Caffeine Modulates Ferroptosis Through the PI3K/Akt Signaling Pathway to Inhibit Invasion and Migration of U-373 Glioblastoma Cells

Caffeine Modulates Ferroptosis Through the PI3K/Akt Signaling Pathway to Inhibit Invasion and Migration of U-373 Glioblastoma Cells | 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 Caffeine Modulates Ferroptosis Through the PI3K/Akt Signaling Pathway to Inhibit Invasion and Migration of U-373 Glioblastoma Cells Xu Jiazhi, Zhang Yun, Chen Dagang, Gao Fengquan, Ren Deshuai, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5015415/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 Objective This study aimed to investigate the effects of caffeine on the invasion and migration of human glioblastoma U-373 cells and to elucidate its mechanism of action through the regulation of the PI3K/Akt signaling pathway and ferroptosis. Methods U-373 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM). The effects of caffeine on cell invasion, migration, apoptosis, and ferroptosis were assessed using Transwell assays, flow cytometry, cell scratch assays, and Western blotting techniques. Western blot analysis was specifically used to evaluate the expression of PI3K/Akt-related proteins and markers of ferroptosis. Results Caffeine significantly inhibited the invasion and migration of U-373 cells in a concentration-dependent manner. It also promoted apoptosis and decreased the expression of p-PI3K/PI3K and p-Akt/Akt proteins, as well as markers associated with ferroptosis. Conclusion Caffeine, through its regulation of the PI3K/Akt signaling pathway and modulation of ferroptosis, effectively suppresses the invasion and migration of U-373 glioblastoma cells. These findings suggest that caffeine could be a promising anti-glioblastoma agent and offer new insights into its potential applications in cancer treatment. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Glioblastoma is one of the most prevalent and malignant tumors of the central nervous system, contributing to significant morbidity and mortality rates. This type of tumor is notorious for its high invasiveness, often spreading rapidly within the brain, which drastically shortens patient survival time. U-373MG cells, a human brain glioblastoma cell line, are frequently used as an experimental model for studying glioblastomas due to their high degree of invasiveness and proliferative capacity. These cells effectively simulate the proliferation and spread characteristics of glioblastomas in the human body. Therefore, research on U-373MG cells not only enhances our understanding of glioblastomas' biological characteristics but also provides valuable insights for developing new treatment strategies. The PI3K/Akt signaling pathway is crucial in various biological processes, including cell growth, proliferation, migration, and survival. Overactivation of the PI3K/Akt pathway has been implicated in the development and progression of several cancers, including glioblastomas. Consequently, targeting this pathway presents a promising approach for glioblastoma treatment. Caffeine, a natural compound commonly found in food and beverages, has garnered interest for its potential therapeutic applications in central nervous system diseases due to its ability to cross the blood-brain barrier. Caffeine is known to exhibit various biological activities, including anti-tumor effects. Previous studies have shown that caffeine can inhibit inositol phosphate metabolism and suppress Akt enzymatic activity in certain cell models. Given these properties, it is plausible to hypothesize that caffeine may influence the biological behavior of U-373 glioblastoma cells by regulating the PI3K/Akt signaling pathway and inducing ferroptosis, a form of regulated cell death characterized by iron-dependent lipid peroxidation. In this study, we aim to explore the impact of caffeine on the invasion and migration of U-373 cells, focusing on its regulation of the PI3K/Akt signaling pathway and its role in mediating ferroptosis. This research may provide new insights into potential therapeutic strategies for glioblastoma treatment. 2. Materials and Methods Cell Culture The glioblastoma U-373 cell line was purchased from the Cell Resource Center of the Shanghai Institute of Life Sciences, Chinese Academy of Sciences. Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, 100 IU/ml penicillin, and streptomycin (pH 7.4) (all from Gibco BRL, Grand Island, NY), with 5% CO2 at 37°C. CCK-8 Cell proliferation was determined using the Cell Counting Kit-8 (CCK-8) assay (Sigma, USA) according to the manufacturer's instructions. Cells were treated with 0.25 mmol/L caffeine, 0.5 mmol/L caffeine, 0.5 mmol/L caffeine, and FAK inhibitor (PF573228), respectively, for 24 h. CCK-8 was then added to the wells, and the assay was performed 1 h later. Absorbance was measured at 450 nm. Transwell assay To detect cell migration ability, we used the transwell method. U-373 cancer cells in good growth condition were cultured in a 6-well plate, and when the cell density reached about 85%, cell transfection was performed. Then, based on the reference steps of the Transwell experiment, an invasion experiment was conducted to detect cell migration ability. Flow Cytometry Detection U-373 single-cell suspension was inoculated onto 4 pieces of 12-well plates (10 × 104/well, 1ml/well) and cultured in an incubator for 24h. Once the cells adhered, 1 control group and 3 experimental groups were set (0.25mmol/L, 0.5mmol/L, and 0.5mmol/L + P13K/AKT inhibitor). The 12-well plate with added caffeine was placed in an incubator at 37°C, 5% CO2, and saturated humidity for 24h. Early apoptosis of tumor cells was detected using the Annexin V + PI double staining flow cytometry method. Specific operation method: Take out the 12-well plate at the time point of caffeine action. Add 0.25% trypsin (200 