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Chemopreventive potential of phenolic compounds in oral carcinogenesis
archives of oral biology 59 (2014) 1101–1107
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Chemopreventive potential of phenolic compounds in oral carcinogenesis B. Baldasquin-Caceres a, F.J. Gomez-Garcia a, P. Lo´pez-Jornet b,*, J. Castillo-Sanchez c, V. Vicente-Ortega a a
Department of Pathology and Anatomical Sciences, Faculty of Medicine and Dentistry, Ageing Research Institute, University of Murcia, Murcia, Spain b Oral Medicine Ageing Research Institute, Faculty of Medicine and Dentistry, University of Murcia, Murcia, Spain c I+D+I Nutrafur SA Murcia Spain Ageing Research Institute, University of Murcia, Murcia, Spain
article info
abstract
Article history:
Objective: To evaluate the chemopreventive potential of phenolic compounds – potassium
Accepted 18 June 2014
apigenin, cocoa, catechins, eriocitrin and rosmarinic acid in oral carcinogenesis induced in hamsters by means of the topical application of 7,12-dimethylbenz(a)anthracene(DMBA).
Keywords:
Study design: An experimental study at the University of Murcia.
Oral carcinogenesis
Methods: 50 male Syrian hamsters (Mesocricetus auratus) were divided into five groups of ten:
Apigenin
Group I (control group): 0.5% DMBA; Group II: 0.5% DMBA + 1.1 mg/15 ml potassium api-
Cocoa cathechin
genin; Group III: 05% DMBA + 2.5 mg/15 ml cocoa catechins; Group IV: 0.5% DMBA + 6 mg/
Eriocitrin and rosmarinic acid
15 ml eriocitrin; Group V: 0.5% DMBA + 1.3 mg/15 ml rosmarinic acid. The flavonoids were
DMBA
administered orally. All the animals were sacrificed after 12 weeks. Macroscopic, micro-
Hamster
scopic and immunohistochemical (PCNA and p53) analyses of the lesions were performed. Results: All the groups treated with phenolic compounds showed lower incidences of tumour, greater differentiation and lower scores in the tumour invasion front grading system in comparison with the control group. Potassium apigenin and rosmarinic acid achieved the best results, the former considerably reduced the carcinoma tumour volumes developed and both significantly reduced the intensity and aggression of the tumours. Immunoexpression of PCNA and p53 were significantly altered during DMBA-induced oral carcinogenesis. Conclusions: Animals treated with phenolic compounds, particularly potassium apigenin and rosmarinic acid, showed a lower incidence of tumours. # 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
Oral squamous cell carcinoma is the most frequently occurring cancer of the head and neck and is characterized by poor prognosis and a low patient survival rate.1,2
Recent efforts to control the incidence of oral squamous cell carcinoma, have focused on developing effective chemopreventive strategies. Chemoprevention by natural products and dietary and lifestyle changes have evolved as promising strategies for the management of cancer. In this way, dietary phytochemicals have gained significant recognition in recent
* Corresponding author at: Clı´nica Odontolo´gica Universitaria, Hospital Morales Meseguer, Avda. Marque´s de los Ve´lez s/n, Murcia 30008, Spain Tel.: +34 968 398588; fax: +34 968 398576. E-mail address: [email protected] (P. Lo´pez-Jornet). http://dx.doi.org/10.1016/j.archoralbio.2014.06.007 0003–9969/# 2014 Elsevier Ltd. All rights reserved.
