Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Department of Biotechnology , School of Life Sciences and Food Engineering , Hanshan Normal University , Chaozhou , 521041 , Guangdong, China , hstc.edu.cn
Background: Proinflammatory cytokines TNFα and IL1β drive esophageal squamous cell carcinoma (ESCC) cell proliferation. However, the underlying molecular mechanism and potential therapeutic interventions to target this inflammatory signaling remain unclear.
Methods: Plasminogen activator urokinase (PLAU) expression was analyzed using the public database (GEO and iProX) and molecular experiments (qRT-PCR and Western blotting). The DNA-binding activity of nuclear factor κB (NFκB) at the promoter of PLAU was analyzed using several online servers (AnimalTFDB, JASPAR, PROMO, Cistrome, and UCSC) and confirmed through ChIP-qPCR. The role of PLAU in ESCC proliferation was investigated through PLAU overexpression experiments, GO annotation, CCK8 assay, and 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay.
Results: PLAU expression was significantly higher in ESCC tissues compared to normal tissues and in ESCC cells compared to immortalized esophageal epithelial cells. Treatment with TNFα and IL1β induced NFκB binding at the PLAU promoter in ESCC cells, leading to increased PLAU expression. Conversely, treatment with BAY11-7082, an NFκB inhibitor, significantly blocked this upregulation. Overexpression of PLAU promoted ESCC cell proliferation. Thus, our findings demonstrate that the TNFα/IL1β-NFκB-PLAU axis promotes ESCC cell proliferation. Moreover, EGCG inhibited NFκB binding to the PLAU promoter, thereby preventing PLAU upregulation in TNFα/IL1β-treated ESCC cells and inhibiting ESCC cell proliferation induced by PLAU overexpression.
Conclusion: EGCG effectively blocks the inflammatory signaling TNFα/IL1β-NFκB-PLAU, thereby inhibiting ESCC cell proliferation. Our study provides new insights into blocking the pro-proliferative role of inflammation in ESCC and highlights EGCG as a potential therapeutic agent.
1. Introduction
Esophageal cancer (EC) is a prevalent malignant tumor of the digestive tract. Although multiple treatment options such as surgery, chemotherapy, radiotherapy, and targeted therapies are available, EC patients still face significant challenges. Post-treatment recurrence, metastasis, development of resistance to therapy, chemotherapeutic drug toxicity, and the limited availability of targets for EC treatment contribute to a poor prognosis, with a five-year survival rate of 15%–25% [1, 2]. In 2020, there were approximately 604,000 new cases and 544,000 deaths of EC worldwide, ranking it seventh and sixth in incidence and mortality, respectively [3]. In China, there were about 324,000 new cases and 301,000 deaths from EC in the same year [4], accounting for more than half of the worldwide. The predominant subtype of EC is esophageal squamous cell carcinoma (ESCC), followed by esophageal adenocarcinoma (EAC) [5]. ESCC primarily occurs in Africa, Southeast Asia, and China, whereas EAC is more common in Western countries. Over ninety percent of EC cases in China are ESCC [5].
ESCC often begins with chronic inflammation and eventually progresses to a lethal cancer [6]. An esophageal endoscopic biopsy screening study (1191 cases) showed that in the Chaoshan region of China, where the population is at high risk of ESCC, 68.9% of the population suffers from chronic inflammation [7]. Inflammation may be a crucial driver of ESCC, leading to esophageal lesions and DNA damage [8]. ESCC microenvironment harbors cancer cells, infiltrating inflammatory cells, proinflammatory cytokines, and other components [9–11]. In advanced tumors, many proinflammatory cytokines such as TNFα and IL1β promote tumor growth, while cytokines that inhibit tumor growth are often absent [9–11]. TNFα and IL1β lead to the constitutive activation of pro-cancer signals, notably nuclear factor κB (NFκB), which is often hyperactivated in ESCC cells [12, 13]. NFκB is a ubiquitous inducible DNA-binding transcription factor (TF). The NF-κB family consists of five members: NFκB1 (p50), NFκB2 (p52), p65/RelA, c-Rel, and RelB, forming homodimers or heterodimers that directly regulate gene expression [14, 15]. Activated NFκB promotes the expression of target genes like MYC and MMP9, driving cancer cell proliferation, invasion, and metastasis [14, 15]. Moreover, activated NFκB transcriptionally upregulates procancer cytokines such as TNFα and IL1β, thereby establishing a molecular bridge linking inflammation to cancer [14, 15]. In ESCC, TNFα or IL1β activates NFκB, leading to the upregulation of target genes SLC52A3 and DGKα, which promote ESCC cell proliferation and colony formation [16, 17].
Given the limited efficacy of ESCC treatment, there is a pressing need to develop novel therapeutic approaches. Epigallocatechin gallate (EGCG) is a polyphenolic compound found in green tea and oolong tea and has well-documented anti-inflammatory and anti-tumorigenic effects [18, 19]. Importantly, tea-derived EGCG is safe for humans. The cytotoxic effects of EGCG on normal myoepithelial cells were much less than those on cancer cells, potentially reducing side effects [20, 21]. A daily intake of 800 mg of EGCG (equivalent to 8 to 16 cups of green tea) for 4 weeks is beneficial for humans [22]. These findings suggest the practical application of EGCG as a dietary supplement or adjunct therapy for ESCC treatment. In ESCC, EGCG’s ability to inhibit inflammation is particularly relevant, as chronic inflammation plays a significant role in the progression of this disease. For instance, EGCG may exert anticancer effects by reducing proinflammatory markers that were triggered by extracellular vesicles in triple-negative breast cancer cells [23]. EGCG inhibits inflammation in LPS-induced RAW 264.7 macrophages by blocking NFκB activation and reducing NO and ROS production [24]. EGCG also blocked TNFα-induced NFκB activation in human synovial fibroblasts [25]. Thus, EGCG may be beneficial in treating ESCC, especially in patients with abnormal NFκB activation. Since NFκB-activated target genes (e.g., SLC52A3 and DGKα) promote ESCC cell proliferation, the potential of EGCG to target this pathway is particularly noteworthy. However, the specific NFκB target genes regulated by EGCG remain poorly understood. Identifying these target genes will provide valuable insights into the therapeutic potential of EGCG and pave the way for developing more effective treatments for ESCC.
The plasminogen activator urokinase (PLAU) gene encodes for plasminogen activator, urokinase (uPA), a secret serine protease responsible for converting plasminogen to plasmin [26]. High expression of PLAU has been reported in several tumors, including head and neck squamous cell carcinoma (HNSCC) [27, 28], gastric adenocarcinoma [29], breast cancers [30], cervical cancer [31], and ESCC [32]. In these tumors, PLAU, which has oncogenic properties, contributes to cancer cell proliferation, migration, invasion, and epithelial–mesenchymal transition. However, the regulatory mechanisms underlying PLAU’s high expression in cancers remain unclear. In this study, we found that TNFα and IL1β induce the upregulation of PLAU through NFκB activation, thereby promoting cell proliferation in ESCC. Conversely, EGCG inhibits both the upregulation of PLAU and its associated promotion of cell proliferation. These findings deepen the understanding of how proinflammatory cytokines contribute to ESCC progression and may inform the development of novel therapeutic strategies for this lethal disease.
2. Materials and Methods
2.1. Bioinformatics Analysis
PLAU acts as an immune-related hub gene with a high diagnostic value for ESCC in integrated bioinformatics analysis [33, 34]. To better understand the role of PLAU in ESCC, we analyzed its expression using data from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). We first established criteria for data selection: (a) studies involving Homo sapiens ESCC, (b) datasets including tumor and paired adjacent nontumor tissues, and (c) datasets with at least 30 samples. Subsequently, we obtained microarray datasets GSE20347, GSE23400, GSE38129, GSE44021, GSE53625, GSE161533, GSE29001, GSE45670, GSE70409, and GSE75241. The basic information for these 10 GEO datasets is provided in Supporting information (Table S1). In addition, proteomic data from 124 paired ESCC tissues and adjacent nontumor tissues were obtained from Professor Xu Liyan at Shantou University Medical College for analyzing PLAU protein expression [35]. These data are stored in the iProX database with accession number IPX0002501000 (https://www.iprox.cn/). The online servers AnimalTFDB (https://bioinfo.life.hust.edu.cn/AnimalTFDB/#!/), JASPAR (https://jaspar.genereg.net/analysis), PROMO (https://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3), Cistrome (https://cistrome.org/db/#/), and UCSC Genome Browser (https://genome.ucsc.edu/) were used for analyzing the potential TFs that regulate PLAU gene. UALCAN and GEPIA2 databases were used for analyzing the co-expressed genes with PLAU in ESCC. DAVID database was used for the functional annotation.
