Rosmarinic Acid Potentiates Cisplatin-Induced Antitumour Activity Through ROS-Associated Apoptotic Signalling in Two-and Three-Dimensional Breast Cancer Models

Sep 22, 2026

Triple-negative breast cancer (TNBC) is highly aggressive and often resistant to platinum-based chemotherapy. This study evaluates rosmarinic acid (RA) combined with cisplatin (CDDP) in 4T1 breast cancer cells, alongside cytotoxicity profiling in non-cancerous HaCaT keratinocytes. Using MTT-based combination indices, ROS detection with DCFH-DA and NAC rescue, and apoptosis assays by flow cytometry and imaging, RA+CDDP shows strong synergistic cytotoxicity, ROS-associated apoptotic signalling, and improved selectivity.

LanguageEnglish

CategoryResearch & Report

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Pages35

Reading Time1 h 28 min

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Main finding and implications

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1. Introduction

Article Rosmarinic Acid Potentiates Cisplatin-Induced Antitumour Activity Through ROS-Associated Apoptotic Signalling in Two-and Three-Dimensional Breast Cancer Models Co¸skun Orhaner 1, Aylin Orhaner 2, Mehmet Cudi Tuncer 3, * and ˙Ilhan Özdemir 4 Academic Editors: Dragana Z. Jakovljevi´c, Edyta Skrzypek and Marzena Warchoł Received: 8 July 2026 Revised: 30 July 2026 Accepted: 31 July 2026 Published: 5 August 2026 Copyright: © 2026 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license. 1 Department of Gynaecology and Obstetrics, Soranus IVF Center, Bursa 16070, Turkey; [orhanercoskun@gmail.com](orhanercoskun@gmail.com) 2 Department of Gynaecology and Obstetrics, Medicana Bursa Hospital, Bursa 16150, Turkey; [draylinorhaner@hotmail.com](draylinorhaner@hotmail.com) 3 Department of Anatomy, Faculty of Medicine, Dicle University, Diyarbakır 21280, Turkey 4 Department of Histology and Embryology, Faculty of Medicine, Kahramanmara¸s Sütçü ˙Imam University, Kahramanmara¸s 46000, Turkey; [ilhanozdemir32@hotmail.com](ilhanozdemir32@hotmail.com) * Correspondence: [drcudi@hotmail.com](drcudi@hotmail.com) Highlights What is the main finding? • Rosmarinic acid potentiated the antitumor efficacy of cisplatin in models of twoand three-dimensional 4T1 breast cancer by synergistically decreasing cell viability, inhibiting spheroid expansion, and inducing apoptosis. The combined treatment substantially elevated intracellular ROS levels, while NAC-driven ROS scavenging reduced cytotoxicity and partially rescued cell viability, indicating a functional role of ROS in mediating the response to treatment. What are the implications of the main finding? • This study provides the first comprehensive evaluation of the RA + CDDP combination in an aggressive 4T1 breast cancer model by integrating 2D and 3D tumour systems, ROS functional validation using NAC rescue experiments, apoptosis-related gene expression profiling, and network pharmacology analyses. • Integrated experimental and network pharmacology analyses identified the PI3K/Akt signalling pathway as a key molecular network potentially associated with the synergistic anticancer effects of the combination of RA + CDDP. • These findings support the potential of rosmarinic acid as an adjuvant to CDDP for breast cancer treatment and provide a rationale for future protein-level validation and in vivo studies. Abstract 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

FAQs

What main effect does RA have when combined with cisplatin in the 4T1 breast cancer model?

Rosmarinic acid potentiates cisplatin’s antitumour activity, showing synergistic cytotoxicity, inhibited spheroid expansion, and enhanced apoptotic cell death in the 4T1 model.

How is the role of ROS in the RA + cisplatin response tested?

Intracellular ROS is measured using DCFH-DA imaging, and the functional role is validated by N-acetyl-L-cysteine (NAC) rescue experiments that reduce ROS accumulation and partially restore viability.

What apoptosis-related molecular changes are reported after combined treatment?

Combined treatment increases expression of pro-apoptotic genes such as Bax, Casp9, Cycs, and Trp53 and decreases Bcl2, consistent with intrinsic apoptotic signalling regulation.

Sep 22, 2026

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