Ethyl 2,2-difluoro-2-(2-oxo-2H-chromen-3-yl) acetate inhibits the malignant biological behaviors of colorectal cancer by restricting the phosphorylation and nuclear translocation of STAT3

Ethyl 2,2-difluoro-2-(2-oxo-2H-chromen-3-yl) acetate inhibits the malignant biological behaviors of colorectal cancer by restricting the phosphorylation and nuclear translocation of STAT3 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ethyl 2,2-difluoro-2-(2-oxo-2H-chromen-3-yl) acetate inhibits the malignant biological behaviors of colorectal cancer by restricting the phosphorylation and nuclear translocation of STAT3 JIE LIN, Weijing LIU, Xiaodan LI, Jiansuo LIN, Xuehong FANG, Yanwen LIANG, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4376706/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract To investigate the effect of a novel coumarin derivative, ethyl 2,2-difluoro-2 - (2-oxo-2H-chromen-3-yl) acetate (C2F), on the malignant biological behaviors of colorectal cancer (CRC) and elucidate its mechanism. In vitro , the effects of C2F on the proliferation, apoptosis, migration, invasion, and cell cycle of CRC cells were analyzed by MTT assay, EdU stainning, colony formation assay, flow cytometry, wound healing and transwell assay. The anti-CRC activity of C2F was evaluated in a nude mice xenograft model in vivo . Western blot was conducted to detect the expression of protein in cells and mice tissue. Then, the potential targets of C2F in CRC were predicted by network pharmacology analysis and molecular docking. The localization of STAT3 was observed through immunofluorescence experiment. C2F inhibits CRC cell proliferation, promotes CRC cell apoptosis, hinders CRC cell migration and invasion, and prevents the cell cycle from entering the G2/M phase. In vivo , C2F inhibited tumor growth in xenograft model. C2F inhibited signal transduction and activator of transcription 3 (STAT3) phosphorylation and blocked interleukin-6 (IL-6)-induced STAT3 nuclear translocation. C2F inhibits the malignant biological behavior of CRC by limiting STAT3 phosphorylation and entry into the nucleus. Ethyl 2 2-difluoro-2-(2-oxo-2H-chromen-3-yl) acetate Colorectal cancer Malignant biological behavior STAT3 phosphorylation Nuclear transfer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Global cancer statistics show that CRC is the second most common cause of cancer death in both males and females(Siegel et al., 2024). With the advancement of colonoscopy technology, surgical resection of intestinal tumors has arisen as an effective treatment program for patients in the initial stages of CRC(Ainhoa et al., 2019). However, most patients are usually diagnosed in the late stage, and combination chemotherapy remains the main treatment option that can provide systematic and sustained efficacy. Unsatisfactorily, the predominant hurdles in achieving successful CRC treatment lie in the adverse toxic effects and development of therapeutic resistance associated with chemotherapy medications(Benoist et al., 2015; Peeters et al., 2010). Hence, there is a pressing need for continued investigation into targets or agents that have minimal side effects in CRC therapy, ultimately enhancing the treatment results for CRC patients. Up to this point, approximately 85% of sanctioned small molecule anticancer drugs have originated either directly or indirectly from natural sources(J and M, 2016). Coumarin (benzopyran-2-one) is a typical natural product, which is derived from plants, bacteria and fungi(Maria et al., 2016; Saleta et al., 2015). Studies have found that coumarin compounds have various biological activities, such as anti-inflammatory(Chaoyu et al., 2018), antiviral(Lei et al., 2020), antibacterial(Ivana et al., 2021; Lipeeva et al., 2019), antioxidant(Jinxiu et al., 2020) and antitumor(Li et al., 2015; Lingaraju et al., 2018; Luo et al., 2017; Zhou et al., 2016), etc. A series of coumarin derivatives effective for different cancers can be obtained by structural modification on the basis of the coumarin nucleus(Mintas et al., 2012; Nadia et al., 2011). In our preliminary research, we found that C2F has significant anti-cancer activity, however, the exact mechanism of cancer treatment has not yet been elucidated. Therefore, C2F shows potential for development as an anti-cancer drug. This study investigates the impact of C2F on the malignant biological behaviors of CRC through ex vivo experiments. It also predicts potential targets of C2F activity in CRC using network pharmacology and molecular docking analysis. The results demonstrate that C2F significantly suppresses the malignant biological behaviors of CRC both in vitro and in vivo by inhibiting STAT3 phosphorylation and constraining its nuclear translocation. To our knowledge, this is the first investigation into the role and mechanism of C2F in the treatment of CRC. Thus, this study will establish the theoretical foundation for the potential development of C2F as a therapeutic agent for CRC. 2. Materials And Methods 2.1 Antibodies and reagents C2F, chemical structure shown as Fig. 1 A, was synthesized by Shenzhen Small Molecule New Drug Innovation Center Co., Ltd. Antibodies of β-actin, PCNA, Bax, Bcl-2, Cleaved Caspase-9, Cleaved Caspase-3, E-Cadherin, Vimentin, N-Cadherin, PI3K were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies of cyclin B1, CDK1, STAT3, Phosphorylated STAT3 (Tyr705), Phosphorylated STAT3 (Ser727) were purchased from Zen bioscience (Chengdu, China). BeyoClick™ EdU-594 and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were supplied by Beyotime Biotechnology. The recombinant human IL-6 was purchased from Peprotech (London, UK). The STAT inhibitor Stattic was purchased from MedChem Express (NJ, USA). Matrigel was purchased from ABW (CAT: 082704). Exkine™ Nuclear and Cytoplasmic Protein Extraction Kit was supplied by Abbkine (Wuhan, China). Alanine aminotransferase (ALT) test kit and aspartate aminotransferase (AST) test kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 2.2 Cell cultures The CRC cell lines DLD-1, SW620 and human normal colon cells HCoEpiC used in this study were developed and provided by Shenzhen Ruike Biotechnology Co., Ltd. Cell culture has been described in detail in our previous research(Xuehong et al., 2022). 2.3 MTT cytotoxicity assay After overnight attachment, the DLD-1, SW620 and HCoEpiC cells were processed with a multi-Concentrationn of C2F (0, 1, 5, 10, 15, and 20 µM) and 5-FU (5, 10 and 15 µM). We followed the MTT experimental steps for subsequent operations(Priti et al., 2018). 2.4 Colony formation assay DLD-1 and SW620 cells (500 per well) were seeded in 6-well plates overnight and then the medium was replaced with different concentrations of C2F. Two weeks later, 4% paraformaldehyde was added for 15 min for cell fixation and then aspirated, washed twice with PBS and finally stained with 0.1% crystal violet for 15 min. 2.5 EdU cell proliferation detection The proliferative capacity of DLD-1 and SW620 cells were treated by EdU method according to the protocol provided by the manufacturer. Briefly, seeded of cells into 24-well plates and after adhesion, cells were stimulated with different concentrations of C2F for 24 hours. Then the reaction system was prepared according to BeyoClick™ EdU-594, and the reaction system was incubated at room temperature for 30 min, protected from light. Finally, the nuclei were stained with Hoechst33342 reaction solution. The results were observed by an inverted fluorescence microscope (200 × magnification, Olympus, Tokyo, Japan). 2.6 Cell cycle and apoptosis analysis The methods for analyzing cell cycle and apoptosis have been specifically described in our previous research(Xuehong et al., 2022). 2.7 Hoechst 33342 staining DLD-1 and SW620 cells were seeded in 12-well plates at a density of 1 × 10 4 cells per well. After 24 hours, we followed the Hoechst 33342 staining experimental steps for subsequent operations(Yao et al., 2020). Fluorescence microscopy was used for analysis. 2.8 Wound-healing assay The wound healing assay was conducted with reference to our previous study(Xuehong et al., 2022). 2.9 Transwell migration/invasion assay To rule out the effects of cell proliferation inhibition and induction of cell apoptosis on migration and invasion assay, we first set 4 × 10 4 DLD-1 cells and SW620 cells were suspended in 200 µl serum-free medium and then inoculated into the upper chamber, while the lower chamber was filled with 600 µl of medium containing 20% FBS. After 48 hours, fixed the cells on the pore membrane at the bottom of the chamber, and then soaked the cells in crystal violet staining solution for staining. Finally, the chamber was observed and photographed under an optical microscope, and the inhibitory effect of C2F on the migration and invasion of DLD-1 cells and SW620 cells was statistically analyzed using Image J. 2.10 Western blot analysis After overnight attachment, the cells were processed with a gradient concentration of C2F (0, 1, 5, 10, 15, and 20 µM). The cells were then washed gently with PBS 2 times. The CRC cells were lysed using cell lysates for 30 min on ice, which were transferred to new tubes and then centrifuged. Protein concentration was determined by Coomassie Brilliant Blue staining to configure the reaction system. Protein was separated by sodium dodecyl sulfate- polyacryl- amide gel electrophoresis gel and transferred onto polyvinylidene difluoride membranes. The membranes were then blocked with 5% skim milk for 1.5 h and then washed with Tris Buffered Saline with Tween 20. The blots were ultimately incubated with specific primary antibodies and incubated with secondary antibodies on the following day. The membranes were imaged using the BIO-RAD high sensitivity chemiluminescence imaging system ChemiDoc touch and the software Image J was used to analyze the reactive bands. 2.11 Predicting the therapeutic targets of C2F for CRC by bioinformatic analysis We downloaded the structure of C2F (PubChem CID: 132961735) from the PubChem(Kim et al., 2019) database ( https://pubchem.ncbi.nlm.nih.gov/),an d input it into the SwissTarget-Prediction(David et al., 2014) ( http://www.swisstargetprediction.ch/ ) and Pharmmapper(Xiaofeng et al., 2010) ( http://www.swisstargetprediction.ch/ ) platforms to predict the potential targets of C2F. The Uniprot(2020) database ( https://www.uniprot.org/ ) was used to convert the protein target name into the corresponding gene symbol. We merged targets from two databases and removed duplicates to obtain targets related to C2F. 2.12 Determination of potential CRC-related targets Four databases were then searched using Genes related to CRC: Genecards(Gil et al., 2016) database ( https://www.genecards.org/ ), OMIM(S et al., 2019) database ( https://omim.org/ ), TTD(Yunxia et al., 2020) database ( https://db.idrblab.net/ttd/ ) and DrugBank(S et al., 2018) database ( https://www.drugbank.com/ ). After the removal of repeated targets, Venny2.1.0 ( http://www.liuxiaoyuyuan.cn/ ) was used to study the intersection of C2F with pathologic targets to identify potential targets for C2F in the treatment of colorectal cancer. 2.13 Network construction and enrichment analysis To identify the possible interactions between all target genes in the pharmacological network, we constructed the PPI network using the STRING(Damian et al., 2019) database. When we used the database, we selected "Homo sapiens" for the species, chose 0.4 as the cutoff value for the confidence score, and left all other settings unchanged. Cytoscape(Paul et al., 2003) software version 3.8.2 was used to analyze and visualize the network topology of the results obtained by STRING. Nodes in the network diagram were intersection targets, and edges represent the relationship between targets and targets. We used the built-in Network Analyzer program to analyze network topology parameters such as degree, betweenness centrality, closeness centrality, etc.