Aerobic glycolysis in colon cancer is repressed by naringin via the HIF1Α pathway

Aerobic glycolysis in colon cancer is repressed by naringin via the HIF1Α pathway 柚皮苷通过 HIF1A 通路抑制结肠癌的有氧糖酵解 1 equal first-author 2 equal 3 2 1 1 1 2 4 corresp equal Yanran WU, wuyanran196512@163.com Yanran WU, wuyanran196512@163.com wuyanran196512@163.com https://orcid.org/0000-0003-0816-5240 5 corresp equal Sen LI, lisentome728@hotmail.com Sen LI, lisentome728@hotmail.com lisentome728@hotmail.com https://orcid.org/0000-0003-3297-6270 南京中医药大学附属盐城中医院，中国盐城市， 224000 南京中医药大学附属盐城中医院，中国盐城市， 224000 Yancheng TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Yancheng 224000, China Yancheng TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Yancheng 224000, China 湖北中医药大学，中国武汉市， 430000 湖北中医药大学，中国武汉市， 430000 Hubei University of Chinese Medicine, Wuhan 430000, China Hubei University of Chinese Medicine, Wuhan 430000, China 南京中医药大学，中国南京市， 210033 南京中医药大学，中国南京市， 210033 Nanjing University of Chinese Medicine, Nanjing 210033, China Nanjing University of Chinese Medicine, Nanjing 210033, China 华中科技大学同济医学院附属同济医院中西医结合科，中国武汉市， 430000 华中科技大学同济医学院附属同济医院中西医结合科，中国武汉市， 430000 Department of Integrated Traditional Chinese and Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000, China Department of Integrated Traditional Chinese and Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000, China 华中科技大学同济医学院附属协和医院药学部，中国武汉市， 430000 华中科技大学同济医学院附属协和医院药学部，中国武汉市， 430000 Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000, China Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000, China Metabolic reprogramming is a common phenomenon in cancer, with aerobic glycolysis being one of its important characteristics. Hypoxia-inducible factor-1α ( HIF1Α ) is thought to play an important role in aerobic glycolysis. Meanwhile, naringin is a natural flavanone glycoside derived from grapefruits and many other citrus fruits. In this work, we identified glycolytic genes related to HIF1Α by analyzing the colon cancer database. The analysis of extracellular acidification rate and cell function verified the regulatory effects of HIF1Α overexpression on glycolysis, and the proliferation and migration of colon cancer cells. Moreover, naringin was used as an inhibitor of colon cancer cells to illustrate its effect on HIF1Α function. The results showed that the HIF1Α and enolase 2 ( ENO2 ) levels in colon cancer tissues were highly correlated, and their high expression indicated a poor prognosis for colon cancer patients. Mechanistically, HIF1Α directly binds to the DNA promoter region and upregulates the transcription of ENO2 ; ectopic expression of ENO2 increased aerobic glycolysis in colon cancer cells. Most importantly, we found that the appropriate concentration of naringin inhibited the transcriptional activity of HIF1Α , which in turn decreased aerobic glycolysis in colon cancer cells. Generally, naringin reduces glycolysis in colon cancer cells by reducing the transcriptional activity of HIF1Α and the proliferation and invasion of colon cancer cells. This study helps to elucidate the relationship between colon cancer progression and glucose metabolism, and demonstrates the efficacy of naringin in the treatment of colon cancer. 代谢重编程是癌症中一种常见的现象，而有氧糖酵解是其重要特征之一。缺氧诱导因子1亚基（ HIF1Α ）被认为在有氧糖酵解中发挥重要作用。柚皮苷是一种从葡萄柚和柑橘类水果中提取的天然的黄酮糖苷。在本研究中，我们通过分析结肠癌数据库，确定了与 HIF1Α 相关的糖酵解基因。通过进行细胞外酸化率和细胞功能的实验，证实了过表达 HIF1Α 对糖酵解以及结肠癌细胞增殖和迁移的调控作用。此外，我们用柚皮苷作为结肠癌细胞的抑制剂来阐明其对 HIF1Α 功能的影响。结果显示，结肠癌组织中 HIF1Α 和烯醇酶-2（ ENO2 ）水平高度相关，其高表达提示结肠癌患者预后较差。 HIF1Α 直接与DNA启动子区域结合，上调 ENO2 的转录；而 ENO2 的异常表达增加了结肠癌细胞的有氧糖酵解水平。最重要的是，一定浓度的柚皮苷抑制了 HIF1Α 的转录活性，从而降低了结肠癌细胞的有氧糖酵解水平。总之，柚皮苷通过降低 HIF1Α 的转录活性来减少结肠癌细胞的糖酵解以及结肠癌细胞的增殖和侵袭。本研究有助于阐明结肠癌的进展与糖代谢之间的关系，并证明柚皮苷对结肠癌的治疗效果。 