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.