Bilberry (Vaccinium myrtillus L.) Peel-Enriched Pomace as Natural Anticancer Agents: Preclinical Evidence from Breast Carcinoma Models Supporting Preventive and Personalized Treatment Strategies

BACKGROUND: Breast cancer (BC) is the most commonly diagnosed malignancy in women (∼25% of new cases). Plants adapted to specific niches accumulate secondary metabolites with bioactivity, including anticancer effects.

OBJECTIVE: The objective of this study was to evaluate the chemopreventive and treatment potential of Vaccinium myrtillus (bilberry) peel-enriched pomace powder in BC models, hypothesizing system-level effects and synergy with standard therapy.

METHODS: Chemistry was profiled by liquid chromatography with diode-array detection-mass spectrometry (LC-DAD-MS)/liquid chromatography with diode-array detection (LC-DAD) from 2 extracts: BILHEX (hexane) and BILMeOH (methanol). Biology was tested in the following: 1) 4T1 triple-negative mouse model (treatment); 2) NMU rat mammary carcinogenesis (chemoprevention); and 3) in vitro assays with MCF-7 and MDA-MB-231, including 3D spheroids and cisplatin combinations.

RESULTS: BILMeOH was enriched for phenolics; BILHEX for unsaturated triacylglycerols/triterpene acids. Compared with control, bilberry diet reduced 4T1 tumor volume by 51% and 91% (low/high dose; P < 0.05/0.001), lowered mitotic index (P < 0.001), and reduced necrosis:tumor ratio (P < 0.05). In NMU rats, chemoprevention increased cleaved caspase-3 (P < 0.05) and Bax/Bcl-2 (P < 0.001), and decreased Ki-67 (P < 0.05), MDA, and CD133 (P < 0.01). Histone marks shifted (↓H3K4me3 P < 0.05; ↑H4K20me3 P < 0.01; ↑H4K16ac P < 0.001). Promoter methylation was gene-specific (e.g., RASSF1 +12% absolute, P < 0.05); low-baseline loci were treated as low magnitude. miRNA profiling (758 features) identified significant dysregulation (|FC| ≥ 2) of let-7d-5p, let-7i-5p, miR-1224-3p, miR-494-3p, and miR-6216. TGF-β and IL-10 increased (both P < 0.001), IL-6 rose (P < 0.05), indicating selective immunomodulation. Metabolomics showed shifts in lactate (P < 0.05), branched-chain ketoacids (P < 0.05/0.01), and histidine (P < 0.001). In vitro, BILHEX suppressed proliferation, induced G2/M arrest, activated intrinsic apoptosis (cytochrome-c → caspase-9 → caspase-3/7 → PARP), and synergized with cisplatin in 3D spheroids (Bliss).

CONCLUSIONS: Bilberry pomace shows multitarget anticancer activity across BC models; translational studies should confirm mechanisms, refine dosing, and explore biomarker-guided use.