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Antitumor effect of Croatian propolis as a consequence of diverse sex-related dihydropyrimidine dehydrogenase (DPD) protein expression.


Keywords: Propolis Mammary carcinoma DPD Tumor growth Metastasis Gender-related


The aim of this study was to detect the antitumor properties of Croatian propolis in BALB/c male and female mice injected with 4T1 mammary carcinoma. Furthermore, the gender-dependence of this effect and the possible involvement of combined effect of propolis and 5-Fluorouracil (5FU) on dihydropy-rimidine dehydrogenase (DPD) transcriptional and translational level, were determined. In combination with 5FU propolis treatment induced gender-related effects. The results of the study revealed that pre-treatment of mice with propolis combined with 5FU treatment prolonged the suppressive effect of 5FU on tumor growth and reduced the number of metastasis only in male mice. Only males pretreated with propolis prior to 5FU administration had decreased DPD protein level indicating higher sensitivity to 5FU. Thus, benefitial effects of propolis in male tumor-bearing mice treated with 5FU might be explained by increased sensitivity to 5FU as the result of translationally downregulated DPD.

[C] 2011 Elsevier GmbH. All rights reserved.


Propolis has been used in folk medicine since ancient times. Nowadays its usage has reached the stage of scientific medicine. Propolis is considered to have antiviral, antibacterial, antifungal and antioxidative properties due to its scavenging capacity of free radicals. Beside other compounds, it is composed of flavonoids which have high antioxidative potential and are able to influence various pathological processes (tumors, osteoporosis, cardiovascular and neurodegenerative diseases) (van Acker et al. 2000). It has been shown, that crude Egyptian propolis extract markedly reduced tumor volume of mice Ehrlich ascites carcinoma (El-khawaga et al. 2003). Water-soluble derivates of propolis markedly reduced metastasis induced with transplantable mammary carcinoma cells (Orsolic et al. 2004). A large number of enzymes that are involved in oncogenesis are influenced by flavonoides. It has been recently recognized that flavones isolated from radix Scutellariae demonstrate a new source of anticancer drugs and new chemotherapy adjuvant (Li-Weber 2009). Besides direct effect, some flavonoids can affect tumors by reducing toxicity of cyclophosphamide and vinblastine (Lahouel et al. 2004). 5-Fluorouracil (5FU) is widely used to treat solid tumors, i.e. as a single agent to treat colorectal cancer (Meropol 1998) and as a significant component of combination therapy for breast, head/neck and upper gastrointestinal malignancies (Diasio and Harris 1989). 5FU is initially catabolized to 5-fluorodihydrouracil by dihydropyrimidine dehydrogenase (DPD) mainly in the liver. DPD has variable activity in tumors and this may potentially influence the efficiency of 5FU (Milano and Etienne 1994). Pharmacokinetic studies have demonstrated that 85% of clinically administrated 5FU is inactivated and eliminated through the catabolic pathway. Thus, DPD has been recognized as an important factor in 5FU pharmacokinetics, clinical toxicity and tumor resistance. A high level of DPD would metabolize 5FU to inactive products before cytotoxic nucleotides can be formed - the lower the enzymatic activity, the greater the cytotoxicity, while increased DPD activity is associated with the resistance to 5FU (Diasio et al. 1998).

Thus, based on our previous studies of antioxidant properties of Croatian propolis (Sobocanec et al. 2006), the effect of propolis on gene expression (Sobocanec et al. 2008) and high concentration of some phenolics in ethanolic extract of propolis presented in our study (Sobocanec et al. 2006), we aimed to detect antitumor properties of Croatian propolis in vivo, the gender-dependence of this effect and the possible involvement of DPD in the combined effect of propolis and 5FU treatment of mouse mammary carcinoma in vivo.