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years as potential candidates for cancer chemoprevention.1–9 In vitro studies have shown that flavonoids significantly increase p53, E-cadherin and b-catenin protein expression and caspase 3, fas ligand and Fas/Apo-1 receptor activation which are strongly associated with the signal transduction pathways involved in apoptosis and cellular adhesion that affect the chemosensitivity of tumour cells. The biological behaviour of flavonoids depends on chemical structure and epidemiological studies support their role in cancer prevention.10–14 Apigenin, a flavonoid compound, has the capacity to inhibit the proliferation and cell viability in numerous cancer cell lines.14–16 Apigenin has shown chemoprotective effects in various in vivo mouse models of cancer (lung, skin, neck of the uterus, ovarian, prostate, leukaemia, and colon), in which it has been seen to reduce the size of primary tumors.17 It is also able to inhibit the mobility of tumour cells (inhibition of FAK expression)18 and to reduce the number and size of metastatic nodes in ovarian cancer, breast cancer, and melanoma.19 Initial observations have shown that procyanidin-rich fractions prepared from the seeds of Theobroma cacao, which are used to prepare cocoa, inhibit in vitro growth of several human breast cancer cell lines.20 This research has revealed oligomeric procyanidins, such as the pentameric procyanidin, to be cytotoxic to breast cancer cells. Rosmarinic acid (RA) ((2R)-2-[[(2E)-3-(3,4-dihydroxyphenyl)-1oxo-2-propenyl]]oxy-3-(3,4-dihydroxyphenyl) propanoic acid) has a number of interesting biological activities, including antiviral, antibacterial, anti-inflammatory and antioxidant effects.21–23 RA prevents mutagenic activity, which has been observed by means of micronuclear tests in Swiss mice. In addition, the reduction in tumorigenesis in a murine two-stage skin carcinogenesis model with topical application of Perilla Frutescens extract has also been attributed to the presence of RA. It has been shown to delay colorectal carcinogenesis in rodent models. The pharmacological effect of RA has been seen to act through the inhibition of several complement-dependent inflammatory processes. Recent studies confirm that caffeoyl esters such as RA show high specific antioxidant activity, delay vitamin E depletion, decrease pro-inflammatory lysophosphatidyl choline production and prevent the oxidation of low-density lipoprotein (LDL), which is compatible with its anti-inflammatory and antiatherosclerotic role in pathophysiological conditions.21–23 Among the glycoside flavonoids present in lemons, eriocitrin shows the highest antioxidant activity and has been shown to inhibit proliferation and apoptosis in different cancer cell lines.24–26 Ogata et al.27 demonstrated that lemon flavonoids and their metabolites induce apoptosis in leukaemia cell lines HL-60 dose- and time-dependently. The aim of this study was to evaluate the chemopreventive efficacy of phenolic compounds potassium apigenin, cocoa catechins, eriocitrin and rosmarinic acid on oral carcinogenesis induced in hamsters by means of topical application of DMBA.
2.
Materials and methods
2.1.
Animals
The study used 50 male Syrian hamsters (Mesocricetus auratus), with an average weight of 100 g and an average
age of five months. All the animals were supplied by the University of Murcia (Spain) Research Support Services Animal Laboratory (License No. REGAES 300305440012). The hamsters were housed in cages and received food and water ad libitum. The room in which they were housed had a controlled 12/12 light/darkness cycle and a temperature of 22 8C, in compliance with European Union norms for the protection of animals used in experimentation (2010/63/UE). The experiment was approved by the University of Murcia Bioethics Committee.
2.2.
Agents
- Polycyclic aromatic hydrocarbon 7,12-dimethyl-1,2-bezantracene (DMBA) (Sigma Aldrich Co., Madrid, Spain). The DMBA was dissolved in acetone (Sigma Aldrich Co., Madrid, Spain) at 0.5%. The anaesthetic used was ketamine (Imalgene 1000 Merial, Barcelona, Spain) and xylazine (Xylagesic 2% [20 mg/ml] Laboratorios Calier S.A, Barcelona, Spain). - Concentrated extract of apigenin (90% HPLC purity) (Nutrafur, S.A., Murcia, Spain) dissolved in distilled water at a concentration of 1.1 mg/15 ml. - Concentrated extract of cocoa catechins (40% HPLC purity) (Nutrafur, S.A., Murcia, Spain) dissolved in distilled water at a concentration of 2.5 mg/15 ml. - Concentrated extract of eriocitrin (20% HPLC purity) (Nutrafur, S.A., Murcia, Spain) dissolved in distilled water at a concentration of 6 mg/15 ml. - Concentrated extract of rosmarinic acid (15% HPLC purity) (Nutrafur, S.A., Murcia, Spain) dissolved in distilled water at a concentration of 1.3 mg/15 ml.
