Betulinic Acid and Oleanolic Acid Modulate CD81 Expression and Induce Apoptosis in Triple-Negative Breast Cancer Cells through ROS Generation

Betulinic Acid and Oleanolic Acid Modulate CD81 Expression and Induce Apoptosis in Triple-Negative Breast Cancer Cells through ROS Generation | 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 Betulinic Acid and Oleanolic Acid Modulate CD81 Expression and Induce Apoptosis in Triple-Negative Breast Cancer Cells through ROS Generation Dian Yuliartha Lestari, Gondo Mastutik, Indri Safitri Mukono This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5208187/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Dec, 2024 Read the published version in Medical Oncology → Version 1 posted 9 You are reading this latest preprint version Abstract Background Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by a lack of hormones and the HER2 receptor, making it unresponsive to targeted therapy. Triterpenoids such as betulinic acid (BA) and oleanolic acid (OA) have anticancer effects by inducing apoptosis in TNBC cells. CD81 is a tetraspanin that affects the growth and metastasis of cancer cells. Aim To evaluate the effect of BA and OA on viability MDA-MB 231 by analyzed of CD81 expression, intracellular ROS and apoptosis. Materials and Methods The TNBC cell, MDA-MB 231, was cultured and exposed by BA dan OA. The viability cell was evaluated by the CCK-8 assay. The binding of BA dan OA to CD81 was analyzed using molecular docking. This study evaluated CD81 expression, intracellular ROS, and apoptosis by flow cytometri. Results The result showed that BA and OA inhibited viability of MDA-MB-231 cells. BA and OA bind to CD81 in silico, with binding affinities of 9.0 kcal/mol for BA and 7.2 kcal/mol for OA. Flow cytometry results revealed that BA can downregulate CD81 expression. BA and OA also increased intracellular ROS levels and induced apoptosis. Conclusion These findings suggest that BA and OA, especially BA, can modulate CD81 expression and promote apoptosis in TNBC cells through the generation of ROS, thereby offering a potential therapeutic strategy for the treatment of TNBC. Triple-negative breast cancer Betulinic acid Oleanolic acid CD81 ROS Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by a lack of expression of estrogen receptors, progesterone receptors, and HER2 receptors [ 1 ]. Breast cancer represents approximately 10‑15% of all breast cancers and is associated with aggressive clinical behavior and poor prognosis [ 2 ]. Owing to its characteristics, TNBC is unresponsive to hormonal or HER2-targeted therapy, so the main treatment option is chemotherapy [ 3 ]. However, resistance to chemotherapy and a high risk of metastasis are major challenges in the treatment of TNBC. Therefore, research into new agents that can target specific molecular pathways in TNBC, such as CD81 expression, is highly important. Triterpenoids are known to promote the apoptosis of cancer cells in different types of cancers, including TNBC [ 4 ]. Natural derivatives such as betulinic acid (BA) or oleanolic acid (OA) are two chemically distinct triterpenoids [ 5 ]. BA has previously been reported to induce apoptosis and inhibit tumorigenesis by affecting important signaling pathways in TNBC, such as the PI3K/Akt pathway [ 6 ]. BA also has the ability to increase the generation of reactive oxygen species (ROS) in cancer cells, leading to excessive oxidative stress [ 7 ]. The increase in ROS might lead to oxidative damage to the DNA, proteins, and lipids of cancer cells, ultimately initiating pathways that result in cell death [ 8 ]. OA has been shown to exert anticancer effects on TNBC. Studies have shown that OA can inhibit one crucial step of epithelial‒mesenchymal transition (EMT), a chief process for generating PT-bearing cell populations, and consequently decrease the invasive ability of TNBC cells [ 9 ]. Tetraspanins are transmembrane proteins present in all eukaryotic cells and organs. They are involved in a multitude of biological processes, including cell adhesion and mobility, cell signaling, protein trafficking, cell proliferation, neurotransmission, immunological activation, reproduction, and extracellular activities. In tumorigenesis, tetraspanins play a role in tumor growth, invasion and angiogenesis. In humans, more than 33 tetraspanins are present; CD81, or target antiproliferative 1 (TAPA1), is a tetraspanin [ 10 ]. Normally, CD81 plays a role in the immune system and plays an important role in the activation of B lymphocyte cells [ 11 , 12 ]. CD81 has been shown to promote tumor cell cluster formation and metastasis in TNBC through interactions with other cell surface proteins, such as CD44. CD81 expression in TNBC is also correlated with poor prognosis and increased metastatic ability [ 13 ]. CD81 downregulation can inhibit cancer cell communication via exosomes, which are essential for tumor growth and metastasis [ 14 ]. Therefore, understanding the potential effects of BA and OA on CD81 expression in TNBC cells is important for developing more potential therapeutic strategies. This study aims to uncover the impact of BA and OA on CD81 regulation in TNBC, paving the way for a novel therapeutic approach. Methods Molecular docking The 3D structure of the CD81 protein was obtained from the UniProt database with ID A6NMH8 via the AlphaFold database (AlphaFold Protein Structure Database). 3D structures of BA (64971) & OA (10494) were obtained from the PubChem database ( https://www.pubchem.ncbi.nlm.nih.gov ). A binding site prediction approach was used for docking with CD81. PrankWEB tools ( https://prankweb.cz/ ) were used to predict the position of the CD81 binding site. Cell Lines and Culture MDA-MB 231 triple-negative breast cancer cells were obtained from the cell culture division of the biomedical laboratory, Faculty of Medicine, Pajajaran University, Indonesia. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (fetal bovine serum) and 1% antibiotic solution (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cells were maintained in 5% CO 2 and humidified air at 37°C. Cell viability assay The cytotoxic activity of BA and OA against MDA-MB 231 cells was determined via the CCK8 assay (GLPBio, USA). BA and OA were purchased from Chemfaces (Wuhan, China) and dissolved in dimethyl sulfoxide (DMSO) as stock solutions. Viable cancer cells were grown in 96-well plates at a density of 100 µl/well in 100 µl of DMEM. The cells were incubated for 24 hours at 37°C in an environment containing 95% air humidity and 5% CO2. The BA concentration used was 20 µg/mL, while the OA concentration was 50 µg/mL. Each treatment was repeated 4 times. After 10 µL of CCK8 solution was added to each well of the plate via a repeating pipettor, the plate was incubated for 1–4 hours. The absorbance was determined at 450 nm via a microplate reader, and the percentage of viable cells was computed via the following formula: Cell viability (% ) = [(As-Ab)/(Ac-Ab)]x100 Analysis of CD81 expression by flow cytometry Primary antibodies against CD81 were purchased from Biolegend (San Diego, California). Fifty microliters of the MDA-MB-231 cell suspension was removed and placed into a clean 1.5 mL centrifuge tube. Each sample was diluted by adding 1 µL of the fluorescent antibody CD81 to each 50 µL of cell staining buffer. Cell surface antigen staining was carried out, and the samples were incubated in the dark for 20 minutes at room temperature. Then, 300–500 µL of cell staining buffer was added, and the mixture was stirred by pipetting gently. Finally, it was transferred into a 5 cm round bottom tube and analyzed via flow cytometry. Intracellular ROS detection assay The production of ROS was detected by 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. DCFH-DA was purchased from GLPBio (Montclair, USA). After the cultured cells were treated, they were stained with DCFH-DA (GLPBio, USA), incubated at 37°C for 30 minutes, and then rinsed 2–3 times with PBS. ROS were measured via flow cytometry at a 525 nm wavelength. Cell Apoptosis Cell apoptosis was analyzed with an annexin V-FITC/PI apoptosis detection kit (BioLegend, USA) with a FACSCalibur flow cytometer (BD). The cells were harvested, washed twice, and then resuspended. The cell suspension was fixed with 400 µL of Annexin binding buffer after incubation with 5 µL of FITC-Annexin V and 10 µL of PI for 15 minutes at room temperature in the dark (25°C). Apoptotic cells were detected via microscopy or FACS. Statistical analysis The in vitro research data are presented as the means ± standard deviations (SDs), with four repetitions. One-way ANOVA was used for analysis, and p < 0.005 was considered significant. Results BA and OA bound to CD81 in silico In this study, in silico analysis revealed the binding between BA and OA with CD81. The docking results obtained using AutoDock, an integrated PyRx tool, demonstrated that BA and OA interact with CD81 in similar positions (Fig. 1 a,b). There were binding affinities of -9.0 kcal/mol for BA through ALA59, LYS56, PHE49, ALA52, LEU260, and LEU264. On the other hand, there were binding affinities of -7.2 kcal/mol for OA through MET110, LYS56, PHE132, and VAL113. The more negative the binding affinity value, the stronger the interaction between the receptor and ligand. These results indicated that BA interacts more strongly with CD81 than OA does because the surface area of BA structure is larg er than OA. BA and OA have the same number of amino acids as the PRANKWEB binding prediction, which is 5 amino acids. However, OA interactions are more diverse because they include not only hydrophobic interactions but also hydrogen interactions. LYS56 on CD81 is an amino acid that binds equally with BA and OA (Fig. 1 c,d). BA and OA inhibited proliferation MDA-MB-231 cells The CCK-8 assay was used to determine the impact of BA and OA on TNBC proliferation, and the results were compared with those of controls. The CCK-8 assay results were significantly reduced by BA and OA at 24 (p = 0.022) and 48 (p = 0.019) hours. BA cell viability at the 48th hour was 48.29%, and OA viability at the 48th hour was 72.80% (Fig. 2 ). The results of this study revealed that BA reduced TNBC cell proliferation more significantly than OA did. CD81 expression post BA and OA administration The effects of BA and OA on CD81 expression were analyzed by flow cytometry. MDA-MB-231 cells treated with BA showed a decrease in CD81 expression, while OA increased it. (Fig. 3 ). These results indicate that BA and OA affect CD81 levels in TNBC cell types (p = 0,000). BA and OA Increased ROS The enzymatic cleavage of DCFH-DA to DCF was measured in TNBC cells to evaluate intracellular ROS levels and characterize the mechanism of BA and OA-induced cytotoxicity. We found that BA significantly increased the fluorescence intensity in MDA-MB 231 cells (p = 0.000). The results revealed that BA was increased 1.15-fold in the MDA-MB 231 cells compared with the control cells. OA increased fluorescence 1.08-fold in the MDA-MB 231 cells compared with the control cells (Fig. 4 ). These results indicate that BA and OA trigger the formation of ROS in TNBC cells. BA and OA induced apoptosis in MDA-MB-231 cells We measured the percentage of apoptotic TNBC cells via Annexin-V/FITC staining. In this study, BA increased early apoptosis by 5.18-fold and late apoptosis by 8.22-fold in MDA-MB-231 cells compared with the control group (Fig. 5 ). Moreover, OA increased early apoptosis by 2.18-fold and late apoptosis by 1.9-fold in MDA-MB-231 cells compared with controls group. Discussion CD81 is involved in a wide range of critical cellular functions, including cell-to-cell contacts, cellular fusion, protein tracking, and membrane organization. Since CD81 is present on the surface of the cell membrane and plays a role in signal transmission by generating TEMs and interacting with other molecules, suppression of some of these activities will result in inhibition of cell growth [ 21 ]. Recent evidence suggests that CD81 has a significant effect on the progression of human cancer and might be a promising target for chemotherapy [ 11 , 13 ]. The inhibition of cell proliferation by the suppression of CD81 expression has been reported in parotid cancer [ 21 ], breast cancer [ 15 ], prostate cancer [ 18 ], bladder cancer [ 19 ], and osteosarcoma [ 17 ]. CD81 expression in breast cancer cells, particularly MDA-MB 231 cells, has been studied previously [ 15 , 16 ]. CD81 is overexpressed in breast carcinoma tissue compared with normal breast tissue. The overall survival (OS) of breast cancer patients is substantially reduced when tumor tissue has elevated CD81 expression [ 15 ]. Downregulation of CD81 in various malignancies can decrease tumor growth, migration, and cell invasion [ 17 – 19 ], whereas overexpression of CD81 in breast cancer and melanoma enhances metastasis [ 13 , 20 ]. Therefore, in this study, we evaluated the potential of triterpenoid acids especially BA and OA in CD81 regulation. In addition, we showed that binding between BA and OA can reduce CD81 expression, thereby suppressing cell proliferation. Hence, our results indicate that focusing on CD81 might provide therapeutic advantages in the management of TNBC. Our in silico study predicted the interaction of BA and OA with CD81. The AutoDock integrated PyRx study used an estimated BA affinity of -9.0 kcal/mol and an OA affinity of -7.2 kcal/mol with CD81. This study investigated the ability of BA and OA to sensitize TNBC cells by targeting CD81 and provided an experimental basis for their clinical application. The study also showed that treatment of TNBC cells with BA reduced the cell surface expression of CD81 via cytometric analysis. Researchers have reported promising reviews of experimental investigations on the anticancer mechanisms of triterpenoid acids, particularly BA and OA, which include inducing cell cycle arrest and apoptosis, reducing angiogenesis, and repressing oncogenic signaling pathways. Studies have also shown that BA can inhibit antiapoptotic protein, NFκB, and β-catenin signaling, MMP-9 and Ras/ERK pathway-dependent growth in MDA-MB 231 cells [ 22 ], whereas OA can induce apoptosis and inhibit NFκB and COX-2 pathways and MAPK signaling [ 23 ]. Liang et al. reported that the p53 signaling pathway, the TNF signaling pathway, and the mTOR signaling pathway all play a role in how OA treatment affects the growth of breast tumors [ 24 ]. In our study, MDA-MB 231 cells treated with BA were 50% viable at 48 hours of exposure. Moreover, 70% of the OA-treated plants remained viable after 48 hours of exposure. These results suggest that BA is more efficacious in sensitizing TNBC cells than OA. This study observed the number of apoptotic cells via Annexin V-FITC, and the results of the number of cells detected both early and late in apoptosis revealed that BA was more effective than OA. Apoptosis in mammalian cells is mediated by two distinct pathways: the cell surface death receptor pathway (extrinsic) and the mitochondrial pathway (intrinsic). Cell surface receptors activate caspase-8, which triggers the extrinsic pathway, eventually delivering signals associated with cell death. Caspase-9 activation and loss of mitochondrial integrity are defining features of the intrinsic route [ 25 ]. Impaired mitochondrial integrity significantly contributes to the initiation of mitochondrial apoptosis [ 26 ]. This process can be induced by ROS, which are biochemically small molecules containing reactive oxygen [ 27 ]. Previous studies have shown that BA can increase intracellular ROS in cervical cancer [ 28 ], oral squamous cell carcinoma [ 29 ], colorectal cancer [ 30 ], hepatocellular carcinoma [ 31 ], and breast cancer [ 32 , 33 ]. This study also revealed that BA and OA can cause TNBC cells to produce intracellular ROS. Liang et al. reported that BA stopped the growth of MCF-7 cells, which in turn stopped the cell cycle at the G2/M phase and killed the cells through the mitochondrial transfer route. Gene and protein studies revealed that BA greatly reduced the expression of Bcl-2 and increased the expression of Bax. This causes the caspase-3 and caspase-9 cascades to start [ 34 ]. To strengthen the antitumor effects of BA, Guo et al. created Soluplus BA micelles that were covered in a graft copolymer made of polyvinyl caprolactam, polyvinyl acetate, and polyethylene glycol (PVCL-PVA-PEG). BA Soluplus micelles improved the ability of BA to stop MDA-MB-231 cells, mostly because they increase the production of ROS and damage the mitochondrial membrane potential (MMP) [ 35 ]. OA caused apoptosis in MCF-7 and MDA-MB-231 cells by decreasing the expression of Bcl-2 and increasing the expression of Bax and Bad. A new study revealed that SZC015, a type of OA, leads to caspase-dependent apoptosis. This is associated with increased Bax levels and decreased Bcl-2 and Bcl-xL levels [ 36 ]. One type of OA called HIMOXOL (methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate) kills cells by initiating apoptosis through NFκB/p53 signaling and the mitogen-activated protein kinase pathway [ 37 ]. This result provides evidence for our hypothesis that triterpenoid acids, particularly BA, might increase the sensitivity of TNBC cells to the ROS-induced suppression of cell survival and initiation of apoptosis by reducing CD81 expression. CD81 interacts with EGFR and, in conjunction with CD151, controls the transmission of EGFR signals by concentrating signaling intermediates in the tetraspanin domain [ 38 ]. Unfortunately, the mechanism is not yet clear, which is a limitation of this study. We suggest that BA disrupts the CD81/EGFR complex interaction, resulting in elevated levels of ROS that inhibit cell survival, metastasis, and metabolism and induce TNBC cell apoptosis. In summary, we present evidence for the first time that BA administration can induce apoptosis by generating ROS through targeting CD81. Therefore, our findings suggest that BA may possess anticancer properties for the treatment of TNBC. Declarations Competing Interest The authors have no relevant financial or non-financial interest to disclose Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [DYL], [GM] and [ISM]. The first draft of manuscript was written by [DYL] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgement Thanks to all partners who were helpful to the article, the guidance provided by the teacher, and the platform provided by the school. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Lehmann BD, Abramson VG, Sanders ME, Mayer EL, Haddad TC, Nanda R et al. TBCRC 032 IB/II Multicenter Study: Molecular Insights to AR Antagonist and PI3K Inhibitor Efficacy in Patients with AR + Metastatic Triple-Negative Breast Cancer. 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Cite Share Download PDF Status: Published Journal Publication published 07 Dec, 2024 Read the published version in Medical Oncology → Version 1 posted Editorial decision: Revision requested 08 Nov, 2024 Reviews received at journal 07 Nov, 2024 Reviews received at journal 07 Nov, 2024 Reviewers agreed at journal 23 Oct, 2024 Reviewers agreed at journal 23 Oct, 2024 Reviewers invited by journal 21 Oct, 2024 Editor assigned by journal 07 Oct, 2024 Submission checks completed at journal 07 Oct, 2024 First submitted to journal 05 Oct, 2024 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. 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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-5208187","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":375739645,"identity":"e175f962-948a-417c-80bf-6c83aed30e03","order_by":0,"name":"Dian Yuliartha Lestari","email":"","orcid":"","institution":"Airlangga University","correspondingAuthor":false,"prefix":"","firstName":"Dian","middleName":"Yuliartha","lastName":"Lestari","suffix":""},{"id":375739646,"identity":"cf75fbc9-66fa-4e7c-8eb1-950564b25fc0","order_by":1,"name":"Gondo Mastutik","email":"","orcid":"","institution":"Airlangga University","correspondingAuthor":false,"prefix":"","firstName":"Gondo","middleName":"","lastName":"Mastutik","suffix":""},{"id":375739647,"identity":"2676a93f-2650-43c5-ba62-fd7e0731577a","order_by":2,"name":"Indri Safitri Mukono","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIie2RsWrDMBCG/yJQFmOvNi3VKyh06RDyLDIGZXEhkMUQSAQGT+3uxygEOscInKUPYGiH5g3czYOHynaXtm5aOnXQN0jHHR93JwEWyz+EAhwC8EyAfT0TfXbfn8SUTihBZqIil79QgCEfKKMQ5135UPqMe54+1C/t3HfZ3VHPhAR/So86wZxhQu5HB7soV3mYRT6lE65jEYM/l7x4RDRVhC5HFT++QqjIhlIKfdMk4JXghQIRZs7x9TtFtFu/V65Fpyxqo2x/UKgeFHSDVXHXRZ9Q5AphdjCKRHErpBNU8bJQ/DDNvtmF5dHurGnXPktLUjciunSrxe5VJWvmeXr0xb7gDBfvf9lisVgsf+QNMh5UR5c4IS8AAAAASUVORK5CYII=","orcid":"","institution":"Airlangga University","correspondingAuthor":true,"prefix":"","firstName":"Indri","middleName":"Safitri","lastName":"Mukono","suffix":""}],"badges":[],"createdAt":"2024-10-05 09:38:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5208187/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5208187/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12032-024-02574-4","type":"published","date":"2024-12-07T15:57:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69773039,"identity":"fb3244d7-9b81-47c9-842f-a3822031dc11","added_by":"auto","created_at":"2024-11-25 06:58:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":42516,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking results of BA and OA interaction with CD81. \u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003eb\u003c/strong\u003e. Interaction of betulinic acid (blue) and oleanolic acid (dark red) with CD81 (wheat). \u003cstrong\u003ec\u003c/strong\u003e Interaction of CD81 amino acid residues with betulinic acid \u003cstrong\u003ed\u003c/strong\u003e oleanolic acid.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5208187/v1/e7a0b9d0da7ece4069aa8dac.jpg"},{"id":69771716,"identity":"ad55cc50-2727-4350-830a-4dc856762824","added_by":"auto","created_at":"2024-11-25 06:50:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":267263,"visible":true,"origin":"","legend":"\u003cp\u003eThe viability of MDA-MB 231 cells was assessed after treatment with BA and OA at 24 and 48 hours. \u003cstrong\u003ea\u003c/strong\u003e. Negative control. \u003cstrong\u003eb\u003c/strong\u003e. BA. \u003cstrong\u003ec\u003c/strong\u003e. OA. \u003cstrong\u003ed\u003c/strong\u003e. DMSO. \u003cstrong\u003ee\u003c/strong\u003e. Diagram of the average number of cell viability in 24 and 48 hours.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5208187/v1/c2d7e9d423eeaa0aa92a2afb.jpg"},{"id":69771714,"identity":"5a0894ae-f4b6-4ab5-9403-f6e4aed8cd24","added_by":"auto","created_at":"2024-11-25 06:50:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":106398,"visible":true,"origin":"","legend":"\u003cp\u003eCD81 expression in MDA-MB 231 cells after BA and OA administration. \u003cstrong\u003ea\u003c/strong\u003e. Negative control. \u003cstrong\u003eb\u003c/strong\u003e. BA. \u003cstrong\u003ec\u003c/strong\u003e. OA. \u003cstrong\u003ed\u003c/strong\u003e. DMSO. \u003cstrong\u003ee\u003c/strong\u003e. Diagram of the average number of CD81 expression. (*p=0,000)\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5208187/v1/ff53781f0355e833ec41fffa.jpg"},{"id":69771713,"identity":"6ae1ec1e-09b2-4e35-b07f-1ec38cad270c","added_by":"auto","created_at":"2024-11-25 06:50:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":113065,"visible":true,"origin":"","legend":"\u003cp\u003eIntracellular ROS levels of MDA-MB 231 cells after BA and OA administration. \u003cstrong\u003ea\u003c/strong\u003e. Negative control. \u003cstrong\u003eb\u003c/strong\u003e. BA. \u003cstrong\u003ec\u003c/strong\u003e. OA. \u003cstrong\u003ed\u003c/strong\u003e. DMSO. \u003cstrong\u003ee\u003c/strong\u003e. Diagram of the average number of intracellular ROS levels. (*p=0,000)\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5208187/v1/95b5ae1752a0a4cc0d197929.jpg"},{"id":69771719,"identity":"02b456fa-4d59-4640-97f1-db31114dd833","added_by":"auto","created_at":"2024-11-25 06:50:35","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":163039,"visible":true,"origin":"","legend":"\u003cp\u003eEarly and late apoptosis of MDA-MB 231 cells after BA and OA administration. \u003cstrong\u003ea\u003c/strong\u003e. Negative control. \u003cstrong\u003eb\u003c/strong\u003e. BA. \u003cstrong\u003ec\u003c/strong\u003e. OA. \u003cstrong\u003ed\u003c/strong\u003e. DMSO. \u003cstrong\u003ee\u003c/strong\u003e. Diagram of the average number of early and late