{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T21:09:42Z","timestamp":1792789782301,"version":"4.5.0"},"reference-count":44,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T00:00:00Z","timestamp":1790294400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Medical University of Silesia","award":["BNW-2-026\/N\/4\/I"],"award-info":[{"award-number":["BNW-2-026\/N\/4\/I"]}],"id":[{"id":"https:\/\/ror.org\/005k7hp45","id-type":"ROR","asserted-by":"crossref"}]},{"award":["BNW-2-026\/N\/4\/I"],"award-info":[{"award-number":["BNW-2-026\/N\/4\/I"]}],"id":[{"id":"https:\/\/ror.org\/005k7hp45","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Colorectal cancer (CRC) remains one of the leading causes of cancer-related morbidity and mortality worldwide. Chronic inflammation plays a pivotal role in CRC development and progression, with cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and interleukin-8 (IL-8) representing key mediators of tumor-promoting inflammation. This study aimed to evaluate the effects of thymoquinone (TQ), 5-fluorouracil (5-FU), and their combination on the expression of COX-2, IL-6, and IL-8 at both the mRNA and protein levels in colorectal cancer cell lines (SW1116 and RKO) and normal colon epithelial cells (CCD-841CoN). Although numerical changes in inflammation-associated mediator expression were observed following TQ monotherapy, none of the TQ-monotherapy comparisons versus the untreated control reached statistical significance after correction for multiple comparisons. Notably, combined TQ\/5-FU treatment significantly reduced COX-2 protein levels in SW1116 cells compared with the untreated control. Differences between transcript and protein responses were observed across the investigated cell lines and treatment conditions. However, since both endpoints were assessed at a single time point, the mechanisms underlying these differences cannot be conclusively determined from the present data. Exploratory molecular docking analysis was additionally performed to examine potential direct interactions of TQ with COX-2, IL-6, and IL-8. The predicted docking scores ranged from \u22125.1 to \u22126.6 kcal\/mol, with the most favorable predicted interaction observed for COX-2. These computational findings are hypothesis-generating and should be interpreted independently of the experimentally observed changes in target expression. Collectively, the present findings demonstrate treatment- and cell-line-dependent response patterns in inflammation-associated mediators, while providing no statistically significant evidence of an effect of TQ monotherapy versus the untreated control under the present experimental conditions.<\/jats:p>","DOI":"10.3390\/molecules31193415","type":"journal-article","created":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T08:09:02Z","timestamp":1790323742000},"page":"3415","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Exploratory Analysis of the Effects of Thymoquinone Alone and in Combination with 5-Fluorouracil on Inflammatory Mediators in Colorectal Cancer Cells: An In Vitro and In Silico Study"],"prefix":"10.3390","volume":"31","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9515-0834","authenticated-orcid":false,"given":"Natalia","family":"Kurowska","sequence":"first","affiliation":[{"name":"Department of Molecular Biology, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia, 41-200 Sosnowiec, Poland"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9815-3149","authenticated-orcid":false,"given":"Marcel","family":"Madej","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia, 41-200 Sosnowiec, Poland"},{"name":"Silesia LabMed, Centre for Research and Implementation, Medical University of Silesia, 40-752 Katowice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"software","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Maria","family":"Ksi\u0105\u017cek","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia, 41-200 Sosnowiec, Poland"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"software","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9854-2569","authenticated-orcid":false,"given":"Barbara","family":"Strza\u0142ka-Mrozik","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia, 41-200 Sosnowiec, Poland"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,25]]},"reference":[{"key":"ref_1","first-page":"e70067","article-title":"Colorectal Cancer Statistics, 2026","volume":"76","author":"Siegel","year":"2026","journal-title":"CA Cancer J. Clin."