Eugenol's Molecular Warfare against Human Leukemia K562 cells: In Vitro Insights to Chemotherapeutic Potentials

Abstract

Background: The search for effective anticancer agents has led to an increased focus on natural compounds, given their role as the foundation for many chemotherapeutic drugs. Eugenol (EUG) and Bis-Eugenol (Bis-EUG), derived from clove oil, have shown promise due to their antioxidative, anti-inflammatory, pro-apoptotic, and anti-proliferative properties across various cancer cell lines. This study aims to elucidate the chemotherapeutic potential of EUG and Bis-EUG on human leukemia K562 cells, exploring their ability to modulate apoptosis and cell proliferation. Objective: To assess the in vitro anticancer effects of EUG and Bis-EUG on K562 cell lines through various assays measuring cell viability, apoptosis, gene expression, and nitric oxide release, with a focus on elucidating the mechanisms underlying their anticancer activity. Methods: The study employed an inter-collaborative approach, utilizing MTT assays to determine the IC50 values of EUG and Bis-EUG on K562 cells. Gene expression analysis of pro-apoptotic genes Caspase-3 and Caspase-9 was performed using RT-qPCR. Apoptotic changes were further analyzed through flow cytometry and Hoechst 33258 staining to observe morphological alterations in treated cells. The release of nitric oxide (NO) as an indicator of anti-proliferative activity was measured using a Griess assay. Statistical analysis was conducted using GraphPad Prism Software, Version 9, and Microsoft Excel, with a significance threshold of p ≤ 0.05. Results: The IC50 values for EUG and Bis-EUG were determined to be 16.7 µM and 0.14 mM, respectively. Gene expression profiling revealed a significant increase in Caspase-3 and Caspase-9 expression, indicating the activation of apoptotic pathways. Flow cytometry and Hoechst 33258 staining confirmed the induction of apoptosis, with noticeable changes in cell morphology and nuclear fragmentation. The NO release assay demonstrated a dose-dependent increase in NO levels, highlighting the compounds' anti-proliferative effects. Conclusion: EUG and Bis-EUG exhibit potent anticancer effects on leukemia K562 cells by inducing apoptosis and inhibiting cell proliferation. The significant increase in NO levels and the activation of caspases suggest that these compounds may serve as effective chemotherapeutic agents. Further in vivo studies are warranted to explore their potential in cancer treatment regimens.