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
- 418 Downloads
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), Ca2+, 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 Ca2+, ROS, and upregulation of soluble histone H2AX. Our results suggest that zerumbone holds promise as a potential candidate drug for CML.
KeywordsApoptosis K562 cells ROS Soluble H2AX Zerumbone
The authors wish to thank the Director, Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, for allowing the use of their FACS facility. Thanks are due to Dr. Ruby John Anto (RGCB) for gifting us with K562 cells, Mrs Indu Ramachandran (RGCB) for the technical help with FACS, and the National Institute of Technology, Calicut, for the SEM analysis. Financial assistance to JPR through Govt. of India—Dept of Biotechnology—BioCARe Scheme is gratefully acknowledged. Technical assistance by Ms. Soumya T. is also acknowledged.
Conflicts of interest
- 8.Zhang S, Liu Q, Liu Y, Qiao H, Liu Y. Zerumbone, a southeast Asian Ginger Sesquiterpene, induced apoptosis of pancreatic carcinoma cells through p53 signaling pathway. Evid Based Complement Alternat Med. 2012;2012:8.Google Scholar
- 9.Murakami A, Takahashi D, Kinoshita T, Koshimizu K, Kim HW, Yoshihiro A, et al. Zerumbone, a Southeast Asian ginger sesquiterpene, markedly suppresses free radical generation, proinflammatory protein production, and cancer cell proliferation accompanied by apoptosis: the α, β-unsaturated carbonyl group is a prerequisite. Carcinogenesis. 2002;23:795–802.CrossRefPubMedGoogle Scholar
- 20.Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman J, Smith JA, et al. Short protocols in molecular biology. USA: Wiley; 1992. p. 10.8.1–10.8.23.Google Scholar
- 22.Speit GN, Rothfuss A. The comet assay: a sensitive genotoxicity test for the detection of DNA damage and repair. In: Henderson DS, editors. DNA repair protocols. Totowa, New Jersy: Humana press; 2005. p. 79–90.Google Scholar
- 31.Salido G. Oxidative stress, intracellular calcium signals and apoptotic processes. In: Salido GM, Rosasdo JA, editors. Apoptosis: involvement of oxidative stress and intracellular Ca2+ homeostasis. Netherlands: Springer; 2009. p. 1–16.Google Scholar