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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Immunomodulatory efficacy of ethanol extract of propolis on tumor-bearing mice with disseminated candidiasis ´ immunomodulatrice de l’extrait ´ethanolique de Efficacite propolis sur les souris porteuses de tumeurs avec une candidose ´ mine ´e disse A.R. Khosravi a,*, H. Shokri b, S. Darvishi a, M. Taghavi a a
Mycology Research Center, Faculty of Veterinary Medicine, University of Tehran, PO Box 14155-6453, Azadi street, Tehran, Iran b Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, Iran Received 1 January 2014; received in revised form 26 April 2014; accepted 15 May 2014
KEYWORDS Disseminated candidiasis; Candida albicans; Cancer; Propolis; Immunomodulation
Summary Objective. — This study was aimed at investigating the effect of propolis on immunosurveillance by measuring the levels of serum interleukin (IL)-4, IL-10, IL-17, tumor necrosis factor (TNF)-a and interferon (IFN)-g in tumor-bearing mice with disseminated candidiasis. Methods. — The ethanol extract of propolis was selected for this study. Balb/C female mice were infected with Candida albicans (C. albicans) and inoculated with spontaneous mouse mammary tumor (SMMT). The serum levels of tissue inhibitor of metalloproteinase-1(TIMP-1) were assessed by enzyme- linked immunosorbent assay (ELISA). Mice were treated daily with propolis solution (100 mg/kg, 0.1 mL, orally) for 3 days before IV challenge with C. albicans and SC challenge with SMMT and continued for 10 days. The rates of survival and tumor growth of understudy mice were investigated as well. The levels of TNF-a, IFN-g, IL-4, IL-10 and IL-17 cytokines in culture supernatants were determined by ELISA. Results. — The mean tumor size was significantly increased in tumor-bearing mice infected with C. albicans (16.98 0.49 mm2) as compared to other mice groups (P < 0.05). The results showed a significant decline of IL-4 and IL-10 levels after propolis administration to tumor-bearing mice infected with C. albicans (53.41 pg/mL, 156.81 pg/mL and 63.45 pg/mL) (P < 0.05). The increment of TNF-a (433.85 pg/mL) and IFN-g (120.43 pg/mL) levels were also observed.
* Corresponding author. Tel.: +0098 21 61117151; fax: +0098 21 66933222. E-mail address: [email protected] (A.R. Khosravi). http://dx.doi.org/10.1016/j.mycmed.2014.05.002 1156-5233/# 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Khosravi AR, et al. Immunomodulatory efficacy of ethanol extract of propolis on tumor-bearing mice with disseminated candidiasis. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.05.002
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A.R. Khosravi et al. Conclusion. — Data revealed that propolis has remarkable immunomodulatory effect, which provides a scientific validation for the popular use of this natural substance, and further investigation will help to understand propolis usefulness during immunosuppressive conditions. # 2014 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Candidose disséminée ; Candida albicans ; Cancer ; Propolis ; Immunomodulation
Re ´sume ´ Objectif. — Cette étude visait à étudier l’effet de propolis sur l’immunosurveillance en mesurant les niveaux sériques de l’interleukine (IL) -4, IL-10, IL-17, du facteur de nécrose tumorale (TNF)-a et de l’interféron (IFN)-g de souris porteuses de tumeurs avec une candidose disséminée. Mate´riel et me´thodes. — L’extrait l’éthanolique de propolis a été sélectionné pour cette étude. Des souris femelles Balb/c étaient infectées avec Candida albicans (C. albicans) et inoculées avec une tumeur mammaire spontanée de souris (SMMT). Les taux sériques d’une métalloprotéinase-1 (TIMP-1) inhibiteur cellulaire ont été évalués par enzyme-linked immunosorbent assay (ELISA). Les souris ont été traitées quotidiennement avec la solution de propolis (100 mg/kg, 0,1 mL, oralement) pendant 3 jours avant l’injection IV avec C. albicans et SC avec la SMMT, traitement poursuivi pendant 10 jours après challenge. Les taux de survie et la croissance des tumeurs des souris ont été enregistrés. Les niveaux de TNF-a, IFN-g, et des cytokines IL-4, IL-10, IL-17 dans les surnageants de culture ont été déterminés par la technique ELISA. Re´sultats. — La taille de la tumeur a été augmentée de manière significative chez les souris porteuses de tumeurs et infectées par C. albicans (16,98 0,49 mm2) par rapport aux autres groupes de souris ( p < 0,05). Les résultats ont montré une diminution significative des niveaux de l’IL-4 et de l’IL-10 après administration de propolis aux souris porteuses de tumeurs et infectées par C. albicans (53,41 pg/mL, 156,81 pg/mL et 63,45 pg/mL) ( p < 0,05). L’incrément des niveaux de TNF-a (433,85 pg/mL) et IFN-g (120,43 pg/mL) a également été observé. Conclusion. — Ces données ont révélé que la propolis a un remarquable effet immunomodulateur, et représentent une validation scientifique pour l’utilisation de cette substance naturelle. Des études complémentaires aideront à comprendre l’intérêt de la propolis lors de conditions immunosuppressives. # 2014 Elsevier Masson SAS. Tous droits réservés.
Introduction Propolis has gained popularity in alternative medicine, and it is used in food and beverages to improve health and to prevent diseases. Propolis is a chemically complex bee product, containing material collected from buds or exudates of plants, volatile substances and wax [3]. It is composed of 30% wax, 50% resins and vegetable balsams, 10% essential oils, 5% pollen and other substances [5]. Because of its various bioactivities, including antibacterial [21], antiviral [9], antifungal [7], anti-inflammatory [28] and antitumoral [4,22] effects, propolis has been used in folk medicine and has attracted many investigators’ interest. However, little is known about propolis effect on various cytokines production by immune cells. In vitro and in vivo assays demonstrated that propolis may activate macrophages, increasing their microbicidal activity and enhance the lytic activity of natural killer cells against tumor cells [20,26]. The polymorphic yeast Candida albicans (C. albicans) is an important opportunistic human pathogen causing infections that range from superficial mucosal lesions to life-threatening systemic disease. It is by far the most common cause of fungal invasive infections, which could be attributed to the little immunosuppression required to predispose an individual to invasive Candida infections [23]. Resistance to disseminated candidiasis requires the coordinated action of
innate and adaptive immune defenses. Neutrophils and macrophages can clear the yeast via phagocytosis, and macrophage activation also leads to the release of several key mediators, which are important for protecting the host against disseminated candidiasis. Cytokines appear to play a major role, acting not only as modulators of antifungal effector functions but also as key regulators in the development of the different Th subsets from precursor Th cells. Studies in mice have shown that development of protective anticandidal Th1 responses requires the concerted actions of several cytokines, such as IFN-g and TNF-a in the relative absence of inhibitory Th2 cytokines, such as IL-4 and IL-10, which inhibit development of Th1 responses [29]. The effect of natural products possessing immunomodulatory properties provided a potential therapeutic modality for reducing the risk of various diseases and cancers [14]. Several studies have indicated the role of immune responses in C. albicans infection [16,24,30], but few or no reports have addressed the dual effect of Candida infection and tumor development. Therefore, this study was aimed at developing an experimental model system for the evaluation of propolis on host — fungal pathogen interplay in Candida-infected tumor-bearing mice. We investigated the immunomodulatory effects of propolis by measuring the levels of IL-4, IL-10, IL-17, TNF-a and IFN-g in culture supernatants, tumor size and TIMP-1 levels in mice understudy.
