Effect of caffeic acid esters on carcinogen-induced mutagenicity and human colon adenocarcinoma cell growth

Chem.-Biol. Interactions, 84 (1992) 277-290 Elsevier Scientific Publishers Ireland Ltd.

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EFFECT OF CAFFEIC ACID ESTERS ON CARCINOGEN-INDUCED MUTAGENICITY AND HUMAN COLON ADENOCARCINOMA CELL GROWTH CHINTHALAPALLY V. RAOa, DHIMANT DESAIb, BIPIN KAULa, SHANTU AMINb and BANDARU S. REDDYa aDivisio~ of Nutritional Carcinogenesis and bChemical Carcinogenesis, American Health Foundation, Dana Road, Valhalla, New York (USA) (Received June 9th, 1992) (Revision received August 14th, 1992) (Accepted August 17th, 1992)

SUMMARY

Propolis, a honey bee hive product, is thought to exhibit a broad spectrum of activities including antibiotic, antiviral, anti-inflammatory and tumor growth inhibition; some of the observed biological activities may be due to caffeic acid (cinnamic acid) esters that are present in propolis. In the present study we synthesized three caffeic acid esters, namely methyl caffeate (MC), phenylethyl caffeate (PEC) and phenylethyl dimethylcaffeate (PEDMC) and tested them against the 3,2'-dimethyl-4-aminobiphenyl, (DMAB, a colon and mammary carcinogen)-induced mutagenecity in Salmonella typhimurium strains TA 98 and TA 100. Also, the effect of these agents on the growth of human colon adenocarcinoma, HT-29 cells and activities of ornithine decarboxylase (ODC) and protein tyrosine kinase (PTK) was studied. Mutagenicity was induced in Salmonella typhimurium strains TA 98 and TA 100 plus $9 activation using 5 and 10 ~g DMAB and antimutagenic activities of 0-150 ~M MC, 0 - 60 ~M PEC and 0 - 80 ~M PEDMC were determined. The results indicate that MC, PEC and PEDMC were not mutagenic in the Salmonella tester system. DMAB-induced mutagenicity was significantly inhibited with 150 ~M MC, 40-60 ~M PEC and 40- 80 ~M PEDMC in both tester systems. Treatment of HT-29 colon adenocarcinoma cells with > 150 ~M MC, 30 ~M PEC and 20 ~M PEDMC significantly inhibited the cell growth and syntheses of RNA, DNA and protein. ODC and PTK activities were also inhibited in HT-29 cells treated with different concentrations of MC, PEC and PEDMC. These results demonstrate that caffeic acid esters which are C o r r e s p ~ e to: B.S. Reddy, Division of Nutritional Carcinogenesis, American Health Foundation, Dana Road, Valhalla, New York, USA. Abbreviations: CA, caffeic acid; DMAB; 3,2'-dimethyl-4-amino-biphenyl; HMPA, hexamethylphosphoric triamide; MC, methylcaffeate; ODC, ornithine decarboxylase; PEC, phenylethyl caffeate; PEDMC, phenylethyl dimethylcaffeate; PTK, protein tyrosine kinase. 0009-2797/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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present in Propolis possess chemopreventive properties when tested in shortterm assay systems.

Key words: Caffeic acid esters -- Mutagenicity -- Colon cancer -- Ornithine decarboxylase -- Protein tyrosine kinase

