103
Cancer Letters, 53 (1990) 103-108 Elsevier Scientific Publishers
Ireland Ltd.
The cytotoxic effect of ajoene, a natural product investigated with different cell lines K. Scharfenberg, GBF (Gesellschaftffir
from garlic,
R. Wagner and K.G. Wagner Biotechnologische
Forschung),
Mascheroder
Weg 1, D-3300 Braunschweig
(F.R.G.)
(Received 22 May 1990) (Accepted 18 June 1990)
Summary The sulfur-containing compound ajoene (4,5,9-trithiadodeca-1,6,11-triene-g-oxide) which arises from alliin, a cysteine derivative stored in garlic bulbs, was produced synthetically by decomposition of allicin. Its cytotoxic effect was tested using human primary fibroblasts (FS4), a permanent, non-tumorgenic cell line derived from baby hamster kidney cells (BHK21) and a tumorgenic lymphoid cell line derived from a Burkitt lymphoma (BJA-B). The cytotoxic action was in the range Z-50 pg/ml depending on the cell density. ED,, values, estimated on the basis of fmol ajoene/ cell, revealed slightly higher doses for the primary cell (FS4) than the permanent line (BHK), whereas the tumorgenic BJA-B cells were most sensitive. Keywords: garlic
ajoene;
cytotoxicity;
cell lines;
Introduction Intact garlic bulbs contain alliin (S-allylcysteine S-oxide) in concentrations up to 1% fresh weight. Injury of the tissue liberates allinase, probably stored in the vacuole, which cleaves alliin to produce mainly allicin (allyl-ZCorrespondence to: K.G. Wagner. 0304.3835/90/$03.50 Published
and Printed
0
1990 Elsevier Scientific Publishers
in Ireland
propenethiosulfinate) . Allicin is not very stable and is further transformed into a variety of sulfur-containing substances. Depending on the during transformation, aloene conditions (4,5,9-trithiadodeca-1,6,11-triene-9-oxid) is obtained as a main component [5,7,17]. Hence in the various garlic preparations consumed and used by the human population different amounts of allicin and ajoene are present. Antitumor and cytotoxic action of garlic extracts [3,13,16,20,22,23,27] and allicin [12] have been probed in several studies; furthermore the bacteriocidic and fungicidic action of allicin [9,10,14] and even its enzymeinactivating property [28] has been tested. Although the antithrombotic properties of ajoene have been thoroughly studied [ 1,2,6,7] there are only few reports on its fungicidic action [29], inhibition of lipoxygenase [4,26], prostaglandin synthetase [26] and mouse skin tumor promotion [4]. In the present work the cytotoxic properties of ajoene have been investigated using 3 different mammalian cell lines along with allicin for comparison. Materials and methods Preparation of ajoene and allicin Allicin was prepared from diallyl disulfide as described [7,18] using perbenzoic acid for the oxidation step. Ajoene was obtained from Ireland Ltd
104
decomposition of allicin [7] and the crude product was purified by column chromatography and analysed by reversed-phase high performance liquid chromatography (H. Baydoun and K.G. Wagner, unpublished results). According to HPLC chromatography and proton NMR measurements the purity of ajoene and allicin was 90%. Allicin and ajoene were dissolved at concentrations of 20 mg/ml in 87% glycerol and stored at - 20°C. Cells and culture conditions
A human foreskin-derived FS4 cell line obtained from Bioferon, Laupheim (F.R. G .) was used during the 16th-22nd subculture. The BHK21 cell line (C-13, ATCC CCLlO) had been transfected by an interleucin 2 gene containing plasmid in the Genetics Dept. of the GBF (BHK 21 pSVIL2) [11,25]. Both cell types were anchorage dependent fibroblasts, whereas the BJA-B cells, originally derived from a Burkitt lymphoma [19] and obtained from Dr. P. Gruss DKFZ (Heidelberg), were grown in suspension. After establishing a cell bank, which was conserved frozen (stored in N,-vapour), thawed cells were used for 3040 passages only. All three cell lines were cultivated in DMEM (from Gibco, Santa Clara, CA), supplemented with 10% inactivated fetal calf serum, at 37 OC and 96% relative humidity with an atmosphere of 93% air and 7% CO,. When the cells (FS4 and BHK) reached confluency, they were harvested by trypsinization and plated for the following passages or for drug treatment. BJA-B cells were treated as a suspension culture. Drug treatment In a 96-well flat bottom microtiter plate every well was inoculated with 103-104 cells in 100 ~1 medium. After incubation for one day, the medium (100 ~1) was adjusted to 20% serum and thereafter 100 ~1 medium (without serum) containing ajoene or allicin were added. Two days after inoculation the number of cells were counted in those wells which had obtained lethal doses of ajoene or allicin (cell number at the time of drug addi-
tion). On day 3 the metabolic activity of the wells was determined by the MTT assay (see below) and related to untreated cells. Assay methods Cells were counted using a hemocytometer (Neubauer, improved version). The percentage of dead cells was determined by the Trypan blue (Boehringer Mannheim) exclusion method. The calorimetric MTT (tetrazolium) assay [15,21] was applied in a modified form (D. Monner, unpublished) with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Chem. Co.) dissolved (5 mg/ml) in buffered saline solution, filtered and stored frozen. After removal of 100 ~1 medium, MTT stock solution was added (10 ~1 per 100 ~1 medium), the plates were incubated at 37OC for 3-5 h, acidic isopropanol (5% formic acid) was added (150 ccl), mixed thoroughly to dissolve the dye crystals and the absorbance was recorded with an immuno-reader (NJ20000 from Nippon Intermed K.K.) at 572 nm at a reference wavelength of 650 nm. Determination of total cell protein was performed with resuspended cells (after trypsinization in the case of FS4 and BHK cells) which were centrifuged for 5 min (Digifuge from Heraeus-Christ) at 2000 rev./min. The cells (approx. 1.5 x 106/100 ~1) were suspended in ice-cold buffer (20 mM EDTA, 0.5% Triton X-100) and homogenized for 30 s in a cooled Douncehomogenizer (Braun, Melsungen) . Protein was determined according to Bradford [8] with the reagent from Biorad (Munich) using bovine serum albumin (Serva, Heidelberg) as reference protein. Ajoene was determined by HPLC using an ODS-Sperisorb 5 pm column (25 x 0.4 cm) (Melz, Berlin), a precolumn (3 cm, 15-25 pm C-18 Lichroprep from Merck, Darmstadt), LDC constametric pump (Milton Roy, Hasselroth), Uvikon 730 LC photometer (Kontron Analytik, Hannover) and C-R 1B integrator (Gynkrotek, Munich). lsocratic elution was performed with 40% acetonitrile in water and detection at 230 nm, Rr value for ajoene was about 6 min at a flow rate of 2 ml/ min.
