Cancer Letters, 63 (1992) 211-219 Elsevier Scientific Publishers Ireland
211 Ltd
Antiproliferative effect of the garlic compound on human neuroblastoma cells in vitro
S-ally1 cysteine
Carrie Welch, Laura Wuarin and Neil Side11 of Pathology (Neuropathology), Brain Research Institute and the Jonsson UCLA School of Medicine, Los Angeles, California 90024 (USA)
Department
(Received (Accepted
1 December 21 February
Cancer Center,
1991) 1992)
Summary
Keywords: antitumorigenesis; cysteine; neuroblastoma
A uariety of compounds derived from garlic bulbs have been shown in animal systems to possess anticancer properties. However, little information is available regarding the effectiveness of garlic in the preuention or treatment of human cancers. In the current study, we have assessed the ability of S-ally/ cysteine (SAC), a derivative of aged garlic extract, to affect the proliferation and differentiation of LA-N-5 human neuroblastoma cells in vitro. Time-and dose-dependent inhibition of cell grow was obserued in cultures treated with SAC for at least 2 days, with a half-maximal response at approximately 600 pg/ml. SAC treatment was unable to induce differentiation in neuroblastoma cells as assessed by morphological, biochemical and molecular markers. In addition, SAC was unable to potentiate the effects of retinoic acid and 8-bromo-cyclic AMP, agents known to promote differentiation of LA-N-5 cells. Our results indicate that SAC can inhibit human neuroblastoma cell growth in vitro. However, the apparent inability of this compound to induce differentiation may limit its therapeutic potential.
Correspondence to: Nell Sidell, Department of Pathology, 18-170 Center for Health Sciences, UCLA School of Medicine, Los Angeles, CA 90024, USA.
0304-3835/92/$05.00 Printed and Published
Comprehensive
0 1992 Elsevier Scientific Publishers in Ireland
garlic; S-ally1
Introduction Weisberger and Pensky [26] first reported the antitumorigenic properties of allicin, a garlic derivative, in 1958. Since then, a variety of garlic compounds have been shown to inhibit chemically-induced and transplanted tumors in experimental animals [13]. Most recently, in a carcinogenesis study using sequential treatment with diethylnitrosamine, Nmethylnitrosourea and N,N-dibutylnitrosamine as the initiation step, Jang et al. [lo] showed that subsequent treatment with ally1 sulfide significantly deceased the incidence of hepatic hyperplastic nodules, adenoma of the lung and thyroid and hyperplasia of the urinary bladder in mice. Although the chemopreventive properties of garlic have been clearly demonstrated in animal models, little information is available regarding the effectiveness of garlic in the prevention or treatment of human cancers. Epidemiological studies have shown inverse associations between the consumption of garlic and incidence of gastric cancer in China [13] and Italy [4] as well as risk of thyroid nodularity among women in China [25]. Scharfenberg et al. [19] recently reported the results of an in vitro study using an established tumorigenic Ireland Ltd
212
cell line derived from a Burkitt lymphoma. Treatment of the lymphoid cells with ajoene, a lipid-soluble oxidation product of raw garlic, caused inhibition of cell proliferation in the lymphoid cell line; however, a toxic effect was also evident in non-tumor cells studied in parallel. Water-soluble compounds derived from garlic may be less cytotoxic than oilsoluble compounds, presumably because they are not stored in body tissues. SAC, a watersoluble component of aged garlic extract, has previously been shown to inhibit 1,2-dimethylhydrazine-induced colon cancer in rats [22]. Takeyama et al. [23] have reported that SAC can inhibit human melanoma cell proliferation and may induce differentiation of tumor cells to a mature, non-tumorigenic phenotype. This apparent antitumoral action on melanoma cells prompted our study of the effects of SAC on human neuroblastoma (NB), a tumor that originates, like melanoma, from tissue of the neural crest. In this study, we tested the ability of SAC to affect the proliferation and differentiation of the human NB cell line LA-N-5, cells shown to be remarkably sensitive to a variety of differentiating agents. In addition, we investigated the potential of SAC to enhance the effects of retinoic acid (RA) and &bromocyclic AMP (8BrcAMP) , compounds known to induce neuronal differentiation in this line. The results of our study confirm the antiproliferative action of SAC on human neural crestderived tumor cells but do not support the hypothesis that this effect results from induction of differentiation. Materials and Methods Chemicals S-allyl-r-cysteine, kindly provided by Wakunaga of America, Mission Viejo, CA, was dissolved in phosphate-buffered saline (pH 7.0) to a stock concentration of 40 mg/ml, filtered with Nalgene disposable syringe filters (Rochester, NY) and stored at - 70°C. All-trans-retinoic acid (Sigma Chemical Co., St. Louis, MO) was dissolved
in dimethyl sulfoxide to a concentration of 10T2 mM and stored at -2OOC. [Methyl3H]thymidine (6.7 Ci/mmol) and [3H]tyrosine (50 Ci/mmol) were obtained from NEN Research Products, Claremont, CA. (6R)-Lerythro-5,6,7,8_tetrahydrobiopterin, which was used in the tyrosine hydroxylase assay, was a generous gift from Dr. John F. Reinhard, Burroughs Wellcome, Research Triangle Park, NC. All other chemicals were purchased from Sigma Chemical Co. Cell Culture LA-N-5 human neuroblastoma cells were grown as monolayers in RPM1 1640 medium (M.A. Bioproducts, Walkersville, MD) supplemented with 10% heat-inactivated fetal calf serum (Gemini Bioproducts, Inc., Calabasas, CA), HEPES buffer, 50 IU/ml penicillin, 50 pg/ml streptomycin and 1 pg/ml amphotericin. Cultures were maintained in a humidified incubator at 37OC with an atmosphere of 95% air and 5% C02. Unless noted otherwise, cells for each experiment were plated into T25 (25 cm*) tissue-culture flasks (Costar Corp., Pleasonton, CA) and incubated overnight with 5 ml of medium to allow attachment and spread of cells. Various treatments were then added directly to the medium as indicated. [3H]thymidine incorporation Cells were plated in 24-well plastic tissue culture trays (Corning, Palo Alto, CA) at a density of 4 x lo3 cells/well. Various concentrations of SAC (200- 1600 pg/ml), RA (5 x lO-‘j M), or SAC + RA were added to the culture medium after 24 h (day 1). On day 7, 1 &i [3H]thymidine/well was added. In separate time course experiments, cells were treated with 800 pg/ml SAC for either 2, 4, or 6 days before the addition of [3H]thymidine. All cells were harvested 16 h after administration of labelled thymidine as previously described [27]. Radioactive incorporation was measured in a liquid scintillation counter according to standard procedures.
