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• W2023405265 abstract "The artemisinin compounds are the frontline drugs for the treatment of drug-resistant malaria. They are selectively cytotoxic to mammalian cancer cell lines and have been implicated as neurotoxic and embryotoxic in animal studies. The endoperoxide functional group is both the pharmacophore and toxicophore, but the proposed chemical mechanisms and targets of cytotoxicity remain unclear. In this study we have used cell models and quantitative drug metabolite analysis to define the role of the mitochondrion and cellular heme in the chemical and molecular mechanisms of cell death induced by artemisinin compounds. HeLa ρ0 cells, which are devoid of a functioning electron transport chain, were used to demonstrate that actively respiring mitochondria play an essential role in endoperoxide-induced cytotoxicity (artesunate IC50 values, 48 h: HeLa cells, 6 ± 3 μm; and HeLa ρ0 cells, 34 ± 5 μm) via the generation of reactive oxygen species and the induction of mitochondrial dysfunction and apoptosis but do not have any role in the reductive activation of the endoperoxide to cytotoxic carbon-centered radicals. However, using chemical modulators of heme synthesis (succinylacetone and protoporphyrin IX) and cellular iron content (holotransferrin), we have demonstrated definitively that free or protein-bound heme is responsible for intracellular activation of the endoperoxide group and that this is the chemical basis of cytotoxicity (IC50 value and biomarker of bioactivation levels, respectively: 10β-(p-fluorophenoxy)dihydroartemisinin alone, 0.36 ± 0.20 μm and 11 ± 5%; and with succinylacetone, >100 μm and 2 ± 5%). The artemisinin compounds are the frontline drugs for the treatment of drug-resistant malaria. They are selectively cytotoxic to mammalian cancer cell lines and have been implicated as neurotoxic and embryotoxic in animal studies. The endoperoxide functional group is both the pharmacophore and toxicophore, but the proposed chemical mechanisms and targets of cytotoxicity remain unclear. In this study we have used cell models and quantitative drug metabolite analysis to define the role of the mitochondrion and cellular heme in the chemical and molecular mechanisms of cell death induced by artemisinin compounds. HeLa ρ0 cells, which are devoid of a functioning electron transport chain, were used to demonstrate that actively respiring mitochondria play an essential role in endoperoxide-induced cytotoxicity (artesunate IC50 values, 48 h: HeLa cells, 6 ± 3 μm; and HeLa ρ0 cells, 34 ± 5 μm) via the generation of reactive oxygen species and the induction of mitochondrial dysfunction and apoptosis but do not have any role in the reductive activation of the endoperoxide to cytotoxic carbon-centered radicals. However, using chemical modulators of heme synthesis (succinylacetone and protoporphyrin IX) and cellular iron content (holotransferrin), we have demonstrated definitively that free or protein-bound heme is responsible for intracellular activation of the endoperoxide group and that this is the chemical basis of cytotoxicity (IC50 value and biomarker of bioactivation levels, respectively: 10β-(p-fluorophenoxy)dihydroartemisinin alone, 0.36 ± 0.20 μm and 11 ± 5%; and with succinylacetone, >100 μm and 2 ± 5%). The endoperoxide antimalarials are a class of semi-synthetic compounds derived from the natural product artemisinin (1) (see Fig. 1) that are currently deployed in the frontline combination treatments for drug-resistant malaria because of their rapid clearance of parasites and high tolerability in humans (1Nosten F. White N.J. Am. J. Trop. Med. Hyg. 2007; 77: 181-192Crossref PubMed Scopus (456) Google Scholar, 2Park B.K. O'Neill P.M. Maggs J.L. Pirmohamed M. Br. J. Clin. Pharmacol. 