105
Results and discussion Ajoene, which is formed via allicin from the garlic stored compound alliin by the action of allinase [5,7,17], can also be obtained from allicin synthesized by oxidation of diallyl disulfide [7,18]. The spontanous transformation of allicin into ajoene which occurs after injuring garlic tissue can be performed in the test tube [7]. The main difficulty arises from the purification of ajoene from substances obtained simultaneously with ajoene. We have produced ajoene, as a mixture of E- and Z-isomers [7], by the chemical route developing a chromatographic purification procedure (K.G. Wagner, H. Baydoun and K. Plank-Schumacher, unpublished results) to give ajoene in gram quantities and at high purity as shown by HPLC and proton-NMR spectroscopy (to be published). The antithrombotic activity of ajoene was originally deduced from its presumable interference with the receptor-mediated signal pathways in the platelets [17], although this hypothesis has not as yet been proved. As regulation of cell proliferation is also correlated with signal transducing properties [24], we assumed that ajoene may also have cytostatic or cytotoxic activities and studied this property with the cell lines available. Human fibroblasts (FS4) were chosen as representing a primary cell line, a BHK line as an example of a permanent and non-tumorgenic cell line and a tumorgenic lymphoid cell line (BJA-B) derived from a Burkitt lymphoma. Figure 1 shows a typical experiment with the surviving fraction plotted versus the ajoene concentration. The BJA-B cell line was rather sensitive revealing an EC,, value of about 2 pg/ml, whereas the other two cell lines were slightly less sensitive. Although the mechanism of ajoene action in cytotoxicity is not known, one could suggest from its chemical structure that reactions with SH-functional groups of cellular molecules of low or high molecular weight might be involved. Evidence for such a hypothesis were elaborated for allicin which has similar functional groups to those of ajoene
0
5
Ajoene
10
15
Concentration
20
pgml-’
Fig. 1.
Effect of ajoene on 3 different cell lines; (0) human foreskin-derived cell line (FS4). (A) baby hamster kidney-derived cell line (BHK) and (0) Burkitt lymphoma-derived cell line (BJA-B) Surviving fraction was determined by the MTT assay (see Materials and methods); cell densities at the time of ajoene application: 5.5 x lo4 (FS4), 1.08 x lo5 (BHK), 9.4 x lo4 (BJA-B) cell/ml. Data are presented as mean + S.D. of one typical experiment with 2 (BJA-B) or 3 (BHK and FS4) parallel determinations (available wells in the microtiter plate). The standard deviations, marked on one side of the points only, are given except where they are within the limits of the symbol; they were all below 6%.
[9,10,28]. Assuming that ajoene reacts with cellular SH-functions and is consumed by this action, the cytotoxic response should not be strictly related to the concentration but to the amount of ajoene consumed per cell. Figure 2 shows the dependence on the cell density of the BJA-B cells. The above expectation is obviously fulfilled, higher cell densities were correlated with higher EC,, values. Table 1 summarizes the results obtained with the different cell lines. With BHK cells the cell densities applied were of higher variance than with the other two cell lines, hence the EC,, values related to the ajoene concentration had a large standard deviation. However, when calculated as the amount of ajoene per cell, the deviation was of the same magnitude as with the other cell lines. This again supports the above conclusion that the cytotoxic activity depends on the amount of ajoene related to
106
1
0.1 Ajoene
10
Concentration
ajoene/mg protein (calculated for the ED values) show that the tumorgenic BJA-B cell: were about twice as sensitive as the FS4 and BHK cells. Allicin which is structurally related to ajoene contains a thiosulfinate groups was also tested; on a mol/cell and mol/mg protein basis ajoene was twice as active as allicin although the mechanism may be similar (Table 2). Also for practical reasons ajoene is a better candidate as it is more stable than allicin which gradually decomposes forming among other substances also ajoene [7]. In the standard assay cell viability was determined after 48 h of ajoene treatment. To determine the influence of the treatment time, BHK cells were exposed to ajoene for different times. It was found that the cytotoxic effect of ajoene required a time span of about 3 h; with tests performed for 3 h treatment the same curvature in the dose-response curves were obtained as with those performed for 46 h, whereas with treatments for shorter times the cytotoxic effect of ajoene was reduced considerably. Uptake of ajoene by the cells was determined with BJA-B cells in serum-free medium as described in the legend of Fig. 3. It is obvious that uptake was accomplished after about 3 h; this is exactly the same time span determined for the establishment of the cytotoxic action; hence action of ajoene must occur parallel with or immediately after its uptake. Figure 3 further shows that an intact cellular metabolism is required for ajoene uptake, as
100
pgml“
Fig. 2. Influence of ajoene on BJA-B cells studied at various cell densities. Data are presented as mean + S.D. of one typical experiment; for further details see legend of Fig. 1. Cell densities in 10s cells/ml: (0) 0.25, (0) 0.5, (A) 1.0, (0) 2.0, (m) 4.0 and (A) 8.0. For clearness the standard deviations were given for only one curve; in total (with one exception) they were all below 10%.