2.3.
Experiment procedure
Fifty animals were divided randomly into five groups of ten: Group I (control group) treated with 0.5% DMBA in acetone (n = 10); Group II treated with 0.5% DMBA + concentrated extract of potassium apigenin (n = 10); Group III treated with 05% DMBA + concentrated extract of cocoa catechins (n = 10); Group IV treated with 0.5% DMBA + concentrated extract of eriocitrin (n = 10); Group V treated with 0.5% DMBA + concentrated extract of rosmarinic acid (n = 10). All the phenolic compounds were administered via the animals’ drinking water, consumed ad libitum. The animals in the study groups treated with polyphenols received pre-treatment for two weeks prior to administering the carcinogenic agent so that the animals would have plasma and tissular concentrations of each of the compounds. When the pretreatment period came to an end, the carcinogenic agent DMBA dissolved in acetone was administered topically, for which the animals were sedated three times a week by means of a mixture of ketamine and xylazine (50%). This was applied to the left cheek mucosa, as follows: the area was dried with a cotton swab, and 40 ml (200 mg) of DMBA solution was applied over a 1-min period using a micropipette. After 12 weeks of DMBA application, the animals were euthanized using a CO2 chamber.
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2.4.
Morphological and histopathological study
washed, counter stained with haematoxylin and covered with a mounting medium. For staining quantification, 3100 mm2 area were selected per slide along the inner border of the tumours, using a Leica DM600B microscope, with Leica DFC280 camera and a Leica image analysis programme supplied by Leica Microsystems AG (Solms, Germany). A histogram of pixel intensity was generated from the image; the number of total cells and strongly possitive stained cells was counted. The data for p53 and PCNA results was expressed as percentage of positive stained cells per area.
Following the procedure used by Feng and Wang28 we recorded the time in weeks required for 50% of the animals in each group to develop tumours (T50). The average number of oral lesions per animal was also calculated at the end of the experiment (no. of tumours/no. of animals). The size of these tumours was measured using a digital calliper (Proinsa, Vitoria, Spain) graduated at 0.01-mm intervals, along three orthogonal axes (A, B and C), and their volume was calculated. Tissue samples were fixed in 10% neutral buffered formalin for at least 48 h before setting in paraffin, sectioned at a thickness of 4 mm and stained using haematoxylin and eosin. Mucosal lesions were diagnosed on the basis of criteria established by the World Health Organization (WHO).29 The samples were examined by an observer blinded to the effects of the study who evaluated the number of carcinomas per animal (in situ and invasive). Bryne’s Invasive Tumour Front (ITF) Grading System was used to establish the invasive carcinomas’ degree of malignancy.30,31 This grading system evaluated four morphological parameters, awarding each a score of one to four to determine the degree of malignancy of each tumour. These registered the degree of keratinization, nuclear polymorphism, pattern of invasion and inflammatory response (lymphoid infiltration). The scores for all parameters were totaled to provide a total malignancy score.
2.5.
2.6.
Statistical analysis
Data were analyzed using the SPSS version 19.0 statistical package (SPSS1 Inc., Chicago, IL, USA). A descriptive study was made for each variable. For comparing qualitative data between groups a contingency table was made. To determine the degree of statistical association, one-way variance analysis was applied (ANOVA), complemented by equality comparison for pairs of means, using the minimum significant difference method, applying Bonferroni correction. Significance was established as p < 0.05.