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1159\/000543921","article-title":"Colorectal Cancer: Epidemiology, Risk Factors, and Public Health Strategies","volume":"106","author":"Matsuda","year":"2025","journal-title":"Digestion"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"789","DOI":"10.1097\/MIB.0b013e31828029c0","article-title":"Declining Risk of Colorectal Cancer in Inflammatory Bowel Disease: An Updated Meta-Analysis of Population-Based Cohort Studies","volume":"19","author":"Lutgens","year":"2013","journal-title":"Inflamm. Bowel Dis."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Burgos-Molina, A.M., T\u00e9llez Santana, T., Redondo, M., and Bravo Romero, M.J. (2024). The Crucial Role of Inflammation and the Immune System in Colorectal Cancer Carcinogenesis: A Comprehensive Perspective. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25116188"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Wu, K.-L., Chang, S.-F., Chen, C.-N., and Lee, K.-C. (2026). Fermented NaDES\u2013Ginger Extract Attenuates Hyperglycemia-Driven Inflammation and Endothelial Adhesion in Colorectal Cancer Through the NF-\u03baB\/COX-2 Axis. Life, 16.","DOI":"10.3390\/life16060927"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Silinskaite, U., Valciukiene, J., Jakubauskas, M., and Poskus, T. (2025). The Immune Environment in Colorectal Adenoma: A Systematic Review. Biomedicines, 13.","DOI":"10.3390\/biomedicines13030699"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1095","DOI":"10.7150\/ijms.44439","article-title":"The Role of Cyclooxygenase-2 in Colorectal Cancer","volume":"17","author":"Sheng","year":"2020","journal-title":"Int. J. Med. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"3142","DOI":"10.1111\/cas.14532","article-title":"5-Fluorouracil resistance mechanisms in colorectal cancer: From classical pathways to promising processes","volume":"111","author":"Blondy","year":"2020","journal-title":"Cancer Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"678","DOI":"10.1200\/JCO.22.01690","article-title":"Treatment of Metastatic Colorectal Cancer: ASCO Guideline","volume":"41","author":"Morris","year":"2023","journal-title":"J. Clin. Oncol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Chauvin, A., Bergeron, D., Vencic, J., L\u00e9vesque, D., Paquette, B., Scott, M.S., and Boisvert, F.-M. (2022). Downregulation of KRAB Zinc Finger Proteins in 5-Fluorouracil Resistant Colorectal Cancer Cells. BMC Cancer, 22.","DOI":"10.1186\/s12885-022-09417-3"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2840","DOI":"10.3892\/ol.2016.4971","article-title":"Thymoquinone Chemosensitizes Colon Cancer Cells through Inhibition of NF-\u03baB","volume":"12","author":"Zhang","year":"2016","journal-title":"Oncol. Lett."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"113213","DOI":"10.1016\/j.phytochem.2022.113213","article-title":"Therapeutic Implications and Clinical Manifestations of Thymoquinone","volume":"200","author":"Alam","year":"2022","journal-title":"Phytochemistry"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"111492","DOI":"10.1016\/j.biopha.2021.111492","article-title":"Anti-Inflammatory Effects of Thymoquinone and Its Protective Effects against Several Diseases","volume":"138","author":"Kohandel","year":"2021","journal-title":"BioMed. Pharmacother."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1171","DOI":"10.3748\/wjg.v23.i7.1171","article-title":"Thymoquinone Suppresses Migration of LoVo Human Colon Cancer Cells by Reducing Prostaglandin E2 Induced COX-2 Activation","volume":"23","author":"Hsu","year":"2017","journal-title":"World J. Gastroenterol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"108783","DOI":"10.1016\/j.biopha.2019.108783","article-title":"Cancer: Thymoquinone Antioxidant\/pro-Oxidant Effect as Potential Anticancer Remedy","volume":"115","author":"Mahmoud","year":"2019","journal-title":"Biomed. Pharmacother."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Madej, M., Halama, A., Chrobak, E., B\u0119benek, E., and Gola, J.M. (2025). Immunomodulatory Effect of Betulin and Its Derivatives on IL-6 Expression in Colorectal Cancer Cell Lines and Molecular Docking Insights. Front. Mol. Biosci., 12.","DOI":"10.3389\/fmolb.2025.1704804"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Madej, M., Halama, A., Chrobak, E., and Gola, J.M. (2025). Time-Dependent Impact of Betulin and Its Derivatives on IL-8 Expression in Colorectal Cancer Cells with Molecular Docking Studies. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26136186"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"S29","DOI":"10.1194\/jlr.R800042-JLR200","article-title":"Cyclooxygenases: Structural and Functional Insights","volume":"50","author":"Rouzer","year":"2009","journal-title":"J. Lipid Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2966","DOI":"10.1016\/j.ejca.2014.08.017","article-title":"Association of Adverse Events and Survival in Colorectal Cancer Patients Treated with Adjuvant 5-Fluorouracil and Leucovorin: Is Efficacy an Impact of Toxicity?","volume":"50","author":"Soveri","year":"2014","journal-title":"Eur. J. Cancer"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1105","DOI":"10.1080\/01635581.2017.1367940","article-title":"The Impact of Dietary Polyphenols on COX-2 Expression in Colorectal Cancer","volume":"69","author":"Owczarek","year":"2017","journal-title":"Nutr. Cancer"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1183","DOI":"10.1016\/0016-5085(94)90246-1","article-title":"Up-Regulation of Cyclooxygenase 2 Gene Expression in Human Colorectal Adenomas and Adenocarcinomas","volume":"107","author":"Eberhart","year":"1994","journal-title":"Gastroenterology"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Khan, Y.S., Farhana, A., Mohammed, G.E.Y., Osman, A.A., Alsrhani, A., Shahid, S.M.A., Kuddus, M., and Rasheed, Z. (2025). Thymoquinone Upregulates microRNA-199a-3p and Downregulates COX-2 Expression and PGE2 Production via Deactivation of P38\/ERK\/JNK-MAPKs and P65\/P50-NF-\u03baB Signaling in Human Lung Cancer Cells. Biology, 14.","DOI":"10.3390\/biology14101348"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Li, W., Chen, F., Gao, H., Xu, Z., Zhou, Y., Wang, S., Lv, Z., Zhang, Y., Xu, Z., and Huo, J. (2023). Cytokine concentration in peripheral blood of patients with colorectal cancer. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1175513"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1038\/s41419-022-04585-3","article-title":"Tumour Cell Apoptosis Modulates the Colorectal Cancer Immune Microenvironment via Interleukin-8-Dependent Neutrophil Recruitment","volume":"13","author":"Schimek","year":"2022","journal-title":"Cell Death Dis."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"258","DOI":"10.1007\/s12032-015-0703-y","article-title":"The Role of Interleukin-8 (CXCL8) and CXCR2 in Acquired Chemoresistance of Human Colorectal Carcinoma Cells HCT116","volume":"32","author":"Dabkeviciene","year":"2015","journal-title":"Med. Oncol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2006","DOI":"10.18632\/oncotarget.28075","article-title":"Pan-Drug and Drug-Specific Mechanisms of 5-FU, Irinotecan (CPT-11), Oxaliplatin, and Cisplatin Identified by Comparison of Transcriptomic and Cytokine Responses of Colorectal Cancer Cells","volume":"12","author":"Carlsen","year":"2021","journal-title":"Oncotarget"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"877","DOI":"10.4251\/wjgo.v12.i8.877","article-title":"Interleukin-1 Receptor Antagonist Enhances Chemosensitivity to Fluorouracil in Treatment of Kras Mutant Colon Cancer","volume":"12","author":"Yan","year":"2020","journal-title":"World J. Gastrointest. Oncol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1016\/j.jss.2003.11.023","article-title":"Inhibition of NF-kappa B augments sensitivity to 5-fluorouracil\/folinic acid in colon cancer","volume":"120","author":"Voboril","year":"2004","journal-title":"J. Surg. Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1007\/s11010-013-1930-1","article-title":"Thymoquinone Suppression of the Human Hepatocellular Carcinoma Cell Growth Involves Inhibition of IL-8 Expression, Elevated Levels of TRAIL Receptors, Oxidative Stress and Apoptosis","volume":"389","author":"Ashour","year":"2014","journal-title":"Mol. Cell. Biochem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1007\/s11010-016-2703-4","article-title":"Thymoquinone Inhibits Growth of Human Medulloblastoma Cells by Inducing Oxidative Stress and Caspase-Dependent Apoptosis While Suppressing NF-\u03baB Signaling and IL-8 Expression","volume":"416","author":"Ashour","year":"2016","journal-title":"Mol. Cell. Biochem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1016\/j.cell.2016.03.014","article-title":"On the Dependency of Cellular Protein Levels on mRNA Abundance","volume":"165","author":"Liu","year":"2016","journal-title":"Cell"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1038\/nrg3185","article-title":"Insights into the Regulation of Protein Abundance from Proteomic and Transcriptomic Analyses","volume":"13","author":"Vogel","year":"2012","journal-title":"Nat. Rev. Genet."