MORPHOLOGICAL AND FLUORESCENCE-BASED EVALUATION OF EPIGALLOCATECHIN GALLATE-INDUCED APOPTOSIS IN MDA-MB-231 TRIPLE-NEGATIVE BREAST CANCER CELLS

Abstract

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited targeted therapeutic options owing to the absence of estrogen receptor, progesterone receptor, and HER2 expression. Epigallocatechin-3-gallate (EGCG), the major polyphenolic catechin of green tea, has demonstrated promising anticancer activity by inducing oxidative stress and apoptosis. Morphological and fluorescence-based techniques provide valuable insights into the cellular events associated with EGCG-induced apoptotic cell death. Objective: To evaluate the morphological and fluorescence-based changes associated with EGCG-induced apoptosis in MDA-MB-231 triple-negative breast cancer cells. Materials and Methods: MDA-MB-231 cells were treated with increasing concentrations of EGCG (0–50 μM) for 24 h. Morphological alterations were assessed using phase-contrast microscopy. Intracellular reactive oxygen species (ROS) generation was qualitatively evaluated using DCFH-DA fluorescence staining. Apoptotic changes were further examined by Acridine Orange/Ethidium Bromide (AO/EB) dual staining and Propidium Iodide (PI) staining. Representative microscopic images were analyzed to characterize cellular and nuclear changes associated with apoptosis. Results: EGCG treatment induced marked morphological alterations in MDA-MB-231 cells, including cell shrinkage, rounding, membrane blebbing, reduced cellular adherence, and loss of normal spindle-shaped morphology. DCFH-DA fluorescence imaging demonstrated enhanced intracellular green fluorescence in EGCG treated cells, indicating increased ROS generation. AO/EB dual staining revealed a progressive increase in early and late apoptotic cells characterized by chromatin condensation, nuclear fragmentation, and orange-red fluorescence. PI staining demonstrated increased membrane permeability with a higher proportion of PI-positive cells following EGCG treatment, indicating late apoptotic cell death. Collectively, the microscopic findings consistently demonstrated concentration-dependent apoptotic changes following EGCG exposure. Conclusion: EGCG induces characteristic morphological and fluorescence-based features of apoptosis in MDA MB-231 triple-negative breast cancer cells. The observed cellular and nuclear alterations, together with enhanced ROS fluorescence and increased PI uptake, provide qualitative evidence supporting oxidative stress-mediated apoptosis. These findings further support the potential of EGCG as a promising naturally derived therapeutic agent for the treatment of triple-negative breast cancer.