Immunohistological study
Immunohistochemical staining was performed using a labelled streptavidin-biotin (LSAB) method. The immunoreactivity of the p53 antigen was enhanced by autoclaving the sections for 15 min in 0.01 M citrate buffer (pH 6.0). The sections were incubated overnight at 4 8C with one of the following antibodies: wild type p53 (Dako Corporation, Glostrup, Denmark) at a 1:200 dilution and PCNA (PC-10; DakoCorporation, Glostrup, Denmark) at a 1:200 dilution. Negative controls were treated with all reagents except the primary antibody. Positive controls of each antibody were also processed simultaneously (mouse intestine). The bound primary antibody was detected by incubation with the secondary antibody conjugated with horseradish peroxidase (BioGenex, San Ramon, CA, USA) for 30 min at room temperature. After rinsing with Tris-buffered saline, the antigen–antibody complex was detected using 3,30 diamminobenzidine, the substrate of horseradish peroxidase. When acceptable colour intensity was reached, the slides were
3.
Results
3.1.
General observations
When the macroscopic characteristics of the lesions were analyzed, it was found that all groups treated with phenolic compounds showed lower incidences of visible tumours than the control group, as well as lower lesion multiplicity, although these differences were not statistically significant. However, significant differences were identified for Groups III and V (Table 1) which showed lower numbers of visible tumours than the rest of the groups (1.1 0.15) ( p = 0.002). The tumour volume presented by the group treated with apigenin (Group II) was noticeably smaller than the control group and the other groups (61.01 29.51). However, these differences did not reach statistical significance ( p > 0.05) (Table 1).
3.2.
Histological study
There were no statistically significant differences in the number of lesions between groups. The control group
Table 1 – Incidence, number of tumours and tumour volume by group (ANOVA). Group
I II III IV V *
p = 0.916. p = 0.870. *** p = 0.002. **
Treatment
Control (DMBA) Apigenin Cocoa cathechin Eriocitrin Rosmarinic acid
No. of animals
10 10 10 10 10
Visible tumours Tumour incidence %
No. of tumours*
Tumour volume (mm3)**
100 90 90 90 90
1.7 0.26 1.5 0.30 2.1 0.37 1.4 0.30 1.1 0.15***
85.35 23.50 61.01 29.51 93.76 46.93 125.63 53.47 97.95 72.12
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Table 2 – Number of carcinomas observed histologically per animal and degree of invasion of microscopic lesions. Group
Treatment
No. of lesions
Microscopic lesions Tumour masses
In situ (%) Degree of tumour invasion
Carcinoma no. Incidence (%) (mean SD) (invasive and in situ) (invasive and in situ) I II III IV V
Control (DMBA) Apigenin Cocoa Cathechin Eriocitrin Rosmarinic Acid
15 8 12 13 10
1.4 0.26 0.9 0.31 1.2 0.38 0.7 0.21 0.6 0.22
presented the highest number of oral carcinomas per animal (1.4 0.26), as well as a higher incidence of tumour. The animals in the groups treated with potassium apigenin (Group II), eriocitrin (Group IV) and rosmarinic acid (Group V) showed lower mean numbers of tumours per animal, as well as lower tumour incidence and most carcinomas were in situ. Comparing groups, the groups treated with apigenin and rosmarinic acid had the highest proportions of in situ carcinomas, 77.78% and 83.33% respectively. The number invading the underlying musculature was greater in the control group (Table 2) (Fig. 1). Applying the Bryne Invasive Tumour Front (ITF) Grading System, the group treated with rosmarinic acid showed the highest proportion of well-differentiated carcinomas (83.33%). The groups treated with apigenin and rosmarinic acid achieved the lowest scores 6.6 0.9 and 6.5 13 respectively. In immunohistochemical determination, PCNA did not identify significant differences between the groups. All carcinomas subjected to PCNA study showed high scores (+++) all presenting intense degrees of proliferation. p-53 was also found to be over-expressed in 100% of carcinomas (both in situ and infiltrative) with severe intensity (+++) (Table 3) (Fig. 2).