INTRODUCTION

In recent years, the biological properties of propolis (natural honey) have become a point of particular interest because it is thought to exhibit a broad spectrum of activities including antibacterial, antifungal, cytostatic and antiinflammatory properties [1- 3]. Propolis contains a variety of compounds including cinnamic acid, benzoic acid and their esters, substituted phenolic acids and esters, flavonoid glycones and bee wax [4]. Some of the observed activities of propolis may be due to its chemical constituents [3,5]. Several naturally-occurring compounds in fruits and vegetables such as phenols, indoles, aromatic isothiocyanates and dithiolethiones have been shown to inhibit several types of cancer including the cancer of the colon [6]. Previously, Wattenberg et al. [7] demonstrated that dietary administration of hydroxycinnamates significantly inhibited benzo[a]pyrene-induced neoplasia of the forestomach in mice. This protective action of hydroxycinnamates appears to be due to their phenyl proponoid group, which is known to have antioxidant properties [8] as well as a potent modulator of many enzyme activities [9]. Caffeic acid (3,4-dihydroxycinnamic acid) ester derivatives present in propolis are more lipophilic and thus easily facilitate their entry into cells [4]. Also, they require small quantities to exhibit their effective inhibitory activities [5]. However, extraction of these compounds requires large quantities of propolis and also the intact structures will be modified during the extraction procedure. To overcome these technical difficulties, we synthesized several caffeic acid esters for further testing of their chemopreventive properties using short-term screening assays. Several cellular components that have been associated with cell proliferation may play a key role in tumor promotion. It is possible that suppression of activities of these cellular components may modulate tumor inhibition. For example, intercellular polyamine levels (putrescine, spermidine and spermine) and polyamine-synthetic enzyme activities such as ornithine decarboxylase (ODC) are high in proliferating normal cells [10], neoplastic cells [11] and cells undergoing neoplastic transformation by carcinogens [12]. Therefore, lowering of polyamine levels by inhibiting ODC activity, may prevent the proliferative activity of neoplastic cells [13]. Several kinases such as protein tyrosine kinases (PTK) mediate proliferative signals as well as metabolic signals and such increased expression of PTK activity may be responsible for unlimited growth [14]. Increased PTK activity has been observed in neoplastic tissues [15]. This realization promoted the hypothesis that PTK blockers can in principle become useful antiproliferative agents [16,17].

279

In the present study, several derivatives of caffeic acid esters namely, methyl caffeate (MC), phenylethyl caffeate (PEC) and phenylethyl dimethylcaffeate (PEDMC) were synthesized (Fig. 1) and tested for their antimutagenic activity in Salmonella typhimurium strains TA 98 and TA 100; also the inhibitory effects of these compounds on the growth of human colon HT-29 cells and on the activities of ODC and PTK were studied. The long-term goal of this study is to develop new agents as potential inhibitors of colon carcinogenesis. MATERIALS AND METHODS~

DMAB was obtained from Ash-Stevens (Detroit, MI) and poly (Glu-Tyr) from Sigma, (St. Louis, MO). [1-14C]Ornithine and [7-32p]ATP were purchased from Amersham (Arlington Heights, IL). [3H]Thymidine, [3H]uridine and [3H]leucine were obtained from New England Nuclear/Dupont (Boston, MA). Human colon HT-29 cells were obtained from ATCC (Rockville, MD). Salmonella typhimuvium strains TA 98 and TA 100 were kindly supplied by Dr. Bruce Ames, University of California at Berkeley, CA.

Synthesis of caffeic acid esters (derivatives). Methyl caffeate [2-propenoic acid, 3-(3,4-dihydroxyphenyl) methyl ester] and Bphenylethyl caffeate [2-propenoic acid, 3-(3,4 dihydroxyphenyl)-2-phenylethyl ester] were prepared according to the methods of Helferich et al. [18] and Grunberger et al. [7]./~-Phenylethyl dimethylcaffeate [2-propeonic acid, 3-(3,4~methoxyphenyl)-2-phenylethyl ester] was prepared by using a modified proce~dure of Hashimoto et al. [19]. Briefly, the method of synthesis of PEDMC is as follows. To a stirring solution of 3,4-dimethoxycinnamic acid (1.04 g, 5 mmol) in hexamethylphosphoric triamide (HMPA, 10 ml), aqueous sodium hydroxide solution (25%, 2.5 ml) was added. The reaction mixture was stirred at room temperature for 1 h. Then a solution of (2-bromoethyl) benzene (2.8 ml) in HMPA (5 ml) was added drop-wise and the stirring was continued for a further 48 h. The H

Caffeicacid (CA) H

H~OO,CH2,CH2,C6Hs

COOH

COOCH3

PhenylethylCaffeate (PEC) []

C H 3 0 ~

COO.CH2.CH2.C6Hs

H

CH30f

MethylCaffeate (MC)

PhenylethyldimethylCaffeate (PEDMC)

Fig. 1. Structural formula of caffeic acid and its esters.

280

mixture was poured into 50 ml of ice water and extracted with ethylacetate (2 x 30 ml). The combined organic layer was washed with 1 N HC1 followed by H20, dried over MgSOa, filtered and evaporated. The residue was chromatographed on silica gel with CH2C12/ethyl acetate (7:3) as an eluent to yield/~-phenylethyl dimethyl caffeate (1.53 g, 97% yield) which was crystallized from hexane, m.p. 95-96°C (lit. m.p., 96-98°C [19]).

