[CANCER RESEARCH 55, 2310-2315. June 1. 1995]

Chemoprevention of Colon Carcinogenesis by Phenylethyl-3-methylcaffeate1

Chinthalapally V. Rao, Dhimant Desai, Abraham Rivenson, Barbara Sinn, Shantu Amin, and Bandaru S. Reddy2

Divisions (tf Nutritional Carcinogenesis 1C. V. R., B. S., B. S. R.I. Chemical Carcinogenesis ID. D.. S. A.], ami Patholog\ and To.\icolot>\ ¡A./?./. American Heallli Foundation.

Valhalla. New York 10595

ABSTRACT

Previous studies from this laboratory have established that caffeic acidesters present in propolis, a natural resin produced by honey bees, arepotent inhibitors of human colon adenocarcinoma cell growth, carcinogen-induced biochemical changes, and preneoplastic lesions in the rat

colon. The present study was designed to investigate the chemopreventiveaction of dietary phenylethyl-3-methylcaffeate (PEMC) on azoxymethane-

induced colon carcinogenesis and to examine the modulating effect ofPEMC on phosphatidylinositol-specific phospholipase C (PI-PLC), phos-

pholipase A,, lipoxygenase (LOX), and cyclooxygenase activities in thecolonie mucosa and tumor tissues in male F344 rats. At 5 weeks of age,groups of rats were fed the control i modified AIN-76A) diet, or a diet

containing 750 ppm of PEMC. At 7 weeks of age, all animals except thosein the vehicle (normal saline)-treated groups were given 2 weekly s.c.

injections of azoxymethane at a dose rate of 15 mg/kg body weight/week.All groups were maintained on their respective dietary regimen until thetermination of the experiment 52 weeks after the carcinogen treatment.Colonie tumors were evaluated histopathologically. Both colonie mucosaand tumors were analyzed for PI-PLC, phospholipase A „cyclooxygenase,

and LOX activities. The results indicate that dietary administration ofPEMC significantly inhibited the incidence and multiplicity of invasive,noninvasive, and total (invasive plus noninvasive) adenocarcinomas of thecolon (/' < 0.05-0.004). Dietary PEMC also suppressed the colon tumor

volume by 43% compared to the control diet. Animals fed the PEMC dietshowed significantly decreased activities of colonie mucosa! and tumorPI-PLC (about 50%), but PEMC diet had no effect on phospholipase A2.The production of 5(S)-, 8(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic

acids via the LOX pathway from arachidonic acid was reduced in coloniemucosa and tumors (30-60% ) of animals fed the PEMC diet as compared

to control diet. PEMC had no effect on the formation of colonie mucosa!cyclooxygenase metabolites but inhibited the formation in colonie tumorsby 15-30%. The precise mechanism by which PEMC inhibits colon tu-

morigenesis remains to be elucidated. It is likely that the chemopreventiveaction may be related, at least in part, to the modulation of PI-PLC-dependent signal transduction and LOX-mediated arachidonic acid

metabolism.

INTRODUCTION

Large bowel cancer is one of the leading neoplastic diseases in bothmen and women in Western countries, including North America (1,2). Although several epidemiological and experimental studies suggest a relationship between colon cancer risk and dietary factors, theetiology of colon cancer is multifactorial and complex in that it mayarise from the combined action of low level environmental (many ofwhich as yet unidentified) and genotoxic agents, and dietary and hostfactors (1-4). Prevention strategies for cancer control involving re

duction or elimination of human exposure to environmental riskfactors may not always be possible; however, agents that can alleviateor diminish the carcinogenic effect of several environmental agentshave been identified and tested for their chemopreventive efficacy (5).

Received 12/15/94; accepted 4/4/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by USPHS Grants CA 17613 and CA 44377 awarded by the National

Cancer Institute.2 To whom requests for reprints should be addressed, at Division of Nutritional

Carcinogenesis, American Health Foundation. Valhalla, NY 105°5.

Wallenberg (5), Boone et al. (6), and Kelloff et al. (7) reviewed Iheresults of many Chemoprevention studies in laboratory animal models,as well as in the human setting.

The use of naturally derived products or their active principles inthe prevention and/or treatment of chronic diseases is based on theexperience of traditional systems of medicine practiced in variousethnic societies, and on epidemiological observations of the relationship of dietary practices and disease patterns. Isolation, identification,and testing of active substances not only provide naturally occurringnovel agents as inhibitors of cancer development but also offer uniqueopportunities to study the mechanisms of carcinogenesis. Propolisfrom honey beehives contains various chemical constituents thatexhibit a broad spectrum of activities including antibacterial, antifun-gal, cytostatic, and anti-inflammatory properties (8-10). Gribel and

Pashinskii (11) have shown that honey possesses moderate antitumorand pronounced antimetastatic effects in tumors in five differentstrains of rats and mice. Caffeic acid (3,4-dihydroxycinnamic acid)and its esters, which are present in propolis at levels of 20-25% (12),

are agents suspected of having a broad spectrum of biological activities including tumor suppression. Because of this potential we andothers have synthesized MC,3 PEC, PEMC, PEDMC, and related

compounds that are present in propolis, and we have examined theirantimutagenic and antitumorigenic activities (13, 14). Our bioassaysconfirmed that these agents are antimutagenic in Salmonella typhi-

murium strains TA98 and TA 100 and that thay inhibited colon adenocarcinoma HT-29 and HCT-116 cell growth (14). Further, theseagents inhibited the AOM-induced colonie mucosa! ornithine decar-

boxylase, tyrosine protein kinase and LOX activities, and colonieaberrant crypt foci formation in F344 rats (15). Among the caffeicacid esters evaluated, PEMC was found to be most effective ininhibiting AOM-induced aberrant crypt foci formation (15).

