1 National Research Laboratories of Natural and Biomimetic Drugs, Peking University, Beijing ROC, and
2 IPBS, CNRS-UMR 5089, 205 Route de Narbonne, 31077 Toulouse Cedex 4, France
Abstract
Ajoene, a garlic stable oil-soluble sulfur rich compound was generally isolated as a mixture of two isomers [(E, Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide]. It has been described essentially as a potent inhibitor of platelet aggregation in vitro and in vivo. The antiproliferative effects of ajoene and experiments using a single isomer had received little attention. The present study aims at defining the antitumor activities of cis-Z-ajoene in vitro and in vivo. Antiproliferative activity of Z-ajoene was demonstrated against a panel of human tumor cell lines with IC50 values varying from 5.2 mM to 26.1 mM and at a lower extent in normal marsupial kidney cells (PtK2). Meanwhile, Z-ajoene arrested HL60 cells in G2/M phase of cell cycle in a dose and time-dependent way. In PtK2 cells, exposure to 20 µM Z-ajoene for 6 h induced a complete disassembly of the microtubule network, that was associated with an increased number of cells blocked in early mitotic stages. An IC50 for microtubule disassembly of 1 µM was determined by a fully automated microplate-based multi-detection reader. In vitro, a reversible inhibition of the microtubule protein assembly was observed with an IC50 of 25 µM Z-ajoene. In vivo, Z-ajoene inhibited tumor growth by 38% and 42% in mice grafted with sarcoma 180 and hepatocarcinoma 22, respectively. For the first time, Z-ajoene was shown to be a potent inhibitor of tumor cell growth both in vitro and in vivo. The microtubule cytoskeleton appeared to be one of the Z-ajoene targets, but the mechanisms by which Z-ajoene interacted with microtubule appeared different from those of other microtubule poisons such as those of the Vinca alkaloids family. The ability of Z-ajoene to preferentially suppress the growth of neoplastic cells could provide a new approach in tumor therapy.
Abbreviations: DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; DMSO, dimethylsulfoxide; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid; FBS, fetal bovine serum; GTP, guanosine triphosphate; IC50, 50% inhibitory concentration; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline; PEG 6000, polyethylene glycol 6000; PEM, (PIPES, EGTA, MgCl2); PI, propidium iodide; PIPES, piperazine-N,N'-bis (2-ethanesulfonic acid)
Introduction
During the past ten years more attention has been given to uncovering the benefits of garlic sulfur compounds in relation to cancer (13). Some garlic constituents have been shown to alter activation of carcinogens (47) and to cause growth inhibition (812) of tumor cells.
It is believed that the products resulting from the breakdown of alliin are responsible for the antiproliferative effects of garlic. Ajoene is considered as a major natural compound derived from garlic through the conversion of alliin (S-allylcystein-S-oxide) into allicin (allyl-2-propenethiosulfinate) by an alliinase-induced cleavage. This enzyme, stored in vacuole of mesophyll cells, is liberated during tissue injuries. Allicin is a labile compound, easily transformed to a number of stable lipid soluble allyl sulfides such as ajoene (4,5, 9-trithiadodeca-1,6,11-triene-9-oxide) (13). Pure ajoene is essential to characterize its bioactivities. Purification of ajoene is rather difficult since the compound is generally obtained in a mixture of cis(Z) and trans(E)-ajoene with a ratio around 2.2 (14). This natural compound was recognized for its ability to improve blood fluidity by suppressing platelet aggregation both in vitro (15,16) and in vivo (17,18). These properties could explain how ajoene might modify cell adhesion and consequently display effects on cell growth inhibition and antitumor activities. Suppression of cell growth has been described with several cancer cell lines (14,19,20). Apoptotic activation has been found to be induced in human promyeloleukemic cells (HL60), accompanied by generation of reactive oxygen species and activation of nuclear factor B (21).