2.2. Cell Culture
Five human ESCC cell lines, KYSE30, KYSE150, KYSE510, SHEEC, and TE3, were generously provided by Dr. Xu Liyan at Shantou University Medical College. SHEE, an immortalized esophageal epithelial cell line, was purchased from MINGZHOU Bio (Ningbo, China). The KYSE series cells and TE3 cells previously described in Xu Liyan’s studies [35, 36] were maintained in RPMI1640 medium (Biosharp Life Sciences, Beijing, China) supplemented with 10% FBS (TransGen, Beijing, China). SHEEC described in Xu Liyan’s study [37] was maintained in DMEM/F12 medium (Biosharp Life Sciences, Beijing, China) supplemented with 10% FBS (TransGen, Beijing, China). All cells were cultured at 37°C with 5% CO2. For activation of NFκB, cells seeded at 6-well plates were treated with 15, 30, or 60 ng/mL TNFα (PeproTech, 300-01A, USA) or 10, 20, or 40 ng/mL IL1β (Beyotime, P5898, Shanghai, China) dissolved in serum-free RPMI1640 medium for 1 h. For inhibition of NFκB, cells were treated with 50 μM BAY11-7082 (Beyotime, SF0011, Shanghai, China) for 30 min followed by TNFα or IL1β treatment. For EGCG treatment, cells were treated with 400 μM EGCG (Yilong, Chaozhou, China) for 48 h followed by treatment with or without TNFα or IL1β. Cells without treatment were used as controls.
2.3. qRT-PCR Assay
The qRT-PCR assay was used to detect gene expression levels. The total RNA of cultured ESCC and immortalized cells was extracted using Trizol reagent (Beyotime, R0016, Shanghai, China) following the manufacturer’s protocol. Specifically, cells were harvested by trypsinization and lysed in Trizol reagent, and the aqueous phase was separated to isolate total RNA. The extracted RNA was then dissolved in RNA storage solution (Thermo Fisher Scientific, AM7001, USA) and stored at −80°C. RNA concentration and purity were assessed using a Biophotometer (METASH, B-500, Shanghai, China). The A260/A280 values of all RNA samples were confirmed to be between 1.8 and 2.0. The integrity of the RNA was evaluated using the Agilent 2100 Bioanalyzer, and the RNA integrity number (RIN) was over 7 to ensure the high quality of the RNA samples. For complementary DNA (cDNA) synthesis, 500 ng total RNA was first treated with a gDNA digester to remove potential DNA contamination and then reversely transcribed into cDNA by using Hifair III first Strand cDNA Synthesis SuperMix (Yeasen, 11141ES60, Shanghai, China). The reverse transcription reaction was performed under the following conditions: 25°C for 2 min, 55°C for 15 min, and 85°C for 5 min. The synthesized cDNA was subjected to a LightCycler96 Real-Time PCR System (Roche, Beijing, China) with a 20 μL reaction mixture constituting 2 μL of cDNA, 10 μL of 2X SYBR Green Master Mix (Yeasen, 11203ES08, Shanghai, China), 1 μL gene-specific primers (Tianyihuiyuan, Guangzhou, China; listed in Supporting information Table S2), and 7 μL sterile, DNase/RNase-free ultrapure water. The qRT-PCR reaction conditions: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 20 s. Melt curve analysis was performed to confirm amplicon specificity, involving a temperature ramp from 95°C for 10 s, 65°C for 60 s, followed by a final increase to 97°C for 1 s. The dissolution curve was unimodal. There was no Cq value for the amplification of the No Template Control (NTC). Gene-specific primers were designed with amplicon lengths of approximately 100 bp (Table S2), confirmed using the NCBI Primer-BLAST program. The qRT-PCR efficiencies were 96%–110%, slope −3.415 ∼ −3.145, and the R2 was approximately 0.990, indicating high amplification efficiency. The gene expression levels were normalized to GAPDH using the 2−ΔΔct method. The Δct values were calculated by subtracting the GAPDH Cq value from the target gene Cq value. The ΔΔct was determined by comparing the Δct of the experimental group to a control group, allowing for relative quantification of gene expression. All PCR experiments were conducted with four biological replicates, and each group underwent two technical replicates to ensure reproducibility.
2.4. Western Blotting
Cells were lysed using ice-cold RIPA lysis buffer (Beyotime, P0013B, Shanghai, China) supplemented with a protease inhibitor (1:100, Beyotime, P1005, Shanghai, China). The lysates were incubated on ice for 30 min and centrifuged at 12,000 rpm for 15 min at 4°C to collect the supernatant. Protein concentrations were quantified using the Detergent Compatible Bradford Protein Assay Kit (Beyotime, P0006C, Shanghai, China) following the manufacturer’s instructions. Equal amounts of protein samples were separated by SDS-PAGE and then transferred to PVDF membranes (Millipore, Burlington, MA, USA) using a wet transfer method at 60 V for 90 min. The membranes were blocked with 5% skim milk in TBST (20 mM Tris, 150 mM NaCl, and 0.1% Tween) for 1 h at room temperature. Following blocking, the membranes were incubated with primary antibodies: PLAU Rabbit Polyclonal Antibody (1:500; Boster, A04352-1, Wuhan, China), NFκB p65 Rabbit Polyclonal Antibody (1:1000; Abcam, ab16502, Shanghai, China), and Actin Mouse Monoclonal Antibody (1:1000; Beyotime, AF2811, Shanghai, China) for 1 h at room temperature. After primary antibody incubation, the membranes were washed three times with TBST for 5 min each to remove unbound antibodies, followed by incubation with a horseradish peroxidase (HRP)–conjugated secondary antibody (dilution 1:5000; Beyotime, Shanghai, China) for 1 h at room temperature. The membranes were washed again with TBST, and protein bands were visualized using an enhanced chemiluminescence substrate (Beyotime, P0018FS, Shanghai, China). Images of the protein bands were captured using the FlourChem HD2 Imaging System (ProteinSimple, California, USA). Western blotting data were analyzed by quantifying the band intensities using ImageJ2X software, with target protein expression levels normalized to Actin for comparative accuracy across samples.
ChIP assays were performed as previously described. For this study, cells were cultured in 10 cm dishes and treated with 30 ng/mL TNFα or 20 ng/mL IL1β for 1 h. After treatment, the cells were fixed with 4% formaldehyde (Sigma, 252549, Shanghai, China) for 10 min to cross-link proteins to DNA, followed by quenching with glycine at the final concentration of 125 mM. Following fixation, nuclear lysate was harvested, and chromatin was fragmented through sonication to obtain DNA fragments of approximately 200–500 bp. For the ChIP assay, the sonicated chromatin was incubated overnight with 2–3 μg ChIP-grade antibodies, i.e., anti-NFκB p65 (Abcam, ab16502, Shanghai, China), Anti-Histone H3 (acetyl K27) antibody (Abcam, ab4729, Shanghai, China), or Rabbit IgG (Beyotime Biotechnology, A7016) and protein A/G magnetic beads (MCE, Shanghai, China). DNA-protein complexes were separated using a magnetic field. Immunoprecipitated DNA was purified using a DNA purification kit (Axygen, Corning Life Sciences, Wujiang, China) and subsequently analyzed by qPCR assay. The results were normalized to the input DNA. The primer sequences for ChIP-qPCR are shown in Table S3. All PCR experiments were three biological replicates, and each group underwent two technical replicates. The fold enrichment of antibody-ChIP DNA to the IgG-ChIP DNA was calculated as we previously described [38]. In brief, the Ct of antibody- or IgG-ChIP DNA was normalized to that of the input DNA using the formula:
(1)
2.6. Gene Overexpression and Cell Proliferation Assay
PLAU (NM_002658) overexpression was conducted using the plasmid EX-F0073-M98 (GeneCopoeia, Guangzhou, China). The vector pEZ-M98 was used as a control. Cell proliferation was measured using the cell counting kit-8 (CCK-8) reagent (Beyotime, C0039, Shanghai, China) and 5-ethynyl-2′-deoxyuridine (EdU) reagent (Beyotime, C0075S, Shanghai, China) according to the manufacturer’s instructions. Briefly, for the CCK-8 assay, 2.0 × 103 cells seeded in 96-well plates were incubated with CCK-8 solution at 37°C for 3 h. The absorbance was measured at 450 nm with a microplate reader (Thermo Fisher Scientific, Shanghai, China). For the EdU incorporation, 2.0 × 105 cells were seeded in 12-well plates and washed with PBS, followed by the addition of fresh medium containing 10 μM EdU solution at 37°C for 2 h, and then fixed in 4% paraformaldehyde at room temperature for 15 min. After incubation with Click Solution and Hoechst 33342 (1000x) at room temperature for 30 min and 10 min, positive cells were observed under a fluorescent microscope (Olympus, IX73, Japan; magnification, × 200). The numbers of EdU-positive and Hoechst 33342-positive cells were calculated from six images of each group. Each experiment was performed with three biological replicates.