(Ziyu et al., 2022). Next, we used CytoHubba, a Cytoscape plugin that can be used for network centrality analysis, to identify the centroids in the network. The 10 genes with the highest degree centrality values were identified as central genes. Analysis of GO functional enrichment and KEGG pathway enrichment for target genes was performed using metascape(Yingyao et al., 2019) ( https://metascape.org/gp/index.html#/main/step1 ) database. Bioinformatics ( http://www.bioinformatics.com.cn/ ) platform was used to draw a bar graph and a bubble graph. 2.14 Targets-pathways network construction Cytoscape software version 3.8.2 was used to build a targets-pathways network. The nodes in the network were targets and pathways, respectively, and the edges represented the interaction relationship between targets and pathways. 2.15 Molecular docking validation of C2F and core targets The most important genes were selected for subsequent molecular docking analysis. The 3D structure of the ligand molecule (C2F) was first downloaded from the PubChem database. The Uniprot database was searched for receptor protein encoded by the input genes. The 3D structure of the protein was obtained from RCSB PDB database ( https://www.rcsb.org/ ) and modified using PyMol software version 2.4.0, including ligand and water removal. Autodocktools was used to calculate protein loads. Finally, receptor proteins were docked to C2F using Autodock Vina and visualized using PyMol. 2.16 Immunofluorescence staining We first inoculated DLD-1 and SW620 cells onto the glass slides, then fixed the cells with 4% paraformaldehyde, penetrated the cells with 0.5% Triton X-100 (in PBS), rinsed several times with PBS, and finally blocked the cells with 1% bovine serum albumin for 1 h. The cells were incubated with the primary antibody in an appropriate proportion, and the second day with the secondary antibody. We stained the cells with DAPI in the dark, then washed the cell slides with PBS again, and sealed them with anti-fluorescence quenching sealing sheets to prevent fluorescence quenching until they were captured by confocal microscopy. 2.17 Cytoplasmic and nuclear protein extraction DLD-1 and SW620 cells were cultured at 80%-90% concentration in 10 cm dishes and treated with C2F (0, 5 and 10 µM). Immediately after IL-6 stimulation for 1 hour, cells were placed on ice. Nuclear and cytoplasmic proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit. The expression of related proteins was detected by western blot. 2.18 Animal experiments Animal experiments are conducted in strict compliance with international standards and the 3R's of animal protection, and through the Shenzhen People's Hospital Laboratory Animal Ethics Committee approval. Approximately 5 weeks old female balb/c nude mice (18–20 g) were acqired from the Laboratory Animal Center of Guangdong Medical University (Dongguan, China) and were housed at the Laboratory Animal Center of Shenzhen People's Hospital. Approximately 1 × 10 7 SW620 cells suspended in 0.1 ml of serum-free culture medium and Matrigel (medium:Matrigel = 1:1) were subcutaneously injected into the right flank of each mouse. Tumor volume was calculated using recognized calculation methods(Yao et al., 2020). In our previous research, we found that C2F has an inhibitory effect on the growth of non-small cell lung cancer cell xenograft tumors. Accordingly, the mice were randomly divided into five groups in this study (control group, mice were intraperitoneally injected with olive oil five times per week. C2F group A were intraperitoneally injected with 10 mg/kg C2F five times per week, C2F group B were intraperitoneally injected with 20 mg/kg C2F five times per week, C2F group C were intraperitoneally injected with 30 mg/kg C2F five times per week, fluorouracil group, mice were intraperitoneally injected with 20 mg/kg fluorouracil three times per week. After 21 days of treatment, blood was collected from the posterior orbital sinus of nude mice for further analysis. Next, the nude mice were sacrificed by spinal dislocation and the tumors were obtained and weighed. The tumors were fixed in formalin and used for histological studies. 2.19 Statistical analysis Each experiment was repeated at least three times independently to ensure reproducibility and reliability and statistically analyzed using Prism (version 8.0, GraphPad Software, San Diego California, U.S.A.). All results are shown as mean ± SD. One -way analysis of variance (ANOVA) was used for comparisons among multiple groups. P < 0.05 was considered to denote statistical significance. 3. Results 3.1 C2F inhibits the proliferation of human CRC cells To evaluate the effect of C2F on the proliferation of CRC cells, DLD-1 and SW620 cells were treated with different concentrations of C2F, and the proliferation of DLD-1 and SW620 cells was detected by MTT assay after 24, 48, and 72 hours. As a result, cell proliferation activity is manifested as a concentration and time dependent decrease (Fig. 1 B and C). Interestingly, C2F treatment had no significant effect on the viability of HCoEpiC cells derived from the human normal colon epithelial cells (Fig. 1 D). In addition, to ensure the reliability and rigor of the experiment, 5-FU was used as a positive control for the MTT experiment (Fig. 1 E and F). Subsequently, we detected the effect of C2F on cell proliferation by EdU staining and colony formation assay. The results indicated that the number of EdU-positive cells and colonies was significantly reduced by C2F treatment, and this inhibitory effect was dose-related (Supplementary Fig. 1 and Fig. 1 G). PCNA expression correlates with malignancy, vascular infiltration, distant metastasis, and survival and has been identified as a biomarker for CRC adenocarcinoma(Katarzyna et al., 2009). We examined the protein expression level of PCNA and showed that PCNA expression decreased with increasing C2F concentration (Fig. 1 H). These data suggested that C2F not only inhibits the proliferation of CRC cells in a doses and time dependent manner but is less toxic to HCoEpiC. 3.2 C2F induces G2/M cell cycle arrest in CRC cells Induction of cell cycle arrest is a promising strategy in cancer therapy(Tobias and Piotr, 2017). To determine whether C2F inhibits cell growth through cell cycle arrest, we performed flow cytometry and western blot analysis. The results showed that C2F increased the number of G2/M phases in DLD-1 and SW620 cells in a concentration-dependent manner (Fig. 2 A and B). The central role of the CDK1/cyclin B1 complex (CCNB1) in the cell cycle of cervical cancer has been well described. The complex phosphorylates the corresponding substrate and promotes the transition of the cell cycle from G2 to M, the mitotic phase(Nagappan et al., 2017). As shown in Fig. 2 C and D, CDK1 and cyclin B1 expression were decreased upon C2F administration. The above data suggested that C2F inhibits the proliferation of DLD-1 and SW620 cells by inducing G2/M phase arrest. 3.3 C2F promotes apoptosis in CRC cells Induction of cancer cell apoptosis is one of the important indicators for evaluating the inhibition of cancer cell growth by antitumor drugs(Bingwu et al., 2021). We then investigated whether the inhibitory effect of C2F on CRC cell proliferation involved the induction of apoptosis. Annexin-V/PI double staining revealed that C2F increased the apoptosis rate of DLD-1 cells from 9.69–30.53%, and SW620 cells from 5.51–30.69% (Fig. 3 A and B). Hoechst 33342 staining showed that after 24 h of C2F treatment, DLD-1 and SW620 cells showed obvious apoptotic morphology, which was manifested as enhanced blue fluorescence of the cells indicated by the arrow (Supplementary Fig. 2). As the Bax/Bcl-2 ratio rises, cytochrome c is released, followed by the cleavage and activation of caspase-3 and caspase-9, which can cause the destruction of intracellular substrates and apoptosis, according to earlier research(E et al., 2014). Data from western blot showed that after 24 hours of C2F treatment, the expression of pro-apoptotic-related proteins such as Bax, cleaved caspase-9 and cleaved caspase-3 were increased while the expression of anti-apoptotic-related proteins such as Bcl-2 was decreased (Fig. 3 C-F). Overall, C2F markedly promoted apoptosis in CRC. 3.4 C2F inhibits migration and invasion of human CRC cells Abnormal differentiation, migration, and metastases are representative biological features of tumor cells(Wen et al., 2019). The effect of C2F on the migration and invasion of CRC cell lines was investigated using a wound healing assay and a transwell assay to determine whether biological processes related to migration and invasion are involved in the anti-tumour effect of C2F in CRC cell lines. Comparison with control group, C2F reduced the cell migration ability of DLD-1 and SW620 cells (Fig. 4 A and B). The results of transwell migration assay showed that C2F effectively suppressed the number of DLD-1 and SW620 cells in the lower lumen (Fig. 4 C and D). We also used the transwell invasion assay to determine the invasive activity of CRC cells. As shown in Fig. 4 E and F, C2F inhibited the invasive potential of CRC cells in a dose-dependent manner. At the same time, we further measured the expression level of major functional proteins during epithelial-mesenchymal transformation (EMT). The results indicated that the epithelial marker E-cadherin was up-regulated after C2F treatment. On the contrary, the expression of N-cadherin and vimentin was decreased, indicating deprivation of mesenchymal phenotype (Fig. 4 G and H). The above results indicated that C2F significantly inhibits the EMT process of DLD-1 and SW620 cells. 3.5 C2F suppresses CRC tumor growth in vivo To investigate the effect of C2F on the growth of CRC in vivo , SW620 cells were transplanted into BALB/c nude mice. The experimental data indicated that the tumor volume of the C2F and 5-FU groups was smaller than that in the control group, confirming the inhibitory effect of C2F on CRC cells in vivo . There was no statistical difference in tumor volume between 5-FU (20 mg/kg) treatment group and C2F (30 mg/kg) treatment group (Fig. 5 A-C). During the whole treatment period, the nude mice did not lose body weight (Fig. 5 D). Accordingly, the liver function test results also indicated that, comparison with control group, the AST of 5-FU group and C2F treatment group showed a downward trend while the ALT did not change significantly (Fig. 5 E and F), suggesting that C2F treatment was safe and did not increase the toxicity in vivo . In order to study the potential mechanism of C2F inhibiting tumor growth, we then detected the expression of PCNA, Bcl-2, Bax in SW620 xenotransplantation tissues by western blot. To sum up, these data indicated that the anti-tumor effects of C2F in tumor xenografts may be associated with anti-proliferative, and pro-apoptotic mechanisms (Fig. 5 G and H). 