Microtubules are essential for cell transport and cell division in all eukaryotes (for review see ref. 22). Accumulated evidences have shown that microtubule dynamics may play a crucial role in the passage through the metaphase/anaphase checkpoint (23). Mitotic block by drugs at concentrations that suppress microtubule dynamics or alter microtubule mass induces apoptosis (for review see ref. 24). It was of interest to investigate whether antiproliferative activity of ajoene involved interference with the microtubule cytoskeleton.
Until now, no experiment has been undertaken to compare the bioactivities of two ajoene isomers. However the study of Yoshida found that the Z-isomer exhibits an antimicrobial activity at least twofold higher than that of the E-isomer (25). Hence, it is important to demonstrate whether Z-ajoene possesses other bioactivities.
Depending on conditions of extraction, Z-ajoene could be obtained as the main component (13). After several further steps of chromatography, we have obtained cis-Z-ajoene, with purity >98% (26). This purified compound has been found to inhibit cell growth and to induce apoptosis (20). However the molecular basis of these two activities which are likely involved in the antitumor activity of Z-ajoene remained elusive. To answer this question, the present work first focused on the effect of Z-ajoene on cell proliferation and cell cycle distribution. Then we investigated its action on tubulin function both in living cells and in the assembly of microtubule proteins. Finally, we checked the in vivo antitumor activity of Z-ajoene, using two transplantable tumor models in mice.
Materials and methods
Chemicals and antibodies
Z-ajoene [(Z) 4,5,9-trithiadodeca-1,6,11-triene-9 oxide] was isolated from a cyclohexane extract of garlic (Allium sativum L.). The compound was purified by silica gel column chromatography, thin-layer chromatography and high performance liquid chromatography, according to a method previously described, and was more >98% pure (26). The structure of Z-ajoene was determined by spectroscopic analysis. It was dissolved in 10% dimethylsulfoxide and further diluted in PBS before use. The final concentration of DMSO in the solution was <0.1%. Unless otherwise stated, all compounds were obtained from Sigma Chemical Co., St Louis, MO.
Cell culture
Human cell lines, breast cancer (MCF-7), nasopharyngeal carcinoma (KB), hepatocellular carcinoma (Bel 7402), gastric carcinoma (BGC 823), colon carcinoma (HCT), Hela cells (Hela), promyeloleukemic cells (HL60), were grown at 37°C and in the presence of 5% CO2, in RPMI-1640 medium supplemented by 10% heat-inactivated fetal bovine serum (FBS), antibiotics (penicillin 100 U/ml, streptomycin 100 µg/ml) and 2 mM L-glutamine (all from GIBCO/BRL, Germany). A marsupial kidney cell line (PtK2), was grown in Dulbecco medium modified by Iscove (Gibco). All the cell lines came from the American Type Culture Collection.
Assay of cytotoxicity
Effect of Z-ajoene on cell proliferation was determined using a standard MTT-based colorimetric assay (27). Briefly, the different cell lines were seeded onto 96-well microplates at 5 x 104 and 104 cells/well for adherent and suspension cells respectively. After 1824 h, exponentially growing cells were exposed to increasing concentrations of Z-ajoene (180 µM) for 48 h, before the addition of MTT. Plates were read at 570 nm using a Bio-Rad 3500 microplate reader. The final steps were followed by computerized data acquisition and processing. IC50 values, i.e. concentrations of compound required to reduce the absorbency to 50% versus control cells, were determined from replicates of 6 wells from at least two independent experiments using linear interpolation between data points.
Flow cytometry
HL60 cells were plated at a density of 5 x 105 cells/ml. The following day, Z-ajoene or solvent was added. At the time indicated, cells were harvested after a centrifugation (300 g for 10 min), washed in PBS and fixed overnight in 80% ethanol at 20°C. The samples were centrifuged, washed with PBS and resuspended in 10 µg/ml of propidium iodide (PI) and 0.1% RNase A in PBS at 4°C overnight. Distribution of the cell cycle phases was determined by analytical flow cytometry using a Coulter Epics XL (Coultronics, France SA) with an excitation/emission of 488/525 nm. All experiments were performed in triplicate and gave similar results.