2.7. Statistical Analysis
All data were analyzed using the Prism software Version 9 (GraphPad Software Inc., San Diego, CA, USA) and shown as the mean ± standard deviation (SD). The statistical differences were analyzed using Student’s t-test. p < 0.05 ( ∗); p < 0.01 ( ∗∗).
3. Results
3.1. High Expression of PLAU in ESCC Cells and Tissues
PLAU as a hub immune-related gene with high diagnostic value for ESCC has been identified in recent studies according to integrated bioinformatics analysis [33, 34]. Indeed, the expression of PLAU mRNA increased significantly in ESCC tissues compared to the adjacent tissues in 10 independent cohorts archived in the GEO database (p < 0.01; Figure 1(a)). We verified the expression of PLAU mRNA in five human ESCC cell lines (KYSE30, KYSE150, KYSE510, TE-3, SHEEC) and one human normal esophageal epithelial cell (SHEE). The qRT-PCR assay results showed significantly higher expression of PLAU mRNA in the five ESCC cells than that in SHEE cells (p < 0.01; Figure 1(b)). The proteomic data from iProX database showed that the PLAU protein is also upregulated in ESCC tissues compared to the adjacent tissues (p < 0.01; Figure 1(c)). These observations show that the expression of PLAU is increased in ESCC, suggesting a potential role in the development of ESCC.
High expression of PLAU in ESCC tissues and cells. (a) PLAU mRNA expression in ESCC and paired adjacent nontumor tissues. The expression levels of PLAU mRNA were evaluated using data from 10 GEO databases (sample information detailed in Table S1). T indicates cancerous tissues and N indicates adjacent nontumor tissues. (b) Relative expression of PLAU mRNA in normal esophageal epithelial cells and ESCC cells. Relative PLAU mRNA expression was performed by qRT-PCR assay. Data are represented as means ± SD from three biological replicates. (c) Protein levels of PLAU in normal and ESCC tissues. Proteomic data obtained from Professor Xu Liyan, stored in the iProX database (IPX0002501000). Statistical differences were analyzed using Student′s t-test. ∗p < 0.05; ∗∗p < 0.01.
High expression of PLAU in ESCC tissues and cells. (a) PLAU mRNA expression in ESCC and paired adjacent nontumor tissues. The expression levels of PLAU mRNA were evaluated using data from 10 GEO databases (sample information detailed in Table S1). T indicates cancerous tissues and N indicates adjacent nontumor tissues. (b) Relative expression of PLAU mRNA in normal esophageal epithelial cells and ESCC cells. Relative PLAU mRNA expression was performed by qRT-PCR assay. Data are represented as means ± SD from three biological replicates. (c) Protein levels of PLAU in normal and ESCC tissues. Proteomic data obtained from Professor Xu Liyan, stored in the iProX database (IPX0002501000). Statistical differences were analyzed using Student′s t-test. ∗p < 0.05; ∗∗p < 0.01.
High expression of PLAU in ESCC tissues and cells. (a) PLAU mRNA expression in ESCC and paired adjacent nontumor tissues. The expression levels of PLAU mRNA were evaluated using data from 10 GEO databases (sample information detailed in Table S1). T indicates cancerous tissues and N indicates adjacent nontumor tissues. (b) Relative expression of PLAU mRNA in normal esophageal epithelial cells and ESCC cells. Relative PLAU mRNA expression was performed by qRT-PCR assay. Data are represented as means ± SD from three biological replicates. (c) Protein levels of PLAU in normal and ESCC tissues. Proteomic data obtained from Professor Xu Liyan, stored in the iProX database (IPX0002501000). Statistical differences were analyzed using Student′s t-test. ∗p < 0.05; ∗∗p < 0.01.
3.2. NFκB Upregulated the Expression of PLAU in TNFα-Treated ESCC Cells
To understand the regulatory mechanism of PLAU high expression in ESCC, we predicted the potential TFs that may regulate PLAU expression by using bioinformatics tools. Four online servers, AnimalTFDB, JASPAR, PROMO, and Cistrome, showed 513, 127, 75, and 77 potential TFs, respectively, to regulate PLAU expression. The common TFs identified were RELA, TP53, GATA1, YY1, and JUN (Figure S1A). We further explored the ChIP-seq clusters for the 5 TFs on the UCSC Genome Browser. The promoter region of PLAU only markedly covers the RELA- and JUN-bound clusters (Figure S1B). The RELA-bound cluster (chr10:75668809–75669242) was detected in seven TNFα-treated lymphoblastoids while the JUN-bound cluster (chr10:75668730–75669117) was detected in the interferon alpha-treated K-562 cells, untreated HUVEC, and HeLa-S3. The scores (out of 1000) of RELA- and JUN-bound clusters are 978 and 533, respectively. Moreover, the RELA-bound cluster contains a canonical NFKB1 motif (Figure S1B). The RELA-bound cluster is also enriched with H3K27Ac, an epigenetic marker positively correlated with transcriptional activation [39]. This enrichment was confirmed by ChIP-qPCR, indicating that the RELA-bound cluster is a crucial regulatory region at TNFα-treated KYSE510 cells (Figure S1C). TNFα is highly expressed in ESCC (Figure S2), consistent with elevated serum TNFα in patients with ESCC [11] who have a poor prognosis (Figure S3A). Based on the above findings, we speculate that NFκB likely regulates PLAU expression upon stimulation of the cytokine.
To investigate the effect of increased TNFα on the PLAU expression in ESCC, we confirm the RELA-bound clusters in TNFα-treated ESCC cells by the ChIP-qPCR assay. According to the genomic loci of a classic NFKB1 motif within the RELA-bound cluster, we inspected the upstream and downstream regions of the motif, respectively. NFκB/RELA binding to the promoter of the PLAU gene was first detected in TNFα-treated KYSE30 and subsequently confirmed in TNFα-treated KYSE150, KYSE50, SHEEC, and TE3 (Figures 2(a) and 2(b)). To see whether NFκB regulates the expression of PLAU, we activated NFκB using TNFα and blocked it using Bay11-7082 in KYSE30 cells. Cells without treatment were used as controls. The results showed that Bay blocked the TNFα-induced expression of PLAU mRNA in KYSE30 cells (Figures 2(c) and 2(d)). The expression patterns of PLAU mRNA were consistent in the KYSE150, KYSE510, SHEEC, and TE3 cells (Figures 2(c) and 2(d)). Moreover, Bay also inhibited the PLAU protein expression in KYSE30 and KYSE150 (Figures 2(e) and 2(f)). Based on these findings, we proposed that PLAU expression is mediated by NFκB in TNFα-treated ESCC cells.
Upregulation of PLAU by NFκB in TNFα-treated ESCC cells. (a, b) Binding of NFκB at the PLAU promoter in TNFα-treated ESCC cells. ChIP-qPCR assay was performed to detect the binding of NFκB at the PLAU promoter. Detection regions are located upstream (a) and downstream (b) of an NFκB1 classical motif in the PLAU promoter. (c, d) Upregulation of PLAU mRNA by NFκB in TNFα-treated ESCC cells. (c) PLAU mRNA expression was enhanced in TNFα-treated ESCC cells. (d) The TNFα-induced upregulation of PLAU mRNA was repressed by the NFκB inhibitor Bay11-7082 (Bay). PLAU mRNA expression was detected by qRT-PCR assay. (e, f) Upregulation of PLAU protein by NFκB in TNFα-treated ESCC cells. Western blot analysis (e) and semi-quantitative statistical analyses (f) show PLAU protein expression in KYSE30 and KYSE150 cells under different treatments. Actin was used as an internal control. The data are presented as means ± SD from three biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗p < 0.05; ∗∗p < 0.01.