Mutagenicity Mutagenicity was assayed by the histidine-reversion plate incorporation method as described by Maron and Ames [20]. The microsomal $9 fraction (9000 × g supernatant) was prepared by standard procedure [20] from the livers of Aroclor 1254-treated male F344 rats (Charles River Breeding Laboratories, Kingston, NY) and stored at -80°C until use. Each test compound was dissolved in DMSO and assayed in quadruplicate with and without $9 activation for its mutagenic and antimutagenic activities. 2Nitrofluorene (5 #g/plate) and sodium azide (1.5 ~g/plate) were used as positive controls for TA 98 and TA 100 without $9 activation. DMAB (5 and 10 t~g/plate) was used as a positive control for both TA 98 and TA 100 with $9 activation. In addition, the rationale for using DMAB has been based on the fact that this compound induces tumors in colon, mammary gland and prostate of rodents [21]. The toxicity of MC, PEC and PEDMC toward TA 98 and TA 100 was determined in complete medium using increasing concentrations upto 250 t~M/plate with or without $9 activation. Similarly, caffeic acid (CA) toxicity on TA 98 and TA 100 was determined using concentrations up to 4000 t~M/plate with or without $9. Toxic effects were evaluated by examination of the background lawn, size and appearance of colonies. Each dose of mutagen was prepared with 2 ml of top agar with and without 0.25 ml of $9 mix and 0.1 ml of overnight bacterial culture with or without the specified doses of CA, MC, PEC and PEDMC. The range of concentrations selected for mutagenicity testing of CA, MC, PEC and PEDMC was based on our preliminary toxicity tests in which the highest concentration of these agents had no inhibitory effect on the TA 98 and TA 100.

Cell line The human colon cell line, HT-29, was maintained in McCoys 5A medium supplemented with L-glutamine and 7.5% heat inactivated fetal bovine serum. Cells were grown at 37°C in a humidified incubator containing 10% CO2. HT-29 cells grow as firmly adherent monolayer; those cells unable to grow or dead riot in the medium. Cells were routinely maintained in exponentially grown monolayer culture by seeding 105 cells/25 cm 2 flasks and splitting every 3 days.

Cytotoxicity and cell growth The cytotoxic effect of these agents was tested in 24-well plates by exposing the cells to various concentrations of CA, MC, PEC and PEDMC for a period of 48 h. Viability was assayed by the trypan blue dye exclusion method [22]. For determination of growth inhibitory effect of CA, MC, PEC and PEDMC, 0.48 × 105 cells were plated in 6-well dishes with several sub-cytotoxic concentra-

281

tions of these agents for 48 h. The concentration of DMSO was adjusted to be less than 0.5%. Cells from quadruplicate dishes were washed once with Hanks' Balanced Salt Solution (HBSS). By this process, the dead cells that are floating will be separated from the live monolayer. Then the monolayer was trypsinized, stained with trypan blue and counted. The total number of viable cells in the control was considered as equal to 100% viability and the agent-treated cells were compared with the control for the determination of % viability. The % viability of agent-treated cells was calculated from the total number of viable cells in the control plate and in the agent-treated plate. In order to relate the cell growth data with macromolecule synthesis, we studied the rate of synthesis of DNA, RNA and protein in HT-29 cells which were estimated by measuring the incorporation of [3H]thymidine, [3H]uridine and [3H]leucine into DNA, RNA and protein, respectively. For each determination, HT-29 cells were incubated with various concentrations of MC, PEC and PEDMC for 48 h. Then the monolayer of cells were pulsed with 1 ~Ci/ml medium of [3H]thymidine (20 Ci/mmol) and [3H]uridine (50 Ci/mmol) and with 2 ~Ci]ml medium with [3H]leucine (140 Ci/mmol) and incubated for another 3 h at 37°C. Radioactive media were then removed and the monolayer was washed with 2 ml of HBSS and 2 ml of ice-cold 10% TCA for 10 min. DNA was extracted with 0.5 ml of 2 N perchloric acid. The DNA extract was placed on hot plate at 60°C for 30 min, then allowed to cool and transferred to a scintillation vial and counted. RNA and protein were extracted with 1.0 ml of cold 0.1 N NaOH and 1.0 ml of hot 1N NaOH. The samples were then neutralized with 6 N HC1 and scintillation cocktail was added prior to counting in the liquid scintillation counter. In order to determine the effect of PEDMC on DNA synthesis at different time periods, HT-29 cells were exposed to 50 #M PEDMC and the [3H]thymidine incorporation into DNA was measured at every 12 h by above procedure until the treated cells formed the monolayer.

