Artemisinin dimer anticancer activity correlates with heme‐catalyzed reactive oxygen species generation and endoplasmic reticulum stress induction

Abstract

Analogs of the malaria therapeutic, artemisinin, possess in vitro and in vivo anticancer activity. In this study, two dimeric artemisinins (NSC724910 and 735847) were studied to determine their mechanism of action. Dimers were >1,000 fold more active than monomer and treatment was associated with increased reactive oxygen species (ROS) and apoptosis induction. Dimer activity was inhibited by the antioxidant L-NAC, the iron chelator desferroxamine and exogenous hemin. Similarly, induction of heme oxygenase (HMOX) with CoPPIX inhibited activity, whereas inhibition of HMOX with SnPPIX enhanced it. These results emphasize the importance of iron, heme and ROS in activity. Microarray analysis of dimer treated cells identified DNA damage, iron/heme and cysteine/ methionine metabolism, antioxidant response, and endoplasmic reticulum (ER) stress as affected pathways. Detection of an ERstress response was relevant because in malaria, artemisinin inhibits pfATP6, the plasmodium orthologue of mammalian sarcoplasmic/endoplasmic reticulum Ca 21 -ATPases (SERCA). A comparative study of NSC735847 with thapsigargin, a specific SERCA inhibitor and ER-stress inducer showed similar behavior in terms of transcriptomic changes, induction of endogenous SERCA and ER calcium mobilization. However, thapsigargin had little effect on ROS production, modulated different ER-stress proteins and had greater potency against purified SERCA1. Furthermore, an inactive derivative of NSC735847 that lacked the endoperoxide had identical inhibitory activity against purified SERCA1, suggesting that direct inhibition of SERCA has little inference on overall cytotoxicity. In summary, these data implicate indirect ER-stress induction as a central mechanism of artemisinin dimer activity. ' UICCKey words: artemisinin dimer; reactive oxygen species (ROS); UPR; thapsigargin; SERCA Artemisinin (Qinghaosu), a traditional Chinese medicine, is an effective chemotherapeutic for the treatment of multi-drug resistant strains of malaria. 1-3 More recently, semi-synthetic derivatives have been shown to have anticancer activity. 4-6 Cancer cells exposed to artemisinin derivatives demonstrate decreased proliferation, increased levels of oxidative stress, induction of apoptosis and inhibition of angiogenesis. Whereas monomeric forms have activity in the nanomolar range for treatment of malaria, activity versus tumor cells is in the upper micromolar range. Conversion of artemisinin to dimeric and trimeric forms was also shown to substantially enhance anticancer activity. 5,6 The mechanism underlying pharmacological activity in both malaria and cancer is still the subject of debate. 7,8 In malaria, the classical mechanism is thought to involve reaction of the endoperoxide bridge with free heme-iron liberated during degradation of hemoglobin inside the parasite food vacuole. 9 Endoperoxide cleavage generates damaging reactive oxygen species (ROS) and carbon-centered radicals leading to parasite death. However, recent work suggests that artemisinins may also function as in...

Artemisinin induces A549 cell apoptosis dominantly via a reactive oxygen species-mediated amplification activation loop among caspase-9, -8 and -3

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Dihydroartemisinin increases temozolomide efficacy in glioma cells by inducing autophagy

“…Screening approaches have established that artemisinin is involved in the inhibition of proliferation, the promotion of apoptosis and the inhibition of angiogenesis (28). Further studies have revealed that artemisinin contributes to the generation of ROS, which leads to heme-mediated endoperoxide cleavage, DNA damage and apoptosis (29). In addition, it has been found that artemisinin enhances the sensitivity of glioma cells to β, γ-rays (17).…”

Endoplasmic-Reticulum Calcium Depletion and Disease

“…Known SERCA blockers like thapsigargin and curcumin have anticancer activity (Denmeade et al 2003;Anand et al 2008;Bakhshi et al 2008). Anticancer drugs like the stable analogue of the Bcl-2 antagonist HA 14-1 (Hermanson et al 2009), artemisinin (Stockwin et al 2009), amiloride analogues (Park et al 2009), and 2,5-dimethyl-celecoxib (Johnson et al 2002;Pyrko et al 2007) also inhibit SERCA with ER stress as a result. SERCA2 expression decreases after photodynamic therapy with hypericin (Buytaert et al 2006).…”