{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T11:43:51Z","timestamp":1785930231894,"version":"3.56.0"},"reference-count":52,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T00:00:00Z","timestamp":1785888000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Triple-negative breast cancer (TNBC) remains a highly aggressive malignancy with limited therapeutic options and frequent resistance to platinum-based chemotherapy. Rosmarinic acid (RA), a naturally occurring polyphenol, has attracted considerable interest as a potential chemosensitising agent. This study investigated the anticancer activity and the underlying mechanisms of RA combined with cisplatin (CDDP) in 4T1 breast cancer cells while assessing the cytotoxic responses of non-cancerous HaCaT keratinocytes as a preliminary indicator of differential treatment sensitivity. Cytotoxicity was assessed using the MTT assay, followed by calculation of the Combination Index (CI), Drug Reduction Index (DRI), and Selectivity Index (SI). The generation of intracellular reactive oxygen species (ROS) was evaluated by DCFH-DA fluorescence imaging, and the functional contribution of oxidative stress was examined using N-acetyl-L-cysteine (NAC) rescue experiments. Apoptosis was analysed by Annexin V\/PI flow cytometry, NucBlue nuclear staining, and Calcein-AM\/propidium iodide (PI) Live\/Dead fluorescence imaging. Three-dimensional (3D) tumour spheroids were used to assess treatment-induced alterations in spheroid morphology, morphometric parameters, viability based on adenosine triphosphate (ATP), and Live\/Dead staining. The expression of genes related to apoptosis was determined by RT-qPCR, and potential molecular mechanisms were explored using the construction of protein\u2013protein interaction (PPI) networks together with Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analyses. The combination of RA + CDDP exhibited strong synergistic cytotoxicity in 4T1 cells while demonstrating comparatively lower toxicity toward HaCaT keratinocytes. Combination treatment markedly increased intracellular ROS generation, whereas NAC significantly reduced ROS accumulation and partially restored cell viability, indicating that oxidative stress is a major but not exclusive mediator of cytotoxicity. Combined treatment significantly enhanced apoptotic cell death, increased chromatin condensation and membrane damage, upregulated the expression of Bax, Casp9, Cycs, and Trp53, and downregulated Bcl2, consistent with transcriptional regulation of intrinsic apoptotic signalling. In 3D tumour spheroids, the combination markedly reduced spheroid size, disrupted structural integrity, decreased ATP-based viability, and substantially increased tumour cell death compared to monotherapy. Bioinformatic analyses identified central genes related to apoptosis and cell survival and predicted significant enrichment of PI3K\/Akt, p53, MAPK, and apoptosis signalling pathways. RA significantly potentiates the antitumor efficacy of CDDP through synergistic induction of ROS-associated apoptotic signalling while showing a more favourable cytotoxic response in 4T1 breast cancer cells than in non-cancerous HaCaT keratinocytes. The integrated findings from two-dimensional (2D) and 3D models, NAC rescue experiments, molecular analyses, and bioinformatics collectively support the potential of RA as a promising chemosensitising adjuvant for CDDP-based breast cancer therapy and warrant further validation in preclinical in vivo models.<\/jats:p>","DOI":"10.3390\/cells15151419","type":"journal-article","created":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T10:41:42Z","timestamp":1785926502000},"page":"1419","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rosmarinic Acid Potentiates Cisplatin-Induced Antitumour Activity Through ROS-Associated Apoptotic Signalling in Two- and Three-Dimensional Breast Cancer Models"],"prefix":"10.3390","volume":"15","author":[{"given":"Co\u015fkun","family":"Orhaner","sequence":"first","affiliation":[{"name":"Department of Gynaecology and Obstetrics, Soranus IVF Center, Bursa 16070, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aylin","family":"Orhaner","sequence":"additional","affiliation":[{"name":"Department of Gynaecology and Obstetrics, Medicana Bursa Hospital, Bursa 16150, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7317-5467","authenticated-orcid":false,"given":"Mehmet Cudi","family":"Tuncer","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Faculty of Medicine, Dicle University, Diyarbak\u0131r 21280, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"\u0130lhan","family":"\u00d6zdemir","sequence":"additional","affiliation":[{"name":"Department of Histology and Embryology, Faculty of Medicine, Kahramanmara\u015f S\u00fct\u00e7\u00fc \u0130mam University, Kahramanmara\u015f 46000, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,5]]},"reference":[{"key":"ref_1","first-page":"209","article-title":"Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries","volume":"71","author":"Sung","year":"2021","journal-title":"CA Cancer J. Clin."