Materials and methods


In our study we used a representative mixture of bee collected propolis obtained from the apiaries located in naturally preserved coastline area of Midlle Dalmatia, Croatia (+44[degrees] 7'24"N and +15[degrees]14"E). Propolis was collected from spring to winter by special ecological net introduced in hive. Nets were taken and frozen to promote propolis removal. Samples of propolis were pooled. To pulverize the gluey crude propolis in powder, an original treatment by firm HEDERA d.o.o. (Stobrec, Croatia) was prepared with no chemical refinement.

Propolis extract

Propolis (1 g) was homogenized in a chilled mortar and mixed vigorously with 10.45 ml of 80% (V:V) ethanol during 72 h at the room temperature. The extract was filtered through Whatman No. 1 paper and the residue was washed with 0.5 ml of 80% ethanol. Such prepared extract was kept at -20[degrees]C, at least for 24 h. This extract was filtered through nylon filter Schleicher &SchnelI (0.2 [micro].m pore) and submitted to HPLC analysis.

HPLC analysis

Qualitative and quantitative chromatographic analysis of phe-nolics was performed on a HPLC system (Agilent 1100 Series) equipped with a quaternary pump, multiwave UV/VIS detector, autosampler and fraction collector. The column used was a 5 p.m Zorbax RX-C18 (250 mm x 4.6 mm, Agilent Technologies). Injection volume was 200u.1, flow rate l.0 ml/min and temperature was 45[degrees] C. The propolis extract was fractionated into nine fractions: fraction 1 ([t.sub.R] 9.8-10.6min), fraction 2 ([t.sub.R] 12.8-13.6min), fraction 3 ([t.sub.R] 18.9-20.3 min), fraction 4 ([t.sub.R] 22.7-23.7 min), fraction 5 ([t.sub.R] 24.3-25.3 min), fraction 6 ([t.sub.R] 26.5-27.8 min), fraction 7 ([t.sub.R] 28-29 min), fraction 8 ([t.subR] 30-31 min), fraction 9 ([t.sub.R] 32.6-34.1 min) were obtained using elution profile consisted of solvent A (5% formic acid) and solvent B (methanol). Linear gradient from 10% to 90% B within 45 min was used.

Phenolic compounds of collected fractions were identified by UV-VIS spectroscopy and chromatography (HPLC) with authentic standards. Fractions 1-8 were analyzed using solvents A (5% formic acid) and B (acetonitrile) on linear gradient from 5% to 53% solvent B within 30 min. Fraction 9 was analyzed using isocratic separation on 33.5% solvent B in 30 min. Chemical analysis of propolis was determined using standard methods previously described (Beythien and Diemair, 1963; Kjeldahlu 1960; Gorsuch 1970).

Phenolic standards

The following compounds representative of various subclasses of phenolics were used in our study: quercetin, isorhamnetin, kaempferol, luteolin, myricetin, taxifolin, naringenin, pinocembrin, galangin, chrysin, genistein, daidzain and phenylpropanoid caffeic acid. All of standards were dissolved in ethanol (96%, V:V) to give 0.01 mg/ml solutions.


Female and male BALB/c mice aged 4 months from breeding colony of the Ruder Boskovic Institute (Zagreb, Croatia) were used. The animals were maintained under the following laboratory conditions: light on from 06:00 to 18:00; 22 [+ or -] 2[degrees]C room temperature; access to food pellets, and tap water ad lib. Experimental groups (tumor bearing, tumor bearing and 5FU treated, tumor bearing and propolis treated, tumor bearing and 5FU and propolis treated) and control groups consisted of 18 mice per group (9 females and 9 males). Mice were fed 14 days before tumor injection either with commercial food pellet (control group, tumor-bearing and tumor-bearing and 5FU treated) or by commercial food mixed with crude propolis powder (300 mg/kg bw) (tumor-bearing and propolis treated and tumor-bearing propolis and 5FU treated). In groups fed with propolis, propolis was added to commercial food until sacrifice. Dose of 300 mg/kg bw of native propolis was used to correspond to the dose usually used in humans with correction for mouse metabolism. Mice were injected (after 14 days of feeding with propolis) in the gluteal region with 1 X 10 (6) (0.1 ml) with 4T1 mammary carcinoma cells which spontaneously induce metastasis in lungs and livers. One day after tumor injection some groups of mice received 200 mg/kg b.wt. (0.5 ml i.p.) 5FU. Tumor response was studied as a tumor growth delay study. In order to get a tumor-growth time (TGT in days), three orthogonal diameters (A, B and C) of a growing tumor were measured with calliper every second day (starting on the 11th day after treatment) until 24th day when they reached endpoint volume (1500 [mm.sup.3]) and were scarified The tumor volume (V) was calculated by the formula V=ABC[pie]/6.

