Anticancer Activity of PLK1 Inhibitor BI-2536 and β-Glucan in HT-29 and AGS Cancer Cell Lines

Anticancer Activity of PLK1 Inhibitor BI-2536 and β-Glucan in HT-29 and AGS Cancer Cell Lines | 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 Anticancer Activity of PLK1 Inhibitor BI-2536 and β-Glucan in HT-29 and AGS Cancer Cell Lines Rabia Gökçe TAKCI, Bülent SARAÇ, Levent HACISÜLEYMAN, Burcu ŞAHİNBAŞ, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6983868/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 Background: Colon and gastric cancers are among the most prevalent gastrointestinal malignancies, often exhibiting poor prognosis due to resistance and recurrence. Polo-like kinase 1 (PLK1), a key regulator of mitosis, is frequently overexpressed in these cancers. BI-2536, a selective PLK1 inhibitor, has shown promising anticancer activity. β-glucan, a natural immunomodulator, has also demonstrated anticancer potential. Objective: This study aimed to evaluate the antiproliferative, apoptotic, and cell cycle effects of BI-2536 alone and in combination with β-glucan on HT-29 colon and AGS gastric cancer cell lines. Methods: Cell viability was assessed by XTT assay. Apoptosis and cell cycle profiles were evaluated by flow cytometry. The combination index (CI) was calculated using the Chou–Talalay method via CompuSyn software. Results: BI-2536 significantly inhibited proliferation and induced G2/M arrest and apoptosis in both cell lines. β-glucan showed moderate cytotoxicity and enhanced BI-2536’s effects in combination. Synergistic antiproliferative activity was observed at lower drug concentrations (CI < 1), with the combination inducing greater apoptosis and G2/M arrest than monotherapy. Mechanistically, this synergy may involve dual restoration of FOXO3 activity via PLK1 inhibition and PI3K/AKT/mTOR suppression. Conclusion: BI-2536 in combination with β-glucan exhibits synergistic anticancer effects in vitro, suggesting a promising strategy for treating colon and gastric cancers. Further in vivo studies are warranted. PLK1 BI-2536 β-glucan colon cancer gastric cancer apoptosis cell cycle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Colon and gastric cancers are among the most commonly diagnosed malignancies globally, posing significant clinical challenges due to their high mortality rates, recurrence, and resistance to conventional therapies (Yıldırım Öztürk & Uyar, 2021; Davies & Goldberg, 2011). While standard treatments such as surgery, chemotherapy, and radiotherapy remain essential, many patients are unable to tolerate these approaches due to severe side effects or limited efficacy, especially in metastatic settings (Harada et al., 2021). These limitations have driven interest in identifying more selective, less toxic therapies. Polo-like kinase 1 (PLK-1), a serine/threonine protein kinase, plays a pivotal role in regulating cell cycle progression, particularly during the G2/M transition. Overexpression of PLK-1 has been documented in various tumor types and is associated with increased tumor aggressiveness and poor clinical prognosis (X. S. Liu et al., 2012; Spänkuch-Schmitt et al., 2002). Targeting PLK-1 using selective inhibitors such as BI-2536 has emerged as a promising anticancer strategy. BI-2536 induces mitotic arrest and apoptosis by inhibiting PLK-1, and its therapeutic effects have been demonstrated in several in vitro and in vivo cancer models (Porta et al., 2014; Pezuk et al., 2013; Choi et al., 2015). In addition to targeted inhibitors, combining chemotherapy with immunomodulatory agents offers another avenue for improving efficacy. β-glucan, a naturally occurring polysaccharide found in the cell walls of yeasts and fungi, has shown potential in enhancing host immune responses and inducing apoptosis in cancer cells (Chang, 2002; Pengkumsri et al., 2017). It acts by engaging receptors such as dectin-1, complement receptor 3 (CR3), and toll-like receptors (TLRs), activating immune cells like macrophages and dendritic cells (Chan et al., 2009). Previous studies have shown that β-glucan, when used in combination with chemotherapeutic agents such as oxaliplatin or trastuzumab, may exert synergistic effects and extend survival in cancer models (Ina et al., 2013; Zhang et al., 2016). This study aims to investigate the anticancer efficacy of BI-2536, both as a monotherapy and in combination with β-glucan, in HT-29 colon and AGS gastric cancer cell lines. The study evaluates antiproliferative activity, apoptosis induction, and cell cycle effects, with the hypothesis that β-glucan may enhance the antitumor potential of BI-2536. The findings may contribute to the development of new combinatory regimens for the treatment of gastrointestinal malignancies. Materials and Methods 2.1. Cell Lines and Reagents Human colon adenocarcinoma (HT-29, ATCC® HTB-38™) and gastric adenocarcinoma (AGS, ATCC® CRL-1739™) cell lines were obtained from the American Type Culture Collection (ATCC, USA). Cells were cultured in RPMI-1640 medium (HT-29) or Ham’s F12 medium (AGS) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Capricorn Scientific, Germany), and maintained at 37°C in a humidified incubator with 5% CO₂. The PLK-1 inhibitor BI-2536 (Cayman Chemical, USA), β-glucan (Sigma-Aldrich), and 5-fluorouracil (5-FU) (Sigma-Aldrich) were used for treatment. BI-2536 and β-glucan were tested individually and in combination. 5-FU was used as a positive control. 2.2. Cell Viability Assay (XTT) The antiproliferative effects of BI-2536, β-glucan, and their combinations were evaluated using the XTT cell proliferation assay (Luminex, USA). HT-29 and AGS cells were seeded in 96-well plates and treated for 24 hours with: BI-2536 alone at concentrations ranging from 1 nM to 64 nM, β-glucan alone at concentrations ranging from 31.25 μg/mL to 500 μg/mL, Combinations of BI-2536 + β-glucan in five fixed-ratio groups: o 2 nM + 31.25 μg/mL o 4 nM + 62.5 μg/mL o 8 nM + 125 μg/mL o 16 nM + 250 μg/mL o 32 nM + 500 μg/mL 5-Fluorouracil (5-FU) at 12.5 μg/mL was used as a positive control. Following treatment, XTT reagent was added, and after incubation, absorbance was measured at 450 nm using a microplate reader (Thermo Scientific) (Dirik & Joha, 2023; Taştemur et al., 23). IC₅₀ values were calculated using nonlinear regression analysis in GraphPad Prism. Drug interactions were assessed with CompuSyn software (version 1.0) using the Chou–Talalay method, and classified as synergistic (CI1) (ÖZER, 2023). 2.3. Apoptosis Analysis Apoptotic cell death was assessed using the Muse Annexin V & Dead Cell Kit (Luminex, USA). After treatment with IC₅₀ concentrations of BI-2536, β-glucan, and their combination for 24 hours, cells were harvested, stained with Annexin V and 7-AAD, and analyzed by flow cytometry (Muse Cell Analyzer, Millipore). Data were evaluated to quantify early apoptotic, late apoptotic, and viable cell populations (Yulak & Ergul, 2024). 