Irisin Mitigates Pro-inflammatory Cytokine Expression in Caco-2 Colon Cancer Cells: An In Vitro Study

Irisin Mitigates Pro-inflammatory Cytokine Expression in Caco-2 Colon Cancer Cells: An In Vitro Study | 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 Irisin Mitigates Pro-inflammatory Cytokine Expression in Caco-2 Colon Cancer Cells: An In Vitro Study ELIF ZEYNEP OZTURK, EBUBEKIR BAKAN, NURCAN KILIC BAYGUTALP, ZAFER BAYRAKTUTAN This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6890787/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 Irisin is a myokine secreted by muscle tissue, recognized for its regulatory effects on inflammation. This study aimed to investigate the potential anti-inflammatory role of irisin in colon cancer by evaluating its effects on the Caco-2 cell line. Specifically, we assessed pro-inflammatory cytokine levels (IL-6 and TNF-α), apoptotic marker Caspase-3 activity, and changes in the NF-κB signaling pathway using ELISA assays. Our results demonstrated a significant increase in IL-6 levels in the 100 nM irisin-treated group compared to controls (p < 0.05), and Caspase-3 activity significantly differed between the 10 nM and 100 nM irisin groups (p < 0.05). While direct antiproliferative effects of irisin on Caco-2 cells were not evident in cytotoxicity analyses, a significant increase in Caspase-3 activity suggested activation of apoptotic pathways. Additionally, the upregulation of NF-κB signaling—typically associated with cancer progression through inhibition of apoptosis—was observed following irisin treatment. In conclusion, these findings suggest that irisin modulates inflammatory and apoptotic responses in colon cancer cells, rather than exerting direct cytotoxic effects. Irisin's regulatory influence on the tumor microenvironment may offer potential therapeutic benefits in oncology. Further preclinical and clinical investigations are warranted to fully elucidate irisin’s role in cancer treatment. apoptosis colon cancer inflammation irisin NF –κB pathway Figures Figure 1 Figure 2 Introduction Adipose tissue in the human body can be categorized into two distinct types: white and brown. White adipose tissue serves primarily as an energy reservoir, storing excess energy in the form of triglycerides and functioning as an endocrine organ contributing to energy homeostasis. In contrast, brown adipose tissue plays a crucial role in thermoregulation by dissipating energy as heat [ 1 ]. Regular exercise is widely recognized for its beneficial effects on metabolism, both in health and disease conditions [ 2 ]. Studies have indicated that a higher ratio of brown adipose tissue correlates with a lower body mass index (BMI), whereas an increase in white adipose tissue is associated with a heightened risk of insulin resistance, obesity, and Type 2 Diabetes [ 2 ]. Skeletal muscle has also been identified as an endocrine organ capable of producing and secreting numerous paracrine and endocrine myokines [ 3 , 4 ]. Among these, irisin, predominantly secreted by skeletal muscle during physical exercise, is known for its thermogenic activity through the conversion of white adipose tissue to brown adipose tissue. Irisin promotes significant changes in subcutaneous white adipose tissue, notably enhancing the expression of uncoupling protein 1 (UCP1), thus facilitating thermogenesis in newly formed brown adipose tissue [ 5 ]. Consequently, irisin contributes to increased total body energy expenditure and reduces obesity-induced insulin resistance. Multiple studies exploring the relationship between irisin and glucose metabolism suggest that irisin could be a potential therapeutic molecule for managing insulin resistance in obesity and Type 2 Diabetes [ 6 ]. Furthermore, physical exercise is recognized for its protective effects against cancer, as it is known to improve cancer prognosis, although the precise mechanisms remain incompletely understood [ 7 ]. Emerging research has indicated that irisin may exert protective roles against various cancers, including breast, lung, pancreatic, renal, and colorectal cancers, suggesting its potential as a biomarker and therapeutic target [ 8 , 9 ]. Maalouf and El Khoury proposed that irisin indirectly reduces cancer risk and improves prognosis by promoting the browning of white adipose tissue (transformation into beige adipose tissue), thereby reducing the secretion of pro-inflammatory cytokines. Conversely, other studies have proposed a direct role for irisin in inhibiting cancer cell proliferation, invasion, and metastasis [ 10 , 11 ]. Nonetheless, the direct mechanisms of irisin in cancer remain unclear and under-investigated. In this study, we evaluated the antiproliferative effects (using the CVDK-8 assay) and apoptotic contributions (via Caspase-3 levels) of irisin on the colon cancer cell line Caco-2. Additionally, we assessed the indirect anti-cancer effects of irisin by measuring key inflammatory mediators such as TNF-α, IL-6, and NF-κB. Our aim was to elucidate both the direct and indirect potential mechanisms of irisin and its prospective contributions to colon cancer treatment strategies. Results and Discussion Regulation of cell proliferation by irisin in Caco-2 cell lines Proliferation rates as a result of the 48-h cytotoxicity