Fluorescence- and TEM-Based Evaluation of Doxorubicin–Zidovudine Combination Therapy in HeLa Cells Using AO/PI Staining

Combination chemotherapy is increasingly investigated to improve anticancer efficacy and mitigate resistance associated with single-agent treatments. In this study, apoptotic responses in HeLa cervical cancer cells exposed to doxorubicin (DOX), zidovudine (AZT), and a fixed-ratio DOX-AZT combination were evaluated using acridine orange/propidium iodide (AO/PI) dual staining, with ultrastructural confirmation by transmission electron microscopy (TEM). Cells were treated for 24 h across a concentration range, enabling discrimination of viable, early apoptotic, late apoptotic, and necrotic populations by fluorescence microscopy. Combination treatment induced a pronounced, concentration-dependent shift toward late apoptosis compared with monotherapies. TEM analysis revealed chromatin condensation, nuclear fragmentation, and membrane blebbing, confirming execution-phase apoptosis. Quantitative analysis demonstrated a statistically significant increase in apoptotic populations following combination exposure (p < 0.05). These findings support the value of integrating fluorescence-based assays with ultrastructural validation and suggest a mechanistic advantage of DOX-AZT combination therapy in cervical cancer models.