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ntimetastatic effects ofpropolis caffeic acid

phenethyl ester andcaffeic acid onmammary carcinoma ofCB mouse

BASIC, N. ORSOLIC, Z. TADIC,N. MACEDO FEREIRE ALCICI*,A. BRBOT-SARANOVIC**, K. BENDELJA***,B. KRSNIK**** and S. RABATIC***

Department Animal Physiology, Faculty ScienceUniversity Zag reb. Croatia

CDNA?, Belo Horizonte. Minas Gerais BR** Department ChemistryVeterinary Faculty. Zagreb. Croatia

***Institute Immunology, Zagreb, Croatia****Department ZoohygieneVeterinary Faculty, Zagreb, Croatia

SUMM RY

We have studied the antimetastatic efficacy of water-soluble derivativeof Brazilian or Croatian propolis WSDP and compared it with antimetastatic effect of caffeic acid phenethyl ester CAPE or with caffeic acid CA . In vitro cytotoxici ty of these compounds on tumor cells were alsostudied. Tumor was a transplantable mammary carcinoma MCA . Metastases in the lung were generated by injecting 2 X 10 5 viable tumorcells iv: tested compounds were given po before or after tumor cell inoculation. In in vitro studies V79 cells, HeLa cells or M e cells wereused. treatment of mice significantly reduced the number of tumor nodules in the lung. In invitro studies WSDP did not affect the growth of

cells. while CAPE and CA expressed strong cytotoxic effect. Antitumoreffect of WSDP was in part due to apoptosis of M cells.

Copyright 1998 by Monduzzi Editore p - Bologna Italy

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INTRODUCTION

Propolis from honey beehives contains various chemical constituents thatexhibit a broad spectrum of activities including antibacterial. antifungal.antineoplastic. cytostatic. and anti-inflammatory propert ies Jeddar et al1985: Hladon et al 1980: Koshihara et al 1984). Propolis also contains avariety of compounds including caffeic and benzoic acid and their esters.substituted phenolic acids and their esters, tlavonoid glycones, andbeeswax Greenaway et al 1987). Some of the above mentioned biological activities of propolis may be due to its chemical const ituents Koshihara et al 1984: Grunberger et al 1988). Several naturally -occurringcompounds in fruits, vegetables and propolis, such as phenols, indoles.aromatic isothiocyanates and dithiolethiones, have been shown to inhibitseveral types cancer including the cancer of the colon Wattenberg1985). It was also demonstrated that dietary administrat ion of hydroxycinnamates which are constituents of propolis, significantly inhibitedbenzo a)pyrene-induced neoplasia in the forestomach of mice Wattenberg et al. 1985).Caffeic acid 3A-dihydroxycinnamic acid) and its esters, caffeic acidphenethyl ester-CAPE), which are present in propolis at levels of 2025 Greenway et al 1987) are the agents suspected of having a broadspectrum of biological activities including tumor suppression. Caffeicacid ester derivatives present in propolis are more lipophilic and thus facil itate their entry into the cells Greenaway et al 1988). Differential cytotoxicity has been observed in normal ratlhuman versus transformedra tlhuman melanoma and breast carc inoma cell lines in the presence ofCAPE.In this studies, we describe the antimetastatic propert ies of the propolisobtained from Brazil and Croatia in the murine mammary carcinomamodel Basic) and compare its antitumor efficiency with the antitumoraction of CA and /o r CAPE. The mode of antitumor actions of thesecompounds v vo and v tro studies are also described

MATERIALS AND METHODS

TumorThe tumor was a transplantable mammary carcinoma MCA , weaklyimmunogenic to syngeneic CBA mouse Basic and Varga 1979). Tumornodules in the lung were generated by injecting 2x10 5 viable tumor cellsintravenously. The mice were killed 21 days after tumor cells inoculation, and the of number of tumor nodules in the lungs was determined.

Water soluble derivative WSD of propolisThe water-soluble derivative WSD) of propolis was prepared by methoddeskribed elsewhere Nikolov et al 1987). Briefly, Brazil ian CDNAP,Belo Horizonte, Minas Gerais, Brazil) or Croatian surroumdings ofZagreb) propolis was extracted with 96 ethanol, filtered and evaporated to dryness in vacuum evaporator. The resultant resinous productwas added to a stirred solution of 8 L-lysine Sigma chernie, Deisenhofen, Germany) and freeze-dried to yield WSD, a yellow-brown powder. The WSD was stored under sterile conditions at 4C. Before use, the


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WS D was dissolved in distilled water. It was given to mice per os tpot atdoses of 50 or 150 rug/kg.