the number of cells within a well. Based on the amount of ajoene per cell, the primary FS4 cells were significantly less sensitive towards ajoene than the permanent cells (BHK), while the tumorgenic cells (BJA-B) were the most sensitive ones. As the cells applied had different sizes, the effective dose was related to the protein content of the cells; the values in pmol
Table 1. Cell line
ES4 BHK BJA-B
A comparison
of the cytotoxic activity of ajoene against the 3 cell lines” Cell density cells/ml lo+
7.9 (2.1) 16.0 (10.2) 14.6 (3.2)
ECmb
ED,,
VM
36 (13) 30 (23) 12 (1.8)
fmol/cell
pmol/mg
450 (64) 190 (49) 85 (23)
0.9 1.0 0.4
protein’
aThe data are mean values of 3 (FS4) or 5 (BHK and BJA-B) separate experiments f S.D. in parenthesis; in each experiment 2-5 parallel determinations were performed. “EC, and ED,, are the concentration and amount of ajoene, respectively, required to reduce viability of the cells to 50%. +mol ajoene per mg cell protein calculated for the ED,, values.
107
Table 2.
Cytotoxic activity of ajoene and allicin against the 3 cell lines”.
Cell line
ED, of allicin
ED,, of ajoene
FS4 BHK BJA-B
fmol/cell
pg/mg
fmol/cell
pgcg/mgb
450 (64) 190 (49) 85 (23)
0.9 1.0 0.4
720 (49) 430 (135) 185 (28)
1.5 2.2 0.8
aThe values for ajoene are derived from Table 1; those for allicin are mean values from 2 (FS4) or 4 (BHK and BJA-B) separate experiments + S.D. in parenthesis; in each experiment 2-5 parallel determinations were performed. “c(gajoene or aIlicin per mg cell protein calculated for the ED, values (cf. Table 1).
0 5
Fig. 3.
2
6
1 -7
(A) Ajoene uptake kinetics with BJA-B cells. Cultivated BJA-B cells were centrifuged and resuspended in DMEM-medium at 37’C (without serum); cell density was 3-4 x 105/ml. At time zero ajoene (25 or 30 pg/ml) was added and after different times cells were centrifuged and ajoene was determined in the supernatant by HPLC. Data are mean values f S.D. from 4 experiments; ajoene concentration was determined twice in each supernatant. (B) Dependence of ajoene uptake on cell viability. Cells were suspended in serum-free DMEMmedium and ajoene was added (30 pg/ml); 4 x lo5 BJA-B cells/ml were used. (0, 0). Two parallel experiments with vital BJA-B cells; (A) BJA-B cells treated for 20 min at 56’ immediately before the experiment; (0) control DMEM-medium without cells.
heat inactivated cells absorbed only a small quantity of ajoene. It is further interesting to note that ajoene absorbed by cells could not be recovered upon ethyl acetate extraction and HPLC analysis. Work is in progress to elucidate the fate of ajoene after absorption by the cells. Although investigated with only three cell lines, the slight but significantly higher sensitivity against ajoene of the tumorgenic line relative to the two non-tumorgenic lines is interesting. This is in accord with the high compatibility of animal skin with ajoene (K.G. Wagner, unpublished results); even in the rabbit eye irritation test, ajoene proved to be a non-irritant. Investigations on the elucidation of the mechanism of the action of ajoene cytotoxitiy are in progress as well as studies to probe ajoene action against tumor formation in animals. Finally, the question arises whether ajoene and allicin ingested with natural and artificial garlic preparations will have similar effects on cell proliferation in the human body as shown with the presented in vitro experiments. Acknowledgements We are grateful to Dr. V. Wray for linguistic advice, Mrs. H. Starke for typing the manuscript and Mrs. C. Lippelt for preparing the
108
graphs. This work has been supported by the Fond der Chemischen Industrie. We very gratefully acknowledge the kind help of Dr. M. K. Jain in the establishment of this subject in our laboratory.
