Mechanistic Evaluation of Zinc Oxide Phyto-Nanoparticles in MDA-MB-231 Cells: Linking Cytotoxicity, Cell cycle Arrest, Apoptosis, and Caspase3 and p53 Gene Modulation

Zinc oxide nanoparticles (ZnONPs) have gained considerable attention as potential nanotherapeutic agents due to their ability to modulate cellular pathways involved in tumour progression. In the present study, ZnONPs were biosynthesized using Azadirachta indica (Neem) and physicochemically characterized. Their anticancer activity was evaluated against the human breast cancer cell line MDA-MB-231. Cytotoxicity was assessed using the MTT assay, while cell cycle progression and apoptosis were investigated by flow cytometry using propidium iodide (PI) and Annexin V-FITC staining, respectively. Cell migration was evaluated using a cell migration assay. The molecular mechanism of ZnONP mediated cytotoxicity was investigated by quantitative PCR analysis of Caspase 3 and p53, followed by western blotting. ZnONPs exhibited pronounced cytotoxicity, producing 88.92% inhibition of cell viability at 10 µg/mL, compared with 77.60% for doxorubicin at 10 µM. Flow-cytometric analysis revealed cell cycle arrest with accumulation of cells in the G0/G1 and G2/M phases. ZnONPs also induced apoptosis, reducing viable cells to 22.33% and increasing early apoptotic cells to 31.88%. Wound closure decreased from 93.48% in controls to 58.49% following ZnONP treatment, compared with 37.81% for doxorubicin. ZnONPs significantly upregulated Caspase-3 (2.48-fold) and p53 (1.56-fold) expression. Western blot further confirmed increased Caspase 3 levels in Doxorubicin (3.25 fold) and ZnONPS (2.95 fold) and p53 levels in Doxorubicin (1.8 fold) and ZnONPs (1.66 fold) treated cells. Collectively, these findings demonstrate that green synthesized ZnONPs suppress MDA-MB-231 cell proliferation through cell cycle arrest, apoptosis induction, and inhibition of cell migration, supporting their potential as candidates for breast cancer nanotherapeutics.