2.4. Cell Cycle Analysis Cell cycle distribution was determined using the Muse Cell Cycle Kit (Luminex, USA). After treatment, cells were fixed in 70% ethanol, stained with the kit reagent, and analyzed by flow cytometry. The percentage of cells in G0/G1, S, and G2/M phases was calculated for each condition (Yulak et al., 2023). 2.5. Statistical Analysis All experiments were conducted in triplicate. Data are expressed as mean ± standard error (SE). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test in SPSS v23. A p-value < 0.05 was considered statistically significant. Results 3.1. BI-2536 and β-Glucan Inhibit Cell Proliferation in Colon (HT-29) and Gastric (AGS) Cancer Cells 3.1.1. BI-2536 Monotherapy Treatment with BI-2536 for 24 hours resulted in dose-dependent antiproliferative effects in both HT-29 (figure 1A) and AGS cells (figure 1B). The calculated IC₅₀ values were: HT-29: 5.083 nM, AGS: 6.865 nM Significant reduction in cell viability was observed at concentrations ≥8 nM for both cell lines ( p < 0.05 vs. control). 5-FU (12.5 μg/mL) served as a positive control and showed comparable antiproliferative activity figure 1. 3.1.2. β-Glucan Monotherapy β-glucan also reduced cell viability in a dose-dependent manner, with IC₅₀ values of: HT-29: 134 μg/mL AGS: 153.2 μg/mL No significant cytotoxicity was observed at ≤62.5 μg/mL. At ≥125 μg/mL, β-glucan caused a significant decrease in cell viability ( p < 0.05) (figure 2). 3.1.3. Combination Therapy Enhances Antiproliferative Effect Combination treatments of BI-2536 + β-glucan were tested at fixed ratio concentrations. The co-treatment significantly enhanced cytotoxicity in both cell lines compared to monotherapy ( p < 0.05) (figure 3). In HT-29 cells, combinations such as: 2 nM BI-2536 + 31.25 μg/mL β-glucan 4 nM BI-2536 + 62.5 μg/mL β-glucan showed strong synergistic effects based on CompuSyn analysis (table 1). Table 1 . CI values of combination drug application to HT29 cell line Drug Concentration CI Value Interpretation 2 nM BI-2536 + 31.25 μg/mL β-glucan 0.379 Synergism 4 nM BI-2536 + 62.5 μg/mL β-glucan 0.487 Synergism 8 nM BI-2536 + 125 μg/mL β-glucan 0.789 Slight synergism 16 nM BI-2536 + 250 μg/mL β-glucan 0.834 Slight synergism 32 nM BI-2536 + 500 μg/mL β-glucan 1.294 Antagonism In AGS cells, all combination groups significantly decreased cell viability compared to BI-2536 alone, with several concentrations exhibiting synergy (table 2) (figure 4). Table 2 . CI values of combination drug application to AGS cell line Drug Concentration CI Value Interpretation 2 nM BI-2536 + 31.25 μg/mL β-glucan 0.501 Synergism 4 nM BI-2536 + 62.5 μg/mL β-glucan 0.803 Slight Synergism 8 nM BI-2536 + 125 μg/mL β-glucan 0.836 Slight Synergism 16 nM BI-2536 + 250 μg/mL β-glucan 0.938 Additive Effect 32 nM BI-2536 + 500 μg/mL β-glucan 1.748 Antagonism 3.2. BI-2536 and β-Glucan Induce Apoptosis Flow cytometry analysis using Annexin V/Dead Cell staining showed that BI-2536 and β-glucan both induced apoptosis, and this effect was significantly enhanced in the combination groups. In HT-29 cells: BI-2536 alone: early + late apoptosis = 31.8% β-glucan alone: early + late apoptosis = 20.1% Combination: 47.9% apoptotic cells ( p < 0.05 vs. BI-2536 alone) (figure 5). In AGS cells, a similar pattern was observed, with combination therapy resulting in the highest apoptotic cell percentage (figure 6). 3.3. Cell Cycle Arrest at G2/M Phase Cell cycle analysis revealed that BI-2536 monotherapy induced significant G2/M phase arrest in both HT-29 and AGS cells, consistent with its role as a PLK-1 inhibitor. β-glucan alone caused modest alterations in phase distribution. However, in combination: G2/M arrest was significantly enhanced, Accompanied by a reduction in G0/G1 and S phases, Suggesting potentiation of BI-2536-induced cell cycle blockade. Discussion Colon and gastric cancers remain among the most prevalent and deadly malignancies globally, particularly when diagnosed at metastatic stages, where five-year survival rates can drop below 10% (Davies & Goldberg, 2011; Li et al., 2022). Despite available therapeutic strategies such as surgery, radiotherapy, and chemotherapy, these treatments often face limitations including toxicity, drug resistance, and high recurrence rates (Harada et al., 2021). Therefore, novel targeted therapies with improved efficacy and tolerability are critically needed. In this context, Polo-like kinase 1 (PLK1) has emerged as an attractive therapeutic target due to its pivotal role in regulating mitosis, centrosome maturation, and the G2/M cell cycle transition. PLK1 is frequently overexpressed in various malignancies, including colorectal and gastric cancers, and its inhibition has been associated with mitotic arrest and induction of apoptosis (Liu et al., 2012; Moolmuang et al., 2024). The selective PLK1 inhibitor BI-2536 has demonstrated promising anticancer effects in various cancer cell lines including non-small cell lung cancer and glioblastoma cells (Choi et al., 2015; Pezuk et al., 2013). In colon cancer models, BI-2536 has shown significant antiproliferative effects, particularly in HT-29 cells, where it sensitized cells to apoptotic signals and enhanced caspase activation when combined with tumor necrosis factor (TNF) (Chopra et al., 2013). Indole-substituted quinolines (ISQs) showed remarkable synergistic tumor remission in vivo when administered together with BI-2536 (Xie et al., 2021). However, despite documented overexpression of PLK1 in gastric tumors and its association with poor prognosis (Takai et al., 2005; Lin et al., 2017), the activity of BI-2536 in gastric cancer cell lines, such as AGS, remains largely unexplored. Moreover, there is currently no available literature evaluating the combinatorial use of BI-2536 with immunomodulatory agents such as β-glucan. Therefore, our study is among the first to investigate the antiproliferative and pro-apoptotic effects of BI-2536 in both colon and gastric cancer models and to assess its potential synergy with β-glucan—a novel combination that may enhance therapeutic efficacy while minimizing toxicity. In this study, we investigated the anticancer effects of BI-2536, both as a monotherapy and in combination with β-glucan, in human HT-29 colon and AGS gastric cancer cell lines. Our results demonstrated that BI-2536 significantly inhibited proliferation and induced apoptosis in a dose-dependent manner, with calculated IC₅₀ values of 5.083 nM for HT-29 and 6.865 nM for AGS cells. Furthermore, BI-2536 treatment resulted in cell cycle arrest at the G2/M phase, consistent with its known mechanism of action (Fu et al., 2008). β-glucan is a well-known immunomodulatory polysaccharide with documented anti-cancer potential. It binds to Dectin-1, CR3, and TLR-2/6 receptors on immune cells, enhancing macrophage, monocyte, and dendritic cell function (Chan