Please cite this article in press as: Khosravi AR, et al. Immunomodulatory efficacy of ethanol extract of propolis on tumor-bearing mice with disseminated candidiasis. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.05.002
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Materials and methods Yeast strain C. albicans standard strain (ATCC 10231) was cultured on Sabouraud dextrose agar (SDA, Merck Co., Darmadstat, Germany) at 35 8Cfor 20 h. Yeast colonies were washed twice with phosphate-buffered saline (PBS) and adjusted to 2 105 cells/mL.
Preparation of propolis solution Propolis was produced by Apis mellifera L. bees in the apiary located in Lorestan Province, Iran, using plastic nets. Propolis was ground and a 30% ethanol extract was prepared in sterile conditions (30 g of propolis added to a 70% ethanol solution totaling 100 mL), in the absence of bright light, at room temperature and shaken moderately [27]. After a week, the extract was filtered and final concentration was calculated, obtaining the dry weight of the solution (120 mg/mL).
Spontaneous mouse mammary tumor (SMMT) SMMT is an invasive ductal carcinoma [10]. Tumor transplants were surgically removed from a mature breast cancer-bearing Balb/C mouse and cut into pieces of less than 0.5 cm3 with forceps and scalpel. Each piece was transplanted subcutaneously (SC) to the female Balb/C mice. All transplanted animals were daily palpated for tumor formation, and the size of the tumor mass was measured using a digital caliper, starting from the first day until the end of the experiment. Tumor size was calculated by the following formula: V = 1/ 2 LW2, where V represents volume, L is length, and W is width.
Experimental design A total of 70, 4- to 6-week female inbred BALB/c mice were obtained from Razi Institute (Karaj, Iran). The mice were reared, before and after treatment, under similar environmental conditions, with the same availability of food and drinking water, with separate ventilation systems, and were offered food and drinking water ad libitum at the animal research unit of Tehran University. Before the experiment, rooms and cages were vigorously washed and fumigated using formaldehyde. In this study, mice were divided into 7 groups (10 mice per each group) as follows: group 1: mice administered with normal saline (NaCl 0.9%, 0.1 mL/day, orally, as healthy mice); group 2: mice engrafted with tumor (NaCl 0.9%, 0.1 mL/ day, orally, as tumor control mice); group 3: mice infected with C. albicans (2 105 cell/mL, 0.1 mL, IV, as infection control mice); group 4: tumor-bearing mice infected with C. albicans (2 105 cell/mL, 0.1 mL, IV); one week after the initial surgery, when tumor diameter appeared to be 4—6 mm, the animals were infected with C. albicans; group 5: tumor-bearing mice treated with propolis (100 mg/kg, 0.1 mL/day, orally);
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group 6: mice infected with C. albicans and treated with propolis (100 mg/kg, 0.1 mL/day, orally); group 7: tumor-bearing mice infected with C. albicans and treated with propolis (100 mg/kg, 0.1 mL/day, orally). Orally administration of propolis solution to each mouse was performed by gavage for 3 days before IV challenge with C. albicans and SC challenge with SMMT and continued for 10 days.
Survival analysis The animals of different groups were observed for 30 days and evaluated by monitoring their survival status (%).
Measurement of TIMP-1 in serum Serum levels of TIMP-1 were assayed by Quantikine Mouse TIMP-1 Immunoassay (R&D Systems, Wiesbaden-Nordenstadt, Germany) according to the manufacturer’s instructions. The blood of mice groups 1 to 5 was collected from orbital veins, clotted at room temperature, and left at 4 8C overnight. Sera were collected by centrifugation at 3000g for 10 min at 4 8C and stored at 20 8C until use.
Measurement of different cytokines in spleen cell cultures Mice were euthanized; the spleen was removed in sterile conditions. Mononuclear cells were separated by ficollHypaque reagent and centrifugation at 600 g for 20 min. Cells were washed in Dulbecco’s Modified Eagle Medium (DMEM) medium and centrifuged at 200 g for 5 min. Cell pellet was then resuspended in 1 mL endotoxin-free DMEM medium (Gibco RBL, Grand Island, NY) supplemented with 10% fetal bovine serum and 1% L-glutamine DMEM medium. They were counted by hemocytometer and a final concentration of 3 107 cell/mL was made. A suspension of 3 105 cells was cultured in flat-bottomed 96-well plates. Levels of IL-4, IL-10, IL-17, TNF-a and IFN-g cytokines in supernatants were determined using commercially available ELISA kits (Pierce Endogen, Rockford, IL, USA) according to manufacturers’ recommendation.
Statistical analysis Data were analyzed using SPSS for Windows version 15. Comparisons of the data for each group were performed to test for significance (P < 0.05) by using one-way analyses of variance when variance between groups was homogeneous, and distribution of the data was normal or a nonparametric test (Kruskal—Wallis) when the normality test or homogeneity of variance test failed.
Results Survival rate The results showed that the survival rates were 100%, 33.3%, 33.3%, 0%, 33.3%, 66.7% and 33.3% in groups 1 to 7, respec-
Please cite this article in press as: Khosravi AR, et al. Immunomodulatory efficacy of ethanol extract of propolis on tumor-bearing mice with disseminated candidiasis. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.05.002
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Figure 1 The efficacy of propolis on tumor growth. Group 2: mice engrafted with tumor (tumor control mice). Group 4: tumorbearing mice infected with C. albicans; Group 5: tumor-bearing mice treated with propolis; Group 7: tumor-bearing mice infected with C. albicans and treated with propolis. ˆ le tumeur) groupe 4 : ´ de propolis sur la croissance de la tumeur. Groupe 2 : souris avec greffe de tumeur (groupe contro L’efficacite ´ es avec C. albicans ; groupe 5 : souris avec tumeur et traite ´ es avec propolis ; groupe 7 : souris avec souris avec tumeur et infecte ´ es avec C. albicans et traite ´ es avec propolis. tumeur infecte
tively. There were significant differences between group 1 and other groups (P < 0.05) as well as between group 4 and other treated groups (P < 0.05).
(7.57 0.39 mm2) and tumor-bearing mice treated with propolis (7.4 0.38 mm2).
Measurement of serum TIMP-1 Measurement of tumor growth Approximately 3—4 days after transplantation, the tumor mass was appeared. As illustrated in Fig. 1, the mean tumor size was greatly increased in tumor-bearing mice infected with C. albicans (16.98 0.49 mm2), mice engrafted with tumor (tumor control mice) (9.64 0.27 mm2), tumor-bearing mice infected with C. albicans and treated with propolis
As shown in Fig. 2, although the level of TIMP-1 was significantly elevated in tumor-bearing mice infected with C. albicans (345 32.6 ng/mL) (P < 0.05), the levels were not significantly different in mice infected with C. albicans (127.64 15.42 ng/mL), tumor-bearing mice (197 8.27 ng/mL), tumor-bearing mice infected with C. albicans and treated with propolis (65.94 12.13 ng/ mL) and tumor-bearing mice treated with propolis (89.45 13.48 ng/mL).
Measurement of IL-4, IL-10, IL-17, TNF-a and IFN-g levels in different mice groups
Figure 2 Effect of propolis on serum levels of TIMP-1 in different mice groups. *Significant difference; Group 2: mice engrafted with tumor (tumor control mice); Group 4: tumorbearing mice infected with C. albicans; Group 5: tumor-bearing mice treated with propolis; Group 7: tumor-bearing mice infected with C. albicans and treated with propolis. ´ riques de TIMP-1 dans diffe ´ rents Effet de propolis sur les taux se groupes de souris.