结肠癌 柚皮苷 缺氧诱导因子1亚基（ HIF1Α ） 烯醇酶-2（ ENO2 ） 糖酵解 代谢重编程 Colon cancer Naringin Hypoxia inducible factor-1α ( HIF1Α ) Enolase 2 ( ENO2 ) Glycolysis Metabolic reprogramming 1 Introduction 1 The most common gastrointestinal tumors are those of the colon and rectum. More than 95% of colorectal cancer patients are over 50 years of age at diagnosis, and the five-year relative survival rate in colorectal cancer is 65% ( Miller et al., 2019 Miller et al., 2019 ). For earlier stages of colorectal cancer, surgery is still the main effective treatment. However, chemotherapy and radiotherapy are beneficial for patients with middle and advanced colon cancer ( Denlinger and Barsevick, 2009 Denlinger and Barsevick, 2009 ; Cartwright, 2012 Cartwright, 2012 ). In addition, according to the molecular characteristics of colorectal cancer, some targeted drugs, such as inhibitors of B-Raf proto-oncogene, serine/threonine kinase ( BRAF ) and Kirsten rat sarcoma viral oncogene homolog ( KRAS ), have better curative effects ( André et al., 2015 André et al., 2015 ; Dienstmann et al., 2015 Dienstmann et al., 2015 ; Taieb et al., 2017 Taieb et al., 2017 ). Despite that our understanding of colon cancer is increasing, the current treatment efficacy remains unsatisfactory. The Warburg effect is a specific means of metabolic reprogramming in cancer cells. Briefly, in an oxygen-rich environment, cancer cells still undergo aerobic glycolysis to break down glucose for energy ( Warburg et al., 1927 Warburg et al., 1927 ; Koppenol et al., 2011 Koppenol et al., 2011 ). The Warburg effect is the result of energy metabolism evolution in cancer cells to meet their substantial energy demands; thus, it is considered a hallmark of cancer ( Hsu and Sabatini, 2008 Hsu and Sabatini, 2008 ; Schwartz et al., 2017 Schwartz et al., 2017 ). Recent research has suggested that abnormally high expression and activation of multiple key kinases of the glycolytic metabolic pathway are the direct causes of the Warburg effect ( Ramzy et al., 2023 Ramzy et al., 2023 ). Phosphoglycerate kinase 1 (PGK1) is the first rate-limiting enzyme in glycolysis and is highly expressed in various cancers ( Ahmad et al., 2013 Ahmad et al., 2013 ; Hu et al., 2017 Hu et al., 2017 ). Previous research has shown that O -GlcNAcylation of PGK1 contributes to cancer progression by regulating the ratio of aerobic glycolysis to tricarboxylic acid cycle ( Ahmad et al., 2013 Ahmad et al., 2013 ). Similarly, enolase 2 ( ENO2 ) is highly expressed in lung, colon, and breast cancers and is strongly associated with poor cancer prognosis ( Soh et al., 2011 Soh et al., 2011 ; Liu et al., 2020 Liu et al., 2020 ). However, the networks in which these genes function and their contributions to tumor progression remain unclear. In recent years, a number of natural compounds have been shown to have apoptotic activity against a variety of cancers ( Rajedadram et al., 2021 Rajedadram et al., 2021 ; Sun et al., 2021 Sun et al., 2021 ; Du et al., 2022 Du et al., 2022 ). Naringin, chemically known as 4',‍5,‍7-trihydroxyflavanone 7- O -rhamnoglucoside, is a natural flavanone glycoside derived from grapefruits and many other citrus fruits ( Chen et al., 2016 Chen et al., 2016 ). It is one of the main active ingredients from the dried and immature fruit of Citrus aurantium L. ( Zhang et al., 2014 Zhang et al., 2014 ). Citrus peel extracts have been shown to reduce metallothionein activity in colon cancer cell lines, thereby inhibiting the progression of colon cancer ( Ademosun et al., 2015 Ademosun et al., 2015 ). The consumption of grapefruit has also been shown to inhibit the development of colon cancer ( Vanamala et al., 2006 Vanamala et al., 2006 ). Naringin is believed to inhibit the formation of inflammatory factors and inflammatory signaling pathways ( Habauzit et al., 2011 Habauzit et al., 2011 ; Gopinath and Sudhandiran, 2012 Gopinath and Sudhandiran, 2012 ). In addition, in vivo experiments showed that naringin improves mitochondrial dysfunction by regulating