Determination of protein concentration

Protein concentration in the tissue samples (mg/g) was estimated by the method of Lowry et al. (1951), using bovine serum albumin as the standard.

Total RNA isolation

Total RNA was extracted from three pools of three individual mouse livers per pool using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The integrity and concentration of RNA was determined by measuring absorbance at 260 nm followed by electrophoresis on 1% agarose gel.


For the first strand cDNA synthesis, the reverse transcription reaction was performed with 1 [micro]g of total RNA prepared from each pool using Superscript[TM] II Rnase H- Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). cDNAs were subjected to PCR using the specific primers for the DPD and ((beta)actin genes, respectively. The PCR reactions were performed with HotMaster, Taq DNA Polymerase (Eppendorf, Hamburg, Germany) and forward and reverse primers (Invitrogen, Carlsbad.CA.USA) as listed in Table 1. Initial incubation was at 95[degrees]C for 15 min, followed by 28 cycles, denat-uration at 94[degrees]C for 30s, annealing at 57[degrees]C for 30 s, and extension at 70[degrees]C for 30 s for fi-actin and 26 cycles denaturation at 94[degrees]C for 45 s, annealing at 55[degrees]C for 30 s, and extension at 70[degrees]C for 40 s for DPD. The number of cycles was optimized to ensure that the PCR has not reached its plateau.
Table 1

Oligonucleotid primers used in this study.

Gene               Primer sequence      Product size (bp)


  Sens      5'-CCAAAGTGAAAGAAGCATTG-3'          418


  Sens      5'-ATGGATGACGATATCGCTG-3'           568

Western blot analysis

Fifty micrograms of total, denaturized, proteins from each sample were subjected, in triplicates to 10% SDS-PAGE according to the method of Laemmli (1970). The proteins were then transferred to the Immun-Blot[R] PVDF membrane (BIO-RAD, Hercules, CA). Membranes were blocked overnight at 4[degrees]C in 5% non-fat dry milk in 50 mM phosphate buffer (pH 7.8 and 0.1% Tween 20). Western blotting was performed using the primary polyclonal goat IgG anti-DPD (C-14, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) antibody, diluted 1:250, followed by donkey anti-goat IgG horseradish peroxidase-conjugated secondary antibody (C-14, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted 1:5000. Anit-ERK-2 (C-14, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), diluted 1:1000 was used as a loading control. Bands were visualized using BM Chemiluminescence blotting substrate (POD) (Roche Applied Science, Mannheim, Germany), exposed to film and digitized.

Statistical analysis

Statistical analyses were performed using SPSS for Windows (17.0). Obtained data were tested by one-way ANOVA to assess the overall difference among experimental groups. When significant differences occurred then differences among the means were assessed by the post hoc LSD test. For nonparametric analysis Wilcoxon signed Rank test and Mann-Whitney L/-test were used. Statistical significance was defined as p < 0.05.