1998; 46: 521-529Crossref PubMed Scopus (49) Google Scholar). Despite the widespread use of artemisinin-based compounds (ARTs) 2The abbreviations used are: ART, artemisinin-based compound; PFDHA, 10β-(p-fluorophenoxy) dihydroartemisinin; ETC, electron transport chain; THF, terahydrofuran; SA, succinylacetone; PPIX, protoporphyrin IX; HTF, holotransferrin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PI, propidium iodide; TMRE, tetramethylrhodamine ethyl ester; HBSS, Hanks' balanced salt solution; DCFH-DA, dichlorofluorscein diacetate; MRM, multiple-reaction monitoring; ROS, reactive oxygen species; MMP, mitochondrial membrane potential. (2Park B.K. O'Neill P.M. Maggs J.L. Pirmohamed M. Br. J. Clin. Pharmacol. 1998; 46: 521-529Crossref PubMed Scopus (49) Google Scholar), there have been persistent reports of neurotoxicity and embryotoxicity in cross-species animal studies, although this hazard has not translated to clinical use (2Park B.K. O'Neill P.M. Maggs J.L. Pirmohamed M. Br. J. Clin. Pharmacol. 1998; 46: 521-529Crossref PubMed Scopus (49) Google Scholar, 3Clark R.L. White T.E. Clode S.A. Gaunt I. Winstanley P. Ward S.A. Birth Defects Res. B Dev. Reprod. Toxicol. 2004; 71: 380-394Crossref PubMed Scopus (112) Google Scholar, 4Toovey S. Toxicol. Lett. 2006; 166: 95-104Crossref PubMed Scopus (32) Google Scholar, 5Clark R.L. Reprod. Toxicol. 2009; 28: 285-296Crossref PubMed Scopus (94) Google Scholar). It is currently perceived that the benefits of these drugs in treating malaria outweigh the risks when administered in the second and third trimesters of pregnancy; however, the World Health Organization have contraindicated the use of ARTs during the first trimester (6World Health Organization WHO/CDS/MAL/2003.1094. 2003; Google Scholar). The emergence of resistance to the ARTs is a pressing problem (7Muller O. Sie A. Meissner P. Schirmer R.H. Kouyate B. Lancet. 2009; 374: 1419-1420Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 8Taylor S.M. Juliano J.J. Meshnick S.R. N. Engl. J. Med. 2009; 361: 1807-1808Crossref PubMed Scopus (54) Google Scholar), and the development of novel synthetic endoperoxides with improved efficacy and pharmacokinetics is therefore a priority area (9Noedl H. Se Y. Schaecher K. Smith B.L. Socheat D. Fukuda M.M. Consortium A.S. N. Engl. J. Med. 2008; 359: 2619-2620Crossref PubMed Scopus (1272) Google Scholar, 10Jefford C.W. Drug Discovery Today. 2007; 12: 487-495Crossref PubMed Scopus (137) Google Scholar). The major structural differences between the novel synthetic derivatives, resulting in increased systemic exposure, have the potential to induce highly variable toxicology and pharmacology (11Mercer A.E. Curr. Opin. Drug Discovery Dev. 2009; 12: 125-132PubMed Google Scholar). Therefore, the elucidation of the factors that define cell susceptibility to the ARTs is essential to facilitate the safe use of existing ARTs therapies; to inform the design of safe, novel, endoperoxide compounds in which pharmacological activity is dissociated from toxicological effects and to define the molecular target to enable their consideration and potential development as anticancer agents (12Posner, G. H., D'Angelo, J., O'Neill, P. M., Mercer, A., (2006) Exp. Opin. Therapeutic Patents 16, 1665–1672.Google Scholar). The endoperoxide group contained within the artemisinin (1) framework functions as both the pharmacophore and toxicophore. It is hypothesized that its one-electron reductive activation by an Fe(II) species to toxic carbon-centered radicals is essential for activity (Fig. 1) (13O'Neill P.M. Posner G.H. J. Med. Chem. 2004; 47: 2945-2964Crossref PubMed Scopus (504) Google Scholar). The parasiticidal mechanism of action is not fully understood, and the chemical source of activation and the parasite target remain under investigation. However, it has been postulated that the endoperoxides have multiple targets including the parasite sarco/endoplasmic reticulum Ca2+-ATPase, PfATP6, and heme- and non-heme-containing proteins while also inducing reactive oxygen species (ROS) and lipid peroxidation (13O'Neill P.M. Posner G.H. J. Med. Chem. 2004; 47: 2945-2964Crossref PubMed Scopus (504) Google Scholar, 14Eckstein-Ludwig U. Webb R.J. Van Goethem I.D. East J.M. Lee A.G. Kimura M. O'Neill P.M. Bray P.G. Ward S.A. Krishna S. Nature. 