et al., 2009). In our study, β-glucan alone exhibited moderate antiproliferative effects at higher concentrations and significantly enhanced BI-2536 efficacy when used in combination. This synergistic interaction, confirmed by CompuSyn software (CI < 1), was particularly notable at lower drug concentrations. These findings are in agreement with previous studies where β-glucan enhanced chemotherapy responses in various cancer models (Zhang et al., 2016; Liu et al., 2015; Ina et al., 2013). Mechanistically, β-glucan may contribute to this synergy by promoting immune-mediated clearance of tumor cells or through indirect modulation of intracellular pathways, such as PI3K/AKT/mTOR, which interact with PLK1-mediated mitotic control. β-glucan has been shown to suppress the PI3K/AKT/mTOR signaling pathway, which is commonly overactivated in many cancers and contributes to cell survival and proliferation (Hu et al., 2024). One downstream effect of AKT activation is the phosphorylation and subsequent inactivation of the tumor suppressor FOXO3, preventing its nuclear localization and pro-apoptotic functions (Yao et al., 2018). Concurrently, PLK1 also negatively regulates FOXO3 by promoting its cytoplasmic retention and degradation. Thus, BI-2536-mediated PLK1 inhibition and β-glucan-mediated suppression of PI3K/AKT signaling may act in concert to restore FOXO3 activity. This dual targeting of FOXO3 derepression may explain the enhanced apoptotic and antiproliferative effects observed in the combination treatment (figure 9). Apoptosis analysis revealed that the BI-2536 + β-glucan combination significantly increased both early and late apoptotic cell populations compared to monotherapies. Flow cytometry analysis of the cell cycle further showed that the combination therapy led to a more pronounced G2/M arrest, especially in HT-29 cells. These results reinforce the hypothesis that combining a mitotic inhibitor with an immune-enhancing agent can intensify cell death and disrupt cancer cell proliferation more effectively than either agent alone. Nevertheless, this study has some limitations. All experiments were conducted in vitro; thus, pharmacokinetic properties, immune system interactions, and potential off-target effects remain unassessed. In vivo studies are necessary to confirm these findings and to evaluate the safety and efficacy of the combination in tumor-bearing models. Additionally, mechanistic investigations such as PLK1 knockdown or PI3K/mTOR inhibition would help to delineate the specific signaling pathways involved. Conclusion This study demonstrated that the selective PLK1 inhibitor BI-2536 exhibits strong antiproliferative and pro-apoptotic effects in HT-29 colon and AGS gastric cancer cell lines, primarily through G2/M cell cycle arrest. Importantly, the combination of BI-2536 with the immunomodulator β-glucan enhanced these effects, showing synergistic interactions and allowing for efficacy at lower drug concentrations. These findings support the therapeutic potential of combining mitotic inhibitors with immune-activating agents to overcome current limitations in cancer treatment, such as toxicity and resistance. However, further mechanistic studies and in vivo validations are necessary to fully establish the clinical applicability of this combination approach in gastrointestinal cancers. Declarations Funding Information This study was funded by Sivas Cumhuriyet University Scientific Research Projects Commission (Project No: T-2022-987, Doctoral Thesis Project, CUBAP, Türkiye). Conflict of Interest The author declares that there are no conflicts of interest related to this work. Ethical Approval This article does not contain any studies with human participants or animals performed by any of the authors. Data Availability The data supporting the findings of this study are available from the corresponding author upon reasonable request. 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Anticancer activity of sinapic acid by inducing apoptosis in HT-29 human colon cancer cell line. Canadian journal of physiology and pharmacology , 101 (7), 361–368. https://doi.org/10.1139/cjpp-2022-0523 Additional Declarations No competing interests reported. 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-6983868","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":492891277,"identity":"eaab3976-30ac-4f42-a046-15e0fbd7eba6","order_by":0,"name":"Rabia Gökçe TAKCI","email":"","orcid":"","institution":"Sivas Cumhuriyet University","correspondingAuthor":false,"prefix":"","firstName":"Rabia","middleName":"Gökçe","lastName":"TAKCI","suffix":""},{"id":492891282,"identity":"14ce309c-cd4d-4473-b754-f1d379261b3e","order_by":1,"name":"Bülent SARAÇ","email":"","orcid":"","institution":"Sivas Cumhuriyet University","correspondingAuthor":false,"prefix":"","firstName":"Bülent","middleName":"","lastName":"SARAÇ","suffix":""},{"id":492891283,"identity":"1cb492df-dfd8-4ba5-ab0a-18b64a9aa79e","order_by":2,"name":"Levent HACISÜLEYMAN","email":"data:image/png;base64,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","orcid":"","institution":"Fenerbahçe University","correspondingAuthor":true,"prefix":"","firstName":"Levent","middleName":"","lastName":"HACISÜLEYMAN","suffix":""},{"id":492891284,"identity":"fcf0d2ea-56a7-4053-8c15-f42018ed4d6c","order_by":3,"name":"Burcu ŞAHİNBAŞ","email":"","orcid":"","institution":"Sivas Cumhuriyet University","correspondingAuthor":false,"prefix":"","firstName":"Burcu","middleName":"","lastName":"ŞAHİNBAŞ","suffix":""},{"id":492891285,"identity":"efb7f089-b244-4f62-be9b-df769d603a9f","order_by":4,"name":"Ziad JOHA","email":"","orcid":"","institution":"Sivas Cumhuriyet University","correspondingAuthor":false,"prefix":"","firstName":"Ziad","middleName":"","lastName":"JOHA","suffix":""}],"badges":[],"createdAt":"2025-06-26 13:23:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6983868/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6983868/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88003180,"identity":"08fa1fc7-718c-45d1-9cda-aaea646cc7d5","added_by":"auto","created_at":"2025-07-31 10:31:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":394312,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntiproliferative Effects of BI-2536 on HT-29 and AGS Cancer Cell Lines.\u003c/strong\u003e\u003cbr\u003e\n(A) BI-2536 treatment significantly inhibited the proliferation of HT-29 colon cancer cells in a dose-dependent manner (n = 6; data are presented as mean ± standard error). Statistically significant inhibition was observed at concentrations of 8 nM and above compared to the control (p \u0026lt; 0.01).\u003cbr\u003e\n(B) BI-2536 exhibited a similar antiproliferative effect on AGS gastric cancer cells, with significant inhibition at concentrations ≥ 8 nM compared to the control (p \u0026lt; 0.01).\u003cbr\u003e\n5-Fluorouracil (5-FU) was used as a positive control in both experiments.