immunity 14
2
3
Apitz-Castro, R., Cabrera, S., Cruz, M.R., Ledezma, E. and Jain, M.K. (1983) Effects of garlic extract and of three pure components isolated from it in human platelet aggregation, arachidonate metabolism, release reaction and platelet ultrastructure. Thrombosis Res., 32, 155- 169. Apitz-Castro, R., Ledezma, E., Escalante, J. and Jain, M.K. (1986) The molecular basis of the antiplatelet action of ajoene: Direct interaction with the fibrinogen receptor. Biochem. Biophys. Res. Commun., 141.145-150. Belman, S. (1983) Onion and garlic oils inhibit tumor pro-
6
motion. Carcinogenesis, 4, 1063-1065. Belman, S., Solomon, J., Segal, A., Block, E. and Barany, G. (1989) Inhibition of soybean lopoxygenase and mouse skin tumor promotion by onion and garlic components. J. Biochem. Toxicol., 4, 151-160. Block, E. (1985) The chemistry of garlic and onions. Sci. Am. 252,94-99. Block, E., Ahmad, S., Jain, M.K., Crecely, R.W., Apitz-
7
Castro, R. and Crux, M.R. (1984) (E,Z)-Ajoene: A potent antithrombotic agent from garlic. J. Am. Chem. Sot., 106,8295-8296. Block, E., Ahmad, S., Catalfamo. J.L., Jain, M.K. and
a
Apitz-Castro, R. (1986) Antithrombotic organosulfur compounds from garlic: Structural, mechanistic and synthetic studies. J. Am. Chem. Sot., 108, 7045-7055. Bradford, M.M. (1976) A rapid and sensitive method for
9
quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem., 72, 248-254. Cavallito, C.J. and Bailey, J.H. (1944) Allicin, the antibac-
4
5
10
11
terial principle of Allium sativum. 1. Isolation, physical properties and antibacterial action. J. Am. Chem. Sot., 66.1950-1951. Cavallito, C.J., Buck, J.S. and Suter, C.M. (1944) Allicin. the antibacterial principle of Allium sativum. II. Determination of the chemical structure. J. Am. Chem. Sot., 66, 1952-1954. Conradt, H.S. Nimtz, M., Dittmar, K.E.J.. Lindenmaier, W., Hoppe, J. and Hauser, H. (1989) Expression of human interleukin-2 in recombinant BHK LTK( - ) CHO cells: Structure of O-linked carbohydrates and their location within the polypeptide. J. Biol. Chem., 264, 1479-
12
13
1486. Feldberg, Neuwirth,
cells treated
with extract
of garlic
15
Green, L.M., Reade, J.L. and Ware, C.F. (1984) Rapid calorimetric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lymphokines. J. Immunol. Methods, 70,257-268.
16
Hussain, S.P., Jannu, L.N. and Rao, A.R. (1990) Chemopreventive action of garlic on methylcholanthreneinduced carcinogenesis in the uterine cervix of mice. Cancer Lett., 49, 175-180. Jain, M.K. and Apitz-Castro, R. (1987) Garlic: Molecular basis of the putative “vampire-repellant” action and other matters related to heart and blood. Trends Biochem. Sci., 12,252-254.
References 1
with tumour
(Allium sativum). Nature, 216, 83-84. Ghannoum, M.A. (1988) Studies on the anticandidal mode of action of Allium sativum (garlic). J Gen, Microbiol., 134, 2917-2924.
17
18
19
Jansen, H., Miiller, 6. and Knobloch, K. (1987) Allicin characterization and its determination by HPLC. Planta Med., 559-562. Klein, G., Lindahl. T., Jondal, M., Leibold, W., Menezes, J., Nilsson, K. and SundstrGm. C. (1974) Continuous lymphoid cell lines with characteristics of B cells (bonemarrow-derived), lacking the Epstein-Barr virus genome and derived from three human lymphomas. Proc. Natl.
20 21
22
23
24 25
26
27
R.S., Chang, S.C., Kotik, A.N., Nadler. M., Z., Sundstrom, D.C. and Thompson, N.H.
28
(1988) In vitro mechanism of inhibition of bacterial cell growth by allicin. Antimicrob. Agents Chemother.. 32, 1763-1768. Fujiwara, M. and Natata, T. (1967) Induction of tumour
29
Acad. Sci. Konvicka, von Allium Mosmann,
USA, 71,3283-3286. 0. (1984) Zum mitotischen Hemmungseffekt sativum-Extrakt. Cytologia, 49, 761-769. T. (1983) Rapid calorimetric assay for cellular
growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Meth., 65, 55-63. Nishino, H., Fujita, K., Fukuda, T., Tanaka, H. and Okuyama, T. (1988) Inhibition of tumor promoter-induced phenomena by the principles obtained from Allium SPP. Abstracts 16th Internat. Symp. Chemistry of Natural Products (IUPAC) Kyoto, Japan, 507. Nisino, H., Iwashima, A., Matsuura, H., Itakura, Y., Fuwa, T., Fujita, K., Okuyama, T. and Shibata, S. (1988) Antitumor-promoting activity of garlic extract. Abstracts 16th Internat. Symp. Chemistry of Natural Products (IUPAC) Kyoto, Japan, 508. Rozengurt. E. (1986) Early signals in the mitogenic response. Science, 234, 161-164. Stoker, M. and MacPherson, I. (1964) Syrian hamster fibroblast cell line BHK21 and its derivatives. Nature, 203 1355-1357. Wagner, H., Wierer, M. and Fessler, B. (1987) Effects of garlic constituents on arachidonate metabolism. Planta Med., 53,305-306. Weisberger, A.S. and Pensky, J. (1958) Tumor inhibition by a sulfhydryl-blocking agent related to an active principle of garlic (Allium sativum). Cancer Res., 18, 1301-1308. Wills, E.D. (1956) Enzyme inhibition by allicin, the active principle of garlic. Biochem. J., 63, 514-520. Yoshida, S., Kasuga, S., Hayashi, N., Ushiroguchi, T., Matsuura, H. and Nakagawa, S. (1987) Antifungal activity of ajoene derived from garlic. Appl. Environ. Microbial., 53,615-617.