apoptosis. (*p=0,000)\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5208187/v1/488501b8cf9ff00b2d919644.jpg"},{"id":70964626,"identity":"615fc116-6ca8-42cf-9a06-cf0a7ee80d82","added_by":"auto","created_at":"2024-12-09 16:12:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1117674,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5208187/v1/d3537929-7f9b-46ab-9547-629f9ebc84c1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Betulinic Acid and Oleanolic Acid Modulate CD81 Expression and Induce Apoptosis in Triple-Negative Breast Cancer Cells through ROS Generation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTriple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by a lack of expression of estrogen receptors, progesterone receptors, and HER2 receptors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Breast cancer represents approximately 10‑15% of all breast cancers and is associated with aggressive clinical behavior and poor prognosis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Owing to its characteristics, TNBC is unresponsive to hormonal or HER2-targeted therapy, so the main treatment option is chemotherapy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, resistance to chemotherapy and a high risk of metastasis are major challenges in the treatment of TNBC. Therefore, research into new agents that can target specific molecular pathways in TNBC, such as CD81 expression, is highly important.\u003c/p\u003e \u003cp\u003eTriterpenoids are known to promote the apoptosis of cancer cells in different types of cancers, including TNBC [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Natural derivatives such as betulinic acid (BA) or oleanolic acid (OA) are two chemically distinct triterpenoids [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. BA has previously been reported to induce apoptosis and inhibit tumorigenesis by affecting important signaling pathways in TNBC, such as the PI3K/Akt pathway [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. BA also has the ability to increase the generation of reactive oxygen species (ROS) in cancer cells, leading to excessive oxidative stress [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The increase in ROS might lead to oxidative damage to the DNA, proteins, and lipids of cancer cells, ultimately initiating pathways that result in cell death [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. OA has been shown to exert anticancer effects on TNBC. Studies have shown that OA can inhibit one crucial step of epithelial‒mesenchymal transition (EMT), a chief process for generating PT-bearing cell populations, and consequently decrease the invasive ability of TNBC cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTetraspanins are transmembrane proteins present in all eukaryotic cells and organs. They are involved in a multitude of biological processes, including cell adhesion and mobility, cell signaling, protein trafficking, cell proliferation, neurotransmission, immunological activation, reproduction, and extracellular activities. In tumorigenesis, tetraspanins play a role in tumor growth, invasion and angiogenesis. In humans, more than 33 tetraspanins are present; CD81, or target antiproliferative 1 (TAPA1), is a tetraspanin [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Normally, CD81 plays a role in the immune system and plays an important role in the activation of B lymphocyte cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. CD81 has been shown to promote tumor cell cluster formation and metastasis in TNBC through interactions with other cell surface proteins, such as CD44. CD81 expression in TNBC is also correlated with poor prognosis and increased metastatic ability [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. CD81 downregulation can inhibit cancer cell communication via exosomes, which are essential for tumor growth and metastasis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, understanding the potential effects of BA and OA on CD81 expression in TNBC cells is important for developing more potential therapeutic strategies. This study aims to uncover the impact of BA and OA on CD81 regulation in TNBC, paving the way for a novel therapeutic approach.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMolecular docking\u003c/h2\u003e \u003cp\u003eThe 3D structure of the CD81 protein was obtained from the UniProt database with ID A6NMH8 via the AlphaFold database (AlphaFold Protein Structure Database). 3D structures of BA (64971) \u0026amp; OA (10494) were obtained from the PubChem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.pubchem.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"https://www.pubchem.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A binding site prediction approach was used for docking with CD81. PrankWEB tools (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://prankweb.cz/\u003c/span\u003e\u003cspan address=\"https://prankweb.cz/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used to predict the position of the CD81 binding site.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Lines and Culture\u003c/h3\u003e\n\u003cp\u003eMDA-MB 231 triple-negative breast cancer cells were obtained from the cell culture division of the biomedical laboratory, Faculty of Medicine, Pajajaran University, Indonesia. The cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 10% FBS (fetal bovine serum) and 1% antibiotic solution (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cells were maintained in 5% CO\u003csub\u003e2\u003c/sub\u003e and humidified air at 37\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eCell viability assay\u003c/h3\u003e\n\u003cp\u003eThe cytotoxic activity of BA and OA against MDA-MB 231 cells was determined via the CCK8 assay (GLPBio, USA). BA and OA were purchased from Chemfaces (Wuhan, China) and dissolved in dimethyl sulfoxide (DMSO) as stock solutions. Viable cancer cells were grown in 96-well plates at a density of 100 \u0026micro;l/well in 100 \u0026micro;l of DMEM. The cells were incubated for 24 hours at 37\u0026deg;C in an environment containing 95% air humidity