Upregulation of PLAU by NFκB in TNFα-treated ESCC cells. (a, b) Binding of NFκB at the PLAU promoter in TNFα-treated ESCC cells. ChIP-qPCR assay was performed to detect the binding of NFκB at the PLAU promoter. Detection regions are located upstream (a) and downstream (b) of an NFκB1 classical motif in the PLAU promoter. (c, d) Upregulation of PLAU mRNA by NFκB in TNFα-treated ESCC cells. (c) PLAU mRNA expression was enhanced in TNFα-treated ESCC cells. (d) The TNFα-induced upregulation of PLAU mRNA was repressed by the NFκB inhibitor Bay11-7082 (Bay). PLAU mRNA expression was detected by qRT-PCR assay. (e, f) Upregulation of PLAU protein by NFκB in TNFα-treated ESCC cells. Western blot analysis (e) and semi-quantitative statistical analyses (f) show PLAU protein expression in KYSE30 and KYSE150 cells under different treatments. Actin was used as an internal control. The data are presented as means ± SD from three biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗p < 0.05; ∗∗p < 0.01.
Upregulation of PLAU by NFκB in TNFα-treated ESCC cells. (a, b) Binding of NFκB at the PLAU promoter in TNFα-treated ESCC cells. ChIP-qPCR assay was performed to detect the binding of NFκB at the PLAU promoter. Detection regions are located upstream (a) and downstream (b) of an NFκB1 classical motif in the PLAU promoter. (c, d) Upregulation of PLAU mRNA by NFκB in TNFα-treated ESCC cells. (c) PLAU mRNA expression was enhanced in TNFα-treated ESCC cells. (d) The TNFα-induced upregulation of PLAU mRNA was repressed by the NFκB inhibitor Bay11-7082 (Bay). PLAU mRNA expression was detected by qRT-PCR assay. (e, f) Upregulation of PLAU protein by NFκB in TNFα-treated ESCC cells. Western blot analysis (e) and semi-quantitative statistical analyses (f) show PLAU protein expression in KYSE30 and KYSE150 cells under different treatments. Actin was used as an internal control. The data are presented as means ± SD from three biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗p < 0.05; ∗∗p < 0.01.
Upregulation of PLAU by NFκB in TNFα-treated ESCC cells. (a, b) Binding of NFκB at the PLAU promoter in TNFα-treated ESCC cells. ChIP-qPCR assay was performed to detect the binding of NFκB at the PLAU promoter. Detection regions are located upstream (a) and downstream (b) of an NFκB1 classical motif in the PLAU promoter. (c, d) Upregulation of PLAU mRNA by NFκB in TNFα-treated ESCC cells. (c) PLAU mRNA expression was enhanced in TNFα-treated ESCC cells. (d) The TNFα-induced upregulation of PLAU mRNA was repressed by the NFκB inhibitor Bay11-7082 (Bay). PLAU mRNA expression was detected by qRT-PCR assay. (e, f) Upregulation of PLAU protein by NFκB in TNFα-treated ESCC cells. Western blot analysis (e) and semi-quantitative statistical analyses (f) show PLAU protein expression in KYSE30 and KYSE150 cells under different treatments. Actin was used as an internal control. The data are presented as means ± SD from three biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗p < 0.05; ∗∗p < 0.01.
Upregulation of PLAU by NFκB in TNFα-treated ESCC cells. (a, b) Binding of NFκB at the PLAU promoter in TNFα-treated ESCC cells. ChIP-qPCR assay was performed to detect the binding of NFκB at the PLAU promoter. Detection regions are located upstream (a) and downstream (b) of an NFκB1 classical motif in the PLAU promoter. (c, d) Upregulation of PLAU mRNA by NFκB in TNFα-treated ESCC cells. (c) PLAU mRNA expression was enhanced in TNFα-treated ESCC cells. (d) The TNFα-induced upregulation of PLAU mRNA was repressed by the NFκB inhibitor Bay11-7082 (Bay). PLAU mRNA expression was detected by qRT-PCR assay. (e, f) Upregulation of PLAU protein by NFκB in TNFα-treated ESCC cells. Western blot analysis (e) and semi-quantitative statistical analyses (f) show PLAU protein expression in KYSE30 and KYSE150 cells under different treatments. Actin was used as an internal control. The data are presented as means ± SD from three biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗p < 0.05; ∗∗p < 0.01.
Upregulation of PLAU by NFκB in TNFα-treated ESCC cells. (a, b) Binding of NFκB at the PLAU promoter in TNFα-treated ESCC cells. ChIP-qPCR assay was performed to detect the binding of NFκB at the PLAU promoter. Detection regions are located upstream (a) and downstream (b) of an NFκB1 classical motif in the PLAU promoter. (c, d) Upregulation of PLAU mRNA by NFκB in TNFα-treated ESCC cells. (c) PLAU mRNA expression was enhanced in TNFα-treated ESCC cells. (d) The TNFα-induced upregulation of PLAU mRNA was repressed by the NFκB inhibitor Bay11-7082 (Bay). PLAU mRNA expression was detected by qRT-PCR assay. (e, f) Upregulation of PLAU protein by NFκB in TNFα-treated ESCC cells. Western blot analysis (e) and semi-quantitative statistical analyses (f) show PLAU protein expression in KYSE30 and KYSE150 cells under different treatments. Actin was used as an internal control. The data are presented as means ± SD from three biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗p < 0.05; ∗∗p < 0.01.
3.3. NFκB Upregulated the Expression of PLAU in IL1β-Treated ESCC Cells
IL1β is also a crucial cancer-related proinflammatory cytokine increased in ESCC (Figure S2) [11]. ESCC patients with elevated IL1β levels have a poor prognosis (Figure S3B). To detect whether this cytokine could increase PLAU expression dependent on the NFκB signal, we performed ChIP and qPCR and found that NFκB/RELA again bound at the promoter of PLAU gene in IL1β-treated KYSE30, KYSE150, KYSE50, SHEEC, and TE3 cells (Figures 3(a) and 3(b)). IL1β induced the expression of PLAU mRNA in the five ESCC cell lines (Figure 3(c)). Furthermore, Bay treatment inhibited the increase of PLAU mRNA and protein levels induced by IL1β treatment in ESCC cells (Figures 3(d), 3(e), 3(f), and 3(g)). Therefore, NFκB upregulates PLAU expression in TNFα/IL1β-treated ESCC cells. Consistently, PLAU mRNA expression was found to be positively correlated with NFκB/RELA in ESCC (Figure S4).
Upregulation of PLAU by NFκB in IL1β-treated ESCC cells. (a-b) Binding of NFκB at the PLAU promoter in IL1β-treated ESCC cells. ChIP-qPCR assay was performed to detect the binding of NFκB at the PLAU promoter. Detection regions are located upstream (a) and downstream (b) of a canonical NFκB1 motif at the PLAU promoter. (c–e) Upregulation of PLAU mRNA by NFκB in IL1β-treated ESCC cells. (c) PLAU mRNA expression was induced in IL1β-treated ESCC cells. (d-e) The induction of PLAU mRNA by IL1β at 20 ng/mL (d) and 40 ng/mL IL1β (e) was blocked by the NFκB inhibitor Bay11-7082 (Bay). qRT-PCR assay was performed to detect PLAU mRNA expression. (f-g) Upregulation of PLAU protein by NFκB in IL1β-treated ESCC cells. (f) Western blot assays were performed to detect PLAU protein levels in KYSE30 and KYSE150 cells under different treatments. (g) Semiquantitative statistical analyses were performed to quantify PLAU protein levels. Actin was used as an internal control. Data are presented as means ± SD from three biological replicates. The significance of differences was analyzed by Student’s t-test. ∗∗p < 0.01.
