title: 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 ER-stress response was relevant because in malaria, artemisinin inhibits pfATP6, the plasmodium orthologue of mammalian sarcoplasmic/endoplasmic reticulum Ca(2+)-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.
authors.affiliation: Developmental Therapeutics Program, SAIC-Frederick, NCI-Frederick, Frederick, MD 21702, USA.
authors.forename: Luke H
authors.initials: LH
authors.lastname: Stockwin
authors.forename: Bingnan
authors.initials: B
authors.lastname: Han
authors.forename: Sherry X
authors.initials: SX
authors.lastname: Yu
authors.forename: Melinda G
authors.initials: MG
authors.lastname: Hollingshead
authors.forename: Mahmoud A
authors.initials: MA
authors.lastname: ElSohly
authors.forename: Waseem
authors.initials: W
authors.lastname: Gul
authors.forename: Desmond
authors.initials: D
authors.lastname: Slade
authors.forename: Ahmed M
authors.initials: AM
authors.lastname: Galal
authors.forename: Dianne L
authors.initials: DL
authors.lastname: Newton
authors.forename: Maja A
authors.initials: MA
authors.lastname: Bumke
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D019306:Artemisia / Q000737:chemistry; D037621:Artemisinins / Q000494:pharmacology*; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020869:Gene Expression Profiling; D006418:Heme / Q000378:metabolism*; D051547:Heme Oxygenase-1 / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D000975:Antioxidants; D037621:Artemisinins; D015415:Biomarkers; D004791:Enzyme Inhibitors; C086501:N-acetylcysteine lysinate; D017382:Reactive Oxygen Species; D006418:Heme; D019284:Thapsigargin; C031327:artemisinin; D051547:Heme Oxygenase-1; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D008239:Lysine; D002118:Calcium; D000111:Acetylcysteine