3.6 Bioinformatics analysis of the anti-CRC mechanism of C2F Through searching, a total of 86 C2F-related targets and 2831 CRC-related targets were screened after deleting duplicates. Then, 40 potential C2F targets for the treatment of CRC were obtained by intersection (Fig. 6 A). The protein-protein interaction network from the STRING database showed that there are complex interactions between proteins encoded by these target genes (Fig. 6 B). We studied the relationship between nodes and edges by referring to previous research methods of Zhenshuang Yuan(Zhenshuang et al., 2023). Using Cytoscape, we performed further analysis of the imported PPI network for more information. Nodes were sorted based on their size and color, with larger nodes and darker colors indicating more critical targets (Fig. 6 C). Finally, 10 core target genes were obtained by using CytoHubba, including epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), estrogen receptor 1 (ESR1), matrix metalloprotein 9 (MMP9), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase 8 (MAPK8), mechanistic target of rapamycin (MTOR), androgen receptor (AR), progesterone receptor (PGR), nuclear receptor subfamily 3 group C member 1 (NR3C1). GO functional enrichment analysis indicated that a total of 589 GO items were screened, including 507 items for biological process and 54 items for molecular function and 28 items for cellular component. In the GO analysis results (Fig. 6 D), the changes of BP were mainly reflected in the regulation of intracellular receptors, inflammatory responses, responses to reactive oxygen species, rhythmic process and responses to estradiol. In terms of CC, C2F is closely related to all parts of the cell, including intracellular matrix, cell membrane, extracellular matrix, receptor complexes, dendrites, vacuoles. In terms of MF, C2F is mainly related to the binding of enzymes, proteins, phosphatases, and kinases, protein serine/threonine/tyrosine kinase activity, and nuclear receptor activity regulation. This result confirms that C2F can treat CRC by modulating multiple biological pathways. KEGG pathway analysis further showed that C2F targeted proteins are involved in pathways in cancer, endocrine resistance, chemical carcinogenesis - receptor activation, lipid and atherosclerosis, phospholipase D signaling pathway, transcriptional misregulation in cancer, PPAR signaling pathway, which indicated that C2F can exert anti-CRC through multiple signaling pathways, and its targets may be the common targets of various malignant cancer (Fig. 6 E). We next used Cytoscape to analyze the network topology parameters of signaling pathways and targets, and the core targets were EGFR, STAT3, etc; the main pathways were pathways in cancer signaling pathway, endocrine resistance, Chemical carcinogenesis - receptor activation, etc (Fig. 6 F and G). Molecular docking was used to verify whether the top 10 core targets had a key role in the treatment of CRC. The docking scores were recorded in the Supplementary Table 1. The docking results were visualized in the PyMol software, marked with Amino acid residues in somatic proteins that are linked to C2F, resulting in the corresponding amino acid number and number of hydrogen bonds. The 3D structure of the docking results was shown in Fig. 7 A and the 2D structure of the docking results was shown in Fig. 7 B. For instance, C2F can form hydrogen bonds with EGFR through GLN-849, TRY-813. The binding energies of C2F to all core targets were lower than − 5 kcal·mol − 1 , showing that C2F has a high biological affinity with the core targets of CRC and has high pharmacodynamic activity. Overall, C2F maybe play a significant role under the treatment of CRC by acting on the above core targets. 3.7 C2F inhibits the malignant biological behaviors of CRC by suppressing STAT3 Prior research has demonstrated that coumarin derivatives possess the capability to impede tumor growth by targeting the STAT3 signaling pathway(Cai et al., 2019a). Moreover, our previous bioinformatics prediction showed that EGFR and STAT3 are the core targets of C2F in the treatment of CRC. In addition, the molecular functions involved in GO function enrichment related targets mainly involved the activity of nuclear receptor. Among the top ten core targets, STAT3 belongs to the signal transducers and activators of transcription (STATs) family. This protein family, present in the cytoplasm, orchestrates a range of biological functions through nuclear activation for DNA binding. These factors collectively indicated that STAT3 could potentially be the primary target for C2F in CRC treatment. We next conducted western blot analysis to determine whether C2F inhibited CRC cells growth by suppressing STAT3 signaling pathway. Western blot analysis results demonstrated that SW620 cells did not express EGFR (Supplementary Fig. 3). The results further demonstrated that total STAT3 and phosphorylated STAT3 (Ser727) expression remained relatively stable with C2F treatment (0, 5 and 10 µM), whereas phosphorylated STAT3 (Tyr705) decreased considerably (Fig. 8 A). The expression level of p-STAT3 (Tyr705) in tumors of C2F experimental group mice was much lower than that of control group mice (Fig. 8 B). This result suggested that inhibition of STAT3 expression by C2F may be associated with reduced p-STAT3 (Tyr705) phosphorylation. To further confirm that STAT3 is the target for C2F to inhibit the biological behavior of CRC, we used the specific STAT3 agonist IL-6 (50 ng/mL) and inhibitor stattic (20 µM) in combination with C2F in CRC cells to re-detected the expression levels of STAT3 and p-STAT3 (Tyr705) again. Compared with the control group, the expression level of p-STAT3 (Tyr705) in DLD-1 and SW620 cells were significantly decreased after the treatment of C2F for 24 hours, while the expression level of total STAT3 did not change significantly. STAT3 agonist increased the expression level of p-STAT3 (Tyr705). When it was combined with C2F, the expression level of p-STAT3 (Tyr705) did not increase, which means that C2F reversed the agonistic effect of STAT3 agonist on STAT3 phosphorylation. STAT3 inhibitor decreased the expression level of p-STAT3 (Tyr705), and when used in conjunction with C2F, the expression level of p-STAT3 (Tyr705) decreased more significantly (Fig. 8 C and D). The above results indicated that C2F inhibits the biological behavior of CRC through suppressing phosphorylation of STAT3 (Tyr705). Moreover, immunofluorescence staining revealed that upon IL-6 stimulation, there was an augmentation of green fluorescent protein within the nucleus, accompanied by a reduction of green fluorescent protein in the cytoplasm (Fig. 9 A), indicated that STAT3 was localized in the cytoplasm and translocated to the nucleus upon IL-6 stimulation C2F could reverse this role of IL-6 in CRC (Fig. 9 A). Furthermore, the STAT3 protein was extracted from both the nucleus and cytoplasm for analysis. Following an hour stimulation with IL-6, there was a noticeable upregulation of STAT3 protein expression within the nucleus, surpassing the levels observed in unstimulated cells. Conversely, there was a discernible reduction in the expression of STAT3 protein within the cytoplasm subsequent to the stimulation. We also found that C2F inhibited IL-6-induced STAT3 nuclear translocation, and the inhibitory effect was more significant with increasing concentration (Fig. 9 B). These results strongly suggested that C2F inhibits STAT3 (Tyr705) phosphorylation and nuclear translocation. 4. Discussion With the increasing understanding of the molecular mechanisms of tumorigenesis and progression, active pharmaceutical ingredients from natural product sources for the treatment of a wide range of malignancies has become a major goal of modern healthcare professionals(Serge et al., 2018). C2F is a derivative resulting from structural modification built upon the foundation of a natural product, coumarin. Medicinal chemists have been actively exploring coumarin-derived pharmacophoric substituents as a promising avenue for the development of anti-cancer drugs(Jing-Jing and Jian-Guo, 2018; Lin et al., 2014). More and more evidences confirm that coumarin derivatives have obvious cytotoxic effects on CRC cells, but they did not mention whether these coumarin derivatives have effects on normal human colon epithelial cells(Lin et al., 2019; Luo et al., 2017). At present, fluoride-containing drugs occupy a considerable proportion in clinical treatment drugs. Modern drugs containing fluorine atoms account for about 35% of all drugs, and these drugs treat fields such as tumors, infections, and metabolic diseases(K, 2008; Sophie et al., 2008). The earliest synthetic fluorine-containing drug was 5-FU, an antimetabolite synthesized for the first time in 1957(C et al., 1957). The drug has high antitumor activity by inhibiting thymidine synthase, and is currently one of the most effective drugs for clinical treatment of CRC(Yushan et al., 2020). Since the emergence of 5-FU, the introduction of fluorine atoms has been widely used in drug modification, and fluorine substitution strategies in drug design have become one of the main research strategies to change the structure of drugs(Bao-Cheng et al., 2017). The present study demonstrated that C2F, a coumarin derivative containing a fluorine-substituted group, exerted inhibitory effects against DLD-1 (IC 50 : 9.33 µM) and SW620 (IC 50 : 10.31 µM) cell line for 72 hours. As one of the main chemotherapy drugs for CRC, the chemotherapy resistance and side effects of 5-FU often limit its efficacy in treating CRC(Wilhelm et al., 2016). However, C2F has no significant inhibitory effect on non-cancerous human colon epithelial cells, suggesting that it has anti-colon cancer potential. In vitro , we observed that C2F exerted anti-CRC effects by inhibiting CRC cell proliferation, inducing CRC cell apoptosis, impeding migration and invasion, and blocking the G2/M phase of the cell cycle. Meanwhile, we demonstrated that C2F significantly inhibits CRC by constructing a xenograft model in vivo . More importantly, blood sampling analysis of the eyeballs indicated that C2F did not cause liver toxicity to nude mice, indicating the safety of C2F. In addition, detection of the expression levels of tumor related proteins revealed inhibition of PCNA and Bcl-2 expression. In recent years, network pharmacology technology has developed rapidly. It is a new bioinformatics method that uses network database and computer intelligent calculation to establish the relationship network between drug, targets and diseases, and to explore the mechanism of drug action on diseases. Based on PPI network and topological parameter analysis, the core targets of C2F in the treatment of CRC include STAT3. The molecular functions involved in the relevant targets in the GO functional enrichment mainly involve nuclear receptor activity, which also suggests that STAT3 may be a core target for C2F treatment of CRC. Activation of STAT3 is regulated by phosphorylation of tyrosine 705 (Tyr705) by receptor tyrosine kinases, leading to its homodimerization, translocation to the nucleus, DNA binding, and downstream transcriptional activity(B et al., 2009; Cai et al., 2019b). And in addition to phosphorylation of Tyr705, phosphorylation of serine residue 727 (Ser727) has been implicated in STAT3 protein transcription, mitochondrial import, and other biological functions(Meier and Larner, 2014; Rui and Mercedes, 2016). Upon phosphorylation at Tyr705, STAT3 dimers and translocates into the nucleus, where it specifically binds to specific DNA sequences and induces transcription of downstream target genes and EMT. Hyperactivation of STAT3 is responsible for CRC cell proliferation and invasion. Inhibition of STAT3 signaling impedes cancer cell growth, suggesting that STAT3 is a promising target for the treatment of CRC. Our data suggest that by inhibiting STAT3 phosphorylation at the Tyr705 site and nuclear translocation, C2F can modulate STAT3 activity, suppress CRC cell tumors and reduce EMT transformation in CRC cells in vitro . In addition, detection of the expression levels of tumor related proteins revealed inhibition of PCNA and Bcl-2 expression. The expression level of p-STAT3 (Tyr705) in tumors of C2F experimental group mice was significantly lower than that of mice in the control group, indicating that C2F can also inhibit tumor cell proliferation and induce tumor cell apoptosis by suppressing the expression of p-STAT3 (Tyr705) in vivo . Therefore, we conclude that C2F inhibits the overexpression of various oncogenic gene products regulated by STAT3 by regulating the activation of STAT3 in xenograft models, ultimately exerting an effective inhibitory effect on tumor growth. In conclusion, the data from this experiment suggests that C2F has anticancer effects both in vivo and in vitro . Figure 10 illustrates the mechanism by which C2F induces apoptosis and cell cycle arrest in CRC cells by altering the STAT3 signaling pathway. Collectively, these results suggest that C2F may be an attractive lead compound for the development of an anti-CRC drug. Abbreviations ethyl 2,2-difluoro-2 - (2-oxo-2H-chromen-3-yl) acetate (C2F); colorectal cancer (CRC); activator of transcription 3 (STAT3); interleukin-6 (IL-6); fluorouracil group (5-FU). Declarations Ethics statement All the experimentation with animals was approved by the IACUC of Shenzhen People's Hospital Laboratory Animal Center by following the Guide for the Care and Use of Laboratory Animals, and carry out experiments in the animal laboratory of Shenzhen People's Hospital Laboratory Animal Center (Serial number: AUP-210317-LY-0003-01). Author contributions Jie LIN, Weijing LIU: conception and design of experiments, performance of experimental procedures, analysis and interpretation of data, writing a manuscript. Xuehong FANG, Wen ZHANG: performance of experimental procedures. Xiaodan LI, Jiansuo LIN, Yanwen LIANG: experiments in vivo in nude mice. Yi LIU, Liyi ZOU, Jianwei REN, Feng WANG: Conceptualization, Funding acquisition, Writing- Reviewing and Editing. Funding The authors declare that they have not received any funding or other support during the preparation process of the manuscript. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Declarations The authors have no relevant fnancial or non-financial interests to disclose. Acknowledgements The authors would like to thank Shenzhen Ruike Biotechnology Co., Ltd for providing the experimental site and equipment required for this study. References UniProt: the universal protein knowledgebase in 2021. Nucleic acids research. Ainhoa, C.-C., Sarah, T., A, S.M., 2019. Treatment of Patients With Early-Stage Colorectal Cancer: ASCO Resource-Stratified Guideline Summary. Journal of oncology practice. B, A.B., B, K.A., B, H.K., R, G.S., T, T.S., Cemile, K., Sanjit, D., Bokyung, S., 2009. Signal transducer and activator of transcription-3, inflammation, and cancer: how intimate is the relationship? Annals of the New York Academy of Sciences 1171(1). 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Molecular mechanism of QH-BJ drug pair in the treatment of systemic lupus erythematosus based on network pharmacology and molecular docking %J Medicine. 101(48). Additional Declarations No competing interests reported. Supplementary Files supplementarytable.docx supplementaryfig1.tif supplementaryfig2.tif supplementaryfig3.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4376706","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":301631027,"identity":"c467bce7-f527-43c4-99cb-e630450ac95d","order_by":0,"name":"JIE LIN","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"JIE","middleName":"","lastName":"LIN","suffix":""},{"id":301631028,"identity":"53bda743-664c-4b2c-89a2-e3cc6d8f9a3b","order_by":1,"name":"Weijing LIU","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Weijing","middleName":"","lastName":"LIU","suffix":""},{"id":301631029,"identity":"fd339019-fc24-46f2-8854-c00324777845","order_by":2,"name":"Xiaodan LI","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiaodan","middleName":"","lastName":"LI","suffix":""},{"id":301631030,"identity":"3dfd3805-ea5b-4eb5-9a4d-0ac0b83c7d01","order_by":3,"name":"Jiansuo LIN","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jiansuo","middleName":"","lastName":"LIN","suffix":""},{"id":301631031,"identity":"f6ec21e3-0822-42bb-a21d-2f48a1920141","order_by":4,"name":"Xuehong FANG","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xuehong","middleName":"","lastName":"FANG","suffix":""},{"id":301631032,"identity":"43641a7f-e5ad-4683-97b2-a5d1197a4f7d","order_by":5,"name":"Yanwen LIANG","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yanwen","middleName":"","lastName":"LIANG","suffix":""},{"id":301631033,"identity":"343c87ac-1efb-4c77-a07f-dda9ff8f5b1e","order_by":6,"name":"Wen ZHANG","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"ZHANG","suffix":""},{"id":301631034,"identity":"afc632d7-189d-4520-ac2c-1048951e829c","order_by":7,"name":"Jianwei REN","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jianwei","middleName":"","lastName":"REN","suffix":""},{"id":301631035,"identity":"c0543e96-3089-4682-95de-8c58ee64c354","order_by":8,"name":"Feng WAGN","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"WAGN","suffix":""},{"id":301631036,"identity":"018a2df3-2dc1-4f77-8107-8634733dcf03","order_by":9,"name":"Liyi ZOU","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Liyi","middleName":"","lastName":"ZOU","suffix":""},{"id":301631037,"identity":"b725703f-5598-42b1-9cf2-faba5bcb903b","order_by":10,"name":"Yi LIU","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYDACduaGA4z/bOQYmInWwszYcICBLc2YNC0MDGyHExuI1mHOzNh4mIcnLX1+O+/BDww1NtEEtVg2MzYc5pGwyd1wmC9ZguFYWi5B6wwOg7QYpOVuYOYxkACyidWScDhdvpnH+AcJWg4cTmA4zGNGnC0gvxyc25BmuAGoxSKBGL+Yszcf/vC2wUZevv+M8Y0PNTZEOAyFl0BIOaaWUTAKRsEoGAXYAABn4zvveZTq2QAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Yi","middleName":"","lastName":"LIU","suffix":""}],"badges":[],"createdAt":"2024-05-06 12:16:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4376706/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4376706/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56442242,"identity":"51af2ad7-4c2d-4acc-998d-0bd2d6a75c97","added_by":"auto","created_at":"2024-05-14 08:45:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":471284,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC2F suppresses proliferation in CRC cells.\u003c/strong\u003e(A) Chemical structure of C2F. (B, C, D) DLD-1, SW620 and HCoEpiC cells were administered various C2F concentrations for 24, 48 or 72 hours, and cell viability was quantitated by the MTT assay. (E, F) DLD-1 and SW620 cells were administered various 5-FU concentrations for 24, 48 or 72 hours, and cell viability was quantitated by the MTT assay. (G) Colony formation assay was performed to analyze cell proliferation. (H) Western blotting analysis for assessing proliferation-associated proteins in DLD-1 and SW620 cells. The effects of various C2F amounts on PCNA protein levels were assessed. β-actin was employed for normalization. Data are mean ± SD from three assays performed independently. *P \u0026lt; 0.05, **P \u0026lt; 0.01 compare to the control group.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4376706/v1/e4d8c58d4c031dcea23c3e81.png"},{"id":56441279,"identity":"b5270662-088f-4a28-9faf-a4ef6148d680","added_by":"auto","created_at":"2024-05-14 08:37:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":204437,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC2F promotes G2/M arrest in CRC cells.\u003c/strong\u003e (A, B) DLD-1 and SW620 cells underwent treatment with C2F, and cell cycle distribution was assessed by flow cytometry. (C, D) Amounts of the G2/M-associated proteins Cyclin B1 and CDK1, assessed by western blot following C2F administration for 24 hours in CRC cells. β-actin was employed for normalization. Data are mean ± SD from three assays performed independently. *P \u0026lt; 0.05, **P \u0026lt; 0.01 compare to the control group.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4376706/v1/c42a0702ed061c3307557d26.png"},{"id":56441287,"identity":"fce71479-6046-4d0c-967c-39f9408bf582","added_by":"auto","created_at":"2024-05-14 08:37:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":582600,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC2F promotes apoptotic death in CRC cells.\u003c/strong\u003e(A, B) Apoptotic rates of DLD-1 and SW620 cells administered various C2F amounts. (C-F) Western blot for assessing apoptosis-associated proteins in DLD-1 and SW620 cells. The effects of various C2F amounts on Bcl-2, Bax, cleaved Caspase-3 and cleaved Caspase-9 protein levels were assessed. β-actin was employed for normalization. Data are mean ± SD from three assays performed independently. *P \u0026lt; 0.05, **P \u0026lt; 0.01 compare to the control group.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4376706/v1/2d6fb2e7bcae589f1d659704.png"},{"id":56441280,"identity":"d8590eab-01da-477e-ac2a-78566a9906de","added_by":"auto","created_at":"2024-05-14 08:37:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1390785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC2F suppresses migration and invasion in CRC cells.\u003c/strong\u003e(A, B) Wound-healing closure assay was performed to analyze cell migration (magnification, ×100). (C, D) Transwell migration assay to detect the effect of C2F on DLD-1 and SW620 cells migration (magnification, ×200). (E, F) Transwell invasion assay to detect the effect of C2F on DLD-1 and SW620 cells invasion (magnification, ×200). (G, H) Western blot for assessing several markers associated with the EMT process in DLD-1 and SW620 cells. The effects of various C2F amounts on E-cadherin, N-cadherin and vimentin protein levels were assessed. β-actin was employed for normalization. Data are mean ± SD from three assays performed independently. *P \u0026lt; 0.05, **P \u0026lt; 0.01 compare to the control group.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4376706/v1/55633a11247e4d760967d320.png"},{"id":56441289,"identity":"479bf6ec-36d9-4414-a847-0e2dcaf18ecf","added_by":"auto","created_at":"2024-05-14 08:37:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":368195,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of C2F on tumor growth \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e(A) Images of tumors derived from mice in each group. (B, C) Volumes and weights of tumor xenografts in various groups. (D) Weight changes in mice during 21 d. (E, F) Blood from mice was collected at the end of the experiment and the levels of AST and ALT were analyzed. (G, H) Homogenized fresh tumor tissues; western blot was used to detect the expression of proliferation and apoptosis related proteins. β-actin was employed for normalization. Data are mean ± SD from three assays performed independently. *P \u0026lt; 0.05, **P \u0026lt; 0.01 compare to the control group.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4376706/v1/671834a30d7887c990f2b37a.png"},{"id":56441284,"identity":"dbe11c84-a1b5-4dee-bf44-1efb2c46cb82","added_by":"auto","created_at":"2024-05-14 08:37:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":970801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNetwork pharmacological analysis of C2F in the treatment of CRC.\u003c/strong\u003e(A) Venn diagram of C2F target protein and CRC target protein. (B, C) PPI visual network diagram. (D) Precise tests of biological processes (BP), molecular functions (MF) and cellular components (CC) categories were performed using metascape, and graphene oxide terms with enriched p values less than 0.01 were used. The vertical axis represents the amount of protein in each item; the horizontal axis represents GO terms in each category. (E) KEGG pathway enrichment analysis. (F) Target pathway network of C2F therapy for CRC. (G) “KEGG-BP-CC-MF-target” network.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4376706/v1/b14d122940445bb2e2d0bb1f.png"},{"id":56442246,"identity":"e93531f9-6680-4823-9683-2ef1ec9d317a","added_by":"auto","created_at":"2024-05-14 08:45:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1018482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular docking of C2F and core targets.\u003c/strong\u003e (A, B) 3D and 2D structure diagram of molecular docking between C2F and core targets. (green = target protein, blue = amino acid residues interacting with compound, red = C2F, yellow = hydrogen bond)\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4376706/v1/4eb67350d6bbbe79693aa79e.png"},{"id":56441291,"identity":"44623588-3798-4246-a1b0-0b975872cb5d","added_by":"auto","created_at":"2024-05-14 08:37:56","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":373127,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC2F modulates protein expression involved in STAT3 signaling pathway.