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1038\/s41571-021-00565-2","article-title":"Treatment Landscape of Triple-Negative Breast Cancer\u2014Expanded Options, Evolving Needs","volume":"19","author":"Bianchini","year":"2022","journal-title":"Nat. Rev. Clin. Oncol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"364","DOI":"10.1016\/j.ejphar.2014.07.025","article-title":"Cisplatin in Cancer Therapy: Molecular Mechanisms of Action","volume":"740","author":"Dasari","year":"2014","journal-title":"Eur. J. Pharmacol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1351","DOI":"10.3390\/cancers3011351","article-title":"Cisplatin as an Anti-Tumor Drug: Cellular Mechanisms of Activity, Drug Resistance and Induced Side Effects","volume":"3","author":"Florea","year":"2011","journal-title":"Cancers"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Choudhari, A.S., Mandave, P.C., Deshpande, M., Ranjekar, P., and Prakash, O. (2020). Corrigendum: Phytochemicals in Cancer Treatment: From Preclinical Studies to Clinical Practice. Front. Pharmacol., 11.","DOI":"10.3389\/fphar.2020.00175"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/S0031-9422(02)00513-7","article-title":"Rosmarinic Acid","volume":"62","author":"Petersen","year":"2003","journal-title":"Phytochemistry"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1016\/j.tiv.2017.01.016","article-title":"Antioxidant Activity of Rosmarinic Acid and Its Principal Metabolites in Chemical and Cellular Systems: Importance of Physico-Chemical Characteristics","volume":"40","author":"Chan","year":"2017","journal-title":"Toxicol. In Vitro"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Bozzuto, G., Calcabrini, A., Colone, M., Condello, M., Dupuis, M.L., Pellegrini, E., and Stringaro, A. (2024). Phytocompounds and Nanoformulations for Anticancer Therapy: A Review. Molecules, 29.","DOI":"10.3390\/molecules29163784"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"611","DOI":"10.18632\/aging.102644","article-title":"Aspirin Enhances the Sensitivity of Colon Cancer Cells to Cisplatin by Abrogating the Binding of NF-\u03baB to the COX-2 Promoter","volume":"12","author":"Jiang","year":"2020","journal-title":"Aging"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Toprak, V., \u00d6zdemir, \u0130., \u00d6zt\u00fcrk, \u015e., Yanar, O., Kizildemir, Y.Z., and Tuncer, M.C. (2024). Modulation of FOXP3 Gene Expression in OVCAR3 Cells Following Rosmarinic Acid and Doxorubicin Exposure. Pharmaceuticals, 17.","DOI":"10.20944\/preprints202407.2478.v1"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Duman, E., Ma\u00e7in, A., \u00d6zdemir, \u0130., \u00d6zt\u00fcrk, \u015e., and Tuncer, M.C. (2026). Synergistic Antitumor Effects of Rosmarinic Acid and Cisplatin in Retinoblastoma: Evidence from 2D and 3D Tumor Models. Biomedicines, 14.","DOI":"10.3390\/biomedicines14030602"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Han, Y.-H., Kee, J.-Y., and Hong, S.-H. (2018). Rosmarinic Acid Activates AMPK to Inhibit Metastasis of Colorectal Cancer. Front. Pharmacol., 9.","DOI":"10.3389\/fphar.2018.00068"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Huang, J.-Y., Hsu, T.-W., Chen, Y.-R., and Kao, S.-H. (2024). Rosmarinic Acid Potentiates Cytotoxicity of Cisplatin against Colorectal Cancer Cells by Enhancing Apoptotic and Ferroptosis. Life, 14.","DOI":"10.3390\/life14081017"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Naeem, A., Hu, P., Yang, M., Zhang, J., Liu, Y., Zhu, W., and Zheng, Q. (2022). Natural Products as Anticancer Agents: Current Status and Future Perspectives. Molecules, 27.","DOI":"10.3390\/molecules27238367"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"579","DOI":"10.1038\/nrd2803","article-title":"Targeting Cancer Cells by ROS-Mediated Mechanisms: A Radical Therapeutic Approach?","volume":"8","author":"Trachootham","year":"2009","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Marullo, R., Werner, E., Degtyareva, N., Moore, B., Altaweel, G., and Doetsch, P.W. (2013). Cisplatin Induces a Mitochondrial-ROS Response That Contributes to Cytotoxicity Depending on Mitochondrial Redox Status and Bioenergetic Functions. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0081162"},{"key":"ref_17","first-page":"298","article-title":"Associations of Chemo- and Radio-Resistant Phenotypes with the Gap Junction, Mitochondrial and Drug Efflux Functional Properties in Glioblastoma Multiforme","volume":"12","author":"Bai","year":"2014","journal-title":"J. Transl. Med."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"682","DOI":"10.1038\/nchembio.118","article-title":"Network Pharmacology: The Next Paradigm in Drug Discovery","volume":"4","author":"Hopkins","year":"2008","journal-title":"Nat. Chem. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"10800","DOI":"10.1093\/nar\/gkab835","article-title":"Correction to \u2018The STRING database in 2021: Customizable protein-protein networks, and functional characterization of user-uploaded gene\/measurement sets\u2019","volume":"49","author":"Szklarczyk","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1007\/s00204-025-04146-5","article-title":"Interplay of Oxidative Stress and Antioxidant Mechanisms in Cancer Development and Progression","volume":"100","author":"Jomova","year":"2026","journal-title":"Arch. Toxicol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e70268","DOI":"10.1002\/mco2.70268","article-title":"Oxidative Stress: Signaling Pathways, Biological Functions, and Disease","volume":"6","author":"Liu","year":"2025","journal-title":"MedComm"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"101084","DOI":"10.1016\/j.redox.2018.101084","article-title":"ROS and the DNA damage response in cancer","volume":"25","author":"Srinivas","year":"2019","journal-title":"Redox. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.semcdb.2017.05.023","article-title":"ROS signalling in the biology of cancer","volume":"80","author":"Moloney","year":"2018","journal-title":"Semin. Cell Dev. Biol."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Anwar, S., Alharbi, H.O.A., Babiker, A.Y., and Rahmani, A.H. (2026). Oxidative Stress in Health and Disease: Mechanisms and Therapeutic Perspectives. Int. J. Mol. Sci., 27.","DOI":"10.3390\/ijms27062681"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Nagasawa, S., Takahashi, J., Suzuki, G., Hideya, Y., and Yamada, K. (2021). Why Concurrent CDDP and Radiotherapy Has Synergistic Antitumor Effects: A Review of In Vitro Experimental and Clinical-Based Studies. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22063140"},{"key":"ref_26","first-page":"176","article-title":"Apoptosis Induction with Combined Use of Cisplatin and Fisetin in Cisplatin-Resistant Ovarian Cancer Cells (A2780)","volume":"13","author":"Jafarzadeh","year":"2021","journal-title":"Avicenna J. Med. Biotechnol."},{"key":"ref_27","first-page":"1415","article-title":"Rosmarinic Acid Inhibits the Proliferation of Ovarian Carcinoma Cells by Activating the p53\/BAX Signaling Pathway","volume":"40","author":"Tuncer","year":"2025","journal-title":"Histol. Histopathol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"4201","DOI":"10.2174\/1381612822666160601100823","article-title":"Phytochemicals as Adjunctive with Conventional Anticancer Therapies","volume":"22","author":"Farzaei","year":"2016","journal-title":"Curr. Pharm. Des."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1869","DOI":"10.1038\/onc.2011.384","article-title":"Molecular mechanisms of cisplatin resistance","volume":"31","author":"Galluzzi","year":"2012","journal-title":"Oncogene"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Mustafa, M., Ahmad, R., Tantry, I.Q., Ahmad, W., Siddiqui, S., Alam, M., Abbas, K., Moinuddin, A., Hassan, M.I., and Habib, S. (2024). Apoptosis: A Comprehensive Overview of Signaling Pathways, Morphological Changes, and Physiological Significance and Therapeutic Implications. Cells, 13.","DOI":"10.3390\/cells13221838"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"33","DOI":"10.3389\/fmolb.2020.00033","article-title":"Is It Time to Start Transitioning From 2D to 3D Cell Culture?","volume":"7","author":"Jensen","year":"2020","journal-title":"Front. Mol. Biosci."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Hong, C.-E., and Lyu, S.-Y. (2025). Formulation Strategies for Immunomodulatory Natural Products in 3D Tumor Spheroids and Organoids: Current Challenges and Emerging Solutions. Pharmaceutics, 17.","DOI":"10.3390\/pharmaceutics17101258"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Hong, C.-E., and Lyu, S.-Y. (2025). Immunomodulatory Natural Products in Cancer Organoid-Immune Co-Cultures: Bridging the Research Gap for Precision Immunotherapy. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26157247"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"118597","DOI":"10.1016\/j.biopha.2025.118597","article-title":"Factors of bias in spheroid-based drug screening: Fabrication method, spheroid size, and cell viability","volume":"192","author":"Lee","year":"2025","journal-title":"Biomed. Pharmacother."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"106939","DOI":"10.1016\/j.ejps.2024.106939","article-title":"Advances in Understanding Cisplatin-Induced Toxicity: Molecular Mechanisms and Protective Strategies","volume":"203","author":"Elmorsy","year":"2024","journal-title":"Eur. J. Pharm. Sci."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e2413121","DOI":"10.1002\/advs.202413121","article-title":"HDAC2-Mediated METTL3 Delactylation Promotes DNA Damage Repair and Chemotherapy Resistance in Triple-Negative Breast Cancer","volume":"12","author":"He","year":"2025","journal-title":"Adv. Sci."