Upregulation of PLAU by NFκB in IL1β-treated ESCC cells. (a-b) Binding of NFκB at the PLAU promoter in IL1β-treated ESCC cells. ChIP-qPCR assay was performed to detect the binding of NFκB at the PLAU promoter. Detection regions are located upstream (a) and downstream (b) of a canonical NFκB1 motif at the PLAU promoter. (c–e) Upregulation of PLAU mRNA by NFκB in IL1β-treated ESCC cells. (c) PLAU mRNA expression was induced in IL1β-treated ESCC cells. (d-e) The induction of PLAU mRNA by IL1β at 20 ng/mL (d) and 40 ng/mL IL1β (e) was blocked by the NFκB inhibitor Bay11-7082 (Bay). qRT-PCR assay was performed to detect PLAU mRNA expression. (f-g) Upregulation of PLAU protein by NFκB in IL1β-treated ESCC cells. (f) Western blot assays were performed to detect PLAU protein levels in KYSE30 and KYSE150 cells under different treatments. (g) Semiquantitative statistical analyses were performed to quantify PLAU protein levels. Actin was used as an internal control. Data are presented as means ± SD from three biological replicates. The significance of differences was analyzed by Student’s t-test. ∗∗p < 0.01.
Upregulation of PLAU by NFκB in IL1β-treated ESCC cells. (a-b) Binding of NFκB at the PLAU promoter in IL1β-treated ESCC cells. ChIP-qPCR assay was performed to detect the binding of NFκB at the PLAU promoter. Detection regions are located upstream (a) and downstream (b) of a canonical NFκB1 motif at the PLAU promoter. (c–e) Upregulation of PLAU mRNA by NFκB in IL1β-treated ESCC cells. (c) PLAU mRNA expression was induced in IL1β-treated ESCC cells. (d-e) The induction of PLAU mRNA by IL1β at 20 ng/mL (d) and 40 ng/mL IL1β (e) was blocked by the NFκB inhibitor Bay11-7082 (Bay). qRT-PCR assay was performed to detect PLAU mRNA expression. (f-g) Upregulation of PLAU protein by NFκB in IL1β-treated ESCC cells. (f) Western blot assays were performed to detect PLAU protein levels in KYSE30 and KYSE150 cells under different treatments. (g) Semiquantitative statistical analyses were performed to quantify PLAU protein levels. Actin was used as an internal control. Data are presented as means ± SD from three biological replicates. The significance of differences was analyzed by Student’s t-test. ∗∗p < 0.01.
Upregulation of PLAU by NFκB in IL1β-treated ESCC cells. (a-b) Binding of NFκB at the PLAU promoter in IL1β-treated ESCC cells. ChIP-qPCR assay was performed to detect the binding of NFκB at the PLAU promoter. Detection regions are located upstream (a) and downstream (b) of a canonical NFκB1 motif at the PLAU promoter. (c–e) Upregulation of PLAU mRNA by NFκB in IL1β-treated ESCC cells. (c) PLAU mRNA expression was induced in IL1β-treated ESCC cells. (d-e) The induction of PLAU mRNA by IL1β at 20 ng/mL (d) and 40 ng/mL IL1β (e) was blocked by the NFκB inhibitor Bay11-7082 (Bay). qRT-PCR assay was performed to detect PLAU mRNA expression. (f-g) Upregulation of PLAU protein by NFκB in IL1β-treated ESCC cells. (f) Western blot assays were performed to detect PLAU protein levels in KYSE30 and KYSE150 cells under different treatments. (g) Semiquantitative statistical analyses were performed to quantify PLAU protein levels. Actin was used as an internal control. Data are presented as means ± SD from three biological replicates. The significance of differences was analyzed by Student’s t-test. ∗∗p < 0.01.
Upregulation of PLAU by NFκB in IL1β-treated ESCC cells. (a-b) Binding of NFκB at the PLAU promoter in IL1β-treated ESCC cells. ChIP-qPCR assay was performed to detect the binding of NFκB at the PLAU promoter. Detection regions are located upstream (a) and downstream (b) of a canonical NFκB1 motif at the PLAU promoter. (c–e) Upregulation of PLAU mRNA by NFκB in IL1β-treated ESCC cells. (c) PLAU mRNA expression was induced in IL1β-treated ESCC cells. (d-e) The induction of PLAU mRNA by IL1β at 20 ng/mL (d) and 40 ng/mL IL1β (e) was blocked by the NFκB inhibitor Bay11-7082 (Bay). qRT-PCR assay was performed to detect PLAU mRNA expression. (f-g) Upregulation of PLAU protein by NFκB in IL1β-treated ESCC cells. (f) Western blot assays were performed to detect PLAU protein levels in KYSE30 and KYSE150 cells under different treatments. (g) Semiquantitative statistical analyses were performed to quantify PLAU protein levels. Actin was used as an internal control. Data are presented as means ± SD from three biological replicates. The significance of differences was analyzed by Student’s t-test. ∗∗p < 0.01.
Upregulation of PLAU by NFκB in IL1β-treated ESCC cells. (a-b) Binding of NFκB at the PLAU promoter in IL1β-treated ESCC cells. ChIP-qPCR assay was performed to detect the binding of NFκB at the PLAU promoter. Detection regions are located upstream (a) and downstream (b) of a canonical NFκB1 motif at the PLAU promoter. (c–e) Upregulation of PLAU mRNA by NFκB in IL1β-treated ESCC cells. (c) PLAU mRNA expression was induced in IL1β-treated ESCC cells. (d-e) The induction of PLAU mRNA by IL1β at 20 ng/mL (d) and 40 ng/mL IL1β (e) was blocked by the NFκB inhibitor Bay11-7082 (Bay). qRT-PCR assay was performed to detect PLAU mRNA expression. (f-g) Upregulation of PLAU protein by NFκB in IL1β-treated ESCC cells. (f) Western blot assays were performed to detect PLAU protein levels in KYSE30 and KYSE150 cells under different treatments. (g) Semiquantitative statistical analyses were performed to quantify PLAU protein levels. Actin was used as an internal control. Data are presented as means ± SD from three biological replicates. The significance of differences was analyzed by Student’s t-test. ∗∗p < 0.01.
Upregulation of PLAU by NFκB in IL1β-treated ESCC cells. (a-b) Binding of NFκB at the PLAU promoter in IL1β-treated ESCC cells. ChIP-qPCR assay was performed to detect the binding of NFκB at the PLAU promoter. Detection regions are located upstream (a) and downstream (b) of a canonical NFκB1 motif at the PLAU promoter. (c–e) Upregulation of PLAU mRNA by NFκB in IL1β-treated ESCC cells. (c) PLAU mRNA expression was induced in IL1β-treated ESCC cells. (d-e) The induction of PLAU mRNA by IL1β at 20 ng/mL (d) and 40 ng/mL IL1β (e) was blocked by the NFκB inhibitor Bay11-7082 (Bay). qRT-PCR assay was performed to detect PLAU mRNA expression. (f-g) Upregulation of PLAU protein by NFκB in IL1β-treated ESCC cells. (f) Western blot assays were performed to detect PLAU protein levels in KYSE30 and KYSE150 cells under different treatments. (g) Semiquantitative statistical analyses were performed to quantify PLAU protein levels. Actin was used as an internal control. Data are presented as means ± SD from three biological replicates. The significance of differences was analyzed by Student’s t-test. ∗∗p < 0.01.
3.4. Overexpression of PLAU Promoted ESCC Cell Proliferation
To investigate the effect of PLAU upregulation in ESCC, we analyzed genes co-expressed with PLAU using online databases. UALCAN and GEPIA2 showed 61 and 78 co-expressed genes with PLAU, respectively (Pearson correlation coefficient over 0.5), with 53 genes common to both databases (Figure 4(a)). We performed GO annotation for these 53 co-expressed genes using the DAVID database, revealing that 19 genes were significantly enriched in the GO term “cell proliferation” (GO0008283) (Figure 4(b)). To verify the bioinformatic analysis, we overexpressed PLAU in KYSE30 and TE-3 cells and detected the expression of five proliferation-related co-expressed genes: CAV2, CD276, FAP, LAMC2, and SNAI2. Overexpression of PLAU significantly increased the expression of these five genes in both cell lines (Figures 4(c) and 4(d)). Previous studies have shown that these co-expressed genes promote cell proliferation. For example, CD276 and LAMC2 promote the proliferation of endothelial progenitor cells and pancreatic cancer cells, respectively [40, 41]. These findings suggest that PLAU plays a role in ESCC cell proliferation. To further investigate this role, we focused on TE3 cells, which exhibited the lowest baseline PLAU expression among the ESCC cell lines studied (Figure 1(b)). Overexpression of PLAU in TE3 cells significantly promoted cell proliferation, as measured by CCK8 assay (Figure 5(a)). This finding was subsequently confirmed in KYSE30 and KYSE150 cells (Figure 5(a)). The pro-proliferative effect of PLAU overexpression was further validated by the EdU incorporation assay (Figure 5(b)).