213
Morphology Cultures were treated with SAC (ZOO3200 pg/ml), RA (4 x 10m6 M), or SAC + RA. Cells from various regions of the flasks were evaluated by light microscopy at 1, 4, 6, 7, 8 and 14 days post-treatment for morphologic changes. Acetylcholinesterase (AChE) assay Cultures were treated with SAC (400 or 800 pg/ml), RA (5 x 10m6 M), or SAC + RA. On day 10, cells were harvested, pelleted and washed twice with ice-cold PBS. The cells were resuspended in 100 mM sodium phosphate buffer (pH 6.8) containing 0.2% Triton X-100 and sonicated for 10 s. AChE activity was determined photometrically, as described by Ellman et al. [7]. Total sample proteins were measured using the protein-dye binding method of Bradford [3] using bovine serum albumin as a reference protein. Tyrosine hydroxylase
I
I
I
0
I
400
I
800 SAC
I
1200
1600
(m/ml)
Fig.
1. Dose-response curves showing the effects of SAC alone (0) or SAC + 5 x 10e6 M RA (0) on the incorporation of [3H]thymidine in LA-N-5 neuroblastoma cells after 8 days of treatment. Values represent the mean f S.E.M. of triplicate samples of a typical experiment.
(TH) assay
Following a 3-day incubation with medium, cultures were treated with SAC (400 or RA (5 x 10m6 8BrcAMP 800 &ml), (1 mM), SAC + RA, or SAC + 8BrcAMP. On day 6, cells were harvested, washed and homogenized as described above. Aliquots were assayed for TH activity by the method of Reinhard et al. [17], in which [3H]Hs0 is hydrolysis of counted following the [3H]tyrosine. Proteins were determined by the method of Bradford [3].
16
t
blot analysis of N-myc expression Cells were harvested after 4 days of treat-
Northern
ment with either SAC (800pg/ml) or RA (3 x 10 -6 M) RNA was extracted with guanidium isothiocyanate and precipitated through a CsCl gradient [6]. An aliquot of RNA (20 pg) was denatured by glyoxal incubation, fractionated by electrophoresis through a 1% agarose gel and transferred to a Biotrans nylon membrane (ICN, Irvine, CA) using a positive pressure transfer apparatus (Stratagene, La Jolla, CA). The N-myc specific probe pNB-1
0
6
4
2 Days
of
SAC
treatment
Fig. 2. Time-course of [3H]thymidine incorporation by LA-N-5 cells in the absence (0) or presence (0) of 800 &ml SAC. Values represent the mean ZIZS.E.M. of triplicate samples of a typical experiment.
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Morphology of LA-N-5 neuroblastoma cells in the absence and presence of SAC and/or RA. Cells were tre ated for 7 days with vehicle alone (a), 800 pg/ml SAC (b), 4 x 10e6 M RA (c), or 800 pg/ml SAC + 4 x 10-6M RA (d). The addition of SAC to the culture medium did not significantly alter cell morphology. x 200
Fig. 3.
215
216
was labelled by random priming (10’ counts/ min per pg) using a standard kit (Amersham, Arlington Heights, IL). Prehybridization and hybridization medium consisted of 6 x SSC, 5 x Denhardt’s solution, 0.5% SDS and 100 pg/mI salmon sperm DNA. Following 16-24 h of incubation at 68OC, filters were washed once in 1 x SSC, 0.1% SDS at room temperature for 20 min and twice in 0.2 x SSC, 0.1% SDS at 68OC. Autoradiography was performed using Kodak XOMAT film at - 70°C for 2 - 10 days. Be.&@ Cell prohferation The dose effect of SAC on [3H]thymidine uptake in LA-N-5 cells is illustrated in Fig. 1. Cells were treated with 200- 1600 pg/mI SAC, either alone or in combination with RA (5 x 10m6 M) for 8 days. A dose-dependent inhibition of cell proliferation was observed following incubation with SAC doses greater than 400 .pg/rnl SAC. The half-maximal response was observed with approximately 600 pg/ml SAC while greater than 99% inTablo 1.
