Investigating the anticancer potential of phytocompounds isolated from Clinacanthus nutans, as monotherapy and in combination with cisplatin, against triple-negative breast cancer (MDA-MB-231)
Publication Date
July 25, 2026
Creator
Elishea Asudas
Abstract
Triple-negative breast cancer (TNBC) is an aggressive subtype characterised by the absence of hormonal receptors which renders it unresponsive to targeted therapies. As a result, systemic chemotherapy remains the primary form of treatment, with cisplatin being a potent drug which drives anticancer effects through deoxyribonucleic acid (DNA) damage and reactive oxygen species (ROS) generation. Unfortunately, cisplatin’s clinical use is constrained by dose-limiting toxicity and resistance, necessitating the exploration of alternative or adjunct natural therapeutics that could enhance its efficacy while minimizing adverse effects. Clinacanthus nutans (C. nutans), a Southeast Asian medicinal plant, exhibits diverse pharmacological properties, including anticancer activity attributed to bioactive compounds such as phytosterols, triterpenoids and flavonoids. Prior work identified the hexane fraction of C. nutans methanolic extract as highly cytotoxic but poorly selective towards breast cancer cells. Accordingly, four compounds from this fraction, β-sitosterol (BS), stigmasterol, shaftoside and lupeol, were selected for evaluation as potential candidates against TNBC. This study investigated their anticancer potential, individually and in combination with cisplatin, in M D Anderson-Metastatic Breast-231 (MDA-MB-231) TNBC cells, focusing on ROS-mediated toxicity and apoptosis as underlying mechanisms. Cytotoxicity was first determined using cell viability assay to establish half maximal inhibitory concentration (IC50) values for each compound, cisplatin and combination treatments. The mode of cytotoxicity and ROS involvement were evaluated through qualitative fluorescence staining method, apoptotic cell quantification using flow cytometry and ROS quantification assay, conducted in the presence and absence of the antioxidant, N-acetylcysteine (NAC). Molecular analyses were performed to validate transcriptional, translational and functional apoptotic marker expressions and involvement of stress-related signalling activity. Our results found that BS induced delayed but potent, time-dependent cytotoxicity (IC50: 239.6 µM – 71.2 µM, 24 – 72h) through partial ROS-mediated apoptosis, involving potential intrinsic apoptotic signalling and p38 mitogen-activated protein kinase (MAPK) activation. Combination with cisplatin showed synergistic metabolic inhibition likely associated with ROS-mediated membrane disruption; however, this synergy did not extend to apoptotic enhancement, with ROS playing a limited role. Stigmasterol demonstrated moderate, time-independent cytotoxicity (IC50: 116 – 135 µM, 24 – 72h), similarly, through partial ROS mediation, potential intrinsic apoptotic activation and p38 MAPK stress-related signalling. Its combination with cisplatin showed partial ROS-mediated synergism at both 48 and 72 hours but lacked apoptotic-related synergy. Shaftoside alone was devoid of cytotoxicity but synergized with cisplatin to inhibit cell viability and disrupt membrane integrity, without enhancing apoptosis or modulating ROS. Lupeol displayed rapid and potent cytotoxicity in a time-independent manner (IC50: 67.3 – 44 µM, 24 – 72h) through apoptosis, potentially influenced by a proposed dual-phase ROS modulatory action, acting as an antioxidant and pro-oxidant at different exposure times. Molecular findings revealed potential intrinsic apoptotic pathway and stress-related p38 MAPK activity. Notably, lupeol-cisplatin combinations were synergistic at both 48 and 72 hours, with only the latter uniquely enhancing apoptosis in an ROS-independent manner. Overall, BS, stigmasterol and lupeol demonstrated the capacity to modulate apoptosis by partially leveraging oxidative processes to varying degrees, highlighting their promise as standalone candidates for TNBC therapy. Lupeol, in particular, showed additional value by synergizing with cisplatin to enhance apoptotic activity, underscoring its potential as an adjuvant therapeutic. These findings provide foundational evidence for C. nutans-based phytoconstituents as prospective TNBC therapeutics and underscore the relevance of targeting ROS and stress-activated kinases as potential strategies for improving TNBC treatment outcomes.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Monash University Malaysia
University of Nottingham, Malaysia
Ling, Sui Kiong
Forest Research Institute Malaysia
Subjects (LC)
Associated Schools / Departments
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