CAPE-Caffeic acid phenethyl esterCAPE was obtained by modified method of Grunberger et al 1988). Inshort. esterification of caffeic acid with phenethyl alcohol molar rat ionsI: 15) in benzene retluxing. 3- 4 days. water removed by dean-stark trap).Following work-up. excess phenethyl alcohol was removed by Kugelrohr distillation 60C < 0.1 mm Hg) to give pure CAPE. rnp l26-128C.needles benzene or H:O). 40 yield. All properties of natural and synthetic CAPE were identical Grunberger et al 1988).

CA-Caffeic acid -3,4 dihydroxycinnymic acidCA was purchased f rom Aldrich-chemie, Mi lwaukee. WI. USA. CAPEand CA were gi ven to mice pe r os p o at doses of 50 or 150 mg/kg.

Cell linesIn v tro experiments. we used human cervical carcinoma cells Hel.a)and Chinese hamster lung V79 f ibroblasts . The average doubling time inlog phase was about 20 h for He La cells and 12 h for V78 cells. HeLaand V79 cells were grown in monolayer cul tures in plast ic disposablePetri dishes Falcon) in Minimal Essential Medium MEM) with 1non-essential amino acids an d 10 fetal calf serum. Cell cul tures wereincubated at 37C in humid atmosphere containing 5 CO : in air.

Cell counting

Cells were counted using Coulter Counter Model B. Coulter Electronics,Dunstable , England). Petri dishes with different concentra-t ions of thestudied compounds were usually incubated for 72 h. Then . the cel ls werecounted in triplicates. Prior to cell number determination medium wasremoved. cel ls were rinsed with phosphate-buffered saline PBS) trypsinized. diluted and counted Maysinger et al 1987).

Long-term experimentsLong-term experiments were performed with proliferating cultures.Hundred cells were seeded into the Petri dishes. After 24 h. different

concentrations of studied compounds were added and cel ls were cul turedon 37 )C for 10 days in air with 5 CO:. The medium was then removedand colonies were stained with 10 Giernsa, Colonies containing 50cells were counted and results were expressed as the percent of coloniesof untreated cells. Th e plating efficiency of untreated control culture was54 .

Dose-response experimentsExponential or plateau phase cel ls were treated with different concentrations of compounds for 24 h. The drugs were then removed and Petridishes were rinsed three times with PBS. The cells were trypsinized,counted and seeded into Petri d ishes l00 cel ls /dish). After 10 days. themedium was removed and cells were stained as described above. Th eplating efficiencies of untreated control of exponential cul tures were 84and 75 . respectively. Mean values. standard errors statistical evaluationby the t-test were ca lculated for each dose or time point.

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Apoptosis analysisApoptosis was determined by techniques described by Tellford et al 1992 . Brietly. bivariant flow cytornetry was performed on cell grownin the presence or absence of WSD of propolis. CA and CAPE for various times 3 and 15 hours . The cells were washed in cold PBS twice andresuspended in a small volume of IX binding buffer. Fluorescein-labeledannexin V and propidium iodide were added to the cells. The were thenanalysed by tlow cytornetry.DNA content of the tested cells was determined by staining withpropidium iodide PI . The cells were incubated in of fixing solution for min at 4C. washed in PBS. resuspended in ~ e r m e i l i z i n gsolution in the presence of of PI and incubated at 4 C for 15 min.The cells were then washed with PBS and immediately analyzed by tlowcytometry.

RESULTS AND CONCLUSIONS

this work. we show that WSD of Brazilian and/or Croatian propolis.CA, and PE prevent the growth of tumor nodules artificial metastases of M in mice. The effect of WSD of propolis, CA and CAPE onthe metastatic ability of tumor cells was tested in lung colony assay.Metastasis in the lung were generated by injecting 2x 10 5 viable tumorcells intravenously. Tasted compounds were given per os before or aftertumor cell inoculation: the dose comprises mglkg or 150mglkg of eachcompound. Mice were killed 21 days after the treatment and the numberof tumor nodules in the lungs was determined. Table shows that mice receiving tested compounds, the number of tumor nodules in thelung was significantly lower p


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Table The effect of WSD of propolis on \ICA lung nodule formation in th Ioternational

N ERCBA mice. Animals were treated with 50 or 150 mg kg of tested compounds ONGRESSbefore or after tumor cell inoculation