With regards to Th2 cytokines production, Table 1 indicated a significant decline of IL-4 and IL-10 levels after propolis administration to mice groups 5 (57.25 pg/mL and 344.2 pg/ mL), 6 (61.22 p/mL and 227.31 pg/mL) and 7 (53.41 pg/mL and 156.81 pg/mL) (P < 0.05). As to IL-17 production, a decline of 9% and 20% were seen in groups 6 and 7, respectively, in comparison with respective controls. Propolis also resulted in a significant increment of TNF-a in groups 5 (345.61 pg/mL), 6 (308.81 pg/mL) and 7 (433.85 pg/mL) in comparison with control groups (P < 0.05). In addition, the levels of IFN-g increased in mice groups 7, 6 and 5 to nearly 120.43 pg/mL, 95.14 p/mL and 127.12 pg/mL, respectively.
Discussion Propolis has been the subject of many studies due to its antibacterial, antifungal, antiviral and hepatoprotective activity. Water- or alcohol soluble propolis and its many compounds have been used in the treatment of inflamma-
Please cite this article in press as: Khosravi AR, et al. Immunomodulatory efficacy of ethanol extract of propolis on tumor-bearing mice with disseminated candidiasis. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.05.002
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Table 1 Th1 (IL-17 and IFN-g), Th2 (IL-4 and IL-10) and macrophage (TNF-a) cytokines basal production (pg/mL) in different mice groups. ´ rents groupes de souris. Production basale de cytokines Th1 (IL-17 et IFN-g), Th2 (IL-4 et IL-10) et macrophage (TNF-a) dans diffe
Group Group Group Group Group Group Group
1 2 3 4 5 6 7
IL-4 (Mean SD)
IL-10 (Mean SD)
IL-17 (Mean SD)
TNF-a (Mean SD)
IFN-g (Mean SD)
57.14 29.00 87.12 32.00 82.31 31.11 92.41 14.00 57.20 14.11 a 61.22 22.40 a 53.41 12.30 a
128.45 19.40 593.62 21.40 339.71 13.20 298.23 21.20 344.21 16.82 a 227.31 14.91 a 156.81 3.20 a
24.12 14.00 46.27 24.00 52.33 21.00 69.75 14.90 52.54 21.10 41.93 17.80 63.45 21.83
169.33 49.20 272.45 53.00 287.21 31.00 311.43 12.30 345.61 39.81 a 308.81 11.45 a 433.85 12.95 a
70.25 33.00 67.56 14.00 79.48 30.00 81.44 39.00 127.14 31.85 95.12 8.72 120.43 19.80
Group 1: mice administered with normal saline (healthy mice); Group 2: mice engrafted with tumor (tumor control mice); Group 3: mice infected with C. albicans (infection control mice); Group 4: tumor-bearing mice infected with C. albicans; Group 5: tumor-bearing mice treated with propolis; Group 6: mice infected with C. albicans and treated with propolis; Group 7: tumor-bearing mice infected with C. albicans and treated with propolis. a Significant difference.
tion, for immunostimulation and as an anticancer agent [25,28]. The above-mentioned properties of propolis make it an unusual material of natural origin, characterized by a specific composition. Some previous reports in experimental models and humans have suggested that tumor growth is stimulated during infection [11,12]. In fungal infection, this may be a result of soluble factor production induced by infection. Moreover, tumor cells can change the microenvironment and cause altered immune responses [33]. Therefore, in the present study, we used experimentally disseminated candidiasis to evaluate the interplay of Candida infection and tumor development and prognosis. This would be a good condition for reinforcement of tumor cells against antitumor defense in infected hosts. Interestingly, our results also revealed that there was the highest mortality rate in tumor-bearing mice with disseminated candidiasis. In order to evaluate the influence of propolis on the lymphoproliferative responses of mice, lymphocyte and macrophage activation of propolis-treated mice were analyzed. Our findings showed a significant decline of IL-4 and IL10 levels after propolis administration to mice groups 7 (42% and 47%), 6 (26% and 33%) and 5 (34% and 42%) as compared to group 1. Since propolis increases TGF-b production, this cytokine could influence cell division as well as decrease the production of other cytokines such as IL-4 and IL-10 [1]. As observed in this study, propolis also resulted in a significant increment of TNF-a in groups 7 (28%), 6 (7%) and 5 (21%) in comparison with control group. In general, propolis increases the inflammation by stimulating the secretion of TNF-a, resulting in increased level of TNF-a in the groups of mice treated with propolis. In addition, the levels of IFN-g increased in mice groups 7, 6 and 5 to nearly 32%, 17% and 47%, respectively. It has been also observed that propolis inhibited both IL-4 and IL-10 production by human PBMC or T cells in vitro [28]. Propolis inhibited IL-10 production by Balb/C mice submitted to experimental asthma model as well [15]. In a study by Fatahinia et al. [6], orally-administered propolis showed that the cytokines production of TNF-a and IFN-g were strongly stimulated in mice receiving propolis altogether with C. albicans. Propolis shows antitumor properties and its anticarcinogenic and antimutagenic potentials are promising, but the
mechanisms involved in the chemoprevention by propolis are still obscure. Iranian propolis is a rich source of phenolic, aromatic and aliphatic substances and the most of them are caffeic acids [31]. These compounds were identified as being responsible for anti-inflammatory, antimicrobial and in particular antifungal actions of propolis [32]. Several researchers have reported the antitumoral property of propolis in vivo and in vitro [2,15]. Matsuno [18] isolated an active substance from Brazilian propolis and characterized it as a new clerodane diterpenoid (PMS-1), which inhibited the growth of hepatoma cells and arrested the tumor cells at ‘‘S’’ phase. In another study conducted by Matsuno et al. [19], an isolated compound (PRF-1) from propolis showed antioxidant activity and it was cytotoxic to human hepatocellular carcinoma. Their group also isolated a tumoricidal compound identical to artepillin C, and its cytoxicity seemed to be partly attributable to apoptosis-like DNA fragmentation. Liao et al. [17] demonstrated the inhibitory effect of caffeic acid (an important compound of propolis) on angiogenesis, tumor invasion and pulmonary metastatic capacity of CT26 cells. Caffeic acid also prolonged the survival of mice implanted with CT26 cells, suggesting its potential as an antimetastatic agent. Taken to gather, propolis and its compounds induce the inhibition of cell proliferation by the suppression of complexes of cyclins and cyclin-dependent protein kinases, as well as by increasing the level of protein inhibitors such as p21, p16 and p27 in tumor cells, and inducing cell cycle arrest by decreasing b-catenin protein expression. IL-17 is a newly described, T cell-derived cytokine with illdefined physiologic properties. T cells secreting IL-17 are among the first to be activated during immune responses, suggesting IL-17 may play an important role in the early stages of candidiasis [8,13]. The present study demonstrated that IL-17 was reduced in both groups 6 and 7 treated with propolis when compared with respective controls. To date, there have been no reports alluding to a role for IL-17 in terms of antitumor activation. In this study, it was demonstrated that TIMP-1 was significantly secreted in the serum of tumor-bearing mice infected with C. albicans. It is speculated that C. albicans can bind to extracellular matrix protein. Furthermore, the decreased
Please cite this article in press as: Khosravi AR, et al. Immunomodulatory efficacy of ethanol extract of propolis on tumor-bearing mice with disseminated candidiasis. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.05.002
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level of TIMP-1 was associated with the decreased mean tumor size. Our study showed that propolis resulted in the decreased TIMP-1 level and tumor size in both tumor-bearing mice and tumor-bearing mice infected with C. albicans. On the basis of these findings, our data suggest that propolis exerted the possible antifungal and tumoricidal actions in vivo enhancing pro-inflammatory (TNF-a) and IFN-g production, but further investigation is still needed to evaluate propolis usefulness in tumor-bearing mice with disseminated candidiasis.