Analysis of the role of PLAU in promoting cell proliferation by GO annotation. (a) Identification of genes co-expressed with PLAU in ESCC. The co-expressed genes were analyzed by using databases UALCAN and GEPIA2. (b) Potential roles of PLAU in promoting cell proliferation analyzed by GO annotation. DAVID database was used for the GO annotation. The bubble chart was created by an online server SangerBox. Experimental verification of genes co-expressed with PLAU in KYSE30 (c) and TE3 (d) by qRT-PCR assay. Ctrl, control cells. PLAU, PLAU-overexpressing cells. The data are presented as means ± SD from four biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗p < 0.05; ∗∗p < 0.01.
Analysis of the role of PLAU in promoting cell proliferation by GO annotation. (a) Identification of genes co-expressed with PLAU in ESCC. The co-expressed genes were analyzed by using databases UALCAN and GEPIA2. (b) Potential roles of PLAU in promoting cell proliferation analyzed by GO annotation. DAVID database was used for the GO annotation. The bubble chart was created by an online server SangerBox. Experimental verification of genes co-expressed with PLAU in KYSE30 (c) and TE3 (d) by qRT-PCR assay. Ctrl, control cells. PLAU, PLAU-overexpressing cells. The data are presented as means ± SD from four biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗p < 0.05; ∗∗p < 0.01.
Analysis of the role of PLAU in promoting cell proliferation by GO annotation. (a) Identification of genes co-expressed with PLAU in ESCC. The co-expressed genes were analyzed by using databases UALCAN and GEPIA2. (b) Potential roles of PLAU in promoting cell proliferation analyzed by GO annotation. DAVID database was used for the GO annotation. The bubble chart was created by an online server SangerBox. Experimental verification of genes co-expressed with PLAU in KYSE30 (c) and TE3 (d) by qRT-PCR assay. Ctrl, control cells. PLAU, PLAU-overexpressing cells. The data are presented as means ± SD from four biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗p < 0.05; ∗∗p < 0.01.
Analysis of the role of PLAU in promoting cell proliferation by GO annotation. (a) Identification of genes co-expressed with PLAU in ESCC. The co-expressed genes were analyzed by using databases UALCAN and GEPIA2. (b) Potential roles of PLAU in promoting cell proliferation analyzed by GO annotation. DAVID database was used for the GO annotation. The bubble chart was created by an online server SangerBox. Experimental verification of genes co-expressed with PLAU in KYSE30 (c) and TE3 (d) by qRT-PCR assay. Ctrl, control cells. PLAU, PLAU-overexpressing cells. The data are presented as means ± SD from four biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗p < 0.05; ∗∗p < 0.01.
Overexpression of PLAU promoted ESCC cell proliferation. Effect of PLAU overexpression on ESCC cell proliferation assessed by CCK8 assay (a) and EdU incorporation assay (b). Data are presented as means ± SD from three biological replicates.
Overexpression of PLAU promoted ESCC cell proliferation. Effect of PLAU overexpression on ESCC cell proliferation assessed by CCK8 assay (a) and EdU incorporation assay (b). Data are presented as means ± SD from three biological replicates.
3.5. EGCG Blocked ESCC Cell Proliferation Induced by TNFα/IL1β-NFκB-PLAU Axis
Given that EGCG inhibits TNFα-induced NFκB activation in human synovial fibroblasts [25], we hypothesized that EGCG might also block the TNFα/IL1β-NFκB-PLAU axis in ESCC. Indeed, treatment with EGCG significantly reduced NFκB protein levels in KYSE150 cells (Figures 6(a) and 6(b)). More importantly, EGCG treatment significantly inhibited TNFα-induced binding of NFκB to the PLAU promoter in KYSE150 cells (Figure 6(c)), an effect that was similarly observed for IL1β-induced NFκB binding (Figure 6(c)). Consistent with these findings, EGCG treatment significantly inhibited the TNFα- and IL1β-induced PLAU expression mediated by NFκB at both mRNA and protein levels (Figures 6(d), 6(e), and 6(f)). Furthermore, the CCK8 assay and EdU incorporation assay both demonstrated that EGCG significantly inhibited KYSE150 cell proliferation induced by PLAU overexpression (Figures 6(g), 6(h), and 6(i)). These results collectively suggest that EGCG can block the inflammatory signaling TNFα/IL1β-NFκB-PLAU, thereby inhibiting ESCC cell proliferation.
EGCG inhibited cell proliferation promoted by the TNFα/IL1β-NFκB-PLAU signaling axis. (a-b) Inhibition of NFκB protein expression by EGCG in KYSE150 cells. Western blot analysis (a) and semiquantitative statistical analyses (b) show the effect of EGCG on NFκB protein level, with Actin used as an internal control. (c) Inhibition of NFκB binding to the PLAU promoter by EGCG in TNFα/IL1β-treated KYSE150 cells. ChIP-qPCR assays were performed to detect the inhibition of NFκB binding by EGCG. The detected regions are located upstream (qDR1) and downstream (qDR2) of a classical NFκB1 motif in the PLAU promoter. (d–f) Inhibition of PLAU mRNA (d) and protein (e and f) expression by EGCG in TNFα/IL1β-treated KYSE150 cells. qRT-PCR (d), Western blot (e), and corresponding semiquantification (f) show the downregulation of PLAU by EGCG in TNFα/IL1β-treated KYSE150 cells. (g–i) The inhibitory effect of EGCG on KYSE150 cell proliferation promoted by PLAU overexpression. Cell proliferation was detected by CCK8 assay (g) and EdU incorporation (h and i), with the latter showing the cell proliferation rate (i). The data are presented as means ± SD from at least three biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗∗p < 0.01.
EGCG inhibited cell proliferation promoted by the TNFα/IL1β-NFκB-PLAU signaling axis. (a-b) Inhibition of NFκB protein expression by EGCG in KYSE150 cells. Western blot analysis (a) and semiquantitative statistical analyses (b) show the effect of EGCG on NFκB protein level, with Actin used as an internal control. (c) Inhibition of NFκB binding to the PLAU promoter by EGCG in TNFα/IL1β-treated KYSE150 cells. ChIP-qPCR assays were performed to detect the inhibition of NFκB binding by EGCG. The detected regions are located upstream (qDR1) and downstream (qDR2) of a classical NFκB1 motif in the PLAU promoter. (d–f) Inhibition of PLAU mRNA (d) and protein (e and f) expression by EGCG in TNFα/IL1β-treated KYSE150 cells. qRT-PCR (d), Western blot (e), and corresponding semiquantification (f) show the downregulation of PLAU by EGCG in TNFα/IL1β-treated KYSE150 cells. (g–i) The inhibitory effect of EGCG on KYSE150 cell proliferation promoted by PLAU overexpression. Cell proliferation was detected by CCK8 assay (g) and EdU incorporation (h and i), with the latter showing the cell proliferation rate (i). The data are presented as means ± SD from at least three biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗∗p < 0.01.
EGCG inhibited cell proliferation promoted by the TNFα/IL1β-NFκB-PLAU signaling axis. (a-b) Inhibition of NFκB protein expression by EGCG in KYSE150 cells. Western blot analysis (a) and semiquantitative statistical analyses (b) show the effect of EGCG on NFκB protein level, with Actin used as an internal control. (c) Inhibition of NFκB binding to the PLAU promoter by EGCG in TNFα/IL1β-treated KYSE150 cells. ChIP-qPCR assays were performed to detect the inhibition of NFκB binding by EGCG. The detected regions are located upstream (qDR1) and downstream (qDR2) of a classical NFκB1 motif in the PLAU promoter. (d–f) Inhibition of PLAU mRNA (d) and protein (e and f) expression by EGCG in TNFα/IL1β-treated KYSE150 cells. qRT-PCR (d), Western blot (e), and corresponding semiquantification (f) show the downregulation of PLAU by EGCG in TNFα/IL1β-treated KYSE150 cells. (g–i) The inhibitory effect of EGCG on KYSE150 cell proliferation promoted by PLAU overexpression. Cell proliferation was detected by CCK8 assay (g) and EdU incorporation (h and i), with the latter showing the cell proliferation rate (i). The data are presented as means ± SD from at least three biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗∗p < 0.01.
