyotime Biotechnology, Shanghai, China) was added to each well and incubated for 2 h at 37°C, and the absorbance was measured by a spectrophotometer at a wavelength of 450 nm.

5-Bromo-2-deoxyuridine assay

The 5-Bromo-2-deoxyuridine (BrdU) assay was performed using a BrdU proliferation kit (#2750, Millipore, Billerica, MA, USA) following the manufacturer's instructions. In brief, A549 cells were separately seeded in 96-well plates (5000 cells/well), and then treated with different agents in 5% FBS DMEM with 10 ng/mL BrdU labeling solution. Next, the cells were incubated with anti-BrdU monoclonal antibody for 1 h and subsequently conjugated with goat anti-mouse IgG. Finally, after adding 100 μL of stop solution, the absorbance was measured by a spectrophotometer at a wavelength of 450 nm.

Cell cycle analysis

Cell cycle progression was detected using the Cycle TEST PLUS DNA reagent kit according to the instruction manual. Briefly, A549 cells were collected and fixed with 70% ethanol for overnight at 4°C. Then, the cells were incubated with propidium iodide at 37°C in the dark. DNA fluorescence was measured, and flow cytometry was performed using a BD FACSCalibur flow cytometer (Bedford, MA, USA).

Western blot analysis

Protein samples were extracted from A549 cells using lysates (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and 2 mM PMSF) on ice and then centrifuged at 13500 rpm for 15 min at 4 °C. The protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., Berkeley, CA, USA). Protein samples (30 μg) were separated on 10% SDS-PAGE gels, transferred onto nitrocellulose membranes, and subsequently blocked with 5% nonfat milk at room temperature for 1 h. Primary antibodies against cyclin A (PB0515, Boster, 1:500), cyclin E (BA0774, Boster, 1:500), ACADM (ab110296, Abcam, 1:1000), METTL3 (ab195352, Abcam, 1:1000), FTO (bs-7056R, Bioss, 1:500), YTHDC1 (29441-1-AP, Proteintech, 1:500), YTHDF1 (ab290734, Abcam, 1:1000) and β-actin (sc-47778, Santa Cruz, 1:6000). Blots were then sequentially incubated with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescent reagent imaging.

EdU assay

EdU assays were carried out using a BeyoClick EdU cell proliferation kit with Alexa Fluor 488 following the manufacturer's instructions (C0075S, Beyotime, Shanghai, China). In brief, A549 cells exposed to 50 μmol/L 5-ethynyl-2'-deoxyuridine for an additional 2 h at 37°C. After incubation for 2 h, cells were fixed with 4% formaldehyde for 10 min and treated with 0.3% Triton X-100 for 10 min at room temperature. Next, the cells were reacted with Click Additive Solution for 30 min. Subsequently, cells were stained with Hoechst 33342, and visualized under a confocal microscope at a wavelength of 530 nm.

Glycolysis assays

Glycolysis was analyzed using a Seahorse XF24 extracellular flux analyzer (Seahorse Biosciences, North Billerica, MA, USA) according to the manufacturer's instructions. Briefly, 10⁴ A549 cells were seeded in Seahorse XF 96-well cell culture microplate, and then the cells were treated with different concentrations of paeonol. After 24 h of incubation in 37°C, the cells were glucose-starved in XF assay medium in a CO₂-free XF prep station at 37°C for 60 min, and then glucose (10 mM), oligomycin (1 μM) and 2-Deoxy-D-Glucose (2-DG, 50 mM) were added to each well at the indicated time points.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8 (La Jolla, CA, USA). Data are expressed as the mean ± standard error of the mean. Statistical comparisons were performed with Student's t-test or one-way analysis of variance followed by Dunnett tests where appropriate. A value of P < 0.05 was considered significant.

