Leukemia (2002) 16, 74–83 2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Ajoene, an experimental anti-leukemic drug: mechanism of cell death VM Dirsch1, DSM Antlsperger1, H Hentze2,3 and AM Vollmar1 1Department of Pharmacy, Center of Drug Research, University of Munich, Munich, Germany; and 2Biochemical Pharmacology, University of Konstanz, Konstanz, Germany The organosulfur compound ajoene, a constitutent of garlic, Thus, evidence is provided that certain OSC may have anti- has been shown to induce apoptosis in a leukemic cell line as leukemic potential. The molecular pathway, however, by well as in blood cells of a leukemic patient. The mechanisms of action of ajoene, however, are unknown. The present study which OSC trigger apoptosis, needs to be clarified. aims to characterize the molecular events leading to ajoene- Two major pathways mediating drug-induced apoptosis triggered apoptosis. We show here that ajoene (20 ␮M) leads to have been characterized. One involves the triggering of cell a time-dependent activation of caspase-3-like activity as well surface death receptors and the other targets mitochondria as to the proteolytic processing of procaspase-3 and -8. Acti- without the involvement of a receptor/ligand system.13 vation of caspases was necessary for ajoene-induced apoptosis since the broad-range caspase inhibitor zVAD-fmk In certain cell types, the CD95 (APO-1/Fas)receptor system completely abrogated ajoene-mediated DNA fragmentation. was suggested to be involved in apoptosis evoked by chemo- Although the initiator caspase-8 was activated, the CD95 death therapeutic drugs. Drug-mediated activation of the CD95 sig- receptor was not involved in death signaling since the HL-60 naling pathway was shown to occur by induction of the CD95 clone used was shown to express a functionally inactive CD95 ligand (CD95-L)or increased expression of the CD95 recep- receptor. Furthermore, ajoene induced the release of cyto- 14–17 chrome c, which was not inhibited by zVAD-fmk indicating that tor. CD95 is a cell surface receptor belonging to the cytochrome c release precedes caspase activation. Ajoene also TNF/nerve growth factor-receptor superfamily. Activation of led to a dissipation of the mitochondrial transmembrane poten- this receptor leads to trimerization and subsequent recruit- tial. Overexpression of Bcl-xL clearly diminished ajoene- ment of FADD (Fas-associated protein with death domain)and induced caspase activation as well as apoptosis. These results procaspase-8 forming a death-inducing signaling complex indicate that apoptosis in leukemia cells triggered by ajoene is 18 based on the activation of a mitochondria-dependent caspase (DISC). Depending on the cell type, either large amounts of cascade which includes also the activation of the initiator cas- procaspase-8 are activated directly at the DISC leading to the pase-8. activation of a caspase cascade independent of mitochondria Leukemia (2002) 16, 74–83. DOI: 10.1038/sj/leu/2402337 (type I cells)or small amounts of activated caspase-8 lead to Keywords: apoptosis; organosulfur compound; caspases; mito- a pathway which needs to be amplified by a mitochondria- chondria; HL-60 controlled caspase cascade (type II cells).19 Besides the view that chemotherapeutic drugs act through triggering the CD95 receptor/ligand system, there are also Introduction studies demonstrating that anticancer drug-induced apoptosis occurs in the absence of a functional CD95 receptor/ligand Organosulfur compounds (OSC)from natural sources (eg gar- system.20–23 These apoptotic signals seem to be integrated by lic, onion, broccoli)as well as related synthetic compounds mitochondria leading to a pathway involving a caspase cas- have been shown to possess anticarcinogenic activity.1–4 The mechanism discussed to mediate the chemopreventive effect cade downstream of these organelles. A central event in mito- of OSC is an increase in the activity of phase II detoxication chondria-controlled cell death is the mitochondrial membrane enzymes.1–4 On the other hand, OSC were also demonstrated permeabilization (MMP). MMP of the inner mitochondrial to have direct antiproliferative effects on tumor cells.5–7 membrane causes loss of the mitochondrial transmembrane ⌬⌿ Importantly, several reports demonstrated triggering of potential ( m). MMP of the outer membrane leads to the apoptosis by diverse OSC in leukemia cells.8–11 Wong et al11 release of apoptogenic factors such as cytochrome c into the studied the structural requirements of synthetic OSC to induce cytosol. Released cytochrome c binds Apaf-1 forming a com- apoptosis in human leukemia cells. They found that a disulfide plex which in turn recruits and subsequently activates procas- moiety seems to be necessary to trigger cell death whereas pase-9. Activated caspase-9 is released from the complex to adjacent groups contribute to the specificity of cell killing. cleave and activate effector caspases, eg caspase-3. Important Studies from our