and 5% CO2. The BA concentration used was 20 \u0026micro;g/mL, while the OA concentration was 50 \u0026micro;g/mL. Each treatment was repeated 4 times. After 10 \u0026micro;L of CCK8 solution was added to each well of the plate via a repeating pipettor, the plate was incubated for 1\u0026ndash;4 hours. The absorbance was determined at 450 nm via a microplate reader, and the percentage of viable cells was computed via the following formula: Cell viability (% ) = [(As-Ab)/(Ac-Ab)]x100\u003c/p\u003e\n\u003ch3\u003eAnalysis of CD81 expression by flow cytometry\u003c/h3\u003e\n\u003cp\u003ePrimary antibodies against CD81 were purchased from Biolegend (San Diego, California). Fifty microliters of the MDA-MB-231 cell suspension was removed and placed into a clean 1.5 mL centrifuge tube. Each sample was diluted by adding 1 \u0026micro;L of the fluorescent antibody CD81 to each 50 \u0026micro;L of cell staining buffer. Cell surface antigen staining was carried out, and the samples were incubated in the dark for 20 minutes at room temperature. Then, 300\u0026ndash;500 \u0026micro;L of cell staining buffer was added, and the mixture was stirred by pipetting gently. Finally, it was transferred into a 5 cm round bottom tube and analyzed via flow cytometry.\u003c/p\u003e\n\u003ch3\u003eIntracellular ROS detection assay\u003c/h3\u003e\n\u003cp\u003eThe production of ROS was detected by 2\u0026rsquo;,7\u0026rsquo;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. DCFH-DA was purchased from GLPBio (Montclair, USA). After the cultured cells were treated, they were stained with DCFH-DA (GLPBio, USA), incubated at 37\u0026deg;C for 30 minutes, and then rinsed 2\u0026ndash;3 times with PBS. ROS were measured via flow cytometry at a 525 nm wavelength.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell Apoptosis\u003c/h2\u003e \u003cp\u003eCell apoptosis was analyzed with an annexin V-FITC/PI apoptosis detection kit (BioLegend, USA) with a FACSCalibur flow cytometer (BD). The cells were harvested, washed twice, and then resuspended. The cell suspension was fixed with 400 \u0026micro;L of Annexin binding buffer after incubation with 5 \u0026micro;L of FITC-Annexin V and 10 \u0026micro;L of PI for 15 minutes at room temperature in the dark (25\u0026deg;C). Apoptotic cells were detected via microscopy or FACS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe in vitro research data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SDs), with four repetitions. One-way ANOVA was used for analysis, and p\u0026thinsp;\u0026lt;\u0026thinsp;0.005 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBA and OA bound to CD81 in silico\u003c/h2\u003e \u003cp\u003eIn this study, in silico analysis revealed the binding between BA and OA with CD81. The docking results obtained using AutoDock, an integrated PyRx tool, demonstrated that BA and OA interact with CD81 in similar positions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea,b). There were binding affinities of -9.0 kcal/mol for BA through ALA59, LYS56, PHE49, ALA52, LEU260, and LEU264. On the other hand, there were binding affinities of -7.2 kcal/mol for OA through MET110, LYS56, PHE132, and VAL113. The more negative the binding affinity value, the stronger the interaction between the receptor and ligand. These results indicated that BA interacts more strongly with CD81 than OA does because the surface area of BA structure is larg er than OA. BA and OA have the same number of amino acids as the PRANKWEB binding prediction, which is 5 amino acids. However, OA interactions are more diverse because they include not only hydrophobic interactions but also hydrogen interactions. LYS56 on CD81 is an amino acid that binds equally with BA and OA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec,d).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBA and OA inhibited proliferation MDA-MB-231 cells\u003c/h2\u003e \u003cp\u003eThe CCK-8 assay was used to determine the impact of BA and OA on TNBC proliferation, and the results were compared with those of controls. The CCK-8 assay results were significantly reduced by BA and OA at 24 (p\u0026thinsp;=\u0026thinsp;0.022) and 48 (p\u0026thinsp;=\u0026thinsp;0.019) hours. BA cell viability at the 48th hour was 48.29%, and OA viability at the 48th hour was 72.80% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The results of this study revealed that BA reduced TNBC cell proliferation more significantly than OA did.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCD81 expression post BA and OA administration\u003c/h2\u003e \u003cp\u003eThe effects of BA and OA on CD81 expression were analyzed by flow cytometry. MDA-MB-231 cells treated with BA showed a decrease in CD81 expression, while OA increased it. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results indicate that BA and OA affect CD81 levels in TNBC cell types (p\u0026thinsp;=\u0026thinsp;0,000).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBA and OA Increased ROS\u003c/h2\u003e \u003cp\u003eThe enzymatic cleavage of DCFH-DA to DCF was measured in TNBC cells to evaluate intracellular ROS levels and characterize the mechanism of BA and OA-induced cytotoxicity. We found that BA significantly increased the fluorescence intensity in MDA-MB 231 cells (p\u0026thinsp;=\u0026thinsp;0.000). The results revealed that BA was increased 1.15-fold in the MDA-MB 231 cells compared with the control cells. OA increased fluorescence 1.08-fold in the MDA-MB 231 cells compared with the control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results indicate that BA and OA trigger the formation of ROS in TNBC cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBA and OA induced apoptosis in MDA-MB-231 cells\u003c/h2\u003e \u003cp\u003eWe measured the percentage of apoptotic TNBC cells via Annexin-V/FITC staining. In this study, BA increased early apoptosis by 5.18-fold and late apoptosis by 8.22-fold in MDA-MB-231 cells compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Moreover, OA increased early apoptosis by 2.18-fold and late apoptosis by 1.9-fold in MDA-MB-231 cells compared with controls