Evidence for the Involvement of Carbon-centered Radicals in the Induction of Apoptotic Cell Death by Artemisinin Compounds
Artemisinin and its derivatives are currently recommended as first-line antimalarials in regions where Plasmodium falciparum is resistant to traditional drugs. The cytotoxic activity of these endoperoxides toward rapidly dividing human carcinoma cells and cell lines has been reported, and it is hypothesized that activation of the endoperoxide bridge by an iron(II) species, to form C-centered radicals, is essential for cytotoxicity. The studies described here have utilized artemisinin derivatives, dihydroartemisinin, 10β-(p-bromophenoxy)dihydroartemisinin, and 10β-(p-fluorophenoxy)dihydroartemisinin, to determine the chemistry of endoperoxide bridge activation to reactive intermediates responsible for initiating cell death and to elucidate the molecular mechanism of cell death. These studies have demonstrated the selective cytotoxic activity of the endoperoxides toward leukemia cell lines (HL-60 and Jurkat) over quiescent peripheral blood mononuclear cells. Deoxy-10β-(p-fluorophenoxy)dihydroartemisinin, which lacks the endoperoxide bridge, was 50- and 130-fold less active in HL-60 and Jurkat cells, respectively, confirming the importance of this functional group for cytotoxicity. We have shown that chemical activation is responsible for cytotoxicity by using liquid chromatography-mass spectrometry analysis to monitor endoperoxide activation by measurement of a stable rearrangement product of endoperoxide-derived radicals, which was formed in sensitive HL-60 cells but not in insensitive peripheral blood mononuclear cells. In HL-60 cells the endoperoxides induce caspase-dependent apoptotic cell death characterized by concentration- and time-dependent mitochondrial membrane depolarization, activation of caspases-3 and -7, sub-G0/G1 DNA formation, and attenuation by benzyloxycarbonyl-VAD-fluoromethyl ketone, a caspase inhibitor. Overall, these results indicate that endoperoxide-induced cell death is a consequence of activation of the endoperoxide bridge to radical species, which triggers caspase-dependent apoptosis. Artemisinin and its derivatives are currently recommended as first-line antimalarials in regions where Plasmodium falciparum is resistant to traditional drugs. The cytotoxic activity of these endoperoxides toward rapidly dividing human carcinoma cells and cell lines has been reported, and it is hypothesized that activation of the endoperoxide bridge by an iron(II) species, to form C-centered radicals, is essential for cytotoxicity. The studies described here have utilized artemisinin derivatives, dihydroartemisinin, 10β-(p-bromophenoxy)dihydroartemisinin, and 10β-(p-fluorophenoxy)dihydroartemisinin, to determine the chemistry of endoperoxide bridge activation to reactive intermediates responsible for initiating cell death and to elucidate the molecular mechanism of cell death. These studies have demonstrated the selective cytotoxic activity of the endoperoxides toward leukemia cell lines (HL-60 and Jurkat) over quiescent peripheral blood mononuclear cells. Deoxy-10β-(p-fluorophenoxy)dihydroartemisinin, which lacks the endoperoxide bridge, was 50- and 130-fold less active in HL-60 and Jurkat cells, respectively, confirming the importance of this functional group for cytotoxicity. We have shown that chemical activation is responsible for cytotoxicity by using liquid chromatography-mass spectrometry analysis to monitor endoperoxide activation by measurement of a stable rearrangement product of endoperoxide-derived radicals, which was formed in sensitive HL-60 cells but not in insensitive peripheral blood mononuclear cells. In HL-60 cells the endoperoxides induce caspase-dependent apoptotic cell death characterized by concentration- and time-dependent mitochondrial membrane depolarization, activation of caspases-3 and -7, sub-G0/G1 DNA formation, and attenuation by benzyloxycarbonyl-VAD-fluoromethyl ketone, a caspase inhibitor. Overall, these results indicate that endoperoxide-induced cell death is a consequence of activation