2003; 424: 957-961Crossref PubMed Scopus (863) Google Scholar, 15Berman P.A. Adams P.A. Free Radic. Biol. Med. 1997; 22: 1283-1288Crossref PubMed Scopus (107) Google Scholar, 16Robert A. Benoit-Vical F. Claparols C. Meunier B. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 13676-13680Crossref PubMed Scopus (147) Google Scholar). Our previous studies in mammalian cells have demonstrated that activation of the endoperoxide bridge to carbon-centered radicals only occurs in sensitive proliferating cells and not their primary counterparts and therefore that this differential activation is the chemical basis of selective cytotoxicity (17Mercer A.E. Maggs J.L. Sun X.M. Cohen G.M. Chadwick J. O'Neill P.M. Park B.K. J. Biol. Chem. 2007; 282: 9372-9382Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The mechanism of endoperoxide-induced apoptotic cell death via ROS generation, mitochondrial membrane depolarization, caspase-3 and -7 activation, and DNA degradation has been well characterized in several cell types (17Mercer A.E. Maggs J.L. Sun X.M. Cohen G.M. Chadwick J. O'Neill P.M. Park B.K. J. Biol. Chem. 2007; 282: 9372-9382Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 18Disbrow G.L. Baege A.C. Kierpiec K.A. Yuan H. Centeno J.A. Thibodeaux C.A. Hartmann D. Schlegel R. Cancer Res. 2005; 65: 10854-10861Crossref PubMed Scopus (138) Google Scholar, 19Lu J.J. Meng L.H. Cai Y.J. Chen Q. Tong L.J. Lin L.P. Ding J. Cancer Biol. Ther. 2008; 7: 1017-1023Crossref PubMed Scopus (102) Google Scholar). Nevertheless, crucial information defining the primary cellular target of cytotoxicity and the exact intracellular mechanism of chemical activation remains unclear. Intriguingly, the ARTs display a range of cytotoxic activity among mammalian cells (17Mercer A.E. Maggs J.L. Sun X.M. Cohen G.M. Chadwick J. O'Neill P.M. Park B.K. J. Biol. Chem. 2007; 282: 9372-9382Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 18Disbrow G.L. Baege A.C. Kierpiec K.A. Yuan H. Centeno J.A. Thibodeaux C.A. Hartmann D. Schlegel R. Cancer Res. 2005; 65: 10854-10861Crossref PubMed Scopus (138) Google Scholar, 20Efferth T. Dunstan H. Sauerbrey A. Miyachi H. Chitambar C.R. Int. J. Oncol. 2001; 18: 767-773PubMed Google Scholar). The iron activation hypothesis provides an explanation for this selectivity, proposing that cytotoxicity is dependent upon the higher concentration of iron required by malignant cells and neuronal cells to sustain continued growth and proliferation (21Kwok J.C. Richardson D.R. Crit. Rev. Oncol. Hematol. 2002; 42: 65-78Crossref PubMed Scopus (173) Google Scholar). Accordingly, sensitive cells have an increased number of transferrin receptors responsible for cellular iron uptake and higher intracellular iron levels, and pretreatment with iron chelators can reduce cytotoxicity (18Disbrow G.L. Baege A.C. Kierpiec K.A. Yuan H. Centeno J.A. Thibodeaux C.A. Hartmann D. Schlegel R. Cancer Res. 2005; 65: 10854-10861Crossref PubMed Scopus (138) Google Scholar, 19Lu J.J. Meng L.H. Cai Y.J. Chen Q. Tong L.J. Lin L.P. Ding J. Cancer Biol. Ther. 2008; 7: 1017-1023Crossref PubMed Scopus (102) Google Scholar). In addition, recent biochemical evidence has been presented suggesting that heme may be a physiologically relevant mediator of the ARTs in cancer cells, and the authors have used biomimetic chemistry to speculate that this may be due to the formation of heme adducts (22Zhang S.M. Gerhard G.S. PLoS One. 2009; 4: e7472Crossref PubMed Scopus (68) Google Scholar). Several investigations have implicated the mitochondrion in the antiparasitic (23Jiang J.B. Jacobs G. Liang D.S. Aikawa M. Am. J. Trop. Med. Hyg. 1985; 34: 424-428Crossref PubMed Scopus (45) Google Scholar, 24Maeno Y. Toyoshima T. Fujioka H. Ito Y. Meshnick S.R. Benakis A. Milhous W.K. Aikawa M. Am. J. Trop. Med. Hyg. 1993; 49: 485-491Crossref PubMed Scopus (79) Google Scholar), cytotoxic (17Mercer A.E. Maggs J.L. Sun X.M. Cohen G.M. Chadwick J. O'Neill P.M. Park B.K. J. Biol. Chem. 2007; 282: 9372-9382Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), and embryotoxic (25Longo M. Zanoncelli S. Della Torre P. Rosa F. Giusti A. Colombo P. Brughera M. Mazué G. Olliaro P. Reprod. Toxicol. 