ODC and P T K activities The effect of various sub-cytotoxic concentrations of MC, PEC and PEDMC on ODC and PTK activities of HT-29 cells was determined by exposing cells to the test agents in 25 c m 2 flasks for a period of 72 h. After incubation, the cells were washed twice with cold HBSS, harvested with a cell scraper and centrifuged at 500 x g for 10 min at 4°C. The cytosol fraction was prepared by homogenizing the cells in 1 ml of homogenizing buffer (50 mM sodium phosphate buffer (pH 7.2), containing 5 mM dithiothretol, 0.1 mM EDTA, 0.1 mM pyridoxal 5-phosphate) using a polytran homogenizer (Brinkman Instruments, Westbury, NY). The homogenate was centrifuged at 100 000 × g at 4°C for 1 h and the ODC activity was determined in the cytosol fraction [23]. Briefly, the reaction mixture containing 50 mM sodium phosphate, 2 mM L-ornithine and 0.25 #Ci of D,L-[1-14C]ornithine (58 Ci/mmol) in a final volume of 250 ~l was incubated for 1 h at 37°C. The reaction was stopped by adding 200 ~l of 2 N sulfuric acid and the 1aco2 released was collected for another 45 min. Radioactivity was counted in a Beckman Scintillation counter model LD6800.

282

PTK activity was measured in membrane fraction of HT-29 cells. Membrane fractions were prepared from HT-29 cells by homogenizing in ice-cold buffer containing 20 mM Tris-HC1 (pH 7.4), 0.25 M surcose, 1 mM MgCI2, 1 mM EDTA, 50 gg/ml trypsin inhibitor, 10 ttg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride and centrifuged at 100 000 × g at 4°C for 1 h. PTK activity was measured using poly Glu-Tyr, 4.1:0.9, (Sigma Chemical Co, St. Louis, MO) as substrate, which has been shown to be highly specific for PTK [23,24]. Phosphorylation of membrane proteins (5-10/~g/assay) was carried out at 24°C in a total volume of 50 ttl reaction mixture, containing 50 mM Tris- HC1 (pH 7.4), 20 mM MgC12, 0.02% Triton X-100, 50 gM sodium o-vanadate, and 20 #g of GhTyr polymer. The reaction was initiated with 50 gM ATP and 0.4 gCi [~¢82P]ATP (11.7 Ci/mmol). The reaction was terminated by applying 25 gl of reaction mixture on a 2.3 cm 2 Whatman No. 3 filter paper disc. The filters were washed several times in 10% trichloroacetic acid containing 10 mM sodium pyrophosphate, rinsed with ethanol and petroleum ether, dried and counted in 10 ml of scintillation cocktail. Protein content was determined by the Bio-Rad method using bovine serum albumin as standard. Results are expressed as pmol 82p-incorporated]mg protein/min.

Statistical analysis Statistical analysis of data was performed by Student's t-test and P < 0.05 was considered significant. RESULTS

The survival rates for Salmonella typhimurium strains TA 98 and TA 100 were over 98% at concentrations of 2,500 gM CA, 150 #M MC, 70 ttM PEC and 80 ttM PEDMC/plate. Higher concentrations of these compounds significantly decreased the survival of both TA 98 and TA 100. CA, MC, PEC and PEDMC were not mutagenic toward Salmonella typhimurium TA 98 and TA 100 with or without $9 activation at all the concentrations employed in this study (Data not shown). The effect of various concentrations of MC, PEC and PEDMC on DMAB-induced mutagenicity is shown in Fig. 2. In strains TA 98 and TA 100, 50 gM MC, 20 gM PEC and 20 gM PEDMC had no inhibitory effect on DMABinduced mutagenicity. However, 150 gM MC significantly inhibited DMABinduced mutagenicity in both strains. Similarly increasing concentrations of PEC (40-60 gM) and PEDMC (40-80 gM) significantly inhibited DMABinduced mutagenicity in both strains in a dose-dependent manner. The toxic effects of various concentrations of CA, MC, PEC and PEDMC on HT-29 cells were also tested. The results indicate that MC at a concentration greater than 225 gM and PEC and PEDMC at a level greater than 60 gM were toxic. CA exhibited significant toxicity only at above 2500 gM concentration. The growth inhibitory effect of these compounds measured by cell number after exposing cells for a period of 48 h is shown in Fig. 3. CA was found to be the least effective growth inhibitor of HT-29 cells when compared to its ester analogs. CA significantly inhibited the growth of HT-29 cells at above 2225 gM concentration

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Fig. 3. Effect of caffeicacid (CA), methyl caffeate(MC),phenylethylcaffeate(PEC) and phenylethyl dimethylcaffeate (PEDMC)on human colon HT-29 growth.