Cancer Letters, 53 (1990) 103-108 Elsevier Scientific Publishers
Ireland Ltd.
The cytotoxic effect of ajoene, a natural product investigated with different cell lines K. Scharfenberg, GBF (Gesellschaftffir
from garlic,
R. Wagner and K.G. Wagner Biotechnologische
Forschung),
Mascheroder
Weg 1, D-3300 Braunschweig
(F.R.G.)
(Received 22 May 1990) (Accepted 18 June 1990)
Summary The sulfur-containing compound ajoene (4,5,9-trithiadodeca-1,6,11-triene-g-oxide) which arises from alliin, a cysteine derivative stored in garlic bulbs, was produced synthetically by decomposition of allicin. Its cytotoxic effect was tested using human primary fibroblasts (FS4), a permanent, non-tumorgenic cell line derived from baby hamster kidney cells (BHK21) and a tumorgenic lymphoid cell line derived from a Burkitt lymphoma (BJA-B). The cytotoxic action was in the range Z-50 pg/ml depending on the cell density. ED,, values, estimated on the basis of fmol ajoene/ cell, revealed slightly higher doses for the primary cell (FS4) than the permanent line (BHK), whereas the tumorgenic BJA-B cells were most sensitive. Keywords: garlic
ajoene;
cytotoxicity;
cell lines;
Introduction Intact garlic bulbs contain alliin (S-allylcysteine S-oxide) in concentrations up to 1% fresh weight. Injury of the tissue liberates allinase, probably stored in the vacuole, which cleaves alliin to produce mainly allicin (allyl-ZCorrespondence to: K.G. Wagner. 0304.3835/90/$03.50 Published
and Printed
0
1990 Elsevier Scientific Publishers
in Ireland
propenethiosulfinate) . Allicin is not very stable and is further transformed into a variety of sulfur-containing substances. Depending on the during transformation, aloene conditions (4,5,9-trithiadodeca-1,6,11-triene-9-oxid) is obtained as a main component [5,7,17]. Hence in the various garlic preparations consumed and used by the human population different amounts of allicin and ajoene are present. Antitumor and cytotoxic action of garlic extracts [3,13,16,20,22,23,27] and allicin [12] have been probed in several studies; furthermore the bacteriocidic and fungicidic action of allicin [9,10,14] and even its enzymeinactivating property [28] has been tested. Although the antithrombotic properties of ajoene have been thoroughly studied [ 1,2,6,7] there are only few reports on its fungicidic action [29], inhibition of lipoxygenase [4,26], prostaglandin synthetase [26] and mouse skin tumor promotion [4]. In the present work the cytotoxic properties of ajoene have been investigated using 3 different mammalian cell lines along with allicin for comparison. Materials and methods Preparation of ajoene and allicin Allicin was prepared from diallyl disulfide as described [7,18] using perbenzoic acid for the oxidation step. Ajoene was obtained from Ireland Ltd
104
decomposition of allicin [7] and the crude product was purified by column chromatography and analysed by reversed-phase high performance liquid chromatography (H. Baydoun and K.G. Wagner, unpublished results). According to HPLC chromatography and proton NMR measurements the purity of ajoene and allicin was 90%. Allicin and ajoene were dissolved at concentrations of 20 mg/ml in 87% glycerol and stored at - 20°C. Cells and culture conditions
A human foreskin-derived FS4 cell line obtained from Bioferon, Laupheim (F.R. G .) was used during the 16th-22nd subculture. The BHK21 cell line (C-13, ATCC CCLlO) had been transfected by an interleucin 2 gene containing plasmid in the Genetics Dept. of the GBF (BHK 21 pSVIL2) [11,25]. Both cell types were anchorage dependent fibroblasts, whereas the BJA-B cells, originally derived from a Burkitt lymphoma [19] and obtained from Dr. P. Gruss DKFZ (Heidelberg), were grown in suspension. After establishing a cell bank, which was conserved frozen (stored in N,-vapour), thawed cells were used for 3040 passages only. All three cell lines were cultivated in DMEM (from Gibco, Santa Clara, CA), supplemented with 10% inactivated fetal calf serum, at 37 OC and 96% relative humidity with an atmosphere of 93% air and 7% CO,. When the cells (FS4 and BHK) reached confluency, they were harvested by trypsinization and plated for the following passages or for drug treatment. BJA-B cells were treated as a suspension culture. Drug treatment In a 96-well flat bottom microtiter plate every well was inoculated with 103-104 cells in 100 ~1 medium. After incubation for one day, the medium (100 ~1) was adjusted to 20% serum and thereafter 100 ~1 medium (without serum) containing ajoene or allicin were added. Two days after inoculation the number of cells were counted in those wells which had obtained lethal doses of ajoene or allicin (cell number at the time of drug addi-
tion). On day 3 the metabolic activity of the wells was determined by the MTT assay (see below) and related to untreated cells. Assay methods Cells were counted using a hemocytometer (Neubauer, improved version). The percentage of dead cells was determined by the Trypan blue (Boehringer Mannheim) exclusion method. The calorimetric MTT (tetrazolium) assay [15,21] was applied in a modified form (D. Monner, unpublished) with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Chem. Co.) dissolved (5 mg/ml) in buffered saline solution, filtered and stored frozen. After removal of 100 ~1 medium, MTT stock solution was added (10 ~1 per 100 ~1 medium), the plates were incubated at 37OC for 3-5 h, acidic isopropanol (5% formic acid) was added (150 ccl), mixed thoroughly to dissolve the dye crystals and the absorbance was recorded with an immuno-reader (NJ20000 from Nippon Intermed K.K.) at 572 nm at a reference wavelength of 650 nm. Determination of total cell protein was performed with resuspended cells (after trypsinization in the case of FS4 and BHK cells) which were centrifuged for 5 min (Digifuge from Heraeus-Christ) at 2000 rev./min. The cells (approx. 1.5 x 106/100 ~1) were suspended in ice-cold buffer (20 mM EDTA, 0.5% Triton X-100) and homogenized for 30 s in a cooled Douncehomogenizer (Braun, Melsungen) . Protein was determined according to Bradford [8] with the reagent from Biorad (Munich) using bovine serum albumin (Serva, Heidelberg) as reference protein. Ajoene was determined by HPLC using an ODS-Sperisorb 5 pm column (25 x 0.4 cm) (Melz, Berlin), a precolumn (3 cm, 15-25 pm C-18 Lichroprep from Merck, Darmstadt), LDC constametric pump (Milton Roy, Hasselroth), Uvikon 730 LC photometer (Kontron Analytik, Hannover) and C-R 1B integrator (Gynkrotek, Munich). lsocratic elution was performed with 40% acetonitrile in water and detection at 230 nm, Rr value for ajoene was about 6 min at a flow rate of 2 ml/ min.