Metabolites of AA exert a variety of biological activities. Severalstudies have shown that COX metabolites (particularly PCs derivedfrom (0-6 fatty acids) modulate cell proliferation, tumor growth, and

immune responses (16, 17), whereas LOX metabolites can influencevarious biological responses including chemotaxis, hormone secretion, ion transport, stimulation of tumor cell adhesion, tumor cellspreading, and regulation of the metastatic potential of tumor cells(18-20). The generation of AA for biosynthesis of COX and LOX

metabolites involves degradation of phosphatidylinositol via a sequence of reactions regulated by PLC (17, 21). The second pathwayin the generation of AA involves direct action of a PLA2 on aphospholipid (17, 22). Several forms of PLC and PLA,, among themPI-PLC, membrane-bound a PlP^-specific, and cytosolic PLA-,, have

been implicated in the regulation of eicosanoid biosynthesis and cellproliferation (22, 23). Increased levels of PLA2 and PI-PLC activities

have been observed in human colon and breast tumors and in melanomas compared to normal tissues (24-26). It is also noteworthy that

the COX metabolite, PGE2, which significantly affects tumor growth

1The abbreviations used are: MC, methyl caffeate; PEC, phenylethyl caffeate; PEMC,

phenvlcthyl-3-mcthylcaffeate; PEDMC, phenylethyl dimethvlcaffeate; AOM, azoxymethane; TPA. 12-O-tetradecanoylphorbol-13-acetate; COX, cyclooxygenase; LOX, lipoxygenase; PLA2- phospholipase A2; PI-PLC, phosphatidylinositol-specific phopholipase C;AA. arachidonic acid; PAPC. L-a-l-palmiloyl-2-arachidonyl phosphatidylcholine; HETE,hydroxyeicosatetraenoic acid; PG, prostaglandin; Tx. thromboxanc; PIP>, I -3-phophati-dylinositol 4,5-biphosphate.

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ntEMOPRtVKNTION OF COLON CANCER BY CAFFEIC ACID ESTERS

and immune responses, has been found at high levels in tumors inlaboratory animals, as well as in neoplasms in humans (16, 27, 28).We and others have shown that inhibitors of COX such as aspirin,indomethacin, piroxicam, sulindac, and ibuprofen suppress colon car-cinogenesis in laboratory animal models (16, 28-30). Thus, it ispossible that changes in the activities of PLA2 and PI-PLC that are

involved in signal transduction and AA release, as well as COX andLOX pathways of AA metabolism produced by exogenous agents,may alter the tumorigenesis.

It was therefore of interest to evaluate the chemopreventive efficacyof PEMC (Fig. 1) in an established colon cancer model. PEMC, alsoknown as phenylethyl ferulate, is closely related to the naturallyoccurring turmeric compound curcumin, or diferuloylmethane. In thepresent study, we report on the chemopreventive efficacy of dietaryadministration of PEMC on AOM-induced colon tumorigenesis in

male F344 rats. In addition, the effects of dietary PEMC on PLA2,PI-PLC, COX, and LOX activities in colonie mucosa! and tumor

tissues were analyzed to better understand the modulating role of thisagent in colon tumorigenesis.

MATERIALS AND METHODS

Materials

AOM (CAS:25843-45-2) was purchased from Ash Stevens (Detroit, MI).[I4C]-AA and PAPC were bought from DuPont New England Nuclear (Bos

ton, MA). PIP-, was obtained from Amersham (Arlington Heights, IL). AA,5(S)-HETE, 8(S)-HETE, 9(S)-HETE, 11(S)-HETE, 12(5)-HETE 15(5)-

HETE, PGE2, PGF2„,6-Keto PGF,„,PGD2 AND TxB2 were procured from

the Cayman Chemical Co. (Ann Arbor. MI). PEMC was prepared from4-hydroxy-3-methoxycinnamic acid by a procedure similar to one reported

earlier (14). Chromatography on silica gel with hexane:ethyl acetate (1:1) as aneluent gave PEMC (71%), melting point 57-59°C, and a 'H nuclear magnetic

resonance (CDC1,) with following characterstics: 8 3.02 (t, 2H, COOCH2,J = 7.05 Hz), 3.93 (s, 3H, OCH,), 4.43 (t, 2H, CH2Ph, J = 7.06 Hz), 6.28 (d,IH, CH, J = 15.90 Hz), 6.92 (d, IH, aromatic, J = 8.18 Hz), 7.00-7.09 (m,2H, aromatic), 7.21-7.35 (m, 5H, aromatic), 7.61 (d, IH, CH, J = 15.92 Hz);

The normal phase HPLC (4.6 x 250 mm) silica columns were obtained fromAlltech Associates Inc. (Deerfield, IL), and the reverse phase HPLC (3.9 x 300mm) n Bondpak C,x column was purchased from Waters Associates (Milford.MA). Precoaled Silica G plastic TLC plates were bought from Fisher ScientificCo. (Springfield, NJ).

Animals and Diets

Weanling male F344 rats were purchased from Charles River BreedingLaboratories (Kingston, NY). All ingredients of the semipurified diet wereobtained from Dyets, Inc. (Bethlehem, PA) and were stored at 4°Cprior to

preparation of diets. Male F344 rats received as weanlings were quarantinedfor 10 days and had access to modified AIN-76A control diet (31). Following

the quarantine, the animals were randomized by weight into various study andcontrol groups and housed in plastic cages with filter tops (3/cage) undercontrolled conditions of a 12-h light/12-h dark cycle, at 50% relative humidity,and at 21°C.The experimental diet was prepared by adding PEMC to the

control diet at the expense of dextrose. PEMC was incorporated into thecontrol diet in a Hobart mixer after it had been premixed with a smallerquantity of diet in a food mixer to ensure its uniform distribution. All controland experimental diets were prepared weekly in our laboratory and were stored

CH3O.

Wk 5 Wk / Wk 852 Weeks•tterAOM

1 J|1,11l

tQuarantine' TTAOMAOM

Fig. 1. Structure of PEMC (pcnylelhyl-3-mclhylcaffeatc or phenylethyl ferulate).

HistopathologyBiochemical assay

CARCINOGEN : AzoxymMhtn« (AOM). B.c.. 15 mgAg body w«4flhtt. wrMkly for 2 MM*»

EXPERIMENTAL DIET: 7SO ppm PEUC In modlflM «IN-/«* HI«

Fig. 2. Experimental design for evaluation of chemopreventive efficacy by PEMC.Animals were fed PEMC 2 weeks prior to exposure to AOM, during treatment, and untilthe termination of experiment. AOM (15 mg/kg body weight/week) was given to theanimals s.c. at beginning of 7 and 8 weeks of age. Wk, week.

in a cold room. The rats had access to food and water at all times; food cupswere replenished with fresh diet three times weekly.