Staining of microtubule network by indirect immunofluorescence
For microscopic observation, adherent PtK2 cells were plated onto coverslips in six-well plates at 6 x 104 cells per well, and allowed to attach overnight. Cells were then treated with 120 µM Z-ajoene for 6 h. Indirect immunofluorescence of the microtubule network was performed as previously described (28). Briefly, the cells were permeabilized at room temperature, with 0.05% Triton X-100 in 2% PEG/PEM buffer (100 mM PIPES pH 6.9, 1 mM EGTA, 2 mM MgCl2) for 1 min, then fixed in PEM buffer containing 3% formaldehyde and 1% DMSO for 45 min. After washing, cells were incubated at 37°C for 90 min with a mouse monoclonal antibody against -tubulin (1:2000) in PBS supplemented with 25% fetal bovine serum. Binding of the primary antibody was visualized subsequently with the secondary antibody (goat against mouse IgG, labeled with FITC purchased from Nordic, Tiburg, Netherlands) in PBS supplemented with 40% fetal bovine serum. Nuclei and chromosomes were stained with DAPI (0.2 µg/ml). Preparations were then observed using a Zeiss Axioskop epifluorescent microscope.
The intensity of the fluorescent microtubule network was quantified by the FLUOstar Galaxy (BMG Labtechnologies GmbH, Offenburg, Germany), a fully automated microplate-based multi-detection reader. PtK2 cells (104/well) were plated in 96-well black microplate with a clear bottom (3603, Costar MA). They were treated by increasing amounts of Z-ajoene (0.1 to 20 µM) for 6 h; then the cellular microtubule network was immunostained as above. Microtubular fluorescence intensity was measured according to the excitation/emission at 485/520 nm and given as the mean of eight replicates. Fluorescence background was given by the cells treated only with FITC-labeled second antibody.
Microtubule assembly or disassembly
Pig brain microtubule proteins were obtained by two cycles of assembly/disassembly (29). This preparation was resuspended in a PIPES buffer containing 1 mM GTP and stored in liquid nitrogen. Prior to use, aliquots were thawed and centrifuged for 5 min at 21 000 g. SDSPAGE showed that the microtubule protein preparations contained 65% tubulin and 35% microtubule associated proteins (MAPs). In vitro microtubule assembly or disassembly was followed as described by Gaskin et al. (30) by quantitating the variations of absorption at 400 nm using a spectrophotometer (Beckman) equipped with thermostatically controlled cuvettes. Turbidity experiments were conducted with 2.5 mg/ml microtubular proteins in assembly buffer (100 mM PIPES pH 6.6, 0.5 mM MgCl2, 0.1 mM EDTA, 1mM EGTA and 2 mM GTP). The microtubular protein preparations initially at 0°C with or without the tested compounds, were warmed to 37°C in order to initiate microtubule assembly. After 10 min at 37°C, temperature was decreased to 0°C to induce disassembly.
Effect of Z-ajoene on tumor growth
Kunming mice (Swiss mice origin), 46 weeks old, initially weighing 1820 g, were obtained from Beijing Health Science Center (Peking University). Two transplantable tumors, hepatocarcinoma 22 (H22) and sarcoma 180 (S180), were implanted (31). For each model, tumors were induced in 30 mice by subcutaneous injection of 0.2 ml PBS containing 5 x 106 tumor cells, on the right flank at day one. The animals were then divided into three groups of 10, two experimental groups and one control group. Beginning at day two, the mice in two experimental groups injected with hepatocarcinoma 22 cells received 2 or 4 mg/kg of Z-ajoene every day intraperitoneously. Similarly, the two experimental groups injected with sarcoma 180 cells, received 4 and 8 mg/kg of Z-ajoene in the same conditions. These doses of Z-ajoene were previously checked to be non-toxic for the animals. Solvent (0.1% DMSO in PBS) was injected in the same conditions in the control group. Twelve days later, the mice were killed. Harvested tumors were weighed and the inhibition rate was calculated in comparison with the control group.