Results

Paeonol depressed A549 cell proliferation and glycolytic reprogramming

First, the structure of paeonol is shown in [Figure 1]. Then, the CCK8 assay was used to identify the effects of paeonol on A549 cells, various concentrations of paeonol (0, 10, 20, 50, 100, 200, 400 and 600 μM) were used to treat A549 cells. As shown in [Figure 2]a, the results suggested that paeonol can inhibit the viability of A549 cells [Figure 2]a. The IC₅₀ value of A549 cells was 182.3 ± 11.79 μM, so 100 and 200 μM paeonol were choose for subsequent experiments. BrdU assay showed that paeonol can inhibit A549 cell proliferation [Figure 2]b. Next, the effect of paeonol on the cell cycle was determined using flow cytometry analysis. At the higher concentrations (100 and 200 μM), paeonol suppressed the increase in the percentage of cells in S + G2/M phases in A549 cells [Figure 3]a. Meanwhile, the EdU incorporation assay showed that the percentage of proliferating cells was increased in A549 cells, which was decreased by paeonol application [Figure 3]b. Moreover, western blotting assays demonstrated that the expression of the cell cycle regulatory proteins Cyclin A and Cyclin E were blocked by paeonol in A549 cells [Figure 3]c and [Figure 3]d. The above results showed that paeonol had profound antiproliferation efficacy in A549 cells.

Figure 1: Chemical structure of paeonol.

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Figure 2: Paeonol inhibited A549 cell proliferative. (a) A549 cells were treated with paeonol (0, 10, 20, 50, 100, 200, 400, and 600 μM) for 24 h, and cell viability was determined by CCK8 assay (n = 6). (b) BrdU assay showed that the cell proliferation decreased with paeonol treatment (n = 6). All tests were performed at least three times, and the values are presented as the mean ± SEM. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared with DMSO (0 μM paeonol). CON: Control, CCK8: Cell counting kit 8, DMSO: Dimethyl sulfoxide, SEM: Standard error of the mean.

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Figure 3: Effects of paeonol on A549 cell cycle progression and aerobic glycolysis. (a) Cell cycle analysis by flow cytometry indicated that paeonol inhibited the acceleration of the cell cycle in A549 cells. (b) Fluorescent EdU assay proved that A549 cell proliferation was blocked by paeonol. Scale bar = 50 μm. (c and d) Cyclin A and Cyclin E expression were increased in A549 cells, and these changes were reversed by paeonol (n = 5). (e) ECAR of cultured A549 cells measured by Seahorse XFe24. (f) Paeonol decreased glycolysis, glycolytic reserve and nonglycolytic acidification of A549 cells (n = 3). All tests were performed at least three times, and the values are presented as the mean ± SEM. ^#P < 0.05, ^##P < 0.01 compared with DMSO (0 μM paeonol). Pae: Paeonol, ECAR: Extracellular acidification rates, SEM: Standard error of the mean.

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It is well known that continuously uncontrolled proliferation of cancer cells is usually accompanied by disturbed glucose metabolism.^[21] To investigate the role of paeonol in regulating aerobic glycolysis, we measured the aerobic glycolysis capability of A549 cells treated with paeonol by using a Seahorse XFe24 extracellular flux analyzer, in which the extracellular acidification rate (ECAR) was monitored in real time to indicate glycolysis [Figure 3]e. The results showed that paeonol treatment decreased glycolysis, the glycolytic reserve and the glycolytic capacity of A549 cells [Figure 3]f.

Inhibition of the glycolytic reprogramming pathway prevented A549 cell proliferation

To further explore the effect of aerobic glycolysis on the induction of A549 cell proliferation, we simultaneously treated cells with paeonol and the glycolysis inhibitor 2-DG. Flow cytometry analysis demonstrated that 2-DG alone abrogated the proliferative process of A549 cells, and after inhibition of glycolysis, the inhibitory effect of paeonol on proliferation was not further enhanced [Figure 4]a. The same results were also observed by CCK8 assay [Figure 4]b. In addition, we detected two key regulatory molecules in the cell cycle, Cyclin A and Cyclin E by western blotting, and decreased expression of Cyclin A and Cyclin E was observed in A549 cells after treatment with paeonol and 2-DG [Figure 4]c and [Figure 4]d. The above results showed that paeonol regulated A549 cell proliferation and cell cycle progression partly via glycolytic reprogramming.