The purity and stability of PEMC in the feed samples was determined byHPLC, for which the compound was extracted with 3 volumes of ethylacetate.The organic layer was dried, redissolved in mobile phase containing 0.1 Msodium phosphate (pH 4) and acetonitrilc. and injected into the HPLC. Theseparation on a Waters Cls column with gradient elution was monitored at 254nm wavelength in a Waters 990 photodiode array detector, indicating that>97% of PEMC can be accounted for in feed samples stored in a cold room forup to 14 days.

Experimental Procedure

The study was designed to determine the chemopreventive efficacy of 750ppm PEMC on colon carcinogenesis. The rationale for the selection of thisdose was based on our biochemical and short-term bioassay results in F344 rats

(14, 15). The experimental procedure was described previously (27). Asindicated in Fig. 2, beginning at 5 weeks of age, groups of rats were fed thecontrol diet or the experimental diet containing 750 ppm of PEMC. Two weekslater, groups of animals intended for carcinogen treatment received AOM s.c.,once weekly for 2 weeks, at a dose rate of 15 mg/kg body weight, whereasthose intended for vehicle treatment were administered an equal volume ofnormal saline. Animals were maintained on control or experimental diets untilthe termination of the experiment. Body weights were recorded every 2 weeksfor the first 10 weeks and every 4 weeks there after. All animals weresacrificed by CO2 euthanasia 52 weeks after the second AOM treatment. Afterlaparotomy, the entire gastrointestinal tract was resected and opened longitudinally, and the contents were flushed with normal saline. With the use of adissection microscope, colonie and small intestinal tumors were noted grosslyfor their location, number, and size. For each tumor, the length, width, anddepth were measured with calipers. Estimates of tumor volume were madewith the use of the formula V=LXWXDXir/6 (32). All other organs,

including kidney and liver, were also examined grossly under the dissectionmicroscope for abnormalities. Colon tumors with a diameter of more than 0.4cm were cut into two halves; one portion of the tumor was used for analysesof PLA,, PI-PLC, and COX and LOX metabolites, the other half for his-topathological examination. Mucosa that was free of tumors in AOM-treatedanimals and from saline-treated animals was scraped from each colon for

comparative biochemical analysis according to our previously describedmethod (32). Colonie mucosa and portions of tumors intended for biochemicaldeterminations were quickly frozen in liquid nitrogen and stored at -80°C

until analysis.For histopathological evaluation, tumors were fixed in 10% buffered for

malin, embedded in paraffin blocks, and processed with hematoxylin and eosinstaining. The stained sections were examined histologically according to thetumor classification of Pozharisski (33) with minor modifications. All colontumors in this experiment were adenocarcinomas, either invasive or noninva-

sive. The invasive adenocarcinomas were mostly signet ring mucinous type,invading muscularis mucosa deep into the intestinal wall and beyond. Thenoninvasive adenocarcinomas were those growing outward toward the intestinal lumen and not invading the muscularis mucosa. They were usually welldifferentiated adenocarcinomas.

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Biochemical Analysis

Samples of colonie mucosa and tumors intended for PLA2 and PI-PLC

assay were homogenized in 1:3 (w/v) volumes of homogenizing buffer containing 30 mM Tris-HCl (pH 7.4), 140 mM NaCl, 5 ITIMKC1, 20 JU.MEDTA, 10

fig/ml leupcptin, 50 /¿g/mltrypsin inhibitor, and 1 mM phenylmethylsulfonylfluoride, and the homogenatcs were centrifuged at 100,000 x g at 4°Cfor 1 h.

The resulting supernatant fraction was used for cytosolic PLA2 activity, andthe pellet fraction was redissolved in 30 mM HEPES-NaOH buffer (pH 7.2)containing 0.2% Triton X-100, which was used for the analysis of membrane-bound Pl-PLC activity.

PI-PLC Activity. Membrane-bound Pl-PLC activity was measured by themethod of Bleasdale et al (34) with some modifications using [3H]-PIP, (5

Ci/mmol) as a substrate. PI-PLC activity of membrane proteins (100-200 /xg)

was determined in a total volume of 250 ¡ureaction mixture containing 30 mMHEPES-NaOH buffer (pH 7.2), 5 mM DDT, 4 mM CaCU, 2 mM EGTA, 0.9 mMMgS04, and 50 JJ.M[3H]-PIP2 (50 /xCi/mmol). The reaction was initiated byadding substrate to the mixture and incubating at 37°Cfor 20 min in a shaking

water bath. The reaction was terminated by addition of 0.2 ml of chloroform:methanol (1:2, v/v) and then 0.3 ml of l M HC1. The incubation mixture wasmixed vigorously and centrifuged to yield two phases. An aliquot of 0.3 ml ofthe aqueous layer containing ['Hjinositol 1,4,5-triphosphate was transferred

into a scintillation vial containing 10 ml of scintillation cocktail. Radioactivitywas counted in a Beckman Model LD6800 scintillation counter. The activityis expressed as pmol of [3H]inositol 1,4,5-triphosphate formed from [3H]-PIP2/

mg protein/15 min.PLA2 Activity. Cytosolic PLA, activity was measured by the method of

Leslie (35) with some modifications with the use of [14C]-PAPC (40-60

mCi/mmol) as substrate. PLA, activity of cytosolic protein was carried out ina total volume of 100 fil reaction mixture containing 50 mM sodium HEPES(pH 7.3), 0.8 mM CaCl2, 0.02% Triton X-100, and 20-30 ¿igof cytosolic

protein. The reaction was initiated by adding 40 JIM PAPC (10 j¿Ci//j.mol,adjusted with cold substrate), and the reaction mixture was incubated at 37°C

in a shaking water bath for 30 min. Reaction was terminated by adding 300 ¡úchloroform:methanol (3:2,v/v). An additional 200 ¡uof chloroform wereadded to each sample and mixed thoroughly. The samples were then centrifuged, and the chloroform layer was separated and evaporated to dryness underN,. Five fig of AA were added to the dried extract and redissolved inchloroform. An aliquot of the chloroform extract was then subjected tochromatography on precoated plastic TLC plates (Silica G). The TLC plateswere developed with a solvent system containing chloroform:methanol:aceticacid:water (90:12:2:1, v/v/v/v) and exposed in an iodide chamber for 5 min forvisualization of AA. The area of each [ 14C]-AA metabolite was determined

with a Bioscan System 200 image-scanning counter (Bioscan, Inc., Washington DC) equipped with a ß-detector. Protein content was determined bythe Bio-Rad method. Results are expressed as pmol [I4C]-AA released/mg

protein/min.