of the endoperoxide bridge to radical species, which triggers caspase-dependent apoptosis. Artemisinin (ART, 2The abbreviations used are: ART, artemisinin; DHA, dihydroartemisinin; PBrDHA, 10β-(p-bromophenoxy)dihydroartemisinin; PFDHA, 10β-(p-fluorophenoxy)dihydroartemisinin; dPFDHA, deoxy-10β-(p-fluorophenoxy) dihydroartemisinin; PBMC, peripheral blood mononuclear cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MMP, mitochondrial membrane potential; TMRE, tetramethylrhodamine ethyl ester; PI, propidium iodide; LC-MS, liquid chromatography-mass spectrometry; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; THF, tetrahydrofuran; LDH, lactate dehydrogenase; HBSS, Hanks' balanced salt solution. 1; see Fig. 1) is a sesquiterpene lactone endoperoxide found in the traditional Chinese medicinal plant Artemisia annua (1Klayman D.L. Science. 1985; 228: 1049-1055Crossref PubMed Scopus (2046) Google Scholar). The activity against multidrug-resistant malaria parasites and a rapid therapeutic response (2Van Agtmael M.A. Eggelte T.A. Van Boxtel C.J. Trends Pharmacol. Sci. 1999; 20: 199-205Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar) reported for ART and semi-synthetic ART compounds combined with an absence of significant toxicity in patients (3Park B.K. O'Neill P.M. Maggs J.L. Pirmohamed M. Br. J. Clin. Pharmacol. 1998; 46: 521-529Crossref PubMed Scopus (49) Google Scholar, 4Ribierio I.R. Olliaro P. Med. Trop. 1998; 58: 50-53PubMed Google Scholar) have led the World Health Organization to recommend the use of ART-based combination therapies to all countries experiencing resistance to conventional monotherapies. The cytotoxic properties of ART compounds in cancer cell lines were first observed against Ehrlich ascites tumor cells (5Woerdenbag H.J. Moskal T.A. Pras N. Malingre T.M. El-Feraly F.S. Kampinga H.H. Konings A.W.T. J. Nat. Prod. 1993; 56: 849-856Crossref PubMed Scopus (269) Google Scholar) and have subsequently been reported in many other human cell lines of the 60 cell line test panel at the NCI, National Institutes of Health (6Beekman A.C. Barentsen A.R.W. Woerdenbag H.J. Van Uden W. Pras N. Konings A.W.T. El-Feraly F.S. Galal A.M. Wikstrom H.V. J. Nat. Prod. 1997; 60: 325-330Crossref PubMed Scopus (118) Google Scholar, 7Jung M. Bioorg. Med. Chem. Lett. 1997; 7: 1091-1094Crossref Scopus (53) Google Scholar, 8Beekman A.C. Wierenga P.K. Woerdenbag H.J. Van Uden W. Pras N. Konings A.W.T. El-Feraly F.S. Galal A.M. Wikstrom H.V. Planta Med. 1998; 64: 615-619Crossref PubMed Scopus (107) Google Scholar, 9Posner G.H. Ploypradith P. Parker M.H. O'Dowd H. Woo S.H. Northrop J. Krasavin M. Dolan P. Kensler T.W. Xie S. Shapiro T.A. J. Med. Chem. 1999; 42: 4275-4280Crossref PubMed Scopus (151) Google Scholar, 10Efferth T. Dunstan H. Sauerbrey A. Miyachi H. Chitambar C.R. Int. J. Oncol. 2001; 18: 767-773PubMed Google Scholar, 11Posner G.H. Paik I.H. Sur S. McRiner A.J. Borstnik K. Xie S. Shapiro T.A. J. Med. Chem. 2003; 46: 1060-1065Crossref PubMed Scopus (151) Google Scholar, 12Jeyadevan J.P. Bray P.G. Chadwick J. Mercer A.E. Byrne A. Ward S.A. Park B.K. Williams D.P. Cosstick R. Davies J. Higson A.P. Irving E. Posner G.H. O'Neill P.M. J. Med. Chem. 2004; 47: 1290-1298Crossref PubMed Scopus (109) Google Scholar). ART and its derivatives exhibit selective cytotoxicity toward rapidly proliferating neoplastic cells, with the highest activity reported against colon and leukemia cell lines and the least against non-small cell lung cancer cells (10Efferth T. Dunstan H. Sauerbrey A. Miyachi H. Chitambar C.R. Int. J. Oncol. 2001; 18: 767-773PubMed Google Scholar). The most sensitive cell lines are characterized by their rapid proliferation, often accompanied by a high intracellular iron concentration to sustain continued proliferation (13Disbrow G.L. Baege A.C. 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PubMed Google Scholar). was and human was was The human cell lines HL-60 and Jurkat were the of The cytotoxicity was Science. was National and were as described M. M. J. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). to was was was other and were and the of and Jurkat cell lines were in with and The cells were at was to and to of HL-60 cells, which were used and blood was in and were as described D.P. Pirmohamed M. Maggs J.L. Park B.K. J. Pharmacol. 1997; Google Scholar). were to the in with human and was for all PubMed Scopus Google Scholar). were in and the concentration was in of the and cells and Jurkat cells were in in and were to to of for at cell using the were by the of of in to and for at of a was to to the and for an The of the were at a test of and a of with a lactate was using the cytotoxicity to the results are as a of cells. The were by of of of by was to a for and iron salt S.A. J. Pharmacol. 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In sensitive HL-60 cells, and a time-dependent which was first but by and these cells were used in of the endoperoxides against HL-60 and Jurkat cell lines and human in a cytotoxicity of and against HL-60 in a The as a of of the endoperoxide bridge was using in HL-60 cells and resistant of endoperoxide bridge C-centered radical formation, in HL-60 cells was by analysis of endoperoxide-derived time-dependent of was demonstrated over accompanied by the of the Fig. of endoperoxide bridge activation the radical The were not in the of cells in that these a of the and the using that a of the to and of the activation the C-centered radical to form the for a The analysis was a of resistant with in with sensitive HL-60 cells In these studies of were which were the of the of was (3Park B.K. O'Neill P.M. Maggs J.L. Pirmohamed M. Br. J. Clin. Pharmacol. 1998; 46: 521-529Crossref PubMed Scopus (49) Google Scholar) is not to (6Beekman A.C. Barentsen A.R.W. Woerdenbag H.J. Van Uden W. Pras N. Konings A.W.T. El-Feraly F.S. Galal A.M. Wikstrom H.V. J. Nat. Prod. 1997; 60: 325-330Crossref PubMed Scopus (118) Google Scholar) in of cells were with and the of and was by LC-MS, as described of and in HL-60 and is shown are the of of of with HL-60 as by the test for The in HL-60 of the endoperoxides to induce was by measurement of mitochondrial membrane depolarization, analysis of and and DNA which are with the apoptotic was used to cells with high J. 1999; Full Text Full Text PDF PubMed Scopus Google and in was to mitochondrial membrane DHA, PBrDHA, and and and The endoperoxides of depolarization, with significant at the was at of cells were not mitochondrial membrane with not analysis of DNA with A. J. PubMed Scopus Google Scholar) was used to the of a sub-G0/G1 of apoptotic cells with DNA endoperoxide DHA, PBrDHA, and and all of and of a sub-G0/G1 which significant a of of cells in the sub-G0/G1 The of PFDHA, dPFDHA, not induce mitochondrial membrane DNA in HL-60 cells, that the bridge is essential for the of apoptotic cell death. The activation of caspases-3 and in HL-60 cells was by that the of the caspase to the active In HL-60 cells, was as the and to the a of was by the of the and of to its active and the endoperoxides a time-dependent of to its and In the to induce of and was as the in cells and PBrDHA, DHA, and all a of to its active and of was observed with the the active but not the dPFDHA, a time-dependent of to its active endoperoxides induce and of a sub-G0/G1 in HL-60 cells. The of a sub-G0/G1 endoperoxide was by and as described of cells. with in the and the sub-G0/G1 of this is cells are in the sub-G0/G1 with and the sub-G0/G1 The results were to determine the and of the of a sub-G0/G1 in HL-60 cells. concentration of sub-G0/G1 of sub-G0/G1 of cells mitochondrial the cells in were and results are the of of of PBrDHA, DHA, and with as by the test for endoperoxides induce and of and in HL-60 cells. HL-60 cells were with the as and were by as described to caspase were and are concentration and of the to the and concentration of the to the in HL-60 caspase was used to the of activation of the caspase in HL-60 cells. In HL-60 cells, the and with of the to PFDHA, a of was with cells with and and not cells of of that against the cells a with of the but were These were by most cells but a of their which against the as that