Figure 4: Impact of hyperactive aerobic glycolysis on A549 cell proliferation. (a) Cellcycle analysis indicated that 2-DG and paeonol inhibited A549 cell progression into G2/M + S phase. (b) The proliferation rate of A549 cells following treatment of 2-DG and paeonol was studied by CCK8 assay (n = 8). (c and d) Effects of 2-DG and paeonol on the expression of Cyclin A and Cyclin E (n = 3). All tests were performed at least three times, and the values are presented as the mean ± SEM. ^##P < 0.01 compared with DMSO (0 μM paeonol). CCK8: Cell counting kit 8, DMSO: Dimethyl sulfoxide, SEM: Standard error of the mean.

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ACADM was implicated in paeonol regulated glycolytic reprogramming and proliferation in A549 cells

Previous studies have reported that ACADM is a central modulator of fatty acid metabolism. However, whether ACADM is involved in regulating glycolytic reprogramming remains unclear. Therefore, we first measured the effect of paeonol on ACADM expression in A549 cells. The results showed that the expression of ACADM was decreased in A549 cells treated with paeonol [Figure 5]a.

Figure 5: Effect of ACADM on A549 cell proliferation and aerobic glycolysis. (a) Paeonol decreased the expression of ACADM in A549 cells (n = 3). (b) The expression level of ACADM in siRNA and negative control (siNC)-transfected cells (n = 6). (c) Cellcycle analysis indicated that siACADM inhibited A549 cell progression into G2/M + S phase. (d and e) siACADM decreased the expression of Cyclin A and Cyclin E (n = 10). (f) Fluorescent EdU assay proved that A549 cell proliferation was blocked by siACADM. Scale bar = 50 μm. (g) The proliferation rate of A549 cells following treatment of siACADM was studied by CCK8 assay (n = 8). (h) ECAR of cultured A549 cells measured by Seahorse XFe24. (i) siACADM decreased glycolysis, glycolytic reserve and nonglycolytic acidification of A549 cells (n = 3). All tests were performed at least three times, and the values are presented as the mean ± SEM. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared with DMSO (0 μM paeonol) or NC. NC: negative control, siRNA: small interfering RNA, ACADM: Acyl-CoA dehydrogenase, ECAR: extracellular acidification rate, SEM: Standard error of the mean.

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We next investigated the involvement of ACADM in the glycolytic reprogramming and proliferation of A549 cells. ACADM protein expression was knocked down by transfecting ACADM siRNA into A549 cells [Figure 5]b. Flow cytometry analysis demonstrated that siACADM inhibited the number of A549 cells entering the G2/M + S phase and arrested the cells in the G1 phase [Figure 5]c. The western blotting assay showed decreased expression of Cyclin A and Cyclin E in siACADM-treated A549 cells [Figure 5]d and [Figure 5]e. Similar results were also found in the EdU incorporation and CCK8 assays [Figure 5]f and [Figure 5]g. Furthermore, we measured the aerobic glycolysis capability of A549 cells treated with siACADM by using a Seahorse XFe24 extracellular flux analyzer, in which the ECAR was monitored in real time to indicate glycolysis [Figure 5]h and [Figure 5]i. These results demonstrated that ACADM plays a prominent role in paeonol regulated glycolytic reprogramming and proliferation.

Paeonol inhibited m6A modification of ACADM via m6A reader YTH domain containing 1