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6983868/v1/ff4e8d2e4a7df3b134ab0ab9.png"},{"id":87999117,"identity":"b3891c42-80a8-4a94-b519-2caf4fdaf037","added_by":"auto","created_at":"2025-07-31 10:07:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":354376,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntiproliferative Effects of β-Glucan on HT-29 and AGS Cancer Cell Lines.\u003c/strong\u003e\u003cbr\u003e\n(A) β-Glucan significantly inhibited the proliferation of HT-29 colon cancer cells in a dose-dependent manner (n = 6; data are presented as mean ± standard error). Significant inhibition was observed at concentrations of 125 μg/mL (*p \u0026lt; 0.05) and at 250 μg/mL and above (**p \u0026lt; 0.01) compared to the control.\u003cbr\u003e\n(B) β-Glucan also inhibited proliferation of AGS gastric cancer cells, with statistically significant effects at 125 μg/mL (*p \u0026lt; 0.05) and at 250 μg/mL and above (**p \u0026lt; 0.01) compared to control.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6983868/v1/d510fa044359568b2eee7717.png"},{"id":87999893,"identity":"c8729450-4b4a-4303-950f-cf7d29344e80","added_by":"auto","created_at":"2025-07-31 10:15:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":289792,"visible":true,"origin":"","legend":"\u003cp\u003eAntiproliferative effects of BI-2536 and β-glucan combinations on HT-29 cells.\u003c/p\u003e\n\u003cp\u003eHT-29 cells were treated for 24 h with BI-2536 and β-glucan at increasing concentrations, and viability was assessed by XTT assay. Panels show:\u003c/p\u003e\n\u003cp\u003e(A) 2 nM BI-2536 + 31.25 μg/mL β-glucan, (B) 4 nM BI-2536 + 62.5 μg/mL β-glucan, (C) 8 nM BI-2536 + 125 μg/mL β-glucan, (D) 16 nM BI-2536 + 250 μg/mL β-glucan, (E) 32 nM BI-2536 + 500 μg/mL β-glucan.\u003c/p\u003e\n\u003cp\u003eSignificant reductions in viability were observed vs. control and BI-2536 alone at several concentrations.\u003c/p\u003e\n\u003cp\u003e* p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001 vs. control;\u003c/p\u003e\n\u003cp\u003e# p \u0026lt; 0.05, ## p \u0026lt; 0.01 vs. BI-2536 alone.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6983868/v1/1895d192aed7f663eedfae32.png"},{"id":87999125,"identity":"9b43ff8a-5145-4fe7-ab95-489263a47b32","added_by":"auto","created_at":"2025-07-31 10:07:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":280457,"visible":true,"origin":"","legend":"\u003cp\u003eAntiproliferative effects of BI-2536 and β-glucan combinations on AGS cells. AGS cells were treated for 24 h with BI-2536 and β-glucan at increasing concentrations, and viability was assessed by XTT assay. Panels show:\u003c/p\u003e\n\u003cp\u003e(A) 2 nM BI-2536 + 31.25 μg/mL β-glucan, (B) 4 nM BI-2536 + 62.5 μg/mL β-glucan, (C) 8 nM BI-2536 + 125 μg/mL β-glucan, (D) 16 nM BI-2536 + 250 μg/mL β-glucan, (E) 32 nM BI-2536 + 500 μg/mL β-glucan.\u003c/p\u003e\n\u003cp\u003eSignificant reductions in viability were observed vs. control and BI-2536 alone at several concentrations.\u003c/p\u003e\n\u003cp\u003e* p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001 vs. control;\u003c/p\u003e\n\u003cp\u003e# p \u0026lt; 0.05, ## p \u0026lt; 0.01 vs. BI-2536 alone.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6983868/v1/d5c0f332c2f39349173c0fbe.png"},{"id":87999123,"identity":"ffab4121-b90e-4fc1-aa01-6bb6f94e3a25","added_by":"auto","created_at":"2025-07-31 10:07:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":676572,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometric analysis of the apoptosis profile in HT-29 cells treated with BI-2536, β-glucan, and their combination. (n = 6, data are presented as mean ± standard error).\u003cbr\u003e\n‘A’: Compared to the control, BI-2536, β-glucan, and their combination significantly increased the percentage of early and late apoptotic cells, while significantly reducing the percentage of viable cells (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003cbr\u003e\n‘+’: The combination treatment significantly increased early and late apoptotic cell percentages and decreased viable cells compared to BI-2536 monotherapy (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05).\u003cbr\u003e\n(BI-2536 IC₅₀ = 5.083 nM, β-glucan IC₅₀ = 134.5 μg/mL)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6983868/v1/c0ec7e4a25ce90f7bf15b8bd.png"},{"id":87999895,"identity":"0112eca4-404e-4eb0-8d63-6212231806e7","added_by":"auto","created_at":"2025-07-31 10:15:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":557542,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometric analysis of the apoptosis profile in AGS cells treated with BI-2536, β-glucan, and their combination. (n = 6, data are presented as mean ± standard error).\u003cbr\u003e\n‘A’: Compared to the control, BI-2536, β-glucan, and their combination significantly increased the percentage of early and late apoptotic cells, while significantly reducing the percentage of viable cells (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003cbr\u003e\n‘+’: The combination treatment significantly increased early and late apoptotic cell percentages and decreased viable cells compared to BI-2536 monotherapy (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05).\u003cbr\u003e\n(BI-2536 IC₅₀ = 6.865 nM, β-glucan IC₅₀ = 153.2 μg/mL)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6983868/v1/42b346669ea72769732032d5.png"},{"id":87999896,"identity":"cb13c63d-8867-4953-a277-4139ea181ecc","added_by":"auto","created_at":"2025-07-31 10:15:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":630678,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometric analysis of cell cycle distribution in HT-29 cells following treatment with BI-2536, β-glucan, and their combination (n = 6; data are presented as mean ± standard error).\u003cbr\u003e\n*** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. control.\u003c/p\u003e\n\u003cp\u003e++ \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. BI-2536 monotherapy.\u003cbr\u003e\n+++ \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. BI-2536 monotherapy.\u003cbr\u003e\n(BI-2536 IC₅₀ = 5.083 nM, β-glucan IC₅₀ = 134.5 μg/mL)\u003cbr\u003e\n*** At 5.083 nM, BI-2536 significantly reduced the percentage of cells in the G0/G1 and S phases while significantly increasing the percentage of cells in the G2/M phase compared to the control (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003cbr\u003e\n+++ The combination therapy further increased the proportion of cells in the G2/M phase and decreased those in the G0/G1 phase compared to BI-2536 monotherapy (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6983868/v1/5157987439ada67c907a4677.png"},{"id":87999120,"identity":"f59a27d3-becc-4307-8799-3e3ab7c5cbb0","added_by":"auto","created_at":"2025-07-31 10:07:25","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":665564,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometric analysis of cell cycle distribution in AGS cells following treatment with BI-2536, β-glucan, and their combination (n = 6; data are presented as mean ± standard error).