Inhibitory effect of Costus speciosus Koen leaf extract on human leukemia cell proliferation via caspase-3–mediated apoptosis

This study evaluated the inhibitory effects of different organic solvent fractions derived from the methanol extract of Costus speciosus Koen (Thebu) leaf on cancer cell growth using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and investigated the underlying mechanism of action. Among the extracts, the ethyl acetate fraction (T-EA) exhibited the most potent anti-proliferative activity against human promyelocytic leukemia (HL-60), mouse melanoma (B16F10), and human lung carcinoma (A549) cells, with IC₅₀ values of 26.06 ± 0.56, 31.02 ± 1.22, and 30.58 ± 1.65 µg mL⁻¹, respectively. HL-60 cell proliferation was suppressed in a dose-dependent manner following 24 h treatment with the T-EA fraction, as measured by flow cytometry after annexin-V fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining. Pre-treatment with the caspase-3 inhibitor z-DEVD-fmk significantly reduced T-EA–induced cytotoxicity, and apoptotic body formation was observed. Western blot analysis revealed dose-dependent upregulation of Bax and downregulation of Bcl-xL through caspase-3 activation. GC-MS analysis identified hexadecanoic acid, methyl ester (59.53%) as the major constituent of the T-EA fraction. These findings suggest that phytochemicals present in the ethyl acetate fraction of C. speciosus leaf may serve as potential natural therapeutics for human leukemia.