test (CVDK − 8) are shown in Fig. 1 . Viability values in the control group with and without irisin were statistically significant in the 5-nM group (p 0.05). We observed that cell proliferation is not regulated by irisin in a dose-dependent manner in human colon cancer cell lines. Figure 2 and Table 1 show the IL -6, caspase − 3, TNF -α, and NF -kB levels of Caco − 2 cells treated with 10 nM and 100 nM irisin and control cells not treated with irisin. There was no difference in IL -6 levels between the group to which 10 nM irisin was applied and the control group (p > 0.05). The difference between the 100 nM irisin-applied group and the control group was significant (p < 0.05). Table 1 Effect of different irisin concentrations (10–100 nM) on IL -6, TNF -α, caspase − 3 and NF -kB levels in Caco − 2 cell line. a: Statistically significant difference at the P < 0.05 level between the control and 10 (nM) irisin groups. b: Statistically significant difference at the P < 0.05 level between the control and 100 (nM) irisin groups. c: 10 (nM) and 100 Statistically significant difference at P < 0.05 level between (nM) irisin groups. Groups IL -6 Levels (Mean (ng/l) ± SD) Groups TNF -α Levels (Mean (ng/l) ± SD) 10-nM irisin 143.77 ± 10.97 10-nM irisin 123.04 ± 6.09 100-nM irisin 157.68 ± 7.65 b 100-nM irisin 125.85 ± 6.81 Control 135.83 ± 7.86 Control 130.25 ± 10.35 Caspase − 3 Levels (Mean (ng/l) ± SD) NF -κB Levels (Mean (ng/l) ± SD) 10-nM irisin 8.26 ± 0.15 c 10-nM irisin 1797.18 ± 35.54 c 100-nM irisin 8.95 ± 0.38 b 100-nM irisin 1505.98 ± 49.99 b Control 8.19 ± 0.16 Control 1236.06 ± 75.51 a Caspase-3 levels in Caco-2 cells treated with 10 nM irisin compared to control cells are illustrated in Fig. 2 . No significant difference was observed between the 10 nM irisin-treated group and the control group (p > 0.05). However, the caspase-3 levels were significantly different between the 100 nM irisin-treated group and both the control and 10 nM irisin-treated groups (p 0.05). Recent studies have focused on the relationship between exercise and cancer [ 13 ], and numerous reports suggest a link between irisin and cancer [ 14 ]. Exerkines, including irisin, are signaling molecules released in response to exercise, exerting their biological effects through endocrine, paracrine, or autocrine pathways [ 15 ]. To date, approximately 2000 exerkines have been identified, with irisin prominently regulating various metabolic processes [ 16 ]. Irisin has been demonstrated to inhibit pancreatic cancer cell growth via the AMPK-mTOR pathway, highlighting its potential as a chemotherapeutic agent [ 17 ]. In our study, instead of preventing cell proliferation, irisin altered cytokine (IL-6), transcription factor (NF-κB), and apoptotic factor (caspase-3) levels. Caspase-3 is a well-established apoptosis marker [ 18 ], and increased caspase-3 levels indicate activation of apoptotic mechanisms. However, the concomitant increase in NF-κB, which plays a significant role in inflammation and carcinogenesis, may hinder apoptosis by enhancing anti-apoptotic mechanisms and promoting cellular proliferation [ 19 ]. Positive effects of irisin on glucose metabolism suggest a potential role in obesity-related cancers, stimulating further exploration of its association with cancer. Few studies have addressed the effects of irisin on colon cancer. Moon et al. reported no significant anticancer effect of irisin in colon cancer cell lines. Similarly, our study, using the Caco-2 cell line, also did not detect direct antiproliferative activity, although we explored indirect effects by examining cytokine levels, a parameter not evaluated in Moon et al.’s research. Additional studies have identified increased irisin expression in colon cancer tissues and cachexia-associated tumors, indicating a complex association between irisin and cancer [ 21 , 22 ]. IL-6 and TNF-α interactions can amplify inflammatory responses, facilitating inflammatory cell infiltration into tissues [ 23 ]. Current cancer research increasingly addresses obesity, tumor microenvironment, and inflammation [ 24 ], emphasizing exercise's therapeutic potential. Exercise reportedly normalizes elevated TNF-α levels, potentially contributing to anticancer effects by reducing inflammation [ 25 ]. In our study, TNF-α levels decreased with irisin treatment, although the difference was not statistically significant (p > 0.05), suggesting limited direct effects of irisin on TNF-α in vitro, though differences may emerge in vivo. Our findings suggest activated apoptotic mechanisms (increased caspase-3) but inadequate antiproliferative effects due to persistent inflammation (increased IL-6 and NF-κB). NF-κB activation not only promotes inflammation but also activates anti-apoptotic pathways. While irisin has demonstrated therapeutic efficacy in cancers such as lung [ 10 ], breast [ 26 ], prostate [ 14 ], hepatocellular carcinoma [ 27 ], osteosarcoma [ 11 ], and pancreatic cancer [ 17 ], antiproliferative effects were absent in colon cancer studies, including ours and that of Moon et al. This discrepancy might be due to distinct mechanistic pathways in colon cancer. Further in vitro, in vivo, and clinical research are required for clearer elucidation. This study uniquely investigates