Synergistic Effects of Alpha-Pinene and D-Limonene on Negative Triple Breast Cancer Cell Death Through Enhanced ROS Level
DOI:
https://doi.org/10.48048/tis.2026.13679Keywords:
α-pinene, Cell survival, d-limonene, Reactive oxygen species, Triple-negative breast cancer cellsAbstract
Triple-negative breast cancer (TNBC) accounts for approximately 10% - 15% of all breast cancers and is associated with high recurrence rates and poor prognosis, contributing disproportionately to breast cancer mortality worldwide. Its limited therapeutic options and aggressive behavior underscore the need for novel combinatorial strategies. α-Pinene and d-limonene have demonstrated anticancer activity in several models; however, their combined effects on TNBC remain unclear. This study aimed to evaluate the synergistic cytotoxic effects of α-pinene and d-limonene and predict their potential molecular targets in TNBC. Cytotoxicity of single and combined treatments was assessed in MDA-MB-231 cells using an MTT assay across defined concentration ranges, followed by combination analysis using the Chou-Talalay method. Intracellular reactive oxygen species (ROS) levels were measured using the DCFDA assay after specified treatment durations. Target prediction was conducted using SMILES-based similarity across STITCH, SEA, and SwissTargetPrediction, while TNBC-associated genes were curated from TCGA (via UALCAN) and literature sources. Overlapping genes were analyzed using WebGestalt for GO and KEGG enrichment, STRING for protein-protein interaction networks, and Cytoscape-CytoHubba for hub gene identification, with survival analysis performed using TCGA datasets. α-Pinene and d-limonene exhibited cytotoxic effects with IC50 values of 0.84 and 1.20 μL/mL, respectively. Combination treatment demonstrated synergism (CI < 1) and significantly increased ROS levels compared to single treatments (p < 0.05), indicating enhanced oxidative stress. Bioinformatics analysis identified the PPAR signaling pathway as enriched among predicted targets. Five hub genes (PPARG, NOS3, ESR1, PPARA, and CYP19A1) were associated with TNBC-related pathways and patient survival. These findings suggest that the combination of α-pinene and d-limonene may exert synergistic cytotoxic effects in TNBC cells, potentially through modulation of ROS and PPAR-related pathways. However, these mechanisms remain predictive and require further experimental validation.
HIGHLIGHTS
- Combined treatment significantly elevates intracellular ROS (DCFDA assay), exceeding single treatments
- The PPAR signaling pathway is enriched among CPTG, indicating critical for TNBC cell survival
- Top 5 hub genes PPARG, NOS3, ESR1, PPARA, CYP19A1, correlated with reduced TNBC survival
- The multitarget mechanism involves ROS accumulation, which disrupts the PPAR signaling pathway and modulates key hub genes (PPARG, NOS3, ESR1, PPARA, CYP19A1), ultimately triggering apoptosis in TNBC cells.
GRAPHICAL ABSTRACT
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