μl/well) to each well to digest and make the cells suspended. Then add DMEM (1 ml/well) to neutralize the trypsin, make a cell suspension, and transfer it to a labeled tube. Centrifuge (1,000 g, 3 min), remove the supernatant, add DMEM (500 μl/tube), and blow into a cell suspension. Then transfer it to a labeled flow tube, successively add Media Binding Reagent 10 μl/tube, 1.25 μl Annexin V-FITC/tube, incubate at room temperature in the dark for 15 min. Centrifuge (1,000 g, 5 min), remove the supernatant, add 4°C 1 × Binding Buffer 0.5 ml/tube, blow into a cell suspension, add PI 10 μl/tube, and then detect on the machine. Test 1 × 104 cells for each sample. Obtain cells and analyze images using FlowJo software (Becton Dickinson). Cell Scratch Assay This study used a cell scratch assay to assess cell migration ability. In brief, before inoculating the cells, three parallel lines were drawn at the bottom of each well of a sterile 6-well plate with a marker pen to locate the wound to be photographed. Cells in the logarithmic growth phase were inoculated into a 6-well plate at a density of 2×105 cells/ml and placed in an incubator for adherent growth. After the cells covered more than 95% of the bottom area of the plate, a wound was formed by scratching the cell monolayer with the tip of a 1 mL pipette. The wound and parallel lines drawn by the marker pen included several intersection points, and then the wound area crossing these lines was selected as the image capture location. Phosphate-buffered saline was used to remove floating cells, and the cells were cultured in a low-serum medium (1% fetal bovine serum) containing chemicals. Images of the selected wound area were obtained 0-24 hours after treatment under an inverted microscope (CKX41, Olympus, Japan) (600D, digital camera, Japan 600D). Six images were collected from each well at each time point and quantified. Cell migration was analyzed using Image-Pro Plus 6 software (Media Cybernetics, USA), and the reduced wound area in each image was divided by the wound length to calculate the migration distance. Western blot assay After collecting cells and obtaining total protein, the protein concentration was determined using a BCA protein quantification kit. The protein concentration was analyzed using a BCA kit. Subsequently, the target protein was separated using SDS-PAGE, transferred to a PVDF membrane, blocked with 5% skim milk for 2 hours, and incubated with the primary antibody overnight at 4°C. The PVDF membrane was then incubated with specific primary antibodies: AKT (1:1000), P-AKT (1:500), PI3K (1:500), Bcl-2 (1:1000), Bax (1:1000), Caspase3 (1:1000), GPX4, ACSL4, and GAPDH (1:10000). After 24 hours, the protein was incubated with the secondary antibody (1:1000) at room temperature for 1 hour and then imaged using ECL reagent 3. Results 3.1 Effect of caffeine on the invasion of U-373 cells. The results showed that caffeine inhibited the proliferative properties of U-373 cells after 24 hours of treatment, and this effect was positively correlated with the concentration of caffeine (P < 0.01)(Fig. 1 C). At the same time, we investigated whether the PI3K/Akt inhibitor mediated the effect of caffeine on the invasion of U-373 cells. The experimental results showed that adding the PI3K/Akt inhibitor further inhibited cell invasion (P < 0.01)(Fig. 1 A and B). 3.2 Effect of caffeine on the apoptosis of U-373 cells. After 24 hours of caffeine treatment, the experimental results showed that U-373 cells treated with different concentrations of caffeine all underwent apoptosis, and this effect increased with the concentration of caffeine (P < 0.01). Moreover, the number of apoptotic cells was higher after adding the PI3K/Akt inhibitor (P < 0.01)(Fig. 2 ). 3.3 Effect of caffeine on the migration of U-373 cells. We conducted a scratch wound healing assay to study the effect of caffeine on the migration of U-373 cells. The results showed that caffeine significantly inhibited cell migration, and this effect was positively correlated with the concentration of caffeine (P < 0.01). Adding the PI3K/Akt inhibitor further inhibited cell migration (P < 0.01)(Fig. 3 ). 3.4 Effect of caffeine on the expression of proteins related to the PI3K/Akt pathway. We detected the expression levels of p-PI3K/PI3K and p-Akt/Akt proteins in each group of U-373 cells using Western blotting. The experimental results showed that the expression of p-PI3K/PI3K and p-Akt/Akt proteins in U-373 cells treated with caffeine was significantly reduced, and this effect was positively correlated with the concentration of caffeine (P < 0.01). After adding the PI3K/Akt inhibitor, the expression of p-PI3K/PI3K and p-Akt/Akt proteins was further inhibited (P < 0.01)(Fig. 4 ). 3.5 Western Blot Analysis of Ferroptosis Markers and PI3K/Akt Pathway Proteins We investigated the role of caffeine in regulating ferroptosis and its effects on the PI3K/Akt signaling pathway in U-373 cells using Western blotting. Our results revealed that caffeine treatment led to a significant reduction in the levels of ferroptosis markers, such as GPX4 and ACSL4, indicating enhanced ferroptosis. Additionally, the expression of p-PI3K/PI3K and p-Akt/Akt proteins was markedly decreased in response to caffeine, with the degree of reduction correlating positively with caffeine concentration (P < 0.01). When a PI3K/Akt inhibitor was added, the suppression of ferroptosis markers and further reduction in p-PI3K/PI3K and p-Akt/Akt levels were observed (P < 0.01) (Fig. 5 ). These findings support the hypothesis that caffeine modulates ferroptosis through the PI3K/Akt signaling pathway in U-373 glioblastoma cells. 