4.
Discussion
The present study investigated the chemopreventive effects of phenolic compounds, potassium apigenin, cocoa catechins, eriocitrin and rosmarinic acid, using doses recommended by other authors.8,14,21–26 Apigenin has been shown to possess anticarcinogenic properties against various cancer cell lines in animal models. The choice of potassium apigenin used in this study was the result of its known chemopreventive properties against oral carcinogenesis.14 The data obtained shows that macroscopically the carcinomas developed in the hamsters treated with this flavonoid were of smaller volume than in the other study groups (61.01 29.51). Furthermore, microscope examination revealed that among this low incidence of squamous cell carcinoma, 77.78% of these were in situ carcinomas. These data agree with earlier findings made by the same research team.32 However, the present study did identify considerably different data in that 60% of samples developed tumours; this may be due to the route of administration. Immunohistochemical protein expression related to cell genesis and oncogenesis appears to be associated with the prognosis of some oral tumours. In this way, oral squamous cell carcinoma has been found to be related to the overexpression of different immunohistochemical markers such
90 60 70 60 50
Invasive Lamina Muscle (%) Propria (%) (%) 50 77.78 50 57.14 83.33
50 22.22 50 42.86 16.67
28.57 11.11 41.67 28.57 12.30
21.43 11.11 8.33 14.28 4.37
as Ki67 (nuclear proliferation antigen), Bcl-2 oncoprotein, P-16 and, as examined in the present study, PCNA and p53. The maximum expression of PCNA occurs in late G1 and S phases of the cell cycle. PCNA expression has been shown to correlate to the proliferative activity and prognosis of cancer patients.33,34 Abnormal expression of PCNA has been shown in precancerous and cancerous lesions of the oral cavity. p53 is a phosphoprotein involved in the cell cycle control, DNA repair, apoptosis and the conservation of genomic integrity. Mutations of p53, the most frequent alterations in human oral squamous cells carcinomas and HBP carcinomas, induce conformational changes that prolong the half-life of the p53 protein enabling immunolocalisation in the nuclei of malignant cells. Overexpression of PCNA and p53 in HBP carcinomas observed in the current study is indicative of increased cell proliferation and is consistent with similar findings in both human malignancies and animal tumour models. Silvan et al.8 observed that oral pretreatment with apigenin (2.5 mg kg1 body weight per day) significantly prevented tumour incidence and load in DMBA-treated hamsters and reduced the frequency of micronucleated polychromatic erythrocytes and abnormalities in the chromosome structure. Choi and Kim12 reported that apigenin-induced apoptosis through a p53-dependent pathway might have played a role in cell cycle arrest in human breast cancer SK-BR-3 cells. They also report that apigenin induced a reversible G2/M and G0/G1 cell cycle arrest by increasing p53 protein stability in a wide array of malignant cells. Recently, Silvan and Manoharan34 have found that the oral administration of apigenin regulates the expression of p53, bcl-2, PCNA, VEGF, c-fos, COX-2, NFkB,
Fig. 1 – DMBA well-defined exophytic tumours.