(Fig. 3). MC was found to be a less effective growth inhibitor than the PEC and PEDMC. MC inhibited the growth of HT-29 cells at concentrations greater than 150 ~M. PEC and PEDMC showed inhibitory effect at much lower concentrations with an IDs0 of 55 and 36 ~M, respectively. Growth inhibition with PEDMC was dose-dependent. Also, the inhibitory effect of PEC and PEDMC on human colon HCT-116 cells (malignant type) were more effective at much lower concentrations (IDs0 < 25 ~M) when compared with HT-29 cells (date not shown). In order to further investigatethe growth inhibitoryproperties of MC, P E C and P E D M C on HT-29 cells,the effect of these agents on polynucleotidesynthesis and protein synthesis was investigated. Figure 4 shows the effect of various concentrations of MC, P E C and P E D M C following a 48-h incubation with 0.5 × 105 cells/mlon DNA, R N A and protein synthesis.M C and P E C at concentrationsgreater than 175 and 40 ~M, respectively,inhibitedsignificantly the DNA, R N A and protein synthesis.The degree of inhibitionwas more pronounced with P E D M C than PEC. P E D M C inhibitedthe polynucleotideand protein synthesis in a dose-dependent manner; at 60 ~ M concentration, the inhibitionreached to about 90%. A similar reduction in viabilitymeasured by trypan blue exclusionmethod was obtained by these agents (Fig. 3), confirming the validityof [SH]thymidine, [3H]uridine and [SH]leucineassays as indicators of metabolic integrity. Figure 5 shows the effect of 50 ~ M concentration of P E D M C on D N A synthesis at various time periods. The results indicate that P E D M C significantlyreduces the HT-29 cell D N A synthesis upto 84 h; this

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Fig. 4. Effect of methylcaffeate (MC), phenylethyi caffeate (PEC) and phenylethyl dimethylcaffeate (PEDMC) on the rate of DNA, RNA and protein synthesis human colon HT-29 cells.

reduction of DNA synthesis at different time points corroborates cell viability at particular time point measured by trypan blue exclusion method (data not shown). Table I shows the effect of MC, P E C and P E D M C on O D C and P T K activities of HT-29 cells.O D C activitywas significantlyinhibitedat concentrations greater

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Hours Fig. 5. Effect of 50 ~M phenylethyl dimethylcaffeate (PEDMC) on D N A s y n t h e s i s h u m a n colon HT29 cells at various time points. TABLE I E F F E C T OF MC, P E C A N D P E D M C ON ODC A N D P T K A C T I V I T I E S OF H U M A N COLON HT29 C E L L L I N E

Concentration (~M)

ODC activity pmol 14CO2/mg protein/h

PTK activity pmol [32p]ATP/mg protcirgmin

0 25 50 100 150 200

42.5 39.4 40.3 35.8 32.4 24.3

± + * ± ± ±

6.8 a 8.1 7.3 8.8 6.1 b,* 4.95'***

95.2 88.1 80.2 75.4 68.3 56.7

0 10 20 30 40 50

48.6 46.3 45.3 41.8 39.3 33.4

~± ± + ± ±

7.2 7.5 6.9 7.2 6.4 b,* 5.6 b,**

104.6 98.7 90.3 80.3 64.2 53.9

± ± ± ± ± ±

0 10 20 30 40 50

48.6 40.5 32.4 19.2 16.4 10.4

± + ± ± ± ±

7.2 7.3 6.6 b,** 3.15'*** 4.25'*** 2.75'***

104.6 84.3 68.4 70.6 62.9 60.1

± 16.1 ± 9.2

MC ± 12.2 a ± 8.9 ± 8.3 ± 9.65'* ± 10.15'** ± 7.2 b'***

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±

7.5 b'**

± 8.3 b'** :t: 6.55'*** ± 7.3 b'***

287

than 150 ~M MC, 40 ~M PEC and 20 #M PEDMC. PEDMC inhibited ODC activity in a dose-dependent manner. PTK activity was also significantly suppressed at concentrations greater than 100 ~M MC, 30 ~M PEC and 20 ~M PEDMC. DISCUSSION