\u003c/strong\u003e(A) Expression of proteins related to STAT3 signaling pathways as detected by western blot analysis. (B) Western blot analysis detected the expression of STAT3 signal pathway related proteins in tumor tissue. (C, D) DLD-1 and SW620 cells exposed to IL-6 and stattic for 1 hour after treatment with C2F for 24 hours; expression levels of proteins related to STAT3 signaling pathways as detected by western blot analysis. β-actin was employed for normalization. Data are mean ± SD from three assays performed independently. *P \u0026lt; 0.05, **P \u0026lt; 0.01 compare to the control group.\u003c/p\u003e","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-4376706/v1/625e8dc119e174d88134b99e.png"},{"id":56441285,"identity":"280e4c43-7b94-45ee-90b4-08981f123165","added_by":"auto","created_at":"2024-05-14 08:37:55","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":445446,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC2F inhibits the malignant biological behaviors of CRC by suppressing STAT3. \u003c/strong\u003e(A) The cellular localization of the protein was observed by immunofluorescence staining in DLD-1 and SW620 cells; analysis of changes in the intracellular position of STAT3 (magnification, ×400). (B) Nuclear and cytosolic proteins extracted from SW620 after treatment; distribution of STAT3 in cells as determined by western blot analysis. LaminB and β-actin was employed for normalization. Data are mean ± SD from three assays performed independently. *P \u0026lt; 0.05, **P \u0026lt; 0.01 compare to the control group. \u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01 compare to the IL-6 group.\u003c/p\u003e","description":"","filename":"fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-4376706/v1/f3d0bab81514d8ef801bd0cd.png"},{"id":56441288,"identity":"700fc599-02a4-4d6c-90ab-f393f40d35e7","added_by":"auto","created_at":"2024-05-14 08:37:55","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":181568,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThis diagram describes the mechanism of C2F inducing CRC cell apoptosis and cell cycle arrest by suppressing STAT3 signal pathway.\u003c/strong\u003eWe speculate that C2F may inhibit the expression of p-STAT3 (Tyr705) and modulate the expression of Bcl-2 family, EMT-related proteins, CDK1, and cyclin B1, which further promotes apoptosis and cell cycle arrest in G2/M phase of CRC cells.\u003c/p\u003e","description":"","filename":"fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-4376706/v1/cdeb0df12b91319c4e49a03e.png"},{"id":57520963,"identity":"f72d52d3-425d-431b-bbe7-04aefe4e390f","added_by":"auto","created_at":"2024-05-31 21:46:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6981802,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4376706/v1/33fe94f9-b37b-4871-86fc-c523a8e16d3e.pdf"},{"id":56441277,"identity":"299cbfd0-2f4c-4742-94e5-60c99b2fada2","added_by":"auto","created_at":"2024-05-14 08:37:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12276,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarytable.docx","url":"https://assets-eu.researchsquare.com/files/rs-4376706/v1/883693f7cdf854d8ab12069b.docx"},{"id":56442244,"identity":"b9983033-e058-46db-81a9-fe6bab3cf1d5","added_by":"auto","created_at":"2024-05-14 08:45:55","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":911806,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4376706/v1/31ffda58e4f3a7df6b93839a.tif"},{"id":56442881,"identity":"4d19806d-1fd6-4265-94d5-e2d87165e6b2","added_by":"auto","created_at":"2024-05-14 08:53:55","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":887372,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfig2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4376706/v1/8fd1542c9d4b5fefcfdcc1a6.tif"},{"id":56442245,"identity":"383ac689-4a61-43f0-9f78-6342b28744a1","added_by":"auto","created_at":"2024-05-14 08:45:55","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":171074,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfig3.tif","url":"https://assets-eu.researchsquare.com/files/rs-4376706/v1/f343a4fcb3f1eda89d7f0cc3.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ethyl 2,2-difluoro-2-(2-oxo-2H-chromen-3-yl) acetate inhibits the malignant biological behaviors of colorectal cancer by restricting the phosphorylation and nuclear translocation of STAT3","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGlobal cancer statistics show that CRC is the second most common cause of cancer death in both males and females(Siegel et al., 2024). With the advancement of colonoscopy technology, surgical resection of intestinal tumors has arisen as an effective treatment program for patients in the initial stages of CRC(Ainhoa et al., 2019). However, most patients are usually diagnosed in the late stage, and combination chemotherapy remains the main treatment option that can provide systematic and sustained efficacy. Unsatisfactorily, the predominant hurdles in achieving successful CRC treatment lie in the adverse toxic effects and development of therapeutic resistance associated with chemotherapy medications(Benoist et al., 2015; Peeters et al., 2010). Hence, there is a pressing need for continued investigation into targets or agents that have minimal side effects in CRC therapy, ultimately enhancing the treatment results for CRC patients.\u003c/p\u003e \u003cp\u003eUp to this point, approximately 85% of sanctioned small molecule anticancer drugs have originated either directly or indirectly from natural sources(J and M, 2016). Coumarin (benzopyran-2-one) is a typical natural product, which is derived from plants, bacteria and fungi(Maria et al., 2016; Saleta et al., 2015). Studies have found that coumarin compounds have various biological activities, such as anti-inflammatory(Chaoyu et al., 2018), antiviral(Lei et al., 2020), antibacterial(Ivana et al., 2021; Lipeeva et al., 2019), antioxidant(Jinxiu et al., 2020) and antitumor(Li et al., 2015; Lingaraju et al., 2018; Luo et al., 2017; Zhou et al., 2016), etc. A series of coumarin derivatives effective for different cancers can be obtained by structural modification on the basis of the coumarin nucleus(Mintas et al., 2012; Nadia et al., 2011). In our preliminary research, we found that C2F has significant anti-cancer activity, however, the exact mechanism of cancer treatment has not yet been elucidated. Therefore, C2F shows potential for development as an anti-cancer drug.\u003c/p\u003e \u003cp\u003eThis study investigates the impact of C2F on the malignant biological behaviors of CRC through \u003cem\u003eex vivo\u003c/em\u003e experiments. It also predicts potential targets of C2F activity in CRC using network pharmacology and molecular docking analysis. The results demonstrate that C2F significantly suppresses the malignant biological behaviors of CRC both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e by inhibiting STAT3 phosphorylation and constraining its nuclear translocation. To our knowledge, this is the first investigation into the role and mechanism of C2F in the treatment of CRC. Thus, this study will establish the theoretical foundation for the potential development of C2F as a therapeutic agent for CRC.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Antibodies and reagents\u003c/h2\u003e \u003cp\u003eC2F, chemical structure shown as Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, was synthesized by Shenzhen Small Molecule New Drug Innovation Center Co., Ltd. Antibodies of β-actin, PCNA, Bax, Bcl-2, Cleaved Caspase-9, Cleaved Caspase-3, E-Cadherin, Vimentin, N-Cadherin, PI3K were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies of cyclin B1, CDK1, STAT3, Phosphorylated STAT3 (Tyr705), Phosphorylated STAT3 (Ser727) were purchased from Zen bioscience (Chengdu, China). BeyoClick\u0026trade; EdU-594 and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were supplied by Beyotime Biotechnology. The recombinant human IL-6 was purchased from Peprotech (London, UK). The STAT inhibitor Stattic was purchased from MedChem Express (NJ, USA). Matrigel was purchased from ABW (CAT: 082704). Exkine\u0026trade; Nuclear and Cytoplasmic Protein Extraction Kit was supplied by Abbkine (Wuhan, China). Alanine aminotransferase (ALT) test kit and aspartate aminotransferase (AST) test kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell cultures\u003c/h2\u003e \u003cp\u003eThe CRC cell lines DLD-1, SW620 and human normal colon cells HCoEpiC used in this study were developed and provided by Shenzhen Ruike Biotechnology Co., Ltd. Cell culture has been described in detail in our previous research(Xuehong et al., 2022).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 MTT cytotoxicity assay\u003c/h2\u003e \u003cp\u003eAfter overnight attachment, the DLD-1, SW620 and HCoEpiC cells were processed with a multi-Concentrationn of C2F (0, 1, 5, 10, 15, and 20 \u0026micro;M) and 5-FU (5, 10 and 15 \u0026micro;M). We followed the MTT experimental steps for subsequent operations(Priti et al., 2018).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Colony formation assay\u003c/h2\u003e \u003cp\u003eDLD-1 and SW620 cells (500 per well) were seeded in 6-well plates overnight and then the medium was replaced with different concentrations of C2F. Two weeks later, 4% paraformaldehyde was added for 15 min for cell fixation and then aspirated, washed twice with PBS and finally stained with 0.1% crystal violet for 15 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 EdU cell proliferation detection\u003c/h2\u003e \u003cp\u003eThe proliferative capacity of DLD-1 and SW620 cells were treated by EdU method according to the protocol provided by the manufacturer. Briefly, seeded of cells into 24-well plates and after adhesion, cells were stimulated with different concentrations of C2F for 24 hours. Then the reaction system was prepared according to BeyoClick\u0026trade; EdU-594, and the reaction system was incubated at room temperature for 30 min, protected from light. Finally, the nuclei were stained with Hoechst33342 reaction solution. The results were observed by an inverted fluorescence microscope (200 \u0026times; magnification, Olympus, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cell cycle and apoptosis analysis\u003c/h2\u003e \u003cp\u003eThe methods for analyzing cell cycle and apoptosis have been specifically described in our previous research(Xuehong et al., 2022).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Hoechst 33342 staining\u003c/h2\u003e \u003cp\u003eDLD-1 and SW620 cells were seeded in 12-well plates at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well. After 24 hours, we followed the Hoechst 33342 staining experimental steps for subsequent operations(Yao et al., 2020). Fluorescence microscopy was used for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Wound-healing assay\u003c/h2\u003e \u003cp\u003eThe wound healing assay was conducted with reference to our previous study(Xuehong et al., 2022).