the activity of metalloproteinases in mitochondria ( Wang et al., 2015 Wang et al., 2015 ). Li et al. (2013) Li et al. (2013) have indicated that naringin inhibits the proliferation of triple-negative breast cancer by blocking the cell cycle. In addition, it has a similar inhibitory effect on bladder cancer cells ( Kim et al., 2008 Kim et al., 2008 ). However, the effect of naringin on aerobic glycolysis in colon cancer remains unexplored. In our study, we identified glycolytic genes related to hypoxia-inducible factor-‍1α ( HIF1Α ) by systematically analyzing the colon cancer database. The analyses of extracellular acidification rate and cell function verified the regulatory effects of HIF1Α overexpression on glycolysis, and the proliferation and migration of colon cancer cells. The natural compound naringin was used as an inhibitor of colon cancer cells to illustrate its effect on HIF1Α function. This study helps elucidate the relationship between colon cancer progression and glucose metabolism and shows the value of naringin in the treatment of colon cancer. 2 Materials and methods 1 2.1 Cell culture and chemical reagents 2 Colon cancer cell lines (LoVo CCL-229 TM and HCT116 CCL-247 TM ) were purchased from the American Type Culture Collection (ATCC; the Global Bioresource Center, Manassas, VA, USA). LoVo cells were cultured in F-12K medium (Life Technologies, Massachusetts, USA), and HCT116 cells were cultured in McCoy’s 5A modified medium (Life Technologies) with 10% (0.1 g/mL) fetal bovine serum (FBS; Life Technologies). The cells were cultured under 5% carbon dioxide (CO 2 ) and a constant temperature of 37 ℃. Trypsin-ethylenediamine tetraacetic acid (EDTA) solution was used to digest and passage these colon cancer cells. Naringin (HY-N0153, 99.79% purity) was purchased from MedChemExpress of China and dissolved in dimethyl sulfoxide (DMSO). 2.2 Vector construction 2 The dual luciferase reporter gene vector was used to clone the HIF1Α motif sequence into the pGL3.0 vector. Specifically, the core binding sequence ACGTGC of HIF1Α was obtained from the JASPAR website ( http://jaspar.genereg.net http://jaspar.genereg.net ), and the sequences were cloned into the pGL3.0 vector four times. 2.3 Cell transfection 2 Fresh medium was added when the cell confluence was 40%–60%. After 2 h, Neofect TM DNA transfection reagent (Neofect Biotech, Beijing, China) was used to treat the plasmid for 20 min. The plasmid:transfection reagent:Opti-MEM (Gibco, Grand Island, NY, USA) ratio was 1 µg:1 µL:200 µL. Cells were cultured for 24‍–‍48 h after they were mixed with the medium. 2.4 Quantitative real-time PCR 2 In order to prepare colon cancer cells for treatment, we first used TRIzol (TaKaRa Biomedical Technology, Kyoto, Japan) to extract total cellular RNA. A total of 2 μg messenger RNA (mRNA) was reversely transcribed into complementary DNA (cDNA) by the PrimeScript RT Master Mix (TaKaRa Biomedical Technology). Then, specific amplification primers for HIF1Α , ENO2 , and β‍-actin ( ACTB ) were designed. Quantitative real-time polymerase chain reaction (qPCR) was performed on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Massachusetts, USA). The qPCR enzyme was a high-fidelity Taq enzyme (Vazyme Biotechnology, Nanjing, China), the final reaction volume was 10 µL, and the qPCR was carried out in a 96-well plate. The primers were as follows: ACTB primers (forward 5'-TCACCAACTGGGACGACA-3', reverse 5'-ACAGCCTGGATAGCAACG-3'); HIF1Α primers (forward 5'-AGAGCAGGAAAAGGAGTC-3', reverse 5'-GCTTGAAAAAGTGAACCA-3'); and ENO2 primers (forward 5'-AAGGACAAATACGGCAAG-3', reverse 5'-CTCTGAGGCAGCAACATC-3'). 