\u003c/p\u003e\n\u003cp\u003e** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. control.\u003cbr\u003e\n*** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. control.\u003cbr\u003e\n+++ \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. BI-2536 monotherapy.\u003cbr\u003e\n(BI-2536 IC₅₀ = 6.865 nM, β-glucan IC₅₀ = 153.2 μg/mL)\u003cbr\u003e\n*** At 6.865 nM, BI-2536 both as monotherapy and combination significantly increased the G2/M phases compared to the control (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001), while in combination significantly decreased the percentage of cells in the G0/G1 and S phases (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003cbr\u003e\n+++ The combination treatment further increased the G2/M phase and reduced the S phase compared to BI-2536 alone (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6983868/v1/093a0417941d687aab7d4c4f.png"},{"id":87999126,"identity":"05b2cbc0-fc55-4f6d-bc78-3980d0c95ca8","added_by":"auto","created_at":"2025-07-31 10:07:25","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":283362,"visible":true,"origin":"","legend":"\u003cp\u003eProposed Mechanism of Synergistic Anticancer Activity of BI-2536 and β-Glucan via Modulation of the PI3K/AKT/PLK1/FOXO3 Pathway.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6983868/v1/82918279f6a413884c7d7893.png"},{"id":90050433,"identity":"d3202b68-5341-4ae6-810b-442eb163177f","added_by":"auto","created_at":"2025-08-27 20:16:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5709556,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6983868/v1/11cba866-2129-4db7-b5fe-8f3e3c6d3449.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anticancer Activity of PLK1 Inhibitor BI-2536 and β-Glucan in HT-29 and AGS Cancer Cell Lines","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColon and gastric cancers are among the most commonly diagnosed malignancies globally, posing significant clinical challenges due to their high mortality rates, recurrence, and resistance to conventional therapies (Yıldırım \u0026Ouml;zt\u0026uuml;rk \u0026amp; Uyar, 2021; Davies \u0026amp; Goldberg, 2011). While standard treatments such as surgery, chemotherapy, and radiotherapy remain essential, many patients are unable to tolerate these approaches due to severe side effects or limited efficacy, especially in metastatic settings (Harada et al., 2021). These limitations have driven interest in identifying more selective, less toxic therapies.\u003c/p\u003e\n\u003cp\u003ePolo-like kinase 1 (PLK-1), a serine/threonine protein kinase, plays a pivotal role in regulating cell cycle progression, particularly during the G2/M transition. Overexpression of PLK-1 has been documented in various tumor types and is associated with increased tumor aggressiveness and poor clinical prognosis (X. S. Liu et al., 2012; Sp\u0026auml;nkuch-Schmitt et al., 2002). Targeting PLK-1 using selective inhibitors such as BI-2536 has emerged as a promising anticancer strategy. BI-2536 induces mitotic arrest and apoptosis by inhibiting PLK-1, and its therapeutic effects have been demonstrated in several in vitro and in vivo cancer models (Porta et al., 2014; Pezuk et al., 2013; Choi et al., 2015).\u003c/p\u003e\n\u003cp\u003eIn addition to targeted inhibitors, combining chemotherapy with immunomodulatory agents offers another avenue for improving efficacy. \u0026beta;-glucan, a naturally occurring polysaccharide found in the cell walls of yeasts and fungi, has shown potential in enhancing host immune responses and inducing apoptosis in cancer cells (Chang, 2002; Pengkumsri et al., 2017). It acts by engaging receptors such as dectin-1, complement receptor 3 (CR3), and toll-like receptors (TLRs), activating immune cells like macrophages and dendritic cells (Chan et al., 2009). Previous studies have shown that \u0026beta;-glucan, when used in combination with chemotherapeutic agents such as oxaliplatin or trastuzumab, may exert synergistic effects and extend survival in cancer models (Ina et al., 2013; Zhang et al., 2016).\u003c/p\u003e\n\u003cp\u003eThis study aims to investigate the anticancer efficacy of BI-2536, both as a monotherapy and in combination with \u0026beta;-glucan, in HT-29 colon and AGS gastric cancer cell lines. The study evaluates antiproliferative activity, apoptosis induction, and cell cycle effects, with the hypothesis that \u0026beta;-glucan may enhance the antitumor potential of BI-2536. The findings may contribute to the development of new combinatory regimens for the treatment of gastrointestinal malignancies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1.\u0026nbsp; Cell Lines and Reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman colon adenocarcinoma (HT-29, ATCC\u0026reg; HTB-38\u0026trade;) and gastric adenocarcinoma (AGS, ATCC\u0026reg; CRL-1739\u0026trade;) cell lines were obtained from the American Type Culture Collection (ATCC, USA). Cells were cultured in RPMI-1640 medium (HT-29) or Ham\u0026rsquo;s F12 medium (AGS) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Capricorn Scientific, Germany), and maintained at 37\u0026deg;C in a humidified incubator with 5% CO₂.\u003c/p\u003e\n\u003cp\u003eThe PLK-1 inhibitor BI-2536 (Cayman Chemical, USA), \u0026beta;-glucan (Sigma-Aldrich), and 5-fluorouracil (5-FU) (Sigma-Aldrich) were used for treatment. BI-2536 and \u0026beta;-glucan were tested individually and in combination. 5-FU was used as a positive control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.\u0026nbsp; Cell Viability Assay (XTT)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antiproliferative effects of BI-2536, \u0026beta;-glucan, and their combinations were evaluated using the XTT cell proliferation assay (Luminex, USA). HT-29 and AGS cells were seeded in 96-well plates and treated for 24 hours with:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eBI-2536 alone at concentrations ranging from 1 nM to 64 nM,\u003c/li\u003e\n \u003cli\u003e\u0026beta;-glucan alone at concentrations ranging from 31.25 \u0026mu;g/mL to 500 \u0026mu;g/mL,\u003c/li\u003e\n \u003cli\u003eCombinations of BI-2536 + \u0026beta;-glucan in five fixed-ratio groups:\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eo 2 nM + 31.25 \u0026mu;g/mL\u003c/p\u003e\n\u003cp\u003eo 4 nM + 62.5 \u0026mu;g/mL\u003c/p\u003e\n\u003cp\u003eo 8 nM + 125 \u0026mu;g/mL\u003c/p\u003e\n\u003cp\u003eo 16 nM + 250 \u0026mu;g/mL\u003c/p\u003e\n\u003cp\u003eo 32 nM + 500 \u0026mu;g/mL\u003c/p\u003e\n\u003cp\u003e5-Fluorouracil (5-FU) at 12.5 \u0026mu;g/mL was used as a positive control.