antiproliferative and anti-inflammatory effects of irisin on Caco-2 cells, complementing prior research. Although our findings parallel those of Moon et al., showing minimal antiproliferative effects, the significant caspase-3 activation suggests a potential apoptotic influence of irisin. It is possible that higher irisin concentrations might reveal antiproliferative properties. Our findings emphasize dosage-dependent variability in irisin’s effectiveness, providing important insights for future therapeutic applications. In summary, physiological and pharmacological concentrations of irisin do not significantly regulate proliferation in the Caco-2 cell line. Our study highlights key cellular responses (caspase-3, IL-6, NF-κB), establishing a foundation for future targeted mechanistic investigations. Further comprehensive in vitro, ex vivo, and in vivo studies are essential to validate our findings and determine irisin's precise therapeutic potential in colon cancer. Conclusions Our study indicates that while irisin triggers apoptotic mechanisms, as evidenced by increased caspase-3 activation, it does not exert a sufficiently potent antiproliferative effect on colon cancer cells. This limited efficacy may be attributed to the concurrent activation of inflammatory responses, characterized by elevated IL-6 levels, insufficient suppression of TNF-α, and enhanced NF-κB signaling. Activation of the NF-κB pathway not only promotes inflammation but also reinforces anti-apoptotic mechanisms, potentially counteracting the pro-apoptotic effects of irisin. Therefore, suppression of NF-κB signaling may be critical for irisin to exert meaningful therapeutic effects in colon cancer. The differential efficacy of irisin observed in other cancer types (e.g., breast, prostate, pancreatic cancers) compared to colon cancer may be explained by the distinct activation of anti-apoptotic pathways specific to gastrointestinal malignancies. Both our findings and previous reports, such as those by Moon et al., suggest that irisin’s mechanisms of action in gastrointestinal cancers differ fundamentally from its effects in other tumor types. Consequently, further detailed investigations incorporating both in vitro and in vivo models are warranted to elucidate the precise molecular mechanisms and therapeutic potential of irisin in colon cancer.. Experimental Section Cell Culture Colon (Caco − 2) cancer cell lines were purchased from the American Type Culture Collection (ATCC®, Manassas, VA). The cells were grown in Dulbecco's Modified Eagle's Medium (Biowest, USA) with 10% (vol/vol) fetal bovine serum. All cells were incubated at 37°C in an atmosphere of 5% CO2 in the air, and sub-cultured beyond 60–70% confluency. Chemicals Human recombinant irisin (Catalog no: BITP3212) was purchased from Apollo Scientific (UK), Cell Viability Detection Kit − 8 (CVDK − 8) from Eco-Tech (TR). IL-6 ELISA kit (Cat. No: E0090Hu) was obtained from BT -Lab (Korea), TNF-alpha ELISA kit (Cat. No: E0082Hu) from BT -Lab (Korea), NF-κB ELISA kit (Cat. No: E3048Hu) from BT -Lab (Korea), and caspase − 3 ELISA kit (Cat. No: E4804 Hu) from BT -Lab (Korea) Proliferation Assay Cells were treated with irisin for 48 hours, and then a CVDK − 8 kit was used for proliferation measurement. In the evaluation of proliferation and viability, 10% CVDK solution was added to the wells according to the CVDK − 8 kit protocol. [ 12 ] Dehydrogenases reduce CVDK in cells to give an orange-colored product (formazan). It was incubated for 2 hours, and the intensity of the color formed was determined with an ELISA reader at a wavelength of 450 nm. Only cells grown in a growth medium were used as a negative control, and 5-fluorouracil (5-FU) at 500 µM concentration was used as a positive control. The proliferation test was performed on cells with 4 different doses of irisin: Group 1: 5nM irisin, Group 2: 10 nM irisin, Group 3: 50 nM irisin, and Group 4: 100 nM irisin, each of the irisin concentrations was added to each respective group. Biochemical Analyses Physiological (10 nM) and pharmacological doses (100 nM) were examined for ELISA tests. After 48 h of incubation with irisin, IL -6, caspase − 3, TNF -alpha, and Nf -kB were analysed according to the kit procedure and measured in a multiple spectrophotometer device. All experiments were performed with duplicate samples and repeated three times. Statistical Analysis Statistical analyses were performed with the SPSS 23.0 package program for Windows. The conformity of the data to the normal distribution was evaluated with the Shapiro-Wilk test. Student's t-test was used to compare the irisin-administered and control groups for cytotoxicity evaluation. A one-way ANOVA test was used to evaluate the ELISA results and to compare the groups. Posthoch analysis was done with the Tukey test. p < 0.05 values were considered statistically significant. Limitations of Study "This study was performed only with the Caco-2 cell line, and its ability to represent CRC may be limited. Additional studies in different colon cancer cell lines and in vivo models are necessary to confirm the anti-inflammatory effect of irisin." Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials All data generated or analysed during this study are included in this published article. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Fundings This study was supported by the Atatürk University Scientific Research Projects Commission (Project no: TDK-2020-8498) Author Contribution Statement EB designed the study, interpreted the data, and edited the paper. EZÖ analysed and interpreted the data for the study and drafted and revised the work. NKB interpreted the data for the study and revised the work. ZB contributed data acquisition and revised the work. 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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-6890787","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472151678,"identity":"fcc27f2d-2fb5-4bbf-a09a-51088747e380","order_by":0,"name":"ELIF ZEYNEP OZTURK","email":"data:image/png;base64,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","orcid":"","institution":"Kutahya Health Sciences University","correspondingAuthor":true,"prefix":"","firstName":"ELIF","middleName":"ZEYNEP","lastName":"OZTURK","suffix":""},{"id":472151680,"identity":"23031bf9-106d-4947-aace-8e10241739e6","order_by":1,"name":"EBUBEKIR BAKAN","email":"","orcid":"","institution":"Agri İbrahim Cecen University","correspondingAuthor":false,"prefix":"","firstName":"EBUBEKIR","middleName":"","lastName":"BAKAN","suffix":""},{"id":472151681,"identity":"b39ad858-dcce-42ad-8692-f46036640218","order_by":2,"name":"NURCAN KILIC BAYGUTALP","email":"","orcid":"","institution":"Ataturk University","correspondingAuthor":false,"prefix":"","firstName":"NURCAN","middleName":"KILIC","lastName":"BAYGUTALP","suffix":""},{"id":472151682,"identity":"1cf93817-8fba-4a0b-a6dd-0631a47b4bc8","order_by":3,"name":"ZAFER BAYRAKTUTAN","email":"","orcid":"","institution":"Ataturk University","correspondingAuthor":false,"prefix":"","firstName":"ZAFER","middleName":"","lastName":"BAYRAKTUTAN","suffix":""}],"badges":[],"createdAt":"2025-06-13 21:38:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6890787/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6890787/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84886469,"identity":"f8587217-a7bd-42a3-8203-f49cad0eb270","added_by":"auto","created_at":"2025-06-18 11:47:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":46431,"visible":true,"origin":"","legend":"\u003cp\u003eRegulation of cell proliferation by irisin in Caco -2 cell lines. The cell culture was performed as described in detail in the Methods section. The cells were treated with irisin at indicated concentrations for 48 h, and cell proliferation assay was then performed as described in detail in the Methods section. Proliferation data were analysed using student-t test. Values are means (n = 3). * are significantly different, p \u0026lt; 0.05. 5-fluorouracil (5 -FU) 500 μg/ml for 48 h was used for a positive control.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6890787/v1/6abc808de48d83f6ccb156c9.png"},{"id":84886471,"identity":"c94b20e2-ec9c-4f34-bfb5-60abfc3fa199","added_by":"auto","created_at":"2025-06-18 11:47:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":75645,"visible":true,"origin":"","legend":"\u003cp\u003eGraph of the mean IL-6, caspase-3, TNF-α and NF-kB of irisin-treated (10-100 nM) Caco-2 cells and control group\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6890787/v1/639e2b9c310b389270e5bba5.png"},{"id":85114531,"identity":"2499f876-9764-49ba-b830-105ec68f41b5","added_by":"auto","created_at":"2025-06-21 13:31:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":618919,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6890787/v1/c25d5764-223b-4d40-a57a-aaf29949d31d.pdf"},{"id":84885876,"identity":"09962e5c-82e3-4cdb-9410-0207a7390e08","added_by":"auto","created_at":"2025-06-18 11:39:14","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":70840,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6890787/v1/5ce1f502bff28fe2d097692c.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Irisin Mitigates Pro-inflammatory Cytokine Expression in Caco-2 Colon Cancer Cells: An In Vitro Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAdipose tissue in the human body can be categorized into two distinct types: white and brown. White adipose tissue serves primarily as an energy reservoir, storing excess energy in the form of triglycerides and functioning as an endocrine organ contributing to energy homeostasis. In contrast, brown adipose tissue plays a crucial role in thermoregulation by dissipating energy as heat [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Regular exercise is widely recognized for its beneficial effects on metabolism, both in health and disease conditions [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Studies have indicated that a higher ratio of brown adipose tissue correlates with a lower body mass index (BMI), whereas an increase in white adipose tissue is associated with a heightened risk of insulin resistance, obesity, and Type 2 Diabetes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSkeletal muscle has also been identified as an endocrine organ capable of producing and secreting numerous paracrine and endocrine myokines [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among these, irisin, predominantly secreted by skeletal muscle during physical exercise, is known for