group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCD81 is involved in a wide range of critical cellular functions, including cell-to-cell contacts, cellular fusion, protein tracking, and membrane organization. Since CD81 is present on the surface of the cell membrane and plays a role in signal transmission by generating TEMs and interacting with other molecules, suppression of some of these activities will result in inhibition of cell growth [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Recent evidence suggests that CD81 has a significant effect on the progression of human cancer and might be a promising target for chemotherapy [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The inhibition of cell proliferation by the suppression of CD81 expression has been reported in parotid cancer [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], breast cancer [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], prostate cancer [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], bladder cancer [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and osteosarcoma [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. CD81 expression in breast cancer cells, particularly MDA-MB 231 cells, has been studied previously [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. CD81 is overexpressed in breast carcinoma tissue compared with normal breast tissue. The overall survival (OS) of breast cancer patients is substantially reduced when tumor tissue has elevated CD81 expression [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Downregulation of CD81 in various malignancies can decrease tumor growth, migration, and cell invasion [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], whereas overexpression of CD81 in breast cancer and melanoma enhances metastasis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, in this study, we evaluated the potential of triterpenoid acids especially BA and OA in CD81 regulation. In addition, we showed that binding between BA and OA can reduce CD81 expression, thereby suppressing cell proliferation. Hence, our results indicate that focusing on CD81 might provide therapeutic advantages in the management of TNBC.\u003c/p\u003e \u003cp\u003eOur in silico study predicted the interaction of BA and OA with CD81. The AutoDock integrated PyRx study used an estimated BA affinity of -9.0 kcal/mol and an OA affinity of -7.2 kcal/mol with CD81. This study investigated the ability of BA and OA to sensitize TNBC cells by targeting CD81 and provided an experimental basis for their clinical application. The study also showed that treatment of TNBC cells with BA reduced the cell surface expression of CD81 via cytometric analysis.\u003c/p\u003e \u003cp\u003eResearchers have reported promising reviews of experimental investigations on the anticancer mechanisms of triterpenoid acids, particularly BA and OA, which include inducing cell cycle arrest and apoptosis, reducing angiogenesis, and repressing oncogenic signaling pathways. Studies have also shown that BA can inhibit antiapoptotic protein, NFκB, and β-catenin signaling, MMP-9 and Ras/ERK pathway-dependent growth in MDA-MB 231 cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], whereas OA can induce apoptosis and inhibit NFκB and COX-2 pathways and MAPK signaling [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Liang et al. reported that the p53 signaling pathway, the TNF signaling pathway, and the mTOR signaling pathway all play a role in how OA treatment affects the growth of breast tumors [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In our study, MDA-MB 231 cells treated with BA were 50% viable at 48 hours of exposure. Moreover, 70% of the OA-treated plants remained viable after 48 hours of exposure. These results suggest that BA is more efficacious in sensitizing TNBC cells than OA.\u003c/p\u003e \u003cp\u003eThis study observed the number of apoptotic cells via Annexin V-FITC, and the results of the number of cells detected both early and late in apoptosis revealed that BA was more effective than OA. Apoptosis in mammalian cells is mediated by two distinct pathways: the cell surface death receptor pathway (extrinsic) and the mitochondrial pathway (intrinsic). Cell surface receptors activate caspase-8, which triggers the extrinsic pathway, eventually delivering signals associated with cell death. Caspase-9 activation and loss of mitochondrial integrity are defining features of the intrinsic route [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Impaired mitochondrial integrity significantly contributes to the initiation of mitochondrial apoptosis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This process can be induced by ROS, which are biochemically small molecules containing reactive oxygen [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Previous studies have shown that BA can increase intracellular ROS in cervical cancer [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], oral squamous cell carcinoma [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], colorectal cancer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], hepatocellular carcinoma [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and breast cancer [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This study also revealed that BA and OA can cause TNBC cells to produce intracellular ROS. Liang et al. reported that BA stopped the growth of MCF-7 cells, which in turn stopped the cell cycle at the G2/M phase and killed the cells through the mitochondrial transfer route. Gene and protein studies revealed that BA greatly reduced the expression of Bcl-2 and increased the expression of Bax. This causes the caspase-3 and caspase-9 cascades to start [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To strengthen the antitumor effects of BA, Guo et al. created Soluplus BA micelles that were covered in a graft copolymer made of polyvinyl caprolactam, polyvinyl acetate, and polyethylene glycol (PVCL-PVA-PEG). BA Soluplus micelles improved the ability of BA to stop MDA-MB-231 cells, mostly because they increase the production of ROS and damage the