4. Discussion Gliomas' invasiveness and migration are critical barriers to effective treatment, highlighting the need for novel therapeutic strategies aimed at inhibiting these processes. In this study, we have demonstrated that caffeine significantly reduces the invasion and migration of U-373 glioblastoma cells and promotes apoptosis. These effects appear to be linked to caffeine's ability to modulate the PI3K/Akt signaling pathway. Our experimental results indicate that caffeine effectively inhibits the invasiveness and migration of U-373 cells in a concentration-dependent manner. This finding is consistent with previous studies, which have reported that caffeine suppresses the invasion and migration of various cancer cells. Additionally, our results show that caffeine promotes apoptosis in U-373 cells, aligning with its antitumor effects observed in other cancer types . The PI3K/Akt signaling pathway plays a pivotal role in glioma development and progression. Our data reveal that caffeine significantly inhibits the phosphorylation of PI3K and Akt, thus disrupting the activation of this critical pathway. This observation corroborates previous research that has found caffeine to inhibit the PI3K/Akt signaling pathway. Furthermore, our study shows that combining caffeine with a PI3K/Akt inhibitor results in an even greater reduction in invasion and migration, supporting the notion that caffeine exerts its effects primarily through this pathway. In addition to its impact on the PI3K/Akt signaling pathway, our study also highlights the role of ferroptosis in the effects observed. Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation, which is distinct from other forms of cell death such as apoptosis or necrosis. Recent research has suggested that ferroptosis is involved in the suppression of cancer cell proliferation and invasion. Our results indicate that caffeine enhances ferroptosis in U-373 cells, as evidenced by increased levels of ferroptosis markers such as GPX4 and ACSL4. This finding suggests that caffeine's effects on glioblastoma cells may be partly mediated through the induction of ferroptosis. The widespread use and favorable safety profile of caffeine make it an attractive candidate for glioma treatment. However, our study is limited to in vitro experiments, and further research is needed to fully evaluate caffeine's efficacy across different grades of glioma and its potential as a complementary treatment in clinical settings. It will be important to conduct in vivo studies to confirm these findings and explore the specific mechanisms by which caffeine modulates both the PI3K/Akt pathway and ferroptosis in glioma cells. In summary, our study provides preliminary evidence that caffeine inhibits U-373 glioblastoma cell invasion and migration, promotes apoptosis, and induces ferroptosis, with these effects largely attributed to its inhibition of the PI3K/Akt signaling pathway. These findings suggest that caffeine holds promise as a potential therapeutic strategy for glioma treatment, warranting further investigation into its clinical applicability and mechanisms of action. 5. Conclusion This study provides strong evidence for caffeine as a potential anti-glioma drug and reveals its potential mechanism of action. This offers a new strategy for the treatment of gliomas and provides clues for the application of caffeine in other cancers. Declarations 6. Funding This study was supported by the Qiqihar Medical Academy Clinical Research Fund, QMSI2020L-14. 7. Authors' contributions Conception and design: XJZ, ZY, CDG Data acquisition: XJZ, GFQ, RDS, WWD. Data analysis and interpretation: XJZ, DY, WN. Laboratory assays: XJZ, WWD, DY. Writing of manuscript and revising it critically: XJZ, DY, WN. All authors read and approved the final manuscript. 8. Conflict of Interest The authors declare no conflict of interest. Acknowledgments Not applicable. Data Availability Statement The datasets analyzed during the current study are available from the corresponding author upon reasonable request. References Ostrom QT, Cote DJ, Ascha M, et al. Adult Glioma Incidence and Survival by Race or Ethnicity in the United States From 2000 to 2014. JAMA Oncol. 2018 Sep 1;4(9):1254-1262. doi: 10.1001/jamaoncol.2018.1789. Ostrom QT, Gittleman H, Liao P, et al. CBTRUS Statistical Report: Primary brain and other central nervous system tumors diagnosed in the United States in 2010-2014. Neuro Oncol . 2017;19(suppl_5):v1-v88. Bhandarkar S, Prabhakar B, Shende P. Quercetin-loaded platelets as a potential targeted therapy for glioblastoma multiforme cell line U373-MG. Biotechnol J . 2021;16(12):e2100271. doi:10.1002/biot.202100271 Manning BD, Toker A. AKT/PKB Signaling: Navigating the Network. Cell . 2017;169(3):381-405. doi:10.1016/j.cell.2017.04.001 Koul D. PTEN signaling pathways in glioblastoma. Cancer Biol Ther . 2008;7(9):1321-1325. doi:10.4161/cbt.7.9.6954 Fredholm BB, Bättig K, Holmén J, et al. Actions of caffeine in the brain with special reference to factors that contribute to its widespread use. Pharmacol Rev . 1999;51(1):83-133. Bonafé GA, Boschiero MN, Sodré AR, et al. Natural Plant Compounds: Does Caffeine, Dipotassium Glycyrrhizinate, Curcumin, and Euphol Play Roles as Antitumoral Compounds in Glioblastoma Cell Lines? Front Neurol. 2022 Feb 17;12:784330. doi: 10.3389/fneur.2021.784330 Miwa S, Sugimoto N, Shirai T, et al. Caffeine activates tumor suppressor PTEN in sarcoma cells. Int J Oncol . 2011;39(2):465-472. doi:10.3892/ijo.2011.1051 Foukas LC, Daniele N, Ktori C, et al. Direct effects of caffeine and theophylline on p110 δ and other phosphoinositide 3-kinases. Differential effects on lipid kinase and protein kinase activities. J Biol Chem 2002;277:37124–30. Kang SS, Han KS, Ku BM, et al. Caffeine-mediated inhibition of calcium release channel inositol 1,4,5-trisphosphate receptor subtype 3 blocks glioblastoma invasion and extends survival. Cancer Res. 2010 Feb 1;70(3):1173-83. doi: 10.1158/0008-5472.CAN-09-2886 Jin Q, Zhao J, Zhao Z, et al. CAMK1D Inhibits Glioma Through the PI3K/AKT/mTOR Signaling Pathway. Front Oncol. 