[14]
[15]
[16]
Disclosure of interest [17]
The authors declare that they have no conflicts of interest concerning this article. [18]
Acknowledgments This study was funded by the Research council of the University of Tehran, Iran.
References [1] Ansorge S, Reinhold D, Lendeckel U. Propolis and some of its constituents down-regulate DNA synthesis and inflammatory cytokine production but induce TGF-ß production of human immune cells. Zeitschrift f¨ur Naturforschung 2003;58c:580—9. [2] Banskota AH, Tezuka Y, Kadota S. Recent progress in pharmacological research of propolis. Phytother Res 2001;15:561—71. [3] Bankova V. Recent trends and important developments in propolis research. ECAM 2005;2:29—32. [4] Bazo AP, Rodrigues MAM, Sforcin JM, Camargo JLV, Ribeiro LR, Salvadori DMF. Protective action of propolis on the rat colon carcinogenesis. Teratog Carcinog Mutagen 2002;22:183—94. [5] Burdock GA. Review of the biological properties and toxicity of bee propolis (propolis). Food Chem Toxicol 1998;36:347—63. [6] Fatahinia M, Khosravi AR, Shokri H. Propolis efficacy on TNF-a, IFN-g and IL2 cytokines production in old mice with and without systemic candidiasis. J Mycol Med 2012;22:237—42. [7] Fernandes FF, Dias AL, Ramos CL, Ikegaki M, Siqueira AM, Franco MC. The ‘‘in vitro’’ antifungal activity evaluation of propolis G12 ethanol extract on Cryptococcus neoformans. Rev Ins Med Trop 2007;49:93—5. [8] Fossiez F, Djossou O, Chomarat L, Fossiez P, Djossou FO, Chomarat P. T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoetic cytokines. J Exp Med 1996;183:2593—603. [9] Gekker G, Hu S, Spivak M, Lokensgard JR, Peterson PK. AntiHIV-1 activity of propolis in CD4(+) lymphocyte and microglial cell cultures. J Ethnopharmacol 2005;102:158—63. [10] Hassan ZM, Yaraee R, Zare N, Ghazanfari T, Sarraf Nejad AH, Nazori B. Immunomodulatory affect of R10 fraction of garlic extract on natural killer activity. Int Immunopharmacol 2003;3:1483—9. [11] Hojilla CV, Mohammed FF, Khokha R. Matrix metalloproteinases and their tissue inhibitors direct cell fate during cancer development. Braz J Cancer 2003;89:1817—21. [12] Hornebeck W, Lambert E, Petitfrère E, Bernard P. Beneficial and detrimental influences of tissue inhibitor of metalloproteinase-1 (TIMP-1) in tumor progression. Biochim 2005;87:377—83. [13] Hovhannisyan Z, Treatman J, Littman DR. Characterization of interleukin-17-producing regulatory T cells in inflamed
[19]
[20]
[21]
[22]
[23]
[24]
[25] [26]
[27]
[28]
[29]
[30] [31]
[32]
[33]
intestinal mucosa from patients with inflammatory bowel diseases. Gastroenterol 2011;140:957—65. Huang CF, Lin SS, Liao PH, Young SC, Yang CC. The immunopharmaceutical effects and mechanisms of herb medicine. Cell Mol Immunol 2008;5:23—31. Huang MT, Ma W, Yen P. Inhibitory effects of caffeic acid phenethyl ester (CAPE) on 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion in mouse skin and the synthesis of DNA, RNA and protein in HeLa cells. Carcinogenesis 1996;17:761—5. Kulberg BJ, van’t Wout JW, Hoogstraten C, van Furth R. Recombinant interferon-g enhances resistance to acute disseminated Candida albicans infection in mice. J Infect Dis 1993;168:436—43. Liao HF, Chen YY, Liu JJ, Hsu ML, Shieh HJ, Liao HJ. Inhibitory effect of caffeic acid phenethyl ester on angiogenesis, tumor invasion, and metastasis. J Agr Food Chem 2003;51:7907—12. Matsuno T. A new clerodane diterpenoid isolated from propolis. Zeitschrift f¨ur Naturforschung 1995;50c:93—7. Matsuno T, Chen C, Basnet P. A tumoricidal and antioxidant compound isolated from an aqueous extract of propolis. Med Sci Res 1997;25:583—4. Orsi RO, Funari SRC, Soares AMVC, Calvi SA, Oliveira SL, Sforcin JM, et al. Immunomodulatory action of propolis on macrophage activation. J Venom Anim Toxins 2000;6:205—19. Orsi RO, Sforcin JM, Funari SRC, Fernandes Jr A, Bankova V. Synergistic effect of propolis and antibiotics on the Salmonella typhi. Braz J Microbiol 2006;37:108—12. Orsolic N, Saranovic AB, Basic I. Direct and indirect mechanism(s) of antitumour activity of propolis and its polyphenolic compounds. Planta Med 2006;72:20—7. Puccetti P, Romani L, Bistoni F. A TH1-TH2-like switch in candidiasis: new perspectives for therapy. Trends Microbiol 1995;3:237—40. Redmond HP, Shou J, Gallagher HJ, Kelly CJ, Daly JM. Macrophage-dependent candidacidal mechanisms in the murine system. Comparison of murine Kupffer cell and peritoneal macrophage candidacidal mechanisms. J Immunol 1993;150: 3427—33. Sforcin JM. Propolis and the immune system: a review. J Ethnopharmacol 2007;113:1—14. Sforcin JM, Kaneno R, Funari SRC. Absence of seasonal effect on the immunomodulatory action of Brazilian propolis on natural killer activity. J Venom Anim Toxins 2002;8:19—29. Sforcin JM, Missima F, Orsatti CL, Pagliarone AC, Kaneno R. Propolis effect on Th1/Th2 cytokine profile in melanoma-bearing mice submitted to stress. Scand J Immunol 2008;68:216—7. Sy LB, Wu YL, Chiang BL, Wang YH, Wu WM. Propolis extracts exhibit animmunoregulatory activity in an OVA-sensitized airway inflammatory animal model. Int Immunopharmacol 2006;6:1053—60. Tonetti L, Spaccapello R, Cenci E, Mencacci A, Pucetti P, Coffmann RL. Interleukin-4 and -10 exacerbate candidiasis in mice. Eur J Immunol 1995;25:1559—65. Trinchieri G, Gerosa F. Immunoregulation by interleukin-12. J Leukocyte Biol 1996;59:505—11. Yaghoubi SMJ, Ghorbani GR, Soleimanian Zad S, Satari R. Antimicrobial activity of Iranian propolis and its chemical composition. Daru 2007;15:45—8. Yarfani R, Khosravi AR, Shokri H. The antifungal activity of Iranian propolis samples against fluconazole-resistant Candida albicans strains isolated from HIV+ patients with oropharyngeal candidiasis. J ApiProd ApiMed Sci 2010;2:160—5. Zou W. Immunosuppressive networks in the tumor environment and their therapeutic relevance. Nat Rev Cancer 2005;5:263—74.