2.5 Chromatin immunoprecipitation (ChIP) 2 The prepared colon cancer cells were treated with a final concentration of 1% (volume fraction) paraformaldehyde at room temperature for 10 min. Next, glycine was added to a final concentration of 0.125 mol/L to stop the crosslinking, and the cells were collected. The cells were washed twice with cold phosphate-buffered saline (PBS) to obtain cell pellets, and then added with protease inhibitor complexes. The cell sonication parameters were as follows: 25% power, 4.5 s impact, and 9 s interval. The samples were centrifuged at 10 000 g for 10 min at 4 ℃, and 300 µL of the supernatant was used to detect the ultrasonic effect by agarose gel electrophoresis. HIF1Α antibody, ChIP dilution buffer, and protein A agarose were added to the lysate and inverted overnight at 4 ℃. After incubation overnight, 60 µL of protein A agarose/salmon sperm DNA was added to each tube, and the samples were inverted at 4 ℃ for 2 h. After 10 min, the samples were centrifuged at 700 r/min for 1 min, followed by removing the supernatant. The precipitated complex was washed sequentially with washing buffer. Subsequently, 250 µL of elution buffer was added to each tube and centrifuged to collect the supernatant. The washing step was repeated, and 20 µL of NaCl (5 mol/L) was added to each tube to decrosslink the sample at 65 ℃ overnight. After decrosslinking, 1 µL of RNase A was added to each tube and incubated at 37 ℃ for 1 h. Finally, 10 µL of 0.5 mol/L EDTA, 20 µL of 1 mol/L Tris-HCl (pH 6.5), and 2 µL of 10 mg/mL proteinase K were added to each tube, followed by the recovery of the DNA fragments. 2.6 Gene expression data analysis 2 We downloaded the colon cancer dataset (GSE14333) from the Gene Expression Omnibus (GEO) database ( https://www. https://www. ‍ncbi.‍nlm.‍nih.‍gov/geo) and obtained the original expression values for many genes. We found 28 glycolysis-related enzymes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database ( https://www. https://www. ‍genome.‍jp/kegg), analyzed the genes related to HIF1Α expression through the Morpheus website ( https://software.broadinstitute. https://software.broadinstitute. ‍org/morpheus), and presented them in a heatmap. GraphPad 5.0 (GraphPad Software, San Diego, USA) was used to evaluate the correlation between the expression value of HIF1Α and that of glycolytic genes, and then fit the correlation curve. After downloading the patient information, such as survival time and survival status, we used GraphPad 5.0 to draw the survival curve and calculate the P value. The supplementary materials and methods described detailed information on JASPAR analysis, western blot, extracellular acidification rate (ECAR), Matrigel invasion, 3-‍(4,5-dimethylthiazol-2-yl)‍-2,‍5-diphenyltetrazolium bromide (MTT) assay, and dual luciferase reporter assay. 3 Results 1 3.1 Inhibition of colon cancer cell proliferation by naringin 2 Naringin is a natural compound derived from certain citrus fruits, and is a traditional Chinese medicine mainly used to fight inflammation ( Fig. 1 Fig. 1 a). Herein, we investigated its effect on the activity of colon cancer cells. Interestingly, the results showed that 10 mmol/L naringin effectively inhibited the proliferation of these cells ( Fig. 1 Fig. 1 b). Aerobic glycolysis is one of the characteristic energy metabolism reprogramming processes of cancer cells. Thus, we next tested the effect of naringin on the aerobic glycolysis of colon cancer cells. As expected, the rate of extracellular acidification confirmed that naringin attenuated the overall level and the reserve level of aerobic glycolysis (Figs. 1 1 c and 1 1 d). The detection results of glycolytic metabolites also demonstrated that naringin reduced glucose uptake and metabolism by colon cancer cells ( Fig. 1 Fig. 1 e). These data suggested that naringin appears to be effective in colon cancer treatment. 