105
Results and discussion Ajoene, which is formed via allicin from the garlic stored compound alliin by the action of allinase [5,7,17], can also be obtained from allicin synthesized by oxidation of diallyl disulfide [7,18]. The spontanous transformation of allicin into ajoene which occurs after injuring garlic tissue can be performed in the test tube [7]. The main difficulty arises from the purification of ajoene from substances obtained simultaneously with ajoene. We have produced ajoene, as a mixture of E- and Z-isomers [7], by the chemical route developing a chromatographic purification procedure (K.G. Wagner, H. Baydoun and K. Plank-Schumacher, unpublished results) to give ajoene in gram quantities and at high purity as shown by HPLC and proton-NMR spectroscopy (to be published). The antithrombotic activity of ajoene was originally deduced from its presumable interference with the receptor-mediated signal pathways in the platelets [17], although this hypothesis has not as yet been proved. As regulation of cell proliferation is also correlated with signal transducing properties [24], we assumed that ajoene may also have cytostatic or cytotoxic activities and studied this property with the cell lines available. Human fibroblasts (FS4) were chosen as representing a primary cell line, a BHK line as an example of a permanent and non-tumorgenic cell line and a tumorgenic lymphoid cell line (BJA-B) derived from a Burkitt lymphoma. Figure 1 shows a typical experiment with the surviving fraction plotted versus the ajoene concentration. The BJA-B cell line was rather sensitive revealing an EC,, value of about 2 pg/ml, whereas the other two cell lines were slightly less sensitive. Although the mechanism of ajoene action in cytotoxicity is not known, one could suggest from its chemical structure that reactions with SH-functional groups of cellular molecules of low or high molecular weight might be involved. Evidence for such a hypothesis were elaborated for allicin which has similar functional groups to those of ajoene
0
5
Ajoene
10
15
Concentration
20
pgml-’
Fig. 1.
Effect of ajoene on 3 different cell lines; (0) human foreskin-derived cell line (FS4). (A) baby hamster kidney-derived cell line (BHK) and (0) Burkitt lymphoma-derived cell line (BJA-B) Surviving fraction was determined by the MTT assay (see Materials and methods); cell densities at the time of ajoene application: 5.5 x lo4 (FS4), 1.08 x lo5 (BHK), 9.4 x lo4 (BJA-B) cell/ml. Data are presented as mean + S.D. of one typical experiment with 2 (BJA-B) or 3 (BHK and FS4) parallel determinations (available wells in the microtiter plate). The standard deviations, marked on one side of the points only, are given except where they are within the limits of the symbol; they were all below 6%.
[9,10,28]. Assuming that ajoene reacts with cellular SH-functions and is consumed by this action, the cytotoxic response should not be strictly related to the concentration but to the amount of ajoene consumed per cell. Figure 2 shows the dependence on the cell density of the BJA-B cells. The above expectation is obviously fulfilled, higher cell densities were correlated with higher EC,, values. Table 1 summarizes the results obtained with the different cell lines. With BHK cells the cell densities applied were of higher variance than with the other two cell lines, hence the EC,, values related to the ajoene concentration had a large standard deviation. However, when calculated as the amount of ajoene per cell, the deviation was of the same magnitude as with the other cell lines. This again supports the above conclusion that the cytotoxic activity depends on the amount of ajoene related to
106
1
0.1 Ajoene
10
Concentration
ajoene/mg protein (calculated for the ED values) show that the tumorgenic BJA-B cell: were about twice as sensitive as the FS4 and BHK cells. Allicin which is structurally related to ajoene contains a thiosulfinate groups was also tested; on a mol/cell and mol/mg protein basis ajoene was twice as active as allicin although the mechanism may be similar (Table 2). Also for practical reasons ajoene is a better candidate as it is more stable than allicin which gradually decomposes forming among other substances also ajoene [7]. In the standard assay cell viability was determined after 48 h of ajoene treatment. To determine the influence of the treatment time, BHK cells were exposed to ajoene for different times. It was found that the cytotoxic effect of ajoene required a time span of about 3 h; with tests performed for 3 h treatment the same curvature in the dose-response curves were obtained as with those performed for 46 h, whereas with treatments for shorter times the cytotoxic effect of ajoene was reduced considerably. Uptake of ajoene by the cells was determined with BJA-B cells in serum-free medium as described in the legend of Fig. 3. It is obvious that uptake was accomplished after about 3 h; this is exactly the same time span determined for the establishment of the cytotoxic action; hence action of ajoene must occur parallel with or immediately after its uptake. Figure 3 further shows that an intact cellular metabolism is required for ajoene uptake, as
100
pgml“
Fig. 2. Influence of ajoene on BJA-B cells studied at various cell densities. Data are presented as mean + S.D. of one typical experiment; for further details see legend of Fig. 1. Cell densities in 10s cells/ml: (0) 0.25, (0) 0.5, (A) 1.0, (0) 2.0, (m) 4.0 and (A) 8.0. For clearness the standard deviations were given for only one curve; in total (with one exception) they were all below 10%.
