A natural flavonoid, apigenin isolated from Clerodendrum viscosum leaves, induces G2/M phase cell cycle arrest and apoptosis in MCF-7 cells through the regulation of p53 and caspase-cascade pathway

Background: With 9.6 million deaths in 2018, cancer remains the second leading cause of death worldwide. Breast cancer is the most deadly type of cancer among females, with 55.2% of crude incidence rate and 16.6% of crude mortality rate. Purpose: The present study was aimed to investigate the anti-breast cancer potential of natural dietary flavonoid, apigenin isolated from Clerodendrum viscosum leaves. Methods: Apigenin was evaluated for in-depth anticancer activity in MCF-7 cells using cell viability assay, cell cycle analysis, Annexin-V-FLUOS staining, ROS induction, morphological analysis, and western blot analysis. Results: Apigenin showed selective cytotoxicity on MCF-7 cells with an IC₅₀-56.72 ± 2.35 µM, while negligible cytotoxicity was observed on WI-38 cells. Further, the flow cytometer-based analysis showed that apigenin halted MCF-7 cells in the G2/M phase arrest followed by dose-dependent apoptosis. Moreover, the FACS and confocal microscopy results confirmed the elevation of intracellular ROS and nuclear fragmentation in apigenin-treated MCF-7 cells. Western blots showed up-regulation of cell cycle regulatory proteins, increased p53 expression, Bax/Bcl-2 ratio, activation of caspases, and cleavage of PARP. Finally, apigenin treatment in the presence of Pifithrin-µ showed decreased apoptotic population and it was further confirmed through western blotting study. The results revealed the vital role of p53 in apigenin-induced apoptosis in MCF-7 cells. Conclusions: In the present findings, treatment of apigenin-induced intracellular ROS in MCF-7 cells followed by induction of G2/M phase cell cycle arrest and further apoptosis through the regulation of p53 and caspase-cascade signaling pathway.