3.2 Role of HIF1Α in the effect of naringin on colon cancer 2 HIF1Α , as a known regulator of aerobic glycolysis, has been reported to be involved in the progression of various cancers. We speculated that naringin has a certain effect on the activity of HIF1Α . Thus, we cloned the DNA sequence containing the HIF1Α binding site into the pGL3.0-Basic vector ( Fig. 2 Fig. 2 a). The dual luciferase assay showed that naringin inhibited the transcriptional activity of HIF1Α ( Fig. 2 Fig. 2 b). To confirm the biological functions of HIF1Α in colon cancer cells, we carried out our experiments using stable colon cancer cell lines transfected with the mock vector, the HIF1Α construct, sh-Scb, or sh- HIF1Α . The ECAR value confirmed that HIF1Α knockdown or overexpression had significant effects on the glycolytic flux ( Fig. 2 Fig. 2 c). In addition, the ectopic expression or silencing of HIF1Α increased or decreased the levels of adenosine triphosphate (ATP), glucose, and lactic acid, which all supported the hypothesis that HIF1Α promoted glycolysis ( Fig. 2 Fig. 2 c). Correspondingly, the key indicators of aerobic glycolysis, i.‍e., glucose uptake, ATP production, and lactic acid production content, also changed with the expression of HIF1Α ( Fig. 2 Fig. 2 d). As expected, the overexpression of HIF1Α significantly increased the proliferation and invasion of colon cancer cells, while the knockdown of HIF1Α effectively inhibited these activities (Fig. S1). The above evidence indicated that HIF1Α improved the aerobic glycolytic capacity of colon cells, which in turn promoted the progression of colon cancer. Subsequently, we sought to explore the role of HIF1Α in the effect of naringin on colon cancer cells. Correspondingly, naringin also counteracted the proliferation of colon cancer cells caused by HIF1Α ( Fig. 2 Fig. 2 e). Matrigel invasion experiments confirmed this conclusion ( Fig. 2 Fig. 2 f). This means that HIF1Α is necessary for naringin to exert its inhibitory effect on colon cancer progression. 3.3 Co-expression of glycolytic gene ENO2 and HIF1Α in colon cancer 2 HIF1Α is a recognized key transcription factor related to glycolysis. To explore the possible mechanism by which HIF1Α regulates aerobic glycolysis in colon cancer, we sought to identify HIF1Α‍ -regulated genes associated with aerobic glycolysis. We established a dataset of validated genes corresponding to glycolysis derived from the KEGG pathway database. Then, we analyzed the human colorectal cancer dataset (GSE14333) by utilizing R2 (Genomics Analysis and Visualization Platform), and generated five candidate genes positively correlated with HIF1Α : ENO2 , phosphoglycerate mutase 1 ( PGAM1 ), bisphosphoglycerate mutase ( BPGM ), pyruvate kinase ( PKM ), and adenosine diphosphate (ADP)‍-dependent glucokinase ( ADPGK ) ( R > 0.3, P < 0.05; Fig. S2). By comparing the correlation between HIF1Α and the expression levels of candidate genes, we identified ENO2 as the target gene of glucose metabolism regulated by HIF1Α ( Fig. 3 Fig. 3 a). In addition, Kaplan-Meier survival analysis indicated that a higher expression of both HIF1Α and ENO2 was significantly associated with poor patient survival ( Fig. 3 Fig. 3 b). DepMap website analysis ( https://depmap. https://depmap. ‍org/portal/interactive) also showed that the expression of HIF1Α and ENO2 in different colon cancer cells was positively correlated ( Fig. 3 Fig. 3 c). These data showed that the expression of the glucose metabolism-related gene ENO2 is positively correlated with HIF1Α , and both can promote cancer. 