the number of cells within a well. Based on the amount of ajoene per cell, the primary FS4 cells were significantly less sensitive towards ajoene than the permanent cells (BHK), while the tumorgenic cells (BJA-B) were the most sensitive ones. As the cells applied had different sizes, the effective dose was related to the protein content of the cells; the values in pmol
Table 1. Cell line
ES4 BHK BJA-B
A comparison
of the cytotoxic activity of ajoene against the 3 cell lines” Cell density cells/ml lo+
7.9 (2.1) 16.0 (10.2) 14.6 (3.2)
ECmb
ED,,
VM
36 (13) 30 (23) 12 (1.8)
fmol/cell
pmol/mg
450 (64) 190 (49) 85 (23)
0.9 1.0 0.4
protein’
aThe data are mean values of 3 (FS4) or 5 (BHK and BJA-B) separate experiments f S.D. in parenthesis; in each experiment 2-5 parallel determinations were performed. “EC, and ED,, are the concentration and amount of ajoene, respectively, required to reduce viability of the cells to 50%. +mol ajoene per mg cell protein calculated for the ED,, values.
107
Table 2.
Cytotoxic activity of ajoene and allicin against the 3 cell lines”.
Cell line
ED, of allicin
ED,, of ajoene
FS4 BHK BJA-B
fmol/cell
pg/mg
fmol/cell
pgcg/mgb
450 (64) 190 (49) 85 (23)
0.9 1.0 0.4
720 (49) 430 (135) 185 (28)
1.5 2.2 0.8
aThe values for ajoene are derived from Table 1; those for allicin are mean values from 2 (FS4) or 4 (BHK and BJA-B) separate experiments + S.D. in parenthesis; in each experiment 2-5 parallel determinations were performed. “c(gajoene or aIlicin per mg cell protein calculated for the ED, values (cf. Table 1).
0 5
Fig. 3.
2
6
1 -7
(A) Ajoene uptake kinetics with BJA-B cells. Cultivated BJA-B cells were centrifuged and resuspended in DMEM-medium at 37’C (without serum); cell density was 3-4 x 105/ml. At time zero ajoene (25 or 30 pg/ml) was added and after different times cells were centrifuged and ajoene was determined in the supernatant by HPLC. Data are mean values f S.D. from 4 experiments; ajoene concentration was determined twice in each supernatant. (B) Dependence of ajoene uptake on cell viability. Cells were suspended in serum-free DMEMmedium and ajoene was added (30 pg/ml); 4 x lo5 BJA-B cells/ml were used. (0, 0). Two parallel experiments with vital BJA-B cells; (A) BJA-B cells treated for 20 min at 56’ immediately before the experiment; (0) control DMEM-medium without cells.
heat inactivated cells absorbed only a small quantity of ajoene. It is further interesting to note that ajoene absorbed by cells could not be recovered upon ethyl acetate extraction and HPLC analysis. Work is in progress to elucidate the fate of ajoene after absorption by the cells. Although investigated with only three cell lines, the slight but significantly higher sensitivity against ajoene of the tumorgenic line relative to the two non-tumorgenic lines is interesting. This is in accord with the high compatibility of animal skin with ajoene (K.G. Wagner, unpublished results); even in the rabbit eye irritation test, ajoene proved to be a non-irritant. Investigations on the elucidation of the mechanism of the action of ajoene cytotoxitiy are in progress as well as studies to probe ajoene action against tumor formation in animals. Finally, the question arises whether ajoene and allicin ingested with natural and artificial garlic preparations will have similar effects on cell proliferation in the human body as shown with the presented in vitro experiments. Acknowledgements We are grateful to Dr. V. Wray for linguistic advice, Mrs. H. Starke for typing the manuscript and Mrs. C. Lippelt for preparing the
108
graphs. This work has been supported by the Fond der Chemischen Industrie. We very gratefully acknowledge the kind help of Dr. M. K. Jain in the establishment of this subject in our laboratory.