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Transwell migration/invasion assay\u003c/h2\u003e \u003cp\u003eTo rule out the effects of cell proliferation inhibition and induction of cell apoptosis on migration and invasion assay, we first set 4 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e DLD-1 cells and SW620 cells were suspended in 200 \u0026micro;l serum-free medium and then inoculated into the upper chamber, while the lower chamber was filled with 600 \u0026micro;l of medium containing 20% FBS. After 48 hours, fixed the cells on the pore membrane at the bottom of the chamber, and then soaked the cells in crystal violet staining solution for staining. Finally, the chamber was observed and photographed under an optical microscope, and the inhibitory effect of C2F on the migration and invasion of DLD-1 cells and SW620 cells was statistically analyzed using Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Western blot analysis\u003c/h2\u003e \u003cp\u003eAfter overnight attachment, the cells were processed with a gradient concentration of C2F (0, 1, 5, 10, 15, and 20 \u0026micro;M). The cells were then washed gently with PBS 2 times. The CRC cells were lysed using cell lysates for 30 min on ice, which were transferred to new tubes and then centrifuged. Protein concentration was determined by Coomassie Brilliant Blue staining to configure the reaction system. Protein was separated by sodium dodecyl sulfate- polyacryl- amide gel electrophoresis gel and transferred onto polyvinylidene difluoride membranes. The membranes were then blocked with 5% skim milk for 1.5 h and then washed with Tris Buffered Saline with Tween 20. The blots were ultimately incubated with specific primary antibodies and incubated with secondary antibodies on the following day. The membranes were imaged using the BIO-RAD high sensitivity chemiluminescence imaging system ChemiDoc touch and the software Image J was used to analyze the reactive bands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Predicting the therapeutic targets of C2F for CRC by bioinformatic analysis\u003c/h2\u003e \u003cp\u003eWe downloaded the structure of C2F (PubChem CID: 132961735) from the PubChem(Kim et al., 2019) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/),an\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/),an\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003ed input it into the SwissTarget-Prediction(David et al., 2014) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://www.swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Pharmmapper(Xiaofeng et al., 2010) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://www.swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) platforms to predict the potential targets of C2F. The Uniprot(2020) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to convert the protein target name into the corresponding gene symbol. We merged targets from two databases and removed duplicates to obtain targets related to C2F.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Determination of potential CRC-related targets\u003c/h2\u003e \u003cp\u003eFour databases were then searched using Genes related to CRC: Genecards(Gil et al., 2016) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), OMIM(S et al., 2019) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://omim.org/\u003c/span\u003e\u003cspan address=\"https://omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), TTD(Yunxia et al., 2020) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://db.idrblab.net/ttd/\u003c/span\u003e\u003cspan address=\"https://db.idrblab.net/ttd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and DrugBank(S et al., 2018) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.drugbank.com/\u003c/span\u003e\u003cspan address=\"https://www.drugbank.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). After the removal of repeated targets, Venny2.1.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.liuxiaoyuyuan.cn/\u003c/span\u003e\u003cspan address=\"http://www.liuxiaoyuyuan.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to study the intersection of C2F with pathologic targets to identify potential targets for C2F in the treatment of colorectal cancer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Network construction and enrichment analysis\u003c/h2\u003e \u003cp\u003eTo identify the possible interactions between all target genes in the pharmacological network, we constructed the PPI network using the STRING(Damian et al., 2019) database. When we used the database, we selected \"Homo sapiens\" for the species, chose 0.4 as the cutoff value for the confidence score, and left all other settings unchanged. Cytoscape(Paul et al., 2003) software version 3.8.2 was used to analyze and visualize the network topology of the results obtained by STRING. Nodes in the network diagram were intersection targets, and edges represent the relationship between targets and targets. We used the built-in Network Analyzer program to analyze network topology parameters such as degree, betweenness centrality, closeness centrality, etc.(Ziyu et al., 2022). Next, we used CytoHubba, a Cytoscape plugin that can be used for network centrality analysis, to identify the centroids in the network. The 10 genes with the highest degree centrality values were identified as central genes. Analysis of GO functional enrichment and KEGG pathway enrichment for target genes was performed using metascape(Yingyao et al., 2019) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metascape.org/gp/index.html#/main/step1\u003c/span\u003e\u003cspan address=\"https://metascape.org/gp/index.html#/main/step1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database. Bioinformatics (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bioinformatics.com.cn/\u003c/span\u003e\u003cspan address=\"http://www.bioinformatics.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) platform was used to draw a bar graph and a bubble graph.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Targets-pathways network construction\u003c/h2\u003e \u003cp\u003eCytoscape software version 3.8.2 was used to build a targets-pathways network. The nodes in the network were targets and pathways, respectively, and the edges represented the interaction relationship between targets and pathways.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Molecular docking validation of C2F and core targets\u003c/h2\u003e \u003cp\u003eThe most important genes were selected for subsequent molecular docking analysis. The 3D structure of the ligand molecule (C2F) was first downloaded from the PubChem database. The Uniprot database was searched for receptor protein encoded by the input genes. The 3D structure of the protein was obtained from RCSB PDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and modified using PyMol software version 2.4.0, including ligand and water removal. Autodocktools was used to calculate protein loads. Finally, receptor proteins were docked to C2F using Autodock Vina and visualized using PyMol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eWe first inoculated DLD-1 and SW620 cells onto the glass slides, then fixed the cells with 4% paraformaldehyde, penetrated the cells with 0.5% Triton X-100 (in PBS), rinsed several times with PBS, and finally blocked the cells with 1% bovine serum albumin for 1 h. The cells were incubated with the primary antibody in an appropriate proportion, and the second day with the secondary antibody. We stained the cells with DAPI in the dark, then washed the cell slides with PBS again, and sealed them with anti-fluorescence quenching sealing sheets to prevent fluorescence quenching until they were captured by confocal microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17 Cytoplasmic and nuclear protein extraction\u003c/h2\u003e \u003cp\u003eDLD-1 and SW620 cells were cultured at 80%-90% concentration in 10 cm dishes and treated with C2F (0, 5 and 10 \u0026micro;M). Immediately after IL-6 stimulation for 1 hour, cells were placed on ice. Nuclear and cytoplasmic proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit. The expression of related proteins was detected by western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.18 Animal experiments\u003c/h2\u003e \u003cp\u003e Animal experiments are conducted in strict compliance with international standards and the 3R's of animal protection, and through the Shenzhen People's Hospital Laboratory Animal Ethics Committee approval. Approximately 5 weeks old female balb/c nude mice (18\u0026ndash;20 g) were acqired from the Laboratory Animal Center of Guangdong Medical University (Dongguan, China) and were housed at the Laboratory Animal Center of Shenzhen People's Hospital. Approximately 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e SW620 cells suspended in 0.1 ml of serum-free culture medium and Matrigel (medium:Matrigel\u0026thinsp;=\u0026thinsp;1:1) were subcutaneously injected into the right flank of each mouse. Tumor volume was calculated using recognized calculation methods(Yao et al., 2020). In our previous research, we found that C2F has an inhibitory effect on the growth of non-small cell lung cancer cell xenograft tumors. Accordingly, the mice were randomly divided into five groups in this study (control group, mice were intraperitoneally injected with olive oil five times per week. C2F group A were intraperitoneally injected with 10 mg/kg C2F five times per week, C2F group B were intraperitoneally injected with 20 mg/kg C2F five times per week, C2F group C were intraperitoneally injected with 30 mg/kg C2F five times per week, fluorouracil group, mice were intraperitoneally injected with 20 mg/kg fluorouracil three times per week. After 21 days of treatment, blood was collected from the posterior orbital sinus of nude mice for further analysis. Next, the nude mice were sacrificed by spinal dislocation and the tumors were obtained and weighed. The tumors were fixed in formalin and used for histological studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.19 Statistical analysis\u003c/h2\u003e \u003cp\u003eEach experiment was repeated at least three times independently to ensure reproducibility and reliability and statistically analyzed using Prism (version 8.0, GraphPad Software, San Diego California, U.S.A.). All results are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. One -way analysis of variance (ANOVA) was used for comparisons among multiple groups. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to denote statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.1 C2F inhibits the proliferation of human CRC cells\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of C2F on the proliferation of CRC cells, DLD-1 and SW620 cells were treated with different concentrations of C2F, and the proliferation of DLD-1 and SW620 cells was detected by MTT assay after 24, 48, and 72 hours. As a result, cell proliferation activity is manifested as a concentration and time dependent decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and C). Interestingly, C2F treatment had no significant effect on the viability of HCoEpiC cells derived from the human normal colon epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In addition, to ensure the reliability and rigor of the experiment, 5-FU was used as a positive control for the MTT experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and F). Subsequently, we detected the effect of C2F on cell proliferation by EdU staining and colony formation assay. The results indicated that the number of EdU-positive cells and colonies was significantly reduced by C2F treatment, and this inhibitory effect was dose-related (Supplementary Fig.\u0026nbsp;1 and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). PCNA expression correlates with malignancy, vascular infiltration, distant metastasis, and survival and has been identified as a biomarker for CRC adenocarcinoma(Katarzyna et al., 2009). We examined the protein expression level of PCNA and showed that PCNA expression decreased with increasing C2F concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). These data suggested that C2F not only inhibits the proliferation of CRC cells in a doses and time dependent manner but is less toxic to HCoEpiC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.2 C2F induces G2/M cell cycle arrest in CRC cells\u003c/h2\u003e \u003cp\u003eInduction of cell cycle arrest is a promising strategy in cancer therapy(Tobias and Piotr, 2017). To determine whether C2F inhibits cell growth through cell cycle arrest, we performed flow cytometry and western blot analysis. The results showed that C2F increased the number of G2/M phases in DLD-1 and SW620 cells in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). The central role of the CDK1/cyclin B1 complex (CCNB1) in the cell cycle of cervical cancer has been well described. The complex phosphorylates the corresponding substrate and promotes the transition of the cell cycle from G2 to M, the mitotic phase(Nagappan et al., 2017). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D, CDK1 and cyclin B1 expression were decreased upon C2F administration. The above data suggested that C2F inhibits the proliferation of DLD-1 and SW620 cells by inducing G2/M phase arrest.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.3 C2F promotes apoptosis in CRC cells\u003c/h2\u003e \u003cp\u003eInduction of cancer cell apoptosis is one of the important indicators for evaluating the inhibition of cancer cell growth by antitumor drugs(Bingwu et al., 2021). We then investigated whether the inhibitory effect of C2F on CRC cell proliferation involved the induction of apoptosis. Annexin-V/PI double staining revealed that C2F increased the apoptosis rate of DLD-1 cells from 9.69\u0026ndash;30.53%, and SW620 cells from 5.51\u0026ndash;30.69% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). Hoechst 33342 staining showed that after 24 h of C2F treatment, DLD-1 and SW620 cells showed obvious apoptotic morphology, which was manifested as enhanced blue fluorescence of the cells indicated by the arrow (Supplementary Fig.\u0026nbsp;2). As the Bax/Bcl-2 ratio rises, cytochrome c is released, followed by the cleavage and activation of caspase-3 and caspase-9, which can cause the destruction of intracellular substrates and apoptosis, according to earlier research(E et al., 2014). Data from western blot showed that after 24 hours of C2F treatment, the expression of pro-apoptotic-related proteins such as Bax, cleaved caspase-9 and cleaved caspase-3 were increased while the expression of anti-apoptotic-related proteins such as Bcl-2 was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-F). Overall, C2F markedly promoted apoptosis in CRC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.4 C2F inhibits migration and invasion of human CRC cells\u003c/h2\u003e \u003cp\u003eAbnormal differentiation, migration, and metastases are representative biological features of tumor cells(Wen et al., 2019). The effect of C2F on the migration and invasion of CRC cell lines was investigated using a wound healing assay and a transwell assay to determine whether biological processes related to migration and invasion are involved in the anti-tumour effect of C2F in CRC cell lines. Comparison with control group, C2F reduced the cell migration ability of DLD-1 and SW620 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). The results of transwell migration assay showed that C2F effectively suppressed the number of DLD-1 and SW620 cells in the lower lumen (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). We also used the transwell invasion assay to determine the invasive activity of CRC cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and F, C2F inhibited the invasive potential of CRC cells in a dose-dependent manner. At the same time, we further measured the expression level of major functional proteins during epithelial-mesenchymal transformation (EMT). The results indicated that the epithelial marker E-cadherin was up-regulated after C2F treatment. On the contrary, the expression of N-cadherin and vimentin was decreased, indicating deprivation of mesenchymal phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG and H). The above results indicated that C2F significantly inhibits the EMT process of DLD-1 and SW620 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.5 C2F suppresses CRC tumor growth in vivo\u003c/h2\u003e \u003cp\u003eTo investigate the effect of C2F on the growth of CRC \u003cem\u003ein vivo\u003c/em\u003e, SW620 cells were transplanted into BALB/c nude mice. The experimental data indicated that the tumor volume of the C2F and 5-FU groups was smaller than that in the control group, confirming the inhibitory effect of C2F on CRC cells \u003cem\u003ein vivo\u003c/em\u003e. There was no statistical difference in tumor volume between 5-FU (20 mg/kg) treatment group and C2F (30 mg/kg) treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). During the whole treatment period, the nude mice did not lose body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Accordingly, the liver function test results also indicated that, comparison with control group, the AST of 5-FU group and C2F treatment group showed a downward trend while the ALT did not change significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and F), suggesting that C2F treatment was safe and did not increase the toxicity \u003cem\u003ein vivo\u003c/em\u003e. In order to study the potential mechanism of C2F inhibiting tumor growth, we then detected the expression of PCNA, Bcl-2, Bax in SW620 xenotransplantation tissues by western blot. To sum up, these data indicated that the anti-tumor effects of C2F in tumor xenografts may be associated with anti-proliferative, and pro-apoptotic mechanisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Bioinformatics analysis of the anti-CRC mechanism of C2F\u003c/h2\u003e \u003cp\u003eThrough searching, a total of 86 C2F-related targets and 2831 CRC-related targets were screened after deleting duplicates. Then, 40 potential C2F targets for the treatment of CRC were obtained by intersection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The protein-protein interaction network from the STRING database showed that there are complex interactions between proteins encoded by these target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). We studied the relationship between nodes and edges by referring to previous research methods of Zhenshuang Yuan(Zhenshuang et al., 2023). Using Cytoscape, we performed further analysis of the imported PPI network for more information. Nodes were sorted based on their size and color, with larger nodes and darker colors indicating more critical targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Finally, 10 core target genes were obtained by using CytoHubba, including epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), estrogen receptor 1 (ESR1), matrix metalloprotein 9 (MMP9), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase 8 (MAPK8), mechanistic target of rapamycin (MTOR), androgen receptor (AR), progesterone receptor (PGR), nuclear receptor subfamily 3 group C member 1 (NR3C1). GO functional enrichment analysis indicated that a total of 589 GO items were screened, including 507 items for biological process and 54 items for molecular function and 28 items for cellular component. In the GO analysis results (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), the changes of BP were mainly reflected in the regulation of intracellular receptors, inflammatory responses, responses to reactive oxygen species, rhythmic process and responses to estradiol. In terms of CC, C2F is closely related to all parts of the cell, including intracellular matrix, cell membrane, extracellular matrix, receptor complexes, dendrites, vacuoles. In terms of MF, C2F is mainly related to the binding of enzymes, proteins, phosphatases, and kinases, protein serine/threonine/tyrosine kinase activity, and nuclear receptor activity regulation. This result confirms that C2F can treat CRC by modulating multiple biological pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKEGG pathway analysis further showed that C2F targeted proteins are involved in pathways in cancer, endocrine resistance, chemical carcinogenesis - receptor activation, lipid and atherosclerosis, phospholipase D signaling pathway, transcriptional misregulation in cancer, PPAR signaling pathway, which indicated that C2F can exert anti-CRC through multiple signaling pathways, and its targets may be the common targets of various malignant cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). We next used Cytoscape to analyze the network topology parameters of signaling pathways and targets, and the core targets were EGFR, STAT3, etc; the main pathways were pathways in cancer signaling pathway, endocrine resistance, Chemical carcinogenesis - receptor activation, etc (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF and G). Molecular docking was used to verify whether the top 10 core targets had a key role in the treatment of CRC. The docking scores were recorded in the Supplementary Table\u0026nbsp;1. The docking results were visualized in the PyMol software, marked with Amino acid residues in somatic proteins that are linked to C2F, resulting in the corresponding amino acid number and number of hydrogen bonds. The 3D structure of the docking results was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and the 2D structure of the docking results was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB. For instance, C2F can form hydrogen bonds with EGFR through GLN-849, TRY-813. The binding energies of C2F to all core targets were lower than \u0026minus;\u0026thinsp;5 kcal\u0026middot;mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, showing that C2F has a high biological affinity with the core targets of CRC and has high pharmacodynamic activity. Overall, C2F maybe play a significant role under the treatment of CRC by acting on the above core targets.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.7 C2F inhibits the malignant biological behaviors of CRC by suppressing STAT3\u003c/h2\u003e \u003cp\u003ePrior research has demonstrated that coumarin derivatives possess the capability to impede tumor growth by targeting the STAT3 signaling pathway(Cai et al., 2019a). Moreover, our previous bioinformatics prediction showed that EGFR and STAT3 are the core targets of C2F in the treatment of CRC. In addition, the molecular functions involved in GO function enrichment related targets mainly involved the activity of nuclear receptor. Among the top ten core targets, STAT3 belongs to the signal transducers and activators of transcription (STATs) family. This protein family, present in the cytoplasm, orchestrates a range of biological functions through nuclear activation for DNA binding. These factors collectively indicated that STAT3 could potentially be the primary target for C2F in CRC treatment.\u003c/p\u003e \u003cp\u003eWe next conducted western blot analysis to determine whether C2F inhibited CRC cells growth by suppressing STAT3 signaling pathway. Western blot analysis results demonstrated that SW620 cells did not express EGFR (Supplementary Fig.\u0026nbsp;3). The results further demonstrated that total STAT3 and phosphorylated STAT3 (Ser727) expression remained relatively stable with C2F treatment (0, 5 and 10 \u0026micro;M), whereas phosphorylated STAT3 (Tyr705) decreased considerably (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The expression level of p-STAT3 (Tyr705) in tumors of C2F experimental group mice was much lower than that of control group mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). This result suggested that inhibition of STAT3 expression by C2F may be associated with reduced p-STAT3 (Tyr705) phosphorylation. To further confirm that STAT3 is the target for C2F to inhibit the biological behavior of CRC, we used the specific STAT3 agonist IL-6 (50 ng/mL) and inhibitor stattic (20 \u0026micro;M) in combination with C2F in CRC cells to re-detected the expression levels of STAT3 and p-STAT3 (Tyr705) again. Compared with the control group, the expression level of p-STAT3 (Tyr705) in DLD-1 and SW620 cells were significantly decreased after the treatment of C2F for 24 hours, while the expression level of total STAT3 did not change significantly. STAT3 agonist increased the expression level of p-STAT3 (Tyr705). When it was combined with C2F, the expression level of p-STAT3 (Tyr705) did not increase, which means that C2F reversed the agonistic effect of STAT3 agonist on STAT3 phosphorylation. STAT3 inhibitor decreased the expression level of p-STAT3 (Tyr705), and when used in conjunction with C2F, the expression level of p-STAT3 (Tyr705) decreased more significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and D). The above results indicated that C2F inhibits the biological behavior of CRC through suppressing phosphorylation of STAT3 (Tyr705).