Flow-cytometric analysis of cell-cycle arrest and apoptosis induced by a novel benzopyran derivative (SK6) in MCF-7 breast cancer cells

Introduction: Breast cancer remains a major global health challenge, accounting for 2.3 million new cases and nearly 670,000 deaths in 2022. The limitations of current therapies, including drug resistance, toxicity, and reduced efficacy, highlight the urgent need for novel anticancer agents. Purpose of the Study: The aim of this study was to evaluate the anticancer potential of a novel benzopyran derivative (SK6) using flow-cytometric analysis. After identifying SK6 as the most potent compound through MTT assay, its mechanism of action was investigated in MCF-7 cells. Flow cytometry was employed to assess SK6-induced apoptosis using Annexin-V/PI staining. Additionally, PI-based DNA content analysis was performed to determine the compound's effect on cell-cycle progression. Experimental Methods: In continuation our research on synthesis of benzopyran derivatives, 5 molecules were synthesized and characterized using UV, IR, ¹HNMR, and mass spectrometry. Their cytotoxicity was evaluated against MCF-7 cells using the MTT assay. Consequently, SK6 was selected for mechanistic investigation. Flow cytometry (Beckman Coulter Gallios analyser, USA) was employed to assess its effects on the cell cycle and apoptosis in treated MCF-7 cells. Results: All five benzopyran derivatives were successfully synthesized and structurally confirmed using spectral analyses. Among them, SK6 showed the lowest IC₅₀ value i.e. 20.99μg/ml in the MTT assay, demonstrating the strongest antiproliferative activity against MCF-7 cells. Flow-cytometric Annexin-V FITC/PI analysis revealed that SK6 treatment induced a measurable apoptotic response in MCF-7 cells. Annexin-V FITC/PI staining revealed that SK6 induced a measurable apoptotic response. Quadrant analysis showed 78.44% viable cells, 8.40% in early apoptosis, 8.15% in late apoptosis, and 5.01% necrotic. Overall, SK6 induced 16.55% total apoptosis, confirming its ability to activate programmed cell death in MCF-7 cells. The SK6 compound caused a marked alteration in the cell-cycle profile, producing a dominant S-phase arrest. Treated cells showed a substantial increase in the S-phase population (54.33% vs. 13.05% in control) with a drastic reduction in G0/G1 cells (1.02% vs. 62.91%). A moderate rise in G2/M cells and minimal sub-G1 events indicate primarily cytostatic rather than DNA-fragmenting effects. These findings suggest that SK6 disrupts DNA replication and halts progression at the S-phase checkpoint. Conclusion: SK6 demonstrated potent cytotoxic activity against MCF-7 cells and caused a pronounced S-phase arrest, indicating disruption of DNA replication. This was accompanied by a moderate induction of apoptosis, supporting its antiproliferative effect. Finally, these results highlight SK6 as a promising lead candidate for further development as a selective and effective therapeutic agent against breast cancer