its thermogenic activity through the conversion of white adipose tissue to brown adipose tissue. Irisin promotes significant changes in subcutaneous white adipose tissue, notably enhancing the expression of uncoupling protein 1 (UCP1), thus facilitating thermogenesis in newly formed brown adipose tissue [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Consequently, irisin contributes to increased total body energy expenditure and reduces obesity-induced insulin resistance. Multiple studies exploring the relationship between irisin and glucose metabolism suggest that irisin could be a potential therapeutic molecule for managing insulin resistance in obesity and Type 2 Diabetes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, physical exercise is recognized for its protective effects against cancer, as it is known to improve cancer prognosis, although the precise mechanisms remain incompletely understood [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Emerging research has indicated that irisin may exert protective roles against various cancers, including breast, lung, pancreatic, renal, and colorectal cancers, suggesting its potential as a biomarker and therapeutic target [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMaalouf and El Khoury proposed that irisin indirectly reduces cancer risk and improves prognosis by promoting the browning of white adipose tissue (transformation into beige adipose tissue), thereby reducing the secretion of pro-inflammatory cytokines. Conversely, other studies have proposed a direct role for irisin in inhibiting cancer cell proliferation, invasion, and metastasis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Nonetheless, the direct mechanisms of irisin in cancer remain unclear and under-investigated.\u003c/p\u003e \u003cp\u003eIn this study, we evaluated the antiproliferative effects (using the CVDK-8 assay) and apoptotic contributions (via Caspase-3 levels) of irisin on the colon cancer cell line Caco-2. Additionally, we assessed the indirect anti-cancer effects of irisin by measuring key inflammatory mediators such as TNF-α, IL-6, and NF-κB. Our aim was to elucidate both the direct and indirect potential mechanisms of irisin and its prospective contributions to colon cancer treatment strategies.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRegulation of cell proliferation by irisin in Caco-2 cell lines\u003c/h2\u003e \u003cp\u003eProliferation rates as a result of the 48-h cytotoxicity test (CVDK \u0026minus;\u0026thinsp;8) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Viability values in the control group with and without irisin were statistically significant in the 5-nM group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not in the 10-, 50-, and 100-nM groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). We observed that cell proliferation is not regulated by irisin in a dose-dependent manner in human colon cancer cell lines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e show the IL -6, caspase \u0026minus;\u0026thinsp;3, TNF -α, and NF -kB levels of Caco \u0026minus;\u0026thinsp;2 cells treated with 10 nM and 100 nM irisin and control cells not treated with irisin. There was no difference in IL -6 levels between the group to which 10 nM irisin was applied and the control group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The difference between the 100 nM irisin-applied group and the control group was significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of different irisin concentrations (10\u0026ndash;100 nM) on IL -6, TNF -α, caspase \u0026minus;\u0026thinsp;3 and NF -kB levels in Caco \u0026minus;\u0026thinsp;2 cell line. a: Statistically significant difference at the P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 level between the control and 10 (nM) irisin groups. b: Statistically significant difference at the P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 level between the control and 100 (nM) irisin groups. c: 10 (nM) and 100 Statistically significant difference at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 level between (nM) irisin groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIL -6 Levels\u003c/p\u003e \u003cp\u003e(Mean (ng/l)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTNF -α Levels\u003c/p\u003e \u003cp\u003e(Mean (ng/l)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10-nM irisin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e143.77\u0026thinsp;\u0026plusmn;\u0026thinsp;10.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10-nM irisin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e123.04\u0026thinsp;\u0026plusmn;\u0026thinsp;6.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100-nM irisin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e157.68\u0026thinsp;\u0026plusmn;\u0026thinsp;7.65\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100-nM irisin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e125.85\u0026thinsp;\u0026plusmn;\u0026thinsp;6.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e135.83\u0026thinsp;\u0026plusmn;\u0026thinsp;7.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e130.25\u0026thinsp;\u0026plusmn;\u0026thinsp;10.