Results
Cytotoxic activity of Z-ajoene
In a first series of experiments, two tumorigenic human cell lines, promyeloleukemic (HL60) and nasopharyngeal carcinoma (KB), and one normal marsupial kidney cell line (PtK2) were chosen to determine the cytotoxic activity of Z-ajoene. A typical dose-dependent inhibition of cell growth is shown in Figure 1 documenting the surviving cell fraction plotted versus Z-ajoene concentration. After a 48 h incubation, increasing concentrations of Z-ajoene (120 µM) led to a gradual decrease of the fraction of viable cells. HL60 cells were more sensitive to Z-ajoene than KB cells. On the contrary, Ptk2 cells were more resistant to the cytotoxic effect of the garlic compound. At a concentration of 20 µM, up to 75% of surviving cells were observed with PtK2, whereas it was 10% and 30% for HL60 and KB cells, respectively. Because of these variations, the cytotoxic activity of Z-ajoene has been determined against a panel of human tumor cells of different origin. Results in Table I
show IC50 values varying from 5.2 µM (HL60) to 26.1 µM (MCF-7) with respect to the cell lines.
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Several pieces of evidence suggested that allyl sulfides found in processed garlic, possess anticancer properties as shown by their ability to suppress tumor proliferation in vivo and in vitro. The ability of allyl sulfides to inhibit the growth of transplanted solid tumors in mice, has been described after diallyl disulfide and allicin administration (6,31,32). In vitro, increasing evidence showed that antiproliferative compounds block the cell cycle progression by stimulating a series of checkpoints that control cell cycle transition (for review see ref. 33). Antiproliferative activity of diallyl sulfides has been related to a block in the progression from G2 to M phase (34,35). Concentration and duration of the exposure to allyl sulfides increased the antiproliferative effects. This antineoplastic effect was greater for lipid soluble than for water-soluble allyl sulfides.
The number of sulfur atoms has been shown to be one main factor in variable efficiency of allyl sulfides (for review see ref. 8). Ajoene, a compound with three sulfur atoms, belongs to the group of biologically active oil-soluble allyl sulfides. Purification of ajoene generally results in a mixture of two isomers in an E to Z ratio depending on the extraction methods (2). Numerous experiments demonstrated that the mixture of the two isomers was a potent inhibitor of platelet aggregation in vitro (15,16) and in vivo (17,18). Removal of the sulfide group at position 4 of ajoene induced a twofold decrease in the ability to suppress collagen-induced aggregation, demonstrating the importance of the disulfide group in mediating suppression of platelet aggregation by ajoene (13). Mechanisms governing inhibition of platelet aggregation by ajoene could imply cell membrane modifications such as decrease of membrane fluidity (15,36) at both cholesterol and arachidonic acid levels (37), change in phospholipid composition (15) and inhibition of fibrinogen binding on the glycoprotein (GP) IIbIIIa, an integrin complex expressed by platelets (38) and by many tumors (39). All these effects could lead to a decrease in cell adhesion. Hence ajoene might elicit its antitumorigenic effects through these mechanisms including an action on the integrin receptors.
The antiproliferative effects of ajoene have received little attention. The BJA-B tumorigenic cells, originally derived from a Burkitt lymphoma, were 25 times more sensitive to ajoene than two normal cell lines. Ajoene was twice as active than allicin (19). Fast uptake of ajoene was accompanied by an immediate variation of glutathione metabolism associated with a decrease of cell viability (14). Promyeloleukemic cells HL60, exposed to ajoene also showed an increase in G2/M population (21), suggesting an effect either in G2 phase or in mitosis.