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Table 3 – Histological grading of invasive carcinomas: invasive tumour front graduation system and immunohistochemical scores. Group
Treatment
Animals (n = 10)
Microscopic lesions Degree of tumour differentiation (% of masses)
Immunohistological detection
PCNA Invasive tumour p53 accumulation front graduation (%) accumulation (%) system
Well Moderately Poorly I II III IV V
Control (DMBA) Apigenin Cocoa cathechin Eriocitrin Rosmarinic acid
10 10 10 10 10
50 66.67 41.67 71.43 83.33
35.71 33.33 50 0 0
cyclin D1 and up regulated the expression of Bax, caspase-3 and 9 in hamsters treated with DMBA. These findings demonstrate the modulating effect of apigenin on the expression pattern of molecular markers of apoptosis, cell proliferation, angiogenesis, and inflammation in DMBA induced buccal pouch carcinogenesis in hamsters. The over-expression of cyclooxygenase-2 (COX-2) has been demonstrated in many malignant and premalignant lesions in humans, including oral lesions. COX-2 is related with more aggressive tumours of worse prognosis and appears to be
14.29 0 8.33 28.57 16.67
8 0.646 6.6 0.98 7 0.707 8.1 1.487 6.5 1.310
81 77 79 77 82
90 91 84 92 89
involved in the maintenance of tumour growth and angiogenesis.35 Feng and Wang28 observed a reduction in the number and volume of tumours developed in animals treated with celecoxib (COX-2 inhibitor) compared with the DMBA-treated animals. COX-2 Inhibition resulting from the administration of the flavones used in this study is probably, at least partly, responsible for the results obtained. Osakabe et al.36 conclude that part of the anticarcinogenic effects of these extracts is due to rosmarinic acid acting via two independent mechanisms: the inhibition of the inflammatory
Fig. 2 – (a) Muscular invasion in an invasive carcinoma of control group, haematoxylin and eosin, original magnification 40T; (b) cords of epithelial cells and keratin pearl detail, haematoxylin and eosin, original magnification 100T; (c) nuclear PCNA and (d) p-53 immunoexpression, 40T and 10T respectively (original magnification).
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response and the scavenging of reactive oxygen radicals. Results of the present study show that rosmarinic acid has an inhibitory effect on oral carcinogenesis. The flavone eriocitrin is abundant in lemon and lime but not in all Citrus fruits.37 It is obtained from the Citrus peel and is used in numerous multivitaminic complexes, in which the antioxidant activity of the ‘‘bioflavonoids’’, for maintaining capillary integrity and peripheral circulation, is of note. Citrus peels and their extracts have been reported to have potent pharmacological activities and health benefits due to the abundance of flavonoids in citrus fruits, particularly in the peels. Previous studies demonstrated that oral administration of Gold Lotion (GL), an extract of multiple varieties of citrus peels containing abundant flavonoids, effectively suppressed azoxymethane (AOM)-induced colonic tumorigenesis.38 Lai et al.39 explored the anti-tumour effects of GL using a human prostate tumour xenograft mouse model and they reported that treatment with GL by both intraperitoneal (i.p.) injection and oral administration dramatically reduced both the weights (57–100% inhibition) and volumes (78–94% inhibition) of the tumours without any observed toxicity. These inhibitory effects were accompanied by mechanistic down-regulation of the protein levels of inflammatory enzymes (inducible nitric oxide synthase, iNOS and cyclooxygenase-2, COX-2), metastasis (matrix metallopeptidase-2, MMP-2 and MMP-9), angiogenesis (vascular endothelial growth factor, VEGF), and proliferative molecules, as well as by the induction of apoptosis in prostate tumours. The effects of cocoa catechins are related to their capacity for improving endothelial function and to increased active nitric oxide. It has been seen that they inhibit the proliferation of colon, breast and prostate cancer and reduce tumour marker enzyme activity during hepatocarcinogenesis.40 In the literature review performed in preparation for the present study, no research into cocoa catechins and oral carcinogenesis was identified. For this reason, further research is required into the action of this flavonoid in cases of oral cancer. Eriocitrin has anti-inflammatory and anticarcinogenic properties and appears to play an important role in cancer; several studies have looked at its anticarcinogenic activity in citric fruits and mint, in which it is an important metabolite.25,26 The present study found that groups treated with cocoa catechins and eriocitrin did not undergo such clearly beneficial effects. The tumours developed were of greater volume than the control group.
5.
Conclusions
All the phenolic compounds tested in this study were found to reduce the incidence of tumour. Potassium apigenin and rosmarinic acid showed the best results, the former considerably reducing tumour volume of the carcinomas developed in the group, and both compounds significantly reduced the incidence and aggression of the tumours developed.
Funding None.
Conflict of interest None declared.