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eCaspase \u0026minus;\u0026thinsp;3 Levels\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(Mean (ng/l)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eNF -κB Levels\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(Mean (ng/l)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10-nM irisin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10-nM irisin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1797.18\u0026thinsp;\u0026plusmn;\u0026thinsp;35.54\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100-nM irisin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100-nM irisin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1505.98\u0026thinsp;\u0026plusmn;\u0026thinsp;49.99\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1236.06\u0026thinsp;\u0026plusmn;\u0026thinsp;75.51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eCaspase-3 levels in Caco-2 cells treated with 10 nM irisin compared to control cells are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. No significant difference was observed between the 10 nM irisin-treated group and the control group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, the caspase-3 levels were significantly different between the 100 nM irisin-treated group and both the control and 10 nM irisin-treated groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). TNF-α levels did not significantly differ between irisin-treated and control groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eRecent studies have focused on the relationship between exercise and cancer [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and numerous reports suggest a link between irisin and cancer [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Exerkines, including irisin, are signaling molecules released in response to exercise, exerting their biological effects through endocrine, paracrine, or autocrine pathways [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To date, approximately 2000 exerkines have been identified, with irisin prominently regulating various metabolic processes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Irisin has been demonstrated to inhibit pancreatic cancer cell growth via the AMPK-mTOR pathway, highlighting its potential as a chemotherapeutic agent [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our study, instead of preventing cell proliferation, irisin altered cytokine (IL-6), transcription factor (NF-κB), and apoptotic factor (caspase-3) levels. Caspase-3 is a well-established apoptosis marker [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and increased caspase-3 levels indicate activation of apoptotic mechanisms. However, the concomitant increase in NF-κB, which plays a significant role in inflammation and carcinogenesis, may hinder apoptosis by enhancing anti-apoptotic mechanisms and promoting cellular proliferation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePositive effects of irisin on glucose metabolism suggest a potential role in obesity-related cancers, stimulating further exploration of its association with cancer. Few studies have addressed the effects of irisin on colon cancer. Moon et al. reported no significant anticancer effect of irisin in colon cancer cell lines. Similarly, our study, using the Caco-2 cell line, also did not detect direct antiproliferative activity, although we explored indirect effects by examining cytokine levels, a parameter not evaluated in Moon et al.\u0026rsquo;s research. Additional studies have identified increased irisin expression in colon cancer tissues and cachexia-associated tumors, indicating a complex association between irisin and cancer [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIL-6 and TNF-α interactions can amplify inflammatory responses, facilitating inflammatory cell infiltration into tissues [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Current cancer research increasingly addresses obesity, tumor microenvironment, and inflammation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], emphasizing exercise's therapeutic potential. Exercise reportedly normalizes elevated TNF-α levels, potentially contributing to anticancer effects by reducing inflammation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In our study, TNF-α levels decreased with irisin treatment, although the difference was not statistically significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), suggesting limited direct effects of irisin on TNF-α in vitro, though differences may emerge in vivo.\u003c/p\u003e \u003cp\u003eOur findings suggest activated apoptotic mechanisms (increased caspase-3) but inadequate antiproliferative effects due to persistent inflammation (increased IL-6 and NF-κB). NF-κB activation not only promotes inflammation but also activates anti-apoptotic pathways. While irisin has demonstrated therapeutic efficacy in cancers such as lung [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], breast [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], prostate [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], hepatocellular carcinoma [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], osteosarcoma [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and pancreatic cancer [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], antiproliferative effects were absent in colon cancer studies, including ours and that of Moon et al. This discrepancy might be due to distinct mechanistic pathways in colon cancer. Further in vitro, in vivo, and clinical research are required for clearer elucidation.