2008; 25: 433-441Crossref PubMed Scopus (30) Google Scholar, 26Laffan S.B. James A.C. Maleeff B.E. Pagana J.M. Clark R.L. White T.E. Birth Defects Res. A Clin. Mol. Teratol. 2006; 76: 329-330Google Scholar) mechanisms of action of the ARTs. Elegant studies performed with a yeast model and isolated mitochondria of parasite and mammalian cell origin have suggested that, in contrast to the iron activation theory, the mitochondrion may be a direct target and mediator of endoperoxide activity and further may underlie selectivity to the parasite (27Wang J. Huang L. Li J. Fan Q. Long Y. Li Y. Zhou B. PLoS One. 2010; 5: e9582Crossref PubMed Scopus (190) Google Scholar, 28Li W. Mo W.K. Shen D. Sun L.B. Wang J. Lu S. Gitschier J.M. Zhou B. PLoS Genet. 2005; 1: 329-334Google Scholar). The authors of these studies hypothesized that the mitochondria play a dual role: the electron transport chain (ETC) delivers reducing equivalents for activation of the endoperoxide function and, as a consequence, the mitochondria are damaged; ROS are generated leading to the eventual induction of parasite death. In the present investigations, we have applied our established techniques to quantify bioactivation of the endoperoxide group via C-centered radicals and cell death in vitro to define more clearly the chemical and molecular mechanisms that determine mammalian cell susceptibility to the ARTs. Specifically we have defined the role of the ETC of the mitochondria, using ρ0 cells that have been depleted of mitochondrial DNA, and that of heme, using chemical modulators of heme synthesis, in the chemical bioactivation of the endoperoxide group and the induction of cell death. The investigations were carried out using two endoperoxide compounds: artesunate (2), which is administered therapeutically as an antimalarial, and 10β-(p-fluorophenoxy)dihydroartemisinin (PFDHA) (3), an artemisinin derivative used as a chemical probe of endoperoxide-induced cell death because of its enhanced metabolic stability (17Mercer A.E. Maggs J.L. Sun X.M. Cohen G.M. Chadwick J. O'Neill P.M. Park B.K. J. Biol. Chem. 2007; 282: 9372-9382Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The human HeLa and HeLa ρ0 cell lines were kindly supplied by Dr E. Bampton (Medical Research Council Toxicology Unit, Leicester, UK). HL-60 cells were purchased from European Collection of Cell Cultures (Salisbury, UK). The annexin V-FITC/propidium iodide (PI) staining kit was from Abd Serotec (Kidlington, Oxford, UK). Artesunate (2) was kindly supplied by Dafra Pharma International (Belgium), PFDHA (3) was synthesized by the method of O'Neill et al. (29O'Neill P.M. Miller A. Bishop L.P. Hindley S. Maggs J.L. Ward S.A. Roberts S.M. Scheinmann F. Stachulski A.V. Posner G.H. Park B.K. J. Med. Chem. 2001; 44: 58-68Crossref PubMed Scopus (105) Google Scholar), and the tetrahydrofuran (THF)-acetate isomer of PFDHA (4) was prepared by an iron-catalyzed rearrangement (17Mercer A.E. Maggs J.L. Sun X.M. Cohen G.M. Chadwick J. O'Neill P.M. Park B.K. J. Biol. Chem. 2007; 282: 9372-9382Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). All other materials and chemicals were purchased from Sigma-Aldrich. HeLa and HeLa ρ0 cell lines were maintained in DMEM high glucose medium supplemented with fetal bovine serum (10% v/v), l-glutamine (1% w/v), and sodium pyruvate, which was supplemented with uridine (50 μm) for HeLa ρ0 cell culture. HL-60 cells were maintained in RPMI 1640 medium supplemented with fetal bovine serum (10% v/v) and l-glutamine (1% w/v). All of the cells were incubated under humidified air containing 5% CO2 at 37 °C. Cell viability was above 95% for all of the experiments based on trypan blue exclusion (30Tennant J.R. Transplantation. 