Lipoxygenase and Cyclooxygenase Activities

Colonie mucosa and tumors from individual animals were homogenized in1:3 (w/v) volumes of 100 mM Tris-HCl buffer (pH 7.2) with the use of a

Polytron tissue homogenizer. The samples were then centrifuged at 9000 X gat 4°Cfor 10 min. The supernatant fraction was centrifuged at 100,000 X g for

1 h. The resulting supernatant was used for determination of LOX activity.Microsomal pellets were resuspended in 50 mM potassium phosphate buffer(pH 7.4) for the assay of COX activity.

Lipoxygenase Activity. LOX activity of sample of the colonie mucosa andof tumors was determined by a modification of the method described by Raoet al. (15). In brief, this method involves HPLC measurement of 14C-labeled

5(S)-, 8(5)-, 9(S)-, 11(5)-, 12(S)-, and 15(S)-HETEs that were formed fromthe [I4C]-AA. The reaction mixture (200 ftl) containing 100 mM Tris-HCl (pH7.2) and 2 mM CaCU [14C]-AA (6 nmol, 480,000 dpm), and cytosol fraction(300-500 fig protein) was incubated for 15 min at 37°C.The reaction wasterminated by adding 12 (j.1of 0.2 M HC1, and the metabolites of [14C]-AA

were extracted with 0.6 ml of ethyl acetate 3 times. The HETEs were analyzedby normal phase HPLC as described previously (15, 36).

Cyclooxygenase Activity. The COX activity of colonie mucosa and oftumors was measured by previously published methods (15, 37). Briefly,150-fil reaction mixture containing 12 JIM [14C]-AA (420,000 dpm), 1 mM

epinephrine, 1 mM glutathione in 50 mM phosphate buffer, and 25-35 fig ofmucosal or tumor microsomal protein was incubated at 37°Cfor 15 min. The

reaction was then terminated by the addition of 40 /xl of 0.2 M HC1. The COXmetabolites of AA were extracted three times with 0.5 ml of ethyl acetate. Thecombined extracts were evaporated to dryness under N2, redissolved in chloroform, and subjected to TLC with Silica G. The TLC plates were developedin a solvent system containing chloroform:methanol:acetic acid:water (100:15:1.25:1, v/v/v/v) and exposed in an iodide chamber for 5 min for visualizationof the standards. The metabolites of [I4C]-AA corresponding to PGE2, PGF2iI,

PGD2, 6-keto PGF,,,, and TxB2 were detected by their comigration (Rf values)

with authentic standards. The area of each metabolite was determined with theBioscan System 200 image-scanning counter equipped with a ß-detector.

Statistical Analysis

Body weights, tumor incidence, tumor multiplicity, tumor volume, andbiochemical parameters were compared between the animals fed the controland PEMC diets. Tumor incidence, expressed as percentage of animals withtumors, was analyzed by x2 test. Tumor multiplicity, expressed as the mean

number of tumors/animal, was analyzed by the unpaired t test accounting forunequal variance. Differences in body weights, tumor volume, and biochemical parameters between the groups were analyzed by Student's t test and

ANOVA. Differences were considered statistically significant at P < 0.05.

RESULTS

General Observations. The body weights of animals treated withvehicle or AOM and fed the control or PEMC diets were comparablethroughout the study (Table 1). In vehicle-treated animals, the feeding

of PEMC did not produce any gross changes in liver, kidney, stomach,intestine, or lungs, nor any kind of histopathological changes in theliver or intestine attributable to toxicity.

Tumor Incidence. Tables 2 and 3 summarize the AOM-induced

colon, small intestinal and ear duct tumor incidence (percentage ofanimals with tumors), and multiplicity (number of tumors/animal).There were no tumors in vehicle-treated animals fed the control or

PEMC diet. Administration of PEMC significantly inhibited theAOM-induced incidences of invasive (P < 0.02), noninvasive

(P < 0.03), and total (invasive plus noninvasive) adenocarcinomas(P < 0.002; Table 2), and multiplicity of invasive (P < 0.01), noninvasive (P < 0.05), and total (P < 0.004) adenocarcinomas of the colon(Table 3). Animals fed PEMC diet had fewer small intestinal tumors

Table 1 Effect of PEMC on body weight gain in male F344 rats

ExperimentalgroupAOM

treatedControl diet(AIN-76A)750 ppmPEMCSaline

treatedControl diet750 ppm PEMCNo.

ofanimals36

3012

12Body

weight of animals on experimental diets (ingWkfl73

±0.8

73 ±0.873

±1.173 ±0.8Wk3171

±1.8

172 ±1.7168

±2.2170 ±1.9Wk6232

±2.5

235 t2.6239

±2.7235±2.2Wk

14317

±3.3

323 ±2.9320

±4.1319 ±4.2Wk22369

±4.0

375 ±3.8378

±4.9375 ±5.1,

mean ±SEM)atWk32428

±4.5

435 ±4.4432

±6.3428 ±6.2Wk42438

±5.6

444±5.3456

±7.2450 ±7.1Wk52449

±5.3

456 ±5.4468

±7.1461 ±7.3

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CHEMOPREVENTION OF COLON CANCER BY CAFFEIC ACID ESTERS

Table 2 Effect of P EM C on AOM-induced inieMiniil unti cur duci Iunior incidence in male F344 rats

Tumor incidence (% of animals withtumors)ExperimentalgroupAOM

treatedControl diet75(1ppmPEMCSaline

treated750 ppm PEMCInvasive38

13' (bS.jf

(P < 0.02)0ColonNoninvasivc67

40' (40.3)

(P < 0.03)0Total"81

43'' (47)

(P < 0.002)0Small

intestineinvasive16

10(37.5)0Intestine*81 50' (38.3)

(P < 0.008)0Ear

dueltumors25

()' (UK))

(P < 0.02)I)

" Total includes invasive plus noninvasive adenocarcinomas." Intestine includes, colon plus small intestinal adenocarcinomas.' Significantly different from control group, by \2 'est using Z-probability.J Values in the parenthesis are percentage inhibition when compared to control group.