2022 Apr 13;12:845036. doi: 10.3389/fonc.2022.845036. PMID: 35494053; PMCID: PMC9043760. Wang M, Zhang W, Liu Y, , et al. PDIA4 promotes glioblastoma progression via the PI3K/AKT/m-TOR pathway. Biochem Biophys Res Commun. 2022 Feb 1;597:83-90. doi: 10.1016/j.bbrc.2022.01.115 Zhou J, Xu N, Liu B, et al. lncRNA XLOC013218 promotes cell proliferation and TMZ resistance by targeting the PIK3R2-mediated PI3K/AKT pathway in glioma. Cancer Sci. 2022 Aug;113(8):2681-2692. doi: 10.1111/cas.15387. Zhong C, Tao B, Tang F, et al. Remodeling cancer stemness by collagen/fibronectin via the AKT and CDC42 signaling pathway crosstalk in glioma. Theranostics. 2021 Jan 1;11(4):1991-2005. doi: 10.7150/thno.50613. Wang H, Guan W, Yang W, et al. Caffeine inhibits the activation of hepatic stellate cells induced by acetaldehyde via adenosine A2A receptor mediated by the cAMP/PKA/SRC/ERK1/2/P38 MAPK signal pathway. PLoS One. 2014 Mar 28;9(3):e92482. doi: 10.1371/journal.pone.0092482. Additional Declarations No competing interests reported. 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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-5015415","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":357386828,"identity":"6e241f28-ec3c-47ff-9f49-4cfc6a6634e7","order_by":0,"name":"Xu Jiazhi","email":"","orcid":"","institution":"Third Affiliated Hospital of Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Jiazhi","suffix":""},{"id":357386829,"identity":"17a9fc0d-5a52-4383-9e44-334bfec3cb24","order_by":1,"name":"Zhang Yun","email":"","orcid":"","institution":"Third Affiliated Hospital of Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhang","middleName":"","lastName":"Yun","suffix":""},{"id":357386830,"identity":"dcdd9a27-23f8-4287-954a-23aa4b6f70c6","order_by":2,"name":"Chen Dagang","email":"","orcid":"","institution":"Third Affiliated Hospital of Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Dagang","suffix":""},{"id":357386831,"identity":"cde56a2d-6704-436c-a8fe-8e43c50616c2","order_by":3,"name":"Gao Fengquan","email":"","orcid":"","institution":"Third Affiliated Hospital of Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Gao","middleName":"","lastName":"Fengquan","suffix":""},{"id":357386832,"identity":"02ac9a2a-ea4a-4e79-ad6a-7690667f68f4","order_by":4,"name":"Ren Deshuai","email":"","orcid":"","institution":"Third Affiliated Hospital of Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ren","middleName":"","lastName":"Deshuai","suffix":""},{"id":357386833,"identity":"e453fd0a-3c00-41f2-a8a0-1a8e3c5a629d","order_by":5,"name":"Wu Weidong","email":"","orcid":"","institution":"Third Affiliated Hospital of Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wu","middleName":"","lastName":"Weidong","suffix":""},{"id":357386834,"identity":"87f28062-2198-4997-a405-ff98362136c6","order_by":6,"name":"Du Yan","email":"","orcid":"","institution":"Third Affiliated Hospital of Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Du","middleName":"","lastName":"Yan","suffix":""},{"id":357386835,"identity":"86fe6175-8500-4525-a0c4-ef1b74e8e09e","order_by":7,"name":"Na Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYFACxuYHCRU2cvzsjY2Pf1RIyMkT1sLcZvDhTJqxZM/hZmOGMxbGhg0EtbA3SM5sOZy44UZ6mzRjW0UiwwECGvjbDzYY8zYAtRxIbDYunCeRwNjA/PDRDTxaJM4kNjzm3ZFuPPPAwcbHM7dJ5LEzsBkb5+Cz5kAi0JYz1rJ9BxubDXi3SRQzNvCwSePTIn/+YYM0bxszY8NhxjYJ3jkSiQ0HCGgxuJEI9H6bs+KEY4xt0rwNRGgxvPEQFsiMzYYzjkkYGzYT8Ivc+fTHkKiUf/7wwYeaOjl59uaHj/F6HxMwk6Z8FIyCUTAKRgEWAABJCVfOnmQRKgAAAABJRU5ErkJggg==","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":true,"prefix":"","firstName":"Na","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-09-02 04:52:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5015415/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5015415/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65710803,"identity":"ef87f872-751c-4d8c-8e40-088e320edc2d","added_by":"auto","created_at":"2024-10-01 14:29:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6721061,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of caffeine on migration and invasion of U-373 glioma cells. \u003c/strong\u003e(A)Representative images of migrated cells in each group. (B).Statistics of U-373 cell migration rate in each group. (C).The effect of Caffeine on the proliferation of U-373 cells was assessed using CCK-8 assay.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5015415/v1/a7bdad77cb3013aa4d9da01c.png"},{"id":65710804,"identity":"8cd683c7-3bcb-424b-ba13-74c7334598fc","added_by":"auto","created_at":"2024-10-01 14:29:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2831422,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of caffeine on apoptosis in U-373 glioma cells.\u003c/strong\u003e (A) . Effect of caffeine on apoptosis of U-373 glioma cells detected by flow cytometry. (B) The corresponding apoptosis rates are summarized.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5015415/v1/7822d43bae4dd504b9bea1bd.png"},{"id":65711069,"identity":"c7873327-5dd8-47e9-bc74-675cb0adb720","added_by":"auto","created_at":"2024-10-01 14:37:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5932420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Caffeine on the proliferation of U-373 glioma cells in vitro.\u003c/strong\u003e(A) Representative light microscopy images showing the migration of U-373 cells with different treatments towards the scratched area of cell stripping within 24 h. B, The corresponding cell migration rates are summarised. (B)The proliferation rate of U-373 cells in each group was counted.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5015415/v1/4584e9f1c748363e19e50265.png"},{"id":65710807,"identity":"24fd7df2-634c-4700-890f-238ef87ac97b","added_by":"auto","created_at":"2024-10-01 14:29:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2907382,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWestern blot analysis showing the protein expression levels in U-373 cells in each group.