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Yan, C., Liu, C., Wu, Z., Dai, Y., Xia, E., Hu, W., and Dai, X. (2022). A Novel Approach: Combining Prognostic Models and Network Pharmacology to Target Breast Cancer Necroptosis-Associated Genes. Front. Genet., 13.","DOI":"10.3389\/fgene.2022.897538"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Bromma, K., Alhussan, A., Perez, M.M., Howard, P., Beckham, W., and Chithrani, D.B. (2021). Three-Dimensional Tumor Spheroids as a Tool for Reliable Investigation of Combined Gold Nanoparticle and Docetaxel Treatment. Cancers, 13.","DOI":"10.3390\/cancers13061465"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Ilyas, S., and Lee, D. (2025). Exploring the Role of BCL2 Interactome in Cancer: A Protein\/Residue Interaction Network Analysis. Biology, 14.","DOI":"10.3390\/biology14030261"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"5115","DOI":"10.1038\/s41598-018-23395-2","article-title":"Interpretation of Biological Experiments Changes with Evolution of the Gene Ontology and Its Annotations","volume":"8","author":"Tomczak","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"D545","DOI":"10.1093\/nar\/gkaa970","article-title":"KEGG: Integrating viruses and cellular orga-nisms","volume":"49","author":"Kanehisa","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"e00457-20","DOI":"10.1128\/MCB.00457-20","article-title":"Correction for Deng et al., \u201cBcl2\u2019s Flexible Loop Domain Regulates p53 Binding and Survival\u201d","volume":"40","author":"Deng","year":"2020","journal-title":"Mol. Cell Biol."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Madera, L., Greenshields, A., Coombs, M.R., and Hoskin, D.W. (2015). 4T1 Murine Mammary Carcinoma Cells Enhance Macrophage-Mediated Innate Inflammatory Responses. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0133385"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Li, Y., Inam, M., Hasan, M.W., Chen, K., Zhang, Z., Zhu, Y., Huang, J., Wu, Z., Chen, W., and Li, M. (2024). Optimizing Antitumor Effect of Triple-Negative Breast Cancer via Rosmarinic Acid-\u03b2-Cyclodextrin Inclusion Complex. Pharmaceutics, 16.","DOI":"10.3390\/pharmaceutics16111408"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"769","DOI":"10.1007\/s10495-011-0609-x","article-title":"Hydroxyl Radical Mediates Cisplatin-Induced Apoptosis in Human Hair Follicle Dermal Papilla Cells and Keratinocytes through a Bcl-2-Dependent Mechanism","volume":"16","author":"Luanpitpong","year":"2011","journal-title":"Apoptosis"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"67","DOI":"10.3892\/ijmm.2021.4900","article-title":"Rosmarinic Acid Inhibits Cell Proliferation, Migration, and Invasion and Induces Apoptosis in Human Glioma Cells","volume":"47","author":"Liu","year":"2021","journal-title":"Int. J. Mol. Med."},{"key":"ref_47","first-page":"1799","article-title":"Therapeutic and Nutraceutical Potential of Rosmarinic Acid: Cytoprotective Properties and Pharmacokinetic Profile","volume":"57","author":"Nunes","year":"2017","journal-title":"Crit. Rev. Food Sci. Nutr."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Zhang, C., Zhou, X., Zhang, H., Han, X., Li, B., Yang, R., and Zhou, X. (2022). Recent Progress of Novel Nanotechnology Challenging the Multidrug Resistance of Cancer. Front. Pharmacol., 13.","DOI":"10.3389\/fphar.2022.776895"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"105341","DOI":"10.5306\/wjco.v16.i5.105341","article-title":"Natural Compound Rosmarinic Acid Displays Anti-Tumor Activity in Colorectal Cancer Cells by Suppressing Nuclear Factor-Kappa B Signaling","volume":"16","author":"Liu","year":"2025","journal-title":"World J. Clin. Oncol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"605","DOI":"10.1016\/j.cell.2017.07.029","article-title":"The PI3K Pathway in Human Disease","volume":"170","author":"Fruman","year":"2017","journal-title":"Cell"},{"key":"ref_51","first-page":"59","article-title":"Triple-Negative Breast Cancer: Advancements in Characterization and Treatment Approach","volume":"28","author":"Hurvitz","year":"2016","journal-title":"Curr. Opin. Obstet. Gynecol."},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Liew, Y.X., Karen-Ng, L.P., and Vincent-Chong, V.K. (2023). A Comprehensive Review of Natural Products as Therapeutic or Chemopreventive Agents against Head and Neck Squamous Cell Carcinoma Cells Using Preclinical Models. Biomedicines, 11.","DOI":"10.20944\/preprints202306.1332.v1"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/15\/1419\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T10:47:54Z","timestamp":1785926874000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/15\/1419"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,5]]},"references-count":52,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["cells15151419"],"URL":"https:\/\/doi.org\/10.3390\/cells15151419","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,5]]}}}