the In cells to and PFDHA, in to the of apoptotic cells, a of cells were observed that not with and these were not by not that was apoptotic its the of the by and and DHA, as as used as a the of to its active and the and of in the of as as a of the form of The most to the form of at the and and is as by an of to the and cells that been with mitochondrial membrane of with and the of and DNA as by of the sub-G0/G1 These results indicate that mitochondrial membrane is not caspase activity but that DNA endoperoxide-induced of a sub-G0/G1 but not mitochondrial membrane HL-60 cells were in the absence of as mitochondrial and sub-G0/G1 were as described of cell of the of a sub-G0/G1 cells in were and results are the of of of in the of with without the as by the test for ART and its derivatives are an of that are active against resistant of P. 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Res. 2003; PubMed Scopus Google Scholar, Chem. Int. 1999; PubMed Scopus Google Scholar, H.H. 2003; Scopus Google Scholar, Chem. Scopus Google Scholar). of of with not of the but it that of the of the were the cells that time-dependent of the endoperoxide bridge in HL-60 cells but not in resistant using these have the first chemical that C-centered the cytotoxicity sensitive and resistant cells. The attenuation by an iron of the of in cells to that intracellular iron a in the activation of ART endoperoxides (13Disbrow 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 (139) Google Scholar). it is that it is that is responsible for the activation of ART its in blood cells, and is to as have the P.M. Posner G.H. J. Med. Chem. 2004; 47: PubMed Scopus Google Scholar). HL-60 cells and the P. K. S. J. Med. 2002; PubMed Scopus Google Scholar, M. M. PubMed Scopus Google a but it is to to the activation of the endoperoxides P.G. W. Lett. PubMed Scopus Google activation P.M. Posner G.H. J. Med. Chem. 2004; 47: PubMed Scopus Google Scholar). It was found that the endoperoxides induce concentration- and time-dependent mitochondrial membrane depolarization, caspase and DNA in sensitive HL-60 cells, at to the studies with that the bridge is essential for the of and that the of cytotoxic activity 1) the of cell death. The of a form of cell by the endoperoxides that the cytotoxic mechanism of by endoperoxide activation a caspase J. D. 1998; PubMed Scopus Google was used to the of caspase activity and to the apoptotic by and that results in the activation of the chemical and the with as the M. K. Res. PubMed Scopus Google but the of cell death the of the In these studies not mitochondrial membrane but DNA and activation this is and used to elucidate the apoptotic by the endoperoxides by the of apoptotic The of of mitochondrial membrane that and not is responsible for the of the caspases-3 and Overall, have demonstrated that the endoperoxides induce the chemical that mitochondrial is an event in and that the of apoptotic cell death is caspase The importance of to the cytotoxicity of endoperoxides in other cell was the of a which reported the of ART the in a ART the of the its to the membrane W. W. D. S. 2005; PubMed Scopus Google Scholar). The that the a in ART and that the formed to the The cytotoxic of the endoperoxides it has been reported that endoperoxide-derived radical are to form with W. Pharmacol. 1993; 46: PubMed Scopus Google Scholar) and DNA K. K. Chem. 18: Scopus Google Scholar). In ART endoperoxides have been shown to the of reactive (13Disbrow 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 (139) Google which have been in the cytotoxic mechanism of In by using ART derivatives as chemical have shown that the cytotoxicity of these endoperoxides and dividing cells is a of selective activation of the endoperoxide bridge C-centered radical in cells. We have demonstrated that selective of active cells results in mitochondrial membrane depolarization, the of the chemical and the activation of and in the of this a chemical and molecular of endoperoxide-induced cell death which used to cell that to these and of the with use of the The was and with the We for with the