Effect SAC alone, SAC + RA
Treatment
Control SAC (400 pg/ml) SAC (800 pg/ml) RA (5 x 10-6) RA + SAC (400 gg/ml) RA + SAC (800Ccg/ml) 8BrcAMP (1 mM) 8BrcAMP + SAC (400 kg/ml) 8BrcAMP + SAC (800 pg/ml)
hibition occurred with the maximum dose given. Combination treatment of cells with SAC + RA produced an additive inhibitory effect but no synergism between the two antiproliferative agents was observed (Fig. 1). Results from time-course studies, shown in Fig. 2, indicate that the growth inhibitory effect of SAC (800 pg/mI) occurred as early as two days following treatment and slowly increased with time. Inhibition of thymidine uptake was 66% after a 2-day incubation and increased to 72% following 6 days of treatment. A small toxic component of the antiproliferative effect of SAC was detected by the inability of some cells to exclude trypan blue dye and an increase in the rounding and detachment of cells from culture flasks. Cell differentiation Cell cultures treated for periods of up to 14 days were observed by light microscopy for changes in morphology. A representative experiment in which cells were treated for 7 days is shown in Fig. 3. Morphological differentiation of NB cells is typically reflected by the extension of long processes, or neurites [18].
and SAC + CAMP on AChE and TH activity. AChE activity (nmol/min per mg) 2.53 zt 0.15”*b 2.23 zt 0.18 2.41 * 0.52 13.76 zt 1.73 13.28 * 1.42 11.25 l 1.30 ND ND ND
TH activity (pmol/h per ccg) 6.69 zt 8.45 zt 6.85 zt
0.29’ 0.86 2.60
NDd ND ND 22.23 zt 3.20 21.18 * 5.60 26.92 zt 12.5
“Mean * S.E.M. from duplicate or triplicate samples. bTwo-way ANOVA: effect of RA, Fnro, = 201, P < 0.0005; effect of SAC, not significant (NS); effect of RA vs. RA + SAC, NS. ‘Two-way ANOVA: effect of 8BrcAMP, F,,, = 33.8, P < 0.001; effect of SAC, NS; effect of 8BrcAMP vs. 8BrcAMP + SAC, NS. dND, not determined.
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In this study, no apparent increase in the number or length of processes was observed in SAC-treated cultures relative to untreated controls (Figs. 3a,b). In contrast, RA-treated cultures showed the expected pattern of morphological change that accompanies differentiation: extension of neurites and ganglion-like clustering within a couple of days followed by bundling of the processes and further cell clustering (Fig. 3~). Similar changes in morphology were observed in cultures treated with SAC + RA, with no enhancement of process extension or cell clustering relative to cultures treated with RA alone (Fig. 3d). The LA-N-5 cell line is comprised of immature cells that have the capacity to express multiple neurotransmitter phenotypes. Cell maturation can be detected, biochemically, by an increase in the specific activities of enzymes involved in neurotransmitter metabolism [27]. We tested the ability of SAC to induce differentiation along either an adrenergic or a cholinergic pathway by assaying for TH and AChE activity, as previously shown for induction of these cells by 8BrcAMP and RA, respectively [27]. As depicted in Table I, concentrations of SAC that were found to be antiproliferative did not result in induction of either enzyme. Furthermore, when given in combination with 8BrcAMP or RA, known inducers of the enzymes, SAC was unable to potentiate their effects,
c
SAC
RA
Fig. 4. Lack of effect of SAC on N-myc mRNA levels in LA-N-5 cells. Total cellular RNA (20 pg) was isolated from 4-day control (C), SAC (800 pg/ml)-, or RA (3 x 10e6 M)-treated LA-N-5 cells and subjected to Northern blot analysis using the pNB-1 N-myc specific probe. Hybridization of the same blots with a @actin probe confirmed that the lanes were loaded with comparable amounts of RNA.
Figure 4 shows the effect of SAC on N-myc expression. Decreased expression of the oncogene N-myc is an earlier marker of neuroblastoma differentiation than biochemical or morphological changes [ 1,12,24]. Treatment of LA-N-5 cells with 800 pg/ml SAC produced no change in N-myc mRNA levels while RA treatment caused a dramatic decrease in the amount of message produced as previously reported [2,24].