1964; 2: 685-694Crossref PubMed Scopus (596) Google Scholar). Drug stock solutions were made up in Me2SO, and the final solvent concentration was below 0.5% (v/v) in each incubation. HeLa/HeLa ρ0 cells (5 × 103/well) were plated in triplicate in flat-bottomed 96-well plates and incubated for 24 h before exposure to each compound (0.005–100 μm). In experiments performed in the presence of modulators of heme synthesis, intracellular iron levels and antioxidants succinylacetone (SA, 0.5 mm), protoporphyrin IX (PPIX, 1 μm), holotransferrin (HTF, 10 μm), and tiron (1 mm) were added to the cells immediately prior to the addition of the drug. Cell viability was measured by the MTT (17Mercer A.E. Maggs J.L. Sun X.M. Cohen G.M. Chadwick J. O'Neill P.M. Park B.K. J. Biol. Chem. 2007; 282: 9372-9382Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) and neutral red (31Repetto G. del Peso A. Zurita J.L. Nat. Protoc. 2008; 3: 1125-1131Crossref PubMed Scopus (1402) Google Scholar) assays as described previously. All of the results are expressed as percentages of the values for vehicle-treated cells. The IC50 values were calculated from individual inhibition curves plotted by Grafit software (Erithacus, West Sussex, UK). Drug-treated cells were stained with annexin V and PI using a commercially available kit according to the manufacturer's instructions. A minimum of 5000 cells were analyzed by flow cytometry (Epics XL; Beckman Coulter, Buckinghamshire, UK). Annexin V-FITC fluorescence was measured on fluorescence channel 1, and PI fluorescence was measured on fluorescence channel 3. The proportions of viable cells (annexin-negative/PI-negative), apoptotic cells (annexin-positive/PI-negative), and necrotic/late apoptotic cells (annexin-positive/PI-positive) were calculated using WinMDI software (version 2.8; Scripps Institute). Tetramethylrhodamine ethyl ester (TMRE) was used to identify cells with a high mitochondrial membrane potential (MMP) as described previously (17Mercer A.E. Maggs J.L. Sun X.M. Cohen G.M. Chadwick J. O'Neill P.M. Park B.K. J. Biol. Chem. 2007; 282: 9372-9382Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). ROS generation was monitored using dichlorofluoroescein diacetate (DCFH-DA). Plated cells were pretreated with DCFH-DA (5 μm, 30 min), the dye solution was then removed, and the cells were washed twice in Hanks' balanced salt solution (HBSS) (1 ml) before drug was added. Following incubation, the cells were washed twice (HBSS, 1 ml) before resuspension (HBSS, 1 ml) and analysis by flow cytometry. A minimum of 5000 cells were analyzed, and the fluorescence intensity was measured using fluorescence channel 1. To correct for any fluorescence of the drug, duplicate samples were prepared without the addition of DCFH-DA for all incubations. In experiments performed in the presence of the antioxidant tiron, 1 mm tiron was added following incubation with DCFH-DA immediately prior to the addition of artesunate. Drug-treated cells (2 × 105) were washed in ice-cold HBSS and resuspended in 50 μl of mitochondrial isolation buffer (250 mm sucrose, 20 mm HEPES, 5 mm MgCl2, 10 mm KCl, pH 7.4) containing 0.05% digitonin. The cells were left on ice for 10 min before centrifugation (13,000 rpm, 3 min). The pellet was retained as the mitochondrially enriched fraction, and the supernatant was retained as the cytosol-containing fraction. Subsequently, both pellets and supernatants were analyzed by Western blotting for cytochrome c as described previously (32Mercer A.E. Regan S.L. Hirst C.M. Graham E.E. Antoine D.J. Benson C.A. Williams D.P. Foster J. Kenna J.G. Park B.K. Toxicol. Appl. Pharmacol. 