EGCG inhibited cell proliferation promoted by the TNFα/IL1β-NFκB-PLAU signaling axis. (a-b) Inhibition of NFκB protein expression by EGCG in KYSE150 cells. Western blot analysis (a) and semiquantitative statistical analyses (b) show the effect of EGCG on NFκB protein level, with Actin used as an internal control. (c) Inhibition of NFκB binding to the PLAU promoter by EGCG in TNFα/IL1β-treated KYSE150 cells. ChIP-qPCR assays were performed to detect the inhibition of NFκB binding by EGCG. The detected regions are located upstream (qDR1) and downstream (qDR2) of a classical NFκB1 motif in the PLAU promoter. (d–f) Inhibition of PLAU mRNA (d) and protein (e and f) expression by EGCG in TNFα/IL1β-treated KYSE150 cells. qRT-PCR (d), Western blot (e), and corresponding semiquantification (f) show the downregulation of PLAU by EGCG in TNFα/IL1β-treated KYSE150 cells. (g–i) The inhibitory effect of EGCG on KYSE150 cell proliferation promoted by PLAU overexpression. Cell proliferation was detected by CCK8 assay (g) and EdU incorporation (h and i), with the latter showing the cell proliferation rate (i). The data are presented as means ± SD from at least three biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗∗p < 0.01.
EGCG inhibited cell proliferation promoted by the TNFα/IL1β-NFκB-PLAU signaling axis. (a-b) Inhibition of NFκB protein expression by EGCG in KYSE150 cells. Western blot analysis (a) and semiquantitative statistical analyses (b) show the effect of EGCG on NFκB protein level, with Actin used as an internal control. (c) Inhibition of NFκB binding to the PLAU promoter by EGCG in TNFα/IL1β-treated KYSE150 cells. ChIP-qPCR assays were performed to detect the inhibition of NFκB binding by EGCG. The detected regions are located upstream (qDR1) and downstream (qDR2) of a classical NFκB1 motif in the PLAU promoter. (d–f) Inhibition of PLAU mRNA (d) and protein (e and f) expression by EGCG in TNFα/IL1β-treated KYSE150 cells. qRT-PCR (d), Western blot (e), and corresponding semiquantification (f) show the downregulation of PLAU by EGCG in TNFα/IL1β-treated KYSE150 cells. (g–i) The inhibitory effect of EGCG on KYSE150 cell proliferation promoted by PLAU overexpression. Cell proliferation was detected by CCK8 assay (g) and EdU incorporation (h and i), with the latter showing the cell proliferation rate (i). The data are presented as means ± SD from at least three biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗∗p < 0.01.
EGCG inhibited cell proliferation promoted by the TNFα/IL1β-NFκB-PLAU signaling axis. (a-b) Inhibition of NFκB protein expression by EGCG in KYSE150 cells. Western blot analysis (a) and semiquantitative statistical analyses (b) show the effect of EGCG on NFκB protein level, with Actin used as an internal control. (c) Inhibition of NFκB binding to the PLAU promoter by EGCG in TNFα/IL1β-treated KYSE150 cells. ChIP-qPCR assays were performed to detect the inhibition of NFκB binding by EGCG. The detected regions are located upstream (qDR1) and downstream (qDR2) of a classical NFκB1 motif in the PLAU promoter. (d–f) Inhibition of PLAU mRNA (d) and protein (e and f) expression by EGCG in TNFα/IL1β-treated KYSE150 cells. qRT-PCR (d), Western blot (e), and corresponding semiquantification (f) show the downregulation of PLAU by EGCG in TNFα/IL1β-treated KYSE150 cells. (g–i) The inhibitory effect of EGCG on KYSE150 cell proliferation promoted by PLAU overexpression. Cell proliferation was detected by CCK8 assay (g) and EdU incorporation (h and i), with the latter showing the cell proliferation rate (i). The data are presented as means ± SD from at least three biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗∗p < 0.01.
EGCG inhibited cell proliferation promoted by the TNFα/IL1β-NFκB-PLAU signaling axis. (a-b) Inhibition of NFκB protein expression by EGCG in KYSE150 cells. Western blot analysis (a) and semiquantitative statistical analyses (b) show the effect of EGCG on NFκB protein level, with Actin used as an internal control. (c) Inhibition of NFκB binding to the PLAU promoter by EGCG in TNFα/IL1β-treated KYSE150 cells. ChIP-qPCR assays were performed to detect the inhibition of NFκB binding by EGCG. The detected regions are located upstream (qDR1) and downstream (qDR2) of a classical NFκB1 motif in the PLAU promoter. (d–f) Inhibition of PLAU mRNA (d) and protein (e and f) expression by EGCG in TNFα/IL1β-treated KYSE150 cells. qRT-PCR (d), Western blot (e), and corresponding semiquantification (f) show the downregulation of PLAU by EGCG in TNFα/IL1β-treated KYSE150 cells. (g–i) The inhibitory effect of EGCG on KYSE150 cell proliferation promoted by PLAU overexpression. Cell proliferation was detected by CCK8 assay (g) and EdU incorporation (h and i), with the latter showing the cell proliferation rate (i). The data are presented as means ± SD from at least three biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗∗p < 0.01.
EGCG inhibited cell proliferation promoted by the TNFα/IL1β-NFκB-PLAU signaling axis. (a-b) Inhibition of NFκB protein expression by EGCG in KYSE150 cells. Western blot analysis (a) and semiquantitative statistical analyses (b) show the effect of EGCG on NFκB protein level, with Actin used as an internal control. (c) Inhibition of NFκB binding to the PLAU promoter by EGCG in TNFα/IL1β-treated KYSE150 cells. ChIP-qPCR assays were performed to detect the inhibition of NFκB binding by EGCG. The detected regions are located upstream (qDR1) and downstream (qDR2) of a classical NFκB1 motif in the PLAU promoter. (d–f) Inhibition of PLAU mRNA (d) and protein (e and f) expression by EGCG in TNFα/IL1β-treated KYSE150 cells. qRT-PCR (d), Western blot (e), and corresponding semiquantification (f) show the downregulation of PLAU by EGCG in TNFα/IL1β-treated KYSE150 cells. (g–i) The inhibitory effect of EGCG on KYSE150 cell proliferation promoted by PLAU overexpression. Cell proliferation was detected by CCK8 assay (g) and EdU incorporation (h and i), with the latter showing the cell proliferation rate (i). The data are presented as means ± SD from at least three biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗∗p < 0.01.
EGCG inhibited cell proliferation promoted by the TNFα/IL1β-NFκB-PLAU signaling axis. (a-b) Inhibition of NFκB protein expression by EGCG in KYSE150 cells. Western blot analysis (a) and semiquantitative statistical analyses (b) show the effect of EGCG on NFκB protein level, with Actin used as an internal control. (c) Inhibition of NFκB binding to the PLAU promoter by EGCG in TNFα/IL1β-treated KYSE150 cells. ChIP-qPCR assays were performed to detect the inhibition of NFκB binding by EGCG. The detected regions are located upstream (qDR1) and downstream (qDR2) of a classical NFκB1 motif in the PLAU promoter. (d–f) Inhibition of PLAU mRNA (d) and protein (e and f) expression by EGCG in TNFα/IL1β-treated KYSE150 cells. qRT-PCR (d), Western blot (e), and corresponding semiquantification (f) show the downregulation of PLAU by EGCG in TNFα/IL1β-treated KYSE150 cells. (g–i) The inhibitory effect of EGCG on KYSE150 cell proliferation promoted by PLAU overexpression. Cell proliferation was detected by CCK8 assay (g) and EdU incorporation (h and i), with the latter showing the cell proliferation rate (i). The data are presented as means ± SD from at least three biological replicates. The significance of the difference was analyzed by Student’s t-test. ∗∗p < 0.01.