mitochondrial membrane potential (MMP) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. OA caused apoptosis in MCF-7 and MDA-MB-231 cells by decreasing the expression of Bcl-2 and increasing the expression of Bax and Bad. A new study revealed that SZC015, a type of OA, leads to caspase-dependent apoptosis. This is associated with increased Bax levels and decreased Bcl-2 and Bcl-xL levels [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. One type of OA called HIMOXOL (methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate) kills cells by initiating apoptosis through NFκB/p53 signaling and the mitogen-activated protein kinase pathway [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis result provides evidence for our hypothesis that triterpenoid acids, particularly BA, might increase the sensitivity of TNBC cells to the ROS-induced suppression of cell survival and initiation of apoptosis by reducing CD81 expression. CD81 interacts with EGFR and, in conjunction with CD151, controls the transmission of EGFR signals by concentrating signaling intermediates in the tetraspanin domain [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Unfortunately, the mechanism is not yet clear, which is a limitation of this study. We suggest that BA disrupts the CD81/EGFR complex interaction, resulting in elevated levels of ROS that inhibit cell survival, metastasis, and metabolism and induce TNBC cell apoptosis.\u003c/p\u003e \u003cp\u003eIn summary, we present evidence for the first time that BA administration can induce apoptosis by generating ROS through targeting CD81. Therefore, our findings suggest that BA may possess anticancer properties for the treatment of TNBC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interest\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interest to disclose\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [DYL], [GM] and [ISM]. The first draft of manuscript was written by [DYL] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThanks to all partners who were helpful to the article, the guidance provided by the teacher, and the platform provided by the school.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLehmann BD, Abramson VG, Sanders ME, Mayer EL, Haddad TC, Nanda R et al. TBCRC 032 IB/II Multicenter Study: Molecular Insights to AR Antagonist and PI3K Inhibitor Efficacy in Patients with AR\u0026thinsp;+\u0026thinsp;Metastatic Triple-Negative Breast Cancer. 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Nat Commun. 2023;14(1).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Triple-negative breast cancer, Betulinic acid, Oleanolic acid, CD81, ROS, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-5208187/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5208187/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTriple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by a lack of hormones and the HER2 receptor, making it unresponsive to targeted therapy. Triterpenoids such as betulinic acid (BA) and oleanolic acid (OA) have anticancer effects by inducing apoptosis in TNBC cells. CD81 is a tetraspanin that affects the growth and metastasis of cancer cells.\u003c/p\u003e\u003ch2\u003eAim\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of BA and OA on viability MDA-MB 231 by analyzed of CD81 expression, intracellular ROS and apoptosis.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eThe TNBC cell, MDA-MB 231, was cultured and exposed by BA dan OA. The viability cell was evaluated by the CCK-8 assay. The binding of BA dan OA to CD81 was analyzed using molecular docking. This study evaluated CD81 expression, intracellular ROS, and apoptosis by flow cytometri.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe result showed that BA and OA inhibited viability of MDA-MB-231 cells. BA and OA bind to CD81 in silico, with binding affinities of 9.0 kcal/mol for BA and 7.2 kcal/mol for OA. Flow cytometry results revealed that BA can downregulate CD81 expression. BA and OA also increased intracellular ROS levels and induced apoptosis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese findings suggest that BA and OA, especially BA, can modulate CD81 expression and promote apoptosis in TNBC cells through the generation of ROS, thereby offering a potential therapeutic strategy for the treatment of TNBC.\u003c/p\u003e","manuscriptTitle":"Betulinic Acid and Oleanolic Acid Modulate CD81 Expression and Induce Apoptosis in Triple-Negative Breast Cancer Cells through ROS Generation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-25 06:50:30","doi":"10.21203/rs.3.rs-5208187/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-08T11:42:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-07T12:56:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-07T05:08:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83026223209168193586396440829277795175","date":"2024-10-24T03:36:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31903639892716371060393749436828127022","date":"2024-10-24T02:44:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-22T01:54:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-07T09:12:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-07T09:11:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Medical Oncology","date":"2024-10-05T09:22:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"70431367-d29d-41f1-b396-1049aedd1096","owner":[],"postedDate":"November 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-09T15:59:26+00:00","versionOfRecord":{"articleIdentity":"rs-5208187","link":"https://doi.org/10.1007/s12032-024-02574-4","journal":{"identity":"medical-oncology","isVorOnly":false,"title":"Medical Oncology"},"publishedOn":"2024-12-07 15:57:03","publishedOnDateReadable":"December 7th, 2024"},"versionCreatedAt":"2024-11-25 06:50:30","video":"","vorDoi":"10.1007/s12032-024-02574-4","vorDoiUrl":"https://doi.org/10.1007/s12032-024-02574-4","workflowStages":[]},"version":"v1","identity":"rs-5208187","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5208187","identity":"rs-5208187","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}