3.4 Transactivation of ENO2 in colon cancer mediated by HIF1Α 2 We next investigated the potential effects and regulatory mechanism of HIF1Α on the expression of ENO2 . The mining of JASPAR revealed that as a key transcription factor, HIF1Α had a 6-nt enrichment peak (ACGTGC) within the promoter of ENO2 ( Fig. 4 Fig. 4 a). Immunofluorescence staining assays revealed that HIF1Α was localized in the nucleus and was not affected by naringin ( Fig. 4 Fig. 4 b). Dual luciferase assays indicated that the overexpression or silencing of HIF1Α enhanced or reduced the activity of the ENO2 promoter in HCT116 and LoVo cells, respectively ( Fig. 4 Fig. 4 c). Consistently, chromatin co-immunoprecipitation demonstrated that transfection with HIF1Α or sh- HIF1Α increased or decreased HIF1Α binding to the promoter of ENO2 , respectively ( Fig. 4 Fig. 4 d). qPCR and western blot analyses further confirmed that HIF1Α promoted the expression of ENO2 at the mRNA and protein levels in HCT116 and LoVo cells (Figs. 4 4 e– 4 4 g). Correspondingly, the expression of other genes ( PGAM1 , BPGM , PKM , and ADPGK ) did not change significantly (Fig. S3a). To further confirm that the HIF1Α regulation of ENO2 expression was based on transcriptional regulation, we deleted the binding site of HIF1Α in the ENO2 promoter region. The results proved that the ENO2 promoter region lacking a binding site was no longer regulated by HIF1Α (Fig. S3b). Subsequently, we overexpressed HIF1Α and knocked down ENO2 to further confirm the necessity of ENO2 for HIF1Α in colon cancer aerobic glycolysis. The results showed that the knockdown of ENO2 partially counteracted the increase in aerobic glycolytic capacity caused by HIF1Α in colon cancer cells (Fig. S4). We believe that other aerobic glycolytic genes exist in colon cancer that are regulated by HIF1Α , and therefore, knocking down ENO2 does not completely suppress the effect of HIF1Α , albeit it is a highly critical player. In addition, we found that naringin did not affect the expression of HIF1Α , but attenuated the transcriptional regulation of ENO2 by HIF1Α ( Figs. 4 Figs. 4 h‍–‍4j, S3c, and S3d). These data suggested that the transcription factor HIF1Α increased colon cancer progression by facilitating the expression of ENO2 at the transcriptional level. In general, naringin at an appropriate concentration was taken up by colon cancer cells, thereby reducing the transcriptional activity of the transcription factor HIF1Α . In this way, this molecule blocked aerobic glycolysis in colon cancer cells. Finally, the proliferation and invasion of colon cancer cells were inhibited by naringin. 4 Discussion 1 Metabolic reprogramming occurs in cancer cells as an adaptation to the substantial energy consumption due to their rapid proliferation. Compared with normal cells, cancer cells obtain ATP faster through glycolysis instead of oxidative phosphorylation in the mitochondria. Even in an environment with sufficient oxygen supply, cancer cells still preferentially utilize aerobic glycolysis, which is known as the Warburg effect ( Warburg et al., 1927 Warburg et al., 1927 ; vander Heiden et al., 2009; Yu et al., 2017 Yu et al., 2017 ). In this study, we found that abnormally elevated levels of aerobic glycolysis existed in colon cancer cells, and this effect was positively correlated with the proliferation and invasion of these cells. Accordingly, the inhibition of aerobic glycolysis effectively reduced the growth of these cells, which constitutes an important finding to understand the metabolic disorders of colon cancer cells. ENO2 is one of the three enolase isoenzymes in mammals and is mainly expressed in mature neurons and cells of neuronal origin ( Kim and Dang, 2005 Kim and Dang, 2005 ; Andreou et al., 2021 Andreou et al., 2021 ). Recent studies have found that ENO2 is highly expressed in various tumor tissues, such as prostate cancer and Ewing sarcoma, which is closely related to the prognosis of cancer patients ( Kim et al., 2017 Kim et al., 2017 ; Jiang et al., 2022 Jiang et al., 2022 ). As a key enzyme in glycolysis, ENO2 promotes the conversion of β‍-glycerophosphate to dihydroxyacetone phosphate. Therefore, the enzymatic activity of ENO2 is crucial for glucose metabolism in cancer cells. Zheng et al. (2020) Zheng et al. (2020) demonstrated that