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Immunomodulatory efficacy of ethanol extract of propolis on tumor-bearing mice with disseminated candidiasis ´ immunomodulatrice de l’extrait ´ethanolique de Efficacite propolis sur les souris porteuses de tumeurs avec une candidose ´ mine ´e disse A.R. Khosravi a,*, H. Shokri b, S. Darvishi a, M. Taghavi a a
Mycology Research Center, Faculty of Veterinary Medicine, University of Tehran, PO Box 14155-6453, Azadi street, Tehran, Iran b Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, Iran Received 1 January 2014; received in revised form 26 April 2014; accepted 15 May 2014
KEYWORDS Disseminated candidiasis; Candida albicans; Cancer; Propolis; Immunomodulation
Summary Objective. — This study was aimed at investigating the effect of propolis on immunosurveillance by measuring the levels of serum interleukin (IL)-4, IL-10, IL-17, tumor necrosis factor (TNF)-a and interferon (IFN)-g in tumor-bearing mice with disseminated candidiasis. Methods. — The ethanol extract of propolis was selected for this study. Balb/C female mice were infected with Candida albicans (C. albicans) and inoculated with spontaneous mouse mammary tumor (SMMT). The serum levels of tissue inhibitor of metalloproteinase-1(TIMP-1) were assessed by enzyme- linked immunosorbent assay (ELISA). Mice were treated daily with propolis solution (100 mg/kg, 0.1 mL, orally) for 3 days before IV challenge with C. albicans and SC challenge with SMMT and continued for 10 days. The rates of survival and tumor growth of understudy mice were investigated as well. The levels of TNF-a, IFN-g, IL-4, IL-10 and IL-17 cytokines in culture supernatants were determined by ELISA. Results. — The mean tumor size was significantly increased in tumor-bearing mice infected with C. albicans (16.98 0.49 mm2) as compared to other mice groups (P < 0.05). The results showed a significant decline of IL-4 and IL-10 levels after propolis administration to tumor-bearing mice infected with C. albicans (53.41 pg/mL, 156.81 pg/mL and 63.45 pg/mL) (P < 0.05). The increment of TNF-a (433.85 pg/mL) and IFN-g (120.43 pg/mL) levels were also observed.
* Corresponding author. Tel.: +0098 21 61117151; fax: +0098 21 66933222. E-mail address: [email protected] (A.R. Khosravi). http://dx.doi.org/10.1016/j.mycmed.2014.05.002 1156-5233/# 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Khosravi AR, et al. Immunomodulatory efficacy of ethanol extract of propolis on tumor-bearing mice with disseminated candidiasis. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.05.002
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A.R. Khosravi et al. Conclusion. — Data revealed that propolis has remarkable immunomodulatory effect, which provides a scientific validation for the popular use of this natural substance, and further investigation will help to understand propolis usefulness during immunosuppressive conditions. # 2014 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Candidose disséminée ; Candida albicans ; Cancer ; Propolis ; Immunomodulation
Re ´sume ´ Objectif. — Cette étude visait à étudier l’effet de propolis sur l’immunosurveillance en mesurant les niveaux sériques de l’interleukine (IL) -4, IL-10, IL-17, du facteur de nécrose tumorale (TNF)-a et de l’interféron (IFN)-g de souris porteuses de tumeurs avec une candidose disséminée. Mate´riel et me´thodes. — L’extrait l’éthanolique de propolis a été sélectionné pour cette étude. Des souris femelles Balb/c étaient infectées avec Candida albicans (C. albicans) et inoculées avec une tumeur mammaire spontanée de souris (SMMT). Les taux sériques d’une métalloprotéinase-1 (TIMP-1) inhibiteur cellulaire ont été évalués par enzyme-linked immunosorbent assay (ELISA). Les souris ont été traitées quotidiennement avec la solution de propolis (100 mg/kg, 0,1 mL, oralement) pendant 3 jours avant l’injection IV avec C. albicans et SC avec la SMMT, traitement poursuivi pendant 10 jours après challenge. Les taux de survie et la croissance des tumeurs des souris ont été enregistrés. Les niveaux de TNF-a, IFN-g, et des cytokines IL-4, IL-10, IL-17 dans les surnageants de culture ont été déterminés par la technique ELISA. Re´sultats. — La taille de la tumeur a été augmentée de manière significative chez les souris porteuses de tumeurs et infectées par C. albicans (16,98 0,49 mm2) par rapport aux autres groupes de souris ( p < 0,05). Les résultats ont montré une diminution significative des niveaux de l’IL-4 et de l’IL-10 après administration de propolis aux souris porteuses de tumeurs et infectées par C. albicans (53,41 pg/mL, 156,81 pg/mL et 63,45 pg/mL) ( p < 0,05). L’incrément des niveaux de TNF-a (433,85 pg/mL) et IFN-g (120,43 pg/mL) a également été observé. Conclusion. — Ces données ont révélé que la propolis a un remarquable effet immunomodulateur, et représentent une validation scientifique pour l’utilisation de cette substance naturelle. Des études complémentaires aideront à comprendre l’intérêt de la propolis lors de conditions immunosuppressives. # 2014 Elsevier Masson SAS. Tous droits réservés.
Introduction Propolis has gained popularity in alternative medicine, and it is used in food and beverages to improve health and to prevent diseases. Propolis is a chemically complex bee product, containing material collected from buds or exudates of plants, volatile substances and wax [3]. It is composed of 30% wax, 50% resins and vegetable balsams, 10% essential oils, 5% pollen and other substances [5]. Because of its various bioactivities, including antibacterial [21], antiviral [9], antifungal [7], anti-inflammatory [28] and antitumoral [4,22] effects, propolis has been used in folk medicine and has attracted many investigators’ interest. However, little is known about propolis effect on various cytokines production by immune cells. In vitro and in vivo assays demonstrated that propolis may activate macrophages, increasing their microbicidal activity and enhance the lytic activity of natural killer cells against tumor cells [20,26]. The polymorphic yeast Candida albicans (C. albicans) is an important opportunistic human pathogen causing infections that range from superficial mucosal lesions to life-threatening systemic disease. It is by far the most common cause of fungal invasive infections, which could be attributed to the little immunosuppression required to predispose an individual to invasive Candida infections [23]. Resistance to disseminated candidiasis requires the coordinated action of
innate and adaptive immune defenses. Neutrophils and macrophages can clear the yeast via phagocytosis, and macrophage activation also leads to the release of several key mediators, which are important for protecting the host against disseminated candidiasis. Cytokines appear to play a major role, acting not only as modulators of antifungal effector functions but also as key regulators in the development of the different Th subsets from precursor Th cells. Studies in mice have shown that development of protective anticandidal Th1 responses requires the concerted actions of several cytokines, such as IFN-g and TNF-a in the relative absence of inhibitory Th2 cytokines, such as IL-4 and IL-10, which inhibit development of Th1 responses [29]. The effect of natural products possessing immunomodulatory properties provided a potential therapeutic modality for reducing the risk of various diseases and cancers [14]. Several studies have indicated the role of immune responses in C. albicans infection [16,24,30], but few or no reports have addressed the dual effect of Candida infection and tumor development. Therefore, this study was aimed at developing an experimental model system for the evaluation of propolis on host — fungal pathogen interplay in Candida-infected tumor-bearing mice. We investigated the immunomodulatory effects of propolis by measuring the levels of IL-4, IL-10, IL-17, TNF-a and IFN-g in culture supernatants, tumor size and TIMP-1 levels in mice understudy.
