Heme synthesis increases artemisinin-induced radical formation and cytotoxicity that can be suppressed by superoxide scavengers

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Abstract

Artemisinin (ART) is a sesquiterpene lactone natural product that is widely used to treat multi-drug resistant strains of malaria. Artemisinin and its derivatives are also selectively cytotoxic to cancer cells, which can be modulated by altering heme synthesis. Cytotoxicity to cancer cells is thought to involve generation of oxidative stress, although conflicting data exist. We have analyzed reactive oxygen species (ROS) generation using the fluorescent probes 2′,7′-dichlorodihydrofluorescein diacetate (DCF) and dihydroethidine (HET) upon exposure to dihydroartemisinin (DHA) in Molt-4 leukemia cells. HET fluorescence correlated with dose-dependent DHA-induced cytotoxicity, increased within 30 min of DHA exposure, and was significantly enhanced by increasing heme synthesis. Protein levels of copper,zinc-superoxide dismutase (CuZnSOD), manganese-superoxide dismutase (MnSOD), catalase, and glutathione peroxidases 1/2 were also found to increase with DHA exposure. 4-hydroxy-tempol (TEMPOL) and DF-Mn, MnSOD mimetics, could significantly inhibit ROS generation and reduce cell death. Production of superoxide appears to be a central mediator of cytotoxicity from DHA.

Introduction

Artemisinin (ART), a sesquiterpene lactone isolated from the plant Artemisia annua, is a drug widely used to treat multi-drug resistant strains of malaria [1]. In addition to its potent antimalarial activity, artemisinin and its derivatives are also selectively toxic to cancer cells in vitro and in vivo. Both of these dual chemotherapeutic activities appear to be mediated by an initial activation step involving its endoperoxide bridge. Various iron containing molecules have been implicated in artemisinin activation [2], [3], although heme iron (Fe2+ protoporphyrin IX) appears to be the kinetically favored activating molecule in vitro [4]. We have recently shown that intracellular heme synthesis can modulate artemisinin cytotoxicity towards cancer cells, providing evidence that heme is also the physiologically relevant activator of artemisinin drugs in cancer cytotoxicity [5].

The molecular events that occur following the activation of artemisinin by heme to induce cytotoxicity to cancer cells is thought to involve generation of oxidative stress following cleavage of the endoperoxide bridge that generates damaging reactive oxygen species (ROS) and carbon-centered radicals to induce cell death [6]. Support for the generation of ROS includes the inverse relationship of ART toxicity to the expression levels of genes involved in antioxidant defenses [7]. Reactive oxygen species (ROS), detected primarily with fluorescent probes, have also been implicated in the mechanism of cytotoxicity. However, conflicting results have been obtained regarding the nature of the radical species involved using the same ROS probes [8], [9]. ROS and cytotoxicity could be suppressed by the chemical antioxidant N-acetyl cysteine but not by the superoxide scavenger MnTE-2-PyP. ROS-induced ER stress induction has also been implicated in the cytotoxicity of artemisinin [10].

We have analyzed ROS generation using two different fluorescent probes upon exposure to DHA in response to increased heme synthesis and have determined the expression pattern of several major antioxidant enzyme (AOEs) and effects of different ROS scavengers.

Section snippets

Reagents

Dihydroartemisinin (DHA) was a generous gift from Dafra Pharma N.V. (Belgium) and prepared in DMSO. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, 97% purity) was from Calbiochem (USA). All other chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA) including δ-aminolevulininc acid (ALA, 98%), succinylacetone (SA, 98%), 4-hydroxy-2,2,6,6-tetra-ethylpiperidine-1-oxyl (TEMPOL, 97% purity), mannitol (≥98% purity), dihydroethidine (HET, ≥95% purity), deferoxamine

HET fluorescence correlates with dose-dependent DHA-induced cytotoxicity

Previous studies on the involvement of ROS in cytotoxicity from artemisinin were primarily conducted with DCF, which is relatively non-specific for a variety of ROS. We compared DCF to HET, a probe with relative specificity for the superoxide anion, in Molt-4 cells treated for 16 h with increasing doses of DHA. HET fluorescence/106 cells increased in proportion to DHA dose and in parallel with the level of cell death (Fig. 1). In contrast, DCF fluorescence/106 cells lagged cytotoxicity at lower

Discussion

The mechanism by which ART is cytotoxic to cancer cells appears to involve oxidative stress as a result of heme-mediated endoperoxide cleavage [6], although the radical species involved have not yet been fully characterized. DCF detectable ROS have been reported to increase within 30 min in both the acute T cell leukemia Jurkat cell line J16 and the acute lymphoblastic leukemia cell line CEM upon exposure to ∼10 μM artesunate [8]. However, no increase in DCF fluorescence was observed in HL-60

Conflict of interest

None.

Acknowledgment

This work was supported by funds from the Geisinger Clinic.

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