Discussion It has been known for many years that garlic and its derivatives exert an anticarcinogenic effect in rodents when administered prior to or simultaneously with carcinogen exposure [9,13,22]. While numerous animal studies have documented that these protective effects could be achieved against a diverse array of chemical carcinogens having different target organ specificities, there have been few experiments designed to investigate the protective or therapeutic potential of garlic in human cancer. In this study we have investigated the ability of a garlic derivative, SAC, to inhibit proliferation and promote differentiation of LA-N-5 human neuroblastoma cells. Our data indicates that SAC, at doses greater than 400 pg/ml, is a potent antiproliferative agent for human neuroblastoma cells. Takeyama et al. [23] have reported similar results for melanoma cells treated with SAC. In addition, they observed significant morphological changes, increased melanin content, and decreased melanoma-associated ganglioside expression in the tumor cells characteristic of a potentially less malignant phenotype [8,16]. Based on these observations, Takeyama suggested that the antiproliferative effect may arise as a result of SAC-induced cell differentiation. To further investigate this hypothesis, we studied NB cell differentiation following treatment with SAC. Neuroblastoma has been a prime model system for the study of neuronal differentiation and tumor cell maturation. The hallmarks of neuron differentiation are neurite
218
extension, growth inhibition, biochemical maturation including increased activity of AChE and decreased N-myc mRNA [2,14]. A variety of compounds have been shown to induce in vitro maturation of NB cells in a manner similar to that of normal differentiating neurons in culture [5,20,21]. In addition, several lines of evidence suggest that the state of tumor cell differentiation is related to the malignant potential of NB tumors [1,5,15]. Although SAC inhibited cell proliferation of LA-N-5 cells, our data provide no evidence of a SACinduced change in differentiation. Whereas processes were observed to extend from RAtreated control cells and form bundles, no outgrowths were seen in the SAC-treated cultures. Similarly, AChE and TH activities were increased by the known inducers RA and 8BrcAMP, respectively; however, SAC treatment produced no changes in these enzyme activity levels. Finally, N-myc expression was significantly decreased in RA-treated cells but unchanged in SAC-treated cultures relative to untreated controls. The differentiation-inducing effect of RA in LA-N-5 cells has been shown to be potentiated by combination treatment with other differentiating agents, including interferon-gamma [27] and CAMP-elevating compounds [ll]. In order to assess the potential of SAC to enhance induction by other agents, we evaluated growth, morphologic and biochemical changes (TH and AChE activities) following treatments with SAC and/or RA (or SAC + 8BrcAMP for TH activity). Although combination treatment resulted in an additive inhibition in the growth rate of LA-N-5 cells, no changes in enzyme activities were detected in combination-treated cultures relative to singly-treated (RA or CAMP) controls. These results are consistent with the notion that SAC induces growth inhibition of LA-N-5 cells independently of differentiation. SAC and other garlic-derived compounds have clearly demonstrated potent anti-proliferative actions in cultured mammalian cells. However, there are conflicting reports regarding their cytotoxic effects. Although we and others [13,19,23] have observed cytotoxicity
in several tumor cell lines, two recent reports suggest that SAC is less toxic [19] or non-toxic [23] to normal cells. Together with these reports, the present results underscore the need to compare the anti-proliferative and cytotoxic effects of SAC and other garlic derivatives on normal and tumorigenic cells in order to fully evaluate their therapeutic potential. Acknowledgements Portions of this work were supported by NIH grants CA43503 and CA30515. The authors wish to thank Dr. Reiko F. Irie for many interesting and helpful discussions. References Abemayor, E. and Sidell, N. (1989) Human neuroblastoma cell lines as models for the in vitro study of neoplastic and neuronal cell differentiation. Environ. Health Perspect., 80, 3 - 15. Amatruda III, T.T., Sidell, N., Ranyard, J. and Koeffler, H.P. (1985) Retinoic acid treatment of human neuroblastoma cells is associated with decreased N-myc expression. Biochem. Biophys. Res. Commun., 126, 1189 - 1195. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248 - 254. Buiatti, E., Palli, D., Decarli, A., Amadori, D., Avellini, C., Bianchi, S., Biserni, R., Cipriani, F., Cocco, P., Giacosa, A., Marubini, E., Puntoni, R., Vindigni, C., Fraumeni, J., Jr. and Blot, W. (1989) A case-control study of gastric cancer and diet in Italy. Int. J. Cancer, 44, 611-616. 0. and Kornhuber, B. Busse, E., Bartsch, Research on the differentiation of human and neuroblastoma cells. Oncology, 48, 196- 201.
(1991) murine
Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18, 5294 - 5299. Ellman, G.L., Courtney, K.D., Andres, V., Jr. and Featherstone, R.M. (1961) A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7, 88 - 95. Hersey, P., Jamal, O., Henderson, C., Zardawi, I. and Alessandro, G.D. (1988) Expression of the gangliosides GM3, GD3 and GD2 in tissue sections of normal skin, naevi, primary and metastatic melanoma. Int. J. Cancer, 41, 336-343. Hussain, S.P., Jannu, L.N. and Rao, A.R. (1990)
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Chemopreventive action of garlic on methylcholanthreneinduced carcinogenesis in the uterine cervix of mice. 10
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Cancer Lett., 49, 175- 180. Jang, J.J., Cho. K.J., Lee, Y.S. and Bae, J.H. (1991) Modifying responses of ally1 sulfide, indole-3carbinol and germanium in a rat multi-organ carcinogenesis model. Carcinogenesis, 12, 691- 695. Lando, M., Abemayor, E., Verity, M.A. and Sidell, N. (1990) Modulation of intracellular cyclic adenosine monophosphate levels and the differentiation response of human neuroblastoma cells. Cancer Res., 50, 722 - 727. Larcher, J.C., Vayssiere, J.L., Lossouarn, L., Gras. F. and Croizat. B. (1991) Regulation of c- and N-myc expression during induced differentiation of murine neuroblastoma cells. Oncogene, 6. 633 - 638. Lau, B.H.S., Tadi, P.P. and Tosk, J.M. (1990) Allium sotiuum (garlic) and cancer prevention. Nutr. Res., 10. 937 - 948. Miki, A. and Mizoguti, H. (1982) Proliferating ability, morphological development and acetylcholinesterase activity of the neural tube cells in early chick embryos. Histochem., 76, 303 - 314. Moss, T.J., Law, Y.M., Slamon, D.J., Brodeur, G.M. and Seeger, R.C. (1988) N-myc protein expression by neuroblastoma cells that have metastasized to bone marrow. In: Advances in Neuroblastoma Research 2, pp.