Recent studies have shown that m6A modification is an important mediator in lung cancer development by regulating diverse transcripts.^{[22],[23],[24]} Therefore, we attempted to explore whether paeonol is involved in regulating the m6A modification of ACADM mRNA in A549 cells. By using the SRAMP prediction server, we found five high confidence m6A modification sites in ACADM mRNA [Figure 6]a. Subsequently, we examined the effect of paeonol on several m6A modification enzymes, including methyltransferase 3 (METTL3), fat mass and obesity-associated gene (FTO), YTH domain containing 1 (YTHDC1) and YTH m6A RNA binding protein 3 (YTHDF3), using western blotting. The results suggested that paeonol significantly inhibited the expression of YTHDC1 [Figure 6]b, [Figure 6]c, [Figure 6]d, [Figure 6]e. Therefore, we chose YTHDC1 for subsequent analyses. Next, we transfected YTHDC1 siRNA into A549 cells to silence YTHDC1, and si-1 had the most obvious effects [Figure 6]f. Western blotting showed decreased expression of ACADM after transfection with YTHDC1 siRNA in A549 cells [Figure 6]g. In addition, MeRIP-PCR results showed that paeonol and YTHDC1 siRNA decreased the m6A level of ACADM mRNA [Figure 6]h and [Figure 6]i. To test the interaction between ACADM mRNA and YTHDC1, we performed a RIP assay, and the results suggested that YTHDC1 was significantly enriched in ACADM mRNA to form an m6A modification complex, which was reversed by paeonol [Figure 6]j. In conclusion, these results showed that paeonol inhibits the expression of ACADM by YTHDC1 in an m6A-dependent manner, providing a novel view for the dysregulation of ACADM in lung cancer development.

Figure 6: Paeonol is involved in regulating the m6A modification of ACADM mRNA in A549 cells. (a) SRAMP predicted m6A sites of ACADM mRNA. (b-e) The expression of paeonol on METTL3, FTO, YTHDC1 and YTHDF3 was detected by western blotting (n = 6). (f) The expression level of YTHDC1 in siRNA and negative control (siNC)-transfected cells (n = 5). (g) siYTHDC1 decreased the expression of ACADM (n = 6). (h) Paeonol inhibited the m6A levels of ACADM mRNA using MeRIP (n = 3). (i) siYTHDC1 inhibited the m6A levels of ACADM mRNA using Me-RIP (n = 3). (j) RIP assay was performed to detect ACADM mRNA interacts with the YTHDC1 protein (n = 3). All tests were performed at least three times, and the values are presented as the mean ± SEM. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared with DMSO (0 μM paeonol) or NC. NC: Negative control, siRNA: small interfering RNA, DMSO: Dimethyl sulfoxide, SEM: Standard error of the mean, ACADM: Acyl-CoA dehydrogenase.

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Discussion

Paeonol, as a flavonoid derivative, is one of the main active ingredients of Paeonia suffruticosa. Paeonol has been reported to possess a wide range of pharmacological properties for the amelioration of inflammation and oxidative stress, and offers beneficial roles antidiabetic, anti-cardiovascular disease and anticancer effects.^[25],[26] Investigations have identified a broad-spectrum antitumor effect of paeonol, which is widely used in the treatment of a range of cancers, including gastric, pancreatic, breast, skin and ovarian cancers.^[27],[28] Liu et al. presented results suggesting that paeonol protects against colorectal cancer, including the induction of cell cycle arrest and initiation of apoptosis by suppressing the Wnt/β-catenin pathway.^[29] Zhang et al. reported that paeonol potently inhibited the migration and invasion of nonsmall cell carcinoma cells by inhibiting the transcriptional activity of the NF-κB and STAT3 pathways.^[8] Consistent with the above findings, our present study found that paeonol concentration-dependently reduced the proliferation of A549 cells. Moreover, paeonol inhibited the cell cycle progression of A549 cells by blocking cyclin A and cyclin E. Thus, it was supposed that paeonol exerted antiproliferative effects by inhibiting the expression of ACADM via YTHDC1 in an m6A-dependent manner.

