Ajoene, a Compound of Garlic, Induces Apoptosis in Human Promyeloleukemic Cells, Accompanied by Generation of Reactive Oxygen Species and Activation of Nuclear Factor kB

The pharmacological role of garlic in prevention and treatment of cancer has received increasing attention, but thorough investigations into the molecular mechanisms of action of garlic compounds are rare. The present study demonstrates that ajoene, a major compound of garlic induces apoptosis in human leukemic cells, but not in peripheral mononuclear blood cells of healthy donors. The effect was dose and time dependent. Apoptosis was judged by three criteria, morphology of cells, quantification of subdiploid DNA content by flow cytometry, and detection of DNA fragmentation by gel electrophoresis. Ajoene increased the production of intracellular peroxide in a doseand time-dependent fashion, which could be partially blocked by preincubation of the human leukemic cells with the antioxidant N-acetylcysteine. Interestingly, N-acetylcysteinetreated cells showed a 50% loss of ajoene-induced apoptosis. Moreover, ajoene was demonstrated to activate nuclear translocation of the transcription factor nuclear factor kB, an effect that was abrogated in N-acetylcysteine-loaded cells. These results suggested that ajoene might induce apoptosis in human leukemic cells via stimulation of peroxide production and activation of nuclear factor kB. This is a novel aspect in the biological profile of this garlic compound and an important step in elucidating the underlying molecular mechanisms of its antitumor action. The role of dietary compounds as drugs in cancer prevention and treatment is widely discussed (Pezzuto, 1993; Agarwal, 1996; Koch and Lawson, 1996; Milner, 1996). In this regard the potential chemopreventive effect of garlic (Allium sativum) was subject of various clinical trials (Dorant et al., 1993; Steinmetz et al., 1994; Agarwal, 1996; Dorant et al., 1996; Koch and Lawson, 1996; Lea, 1996; Milner, 1996). The results, however, were quite contradictory depending on the type of tumor examined and the garlic preparation used (Dorant et al., 1993). Because crude garlic extracts contain numerous pharmacologically active substances. including organosulfur compounds with varying stability and biological activity (Milner, 1996), more detailed studies of the effects of chemically defined garlic compounds on tumor genesis are needed. Some garlic constituents have been shown to alter the activation of several carcinogens and to cause growth inhibition and/or death of tumor cells (Takeyama et al., 1993; Hatono et al., 1996; Koch and Lawson, 1996; Singh et al., 1996). However, the molecular mechanisms underlying the tumor cytotoxicity of garlic substances are poorly defined. The cytotoxicity of most classical antitumor drugs is thought to be mediated by their ability to induce apoptosis (Sen and D’Incalci, 1992). Apoptosis is a form of physiological cell death, characterized by chromatin condensation, cytoplasmatic blebbing, and DNA fragmentation (Wyllie et al., 1980). Apoptosis can be initiated by alterations in signaling pathways (Jones et al., 1989; Hsu et al., 1996) or by oxidative stress mediated by the generation of ROS (Buttke and Sandstrom, 1994). It has been shown that oxidative stress activates the transcription factor NF-kB and that activation of NF-kB is involved in inducing the apoptotic cell death in some cells (Grimm et al., 1996). The aim of the present study was first to examine whether ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide] (Fig. 1), a major compound of crushed garlic (Agarwal, 1996), was able to induce apoptosis in the human promyelocytic leukemia cell line HL-60. This cell line provides a valid model system for testing antileukemic or general antitumoral compounds (Suh et al., 1995). Second, we investigated the mechanisms underlying apoptosis induction. We examined the generation of ROS by ajoene and the effect of ajoene on the This work was supported by Deutsche Forschungsgemeinschaft Grant Vo 376/6–2. ABBREVIATIONS: ROS, reactive oxygen species; DHR, dihydrorhodamine; EGTA, ethylene glycol bis(b-aminoethyl ether)-N, N, N9, N9-tetraacetic acid; EMSA, electrophoretic mobility shift assay; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HL, human leukemic cells; NF, nuclear factor; NAC, N-acetylcysteine; PI, propidium iodide; PBS, phosphate-buffered saline; PMBC, peripheral mononuclear blood cells. 0026-895X/98/030402-06$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 53:402–407 (1998). 402 at A PE T Jornals on Jne 5, 2017 m oharm .aspeurnals.org D ow nladed from activation of the transcription factor NF-kB. Apoptosis was assessed by morphological analysis of cells as well as characterization and quantification of DNA degradation by flow cytometry and gel electrophoresis. The ROS formation of HL-60 cells exposed to ajoene was monitored by oxidation of the dye DHR-123. Activation of NF-kB was examined by its DNA-binding activity using EMSA. Materials and Methods Cell cultures. The human promyelocytic leukemia HL-60 cell line and in addition the human colon adenocarcinoma DLD-1 cell line, human squamous carcinoma cells (A431), and human neuroblastoma cells (SH-SY5Y) were cultured (37° and 5% CO2) in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin/100 mg/ml streptomycin, and L-glutamine (2 mM) (all from GIBCO/BRL, Eggenstein, Germany). For experiments 1–5 3 10 cells/well were seeded (1-ml, 24-well plates; Peske, Aindling-Pichl, Germany) and grown overnight. Human PMBC were recovered from heparin-anticoagulated blood of healthy volunteers by centrifugation with Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) following the manufacturer’s instructions and cultivated as described for the HL-60 cells. In some experiments, PMBC were stimulated with phytohemagglutinin (1 mg/ml) for either 24 or 48 hr. In addition, PMBC of a patient (male, 54 years old) with a chronic myelogenous leukemia undergoing a myeloid blast crisis were purified and cultured as described above. Blood cell differentiation analysis of the patient revealed 70% myeloblasto-