\u003c/p\u003e\n\u003cp\u003eFollowing treatment, XTT reagent was added, and after incubation, absorbance was measured at 450 nm using a microplate reader (Thermo Scientific) (Dirik \u0026amp; Joha, 2023; Taştemur et al., 23). IC₅₀ values were calculated using nonlinear regression analysis in GraphPad Prism. Drug interactions were assessed with CompuSyn software (version 1.0) using the Chou\u0026ndash;Talalay method, and classified as synergistic (CI\u0026lt;1), additive (CI=1), or antagonistic (CI\u0026gt;1) (\u0026Ouml;ZER, 2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.\u0026nbsp; Apoptosis Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApoptotic cell death was assessed using the Muse Annexin V \u0026amp; Dead Cell Kit (Luminex, USA). After treatment with IC₅₀ concentrations of BI-2536, \u0026beta;-glucan, and their combination for 24 hours, cells were harvested, stained with Annexin V and 7-AAD, and analyzed by flow cytometry (Muse Cell Analyzer, Millipore). Data were evaluated to quantify early apoptotic, late apoptotic, and viable cell populations (Yulak \u0026amp; Ergul, 2024).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.\u0026nbsp; Cell Cycle Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell cycle distribution was determined using the Muse Cell Cycle Kit (Luminex, USA). After treatment, cells were fixed in 70% ethanol, stained with the kit reagent, and analyzed by flow cytometry. The percentage of cells in G0/G1, S, and G2/M phases was calculated for each condition (Yulak et al., 2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.\u0026nbsp; Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were conducted in triplicate. Data are expressed as mean \u0026plusmn; standard error (SE). Statistical analysis was performed using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test in SPSS v23. A p-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1.\u0026nbsp;BI-2536 and \u0026beta;-Glucan Inhibit Cell Proliferation in Colon (HT-29) and Gastric (AGS) Cancer Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.1. BI-2536 Monotherapy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTreatment with BI-2536 for 24 hours resulted in dose-dependent antiproliferative effects in both HT-29 (figure 1A) and AGS cells (figure 1B). The calculated IC₅₀ values were:\u003c/p\u003e\n\u003cp\u003eHT-29: 5.083 nM, AGS: 6.865 nM\u003c/p\u003e\n\u003cp\u003eSignificant reduction in cell viability was observed at concentrations \u0026ge;8 nM for both cell lines (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. control). 5-FU (12.5 \u0026mu;g/mL) served as a positive control and showed comparable antiproliferative activity figure 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.2. \u0026beta;-Glucan Monotherapy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026beta;-glucan also reduced cell viability in a dose-dependent manner, with IC₅₀ values of:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eHT-29: 134 \u0026mu;g/mL\u003c/li\u003e\n \u003cli\u003eAGS: 153.2 \u0026mu;g/mL\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eNo significant cytotoxicity was observed at \u0026le;62.5 \u0026mu;g/mL. At \u0026ge;125 \u0026mu;g/mL, \u0026beta;-glucan caused a significant decrease in cell viability (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) (figure 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.3. Combination Therapy Enhances Antiproliferative Effect\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCombination treatments of BI-2536 + \u0026beta;-glucan were tested at fixed ratio concentrations. The co-treatment significantly enhanced cytotoxicity in both cell lines compared to monotherapy (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) (figure 3).\u003c/p\u003e\n\u003cp\u003eIn HT-29 cells, combinations such as:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e2 nM BI-2536 + 31.25 \u0026mu;g/mL \u0026beta;-glucan\u003c/li\u003e\n \u003cli\u003e4 nM BI-2536 + 62.5 \u0026mu;g/mL \u0026beta;-glucan showed strong synergistic effects based on CompuSyn analysis (table 1).\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. CI values of combination drug application to HT29 cell line\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDrug Concentration\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCI Value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eInterpretation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2 nM BI-2536 + 31.25 \u0026mu;g/mL \u0026beta;-glucan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.379\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSynergism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4 nM BI-2536 + 62.5 \u0026mu;g/mL \u0026beta;-glucan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.487\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSynergism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8 nM BI-2536 + 125 \u0026mu;g/mL \u0026beta;-glucan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.789\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSlight synergism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e16 nM BI-2536 + 250 \u0026mu;g/mL \u0026beta;-glucan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.834\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSlight synergism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e32 nM BI-2536 + 500 \u0026mu;g/mL \u0026beta;-glucan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.294\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAntagonism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eIn AGS cells, all combination groups significantly decreased cell viability compared to BI-2536 alone, with several concentrations exhibiting synergy (table 2) (figure 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e. CI values of combination drug application to AGS cell line\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDrug Concentration\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCI Value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eInterpretation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2 nM BI-2536 + 31.25 \u0026mu;g/mL \u0026beta;-glucan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.501\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSynergism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4 nM BI-2536 + 62.5 \u0026mu;g/mL \u0026beta;-glucan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.803\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSlight Synergism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8 nM BI-2536 + 125 \u0026mu;g/mL \u0026beta;-glucan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.836\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSlight Synergism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e16 nM BI-2536 + 250 \u0026mu;g/mL \u0026beta;-glucan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.938\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAdditive Effect\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e32 nM BI-2536 + 500 \u0026mu;g/mL \u0026beta;-glucan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAntagonism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.