Increased aerobic lactate production (glycolytic reprogramming) in tumor cells has been recognized as a “hallmark of cancer.”^[30] In pancreatic cancer cells, a negative correlation between ubiquitin like with plant homeodomain and ring finger domains 1 (UHRF1) and sirtuin 4 negatively regulates aerobic glycolysis to decrease tumor cell proliferation and growth.^[31] Notably, the inhibition of glycolysis by the nonmetabolizable glucose analog 2-DG can convert tumor cells susceptible or sensitive to immunotherapy, thus creating new strategies for immunotherapy.^[32] In addition, several studies have shown that the Wnt/β-catenin, NF-κB and STAT3 pathways are also involved in glycolytic reprogramming, suggesting that paeonol may regulate glycolytic reprogramming in A549 cells.^{[33],[34],[35]} To our knowledge, the current data showed for the first time that paeonol triggered the inhibition of glycolytic reprogramming by m6A modification of ACADM because the glycolytic phenotype can efficiently produce macromolecules and energy for cancer cell proliferation. Therefore, the anticancer effects of paeonol might be due to ACADM-mediated glycolytic inhibition.

ACADM is a key regulator of mitochondrial fatty acid β-oxidation that can maintain metabolic balance physiologically.^[36] ACADM deficiency was previously shown to be associated with acute lung injury and hepatocellular carcinoma via fatty acid metabolism.^[17],[37] Recent studies showed that overexpression of ACADM could reduce hypoxia-induced pulmonary arterial smooth muscle cell proliferation.^[16] Contrary to the notion, we found that ACADM expression was upregulated in A549 cells, ACDAM knockdown can inhibit A549 cell proliferation. One possible reason for these distinct results could be due to the different origins, species and genetic background of cells used in these experiments. Another possibility could be the different culture and treatment conditions of the cells. To investigate whether ACADM may participate in the A549 cell proliferation and glycolytic reprogramming regulated by paeonol, we first showed that ACADM expression was inhibited in A549 cells treated with paeonol. Additionally, ACADM knockdown slowed A549 cell proliferation by inhibiting glycolytic reprogramming. These results indicate that ACADM may act as a vital factor in cell proliferation and is a potential therapeutic target for lung cancer.

Many studies have revealed that m6A modification is involved in multiple biological functions, such as cell proliferation, migration and tumorigenesis.^[38],[39] The m6A “reader” proteins can recognize m6A sites and play multiple biological functions in A549 cells. Among these, YTH domain containing 2 suppressed the proliferation and migration ability of A549 cells.^[40] YTH m6A RNA binding protein 2 recognized the METTL3-induced m6A methylation of XIST to mediate miR-1915-3p expression and suppressed A549 cell migration, invasion and epithelial-mesenchymal transition.^[41] In this study, we revealed that another m6A “reader”, YTHDC1, might be regulated by paeonol in A549 cells. YTHDC1 siRNA significantly decreased the expression of ACADM, which inhibited A549 cell glycolytic reprogramming and proliferation. Thus, our data highlight the effect of paeonol on m6A modification and first elucidate the role of YTHDC1 modified ACADM mRNA in A549 cells.

Conclusion

Our study demonstrated that the potential anticancer agent paeonol induces A549 cell growth suppression by inhibiting glycolytic reprogramming via the m6A “reader” YTHDC1-mediated ACADM, presenting substantial evidence for the potential clinical application of paeonol [Figure 7]. Therefore, the exploration of paeonol derivatives to improve pharmacological and biological activities has important theoretical research and practical value in anticancer strategies.

Figure 7: A schematic diagram to illustrate the hypothetical model. Paeonol decreased the m6A modification of ACADM mRNA by repressing m6A “reader” YTHDC1 and subsequently reduced ACADM expression, and finally inhibiting A549 cell glycolytic reprogramming and proliferation. ACADM: Acyl-CoA dehydrogenase, mRNA: Messenger RNA, YTHDC1: YTH domain containing 1.

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Data availability

The authors declare that all data supporting the findings of this study are available within the paper and any raw data can be obtained from the corresponding author upon request.

Financial support and sponsorship

This work was supported by the National Natural Science Foundation of Heilongjiang Province (grant number LH2020H028 to LZ), The Fundamental Research Funds for the Provincial Universities (grant number JFWLD201901 to LZ), and College Students Innovation Program of Heilongjiang Province (grant number 202110226230 to LZ).

Conflicts of interest

There are no conflicts of interest.

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