\u003c/strong\u003e(A) Expression levels of relevant proteins in different treatment groups. (B) Expression levels of Bcl-2 protein in different groups. (C) Expression levels of BAX protein in different groups. (D) Expression levels of Caspase-3 protein n different groups. (E) Expression levels of P13K protein in different groups. (F) Expression levels of Akt protein in different groups. (G) Expression levels of p-Akt protein in different groups.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5015415/v1/94185ea89218e75d16dba887.png"},{"id":65710805,"identity":"24a88d67-b384-416d-91cf-62816b35f801","added_by":"auto","created_at":"2024-10-01 14:29:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1452551,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWestern Blot Analysis of Ferroptosis Markers and PI3K/Akt Pathway Proteins. \u003c/strong\u003e(A) Western blot images showing the expression levels of p-PI3K/PI3K and p-Akt/Akt proteins in U-373 cells treated with caffeine. (B) Quantitative analysis of PI3K protein levels. (C) Quantitative analysis of Akt protein levels. (D) Quantitative analysis of GPX4 expression. (E) Quantitative analysis of ACSL4 expression..\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5015415/v1/4866b77ee11086b469587b11.png"},{"id":68486784,"identity":"6421d020-d681-4ff8-bd61-95a04cfac044","added_by":"auto","created_at":"2024-11-07 19:46:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":24350979,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5015415/v1/eadd94ee-2566-4382-8d4c-6c742cfdde1d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Caffeine Modulates Ferroptosis Through the PI3K/Akt Signaling Pathway to Inhibit Invasion and Migration of U-373 Glioblastoma Cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGlioblastoma is one of the most prevalent and malignant tumors of the central nervous system, contributing to significant morbidity and mortality rates. This type of tumor is notorious for its high invasiveness, often spreading rapidly within the brain, which drastically shortens patient survival time. U-373MG cells, a human brain glioblastoma cell line, are frequently used as an experimental model for studying glioblastomas due to their high degree of invasiveness and proliferative capacity. These cells effectively simulate the proliferation and spread characteristics of glioblastomas in the human body. Therefore, research on U-373MG cells not only enhances our understanding of glioblastomas' biological characteristics but also provides valuable insights for developing new treatment strategies.\u003c/p\u003e \u003cp\u003eThe PI3K/Akt signaling pathway is crucial in various biological processes, including cell growth, proliferation, migration, and survival. Overactivation of the PI3K/Akt pathway has been implicated in the development and progression of several cancers, including glioblastomas. Consequently, targeting this pathway presents a promising approach for glioblastoma treatment.\u003c/p\u003e \u003cp\u003eCaffeine, a natural compound commonly found in food and beverages, has garnered interest for its potential therapeutic applications in central nervous system diseases due to its ability to cross the blood-brain barrier. Caffeine is known to exhibit various biological activities, including anti-tumor effects. Previous studies have shown that caffeine can inhibit inositol phosphate metabolism and suppress Akt enzymatic activity in certain cell models. Given these properties, it is plausible to hypothesize that caffeine may influence the biological behavior of U-373 glioblastoma cells by regulating the PI3K/Akt signaling pathway and inducing ferroptosis, a form of regulated cell death characterized by iron-dependent lipid peroxidation.\u003c/p\u003e \u003cp\u003eIn this study, we aim to explore the impact of caffeine on the invasion and migration of U-373 cells, focusing on its regulation of the PI3K/Akt signaling pathway and its role in mediating ferroptosis. This research may provide new insights into potential therapeutic strategies for glioblastoma treatment.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003col\u003e\n \u003cli\u003eCell Culture\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe glioblastoma U-373 cell line was purchased from the Cell Resource Center of the Shanghai Institute of Life Sciences, Chinese Academy of Sciences. Cells were grown in Dulbecco\u0026apos;s Modified Eagle\u0026apos;s Medium (DMEM) containing 10% fetal bovine serum, 100 IU/ml penicillin, and streptomycin (pH 7.4) (all from Gibco BRL, Grand Island, NY), with 5% CO2 at 37\u0026deg;C.\u003c/p\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003eCCK-8\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eCell proliferation was determined using the Cell Counting Kit-8 (CCK-8) assay (Sigma, USA) according to the manufacturer\u0026apos;s instructions. Cells were treated with 0.25 mmol/L caffeine, 0.5 mmol/L caffeine, 0.5 mmol/L caffeine, and FAK inhibitor (PF573228), respectively, for 24 h. CCK-8 was then added to the wells, and the assay was performed 1 h later. Absorbance was measured at 450 nm.\u003c/p\u003e\n\u003col start=\"3\"\u003e\n \u003cli\u003eTranswell assay\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eTo detect cell migration ability, we used the transwell method. U-373 cancer cells in good growth condition were cultured in a 6-well plate, and when the cell density reached about 85%, cell transfection was performed. Then, based on the reference steps of the Transwell experiment, an invasion experiment was conducted to detect cell migration ability.