histone deacetylase 3 (HDAC3)-induced deacetylation of ENO2 enhances the activity of ENO2 in glycolysis. A recent study found that the BRAF V600E mutation, which was closely associated with colon cancer progression, exhibited a significant dependence on ENO2 , and mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signalings were also positively correlated with the expression level of ENO2 ( Yukimoto et al., 2021 Yukimoto et al., 2021 ). Our findings suggested that HIF1Α upregulated the ENO2 expression levels and enhanced the aerobic glycolytic capacity of colon cancer cells. Although we had no direct evidence that the enzymatic activity of each ENO2 molecule was altered, the effect of ENO2 on aerobic glycolysis was enhanced. ENO2 not only plays an enzymatic activity in glycolysis, but also it has been shown to be associated with the activation of other intracellular signaling pathways and the mutation of oncogenes. Unfortunately, limited by the research direction and funding support, we did not further deepen this direction of research in this paper. This should be the task of follow-up studies. Naringin, as a natural compound, has been found to regulate various intracellular signaling pathways and physiological processes. Current research suggested that naringin mainly affects the process of inflammation ( Habauzit et al., 2011 Habauzit et al., 2011 ; Gopinath and Sudhandiran, 2012 Gopinath and Sudhandiran, 2012 ). Appropriate concentrations of naringin solutions inhibit nuclear factor-‍κB (NF‍-‍κB) nuclear translocation and reduce inflammation ( Ansari et al., 2022 Ansari et al., 2022 ). This molecule can also suppress inflammation by reducing the production of inflammation-related factors ( Habauzit et al., 2011 Habauzit et al., 2011 ; Luo et al., 2012 Luo et al., 2012 ). Experiments have shown that naringin inhibits the liver 3-hydroxy-3-methyl CoA (HMG-CoA) reductase activity of low-density lipoprotein receptor (LDLR) knockout mice, thereby reducing the plasma lipid concentration of mice ( Luo et al., 2012 Luo et al., 2012 ). However, it is not yet known how naringin affects glycolysis. Our data proved that naringin reduces the transcriptional activity of HIF1Α in colon cancer cells, thereby downregulating the expression of ENO2 , a key gene for glycolysis. Overexpression of HIF1Α counteracted this inhibitory effect. 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This work was supported by the Fund of Hubei Provincial Health Commission (No. ZY2021M080), the Medical Research Project of Jiangsu Provincial Health Commission (No. Z2021068), the Yancheng Medical Science and Technology Development Plan Project (No. YK2021004), and the Young Scientific and Technological Talents Support Project by Jiangsu Association for Science and Technology (No. TJ-2022-097), China. 10.1631/jzus.B2200221 1673-1581（2023）03-0221-11 潘广涛,张平,陈爱莹等.柚皮苷通过 HIF1A 通路抑制结肠癌的有氧糖酵解[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(03):221-231. 潘广涛,张平,陈爱莹等.柚皮苷通过HIF1A通路抑制结肠癌的有氧糖酵解[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(03):221-231. This work was supported by the Fund of Hubei Provincial Health Commission (No. ZY2021M080), the Medical Research Project of Jiangsu Provincial Health Commission (No. Z2021068), the Yancheng Medical Science and Technology Development Plan Project (No. YK2021004), and the Young Scientific and Technological Talents Support Project by Jiangsu Association for Science and Technology (No. TJ-2022-097), China. 2022-04-17 2022-09-06 2023-03-15 2023-03-14 2023-03-08 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2023 浙江大学出版社 Guangtao PAN, Ping ZHANG, Aiying CHEN, Yu DENG, Zhen ZHANG, Han LU, Aoxun ZHU, Cong ZHOU, Yanran WU, and Sen LI declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines Supplementary information 1 Figs. S1‍‒S4; Materials and methods 浙江大学学报（英文版）（B辑：生物医学和生物技术） 3 24