Table 3 Effect of PEMC on AOM-induced intestinal and car duel tumor multiplicity in mail1 F344 rats

ExperimentalgroupAOM

treatedControldiet

751)ppmPEMCSaline

treated750ppm PEMCInvasive0.53

±0.12'0.16 + 0.08''

(P <0.01)0ColonNoninvasive0.97

+ 0.150.60 ±0.11''(P ==0.05)0Adenocarcinomas/animalTotal"

invasive1.5

±0.19 0.25 + 0.090.76 ±0.18a 0.10 ±0.06

(P < (UHM)0

0Intestine''1.75

±0.230.86 + 0.18''

(P <0.00)0Ear

ducttumors0.27

±0.08()''

(P < 0.004)0

" Total includes invasive plus noninvasive adcnocarcinomas.' Intestine includes colon plus small intestinal adenocarcinomas.1 Mean + SEM.' Values are significantly different from control group by Student's / test.

than those fed the control diet. It is noteworthy that administration ofPEMC suppressed 100% of the tumors in the ear duct (Table 2 and 3). Inaddition, AOM-induced colon tumor volume was reduced to about 43%

in animals fed PEMC as compared to those fed the control diet (Table 4).Biochemical Studies. The activities of PI-PLC and PLA, analyzed

in colonie mucosa and tumors are summarized in Table 5. AOMadministration elevated the activities of colonie mucosal PI-PLC and

PLA, irrespective of dietary regimen. It is interesting that there was a3-4-fold increase in the activities of PI-PLC and PLA-, in colontumors when compared to surrounding colonie mucosa. Long-termfeeding of PEMC significantly suppressed PI-PLC activities in co-

Ionic mucosa and in tumors but had no significant effect on the PLA2activity. The effect of dietary PEMC on LOX and COX metabolites inthe colonie mucosa and tumor tissue is shown in Table 6. AOMadministration had a minimal effect on the production of LOX andCOX metabolites in the colonie mucosa compared to those treatedwith vehicle and fed similar diets (data not shown). The levels ofHETEs were significantly higher in colonie tumors than in coloniemucosa. Administration of PEMC in the diet significantly suppressedthe colonie mucosal and tumor 5(5)-, 8(5)-, 12(5)-, and 15(5)-HETEsformation to more than 30-60%. Markedly increased levels (3-5-fold) of

PGs and TxB2 were observed in the colonie tumors of animals fed thecontrol diet when compared to these levels in the colonie mucosa ofsimilarly treated animals. PEMC diet showed no significant inhibitory

Table 4 Effect of dietary PEMC on AOM-induced colon tiinuir si~e and mean volume

in male E344 rats

Tumor size

Table 5 Effect of PEMC on colonie mucosal ami minor PI-PLC and PLA-, activitiesin male F344 rats

Colonie mucosa

ExperimentalgroupControl

diet750 ppm PEMC0.5cm26"

(48.8)''

18 (78.3)0.5-1.0

cm18

(33.3)4(17.4)>1.0cm10(18.5)1 (4.4)Fumor

Volume (mm' )

(mean ±SEM)133

+ 3276 ±18

ExperimentalgroupsPI-PLCactivity"Control

diet750ppmPEMCPLA-,

activity1'Control

diet750ppm PEMCSaline

treated22.5

±2.5''13.4±1.0'I4.5±

1.011.2+1.2AOM

treated32.5

±2.620.4+1.r24.7+

1.819.8+1.6Tumors128

±6.374±4.8'71

±5.657±4.9

" Number of tumors/group.'' Percentage of tumors having particular size.

"PI-PLC activity is expressed as pmol ["HJinositol 1,4,5-triphosphate formed from["HJ-PIPVmg protein/15 min al 37°C

' Values are mean ±SEM (n = 6—8).' Significantly different from control group by Student's t test, P < 0.01 It)0.0001.'yPLA2 activity is expressed as pmol of 14C-labcled arachidonic acid rcleased/mg

protein/min at 37°C.

effect on colonie mucosal COX metabolite formation but had a minimalinhibitory effect on colonie tumor PGs (25-29%) and TxB2 (15%).

DISCUSSION

The major aim of this investigation, which is a part of a large-scale

study on the identification of naturally occurring compounds withpotential chemopreventive properties, was to elucidate the inhibitoryrole of PEMC present in propolis, a natural resin produced by honeybees, on the formation of colon tumors in rats. This investigationclearly demonstrated the colon cancer chemopreventive effect ofPEMC. Previous studies in our laboratory have demonstrated thatdietary administration of PEMC significantly inhibited the formationof AOM-induced preneoplastic aberrant crypt foci in the colon of

F344 rats (15) and also inhibited human colon adenocarcinoma cellgrowth (14). The results of the present study further support ourprevious observations that, indeed, caffeic acid esters such as PEMCpossess antitumorigenic activities. Further, administration of PEMC

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Table 6 Effect of P EMC on AOM-induced colonie mucosa! and tumor lipoxygenaseand cvciooxygenase metabolism in male F344 rats

ColoniemucosaAarachidonic

Controlacid metabolitesdietLipoxygenase

activity"5(5)-HETE8(S)-HETE12(S)-HETE15(S)-HETECyclooxygenasePGE,PGF,„POD",6

Keio-PGFlaTxB2211312266308activity'32935524337812"2114221316131426012PEMC148*

9''(30)''165

±12'(47)122±10r(54)204±13r(34)302+15

(8.2)328

±13(7.6)248±16(0)336±14(11)254±14 (2.3)Control

diet313

±13348±16585±28428

±311577

±38938±29550±201

193 ±46973±33TumorsPEMC228

±12(27)137±11''(60)213

±14r(63)196±13'(54)1178

±35(25)694+ 22(26)423±21(23)847±33C(29)825

±27 (15)"Pmol of HETEs produced from C-labeled arachidonic acid/mg protein/15 min at

37°C.* Mean ±SEM (n = 6).' Values in horizontal rows are significantly different from control group, either in

colonie mucosa or in tumors. P < 0.05 to O.(KK)1.d Values in the parenthesis are percentage inhibition from their respective control diet

group, either in colonie mucosa or tumors." Pmol of PGs/TxB, produced from 14C-labeled arachidonic acid/mg protein/15 min at

37°C.

produced 100% inhibition of ear duct tumors induced by AOM,suggesting that the chemopreventive (antitumorigenic) activities ofthis compound are not limited to cancer of the colon. Although thereare no other studies in animal models to suggest a chemopreventiverole for PEMC or related caffeic acid esters such as MC, PEC, andPEDMC, Wattenberg et al. (38) have demonstrated that dietary administration of hydroxycinnamates, nonesterified caffeic acid derivatives,significantly inhibited benzo[a]pyrene-induced neoplasia of the forestom-

ach of mice. A recent study from our laboratory showed that dietaryadministration of curcumin, structurally similar to PEMC, significantlyinhibited AOM-induced colon carcinogenesis in male F344 rats (36).