\u003c/p\u003e\n\u003col start=\"4\"\u003e\n \u003cli\u003eFlow Cytometry Detection\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eU-373 single-cell suspension was inoculated onto 4 pieces of 12-well plates (10 \u0026times; 104/well, 1ml/well) and cultured in an incubator for 24h. Once the cells adhered, 1 control group and 3 experimental groups were set (0.25mmol/L, 0.5mmol/L, and 0.5mmol/L + P13K/AKT inhibitor). The 12-well plate with added caffeine was placed in an incubator at 37\u0026deg;C, 5% CO2, and saturated humidity for 24h. Early apoptosis of tumor cells was detected using the Annexin V + PI double staining flow cytometry method. Specific operation method: Take out the 12-well plate at the time point of caffeine action. Add 0.25% trypsin (200 \u0026mu;l/well) to each well to digest and make the cells suspended. Then add DMEM (1 ml/well) to neutralize the trypsin, make a cell suspension, and transfer it to a labeled tube. Centrifuge (1,000 g, 3 min), remove the supernatant, add DMEM (500 \u0026mu;l/tube), and blow into a cell suspension. Then transfer it to a labeled flow tube, successively add Media Binding Reagent 10 \u0026mu;l/tube, 1.25 \u0026mu;l Annexin V-FITC/tube, incubate at room temperature in the dark for 15 min. Centrifuge (1,000 g, 5 min), remove the supernatant, add 4\u0026deg;C 1 \u0026times; Binding Buffer 0.5 ml/tube, blow into a cell suspension, add PI 10 \u0026mu;l/tube, and then detect on the machine. Test 1 \u0026times; 104 cells for each sample. Obtain cells and analyze images using FlowJo software (Becton Dickinson).\u003c/p\u003e\n\u003col start=\"5\"\u003e\n \u003cli\u003eCell Scratch Assay\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThis study used a cell scratch assay to assess cell migration ability. In brief, before inoculating the cells, three parallel lines were drawn at the bottom of each well of a sterile 6-well plate with a marker pen to locate the wound to be photographed. Cells in the logarithmic growth phase were inoculated into a 6-well plate at a density of 2\u0026times;105 cells/ml and placed in an incubator for adherent growth. After the cells covered more than 95% of the bottom area of the plate, a wound was formed by scratching the cell monolayer with the tip of a 1 mL pipette. The wound and parallel lines drawn by the marker pen included several intersection points, and then the wound area crossing these lines was selected as the image capture location. Phosphate-buffered saline was used to remove floating cells, and the cells were cultured in a low-serum medium (1% fetal bovine serum) containing chemicals. Images of the selected wound area were obtained 0-24 hours after treatment under an inverted microscope (CKX41, Olympus, Japan) (600D, digital camera, Japan 600D). Six images were collected from each well at each time point and quantified. Cell migration was analyzed using Image-Pro Plus 6 software (Media Cybernetics, USA), and the reduced wound area in each image was divided by the wound length to calculate the migration distance.\u003c/p\u003e\n\u003col start=\"6\"\u003e\n \u003cli\u003eWestern blot assay\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eAfter collecting cells and obtaining total protein, the protein concentration was determined using a BCA protein quantification kit. The protein concentration was analyzed using a BCA kit. Subsequently, the target protein was separated using SDS-PAGE, transferred to a PVDF membrane, blocked with 5% skim milk for 2 hours, and incubated with the primary antibody overnight at 4\u0026deg;C. The PVDF membrane was then incubated with specific primary antibodies: AKT (1:1000), P-AKT (1:500), PI3K (1:500), Bcl-2 (1:1000), Bax (1:1000), Caspase3 (1:1000), GPX4, ACSL4, and GAPDH (1:10000). After 24 hours, the protein was incubated with the secondary antibody (1:1000) at room temperature for 1 hour and then imaged using ECL reagent\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003e3.1 Effect of caffeine on the invasion of U-373 cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe results showed that caffeine inhibited the proliferative properties of U-373 cells after 24 hours of treatment, and this effect was positively correlated with the concentration of caffeine (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01)(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). At the same time, we investigated whether the PI3K/Akt inhibitor mediated the effect of caffeine on the invasion of U-373 cells. The experimental results showed that adding the PI3K/Akt inhibitor further inhibited cell invasion (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01)(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 Effect of caffeine on the apoptosis of U-373 cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter 24 hours of caffeine treatment, the experimental results showed that U-373 cells treated with different concentrations of caffeine all underwent apoptosis, and this effect increased with the concentration of caffeine (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Moreover, the number of apoptotic cells was higher after adding the PI3K/Akt inhibitor (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Effect of caffeine on the migration of U-373 cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe conducted a scratch wound healing assay to study the effect of caffeine on the migration of U-373 cells. The results showed that caffeine significantly inhibited cell migration, and this effect was positively correlated with the concentration of caffeine (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Adding the PI3K/Akt inhibitor further inhibited cell migration (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01)(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 Effect of caffeine on the expression of proteins related to the PI3K/Akt pathway.