The results of the present study demonstrate that naturally-occurring derivatives of caffeic acid esters, namely MC, PEC and PEDMC suppress the mutagenic activity of DMAB, a potent colon, prostate and mammary carcinogen. The results of antimutagenic activity of these compounds are in agreement with earlier studies which indicate that cinnamic acid derivatives inhibit the mutagenicity of benzo[a]pyrene epoxide and N-methyl-N'-nitro-N-nitrosoguanidine [25,26]. Previous studies demonstrated that caffeic acid is not mutagenic to Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 [27]. Mulky et al. [28] showed that CA inhibited the 7,12dimethylbenzanthracene-induced mutagenecity in Salmonella typhimurium TA 98 with $9 mix. Francis et al. [29] demonstrated that CA suppressed the mutagenicity of aflatoxin B1 and N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium strain TA 100. The inhibitory effect of MC, PEC and PEDMC on the $9 dependent mutagenicity of DMAB could be due to modulation of enzyme activities which are involved in their activation and/or detoxification pathways or to alterations in the extent of DNA binding and repairing rate. The results of the present study on human colon HT-29 cell line growth and DNA synthesis suggest that MC, PEC and PEDMC may possess antiproliferative and antitumorigenic properties. The observation that PEC and PEDMC inhibit cell growth and DNA synthesis better than MC, suggests that the increased lipophilicity of PEC and PEDMC which facilitates their transport into the cells may, in part, be responsible for their inhibitory effect. Earlier studies demonstrated that derivatives of hydroxycinnamic acids protected against benzo[a]pyrene-induced neoplasia of mouse forestomach [7] and methylazoxymethanol acetate-induced large intestinal carcinogenesis [30]. Grunberger et al. [5] have shown that PEC has a strong growth inhibitory effect against the various cell lines such as Fischer rat embryo fibroblast (CREF), human breast carcinoma (MCF-7), and melanoma (SK-MEL-28 and SK-MEL-170) cultures. Effect of PEDMC on DNA synthesis at various time intervals indicates that this compound delays the DNA synthesis. Previously Umezawa et al. [31] observed that the epidermal growth factor-induced DNA synthesis was significantly delayed by 2,5-dihydroxycinnamic acid methylester in quiescent normal rat kidney cells. Recently, Su et al. [32] also showed that PEC suppresses the adenovirus type 5E1A-induced transformation and expression of transformed rat embryo fibroblast cells in a dose-dependent manner. ODC is a rate-limiting enzyme in the polyamine biosynthesis and has been correlated with the rate of proliferation in several tissues [10]. ODC has been shown to increase in several tissues upon exposure to carcinogens and promoting agents [11, 12]. It was also demonstrated that agents which inhibit ODC activity, are effective tumor inhibitors [13, 33]. With regard to caffeic acid esters, recently Frenkel et al. [34] had shown that PEC significantly inhibited 12-O-tetra-

288 decanoylphorbol-13-acetate (TPA) induced epidermal ODC activity in mouse. Furthermore, in the present study MC, PEC and PEDMC significantly inhibit the PTK activity. The exact mechanism by which these compounds inhibit PTK activity is not clearly known. There is, however, a possibility that the mechanism of inhibition of PTK activity by these compounds might be similar to that of structurally-related tyrosine analogues such as Erbstatin, Tyrphostin, dihydroxycinnamates and cinnamamides-like specific PTK inhibitors [16,17,35]. These agents inhibit PTK activity by blocking the peptide site of E G F receptor as well as autophosphorylation of E G F receptors and the Src-encoded tyrosine kinase activity [17,36-38]. These results further support the antineoplastic activity of these compounds on human colon HT-29 cells. In summary, the results of present study demonstrate that (a) caffeic acid esters MC, PEC and PEDMC present in propolis, are not mutagenic with or without 8 9 activation but possess the antimutagenic activity against DMABinduced mutagenecity in Salmonella t y p h i m u r i u m strains TA 98 and TA 100, and (b) these compounds significantly inhibit the human colon HT-29 cell growth and the ODC and PTK activities at much lower concentrations when compared to CA. F u r t h e r work is needed to evaluate the efficacy of these compounds as chemopreventive agents against several neoplasia. ACKNOWLEDGEMENTS

We thank Donna Virgil for the preparation of the manuscript. This investigation was supported by USPHS Grant CA-17613 from the National Cancer Institute. REFERENCES

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