immunity 14
2
3
Apitz-Castro, R., Cabrera, S., Cruz, M.R., Ledezma, E. and Jain, M.K. (1983) Effects of garlic extract and of three pure components isolated from it in human platelet aggregation, arachidonate metabolism, release reaction and platelet ultrastructure. Thrombosis Res., 32, 155- 169. Apitz-Castro, R., Ledezma, E., Escalante, J. and Jain, M.K. (1986) The molecular basis of the antiplatelet action of ajoene: Direct interaction with the fibrinogen receptor. Biochem. Biophys. Res. Commun., 141.145-150. Belman, S. (1983) Onion and garlic oils inhibit tumor pro-
6
motion. Carcinogenesis, 4, 1063-1065. Belman, S., Solomon, J., Segal, A., Block, E. and Barany, G. (1989) Inhibition of soybean lopoxygenase and mouse skin tumor promotion by onion and garlic components. J. Biochem. Toxicol., 4, 151-160. Block, E. (1985) The chemistry of garlic and onions. Sci. Am. 252,94-99. Block, E., Ahmad, S., Jain, M.K., Crecely, R.W., Apitz-
7
Castro, R. and Crux, M.R. (1984) (E,Z)-Ajoene: A potent antithrombotic agent from garlic. J. Am. Chem. Sot., 106,8295-8296. Block, E., Ahmad, S., Catalfamo. J.L., Jain, M.K. and
a
Apitz-Castro, R. (1986) Antithrombotic organosulfur compounds from garlic: Structural, mechanistic and synthetic studies. J. Am. Chem. Sot., 108, 7045-7055. Bradford, M.M. (1976) A rapid and sensitive method for
9
quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem., 72, 248-254. Cavallito, C.J. and Bailey, J.H. (1944) Allicin, the antibac-
4
5
10
11
terial principle of Allium sativum. 1. Isolation, physical properties and antibacterial action. J. Am. Chem. Sot., 66.1950-1951. Cavallito, C.J., Buck, J.S. and Suter, C.M. (1944) Allicin. the antibacterial principle of Allium sativum. II. Determination of the chemical structure. J. Am. Chem. Sot., 66, 1952-1954. Conradt, H.S. Nimtz, M., Dittmar, K.E.J.. Lindenmaier, W., Hoppe, J. and Hauser, H. (1989) Expression of human interleukin-2 in recombinant BHK LTK( - ) CHO cells: Structure of O-linked carbohydrates and their location within the polypeptide. J. Biol. Chem., 264, 1479-
12
13
1486. Feldberg, Neuwirth,
cells treated
with extract
of garlic
15
Green, L.M., Reade, J.L. and Ware, C.F. (1984) Rapid calorimetric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lymphokines. J. Immunol. Methods, 70,257-268.
16
Hussain, S.P., Jannu, L.N. and Rao, A.R. (1990) Chemopreventive action of garlic on methylcholanthreneinduced carcinogenesis in the uterine cervix of mice. Cancer Lett., 49, 175-180. Jain, M.K. and Apitz-Castro, R. (1987) Garlic: Molecular basis of the putative “vampire-repellant” action and other matters related to heart and blood. Trends Biochem. Sci., 12,252-254.
References 1
with tumour
(Allium sativum). Nature, 216, 83-84. Ghannoum, M.A. (1988) Studies on the anticandidal mode of action of Allium sativum (garlic). J Gen, Microbiol., 134, 2917-2924.
17
18
19
Jansen, H., Miiller, 6. and Knobloch, K. (1987) Allicin characterization and its determination by HPLC. Planta Med., 559-562. Klein, G., Lindahl. T., Jondal, M., Leibold, W., Menezes, J., Nilsson, K. and SundstrGm. C. (1974) Continuous lymphoid cell lines with characteristics of B cells (bonemarrow-derived), lacking the Epstein-Barr virus genome and derived from three human lymphomas. Proc. Natl.
20 21
22
23
24 25
26
27
R.S., Chang, S.C., Kotik, A.N., Nadler. M., Z., Sundstrom, D.C. and Thompson, N.H.
28
(1988) In vitro mechanism of inhibition of bacterial cell growth by allicin. Antimicrob. Agents Chemother.. 32, 1763-1768. Fujiwara, M. and Natata, T. (1967) Induction of tumour
29
Acad. Sci. Konvicka, von Allium Mosmann,
USA, 71,3283-3286. 0. (1984) Zum mitotischen Hemmungseffekt sativum-Extrakt. Cytologia, 49, 761-769. T. (1983) Rapid calorimetric assay for cellular
growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Meth., 65, 55-63. Nishino, H., Fujita, K., Fukuda, T., Tanaka, H. and Okuyama, T. (1988) Inhibition of tumor promoter-induced phenomena by the principles obtained from Allium SPP. Abstracts 16th Internat. Symp. Chemistry of Natural Products (IUPAC) Kyoto, Japan, 507. Nisino, H., Iwashima, A., Matsuura, H., Itakura, Y., Fuwa, T., Fujita, K., Okuyama, T. and Shibata, S. (1988) Antitumor-promoting activity of garlic extract. Abstracts 16th Internat. Symp. Chemistry of Natural Products (IUPAC) Kyoto, Japan, 508. Rozengurt. E. (1986) Early signals in the mitogenic response. Science, 234, 161-164. Stoker, M. and MacPherson, I. (1964) Syrian hamster fibroblast cell line BHK21 and its derivatives. Nature, 203 1355-1357. Wagner, H., Wierer, M. and Fessler, B. (1987) Effects of garlic constituents on arachidonate metabolism. Planta Med., 53,305-306. Weisberger, A.S. and Pensky, J. (1958) Tumor inhibition by a sulfhydryl-blocking agent related to an active principle of garlic (Allium sativum). Cancer Res., 18, 1301-1308. Wills, E.D. (1956) Enzyme inhibition by allicin, the active principle of garlic. Biochem. J., 63, 514-520. Yoshida, S., Kasuga, S., Hayashi, N., Ushiroguchi, T., Matsuura, H. and Nakagawa, S. (1987) Antifungal activity of ajoene derived from garlic. Appl. Environ. Microbial., 53,615-617.