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe detected the expression levels of p-PI3K/PI3K and p-Akt/Akt proteins in each group of U-373 cells using Western blotting. The experimental results showed that the expression of p-PI3K/PI3K and p-Akt/Akt proteins in U-373 cells treated with caffeine was significantly reduced, and this effect was positively correlated with the concentration of caffeine (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). After adding the PI3K/Akt inhibitor, the expression of p-PI3K/PI3K and p-Akt/Akt proteins was further inhibited (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01)(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Western Blot Analysis of Ferroptosis Markers and PI3K/Akt Pathway Proteins\u003c/h2\u003e \u003cp\u003eWe investigated the role of caffeine in regulating ferroptosis and its effects on the PI3K/Akt signaling pathway in U-373 cells using Western blotting. Our results revealed that caffeine treatment led to a significant reduction in the levels of ferroptosis markers, such as GPX4 and ACSL4, indicating enhanced ferroptosis. Additionally, the expression of p-PI3K/PI3K and p-Akt/Akt proteins was markedly decreased in response to caffeine, with the degree of reduction correlating positively with caffeine concentration (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). When a PI3K/Akt inhibitor was added, the suppression of ferroptosis markers and further reduction in p-PI3K/PI3K and p-Akt/Akt levels were observed (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings support the hypothesis that caffeine modulates ferroptosis through the PI3K/Akt signaling pathway in U-373 glioblastoma cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eGliomas' invasiveness and migration are critical barriers to effective treatment, highlighting the need for novel therapeutic strategies aimed at inhibiting these processes. In this study, we have demonstrated that caffeine significantly reduces the invasion and migration of U-373 glioblastoma cells and promotes apoptosis. These effects appear to be linked to caffeine's ability to modulate the PI3K/Akt signaling pathway.\u003c/p\u003e \u003cp\u003eOur experimental results indicate that caffeine effectively inhibits the invasiveness and migration of U-373 cells in a concentration-dependent manner. This finding is consistent with previous studies, which have reported that caffeine suppresses the invasion and migration of various cancer cells. Additionally, our results show that caffeine promotes apoptosis in U-373 cells, aligning with its antitumor effects observed in other cancer types .\u003c/p\u003e \u003cp\u003eThe PI3K/Akt signaling pathway plays a pivotal role in glioma development and progression. Our data reveal that caffeine significantly inhibits the phosphorylation of PI3K and Akt, thus disrupting the activation of this critical pathway. This observation corroborates previous research that has found caffeine to inhibit the PI3K/Akt signaling pathway. Furthermore, our study shows that combining caffeine with a PI3K/Akt inhibitor results in an even greater reduction in invasion and migration, supporting the notion that caffeine exerts its effects primarily through this pathway.\u003c/p\u003e \u003cp\u003eIn addition to its impact on the PI3K/Akt signaling pathway, our study also highlights the role of ferroptosis in the effects observed. Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation, which is distinct from other forms of cell death such as apoptosis or necrosis. Recent research has suggested that ferroptosis is involved in the suppression of cancer cell proliferation and invasion. Our results indicate that caffeine enhances ferroptosis in U-373 cells, as evidenced by increased levels of ferroptosis markers such as GPX4 and ACSL4. This finding suggests that caffeine's effects on glioblastoma cells may be partly mediated through the induction of ferroptosis.\u003c/p\u003e \u003cp\u003eThe widespread use and favorable safety profile of caffeine make it an attractive candidate for glioma treatment. However, our study is limited to in vitro experiments, and further research is needed to fully evaluate caffeine's efficacy across different grades of glioma and its potential as a complementary treatment in clinical settings. It will be important to conduct in vivo studies to confirm these findings and explore the specific mechanisms by which caffeine modulates both the PI3K/Akt pathway and ferroptosis in glioma cells.\u003c/p\u003e \u003cp\u003eIn summary, our study provides preliminary evidence that caffeine inhibits U-373 glioblastoma cell invasion and migration, promotes apoptosis, and induces ferroptosis, with these effects largely attributed to its inhibition of the PI3K/Akt signaling pathway. These findings suggest that caffeine holds promise as a potential therapeutic strategy for glioma treatment, warranting further investigation into its clinical applicability and mechanisms of action.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study provides strong evidence for caffeine as a potential anti-glioma drug and reveals its potential mechanism of action. This offers a new strategy for the treatment of gliomas and provides clues for the application of caffeine in other cancers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e6. Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Qiqihar Medical Academy Clinical Research Fund, QMSI2020L-14.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Authors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design: XJZ, ZY, CDG Data acquisition: XJZ, GFQ, RDS, WWD. Data analysis and interpretation: XJZ, DY, WN. Laboratory assays: XJZ, WWD, DY. Writing of manuscript and revising it critically: XJZ, DY, WN. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Conflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOstrom QT, Cote DJ, Ascha M, et al. Adult Glioma Incidence and Survival by Race or Ethnicity in the United States From 2000 to 2014. JAMA Oncol. 