Please cite this article in press as: Khosravi AR, et al. Immunomodulatory efficacy of ethanol extract of propolis on tumor-bearing mice with disseminated candidiasis. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.05.002
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Materials and methods Yeast strain C. albicans standard strain (ATCC 10231) was cultured on Sabouraud dextrose agar (SDA, Merck Co., Darmadstat, Germany) at 35 8Cfor 20 h. Yeast colonies were washed twice with phosphate-buffered saline (PBS) and adjusted to 2 105 cells/mL.
Preparation of propolis solution Propolis was produced by Apis mellifera L. bees in the apiary located in Lorestan Province, Iran, using plastic nets. Propolis was ground and a 30% ethanol extract was prepared in sterile conditions (30 g of propolis added to a 70% ethanol solution totaling 100 mL), in the absence of bright light, at room temperature and shaken moderately [27]. After a week, the extract was filtered and final concentration was calculated, obtaining the dry weight of the solution (120 mg/mL).
Spontaneous mouse mammary tumor (SMMT) SMMT is an invasive ductal carcinoma [10]. Tumor transplants were surgically removed from a mature breast cancer-bearing Balb/C mouse and cut into pieces of less than 0.5 cm3 with forceps and scalpel. Each piece was transplanted subcutaneously (SC) to the female Balb/C mice. All transplanted animals were daily palpated for tumor formation, and the size of the tumor mass was measured using a digital caliper, starting from the first day until the end of the experiment. Tumor size was calculated by the following formula: V = 1/ 2 LW2, where V represents volume, L is length, and W is width.
Experimental design A total of 70, 4- to 6-week female inbred BALB/c mice were obtained from Razi Institute (Karaj, Iran). The mice were reared, before and after treatment, under similar environmental conditions, with the same availability of food and drinking water, with separate ventilation systems, and were offered food and drinking water ad libitum at the animal research unit of Tehran University. Before the experiment, rooms and cages were vigorously washed and fumigated using formaldehyde. In this study, mice were divided into 7 groups (10 mice per each group) as follows: group 1: mice administered with normal saline (NaCl 0.9%, 0.1 mL/day, orally, as healthy mice); group 2: mice engrafted with tumor (NaCl 0.9%, 0.1 mL/ day, orally, as tumor control mice); group 3: mice infected with C. albicans (2 105 cell/mL, 0.1 mL, IV, as infection control mice); group 4: tumor-bearing mice infected with C. albicans (2 105 cell/mL, 0.1 mL, IV); one week after the initial surgery, when tumor diameter appeared to be 4—6 mm, the animals were infected with C. albicans; group 5: tumor-bearing mice treated with propolis (100 mg/kg, 0.1 mL/day, orally);
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group 6: mice infected with C. albicans and treated with propolis (100 mg/kg, 0.1 mL/day, orally); group 7: tumor-bearing mice infected with C. albicans and treated with propolis (100 mg/kg, 0.1 mL/day, orally). Orally administration of propolis solution to each mouse was performed by gavage for 3 days before IV challenge with C. albicans and SC challenge with SMMT and continued for 10 days.
Survival analysis The animals of different groups were observed for 30 days and evaluated by monitoring their survival status (%).
Measurement of TIMP-1 in serum Serum levels of TIMP-1 were assayed by Quantikine Mouse TIMP-1 Immunoassay (R&D Systems, Wiesbaden-Nordenstadt, Germany) according to the manufacturer’s instructions. The blood of mice groups 1 to 5 was collected from orbital veins, clotted at room temperature, and left at 4 8C overnight. Sera were collected by centrifugation at 3000g for 10 min at 4 8C and stored at 20 8C until use.
Measurement of different cytokines in spleen cell cultures Mice were euthanized; the spleen was removed in sterile conditions. Mononuclear cells were separated by ficollHypaque reagent and centrifugation at 600 g for 20 min. Cells were washed in Dulbecco’s Modified Eagle Medium (DMEM) medium and centrifuged at 200 g for 5 min. Cell pellet was then resuspended in 1 mL endotoxin-free DMEM medium (Gibco RBL, Grand Island, NY) supplemented with 10% fetal bovine serum and 1% L-glutamine DMEM medium. They were counted by hemocytometer and a final concentration of 3 107 cell/mL was made. A suspension of 3 105 cells was cultured in flat-bottomed 96-well plates. Levels of IL-4, IL-10, IL-17, TNF-a and IFN-g cytokines in supernatants were determined using commercially available ELISA kits (Pierce Endogen, Rockford, IL, USA) according to manufacturers’ recommendation.
Statistical analysis Data were analyzed using SPSS for Windows version 15. Comparisons of the data for each group were performed to test for significance (P < 0.05) by using one-way analyses of variance when variance between groups was homogeneous, and distribution of the data was normal or a nonparametric test (Kruskal—Wallis) when the normality test or homogeneity of variance test failed.
Results Survival rate The results showed that the survival rates were 100%, 33.3%, 33.3%, 0%, 33.3%, 66.7% and 33.3% in groups 1 to 7, respec-
Please cite this article in press as: Khosravi AR, et al. Immunomodulatory efficacy of ethanol extract of propolis on tumor-bearing mice with disseminated candidiasis. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.05.002
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Figure 1 The efficacy of propolis on tumor growth. Group 2: mice engrafted with tumor (tumor control mice). Group 4: tumorbearing mice infected with C. albicans; Group 5: tumor-bearing mice treated with propolis; Group 7: tumor-bearing mice infected with C. albicans and treated with propolis. ˆ le tumeur) groupe 4 : ´ de propolis sur la croissance de la tumeur. Groupe 2 : souris avec greffe de tumeur (groupe contro L’efficacite ´ es avec C. albicans ; groupe 5 : souris avec tumeur et traite ´ es avec propolis ; groupe 7 : souris avec souris avec tumeur et infecte ´ es avec C. albicans et traite ´ es avec propolis. tumeur infecte
tively. There were significant differences between group 1 and other groups (P < 0.05) as well as between group 4 and other treated groups (P < 0.05).
(7.57 0.39 mm2) and tumor-bearing mice treated with propolis (7.4 0.38 mm2).
Measurement of serum TIMP-1 Measurement of tumor growth Approximately 3—4 days after transplantation, the tumor mass was appeared. As illustrated in Fig. 1, the mean tumor size was greatly increased in tumor-bearing mice infected with C. albicans (16.98 0.49 mm2), mice engrafted with tumor (tumor control mice) (9.64 0.27 mm2), tumor-bearing mice infected with C. albicans and treated with propolis
As shown in Fig. 2, although the level of TIMP-1 was significantly elevated in tumor-bearing mice infected with C. albicans (345 32.6 ng/mL) (P < 0.05), the levels were not significantly different in mice infected with C. albicans (127.64 15.42 ng/mL), tumor-bearing mice (197 8.27 ng/mL), tumor-bearing mice infected with C. albicans and treated with propolis (65.94 12.13 ng/ mL) and tumor-bearing mice treated with propolis (89.45 13.48 ng/mL).
Measurement of IL-4, IL-10, IL-17, TNF-a and IFN-g levels in different mice groups
Figure 2 Effect of propolis on serum levels of TIMP-1 in different mice groups. *Significant difference; Group 2: mice engrafted with tumor (tumor control mice); Group 4: tumorbearing mice infected with C. albicans; Group 5: tumor-bearing mice treated with propolis; Group 7: tumor-bearing mice infected with C. albicans and treated with propolis. ´ riques de TIMP-1 dans diffe ´ rents Effet de propolis sur les taux se groupes de souris.