Ethical approval All the animals were supplied by the University of Murcia (Spain) Research Support Services Animal Laboratory (License No. REGAES 300305440012) compliance with European Union norms for the protection of animals used in experimentation (86/609/EEC). The experiment was approved by the University of Murcia Bioethics Committee.
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in normal versus human prostate carcinoma cells. Biochem Biophys Res Commun 2001;287(4):914–20. 16. Gupta S, Afaq F, Mukhtar H. Involvement of nuclear factorkappa B: Bax and Bcl-2 in induction of cell cycle arrest and apoptosis by apigenin in human prostate carcinoma cells. Oncogene 2002;21(23):3727–38. 17. Czernik M, Sroka J, Madeja Z, Czyz J. Apigenin inhibits growth and motility but increases gap junctional coupling intensity in rat prostate carcinoma (MAT-LyLu) cell populations. Cell Mol Biol Lett 2008;13(3):327–38. 18. Lee WJ, Chen WK, Wang CJ, Lin WL, Tseng TH. Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and beta 4 integrin function in MDA-MBB breast cancer cells. Toxicol Appl Pharmacol 2008;226(2):178–91. [19]. Hussain AR, Khan AS, Ahmed SO, Ahmed M, Platanias LC, Al-Kuraya KS, et al. Apigenin induces apoptosis via down regulation of S-phase kinase-associated protein 2mediated induction of p27Kip1 in primary effusion lymphoma cells. Cell Prolif 2010;43(2):170–83. 20. Karim M, McCormik K, Kappagoda C. Effects of cocoa procyanidins on endothelium-dependent relaxation. J Nutr 2000;130(8S Suppl.):2150–8. 21. Furtado MA, Almeida LCF, Furtado RA, Cunha WR, et al. Antimutagenicity of rosmarinic acid in Swiss mice evaluated by the micronucleus assay. Mutat Res 2008;657(2):150–4. 22. del Ban˜o MJ, Lorente J, Castillo J, Benavente-Garcia O, Del Rio JA, Ortun˜o A. Phenolic diterpenes: flavones, and rosmarinic acid distribution during the development of leaves, flowers, stems, and roots of Rosmarinus officinalis. Antioxidant activity. J Agric Food Chem 2003;51(15):4247–53. 23. Cheung S, Tai J. Anti-proliferative and antioxidant properties of rosemary Rosmarinus officinalis. Oncol Rep 2003;17(6):1525–31. 24. Miller EG, Peacock JJ, Bourland TC, Taylor SE, Wright JM, Patil BS, et al. Inhibition of oral carcinogenesis by citrus flavonoids. Nutr Cancer 2008;60(1):69–74. 25. Benavente-Garcı´a O, Castillo J. Update on uses and properties of citrus flavonoids: new findings in anticancer, cardiovascular, and anti-inflammatory activity. J Agric Food Chem 2008;56(15):6185–205. 26. Benavente-Garcı´a O, Castillo J, Alcaraz M, Vicente V, Del Rı´o JA, Ortun˜o A. Beneficial action of Citrus flavonoids on multiple cancer-related biological pathways. Curr Cancer Drug Targets 2007;7(8):795–809. 27. Ogata S, Miyake Y, Yamamoto K, Okumura K, Taguchi H. Apoptosis induced by the flavonoid from lemon fruit and metabolites in HL-60 cells. Biosci Biotechnol Biochem 2000;64(5):1075–8.