own group demonstrated that ajoene, an regulators of cytochrome c release and therefore of mitochon- allylic disulfide originally isolated from garlic extracts,12 dria-mediated caspase activation are members of the Bcl-2 induces apoptosis in the human acute myeloid leukemia cell family proteins. Overexpression of Bcl-2 or Bcl-xL were shown line HL-60 as well as in peripheral blood mononuclear cells to block cytochrome c release in response to various (PBMC)isolated from a patient with a chronic myelogenous apoptotic stimuli.24,25 leukemia undergoing a myeloid blast crisis. In contrast, To date, the molecular pathways leading to OSC-triggered quiescent as well as proliferating PBMCs isolated from healthy apoptosis in leukemia cells remain unknown. The OSC ajoene donors remained unaffected.9 is a promising experimental anti-leukemic agent since it selec- tively induces apoptosis in human promyeloleukemic cells.9 Therefore, the aim of the present study was to characterize Correspondence: V Dirsch, Department of Pharmacy, Center of Drug the molecular signaling involved in ajoene-induced cell Research, Butenandtstraβe 5–13, D-81377 Munich, Germany; Fax: death. +49 +89 2180–7173 3Current address: Institute of Molecular and Cell Biology (IMBC), 30 Medical Drive, 117609 Singapore Received 30 May 2001; accepted 5 September 2001 Ajoene-induced apoptosis VM Dirsch et al 75 Materials and methods fluorogenic substrate DEVD-afc in 50 mM Hepes, pH 7.4, 1% sucrose, 0.1% CHAPS, 10 mM DTT). Blanks contained 10 ␮l Cells and culture conditions extraction buffer and 90 ␮l substrate buffer. Generation of free 7-amino-4-trifluoromethylcoumarin (afc)at 37 °C was deter- The human acute myeloid leukemia cell line HL-60 (DSM mined by fluorescence measurement at t = 0/t = 30 min using ACC 3; DSMZ, German Collection of Microorganisms and the fluorometer plate reader Victor2 (Wallac Instruments, Cell Cultures, Braunschweig, Germany)was cultured (37 °C Turku, Finland)set at an excitation wavelength of 385 nm and and 5% CO2)in RPMI 1640 medium supplemented with 10% an emission wavelength of 505 nm. Protein concentrations of fetal bovine serum and L-glutamine (2 mM)(Life Technologies, the corresponding samples were estimated with the Pierce- Eggenstein, Germany). HL-60 transfected with the pSFFVneo- Assay (Pierce, Rockford, IL, USA)and the activity was calcu- ␮ bcl-xL plasmid (HL-60/bcl-xL)or pSFFV-neo plasmid (HL- lated using serially diluted standards (0–5 M afc). Control 60/neo)(a gift from Dr KN Bhalla, Moffitt Cancer Center and experiments confirmed that the activity was linear with time Research Institute, University of South Florida, Tampa, FL, and with protein concentration under the conditions USA)26 was cultivated as described for HL-60 cells except that described above. 0.1 mM non-essential amino acids and 1 mM sodium pyruvate (Life Technologies)were added to the culture medium. One mg/ml G 418 (Life Technologies)was added every fifth pass- Expression of the CD95 receptor age. Cells exposed to G418 were not used for experiments. The Jurkat clone J16, JurkatR cells27 and Jurkat T cells trans- Cells were seeded (1 × 106/ml)in a 24-well plate. To detect 28 fected with vector control or bcl-xL (kindly provided by Drs CD95 surface expression by flow cytometry cells were incu- PH Krammer and H Walczak, DKFZ, Heidelberg, Germany) bated at 4°C for 30 min with mouse anti-CD95 antibody were cultured as described.29 Ajoene ((E,Z)-4,5,9-trithiado- (clone ZB4, 10 ␮g/ml; MBL, Nagoya, Japan)or the isotype deca-1,6,11-triene-9-oxide)was obtained from Lichtwer control (mouse IgG1; Pharmingen, Becton Dickinson, Heidel- Pharma (Berlin, Germany)and dissolved in PBS. N-acetyl- berg, Germany). After washing the cells twice with phosphate- cysteine (NAC)was obtained from Sigma (Deisenhofen, buffered saline containing 1% fetal calf serum (PBS/1% FCS), Germany), dissolved in PBS and the pH adjusted to pH 7.4. cells were incubated at 4°C for 30 min with fluorescein isothi- ocyanate (FITC)-conjugated rat anti-mouse IgG1 (Pharmingen, Becton Dickinson). Cells were washed again (2 × PBS/1% Quantification of apoptosis FCS)and analyzed by a FACSCalibur flow cytometer (Becton Dickinson). Quantification of apoptosis was performed by flow cytometric analysis of propidium iodide-stained nuclei using CELLQuest software (Becton Dickinson, Heidelberg, Germany)according Analysis of mitochondrial transmembrane potential to Nicoletti et al.30 Briefly, cells were incubated for 12 h in a (⌬⌿) hypotonic buffer, containing 1% sodium citrate, 0.1% Triton- X-100, and 50 ␮g/ml propidium iodide, in order to lyse cells HL-60 were treated with ajoene (20 ␮M)for various time per- and stain nuclei, which were analyzed by flow cytometry. iods (0–24 h)and stained with the fluorochrome 5,5 Ј,6–6’te- Nuclei to the left of the ‘G1-peak’ containing hypodiploid trachloro-1,1Ј,3,3Ј-tetraethylbenzimidazolcarbocyanine iod- DNA were considered apoptotic. Susceptibility of HL-60 cells ide (JC-1; 1,25 ␮M).