With regards to Th2 cytokines production, Table 1 indicated a significant decline of IL-4 and IL-10 levels after propolis administration to mice groups 5 (57.25 pg/mL and 344.2 pg/ mL), 6 (61.22 p/mL and 227.31 pg/mL) and 7 (53.41 pg/mL and 156.81 pg/mL) (P < 0.05). As to IL-17 production, a decline of 9% and 20% were seen in groups 6 and 7, respectively, in comparison with respective controls. Propolis also resulted in a significant increment of TNF-a in groups 5 (345.61 pg/mL), 6 (308.81 pg/mL) and 7 (433.85 pg/mL) in comparison with control groups (P < 0.05). In addition, the levels of IFN-g increased in mice groups 7, 6 and 5 to nearly 120.43 pg/mL, 95.14 p/mL and 127.12 pg/mL, respectively.
Discussion Propolis has been the subject of many studies due to its antibacterial, antifungal, antiviral and hepatoprotective activity. Water- or alcohol soluble propolis and its many compounds have been used in the treatment of inflamma-
Please cite this article in press as: Khosravi AR, et al. Immunomodulatory efficacy of ethanol extract of propolis on tumor-bearing mice with disseminated candidiasis. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.05.002
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Table 1 Th1 (IL-17 and IFN-g), Th2 (IL-4 and IL-10) and macrophage (TNF-a) cytokines basal production (pg/mL) in different mice groups. ´ rents groupes de souris. Production basale de cytokines Th1 (IL-17 et IFN-g), Th2 (IL-4 et IL-10) et macrophage (TNF-a) dans diffe
Group Group Group Group Group Group Group
1 2 3 4 5 6 7
IL-4 (Mean SD)
IL-10 (Mean SD)
IL-17 (Mean SD)
TNF-a (Mean SD)
IFN-g (Mean SD)
57.14 29.00 87.12 32.00 82.31 31.11 92.41 14.00 57.20 14.11 a 61.22 22.40 a 53.41 12.30 a
128.45 19.40 593.62 21.40 339.71 13.20 298.23 21.20 344.21 16.82 a 227.31 14.91 a 156.81 3.20 a
24.12 14.00 46.27 24.00 52.33 21.00 69.75 14.90 52.54 21.10 41.93 17.80 63.45 21.83
169.33 49.20 272.45 53.00 287.21 31.00 311.43 12.30 345.61 39.81 a 308.81 11.45 a 433.85 12.95 a
70.25 33.00 67.56 14.00 79.48 30.00 81.44 39.00 127.14 31.85 95.12 8.72 120.43 19.80
Group 1: mice administered with normal saline (healthy mice); Group 2: mice engrafted with tumor (tumor control mice); Group 3: mice infected with C. albicans (infection control mice); Group 4: tumor-bearing mice infected with C. albicans; Group 5: tumor-bearing mice treated with propolis; Group 6: mice infected with C. albicans and treated with propolis; Group 7: tumor-bearing mice infected with C. albicans and treated with propolis. a Significant difference.
tion, for immunostimulation and as an anticancer agent [25,28]. The above-mentioned properties of propolis make it an unusual material of natural origin, characterized by a specific composition. Some previous reports in experimental models and humans have suggested that tumor growth is stimulated during infection [11,12]. In fungal infection, this may be a result of soluble factor production induced by infection. Moreover, tumor cells can change the microenvironment and cause altered immune responses [33]. Therefore, in the present study, we used experimentally disseminated candidiasis to evaluate the interplay of Candida infection and tumor development and prognosis. This would be a good condition for reinforcement of tumor cells against antitumor defense in infected hosts. Interestingly, our results also revealed that there was the highest mortality rate in tumor-bearing mice with disseminated candidiasis. In order to evaluate the influence of propolis on the lymphoproliferative responses of mice, lymphocyte and macrophage activation of propolis-treated mice were analyzed. Our findings showed a significant decline of IL-4 and IL10 levels after propolis administration to mice groups 7 (42% and 47%), 6 (26% and 33%) and 5 (34% and 42%) as compared to group 1. Since propolis increases TGF-b production, this cytokine could influence cell division as well as decrease the production of other cytokines such as IL-4 and IL-10 [1]. As observed in this study, propolis also resulted in a significant increment of TNF-a in groups 7 (28%), 6 (7%) and 5 (21%) in comparison with control group. In general, propolis increases the inflammation by stimulating the secretion of TNF-a, resulting in increased level of TNF-a in the groups of mice treated with propolis. In addition, the levels of IFN-g increased in mice groups 7, 6 and 5 to nearly 32%, 17% and 47%, respectively. It has been also observed that propolis inhibited both IL-4 and IL-10 production by human PBMC or T cells in vitro [28]. Propolis inhibited IL-10 production by Balb/C mice submitted to experimental asthma model as well [15]. In a study by Fatahinia et al. [6], orally-administered propolis showed that the cytokines production of TNF-a and IFN-g were strongly stimulated in mice receiving propolis altogether with C. albicans. Propolis shows antitumor properties and its anticarcinogenic and antimutagenic potentials are promising, but the
mechanisms involved in the chemoprevention by propolis are still obscure. Iranian propolis is a rich source of phenolic, aromatic and aliphatic substances and the most of them are caffeic acids [31]. These compounds were identified as being responsible for anti-inflammatory, antimicrobial and in particular antifungal actions of propolis [32]. Several researchers have reported the antitumoral property of propolis in vivo and in vitro [2,15]. Matsuno [18] isolated an active substance from Brazilian propolis and characterized it as a new clerodane diterpenoid (PMS-1), which inhibited the growth of hepatoma cells and arrested the tumor cells at ‘‘S’’ phase. In another study conducted by Matsuno et al. [19], an isolated compound (PRF-1) from propolis showed antioxidant activity and it was cytotoxic to human hepatocellular carcinoma. Their group also isolated a tumoricidal compound identical to artepillin C, and its cytoxicity seemed to be partly attributable to apoptosis-like DNA fragmentation. Liao et al. [17] demonstrated the inhibitory effect of caffeic acid (an important compound of propolis) on angiogenesis, tumor invasion and pulmonary metastatic capacity of CT26 cells. Caffeic acid also prolonged the survival of mice implanted with CT26 cells, suggesting its potential as an antimetastatic agent. Taken to gather, propolis and its compounds induce the inhibition of cell proliferation by the suppression of complexes of cyclins and cyclin-dependent protein kinases, as well as by increasing the level of protein inhibitors such as p21, p16 and p27 in tumor cells, and inducing cell cycle arrest by decreasing b-catenin protein expression. IL-17 is a newly described, T cell-derived cytokine with illdefined physiologic properties. T cells secreting IL-17 are among the first to be activated during immune responses, suggesting IL-17 may play an important role in the early stages of candidiasis [8,13]. The present study demonstrated that IL-17 was reduced in both groups 6 and 7 treated with propolis when compared with respective controls. To date, there have been no reports alluding to a role for IL-17 in terms of antitumor activation. In this study, it was demonstrated that TIMP-1 was significantly secreted in the serum of tumor-bearing mice infected with C. albicans. It is speculated that C. albicans can bind to extracellular matrix protein. Furthermore, the decreased
Please cite this article in press as: Khosravi AR, et al. Immunomodulatory efficacy of ethanol extract of propolis on tumor-bearing mice with disseminated candidiasis. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.05.002
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level of TIMP-1 was associated with the decreased mean tumor size. Our study showed that propolis resulted in the decreased TIMP-1 level and tumor size in both tumor-bearing mice and tumor-bearing mice infected with C. albicans. On the basis of these findings, our data suggest that propolis exerted the possible antifungal and tumoricidal actions in vivo enhancing pro-inflammatory (TNF-a) and IFN-g production, but further investigation is still needed to evaluate propolis usefulness in tumor-bearing mice with disseminated candidiasis.