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and sensitive assay for tyrosine-3-mono-oxygenase based upon the release of 3Hz0 and absorption of 3H-tyrosine by charcoal. Life Sci., 39, 2185-2189. Robson, J.A. and Sldell, N. (1985) Ultrastructural features of a human neuroblastoma acid. Neurosci., 14, 1149-
cell line treated 1162.
with retinoic
K., Wagner, R. and Wagner, K.G. (1990) effect of ajoene, a natural product from
garlic, investigated with different cell lines. Cancer Lett.. 53, 103 - 108. Sidell, N. (1982) Retinoic acid-induced growth inhibition and morphologic differentiation of human neuroblastoma cells in vitro. J.Natl. Cancer Inst., 68, 589 - 596. Sidell, N. Lucas, C.A. and Kreutzberg, G.W. (1984) Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line. Exp. Cell Res., 155, 305 - 309. Sumiyoshl, colon cancer
H.
and Wargovich, M.J. (1990) of 1,2-dimethylhydrazine-induced in mice by naturally occurring organosulfur
compounds. Cancer Res., 50. 5084-5087. Takeyama, H., Hoon, D.S.B., Morton, D.L. and Irie, R.F. (1991) Growth inhibition and modulation of cell markers of melanoma by an aged garlic product, sulfur-ally1 cysteine. Proc. Am. Assoc. Cancer Res., 32, 426. Thiele, C.J., Reynolds, C.P. and Israel, M.A. (1985) Decreased expression of N-myc precedes retinoic acidinduced morphological differentiation of human neuroblastoma. Nature, 313,404 -406. Wang, Z., Boice, J.D., Jr., Wei, L., Beebe, G.W., Zha, Y., Kaplan, M.M., Tao, S., Maxon III, H.R., Shang, S., Schneider, A.B., Tan, B., Wesseler, T.A, Chen, D., Ershow, A.G., Kleinerman, R.A., Littlefield, L.G. and Preston, D. (1990) Thyroid nodularity and chromosome aberrations among women in areas of high background radiation in China. J. Natl. Cancer Inst., 82, 478-485.
41- 56. Editors: A.E. Evans, G.J. D’Anglo, A.G. Knudson and R.C. Seeger. Alan R. Liss, Inc., New York. Natoli, E.J., Livingston, P.O., Pukel, C.S., Lloyd, K.O., Wiegandt, H., Szalay, J., Oettgen, H.F. and Old, L.J. (1986) A murine monoclonal antibody detecting N-acetyland N-glycolylGM2: Characterization of cell surface reactivity. Cancer Res., 46, 4116-4120. Reinhard, J.F., Smith, G.K., Nichol, C.A. (1986) A rapid
Scharfenberg, The cytotoxic
26
27
Weisberger, A.S. and Pensky. J. (1958) Tumor inhibition by a sulfhydryl-blocking agent related to an active principle of garlic (Allium satioum). Cancer Res., 18, 1301- 1308. Wuarin, L., Verity, M.A. and Sidell, N. (1991) Effects of Interferon-gamma and its interaction with retinoic acid on human neuroblastoma differentiation. Int. J. Cancer, 48, 136- 141.
211 Ltd
Antiproliferative effect of the garlic compound on human neuroblastoma cells in vitro
S-ally1 cysteine
Carrie Welch, Laura Wuarin and Neil Side11 of Pathology (Neuropathology), Brain Research Institute and the Jonsson UCLA School of Medicine, Los Angeles, California 90024 (USA)
Department
(Received (Accepted
1 December 21 February
Cancer Center,
1991) 1992)
Summary
Keywords: antitumorigenesis; cysteine; neuroblastoma
A uariety of compounds derived from garlic bulbs have been shown in animal systems to possess anticancer properties. However, little information is available regarding the effectiveness of garlic in the preuention or treatment of human cancers. In the current study, we have assessed the ability of S-ally/ cysteine (SAC), a derivative of aged garlic extract, to affect the proliferation and differentiation of LA-N-5 human neuroblastoma cells in vitro. Time-and dose-dependent inhibition of cell grow was obserued in cultures treated with SAC for at least 2 days, with a half-maximal response at approximately 600 pg/ml. SAC treatment was unable to induce differentiation in neuroblastoma cells as assessed by morphological, biochemical and molecular markers. In addition, SAC was unable to potentiate the effects of retinoic acid and 8-bromo-cyclic AMP, agents known to promote differentiation of LA-N-5 cells. Our results indicate that SAC can inhibit human neuroblastoma cell growth in vitro. However, the apparent inability of this compound to induce differentiation may limit its therapeutic potential.
Correspondence to: Nell Sidell, Department of Pathology, 18-170 Center for Health Sciences, UCLA School of Medicine, Los Angeles, CA 90024, USA.