\u0026nbsp;BI-2536 and \u0026beta;-Glucan Induce Apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometry analysis using Annexin V/Dead Cell staining showed that BI-2536 and \u0026beta;-glucan both induced apoptosis, and this effect was significantly enhanced in the combination groups.\u003c/p\u003e\n\u003cp\u003eIn HT-29 cells:\u003c/p\u003e\n\u003cp\u003eBI-2536 alone: early + late apoptosis = 31.8%\u003c/p\u003e\n\u003cp\u003e\u0026beta;-glucan alone: early + late apoptosis = 20.1%\u003c/p\u003e\n\u003cp\u003eCombination: 47.9% apoptotic cells (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. BI-2536 alone) (figure 5).\u003c/p\u003e\n\u003cp\u003eIn AGS cells, a similar pattern was observed, with combination therapy resulting in the highest apoptotic cell percentage (figure 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.\u0026nbsp;Cell Cycle Arrest at G2/M Phase\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell cycle analysis revealed that BI-2536 monotherapy induced significant G2/M phase arrest in both HT-29 and AGS cells, consistent with its role as a PLK-1 inhibitor. \u0026beta;-glucan alone caused modest alterations in phase distribution.\u003c/p\u003e\n\u003cp\u003eHowever, in combination:\u003c/p\u003e\n\u003cp\u003eG2/M arrest was significantly enhanced,\u003c/p\u003e\n\u003cp\u003eAccompanied by a reduction in G0/G1 and S phases,\u003c/p\u003e\n\u003cp\u003eSuggesting potentiation of BI-2536-induced cell cycle blockade.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eColon and gastric cancers remain among the most prevalent and deadly malignancies globally, particularly when diagnosed at metastatic stages, where five-year survival rates can drop below 10% (Davies \u0026amp; Goldberg, 2011; Li et al., 2022). Despite available therapeutic strategies such as surgery, radiotherapy, and chemotherapy, these treatments often face limitations including toxicity, drug resistance, and high recurrence rates (Harada et al., 2021). Therefore, novel targeted therapies with improved efficacy and tolerability are critically needed.\u003c/p\u003e\n\u003cp\u003eIn this context, Polo-like kinase 1 (PLK1) has emerged as an attractive therapeutic target due to its pivotal role in regulating mitosis, centrosome maturation, and the G2/M cell cycle transition. PLK1 is frequently overexpressed in various malignancies, including colorectal and gastric cancers, and its inhibition has been associated with mitotic arrest and induction of apoptosis (Liu et al., 2012; Moolmuang et al., 2024). The selective PLK1 inhibitor BI-2536 has demonstrated promising anticancer effects in various cancer cell lines including non-small cell lung cancer and\u0026nbsp;glioblastoma cells (Choi et al., 2015; Pezuk et al., 2013). In colon cancer models, BI-2536 has shown significant antiproliferative effects, particularly in HT-29 cells, where it sensitized cells to apoptotic signals and enhanced caspase activation when combined with tumor necrosis factor (TNF) (Chopra et al., 2013). Indole-substituted quinolines (ISQs) showed remarkable synergistic tumor remission in vivo when administered together with BI-2536 (Xie et al., 2021). However, despite documented overexpression of PLK1 in gastric tumors and its association with poor prognosis (Takai et al., 2005; Lin et al., 2017), the activity of BI-2536 in gastric cancer cell lines, such as AGS, remains largely unexplored. Moreover, there is currently no available literature evaluating the combinatorial use of BI-2536 with immunomodulatory agents such as \u0026beta;-glucan. Therefore, our study is among the first to investigate the antiproliferative and pro-apoptotic effects of BI-2536 in both colon and gastric cancer models and to assess its potential synergy with \u0026beta;-glucan\u0026mdash;a novel combination that may enhance therapeutic efficacy while minimizing toxicity.\u003c/p\u003e\n\u003cp\u003eIn this study, we investigated the anticancer effects of BI-2536, both as a monotherapy and in combination with \u0026beta;-glucan, in human HT-29 colon and AGS gastric cancer cell lines. Our results demonstrated that BI-2536 significantly inhibited proliferation and induced apoptosis in a dose-dependent manner, with calculated IC₅₀ values of 5.083 nM for HT-29 and 6.865 nM for AGS cells. Furthermore, BI-2536 treatment resulted in cell cycle arrest at the G2/M phase, consistent with its known mechanism of action (Fu et al., 2008).\u003c/p\u003e\n\u003cp\u003e\u0026beta;-glucan is a well-known immunomodulatory polysaccharide with documented anti-cancer potential. It binds to Dectin-1, CR3, and TLR-2/6 receptors on immune cells, enhancing macrophage, monocyte, and dendritic cell function (Chan et al., 2009). In our study, \u0026beta;-glucan alone exhibited moderate antiproliferative effects at higher concentrations and significantly enhanced BI-2536 efficacy when used in combination. This synergistic interaction, confirmed by CompuSyn software (CI \u0026lt; 1), was particularly notable at lower drug concentrations. These findings are in agreement with previous studies where \u0026beta;-glucan enhanced chemotherapy responses in various cancer models (Zhang et al., 2016; Liu et al., 2015; Ina et al., 2013).\u003c/p\u003e\n\u003cp\u003eMechanistically, \u0026beta;-glucan may contribute to this synergy by promoting immune-mediated clearance of tumor cells or through indirect modulation of intracellular pathways, such as PI3K/AKT/mTOR, which interact with PLK1-mediated mitotic control. \u0026beta;-glucan has been shown to suppress the PI3K/AKT/mTOR signaling pathway, which is commonly overactivated in many cancers and contributes to cell survival and proliferation (Hu et al., 2024). One downstream effect of AKT activation is the phosphorylation and subsequent inactivation of the tumor suppressor FOXO3, preventing its nuclear localization and pro-apoptotic functions (Yao et al., 2018). Concurrently, PLK1 also negatively regulates FOXO3 by promoting its cytoplasmic retention and degradation. Thus, BI-2536-mediated PLK1 inhibition and \u0026beta;-glucan-mediated suppression of PI3K/AKT signaling may act in concert to restore FOXO3 activity. This dual targeting of FOXO3 derepression may explain the enhanced apoptotic and antiproliferative effects observed in the combination treatment (figure 9).\u003c/p\u003e\n\u003cp\u003eApoptosis analysis revealed that the BI-2536 + \u0026beta;-glucan combination significantly increased both early and late apoptotic cell populations compared to monotherapies. Flow cytometry analysis of the cell cycle further showed that the combination therapy led to a more pronounced G2/M arrest, especially in HT-29 cells. These results reinforce the hypothesis that combining a mitotic inhibitor with an immune-enhancing agent can intensify cell death and disrupt cancer cell proliferation more effectively than either agent alone.