Structural reprogramming of irradiated ellagic acid suppresses cell cycle regulators and modulates cytokines via activation of the p53/BAX-CASP3/Bcl-2-cyclin D1 pathway: Validation studies across breast cancer cell lines.

[BACKGROUND] EA exhibits anti-cancer potential, limited by bioavailability and solubility. This study evaluated gamma irradiation's effects on EA, characterizing structural and morphological changes, and assessing enhanced selective cytotoxicity.

[METHODS] EA and IEA were compared using SEM, FTIR, UV-Vis, GC-MS, and in vitro cytotoxicity assays (MCF-7 breast cancer cells and WI-38 fibroblasts). Morphological and nuclear changes were examined via microscopy and DAPI staining. Protein expression validation was performed using immunofluorescence microscopy and qRT-PCR analysis targeting key apoptotic genes (p53, BAX, BCL-2, CASP3, Cyclin D1). Cross-validation studies were conducted in MDA-MB-231 triple-negative breast cancer cells to confirm reproducibility across breast cancer subtypes.

[RESULTS] SEM revealed IEA had reduced particle size (93-162 nm) compared to EA (10-26 μm), smoother surfaces, and less aggregation, indicating enhanced dispersibility. FTIR/UV-Vis showed IEA structural alterations, including increased hydroxyl and carbonyl groups and enhanced conjugation. GC-MS indicated compositional shifts with increased lipid-like compounds and decreased impurities. IEA demonstrated enhanced anti-cancer activity (IC50 = 10.24 μg/mL vs. 18.30 μg/mL for EA) and reduced normal cell toxicity (IC50 = 252.3 μg/mL vs. 124.7 μg/mL for EA), with a 3.6-fold improved selectivity index. IEA induced more apoptosis in MCF-7 cells, including chromatin condensation and nuclear fragmentation. Immunofluorescence analysis confirmed enhanced nuclear p53 accumulation, mitochondrial BAX translocation, BCL-2 downregulation, and increased CASP3 activation with IEA treatment. Cross-validation in MDA-MB-231 cells demonstrated consistent anti-cancer efficacy with IEA showing 1.7-fold improved cytotoxicity (IC50 = 13 μg/mL vs. 22 μg/mL for EA) and reproducible protein expression changes across breast cancer subtypes. Western blot analysis of MDA-MB-231 cells revealed that IEA significantly enhanced pro-apoptotic protein expression compared to EA compound, demonstrating 1.3-fold p53 upregulation, 1.1-fold BAX elevation, BCL-2 suppression, and robust cleaved caspase-3 activation (p < 0.01), thereby providing molecular validation of its superior anti-cancer efficacy through mitochondrial apoptotic pathway engagement.

[CONCLUSION] Gamma irradiation transforms EA into a nanoform with enhanced physicochemical and anti-cancer properties. IEA shows improved selectivity toward cancer cells and reduced cytotoxicity toward normal cells, suggesting it is a promising candidate for selective anti-cancer agent development. The validation studies confirm consistent molecular mechanisms and broad therapeutic applicability across different breast cancer cell types.