Concentration of detected phenolics in the ethanofic extract of propolis

Concentration of the HPLC detected phenolics in ethanolic extract od propolis is presented in Table 2. Out of 13 phenolics examined 4 were not detectable (myricetin, daidzein, genistein, taxifolin), chrysin and pinocembrin were the most abundant (9.749 or 8.074 p.mol/g, respectively) followed by galangin (5.335 [micro].mol/g), kaempherol (2.571 p.moI/g), naringenin (2.239 p.mol/g) and caffeic acid (1.592 p.mol/g). In the least amount were detected quercetin (0.483 p,mol/g), luteolin (0.295 ptmol/g) and isorhamnetin (0.102 p.mol/g). Chemical analysis of propolis presented in Table 3 showed that it contains 4.0% water, 25.6% fat, 1.6% protein, 0.7% ashes and minerals as follows: lead (Pb) 2.15 mg/kg, iron (Fe) 344.5 mg/kg, copper (Cu) 1.85 mg/kg, mercury (Hg) 0.0078mg/kg, zinc (Zn) 131 mg/kg, manganese (Mn) 7.61 mg/kg, chromium (Cr) 0.707 mg/kg, calcium (Ca) 770 mg/kg, magnesium (Mg) 271 mg/kg. Analysis of vitamins content in propolis were as follows: vitamin A <0.1 mg/kg, vitamin C 10 mg/kg, vitamin B1 14.5 mg/kg, vitamin B2 0.62 mg/kg, vitamin B6 22.5 mg/kg.
Table 2

Phenol contents of propolis. Qualitative and quantitative
composition of propolis extract was obtained by HPLC analysis,
nd = not detectable.

Flavonoid      Concentration ((mu)mol/g)

Chrysin              9.749
Pinocembrin          8.074
Galangin             5.335
Kaempferol           2.571
Naringenin           2.239
Caffeic acid         1.592
Quercetin            0.483
Luteolin             0.295
Isorliamnetin        0.102
Myricetin            nd
Daidzein             nd
Cenistein            nd
Taxifolin            nd

Table 3

Chemical analysis of propolis.

Compound    Percent (%)

Fat         25.6
Water       4.0
Protein     1.6
Ashes       0.7

                  Concentration (mg/kg)


  Calcium (Ca)        770
  Iron (Fe)           344
  Magnesium (Mg)      271
  Zinc (Zn)           131
  Manganese (Mn)        7.61
  Lead (Pb)             2.15
  Copper(Cu)            1.85
  Chromium (Cr)         0.707
  Mercury (Hg)          0.0078

Vitamin content
  [B.sub.6]            22.50
  [B.sub.1]            14.50
  C                    10.00
  [B.sub.2]             0.62
  A                     0.10

The effect of propolis pretreatment on 5FU inhibited tumor growth

The volume of tumor was measured until the 24th day of growth. As seen in Fig. 1 tumor growth was significantly inhibited by 5FU (p< 0.005) in males and females until the 15th day without significant difference between sexes. After that period 5FU still decreased tumor growth on 17th and 20th day (p< 0.005 or <0.05, respectively) in males while in females 5FU was effective only until the 17th day (p<0.05) (data not shown). Males treated with propolis prior to 5FU had significantly smaller tumors than males treated with 5FU only (p = 0.025,0.005 and 0.005, respectively). On the contrary, in female mice no significant difference was observed in mice treated with 5FU and in mice treated with 5FU and propolis. Propolis per se did not effect tumor growth whatever the treatment or sex.


The effect of propolis on the number of metastasis in lungs and liver of tumor-bearing mice

In lungs of either male (Fig. 2A) or female (Fig. 2B) tumor-bearing mice neither 5FU nor propolis or the combination of both affected the number of metastasis. On the contrary, in the liver of tumor-bearing female mice (Fig. 3B) 5FU significantly decreased the number of metastasis (p = 0.008) while in the liver of male mice (Fig. 3A) the number of metastasis, although decreased, did not reach significance. Opposite effect of propolis on the number of metastasis was observed in the liver of male and female mice. Compared to tumor-bearing mice it was increased in liver of female mice and decreased in the liver of male mice (without reaching statistical significance). Combined treatment with propolis and 5FU significantly reduced number of metastasis in male mice (p = 0.015) while the same treatment in female mice abrogated the effect of 5FU.