TREATMENT DOSE TIME OF No OF TUMOR RANGEmglkg TREATMENT NODULES/

LUNG mean SE t

NO TREATMENT 144.30 IU S 122 - 196

CROATIAN 50 5,37 2,27 0 -7PROPOLIS 150 13,772,21 4 - 2 3

BRAZILIAN 50 15 IOand5 7,75 U S 0- 13PROPOLIS 150 DAYS BEFORE 9,55 2,16 I - 19CAFFEIC 50 TUMOR CELL 15,00 3.55 4 - 3 8

ACID 150 INOCULATION b 15,44 2.07 7 - 29

CAPE 50 10,62 1.94 0-19150 15.5 4.81 5 - 38

CROATIAN 50 15,66 2.97 5 - 2 9PROPOLIS 150 17 423 19 3 - 2 7

BRAZILIAN 50 2, 7 and 12 30,20 3,70 17 - 40PROPOLIS 150 DAYS AFTER 16,11 2,16 8 - 2 6

CAd 50 TUMOR CELL 47,00 6,71 33 - 68CA 150 INOCULATION b 70,00 12,74 50 - 113

CAPE e 50 36.66 7,27 20 - 71

CAPE 150 53,50 5.03 40 -67a Tested compounds were given per as po before and after tumor cellinoculation.

b 2xI0 5 tumor cells per mouse injected iv, the number of tumor nodules in thelung was determined 21 days after tumor cell inoculation.

c Groups comprised 7 - 9 mice each mean standard error)d Caffeic acide Caffeic acid phenethyl ester

togens ill vitro. production of IL-l by peri toneal macrophages, rosseteformation of lymphoid cells with SRBC and plaque forming ability ofsplenocytes to SRBC respectively, correlated well with the antime-tastatic properties, inhibitory effect on tumor growth and tumor take. oftested compounds . These results are in line with previous results fromother laboratory, suggesting that the stimulatory activity of WS D of pro-polis may be assoc ia ted wi th act ivation of rnacrophages, which leads toan increase of their phagocytic capacity Dirnov et al 1992; 1991; Iva-novska 1993; 1995; Tatefuj i et al 1996; Mirzoeva 1996). Increased levelof IL-l activity produced by these ac tiva ted macrophage correlated di-

rectly with tumor cytotoxicity unpublished). Activation of macrophagesis important for the immunogenic property to the extract. as it leads tothe production of factors regulating the functions of Band T-cells Kur-land et al 1977). Th e act ivated macrophage is a major component of hostdefence aga inst infect ious and neoplast ic d isease Maltzer et al 1977).Exam Ie of this act ivitv was demonstrated ill vitro: ethanolic extract of 67


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Figure 1. Growth curves of HeLa cells treated with WSD of Croatian propolis 0 , WSD of Brazilian propolis 6 . caffeic acid x . CAPE 0 . Cells wereseeded lx10 5 per Petri dish and incubated 24 h, then drug was added. Cellswere exposed to the action of the drug for next 72 h. counted and expressed as of number of untreated cells.

proplis EEP increased the cytotoxicity of NK cells. inhibited the devel-opment of HeLa cervix and KB nasopharynx carcinoma cells v troand exerted cytotoxic activity on Ehrlich carcinoma cells Scheller et al1989; Hladon et al 1980 .To study whether the antitumor effect of tested compounds achieved

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Figure 2. Growth curves of V79 cells treated with WSD of Croatian propolis 0 . WSD of Brazilian propolis 6 . caffeic acid x , CAPE 0 . Cells wereseeded Ix10 5 per Petri dish and incubated 24 h. then drug was added. Cellswere exposed to the action of the drug for next 72 h. counted and expressed as of number of untreated cells.


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v vo was due to the direct cytotoxicity to tumor cells or through otherways of action, we tested their influence on the growth of HeLa and V79cells ll v tro Various concentrations of the compounds were used. Theirinfluence was studied during 72 h incubation time. The cells were seededin Petri dishes and 24 h thereafter the different concentration of com

pounds were added to the cultures. igure 1. and igure 2, show the theinfluence of the compounds on the growth of the tested cells in 72 h incubation time.WSD of Croatian or Brazilian propolis did not express cytostatic effecton V79 cells normal cells) and was found to be the least effectivegrowth inhibitor of HeLa cells tumor cells) in comparison to CA andCAPE, respectively. Growth inhibition with CA on HeLa and V79 cellswas dose-dependent. Also, the inhibitory effect of CAPE on HeLa cells umor cells) was more effective at much lower concentrations lC so 19I-tg/ml); inhibitionconcentration lC

so was determined from the corresponding growth

curve indicating the drug concentration causing 50 growth inhibition.However, in colony-forming studies, the cytotoxic efficacy of CA andCAPE on HeLa cells was highly pronounced igure 3,) as compared to

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Figure 3. Survival of proliferating HeLa cells exposed to increasing concentrations of WSD of Croatian propolis D), WSD of Brazilian propolis Mcaffeic acid x), CAPE 0 for days. Cells were seeded for colonies andsupplemented with a drug. Colonies were counted and counts were expressedas of colonies of untreatedcells.