\u003c/p\u003e\n\u003cp\u003eNevertheless, this study has some limitations. All experiments were conducted in vitro; thus, pharmacokinetic properties, immune system interactions, and potential off-target effects remain unassessed. In vivo studies are necessary to confirm these findings and to evaluate the safety and efficacy of the combination in tumor-bearing models. Additionally, mechanistic investigations such as PLK1 knockdown or PI3K/mTOR inhibition would help to delineate the specific signaling pathways involved.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrated that the selective PLK1 inhibitor BI-2536 exhibits strong antiproliferative and pro-apoptotic effects in HT-29 colon and AGS gastric cancer cell lines, primarily through G2/M cell cycle arrest. Importantly, the combination of BI-2536 with the immunomodulator \u0026beta;-glucan enhanced these effects, showing synergistic interactions and allowing for efficacy at lower drug concentrations.\u003c/p\u003e\n\u003cp\u003eThese findings support the therapeutic potential of combining mitotic inhibitors with immune-activating agents to overcome current limitations in cancer treatment, such as toxicity and resistance. However, further mechanistic studies and in vivo validations are necessary to fully establish the clinical applicability of this combination approach in gastrointestinal cancers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Sivas Cumhuriyet University Scientific Research Projects Commission (Project No: T-2022-987, Doctoral Thesis Project, CUBAP, T\u0026uuml;rkiye).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declares that there are no conflicts of interest related to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.G.T., Z.J. and B.S. conceived and designed the study.\u003c/p\u003e\n\u003cp\u003eR.G.T. and B.Ş. performed the experiments and collected the data.\u003c/p\u003e\n\u003cp\u003eR.G.T. and B.Ş. conducted data analysis and interpretation.\u003c/p\u003e\n\u003cp\u003eL.H. wrote the main manuscript text.\u003c/p\u003e\n\u003cp\u003eL.H. prepared Figures 1\u0026ndash;9 and tables.\u003c/p\u003e\n\u003cp\u003eAll authors reviewed and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChan, G. C., Chan, W. K., \u0026amp; Sze, D. M. (2009). The effects of beta-glucan on human immune and cancer cells. \u003cem\u003eJournal of hematology \u0026amp; oncology\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e, 25. https://doi.org/10.1186/1756-8722-2-25\u003c/li\u003e\n\u003cli\u003eChang R. (2002). Bioactive polysaccharides from traditional Chinese medicine herbs as anticancer adjuvants. \u003cem\u003eJournal of alternative and complementary medicine (New York, N.Y.)\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e(5), 559\u0026ndash;565. https://doi.org/10.1089/107555302320825066\u003c/li\u003e\n\u003cli\u003eChoi, M., Kim, W., Cheon, M. G., Lee, C. W., \u0026amp; Kim, J. E. (2015). Polo-like kinase 1 inhibitor BI2536 causes mitotic catastrophe following activation of the spindle assembly checkpoint in non-small cell lung cancer cells. \u003cem\u003eCancer letters\u003c/em\u003e, \u003cem\u003e357\u003c/em\u003e(2), 591\u0026ndash;601. https://doi.org/10.1016/j.canlet.2014.12.023\u003c/li\u003e\n\u003cli\u003eChopra, A. S., Kuratnik, A., Scocchera, E. W., Wright, D. L., \u0026amp; Giardina, C. (2013). Identification of novel compounds that enhance colon cancer cell sensitivity to inflammatory apoptotic ligands. \u003cem\u003eCancer biology \u0026amp; therapy\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(5), 436\u0026ndash;449. https://doi.org/10.4161/cbt.23787\u003c/li\u003e\n\u003cli\u003eDavies, J. M., \u0026amp; Goldberg, R. M. (2011). 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Anticancer activity of sinapic acid by inducing apoptosis in HT-29 human colon cancer cell line. \u003cem\u003eCanadian journal of physiology and pharmacology\u003c/em\u003e, \u003cem\u003e101\u003c/em\u003e(7), 361\u0026ndash;368. https://doi.org/10.1139/cjpp-2022-0523\u003c/li\u003e\n\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":"PLK1, BI-2536, β-glucan, colon cancer, gastric cancer, apoptosis, cell cycle","lastPublishedDoi":"10.21203/rs.3.rs-6983868/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6983868/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eColon and gastric cancers are among the most prevalent gastrointestinal malignancies, often exhibiting poor prognosis due to resistance and recurrence. Polo-like kinase 1 (PLK1), a key regulator of mitosis, is frequently overexpressed in these cancers. BI-2536, a selective PLK1 inhibitor, has shown promising anticancer activity. β-glucan, a natural immunomodulator, has also demonstrated anticancer potential.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eThis study aimed to evaluate the antiproliferative, apoptotic, and cell cycle effects of BI-2536 alone and in combination with β-glucan on HT-29 colon and AGS gastric cancer cell lines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eCell viability was assessed by XTT assay. Apoptosis and cell cycle profiles were evaluated by flow cytometry. The combination index (CI) was calculated using the Chou–Talalay method via CompuSyn software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eBI-2536 significantly inhibited proliferation and induced G2/M arrest and apoptosis in both cell lines. β-glucan showed moderate cytotoxicity and enhanced BI-2536’s effects in combination. Synergistic antiproliferative activity was observed at lower drug concentrations (CI \u0026lt; 1), with the combination inducing greater apoptosis and G2/M arrest than monotherapy. Mechanistically, this synergy may involve dual restoration of FOXO3 activity via PLK1 inhibition and PI3K/AKT/mTOR suppression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eBI-2536 in combination with β-glucan exhibits synergistic anticancer effects in vitro, suggesting a promising strategy for treating colon and gastric cancers. Further in vivo studies are warranted.\u003c/p\u003e","manuscriptTitle":"Anticancer Activity of PLK1 Inhibitor BI-2536 and β-Glucan in HT-29 and AGS Cancer Cell Lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 10:07:21","doi":"10.21203/rs.3.rs-6983868/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":"5cd8446f-1395-48fa-b5fa-b394272d3d2a","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-27T20:08:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-31 10:07:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6983868","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6983868","identity":"rs-6983868","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}