4. Discussion
PLAU, a key enzyme in the uPA system, promotes cancer development [42, 43]. In various cancers, PLAU upregulation occurs through different mechanisms, such as hypomethylation and noncoding RNA regulation [27, 29]. However, the mechanism underlying PLAU upregulation in ESCC remains unclear. In this study, we observed significant upregulation of PLAU in ESCC. Treatment of ESCC cells with TNFα and IL1β induced the binding of NFκB to the promoter of PLAU, resulting in increased PLAU mRNA and protein expression. This effect was blocked by the specific NFκB inhibitor Bay. These findings show that PLAU is a target gene of NFκB in ESCC. Importantly, clinical evidence showing a positive correlation between PLAU expression and RELA levels in ESCC tissues further supports PLAU as an NFκB target gene. Therefore, our study elucidates the regulatory mechanism of PLAU upregulation in ESCC: TNFα and IL1β activate NFκB, which in turn upregulates PLAU expression.
Proinflammatory cytokines play crucial roles in ESCC. Previous studies have shown that both TNFα and IL1β are highly expressed in ESCC and promote cell proliferation [9–11, 44, 45]. Our findings confirm that the elevated levels of TNFα and IL1β correlate with poorer prognosis in ESCC patients. However, the molecular mechanisms underlying the pro-proliferative effect of IL1β and TNFα remain unclear. In this study, we found that both TNFα and IL1β induced the upregulation of PLAU in an NFκB-dependent manner in three KYSE cell lines and two others, SHEEC and TE3. We demonstrated that PLAU overexpression promoted proliferation in three ESCC cell lines. Conversely, silencing PLAU hindered the proliferation of ESCC cells [32]. These findings suggest that the upregulation of PLAU, driven by the TNFα/IL1β-NFκB inflammatory signaling axis, is a primary factor in promoting ESCC cell proliferation. Therefore, this pathway may represent a critical mechanism through which IL1β and TNFα contribute to ESCC cell proliferation.
LPS, produced by gram-negative bacteria, can upregulate PLAU in an NFκB-dependent manner in ESCC [46], and IL8, a typical target of classical NFκB signaling, can also induce PLAU [15, 32]. Therefore, proinflammatory mediators (at least LPS, IL8, TNFα, and IL1β), NFκB, and downstream target gene PLAU form a complex inflammatory signaling network, and PLAU may serve as a key role in this network.
Blocking TNFα and IL-1β signaling may be an effective therapeutic strategy in cancer treatment due to their tumor-promoting actions [47, 48]. EGCG, a polyphenolic substance with antitumor and anti-inflammatory effects, has been shown to inhibit the activity of various oncogenic TFs, including NFκB activity [49, 50]. Studies show that EGCG acts as a covalent inhibitor of the p65 subunit of NFκB [51, 52], reducing cell proliferation and inducing apoptosis in pancreatic cancer cells [51]. Additionally, EGCG represses key NFκB target gene expression such as MMP9, MMP2, cMyc, and BCL2 [51]. Our finding aligns with these observations that EGCG significantly blocked the binding of NFκB to the PLAU promoter induced by TNFα/IL1β, thereby inhibiting the PLAU expression. We propose that by targeting the TNFα/IL1β-NFκB-PLAU signaling axis, EGCG suppresses ESCC cell proliferation. NFκB links tumor development and inflammation [15]. By blocking the NFκB signaling pathway, EGCG also inhibits the inflammatory response in different cancer models [23, 24, 51]. Given the significant role of chronic inflammation in ESCC progression [6, 7], EGCG’s ability to inhibit inflammation further supports its potential therapeutic application.
Furthermore, the molecular mechanisms by which EGCG exerts its antitumor effects go beyond NFκB inhibition. EGCG modulates various cellular signaling pathways involved in cancer progression; for example, by inhibiting Src/JAK/STAT3 signaling, EGCG prevents ovarian tumor spheres from acquiring a cancer-stem-cell phenotype [53, 54]. Additionally, EGCG targets epigenetic modifications, including DNA methylation and histone acetylation, critical for regulating gene expression in cancer cells [55, 56]. These epigenetic modifications can reactivate tumor suppressor genes and silence oncogenes, thereby contributing to EGCG’s antitumor activity. Our findings support this mechanism, as we observed EGCG-downregulated PLAU, a gene with oncogenic properties often upregulated in various cancers, suggesting that EGCG also exerts its antitumor effects by modulating the epigenetic landscape to repress oncogenic gene expression. Overall, EGCG shows promise as a potential clinical drug for ESCC by targeting multiple cancer-related pathways.
While our study provides valuable insights into the TNFα/IL1β-NFκB-PLAU axis in ESCC, several limitations should be acknowledged. First, our findings are primarily based on in vitro experiments using cell lines, and further validation in animal models is necessary to confirm these results in a more complex biological context. Second, although we identified PLAU as an NFκB target gene, the precise mechanisms governing its regulation by other TFs within the ESCC microenvironment warrant further exploration. Third, while EGCG shows promise as a therapeutic agent and previous studies have demonstrated its safety in humans [20–22], additional research is needed to evaluate its long-term efficacy and safety in clinical settings.
5. Conclusions
In this study, we found that the NFκB target gene PLAU promotes ESCC cell proliferation, and EGCG treatment inhibits PLAU upregulation by blocking NFκB activation induced by TNFα and IL1β, and suppresses the pro-proliferative effect of PLAU. In other words, by targeting the TNFα/IL1β-NFκB-PLAU axis, EGCG may provide a novel approach against ESCC cell proliferation.
Disclosure
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conflicts of Interest
The authors declare no conflicts of interest.
Author Contributions
Fei Zhou conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and tables, drafted the work, and approved the final draft.
Yuanduo Li and Yurui Zhang performed the experiments and approved the final draft.
Hui Zhu, Yun Li, Ying Nie, Junjun Sun, Qiulan Luo, Ruixuan Wang, and Xianghui Zou conceived and designed the experiments, analyzed the data, and approved the final draft. Zikai Chen conceived and designed the experiments, analyzed the data, authored or reviewed drafts of the article, and approved the final draft.
Funding
This research was funded by the Project of the Educational Commission of Guangdong Province of China (2021ZDZX2066), the Scientific Research Foundation of Hanshan Normal University (XPY202106), the Special Fund for Science and Technology Innovation Strategy of Guangdong Province (“Climbing Plan” special fund; pdjh2023b0342 and pdjh2024b256), the Doctor’s Start-Up Project of Hanshan Normal University (QD202121), the Scientific and Technological Research Project of Chaozhou City (2020GY01), and Guangdong Provincial Key Laboratory of Functional Substances in Medicinal Edible Resources and Healthcare Products (2021B1212040015).
Supporting Information
Figure S1. Identification of transcription factors (TFs) regulating PLAU expression. (a) Prediction of common TFs by four online servers. (b) TF-bound clusters at the PLAU promoter. (c) Confirmation of H3K27Ac enrichment at the PLAU promoter in TNFα-treated KYSE510 cells via ChIP-qPCR assay. The detection regions are located upstream (qDR1) and downstream (qDR2) of the NFKB1 motif in the PLAU promoter. Data are presented as means ± SD from three biological replicates. The significance of the difference was analyzed by Student′s t-test. ∗∗p < 0.01.
Figure S2. Expression of TNF and IL1B in ESCC and paired normal tissues. The datasets for TNF and IL1B expression levels were obtained from the GEO database. Tumor (T) and adjacent nontumor (N) tissue samples were analyzed. All data are shown as mean ± SD. The statistical differences were analyzed by Student′s t-test. p < 0.05 ( ∗); p < 0.01 ( ∗∗).
Figure S3. Overall survival of ESCC patients. (a) Effect of TNF expression levels on overall survival. The result was analyzed using the KM Plotter online server. (b) Effect of IL1B expression levels on overall survival. The dataset was obtained from the GEO database (GSE53625) and analyzed using the SangerBox online server.
Figure S4. Correlation analysis of PLAU and RELA expression in ESCC. Correlation analysis was performed using the Xiantaozi online server with data from the GSE121931 dataset, sourced from the GEO database.
Table S1. Basic information of the 10 GEO microarray datasets.
Table S2. The sequences of primers for qRT-PCR assay.
Table S3. The sequences of primers for ChIP-qPCR assay.
Supporting Information Additional supporting information can be found online in the Supporting Information section.
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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