Cytotoxicity of Dihydroartemisinin Toward Molt-4 Cells Attenuated by N-Tert-butyl-alpha- phenylnitrone and Deferoxamine

Derivatives of artemisinin, a compound extracted from the wormwood Artemisia annua L, have potent anticancer properties. The anticancer mechanisms of artemisinin derivatives have not been fully-elucidated. We hypothesize that the cytotoxicity of these compounds is due to the free radicals formed by interaction of their endoperoxide moiety with intracellular iron in cancer cells. The effects of N-tert-butyl-alpha-phenylnitrone (PBN), a spin-trap free radical scavenger, and deferoxamine (DX), an iron chelating agent, on the in vitro cytotoxicity of dihyroartemisinin (DHA) toward Molt-4 human T-lymphoblastoid leukemia cells were investigated in the present study. Dihydroartemisinin effectively killed Molt-4 cells in vitro. Its cytotoxicity was significantly attenuated by PBN and DX. Based on the data of our present and previous studies, we conclude that one anticancer mechanism of dihydroartemisinin is the formation of toxic-free radicals via an iron-mediated process. Artemisinin, a compound isolated from the wormwood Artemisia annua L, is a sesquiterpene lactone peroxide. Artemisinin analogs have been used as anti-malarials, with few side-effects. They have also been shown to have potent anticancer properties (1). The anticancer mechanisms of artemisinin are still being investigated. We have hypothesized that one anticancer mechanism of artemisinin is due to the generation of toxic-free radicals via the interaction between its endoperoxide moiety and intracellular iron (2, 3). A high amount of iron is required for DNA synthesis during mitosis in rapidly-dividing cancer cells. Cancer cells have a high concentration of cell surface transferrin receptors that enable a higher iron uptake compared to normal cells (4, 5). The high free iron content in cancer cells makes artemisinin selectively toxic to them in comparison with normal cells. N-Tert-butyl-alpha-phenylnitrone (PBN) is a spin-trap compound that effectively sequesters free radicals both in vivo and in vitro (6, 7). Deferoxamine (DX), an iron- chelating agent, has been shown to inhibit DHA-induced apoptosis in HL-60 leukemia cells (8). Dihydroartemsinin (DHA) has been shown to have significant toxic effects toward cancer cells both in vitro and in vivo (9, 10). In the present study, PBN or DX was added to Molt-4 human lymphoblastoid cells incubated with DHA to inactivate the free radicals generated and to prevent the formation of free radicals, respectively, in order to test our hypothesis.