2009; 239: 297-305Crossref PubMed Scopus (19) Google Scholar). A commercial caspase-glo 3/7 kit (Promega, Madison, WI) was used to measure the combined DEVD-ase activity of caspases-3 and -7. The assay provides a proluminescent caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD, which is cleaved by the caspases to produce a luminescent signal. The kit was used according to the manufacturer's instructions, and the levels of luminescence were measured using a fluorescence microplate reader (BioTek FL600). The method was based on the protocol of Sassa (33Sassa S. J. Exp. Med. 1976; 143: 305-315Crossref PubMed Scopus (215) Google Scholar). To a pellet of endoperoxide-treated cells (1 × 105) was added an aqueous solution oxalic acid (500 μl, 2 m). The samples were shaken before heating (100 °C, 30 min). Standard solutions of hemin (0.01–10 mm) were prepared (water/methanol, MeOH 1:1, v/v containing 1% bovine serum albumin) and heated with oxalic acid, as above. The samples and standard solutions (200 μl) were plated into a white 96-well plate, and the fluorescence of the deferrated heme was measured (excitation, 400 nm; emission, 662 nm). The results were corrected for non-heme endogenous porphyrins by preparing cell blanks in oxalic acid without heating. Intracellular activation of the endoperoxides was monitored by LC-MS/MS. The instrument was an API 2000 triple-quadrupole mass spectrometer (AB Sciex, Warrington, UK) interfaced to a PerkinElmer Series 200 autosampler and a PerkinElmer pump. The data were collected and analyzed by the Analyst 1.3 software (AB Sciex). Cells (1 ml of 1.5 × 104 HeLa cells/ml or 4 ml of 1 × 106 HL-60 cells/ml) were incubated with PFDHA (3) (48 h for HeLa, 24 h for HL-60) at 37 °C. Following incubation, artesunate (2, 1 nmol) was added as an internal standard before the samples were prepared as described previously (17Mercer A.E. Maggs J.L. Sun X.M. Cohen G.M. Chadwick J. O'Neill P.M. Park B.K. J. Biol. Chem. 2007; 282: 9372-9382Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) and were analyzed by LC-MS/MS multiple reaction monitoring (MRM). Chromatographic separation was achieved on an Agilent ZORBAX Eclipse XDB-C8 column (150 × 3.9 mm inner diameter, 5 μm; Agilent Technologies, Santa Clara, CA). The mobile phase consisted of methanol with 10 mm aqueous ammonium acetate (70:30, v/v) delivered at a flow rate of 0.4 ml/min. The mass spectrometer was operated in positive ion mode. The operating parameters were optimized via the quantitative optimization facility in Analyst software as follows: ion spray voltage of +5.0 kV, back pressures for the collision gas of 2 p.s.i., curtain gas of 20 p.s.i., nebulizer gas (GS1) of 30 p.s.i., and turbo gas (GS2) of 65 p.s.i.; the turbo gas temperature was 300 °C. Analyte-specific parameters and fragmentation transitions are detailed in Table 1. All of the gases used were nitrogen. Calibration curves of peak area versus analyte mass (5–5000 pmol) were generated from solutions of synthetic PFDHA, PFDHA THF acetate, and artesunate in methanol, and the limit of quantification was calculated to be 50 pmol using the method of least squares line fit. The efficiency of PFDHA and PFDHA THF acetate recovery was corrected for by the quantification of the internal standard, artesunate.TABLE 1Analyte specific parameters and fragmentation transitions for tandem mass spectrometric analyses of artesunate (2), PFDHA (3), and PFDHA THF acetate (4)ParameterArtesunate (2)PFDHA (3)PFDHA THF acetate (4)Fragmentation transition402.1 to 163.1396.0 to 163.0396.0 to 266.7Declustering potential (V)21621Focusing potential (V)370370370Entrance potential (V)1285Collision energy (V)301730Collision cell entrance potential (V)18.61419Collision cell exit potential (V)261014Dwell time200200100 Open table in a new tab The values are expressed as the means ± S.E. The data were analyzed for non-normality using a Shapiro-Wilk test. Student's t test was used when normality was indicated; a Mann-Whitney U test was used for nonparametric data. All of the calculations were performed using Stats Direct statistical software; the results were considered significant when the p values were less than 0.05. The role of the activity of the mitochondrial ETC in ART-induced cell death was investigated using HeLa ρ0 cells and their normal HeLa cell counterpart. HeLa ρ0 cells have been depleted of mitochondrial DNA, which encodes the majority of the ETC. Therefore, they still contain mitochondria but lack a fully functional ETC and so utilize glycolysis instead of oxidative phosphorylation to generate ATP (34Rodríguez-Enríquez S. Vital-González P.A. Flores-Rodríguez F.L. Marín-Hernández A. Ruiz-Azuara L. Moreno-Sánchez R. Toxicol. Appl. Pharmacol. 