\u003c/p\u003e \u003cp\u003eThis study uniquely investigates antiproliferative and anti-inflammatory effects of irisin on Caco-2 cells, complementing prior research. Although our findings parallel those of Moon et al., showing minimal antiproliferative effects, the significant caspase-3 activation suggests a potential apoptotic influence of irisin. It is possible that higher irisin concentrations might reveal antiproliferative properties. Our findings emphasize dosage-dependent variability in irisin\u0026rsquo;s effectiveness, providing important insights for future therapeutic applications.\u003c/p\u003e \u003cp\u003eIn summary, physiological and pharmacological concentrations of irisin do not significantly regulate proliferation in the Caco-2 cell line. Our study highlights key cellular responses (caspase-3, IL-6, NF-κB), establishing a foundation for future targeted mechanistic investigations. Further comprehensive in vitro, ex vivo, and in vivo studies are essential to validate our findings and determine irisin's precise therapeutic potential in colon cancer.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study indicates that while irisin triggers apoptotic mechanisms, as evidenced by increased caspase-3 activation, it does not exert a sufficiently potent antiproliferative effect on colon cancer cells. This limited efficacy may be attributed to the concurrent activation of inflammatory responses, characterized by elevated IL-6 levels, insufficient suppression of TNF-α, and enhanced NF-κB signaling. Activation of the NF-κB pathway not only promotes inflammation but also reinforces anti-apoptotic mechanisms, potentially counteracting the pro-apoptotic effects of irisin. Therefore, suppression of NF-κB signaling may be critical for irisin to exert meaningful therapeutic effects in colon cancer.\u003c/p\u003e \u003cp\u003eThe differential efficacy of irisin observed in other cancer types (e.g., breast, prostate, pancreatic cancers) compared to colon cancer may be explained by the distinct activation of anti-apoptotic pathways specific to gastrointestinal malignancies. Both our findings and previous reports, such as those by Moon et al., suggest that irisin\u0026rsquo;s mechanisms of action in gastrointestinal cancers differ fundamentally from its effects in other tumor types. Consequently, further detailed investigations incorporating both in vitro and in vivo models are warranted to elucidate the precise molecular mechanisms and therapeutic potential of irisin in colon cancer..\u003c/p\u003e "},{"header":"Experimental Section","content":"\u003ch2\u003eCell Culture\u003c/h2\u003e\u003cp\u003eColon (Caco \u0026minus;\u0026thinsp;2) cancer cell lines were purchased from the American Type Culture Collection (ATCC\u0026reg;, Manassas, VA). The cells were grown in Dulbecco's Modified Eagle's Medium (Biowest, USA) with 10% (vol/vol) fetal bovine serum. All cells were incubated at 37\u0026deg;C in an atmosphere of 5% CO2 in the air, and sub-cultured beyond 60\u0026ndash;70% confluency.\u003c/p\u003e\n\u003ch3\u003eChemicals\u003c/h3\u003e\n\u003cp\u003eHuman recombinant irisin (Catalog no: BITP3212) was purchased from Apollo Scientific (UK), Cell Viability Detection Kit \u0026minus;\u0026thinsp;8 (CVDK \u0026minus;\u0026thinsp;8) from Eco-Tech (TR). IL-6 ELISA kit (Cat. No: E0090Hu) was obtained from BT -Lab (Korea), TNF-alpha ELISA kit (Cat. No: E0082Hu) from BT -Lab (Korea), NF-κB ELISA kit (Cat. No: E3048Hu) from BT -Lab (Korea), and caspase \u0026minus;\u0026thinsp;3 ELISA kit (Cat. No: E4804 Hu) from BT -Lab (Korea)\u003c/p\u003e\n\u003ch3\u003eProliferation Assay\u003c/h3\u003e\n\u003cp\u003eCells were treated with irisin for 48 hours, and then a CVDK \u0026minus;\u0026thinsp;8 kit was used for proliferation measurement. In the evaluation of proliferation and viability, 10% CVDK solution was added to the wells according to the CVDK \u0026minus;\u0026thinsp;8 kit protocol. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] Dehydrogenases reduce CVDK in cells to give an orange-colored product (formazan). It was incubated for 2 hours, and the intensity of the color formed was determined with an ELISA reader at a wavelength of 450 nm. Only cells grown in a growth medium were used as a negative control, and 5-fluorouracil (5-FU) at 500 \u0026micro;M concentration was used as a positive control. The proliferation test was performed on cells with 4 different doses of irisin: Group 1: 5nM irisin, Group 2: 10 nM irisin, Group 3: 50 nM irisin, and Group 4: 100 nM irisin, each of the irisin concentrations was added to each respective group.