0304-3835/92/$05.00 Printed and Published
Comprehensive
0 1992 Elsevier Scientific Publishers in Ireland
garlic; S-ally1
Introduction Weisberger and Pensky [26] first reported the antitumorigenic properties of allicin, a garlic derivative, in 1958. Since then, a variety of garlic compounds have been shown to inhibit chemically-induced and transplanted tumors in experimental animals [13]. Most recently, in a carcinogenesis study using sequential treatment with diethylnitrosamine, Nmethylnitrosourea and N,N-dibutylnitrosamine as the initiation step, Jang et al. [lo] showed that subsequent treatment with ally1 sulfide significantly deceased the incidence of hepatic hyperplastic nodules, adenoma of the lung and thyroid and hyperplasia of the urinary bladder in mice. Although the chemopreventive properties of garlic have been clearly demonstrated in animal models, little information is available regarding the effectiveness of garlic in the prevention or treatment of human cancers. Epidemiological studies have shown inverse associations between the consumption of garlic and incidence of gastric cancer in China [13] and Italy [4] as well as risk of thyroid nodularity among women in China [25]. Scharfenberg et al. [19] recently reported the results of an in vitro study using an established tumorigenic Ireland Ltd
212
cell line derived from a Burkitt lymphoma. Treatment of the lymphoid cells with ajoene, a lipid-soluble oxidation product of raw garlic, caused inhibition of cell proliferation in the lymphoid cell line; however, a toxic effect was also evident in non-tumor cells studied in parallel. Water-soluble compounds derived from garlic may be less cytotoxic than oilsoluble compounds, presumably because they are not stored in body tissues. SAC, a watersoluble component of aged garlic extract, has previously been shown to inhibit 1,2-dimethylhydrazine-induced colon cancer in rats [22]. Takeyama et al. [23] have reported that SAC can inhibit human melanoma cell proliferation and may induce differentiation of tumor cells to a mature, non-tumorigenic phenotype. This apparent antitumoral action on melanoma cells prompted our study of the effects of SAC on human neuroblastoma (NB), a tumor that originates, like melanoma, from tissue of the neural crest. In this study, we tested the ability of SAC to affect the proliferation and differentiation of the human NB cell line LA-N-5, cells shown to be remarkably sensitive to a variety of differentiating agents. In addition, we investigated the potential of SAC to enhance the effects of retinoic acid (RA) and &bromocyclic AMP (8BrcAMP) , compounds known to induce neuronal differentiation in this line. The results of our study confirm the antiproliferative action of SAC on human neural crestderived tumor cells but do not support the hypothesis that this effect results from induction of differentiation. Materials and Methods Chemicals S-allyl-r-cysteine, kindly provided by Wakunaga of America, Mission Viejo, CA, was dissolved in phosphate-buffered saline (pH 7.0) to a stock concentration of 40 mg/ml, filtered with Nalgene disposable syringe filters (Rochester, NY) and stored at - 70°C. All-trans-retinoic acid (Sigma Chemical Co., St. Louis, MO) was dissolved
in dimethyl sulfoxide to a concentration of 10T2 mM and stored at -2OOC. [Methyl3H]thymidine (6.7 Ci/mmol) and [3H]tyrosine (50 Ci/mmol) were obtained from NEN Research Products, Claremont, CA. (6R)-Lerythro-5,6,7,8_tetrahydrobiopterin, which was used in the tyrosine hydroxylase assay, was a generous gift from Dr. John F. Reinhard, Burroughs Wellcome, Research Triangle Park, NC. All other chemicals were purchased from Sigma Chemical Co. Cell Culture LA-N-5 human neuroblastoma cells were grown as monolayers in RPM1 1640 medium (M.A. Bioproducts, Walkersville, MD) supplemented with 10% heat-inactivated fetal calf serum (Gemini Bioproducts, Inc., Calabasas, CA), HEPES buffer, 50 IU/ml penicillin, 50 pg/ml streptomycin and 1 pg/ml amphotericin. Cultures were maintained in a humidified incubator at 37OC with an atmosphere of 95% air and 5% C02. Unless noted otherwise, cells for each experiment were plated into T25 (25 cm*) tissue-culture flasks (Costar Corp., Pleasonton, CA) and incubated overnight with 5 ml of medium to allow attachment and spread of cells. Various treatments were then added directly to the medium as indicated. [3H]thymidine incorporation Cells were plated in 24-well plastic tissue culture trays (Corning, Palo Alto, CA) at a density of 4 x lo3 cells/well. Various concentrations of SAC (200- 1600 pg/ml), RA (5 x lO-‘j M), or SAC + RA were added to the culture medium after 24 h (day 1). On day 7, 1 &i [3H]thymidine/well was added. In separate time course experiments, cells were treated with 800 pg/ml SAC for either 2, 4, or 6 days before the addition of [3H]thymidine. All cells were harvested 16 h after administration of labelled thymidine as previously described [27]. Radioactive incorporation was measured in a liquid scintillation counter according to standard procedures.
213
Morphology Cultures were treated with SAC (ZOO3200 pg/ml), RA (4 x 10m6 M), or SAC + RA. Cells from various regions of the flasks were evaluated by light microscopy at 1, 4, 6, 7, 8 and 14 days post-treatment for morphologic changes. Acetylcholinesterase (AChE) assay Cultures were treated with SAC (400 or 800 pg/ml), RA (5 x 10m6 M), or SAC + RA. On day 10, cells were harvested, pelleted and washed twice with ice-cold PBS. The cells were resuspended in 100 mM sodium phosphate buffer (pH 6.8) containing 0.2% Triton X-100 and sonicated for 10 s. AChE activity was determined photometrically, as described by Ellman et al. [7]. Total sample proteins were measured using the protein-dye binding method of Bradford [3] using bovine serum albumin as a reference protein. Tyrosine hydroxylase
I
I
I
0
I
400
I
800 SAC
I
1200
1600
(m/ml)
Fig.
1. Dose-response curves showing the effects of SAC alone (0) or SAC + 5 x 10e6 M RA (0) on the incorporation of [3H]thymidine in LA-N-5 neuroblastoma cells after 8 days of treatment. Values represent the mean f S.E.M. of triplicate samples of a typical experiment.
(TH) assay
Following a 3-day incubation with medium, cultures were treated with SAC (400 or RA (5 x 10m6 8BrcAMP 800 &ml), (1 mM), SAC + RA, or SAC + 8BrcAMP. On day 6, cells were harvested, washed and homogenized as described above. Aliquots were assayed for TH activity by the method of Reinhard et al. [17], in which [3H]Hs0 is hydrolysis of counted following the [3H]tyrosine. Proteins were determined by the method of Bradford [3].