The precise mechanism by which PEMC inhibits colon tumorigen-

esis has not been established; it would appear that the possible modesof action involve suppression of oxidative processes, tyrosine phos-

phorylation, and modulation of AA metabolism. Frenkel et al. (39)showed that PEC, a demethoxy form of PEMC, inhibits TPA-induced

polymorphonuclear leukocyte infiltration into mouse skin, formationof hydrogen peroxide, and oxidative damage to DNA bases. Our ownstudies have also shown that caffeic acid esters (PEC, PEMC, andPEDMC) significantly inhibit tyrosine protein kinase activity in thecolonie mucosa of male F344 rats (15) and suppress the Mr 70,000 and120,000 phosphotyrosine proteins in human colon adenocarcinomaHT-29 cells in a dose-dependent manner.4

Several previous studies have established that AA metabolites maymodulate the pathogenesis of several immunological and inflammatory diseases (17). In the present experiment, we studied the levels ofPLA2 and Pl-PLC, which are dominant pathways for the AA release,in the colonie mucosa and tumors. Also, PI-PLC is responsible for

diacylglycerol transduction and cell proliferation (40). One of thepathways leading to generation of AA involves a direct action ofPLA-, on a phospholipid that could include 1,2-diacyl- or 1-O-alkyl-2-acyl-phosphatidylinositol, phosphatidylethylamine, or phosphati-dylcholine. The second pathway, mediated by PI-PLC, involves thedegradation of phosphatidylinositol 4,5-biphosphate via a sequence ofreactions beginning with PI-PLC, followed by diglyceride lipase and

monoglyceride lipase (22). Our study demonstrates that dietary PEMCsignificantly inhibits the PI-PLC activity in the colonie mucosa and in

tumor tissue. The exact mechanism by which PEMC inhibits theseenzyme activities is not clear. It may be possible that PEMC exerts

4 Rao el til., unpublished data.

inhibitory activity by directly acting on PI-PLC or, alternatively, byacting on the regulators of PI-PLC, thus decreasing the levels of AAand/or protein kinase C-mediated signal transduction functions and

cell proliferation. Also, the present study demonstrated that PEMChad no significant effect on PLA2 activity. We are not aware of anyprevious studies in which PEMC or other caffeic acid esters weretested on these enzyme activities.

The levels and production of HETEs and PGs in colon tumors arehigher than in colonie mucosa, suggesting an increased synthesis ofLOX and COX metabolites in tumors. LOX metabolites such as12(S)-HETE promote tumor cell adhesion, stimulate tumor cell

spreading, and augment the metastatic potential of the tumor cells(18-20). Also, a positive correlation was observed between the levelsof 8(S)-HETE and degree of inflammation, hyperproliferation, clas-

togenicity, and tumor development induced by TPA (41). In addition,the activities of 5(5)- and 15(5)-HETEs, which are potent modulators

of inflammation, have been shown to be suppressed by lipoxygenaseinhibitors, indicating a mediating role of HETEs in tumor promotion(37, 42). Also, the role of COX metabolites, particularly PGEj, incolon tumor promotion has been established (16, 29, 30). Earlierstudies from our laboratory and others have shown that PG inhibitorssuch as piroxicam, indomethacin, sulindac, and aspirin inhibit colontumorigenesis in rodents (16, 27). Thus, the inhibition of colon ade-

nocarcinomas by PEMC was consistent with the reduction of particular LOX metabolites in the colonie tumors, suggesting that thechemopreventive action of PEMC may be mediated through theinhibition of formation of metabolites of LOX rather than of COX.The exact mechanism by which these agents inhibit predominantlyLOX metabolites is not clearly known. Recently, Glasgow et al. (43)have shown that 2,5-dihydroxycinnamic acid methyl ester nonspecifi-

cally inhibited formation of LOX metabolites but not of COX metabolites in Syrian hamster embryo cells. Previous studies from ourlaboratory showed that administration of caffeic acid esters MC, PEC,PEMC and PEDMC predominantly inhibited AOM-induced LOX

metabolites rather than COX metabolites in the colonie mucosa (15).On the basis of findings, it is reasonable to state that PEMC maymodulate not only PI-PLC to alter endogenous AA levels and proteinkinase C-dependent signal transduction pathways but also the

pathway of LOX. In addition, caffeic acid esters such as PEC andPEMC have been shown to possess preferential cytotoxicity towardmitogen-induced transformed cells and a variety of neoplasms such as

those in human colon, breast, melanoma, and glioblastoma cells(13-15, 44-46).

In conclusion, the study described here demonstrates that dietaryPEMC significantly inhibits AOM-induced colon tumorigenesis in

F344 rats and that this inhibition is mediated through the suppressionof PI-PLC and LOX activities in colonie mucosa and tumor tissues.

Although the exact mechanisms of chemopreventive action of PEMCremain to be elucidated, modulation of PI-PLC-dependent signal

transduction pathways and AA metabolism by PEMC are likelyplaying a role in its inhibitory action. Our current efficacy study andthe availability of PEMC as a natural product strongly suggest thatfurther studies should be pursued to test PEMC for its potentialchemopreventive properties in human clinical trials.

ACKNOWLEDGMENTS

We thank Laura DiSciorio for preparation of the manuscript. Use Hoffmannfor editorial assistance, and the staff of the Research Animal Facility andHistopathology Facility for expert technical assistance.