The effect of propolis on DPD gene expression in tumor-bearing mice

As presented in Fig. 4, no difference in DPD mRNA expression between males and females neither in any treated group of tumor-bearing mice nor in control animals was observed.


The effect of propolis on DPD protein level in tumor-bearing mice

As observed by Western blot analysis, DPD protein level in male mice was decreased by the addition of propolis in the group of tumor-bearing and 5FU treated mice (p = 0.038) (Fig. 5A). On the contrary, the addition of propolis to tumor-bearing and 5FU treated female mice was withouth effect. However, DPD protein level in liver of female mice was significantly lower in the group of mice treated with propolis compared to all other groups (p < 0.001) (Fig. 5B).



In this study, propolis per se did not affect tumor growth neither in males nor in females. However, in combination with 5FU propolis treatment induced gender-related effects on tumor growth. First, only in males the effect of 5FU was prolonged by the addition of propolis up to 24th day of tumor growth (data not shown). Second, males if treated with 5FU and propolis had significantly smaller tumors than females. Finally, in the liver of female mice the addition of propolis abolished 5FU induced reduction of the number of metastasis. In males, 5FU alone was not sufficient to reduce the number of metastasis but in combination with propolis the number of metastasis was significantly decreased. Thus, propolis in combination with 5FU affected tumor growth and metastasis in a gender-related manner, demonstrating favourable effects in males but not in females. The observed combined antitumor effect of native propolis and 5FU could be due to the flavonoid content in propolis. Namely, it has been demonstrated that quercetin participates in cancer prevention (Murakami et al. 2008) while chrysin (Habtemariam 1997) galangin (Tolomeo et al. 2008) caffeic acid and naringenin (Orsolic and Basic 2005) have anticancer activities. In our study the concentration of compounds with anti-tumor activity was specially high for chrysine and galangin (9.75 or 8.07 p.mol/g, respectively) high for kaempferol, naringenin and caffeic acid (2.51, 2.24 and 1.59 [micro].mol/g, respectively) while quercetin concentration was low (0.48 [micro].mol/g). It seems that biological properties of propolis are due to a natural mixture of its components and a single propolis constituent does not have an activity greater than that of the total extract (Kujumgiev et al. 1999).

As for now, multidrug resistance is a major obstacle to the effective treatment of cancer.

One of the major mechanisms of multidrug resistance is the expression of several different ATP-depending drug efflux pumps (Gillet et al. 2007). P-glycoprotein (P-gp) which acts as an efflux pump for various cancer drugs acts as multidrug resistance factor (MDR) in tumor cells by transporting certain anticancer agents out of the cell (Nobili et al. 2006). It has been found that a number of dietary substances could modulate P-gp functions (Honda et al. 2004). Recently, Ampasavate et al. (2010) examined the effects of a Curcuma longa and Curcuma sp. as well as isolated major curcuminoids on intestinal P-gp functions on human colonic adenocarcinoma cell line (Caco-2) in vitro. Their results show that Curcuma longa and Curcuma sp. as well as curcumin and demethoxycurcumin are capable of inhibithing human intestinal P-gp functions. Thus, in our study the interaction of propolis and 5FU might represent another possible mechanism of food-drug interactions and could modify the bioavailability of coadministered P-gp substrate drug.