16
t
blot analysis of N-myc expression Cells were harvested after 4 days of treat-
Northern
ment with either SAC (800pg/ml) or RA (3 x 10 -6 M) RNA was extracted with guanidium isothiocyanate and precipitated through a CsCl gradient [6]. An aliquot of RNA (20 pg) was denatured by glyoxal incubation, fractionated by electrophoresis through a 1% agarose gel and transferred to a Biotrans nylon membrane (ICN, Irvine, CA) using a positive pressure transfer apparatus (Stratagene, La Jolla, CA). The N-myc specific probe pNB-1
0
6
4
2 Days
of
SAC
treatment
Fig. 2. Time-course of [3H]thymidine incorporation by LA-N-5 cells in the absence (0) or presence (0) of 800 &ml SAC. Values represent the mean ZIZS.E.M. of triplicate samples of a typical experiment.
214
Morphology of LA-N-5 neuroblastoma cells in the absence and presence of SAC and/or RA. Cells were tre ated for 7 days with vehicle alone (a), 800 pg/ml SAC (b), 4 x 10e6 M RA (c), or 800 pg/ml SAC + 4 x 10-6M RA (d). The addition of SAC to the culture medium did not significantly alter cell morphology. x 200
Fig. 3.
215
216
was labelled by random priming (10’ counts/ min per pg) using a standard kit (Amersham, Arlington Heights, IL). Prehybridization and hybridization medium consisted of 6 x SSC, 5 x Denhardt’s solution, 0.5% SDS and 100 pg/mI salmon sperm DNA. Following 16-24 h of incubation at 68OC, filters were washed once in 1 x SSC, 0.1% SDS at room temperature for 20 min and twice in 0.2 x SSC, 0.1% SDS at 68OC. Autoradiography was performed using Kodak XOMAT film at - 70°C for 2 - 10 days. Be.&@ Cell prohferation The dose effect of SAC on [3H]thymidine uptake in LA-N-5 cells is illustrated in Fig. 1. Cells were treated with 200- 1600 pg/mI SAC, either alone or in combination with RA (5 x 10m6 M) for 8 days. A dose-dependent inhibition of cell proliferation was observed following incubation with SAC doses greater than 400 .pg/rnl SAC. The half-maximal response was observed with approximately 600 pg/ml SAC while greater than 99% inTablo 1.
Effect SAC alone, SAC + RA
Treatment
Control SAC (400 pg/ml) SAC (800 pg/ml) RA (5 x 10-6) RA + SAC (400 gg/ml) RA + SAC (800Ccg/ml) 8BrcAMP (1 mM) 8BrcAMP + SAC (400 kg/ml) 8BrcAMP + SAC (800 pg/ml)
hibition occurred with the maximum dose given. Combination treatment of cells with SAC + RA produced an additive inhibitory effect but no synergism between the two antiproliferative agents was observed (Fig. 1). Results from time-course studies, shown in Fig. 2, indicate that the growth inhibitory effect of SAC (800 pg/mI) occurred as early as two days following treatment and slowly increased with time. Inhibition of thymidine uptake was 66% after a 2-day incubation and increased to 72% following 6 days of treatment. A small toxic component of the antiproliferative effect of SAC was detected by the inability of some cells to exclude trypan blue dye and an increase in the rounding and detachment of cells from culture flasks. Cell differentiation Cell cultures treated for periods of up to 14 days were observed by light microscopy for changes in morphology. A representative experiment in which cells were treated for 7 days is shown in Fig. 3. Morphological differentiation of NB cells is typically reflected by the extension of long processes, or neurites [18].
and SAC + CAMP on AChE and TH activity. AChE activity (nmol/min per mg) 2.53 zt 0.15”*b 2.23 zt 0.18 2.41 * 0.52 13.76 zt 1.73 13.28 * 1.42 11.25 l 1.30 ND ND ND
TH activity (pmol/h per ccg) 6.69 zt 8.45 zt 6.85 zt
0.29’ 0.86 2.60
NDd ND ND 22.23 zt 3.20 21.18 * 5.60 26.92 zt 12.5
“Mean * S.E.M. from duplicate or triplicate samples. bTwo-way ANOVA: effect of RA, Fnro, = 201, P < 0.0005; effect of SAC, not significant (NS); effect of RA vs. RA + SAC, NS. ‘Two-way ANOVA: effect of 8BrcAMP, F,,, = 33.8, P < 0.001; effect of SAC, NS; effect of 8BrcAMP vs. 8BrcAMP + SAC, NS. dND, not determined.
217
In this study, no apparent increase in the number or length of processes was observed in SAC-treated cultures relative to untreated controls (Figs. 3a,b). In contrast, RA-treated cultures showed the expected pattern of morphological change that accompanies differentiation: extension of neurites and ganglion-like clustering within a couple of days followed by bundling of the processes and further cell clustering (Fig. 3~). Similar changes in morphology were observed in cultures treated with SAC + RA, with no enhancement of process extension or cell clustering relative to cultures treated with RA alone (Fig. 3d). The LA-N-5 cell line is comprised of immature cells that have the capacity to express multiple neurotransmitter phenotypes. Cell maturation can be detected, biochemically, by an increase in the specific activities of enzymes involved in neurotransmitter metabolism [27]. We tested the ability of SAC to induce differentiation along either an adrenergic or a cholinergic pathway by assaying for TH and AChE activity, as previously shown for induction of these cells by 8BrcAMP and RA, respectively [27]. As depicted in Table I, concentrations of SAC that were found to be antiproliferative did not result in induction of either enzyme. Furthermore, when given in combination with 8BrcAMP or RA, known inducers of the enzymes, SAC was unable to potentiate their effects,
c
SAC
RA
Fig. 4. Lack of effect of SAC on N-myc mRNA levels in LA-N-5 cells. Total cellular RNA (20 pg) was isolated from 4-day control (C), SAC (800 pg/ml)-, or RA (3 x 10e6 M)-treated LA-N-5 cells and subjected to Northern blot analysis using the pNB-1 N-myc specific probe. Hybridization of the same blots with a @actin probe confirmed that the lanes were loaded with comparable amounts of RNA.