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical Analyses\u003c/h2\u003e \u003cp\u003ePhysiological (10 nM) and pharmacological doses (100 nM) were examined for ELISA tests. After 48 h of incubation with irisin, IL -6, caspase \u0026minus;\u0026thinsp;3, TNF -alpha, and Nf -kB were analysed according to the kit procedure and measured in a multiple spectrophotometer device. All experiments were performed with duplicate samples and repeated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed with the SPSS 23.0 package program for Windows. The conformity of the data to the normal distribution was evaluated with the Shapiro-Wilk test. Student's t-test was used to compare the irisin-administered and control groups for cytotoxicity evaluation. A one-way ANOVA test was used to evaluate the ELISA results and to compare the groups. Posthoch analysis was done with the Tukey test. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 values were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Limitations of Study","content":"\u003cp\u003e\u0026quot;This study was performed only with the Caco-2 cell line, and its ability to represent CRC may be limited. Additional studies in different colon cancer cell lines and in vivo models are necessary to confirm the anti-inflammatory effect of irisin.\u0026quot;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Atat\u0026uuml;rk University Scientific Research Projects Commission (Project no: TDK-2020-8498)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEB designed the study, interpreted the data, and edited the paper. EZ\u0026Ouml; analysed and interpreted the data for the study and drafted and revised the work. NKB interpreted the data for the study and revised the work. ZB contributed data acquisition and revised the work. All authors read and approved the manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eB. Cannon, J. Nedergaard. \u0026lsquo;Brown adipose tissue: function and physiological significance.\u0026rsquo; Physiol Rev. 2004, 84(1), 277-359. \u003c/li\u003e\n\u003cli\u003eJ. Vina, F. Sanchis-Gomar, V. Martinez-Bello, M. C. Gomez-Cabrera. \u0026lsquo;Exercise acts as a drug; the pharmacological benefits of exercise.\u0026rsquo; Br J Pharmacol. 2012, 167(1),1-12. \u003c/li\u003e\n\u003cli\u003eB. So, H. J. Kim, J. Kim, W. 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Garcia-Smith, M. Bisoffi, K. A. Trujillo. \u0026lsquo;Effects of the exercise-inducible myokine irisin on malignant and non-malignant breast epithelial cell behavior in vitro.\u0026rsquo; Int J Cancer. 2015, 136(4), E197-202. \u003c/li\u003e\n\u003cli\u003eG. Shi, N. Tang, J. Qiu, D. Zhang, F. Huang, Y. Cheng, K. Ding, W. Li, P. Zhang, X. Tan. \u0026lsquo;Irisin stimulates cell proliferation and invasion by targeting the PI3K/AKT pathway in human hepatocellular carcinoma.\u0026rsquo; Biochem Biophys Res Commun. 2017, 493(1), 585-591. \u003c/li\u003e\n\u003cli\u003eG. E. Maalouf, D. El Khoury. \u0026lsquo;Exercise-Induced Irisin, the Fat Browning Myokine, as a Potential Anticancer Agent.\u0026rsquo; J Obes. 2019, 2019, 6561726. \u003c/li\u003e\n\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":"apoptosis, colon cancer, inflammation, irisin, NF –κB pathway","lastPublishedDoi":"10.21203/rs.3.rs-6890787/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6890787/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIrisin is a myokine secreted by muscle tissue, recognized for its regulatory effects on inflammation. This study aimed to investigate the potential anti-inflammatory role of irisin in colon cancer by evaluating its effects on the Caco-2 cell line. Specifically, we assessed pro-inflammatory cytokine levels (IL-6 and TNF-α), apoptotic marker Caspase-3 activity, and changes in the NF-κB signaling pathway using ELISA assays.\u003c/p\u003e \u003cp\u003eOur results demonstrated a significant increase in IL-6 levels in the 100 nM irisin-treated group compared to controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and Caspase-3 activity significantly differed between the 10 nM and 100 nM irisin groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). While direct antiproliferative effects of irisin on Caco-2 cells were not evident in cytotoxicity analyses, a significant increase in Caspase-3 activity suggested activation of apoptotic pathways. Additionally, the upregulation of NF-κB signaling\u0026mdash;typically associated with cancer progression through inhibition of apoptosis\u0026mdash;was observed following irisin treatment.\u003c/p\u003e \u003cp\u003eIn conclusion, these findings suggest that irisin modulates inflammatory and apoptotic responses in colon cancer cells, rather than exerting direct cytotoxic effects. Irisin's regulatory influence on the tumor microenvironment may offer potential therapeutic benefits in oncology. Further preclinical and clinical investigations are warranted to fully elucidate irisin\u0026rsquo;s role in cancer treatment.\u003c/p\u003e","manuscriptTitle":"Irisin Mitigates Pro-inflammatory Cytokine Expression in Caco-2 Colon Cancer Cells: An In Vitro Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-18 11:39:09","doi":"10.21203/rs.3.rs-6890787/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":"9eb5e970-53f5-4a71-b1df-fb2aade2bda4","owner":[],"postedDate":"June 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-21T13:23:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-18 11:39:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6890787","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6890787","identity":"rs-6890787","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}