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, immunofluorescence staining revealed that upon IL-6 stimulation, there was an augmentation of green fluorescent protein within the nucleus, accompanied by a reduction of green fluorescent protein in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA), indicated that STAT3 was localized in the cytoplasm and translocated to the nucleus upon IL-6 stimulation C2F could reverse this role of IL-6 in CRC (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). Furthermore, the STAT3 protein was extracted from both the nucleus and cytoplasm for analysis. Following an hour stimulation with IL-6, there was a noticeable upregulation of STAT3 protein expression within the nucleus, surpassing the levels observed in unstimulated cells. Conversely, there was a discernible reduction in the expression of STAT3 protein within the cytoplasm subsequent to the stimulation. We also found that C2F inhibited IL-6-induced STAT3 nuclear translocation, and the inhibitory effect was more significant with increasing concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). These results strongly suggested that C2F inhibits STAT3 (Tyr705) phosphorylation and nuclear translocation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eWith the increasing understanding of the molecular mechanisms of tumorigenesis and progression, active pharmaceutical ingredients from natural product sources for the treatment of a wide range of malignancies has become a major goal of modern healthcare professionals(Serge et al., 2018). C2F is a derivative resulting from structural modification built upon the foundation of a natural product, coumarin. Medicinal chemists have been actively exploring coumarin-derived pharmacophoric substituents as a promising avenue for the development of anti-cancer drugs(Jing-Jing and Jian-Guo, 2018; Lin et al., 2014). More and more evidences confirm that coumarin derivatives have obvious cytotoxic effects on CRC cells, but they did not mention whether these coumarin derivatives have effects on normal human colon epithelial cells(Lin et al., 2019; Luo et al., 2017). At present, fluoride-containing drugs occupy a considerable proportion in clinical treatment drugs. Modern drugs containing fluorine atoms account for about 35% of all drugs, and these drugs treat fields such as tumors, infections, and metabolic diseases(K, 2008; Sophie et al., 2008). The earliest synthetic fluorine-containing drug was 5-FU, an antimetabolite synthesized for the first time in 1957(C et al., 1957). The drug has high antitumor activity by inhibiting thymidine synthase, and is currently one of the most effective drugs for clinical treatment of CRC(Yushan et al., 2020). Since the emergence of 5-FU, the introduction of fluorine atoms has been widely used in drug modification, and fluorine substitution strategies in drug design have become one of the main research strategies to change the structure of drugs(Bao-Cheng et al., 2017). The present study demonstrated that C2F, a coumarin derivative containing a fluorine-substituted group, exerted inhibitory effects against DLD-1 (IC\u003csub\u003e50\u003c/sub\u003e: 9.33 \u0026micro;M) and SW620 (IC\u003csub\u003e50\u003c/sub\u003e: 10.31 \u0026micro;M) cell line for 72 hours. As one of the main chemotherapy drugs for CRC, the chemotherapy resistance and side effects of 5-FU often limit its efficacy in treating CRC(Wilhelm et al., 2016). However, C2F has no significant inhibitory effect on non-cancerous human colon epithelial cells, suggesting that it has anti-colon cancer potential.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e, we observed that C2F exerted anti-CRC effects by inhibiting CRC cell proliferation, inducing CRC cell apoptosis, impeding migration and invasion, and blocking the G2/M phase of the cell cycle. Meanwhile, we demonstrated that C2F significantly inhibits CRC by constructing a xenograft model \u003cem\u003ein vivo\u003c/em\u003e. More importantly, blood sampling analysis of the eyeballs indicated that C2F did not cause liver toxicity to nude mice, indicating the safety of C2F. In addition, detection of the expression levels of tumor related proteins revealed inhibition of PCNA and Bcl-2 expression.\u003c/p\u003e \u003cp\u003eIn recent years, network pharmacology technology has developed rapidly. It is a new bioinformatics method that uses network database and computer intelligent calculation to establish the relationship network between drug, targets and diseases, and to explore the mechanism of drug action on diseases. Based on PPI network and topological parameter analysis, the core targets of C2F in the treatment of CRC include STAT3. The molecular functions involved in the relevant targets in the GO functional enrichment mainly involve nuclear receptor activity, which also suggests that STAT3 may be a core target for C2F treatment of CRC. Activation of STAT3 is regulated by phosphorylation of tyrosine 705 (Tyr705) by receptor tyrosine kinases, leading to its homodimerization, translocation to the nucleus, DNA binding, and downstream transcriptional activity(B et al., 2009; Cai et al., 2019b). And in addition to phosphorylation of Tyr705, phosphorylation of serine residue 727 (Ser727) has been implicated in STAT3 protein transcription, mitochondrial import, and other biological functions(Meier and Larner, 2014; Rui and Mercedes, 2016). Upon phosphorylation at Tyr705, STAT3 dimers and translocates into the nucleus, where it specifically binds to specific DNA sequences and induces transcription of downstream target genes and EMT. Hyperactivation of STAT3 is responsible for CRC cell proliferation and invasion. Inhibition of STAT3 signaling impedes cancer cell growth, suggesting that STAT3 is a promising target for the treatment of CRC.\u003c/p\u003e \u003cp\u003eOur data suggest that by inhibiting STAT3 phosphorylation at the Tyr705 site and nuclear translocation, C2F can modulate STAT3 activity, suppress CRC cell tumors and reduce EMT transformation in CRC cells \u003cem\u003ein vitro\u003c/em\u003e. In addition, detection of the expression levels of tumor related proteins revealed inhibition of PCNA and Bcl-2 expression. The expression level of p-STAT3 (Tyr705) in tumors of C2F experimental group mice was significantly lower than that of mice in the control group, indicating that C2F can also inhibit tumor cell proliferation and induce tumor cell apoptosis by suppressing the expression of p-STAT3 (Tyr705) \u003cem\u003ein vivo\u003c/em\u003e. Therefore, we conclude that C2F inhibits the overexpression of various oncogenic gene products regulated by STAT3 by regulating the activation of STAT3 in xenograft models, ultimately exerting an effective inhibitory effect on tumor growth.\u003c/p\u003e \u003cp\u003eIn conclusion, the data from this experiment suggests that C2F has anticancer effects both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e illustrates the mechanism by which C2F induces apoptosis and cell cycle arrest in CRC cells by altering the STAT3 signaling pathway. Collectively, these results suggest that C2F may be an attractive lead compound for the development of an anti-CRC drug.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eethyl 2,2-difluoro-2 - (2-oxo-2H-chromen-3-yl) acetate (C2F); colorectal cancer (CRC); activator of transcription 3 (STAT3); interleukin-6 (IL-6); fluorouracil group (5-FU).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the experimentation with animals was approved by the IACUC of Shenzhen People\u0026apos;s Hospital Laboratory Animal Center by following the Guide for the Care and Use of Laboratory Animals, and carry out experiments in the animal laboratory of Shenzhen People\u0026apos;s Hospital Laboratory Animal Center (Serial number: AUP-210317-LY-0003-01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJie LIN, Weijing LIU:\u0026nbsp;\u003c/strong\u003econception and design of experiments, performance of experimental procedures, analysis and interpretation of data, writing a manuscript. \u003cstrong\u003eXuehong FANG, Wen ZHANG:\u0026nbsp;\u003c/strong\u003eperformance of experimental procedures. \u003cstrong\u003eXiaodan LI, Jiansuo LIN, Yanwen LIANG:\u0026nbsp;\u003c/strong\u003eexperiments in vivo in nude mice. \u003cstrong\u003eYi LIU, Liyi ZOU, Jianwei REN, Feng WANG:\u0026nbsp;\u003c/strong\u003eConceptualization, Funding acquisition, Writing- Reviewing and Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have not received any funding or other support during the preparation process of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant fnancial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank\u0026nbsp;Shenzhen Ruike Biotechnology Co., Ltd\u0026nbsp;for providing the experimental site and equipment required for this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eUniProt: the universal protein knowledgebase in 2021. 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Molecular mechanism of QH-BJ drug pair in the treatment of systemic lupus erythematosus based on network pharmacology and molecular docking %J Medicine. 101(48).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ethyl 2,2-difluoro-2-(2-oxo-2H-chromen-3-yl) acetate, Colorectal cancer, Malignant biological behavior, STAT3 phosphorylation, Nuclear transfer","lastPublishedDoi":"10.21203/rs.3.rs-4376706/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4376706/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003ca class=\"FNLink\" href=\"#Fn1\" id=\"#FNLinkFn1\"\u003e\u003c/a\u003eTo investigate the effect of a novel coumarin derivative, ethyl 2,2-difluoro-2 - (2-oxo-2H-chromen-3-yl) acetate (C2F), on the malignant biological behaviors of colorectal cancer (CRC) and elucidate its mechanism. \u003cem\u003eIn vitro\u003c/em\u003e, the effects of C2F on the proliferation, apoptosis, migration, invasion, and cell cycle of CRC cells were analyzed by MTT assay, EdU stainning, colony formation assay, flow cytometry, wound healing and transwell assay. The anti-CRC activity of C2F was evaluated in a nude mice xenograft model \u003cem\u003ein vivo\u003c/em\u003e. Western blot was conducted to detect the expression of protein in cells and mice tissue. Then, the potential targets of C2F in CRC were predicted by network pharmacology analysis and molecular docking. The localization of STAT3 was observed through immunofluorescence experiment. C2F inhibits CRC cell proliferation, promotes CRC cell apoptosis, hinders CRC cell migration and invasion, and prevents the cell cycle from entering the G2/M phase. \u003cem\u003eIn vivo\u003c/em\u003e, C2F inhibited tumor growth in xenograft model. C2F inhibited signal transduction and activator of transcription 3 (STAT3) phosphorylation and blocked interleukin-6 (IL-6)-induced STAT3 nuclear translocation. C2F inhibits the malignant biological behavior of CRC by limiting STAT3 phosphorylation and entry into the nucleus.\u003c/p\u003e","manuscriptTitle":"Ethyl 2,2-difluoro-2-(2-oxo-2H-chromen-3-yl) acetate inhibits the malignant biological behaviors of colorectal cancer by restricting the phosphorylation and nuclear translocation of STAT3","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-14 08:37:50","doi":"10.21203/rs.3.rs-4376706/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"49694a00-50a0-4b8d-b491-dee039e3fbfb","owner":[],"postedDate":"May 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-31T21:38:33+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-14 08:37:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4376706","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4376706","identity":"rs-4376706","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}