Figure 4 shows the effect of SAC on N-myc expression. Decreased expression of the oncogene N-myc is an earlier marker of neuroblastoma differentiation than biochemical or morphological changes [ 1,12,24]. Treatment of LA-N-5 cells with 800 pg/ml SAC produced no change in N-myc mRNA levels while RA treatment caused a dramatic decrease in the amount of message produced as previously reported [2,24].
Discussion It has been known for many years that garlic and its derivatives exert an anticarcinogenic effect in rodents when administered prior to or simultaneously with carcinogen exposure [9,13,22]. While numerous animal studies have documented that these protective effects could be achieved against a diverse array of chemical carcinogens having different target organ specificities, there have been few experiments designed to investigate the protective or therapeutic potential of garlic in human cancer. In this study we have investigated the ability of a garlic derivative, SAC, to inhibit proliferation and promote differentiation of LA-N-5 human neuroblastoma cells. Our data indicates that SAC, at doses greater than 400 pg/ml, is a potent antiproliferative agent for human neuroblastoma cells. Takeyama et al. [23] have reported similar results for melanoma cells treated with SAC. In addition, they observed significant morphological changes, increased melanin content, and decreased melanoma-associated ganglioside expression in the tumor cells characteristic of a potentially less malignant phenotype [8,16]. Based on these observations, Takeyama suggested that the antiproliferative effect may arise as a result of SAC-induced cell differentiation. To further investigate this hypothesis, we studied NB cell differentiation following treatment with SAC. Neuroblastoma has been a prime model system for the study of neuronal differentiation and tumor cell maturation. The hallmarks of neuron differentiation are neurite
218
extension, growth inhibition, biochemical maturation including increased activity of AChE and decreased N-myc mRNA [2,14]. A variety of compounds have been shown to induce in vitro maturation of NB cells in a manner similar to that of normal differentiating neurons in culture [5,20,21]. In addition, several lines of evidence suggest that the state of tumor cell differentiation is related to the malignant potential of NB tumors [1,5,15]. Although SAC inhibited cell proliferation of LA-N-5 cells, our data provide no evidence of a SACinduced change in differentiation. Whereas processes were observed to extend from RAtreated control cells and form bundles, no outgrowths were seen in the SAC-treated cultures. Similarly, AChE and TH activities were increased by the known inducers RA and 8BrcAMP, respectively; however, SAC treatment produced no changes in these enzyme activity levels. Finally, N-myc expression was significantly decreased in RA-treated cells but unchanged in SAC-treated cultures relative to untreated controls. The differentiation-inducing effect of RA in LA-N-5 cells has been shown to be potentiated by combination treatment with other differentiating agents, including interferon-gamma [27] and CAMP-elevating compounds [ll]. In order to assess the potential of SAC to enhance induction by other agents, we evaluated growth, morphologic and biochemical changes (TH and AChE activities) following treatments with SAC and/or RA (or SAC + 8BrcAMP for TH activity). Although combination treatment resulted in an additive inhibition in the growth rate of LA-N-5 cells, no changes in enzyme activities were detected in combination-treated cultures relative to singly-treated (RA or CAMP) controls. These results are consistent with the notion that SAC induces growth inhibition of LA-N-5 cells independently of differentiation. SAC and other garlic-derived compounds have clearly demonstrated potent anti-proliferative actions in cultured mammalian cells. However, there are conflicting reports regarding their cytotoxic effects. Although we and others [13,19,23] have observed cytotoxicity
in several tumor cell lines, two recent reports suggest that SAC is less toxic [19] or non-toxic [23] to normal cells. Together with these reports, the present results underscore the need to compare the anti-proliferative and cytotoxic effects of SAC and other garlic derivatives on normal and tumorigenic cells in order to fully evaluate their therapeutic potential. Acknowledgements Portions of this work were supported by NIH grants CA43503 and CA30515. The authors wish to thank Dr. Reiko F. Irie for many interesting and helpful discussions. References Abemayor, E. and Sidell, N. (1989) Human neuroblastoma cell lines as models for the in vitro study of neoplastic and neuronal cell differentiation. Environ. Health Perspect., 80, 3 - 15. Amatruda III, T.T., Sidell, N., Ranyard, J. and Koeffler, H.P. (1985) Retinoic acid treatment of human neuroblastoma cells is associated with decreased N-myc expression. Biochem. Biophys. Res. Commun., 126, 1189 - 1195. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248 - 254. Buiatti, E., Palli, D., Decarli, A., Amadori, D., Avellini, C., Bianchi, S., Biserni, R., Cipriani, F., Cocco, P., Giacosa, A., Marubini, E., Puntoni, R., Vindigni, C., Fraumeni, J., Jr. and Blot, W. (1989) A case-control study of gastric cancer and diet in Italy. Int. J. Cancer, 44, 611-616. 0. and Kornhuber, B. Busse, E., Bartsch, Research on the differentiation of human and neuroblastoma cells. Oncology, 48, 196- 201.
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