However, these data do not explain the gender-related effect of propolis. One possible reason for the discrepancy among male and female mice in tumor growth and the effect on metastasis might be the fact that flavonoids display estrogenic activity. Among them genistein is the well-known phytoestrogen (McCarty 2006). Genistein, like estrogen, when administrated during tumor development, enhanced tumor growth of estrogen responsive tumors (Hsieh etal. 1998). However, in our study genistein in our propolis sample could be hardly associated with the gender-related effect. First, genistein is because of its bioavailability 20-fold less potent in vivo than in vitro (Birt et al. 2001), second in our sample of propolis the concentration of genistein was under the level of detectability, and finally the cell line 4T1, that we used to induce mammary carcinoma in our study, is estrogen receptor negative. Besides gonadal hormones, gender dimorphism in tumor growth and progression of metastasis might be linked to cytokines. It has been demonstrated that the mechanism of gender dimorphism in growth of T cell lymphoma in mice was associated with cytokines (Singh et al. 2005) DNA chip analysis of our previous study (Sobocanec et al. 2008) found, in the liver of female mice treated with propolis, significant upregulation in gene expression of IL-1 and IL-6. IL-1 was shown to augment metastasis (Giavazzi et al. 1990) while IL-6 is a key growth-promoting and antiapoptotic inflammatory cytokine (Naugler and Karin 2008). Thus, different effect of propolis in male and female tumor-bearing mice might be related to the repertoire of cytokines induced by propolis. However, gender dimorphism in our study was not the result of propolis per se since it appeared only in the group of propolis and 5FU treated mice. It was demonstrated that DPD is the enzyme responsible for 5FU degradation and therefore associated with sensitivity to tumor growth (Milano and Etienne 1994) is also gender-related (Etienne et al. 1994). Thus, by determining DPD in males and females we examined whether the observed gender-related differences could be linked to different regulation of DPD in males and females.

On the transcriptional level no gender-related variation neither in differently treated groups of animals or control mice was noted. At the translational level, only in males was the protein content of DPD significantly decreased in the group of mice treated with 5FU and propolis. It was shown that human tumor xenografts expressing low levels of DPD mRNA and DPD activity showed a significantly better response to 5FU than tumors with a high DPD mRNA level and DPD activity (Ishikawa et al. 1999). Thus, propolis might, in males, by decreasing DPD protein, increase the sensitivity to 5FU. The result would be prolonged 5FU effect and smaller tumors in males.

In summary, propolis induced gender-related effect on tumor growth and number of metastasis in mice liver. Pretreatment of male mice with propolis combined with 5FU prolonged the suppressive effect of 5FU on tumor growth and suppressed the number of metastasis. Thus, propolis in combination with 5FU demonstrated favorable effects in males but not in females. Only in male liver pretreatment with propolis in tumor-bearing and 5FU treated mice decreased DPD protein level. The results suggest that genderrelated propolis effect might be explained by increased sensitivity to 5FU in males as the result of translationally downregulated DPD.


This study was supported by Croatian Ministry of Science, Education, and Sport (grant no. 098-0982464-1647). The authors thank Masakazu Fukushima, Ph.D. Taiho Pharmaceutical Co., Kawauchi Takushima, Japan. The authors also thank Iva Pesun Medimorec for technical assistance.


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SandraSobocanec (a) *,TihomirBaloga (a), AnaSaric (a),Zeljka Macak-Safranko (a), MarinaStroser (a), Kamelija Zarkovicb (a), Neven Zarkovic (a), Ranko Stojkovic (a), Sinisa Ivankovic (a), Tatjana Marottia (a)

(a) Division of Molecular Medicine, Ruder BoSkovic Institute, Bijenitka 54, 10000 Zagreb, Croatia

(b) School of Medicine, University of Zagreb, Department of Pathology, Clinical Center Zagreb, Zagreb, Croatia

Abbreviations: 5FU, 5-fluorouracil; CAPE, caffeic acid phenethyl ester; DPD, dihydropirimidine dehydrogenase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HPLC, high performance liquid chromatography; PBMC, peripheral blood mononuclear cell.

* Corresponding author. Tel.: +385 1 4561 172; fax: +385 1 4561 010.

E-mail address: (S. Sobocanec).

doi: 10.1016/j.phymed.2011.01.015
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Author:Sobocanec, Sandra; Baloga, Tihomir; Saric, Ana; Macak-Safranko, Zeljka; Stroser, Marina; Zarkovicb,
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EXCR
Date:Jul 15, 2011
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