2018 Sep 1;4(9):1254-1262. doi: 10.1001/jamaoncol.2018.1789.\u003c/li\u003e\n\u003cli\u003eOstrom QT, Gittleman H, Liao P, et al. CBTRUS Statistical Report: Primary brain and other central nervous system tumors diagnosed in the United States in 2010-2014. \u003cem\u003eNeuro Oncol\u003c/em\u003e. 2017;19(suppl_5):v1-v88.\u003c/li\u003e\n\u003cli\u003eBhandarkar S, Prabhakar B, Shende P. Quercetin-loaded platelets as a potential targeted therapy for glioblastoma multiforme cell line U373-MG. \u003cem\u003eBiotechnol J\u003c/em\u003e. 2021;16(12):e2100271. doi:10.1002/biot.202100271\u003c/li\u003e\n\u003cli\u003eManning BD, Toker A. AKT/PKB Signaling: Navigating the Network. \u003cem\u003eCell\u003c/em\u003e. 2017;169(3):381-405. doi:10.1016/j.cell.2017.04.001\u003c/li\u003e\n\u003cli\u003eKoul D. PTEN signaling pathways in glioblastoma. \u003cem\u003eCancer Biol Ther\u003c/em\u003e. 2008;7(9):1321-1325. doi:10.4161/cbt.7.9.6954\u003c/li\u003e\n\u003cli\u003eFredholm BB, B\u0026auml;ttig K, Holm\u0026eacute;n J, et al. Actions of caffeine in the brain with special reference to factors that contribute to its widespread use. \u003cem\u003ePharmacol Rev\u003c/em\u003e. 1999;51(1):83-133.\u003c/li\u003e\n\u003cli\u003eBonaf\u0026eacute; GA, Boschiero MN, Sodr\u0026eacute; AR, et al. Natural Plant Compounds: Does Caffeine, Dipotassium Glycyrrhizinate, Curcumin, and Euphol Play Roles as Antitumoral Compounds in Glioblastoma Cell Lines? Front Neurol. 2022 Feb 17;12:784330. doi: 10.3389/fneur.2021.784330\u003c/li\u003e\n\u003cli\u003eMiwa S, Sugimoto N, Shirai T, et al. Caffeine activates tumor suppressor PTEN in sarcoma cells. \u003cem\u003eInt J Oncol\u003c/em\u003e. 2011;39(2):465-472. doi:10.3892/ijo.2011.1051\u003c/li\u003e\n\u003cli\u003eFoukas LC, Daniele N, Ktori C, et al. Direct effects of caffeine and theophylline on p110 \u0026delta; and other phosphoinositide 3-kinases. Differential effects on lipid kinase and protein kinase activities. J Biol Chem 2002;277:37124\u0026ndash;30.\u003c/li\u003e\n\u003cli\u003eKang SS, Han KS, Ku BM, et al. Caffeine-mediated inhibition of calcium release channel inositol 1,4,5-trisphosphate receptor subtype 3 blocks glioblastoma invasion and extends survival. Cancer Res. 2010 Feb 1;70(3):1173-83. doi: 10.1158/0008-5472.CAN-09-2886\u003c/li\u003e\n\u003cli\u003eJin Q, Zhao J, Zhao Z, et al. CAMK1D Inhibits Glioma Through the PI3K/AKT/mTOR Signaling Pathway. Front Oncol. 2022 Apr 13;12:845036. doi: 10.3389/fonc.2022.845036. PMID: 35494053; PMCID: PMC9043760.\u003c/li\u003e\n\u003cli\u003eWang M, Zhang W, Liu Y, , et al. PDIA4 promotes glioblastoma progression via the PI3K/AKT/m-TOR pathway. Biochem Biophys Res Commun. 2022 Feb 1;597:83-90. doi: 10.1016/j.bbrc.2022.01.115\u003c/li\u003e\n\u003cli\u003eZhou J, Xu N, Liu B, et al. lncRNA XLOC013218 promotes cell proliferation and TMZ resistance by targeting the PIK3R2-mediated PI3K/AKT pathway in glioma. Cancer Sci. 2022 Aug;113(8):2681-2692. doi: 10.1111/cas.15387.\u003c/li\u003e\n\u003cli\u003eZhong C, Tao B, Tang F, et al. Remodeling cancer stemness by collagen/fibronectin \u003cem\u003evia\u003c/em\u003e the AKT and CDC42 signaling pathway crosstalk in glioma. Theranostics. 2021 Jan 1;11(4):1991-2005. doi: 10.7150/thno.50613.\u003c/li\u003e\n\u003cli\u003eWang H, Guan W, Yang W, et al. Caffeine inhibits the activation of hepatic stellate cells induced by acetaldehyde via adenosine A2A receptor mediated by the cAMP/PKA/SRC/ERK1/2/P38 MAPK signal pathway. PLoS One. 2014 Mar 28;9(3):e92482. doi: 10.1371/journal.pone.0092482.\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":"","lastPublishedDoi":"10.21203/rs.3.rs-5015415/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5015415/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eObjective\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis study aimed to investigate the effects of caffeine on the invasion and migration of human glioblastoma U-373 cells and to elucidate its mechanism of action through the regulation of the PI3K/Akt signaling pathway and ferroptosis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eU-373 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM). The effects of caffeine on cell invasion, migration, apoptosis, and ferroptosis were assessed using Transwell assays, flow cytometry, cell scratch assays, and Western blotting techniques. Western blot analysis was specifically used to evaluate the expression of PI3K/Akt-related proteins and markers of ferroptosis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCaffeine significantly inhibited the invasion and migration of U-373 cells in a concentration-dependent manner. It also promoted apoptosis and decreased the expression of p-PI3K/PI3K and p-Akt/Akt proteins, as well as markers associated with ferroptosis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCaffeine, through its regulation of the PI3K/Akt signaling pathway and modulation of ferroptosis, effectively suppresses the invasion and migration of U-373 glioblastoma cells. These findings suggest that caffeine could be a promising anti-glioblastoma agent and offer new insights into its potential applications in cancer treatment.\u003c/p\u003e","manuscriptTitle":"Caffeine Modulates Ferroptosis Through the PI3K/Akt Signaling Pathway to Inhibit Invasion and Migration of U-373 Glioblastoma Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-01 14:29:07","doi":"10.21203/rs.3.rs-5015415/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":"fe68cd77-f6f1-480e-82dc-c5e774e87d01","owner":[],"postedDate":"October 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-07T19:38:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-01 14:29:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5015415","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5015415","identity":"rs-5015415","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}