Until now most studies have been performed with the mixture of the two isomers. To characterize the effect of a single form of ajoene, we have purified the Z-isomer (26). In vitro, Z-ajoene clearly showed a cell growth inhibition on several human cancer cell lines at non-toxic concentrations (lower than 50 µM). With respect to the cell lines studied, IC50 values varied from 5.2 in HL60 to 26.1 µM in MCF-7 (Table I). In contrast, Z-ajoene has less cytotoxic effect on a normal marsupial kidney cell (PtK2). Z-ajoene was more efficient in leukemia cells and exhibited a minimal effect on non-neoplastic cells (Figure 1
). Discrepancy in sensitivity between tumor cells of different origin has already been described with another antitumor compound (40). Although cell lines used in our experiments and in the experiments described by Scharfenberg et al. (19) were different, it seemed that the purified Z-ajoene was at least twofold more active than the mixture of E- and Z-ajoene. The oil-soluble sulfur compounds obtained at different steps of the purification procedure, likely produced increasing cytotoxic activities in the following order: allicin < E, Z-ajoene < Z-ajoene.
Z-ajoene was found to block cell cycle in HL60 cells at G2/M phase (Figure 2). To understand the mitotic arrest induced by Z-ajoene, we studied a possible interference with microtubules. The ability of Z-ajoene to alter the microtubule cytoskeleton was investigated by indirect immunofluorescence using PtK2 cells. Microtubule disassembly was observed in cells incubated with 10 µM Z-ajoene for 6 h while entire disassembly was observed in the presence of 20 µM Z-ajoene. Since microscopic observation was not quantitative, analysis of the overall fluorescence raised by the microtubules was performed using a microplate automated reader. This approach which allowed measuring an IC50 value of 1 µM, was interesting for the comparative analysis of several compounds, and appeared useful for the screening of microtubule disassembling compounds.
In agreement with previous results, in vitro assays of microtubule assembly/disassembly showed that 25 µM Z-ajoene inhibited tubulin polymerization by 50%, suggesting that tubulin or microtubules were likely a target of this compound. However, since our tubulin protein preparation contained at least 35% of microtubule associated proteins, it was not excluded that other proteins could also be involved. The discrepancy between the IC50 obtained in the drug-induced inhibition of microtubule assembly and the disappearance of the microtubule cytoskeleton in PtK2 cells was not unusual. This has been already described for Vinca alkaloids which were more potent on the microtubule cytoskeleton than on the in vitro assembly (40). The effects of Z-ajoene seemed to be different from those of Vinca alkaloids which induce spirals and paracrystal insensitive to a temperature decrease (41).
Since the present study is the first one giving evidence of Z-ajoene activity on mammalian tumor cell lines, we assumed that this isomer could exhibit antitumor properties. Using sarcoma 180 and hepatocarcinoma 22, two established transplantable tumor models in mice, we showed that the daily injection of Z-ajoene at non-toxic doses reduced the tumor weight by 32% and 42% at day 12, respectively. These preliminary results demonstrated an inhibition of tumor growth by Z-ajoene, that should be extended to other models of induced-tumors.
The ability of Z-ajoene to preferentially suppress the growth of neoplastic over non-neoplastic cells provides interesting possibilities for the development of new anticancer strategies in humans. Elucidation of the mechanism involved in ajoene cytotoxicity in vitro and in tumor growth inhibition in animals is an important step for a better understanding of the antitumor activity of garlic compounds.
Notes
3 To whom correspondence should be addressed mail: leung{at}ipbs.fr
Acknowledgments
We thank Dr C.Davrinche for his critical reading of the manuscript. We are grateful to Ms F.Viala for preparing photographs and Mr G.Cassar for his technical assistance in flow cytometry analysis.
This work was supported by institutional grants from CNRS, PRA B-9804 (JLT) from AFCRST (Association Franco-Chinoise pour la Recherche Scientifique et la Technologie financed by MAE and MEN/MR) and l'Association pour la Recherche sur le Cancer (MW).
Min Li was supported by CNRS (Poste Rouge) and a thesis fellowship from Université Paul Sabatier, Toulouse, France.
References