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CHEMOPREVENTION OF COLON CANCER BY CAFFEIC ACID ESTERS

REFERENCES

1. Wingo, P. A., Tong, T., and Bolden, S. Cancer Statisucs. CA Cancer J. Clin., 45:8-30, 1995.

2. National Research Council. Inhibitors of carcinogenesis. In: Diet, Nutrition andCancer, pp. 358-370. Washington, DC: National Academy Press, 1982.

3. Wynder, E. L., Kajitani, T., Ishikawa, S ., Dodo, H., and Takano, A. Environmentalfactors in cancer of colon and rectum. Cancer (Phila.), 23: 1210-1220, 1969.

4. Reddy, B. S. Diet and colon cancer: evidence from human and animal model studies.In: B. S. Reddy and L. A. Cohen (eds.). Diet, Nutrition and Cancer: A CriticalEvaluation, pp. 47-65. Boca Raton, FL: CRC Press, 1986.

5. Wattenberg, L. W. Chemoprevention of cancer by naturally occurring and syntheticcompounds. In: M. Wattenberg, C. W. Lipkin, C. W. Boone, and G. J. Kelloff (eds.).Cancer Chemoprevention, pp. 19-39. Boca Raton, FL: CRC Press, 1992.

6. Boone, C. W., Kelloff, G. J., and Malone, W. F. Identification of candidate cancerchemopreventive agents and their evaluation in animal models and human clinicaltrials: a review. Cancer Res., 50: 2-9, 1990.

7. Kelloff, G. J., Boone, C. W., Malone, W. E., and Steele, V. E. Recent results inpreclinical and clinical drug development of chemopreventive agents at the NationalCancer Institute. In: L. W. Wattenberg, M. Lipkin, C. W. Boone, and G. J. Kelloff(eds.), Cancer Chemoprevention, pp. 41-56. Boca Raton, FL: CRC Press, 1992.

8. Jeddar, A., Kharsany, A., Ramsaroop, V. G., Bhamjee, A., Haffejee, I. E., and Moosa,A. The antibacterial action of honey: an in vitro study. S. Afr. Med. J.. 67: 257-258,

1985.9. Hladon, B., Bylka, W., Wojtaszek, M. E., Skrzypczak, L., Szafarek. P., Chodera, A.,

and Kowalewski, Z. In vitro studies on the cytostatic activity of propolis extracts.Arzneim. Forsch. Drug Res., 30: 1847-1848, 1980.

10. Koshihara, Y., Neichi, T., Muróla, S. L., Lao, A., Fujimoti, Y., and Tatsuno, T.Caffeic acid is a selective inhibitor for leukotriene biosynthesis. Biochim. Biophys.Acta, 792: 92-97, 1984.

11. Gribel, N. V., and Pashinskii, V. G. Antitumor properties of honey. Vopr. Onkol. (St.Petersbg.), 36: 704-709, 1990.

12. Greenaway, W., Scaysbrook, T., and Whatley, F. R. The analysis of bud exúdate ofPopulas x euraniericana and of propolis by gas chromatography-mass spectrometry.Proc. R. Soc. Lond., B232: 249-272, 1987.

13. Grunberger, D., Banerjee, R., Eisinger, K., Oltz, E. H., Efros, L., Caldwell, M.,Estevez, V., and Nakanishi, K. Preferential cytotoxicity of tumor cells by caffeic acidphenethyl ester isolated from propolis. Experientia, 44: 230—232,1988.

14. Rao, C. V., Desai, D., Kaul, B., Amin, S., and Reddy, B. S. Effect of caffeic acidesters on carcinogen-induced mutagenicity and human colon adenocarcinoma cellgrowth. Chem. Biol. Interact., 84: 277-290, 1992.

15. Rao, C. V., Desai, D., Simi, B., Kulkarni, N., Amin, S., and Reddy. B. S. Inhibiloryeffecl of caffeic acid eslers on azoxymethane-induced biochemical changes andaberrant crypt foci formalion in ral colon. Cancer Res., 53: 4182-4188, 1993.

16. Marnetl, L. J. Aspirin and the potential role of prostaglandins in colon cancer. CancerRes., 52: 5575-5589, 1992.

17. Smilh, W. L. Proslanoid biosynlhesis and mechanisms of aclion. Am. J. Physiol., 263:F181-191, 1992.

18. Honn, K. V., Grossi, I. M., Steinen, B. W., Chopra, H., Onoda, J., Nelson, K. K., andTaylor, J. D. Lipoxygenase regulalion of membrane expression of lumor cell glyco-

proteins and subsequent metastasis. Adv. Prostaglandin Thromboxane LeukotrieneRes., 19: 439-443, 1989.

19. Timar, J., Chen, Y. Q., Liu, B., Bazaz, R., Taylor, J. D., and Honn, K. V. Thelipoxygenase melabolile 12(S)-HETE promoles a-II b ß3integrin-mediated tumor-cell spreading on fibroneclin. Ini. J. Cancer, 52: 594-603, 1992.

20. Honn, K. V., and Tang, D. G. Adhesion molecules and cancer cell inleraclion withendolhelium and subendolhelial malrix. Cancer Metastasis Rev., //: 353-375, 1992.

21. Neufeid, E. J., and Majerus, P. W. Arachidonate release and phosphalidic acidlurnover in slimulaled human platelets. J. Biol. Chem., 258: 2461-2467, 1983.

22. Dennis, E. A., Rhee, S. G., Billah, M. M., and Hannun, Y. A. Role of phospho-

lipases in generating lipid second messengers in signal transduction. FASEB J., 5:2068-2077, 1991.

23. Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B., Oraziani, A., Kapeller, R.,and Soltoff, S. Oncogenes and signal Iransduclion. Cell, 64: 281-302, 1991.

24. Perrella, F. W., Jankewicz, R., and Dandrow, E. A. Phospholipase C from humanmelanoma: purification and characterizalion of a phosphalidylinosilol-selective enzyme. Biochim. Biophys. Acta., I076: 209-214, 1991.

25. Arteaga, C. L., Johnson, M. D., Todderud, G., Coffey, R. J., Carpenter, G., and Page,D. L. Elevaled coment of the lyrosine kinase subslrale phospholipase Crl in primaryhuman breasl carcinomas. Proc. Nati. Acad. Sci. USA, 88: 10435-10439, 1991.