2006; 215: 208-217Crossref PubMed Scopus (92) Google Scholar, 35King M.P. Attardi G. Science. 1989; 246: 500-503Crossref PubMed Scopus (1448) Google Scholar). The cytotoxicity of PFDHA and artesunate (IC50 values) against HeLa and HeLa ρ0 cells was determined using the MTT assay of total cellular dehydrogenase activity (36Berridge M.V. Tan A.S. Protoplasma. 1998; 205: 74-82Crossref Scopus (76) Google Scholar) and also by measuring the ability of viable cells to accumulate the cationic dye neutral red in the lysosomes via the maintenance of pH gradients through the production of ATP (31Repetto G. del Peso A. Zurita J.L. Nat. Protoc. 2008; 3: 1125-1131Crossref PubMed Scopus (1402) Google Scholar) (Table 2). The neutral red assay was included to confirm that any assessments of ART cytotoxicity made by the MTT assay of dehydrogenase activity were not affected by direct enzyme inhibition or the ETC status of the ρ0 cells. The results demonstrate that the endoperoxide compounds are significantly more cytotoxic to the HeLa cells than the HeLa ρ0 cells, with values three to seven times greater in cells without a functioning ETC.TABLE 2Cytotoxicity of the ARTs against HeLa and HeLa ρ0 cells (48 h)MTT assayNR assayρ+ρ0p valueρ+ρ0p valuePFDHA13 ± 492 ± 90.001215 ± 4>100NDArtesunate6 ± 334 ± 50.011416 ± 644 ± 90.0006 Open table in a new tab The mechanism of cell death induced by artesunate was defined by measuring two diagnostic parameters. The annexin V/PI double staining method was used to identify apoptotic cells in which annexin V binds to the externalized phosphatidylserine residues (annexin-positive) and necrotic and late apoptotic cells in which concomitant PI staining of DNA occurs as a result of leaky cell membranes (annexin- and PI-positive) (Fig. 2A). These results show that in HeLa cells, artesunate induces significant levels of dose-dependent apoptosis, which progresses to low levels of late apoptosis/necrosis at higher concentrations (Fig. 2B). In contrast, in HeLa ρ0 cells, death progresses predominantly via necrosis, with low levels of apoptosis apparent. For example, when treated with artesunate (50 μm, 48 h), HeLa cells are 54 ± 2% apoptotic and 24 ± 9% necrotic/late apoptotic, whereas HeLa ρ0 cells are 21 ± 5% apoptotic and 67 ± 21% necrotic/late apoptotic. The role of the mitochondria was further investigated by examining mitochondria membrane depolarization and the release of cytochrome c into the cytosol. TMRE was used to label cells with high mitochondrial membrane potential, and any reduction in fluorescence was attributed to MMP (Fig. 2C). Artesunate induced dose-dependent mitochondria membrane depolarization in HeLa cells, which reached a maximum of 71 ± 7% of cells (100 μm), but in HeLa ρ0 the fraction of cells with depolarized mitochondrial membranes was lower, with significant levels only seen from 50 μm and a maximum of 49 ± 4% (100 μm) (Fig. 2C). The release of cytochrome c from the mitochondria into the cytosol is a hallmark of apoptosis (37Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar) and was measured by immunoblot analysis of mitochondria and cytosol-containing fractions prepared from artesunate-treated cells (Fig. 2D). Artesunate induced a significant dose-dependent cytochrome c release in HeLa cells, which reached a maximum of 61 ± 4% of total cytochrome c released (48 h; Fig. 2D). Lower levels of cytochrome c release were evident in HeLa ρ0 cells (maximum of 33 ± 17% release), and their response was also more variable (48 h). During the apoptotic pathway, the release of cytochrome c and the formation of the apoptosome trigger the activation of caspases-3 and-7, which are responsible for chromatin condensation and DNA fragmentation. The induction of caspase-3 and -7 activities by artesunate was measured using a luminescent assay (Fig. 