[14]
[15]
[16]
Disclosure of interest [17]
The authors declare that they have no conflicts of interest concerning this article. [18]
Acknowledgments This study was funded by the Research council of the University of Tehran, Iran.
References [1] Ansorge S, Reinhold D, Lendeckel U. Propolis and some of its constituents down-regulate DNA synthesis and inflammatory cytokine production but induce TGF-ß production of human immune cells. Zeitschrift f¨ur Naturforschung 2003;58c:580—9. [2] Banskota AH, Tezuka Y, Kadota S. Recent progress in pharmacological research of propolis. Phytother Res 2001;15:561—71. [3] Bankova V. Recent trends and important developments in propolis research. ECAM 2005;2:29—32. [4] Bazo AP, Rodrigues MAM, Sforcin JM, Camargo JLV, Ribeiro LR, Salvadori DMF. Protective action of propolis on the rat colon carcinogenesis. Teratog Carcinog Mutagen 2002;22:183—94. [5] Burdock GA. Review of the biological properties and toxicity of bee propolis (propolis). Food Chem Toxicol 1998;36:347—63. [6] Fatahinia M, Khosravi AR, Shokri H. Propolis efficacy on TNF-a, IFN-g and IL2 cytokines production in old mice with and without systemic candidiasis. J Mycol Med 2012;22:237—42. [7] Fernandes FF, Dias AL, Ramos CL, Ikegaki M, Siqueira AM, Franco MC. The ‘‘in vitro’’ antifungal activity evaluation of propolis G12 ethanol extract on Cryptococcus neoformans. Rev Ins Med Trop 2007;49:93—5. [8] Fossiez F, Djossou O, Chomarat L, Fossiez P, Djossou FO, Chomarat P. T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoetic cytokines. J Exp Med 1996;183:2593—603. [9] Gekker G, Hu S, Spivak M, Lokensgard JR, Peterson PK. AntiHIV-1 activity of propolis in CD4(+) lymphocyte and microglial cell cultures. J Ethnopharmacol 2005;102:158—63. [10] Hassan ZM, Yaraee R, Zare N, Ghazanfari T, Sarraf Nejad AH, Nazori B. Immunomodulatory affect of R10 fraction of garlic extract on natural killer activity. Int Immunopharmacol 2003;3:1483—9. [11] Hojilla CV, Mohammed FF, Khokha R. Matrix metalloproteinases and their tissue inhibitors direct cell fate during cancer development. Braz J Cancer 2003;89:1817—21. [12] Hornebeck W, Lambert E, Petitfrère E, Bernard P. Beneficial and detrimental influences of tissue inhibitor of metalloproteinase-1 (TIMP-1) in tumor progression. Biochim 2005;87:377—83. [13] Hovhannisyan Z, Treatman J, Littman DR. Characterization of interleukin-17-producing regulatory T cells in inflamed
[19]
[20]
[21]
[22]
[23]
[24]
[25] [26]
[27]
[28]
[29]
[30] [31]
[32]
[33]
intestinal mucosa from patients with inflammatory bowel diseases. Gastroenterol 2011;140:957—65. Huang CF, Lin SS, Liao PH, Young SC, Yang CC. The immunopharmaceutical effects and mechanisms of herb medicine. Cell Mol Immunol 2008;5:23—31. Huang MT, Ma W, Yen P. Inhibitory effects of caffeic acid phenethyl ester (CAPE) on 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion in mouse skin and the synthesis of DNA, RNA and protein in HeLa cells. Carcinogenesis 1996;17:761—5. Kulberg BJ, van’t Wout JW, Hoogstraten C, van Furth R. Recombinant interferon-g enhances resistance to acute disseminated Candida albicans infection in mice. J Infect Dis 1993;168:436—43. Liao HF, Chen YY, Liu JJ, Hsu ML, Shieh HJ, Liao HJ. Inhibitory effect of caffeic acid phenethyl ester on angiogenesis, tumor invasion, and metastasis. J Agr Food Chem 2003;51:7907—12. Matsuno T. A new clerodane diterpenoid isolated from propolis. Zeitschrift f¨ur Naturforschung 1995;50c:93—7. Matsuno T, Chen C, Basnet P. A tumoricidal and antioxidant compound isolated from an aqueous extract of propolis. Med Sci Res 1997;25:583—4. Orsi RO, Funari SRC, Soares AMVC, Calvi SA, Oliveira SL, Sforcin JM, et al. Immunomodulatory action of propolis on macrophage activation. J Venom Anim Toxins 2000;6:205—19. Orsi RO, Sforcin JM, Funari SRC, Fernandes Jr A, Bankova V. Synergistic effect of propolis and antibiotics on the Salmonella typhi. Braz J Microbiol 2006;37:108—12. Orsolic N, Saranovic AB, Basic I. Direct and indirect mechanism(s) of antitumour activity of propolis and its polyphenolic compounds. Planta Med 2006;72:20—7. Puccetti P, Romani L, Bistoni F. A TH1-TH2-like switch in candidiasis: new perspectives for therapy. Trends Microbiol 1995;3:237—40. Redmond HP, Shou J, Gallagher HJ, Kelly CJ, Daly JM. Macrophage-dependent candidacidal mechanisms in the murine system. Comparison of murine Kupffer cell and peritoneal macrophage candidacidal mechanisms. J Immunol 1993;150: 3427—33. Sforcin JM. Propolis and the immune system: a review. J Ethnopharmacol 2007;113:1—14. Sforcin JM, Kaneno R, Funari SRC. Absence of seasonal effect on the immunomodulatory action of Brazilian propolis on natural killer activity. J Venom Anim Toxins 2002;8:19—29. Sforcin JM, Missima F, Orsatti CL, Pagliarone AC, Kaneno R. Propolis effect on Th1/Th2 cytokine profile in melanoma-bearing mice submitted to stress. Scand J Immunol 2008;68:216—7. Sy LB, Wu YL, Chiang BL, Wang YH, Wu WM. Propolis extracts exhibit animmunoregulatory activity in an OVA-sensitized airway inflammatory animal model. Int Immunopharmacol 2006;6:1053—60. Tonetti L, Spaccapello R, Cenci E, Mencacci A, Pucetti P, Coffmann RL. Interleukin-4 and -10 exacerbate candidiasis in mice. Eur J Immunol 1995;25:1559—65. Trinchieri G, Gerosa F. Immunoregulation by interleukin-12. J Leukocyte Biol 1996;59:505—11. Yaghoubi SMJ, Ghorbani GR, Soleimanian Zad S, Satari R. Antimicrobial activity of Iranian propolis and its chemical composition. Daru 2007;15:45—8. Yarfani R, Khosravi AR, Shokri H. The antifungal activity of Iranian propolis samples against fluconazole-resistant Candida albicans strains isolated from HIV+ patients with oropharyngeal candidiasis. J ApiProd ApiMed Sci 2010;2:160—5. Zou W. Immunosuppressive networks in the tumor environment and their therapeutic relevance. Nat Rev Cancer 2005;5:263—74.
Please cite this article in press as: Khosravi AR, et al. Immunomodulatory efficacy of ethanol extract of propolis on tumor-bearing mice with disseminated candidiasis. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.05.002