26. Soydan, A. S., Tavares, I. A., Wecch. P. K., and Bennett. A. High molecular weightphospholipase A2: its occurrence and quantification in human gut cancer and normalmucosa. Eicosanoids and Other Bioactive Lipids in Cancer, Inflammation and Radiation Injury, 3: 66, 1993.

27. Reddy, B. S., Rao, C. V., Rivenson, A., and Kelloff, G. J. Inhibilory effecl of aspirinon azoxymethane-induced colon carcinogenesis in F344 rals. Carcinogencsis (Lond.).14: 1493-1497, 1993.

28. Narisawa, T., Kusaka, H., Yamazaki, Y., Takahashi, M., Koyama, H., Koyama, K.,Fukaura, Y., and Wakizaka, A. Relalionship between blood plasma proslaglandin E2and liver and lung melaslases in coloreclal cancer. Dis. Colon Reclum, 33: 840-845,

1990.29. Verma, A. K., Ashendel, C. L., and Boulwell, R. K. Inhibition by prostaglandin

synthesis inhibilors of Ihe induclion of epidermal ornilhinc dccarboxylasc aclivity, Ihcaccumulation of prostaglandins, and lumor promolion caused by 12-O-letradecanoyl-pohorbol-13-acelale. Cancer Res., 40: 308-315, 1980.

30. Melzger, U., Meier, J., Uhlschmid, G., and Weihe, H. Influence of various prostaglandin synthesis inhibitors on DMH-induced rat colon cancer. Dis. Colon Rectum, 27:366-369, 1984.

31. Reddy, B. S., and Sugie, S. Effect of differenl levels of <o-3fally acids on azoxymelh-ane-induced colon carcinogenesis in F344 rals. Cancer Res., 48: 6642-6647. 1988.

32. Rao, C. V., Rivenson, A.. Kaliwalla, M., Kelloff, G. J., and Reddy, B. S. Chemo-

prevenlive effect of ollipraz during different stages of experimental colon carcinogenesis induced by azoxymelhanc in male F344 rals. Cancer Res., 53: 2502-2506,

1993.33. Pozharisski, K. M. Tumors of Ihe intestines. In: V. Turcusov and V. Mohr (eds.),

Pathology of Tumors in Laboratory Animals, Vol. 1. pp. 159-198. 1ARC ScienlificPub. No. 99. Lyon, France: International Agency for Research on Cancer. 199(1.

34. Bleasdalc, J. E., McGuire, J. C., and Bala, G. A. Measurement of phosphoinositide-specific phospholipase C activity. Methods Enzymol.. /87: 226-237, 1990.

35. Leslie, C. C. Macrophage phospholipase A, specific for jn-2-arachidonic acid.Melhods Enzmol., /87: 216-225, 1990.

36. Rao, C. V., Rivenson, A., Simi, B., and Reddy, B. S. Chemoprevention of coloncarcinogenesis by dietary curcumin, a naturally-occurring plant phenolic compound.Cancer Res., 55: 259-266, 1995.

37. Fischer, S. M., Baldwin, J. K., Jasheway, R. W., Patrick, K. E., and Cameron, G. S.Phorbol ester induction of 8-lipoxygenase in inbred SENCAR (SSIN) but notC57BL/6J Mice correlated with hyperplasia, edema, and oxidant generation but notornithinc decarboxylase induction. Cancer Res., 48: 658-664, 1988.

38. Wallenberg, L. W., Coccia, J. B., and Lam, L. K. Inhibitory effecls of phenolic compounds on benzo(tï)pyrene-induced neoplasia. Cancer Res., 40: 2820-2823, 1980.

39. Frenkel, K., Wei, H., Bhimani, R., Zadunaisky, J. A.. Huang, M. T., Ferrara, T.,Conney, A. H.. and Grunberger, D. Inhibilion of tumor promoter-medialed processes

in mouse skin and bovine lens by caffeic acid phcnelhyl cslcr (CAPE). Cancer Res.,53: 1255-1261, 1993.

40. Nishzuka, Y. The role of prolein kinase C in cell surface signal Iransduction andtumor promolion. Nature (Lond.). 308: 639-698, 1984.

41. Fürstenberger,G., Schurich. B., Kaina, B., Pelrusevska, R. T., Fuesnig, N. E., andMarks, F. Tumor induction in inilialcd mouse skin by phorbol esters and mcthyl-methanesulfonate: correlation belween chromosomal damage and conversion ("slageI of lumor promolion") in VITO.Carcinogenesis (Lond.), 10: 749-752, 1989.

42. Baud, L., Hagege, J., Sraer, J., Rondeau. E., Perez, J., and Ardaillau, R. Reacliveoxygen production by cullured ral glomerular mesangial cells during phagocytosis isassocialed with stimulation of lipoxygenase activity. J. Exp Mod., 158: 1836-1852,

1983.43. Glasgow, W. C., Afshari, C. A., Barrett, J. C., and Eling, T. E. Modulation of the

epidermal growth factor mitogenic response by melaboliles of linolcic and arachi-

donic acid in Syrian Hamsler Embryo Fibroblasls: differential effects in tumorsuppressor gene (+) and (-) phenolypes. J. Biol. Chem., 267: 10771-10779, 1992.

44. Su, Z-Z., Grunberger, D.. and Fisher. P. E. Suppression of adcnovirus type 5ElA-mediated transformation and expression of the Iransformed phenotype by caffeicacid phenelhyl esler (CAPE). Mol. Carcinog., 4: 231-242, 1991.

45. Guarini, L., Su, Z-Z., Zucker, S., Lin, J., Grunberger, D., and Fisher, P. B. Growlhinhibition and modulalion of antigcnic phenolype in human melanoma and glioblas-

toma multiforme cells by caffeic acid phenethyl esler (CAPE). Cell. Mol. Biol., 38:513-527, 1992.

46. Su, Z-Z., Lin, J., Grunberger, D., and Fisher. P. B. Growlh suppression and loxicilyinduced by caffeic acid phenethyl ester (CAPE) in type 5 adcnovirus-transformcd rat

embryo cells correlate directly with transformation progression. Cancer Res., 54:1865-1870, 1994.

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