2E). Artesunate induced a dose-dependent increase in activity at lower concentrations (1–10 μm, 48 h) in HeLa cells. However, the complete death of cells at high concentrations (100 μm) resulted in control levels of caspase activity. Conversely, no significant caspase-3/-7 activity was evident in HeLa ρ0 cells (48 h). The drug-induced generation of ROS was assessed by the pretreatment of cells with DCFH-DA. Esterase cleavage of the acetate groups forms dichlorodihydrofluorescein, which accumulates in the cells and undergoes oxidation by ROS to fluorescent dichlorofluorescin. The investigations of artesunate (100 μm) over a 48-h time course demonstrated that early maximal levels of ROS generation were attained at 16 h (2.3 ± 0.5-fold over control), which is much earlier than cytotoxicity is observed (Fig. 3A). In addition, ROS generation was reduced in HeLa ρ0 cells (Fig. 3A). We have used tiron, a cell membrane-permeable superoxide scavenger (38Greenstock C.L. Miller R.W. Biochim. Biophys. Acta. 1975; 396: 11-16Crossref PubMed Scopus (142) Google Scholar) to define the link between ROS induction and the initiation of cell death. The results show that the addition of tiron significantly inhibits ROS generation at an early 24-h time point (Fig. 3B) and also inhibits the induction of cytotoxicity (48 h) (Fig. 3C). The cellular bioactivation of the endoperoxide bridge was quantified using PFDHA (3, 10" @default.
• W2023405265 created "2016-06-24" @default.
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• W2023405265 creator A5030009340 @default.
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• W2023405265 creator A5060937140 @default.
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• W2023405265 date "2011-01-01" @default.
• W2023405265 modified "2023-10-15" @default.
• W2023405265 title "The Role of Heme and the Mitochondrion in the Chemical and Molecular Mechanisms of Mammalian Cell Death Induced by the Artemisinin Antimalarials" @default.
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• W2023405265 cites W1964066906 @default.
• W2023405265 cites W1965105249 @default.
• W2023405265 cites W1978216857 @default.
• W2023405265 cites W1980941241 @default.
• W2023405265 cites W1981838111 @default.
• W2023405265 cites W1987877328 @default.
• W2023405265 cites W1990321698 @default.
• W2023405265 cites W1995521183 @default.
• W2023405265 cites W1997380789 @default.
• W2023405265 cites W2012733189 @default.
• W2023405265 cites W2013022548 @default.
• W2023405265 cites W2015244174 @default.
• W2023405265 cites W2019276195 @default.
• W2023405265 cites W2020803551 @default.
• W2023405265 cites W2033721665 @default.
• W2023405265 cites W2034275758 @default.
• W2023405265 cites W2040713555 @default.
• W2023405265 cites W2041770695 @default.
• W2023405265 cites W2046623416 @default.
• W2023405265 cites W2046770259 @default.
• W2023405265 cites W2049332203 @default.
• W2023405265 cites W2050506991 @default.
• W2023405265 cites W2051073477 @default.
• W2023405265 cites W2067886822 @default.
• W2023405265 cites W2068599185 @default.
• W2023405265 cites W2070368895 @default.
• W2023405265 cites W2071906583 @default.
• W2023405265 cites W2075937164 @default.
• W2023405265 cites W2084615328 @default.
• W2023405265 cites W2085936370 @default.
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• W2023405265 cites W2111757377 @default.
• W2023405265 cites W2111813303 @default.
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• W2023405265 cites W2113999599 @default.
• W2023405265 cites W2114705206 @default.
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• W2023405265 cites W2133082107 @default.
• W2023405265 cites W2138215247 @default.
• W2023405265 cites W2148794480 @default.
• W2023405265 cites W2151019491 @default.
• W2023405265 cites W2153177866 @default.
• W2023405265 cites W2306306384 @default.
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• W2023405265 doi "https://doi.org/10.1074/jbc.m110.144188" @default.
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