their effects achieved in growth studies. The dose of Sug/ml was shownto be lethal for the cells. In contrast, the effect of WSD of propolis incolony-forming studies was insignificant. igure 4. shows killing action of WSD of propolis, CA or CAPE on V79cells. After long term exposure, V79 cells were more sensitive to WSDof Brazilian propolis than WSD of Croatian propolis. The degree ofkilling action CAPE was more pronounced than CA. The concentration 69


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Figure 4. Survival of proliferating V79 cells exposed to increasing concentra-tions of WSD of Croatian propolis 0 , WSD of Brazilian propolis L\L caffeicacid x , CAPE 0 for 10 days. Cells were seeded for colonies and supple-mented with a drug. Colonies were counted and counts were expressed as ofcolonies of untreated cells.

of lOug/rnl of CA or CAPE was lethal for V79.Since the cytotoxicity of tested compounds was observed, it wa s of im-portance to determine if there wa s a growth-dependent sensitivity. Cellsin exponentially growing phase were treated with tested compounds for24 h Figure 5. an d Figure 6. . Figure 5. shows that survival of HeLa

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Figure 5. Survival of proliferating HeLa cells exposed to increasing concen-trations of WSD of Croatian propolis 0 , WSD of Brazilian propolis L \ .caffeic acid x , CAPE 0 for 24 hours. Cells were seeded for colonies and su-plemented with a drug. Colonies were counted and counts were expresed as of solonies of untreated cells.


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Figure 6. Survival of proliferating V79 cells exposed to increasing concentrations of WSD of Croatian propolis 0 , WSD of Brazilian propolis ~ ,caffeicacid x , CAPE 0 for 24 hours. Cells were seeded for colonies and suplemented with a drug. Colonies were counted and counts were expresed as ofcolonies of untreated cells.

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Figure 7. The effect of WSD of propolis, CA and CAPE on induction ofapoptosis in MCA cells. The cells were analyzed by flow cytometry. l-control,2-Croatian propolis SOllg / ml), 3-Brazilian propolis SOllg / ml), 4-CA 2Sllg/ ml), S CA SOllg / ml), 6-CAPE Sllg / rnl), 7-CAPE (IOug / ml), 8-control, 9Croatian propolis SOllg / ml), IO-Brazilian propolis SOllg / ml), II-CA 2Sllg/ rnl). 12-CA SOllg / ml). 13-CAPE Sllg / rnl), 14-CAPE (l Oug / rnl) 71


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cells was not affected by the presence WSD of Croatian or Brazilianpropolis , while CA and CAPE in concentration of IOug/ml killed all thecells in culture.Figure 6. Shows that the survival of V79 cells was higher by the presence of WSD of Brazilian and Croatian propolis than in the controlgroup. CA and CAPE killed cells in a dose-dependent fashion. CA inconcentrations required to kill 50 of cells in the exponential phase ofgrowth l C so ranged from 12 tol5 ug/rnl, while CAPE killing actionIC so ranged from Sug/m to 7 ~ g m lStudies on apoptotic or necrotic death of MCA cells Figure 7. and 8.)show that the fragmentat ion of tumor cell DNA starts 3 hours after thetreatment of MCA cells with either tested compound. After 15 hours ofincubation of MC A cells, the percentage of apoptotic death was 5-23 .The most pronounced DNA fragmentation was induced by CAPE. Atsome time, the similar rate of MCA cell necrosis was obtained. This suggests that WSD of Brazilian or Croatian propolis as well as CA and

CAPE kill certain proportion of tumor cells by inducing apoptosisThe results of these studies could be summarized as follows:

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F.igure 8. The effect of WSD of propolis, CA and CAPE on induction of necrosis in MCA cells. The cells were analyzed by flow cytometry. l-control, 2Croatian propolis 5 ~ g/ ml , 3-Brazilian propolis 5 ~ g/ ml , 4-CA 2511g /ml , 5-CA 5 11g / rnl), 6-CAPE 5 ~ g/ ml , 7-CAPE I 11g / ml), 8-control, 9Croatian propolis 5 11g / rnl), lO-Brazilian propolis 5 11g / ml , II-CA 251lg/ ml , 12-CA 5 11g / rnl), 13-CAPE 511g / ml , 14-CAPE I 11g / ml)

72a) WSD of Brazilian and Croatian propolis expressed strong antitumoractivity against tmor nodules of MCA in CB A mice.

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b The anti metastatic activity of CAPE and CA was much less pronounced as compared to the effects of the WSD of propolis.

c The antitumor activity of propolis could be partially related to itsapoptotic and immunomodulatory activities.

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