Zerumbone induces mitochondria-mediated apoptosis via increased calcium, generation of reactive oxygen species and upregulation of soluble histone H2AX in K562 chronic myelogenous leukemia cells

Iyyappan Rajan, P. R. Jayasree, P. R. Manish Kumar

Zerumbone, a natural cyclic sesquiterpene, is known to exhibit selective toxicity toward various cancer cells. Sustained efforts to explore the potential of new agents for effective therapy are critical in the context of development of drug resistance especially in cancers like chronic myelogenous leukemia (CML). The present study evaluated the effect of zerumbone on CML-K562 cells. The cell viability of zerumbone-treated K562 cells was detected by MTT assay, and morphological changes were observed by light microscopy and scanning electron microscopy (SEM). Staining with Hoechst 33258, acridine orange/ethidium bromide, and AnnexinV-FITC were used to detect apoptosis. Intracellular reactive oxygen species (ROS), Ca(2+), and changes in mitochondrial membrane potential were measured using Dichloro-dihydro-fluorescein diacetate (DCFH-DA), Fluo-3AM, and Rhodamine-123, respectively. Western blot analysis was carried out to detect key proteins involved in apoptosis. Zerumbone inhibited K562 cell proliferation with an IC50 value of 3.5 μg/mL and colony formation capability (P < 0.001). Interestingly, zerumbone did not affect the growth of normal human peripheral blood lymphocytes (hPBLs). Distinct morphological changes observed by light microscopy and fluorescent staining with Hoechst-33258, AO/EtBr, annexin V-FITC, and cytotoxicity evaluation by comet assay indicated induction of DNA damage and apoptosis. This was further confirmed by demonstration of pro-caspase-3, -9 activation and Poly(ADP-ribose) polymerase (PARP) cleavage on western blots. Apoptosis induction was found to be mitochondria mediated, involving increased free intracellular Ca(2+), ROS, and upregulation of soluble histone H2AX. Our results suggest that zerumbone holds promise as a potential candidate drug for CML.