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"630","DOI":"10.1038\/s41576-020-0258-4","article-title":"mRNAs, Proteins and the Emerging Principles of Gene Expression Control","volume":"21","author":"Buccitelli","year":"2020","journal-title":"Nat. Rev. Genet."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1615\/CritRevImmunol.2014010062","article-title":"Regulation of Chemokine Expression in the Tumor Microenvironment","volume":"34","author":"Gorbachev","year":"2014","journal-title":"Crit. Rev. Immunol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"541","DOI":"10.1111\/j.1476-5381.2010.00874.x","article-title":"Thymoquinone Inhibits Proliferation, Induces Apoptosis and Chemosensitizes Human Multiple Myeloma Cells through Suppression of Signal Transducer and Activator of Transcription 3 Activation Pathway","volume":"161","author":"Li","year":"2010","journal-title":"Br. J. Pharmacol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1038\/cmi.2009.43","article-title":"NF-\u03baB Signaling Pathway, Inflammation and Colorectal Cancer","volume":"6","author":"Wang","year":"2009","journal-title":"Cell. Mol. Immunol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"973","DOI":"10.1080\/10717544.2021.1927245","article-title":"Thymoquinone-Entrapped Chitosan-Modified Nanoparticles: Formulation Optimization to Preclinical Bioavailability Assessments","volume":"28","author":"Rahat","year":"2021","journal-title":"Drug Deliv."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Imam, S.S., Gilani, S.J., Bin Jumah, M.N., Rizwanullah, M., Zafar, A., Ahmed, M.M., and Alshehri, S. (2022). Harnessing Lipid Polymer Hybrid Nanoparticles for Enhanced Oral Bioavailability of Thymoquinone: In Vitro and In Vivo Assessments. Polymers, 14.","DOI":"10.3390\/polym14183705"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"292","DOI":"10.1007\/s13346-022-01193-8","article-title":"Self-Nanoemulsifying Drug Delivery System (SNEDDS) Mediated Improved Oral Bioavailability of Thymoquinone: Optimization, Characterization, Pharmacokinetic, and Hepatotoxicity Studies","volume":"13","author":"Rathore","year":"2023","journal-title":"Drug Deliv. Transl. Res."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Kurowska, N., Ksi\u0105\u017cek, M., Borkowska, P., and Strza\u0142ka-Mrozik, B. (2026). Cell Line\u2013Dependent Cell Death Pathways Induced by Thymoquinone in Colorectal Cancer Cells. Molecules, 31.","DOI":"10.3390\/molecules31030512"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2\u2212\u0394\u0394CT Method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1031","DOI":"10.1105\/tpc.109.066001","article-title":"Real-Time Quantitative RT-PCR: Design, Calculations, and Statistics","volume":"21","author":"Rieu","year":"2009","journal-title":"Plant Cell"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"470","DOI":"10.1091\/mbc.E20-09-0583","article-title":"SuperPlotsOfData\u2014A Web App for the Transparent Display and Quantitative Comparison of Continuous Data from Different Conditions","volume":"32","author":"Goedhart","year":"2021","journal-title":"Mol. Biol. Cell"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"e202001064","DOI":"10.1083\/jcb.202001064","article-title":"SuperPlots: Communicating Reproducibility and Variability in Cell Biology","volume":"219","author":"Lord","year":"2020","journal-title":"J. Cell Biol."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/19\/3415\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T04:18:33Z","timestamp":1790482713000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/19\/3415"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,25]]},"references-count":44,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2026,10]]}},"alternative-id":["molecules31193415"],"URL":"https:\/\/doi.org\/10.3390\/molecules31193415","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,25]]}}}