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28. Feng L, Wang Z. Chemopreventive effect of celecoxib in oral pre-cancers and cancers. Laryngoscope 2006;116(10):1842–5. [29]. Barnes L, Eveson JW, Reichart P, Sidransky D. World Health Organization classification of tumours. Pathology and genetics of head and neck tumours. Lyon (France): IARC Press; 2005. 30. Kristensen GB, Abeler VM, Risberg B, Trop C, Bryne M. Tumor size: depth of invasion, and grading of the invasive tumor front are the main prognostic factors in early squamous cell cervical carcinoma. Gynecol Oncol 1999;74(2):245–51. 31. Bryne M. Is the invasive front of an oral carcinoma the most important area for prognostication? Oral Dis 1998;4(2):70–7. 32. Gomez-Garcia F, Lopez Jornet MP, Alvarez-Sanchez N, Castillo-Sanchez J, Benavente-Garcia O, Vicente Ortega V. Effect of the phenolic compounds apigenin and carnosic acid on oral carcinogenesis in hamsters induced by DMBA. Oral Dis 2013;19(3):279–86. 33. Diniz-Freitas M, Garcia-Caballero T, Antunez-Lopez J, Gandara-Rey JM, Garcia-Garcia A. Pharmaco diagnostic evaluation of EGFR expression in oral squamous cell carcinoma. Oral Dis 2007;13(3):285–90. 34. Silvan S, Manoharan S. Apigenin prevents deregulation in the expression pattern of cell-proliferative: apoptotic, inflammatory and angiogenic markers during 7,12dimethylbenz [a]anthracene-induced hamster buccal pouch carcinogenesis. Arch Oral Biol 2013;58(1):94–101. 35. Nishimura N, Urade M, Hashitani S, Noguchi K, Manno Y, Takaoka K, et al. Increased expression of cyclooxigenase (COX)-2 in DMBA-induced hamster cheek pouch carcinogenesis and chemopreventive effect of a selective COX-2 inhibitor celecoxib. J Oral Pathol Med 2004;33(10):614– 21. 36. Osakabe N, Yasuda A, Natsume M, Yoshikawa T. Rosmarinic acid inhibits epidermal inflammatory responses: anticarcinogenic effect of Perilla frutescens extract in the murine two-stage skin model. Carcinogenesis 2004;25(4):549–57. 37. Fuster MD. Citrus flavonoids distribution, modulation by phytorregulators and their possible physiological function. (Ph.D. thesis) Spain: University of Murcia; 1997. 38. Tanaka T, Kawabata K, Kakumoto M, Makita H, Hara A, Mori H, et al. Citrus auraptene inhibits chemically induced colonic aberrant crypt foci in male F344 rats. Carcinogenesis 1997;18(11):2155–61. 39. Lai CS, Li S, Miyauchi Y, Suzawa M, Ho CT, Pan MH. Potent anti-cancer effects of citrus peel flavonoids in human prostate xenograft tumors. Food Funct 2013;4(6):944–9. 40. Maskarinec G. Cancer protective properties of cocoa: a review of the epidemiologic evidence. Nutr Cancer 2009;61(5):573–9.
Available online at www.sciencedirect.com
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Chemopreventive potential of phenolic compounds in oral carcinogenesis B. Baldasquin-Caceres a, F.J. Gomez-Garcia a, P. Lo´pez-Jornet b,*, J. Castillo-Sanchez c, V. Vicente-Ortega a a
Department of Pathology and Anatomical Sciences, Faculty of Medicine and Dentistry, Ageing Research Institute, University of Murcia, Murcia, Spain b Oral Medicine Ageing Research Institute, Faculty of Medicine and Dentistry, University of Murcia, Murcia, Spain c I+D+I Nutrafur SA Murcia Spain Ageing Research Institute, University of Murcia, Murcia, Spain
article info
abstract
Article history:
Objective: To evaluate the chemopreventive potential of phenolic compounds – potassium
Accepted 18 June 2014
apigenin, cocoa, catechins, eriocitrin and rosmarinic acid in oral carcinogenesis induced in hamsters by means of the topical application of 7,12-dimethylbenz(a)anthracene(DMBA).
Keywords:
Study design: An experimental study at the University of Murcia.
Oral carcinogenesis
Methods: 50 male Syrian hamsters (Mesocricetus auratus) were divided into five groups of ten:
Apigenin
Group I (control group): 0.5% DMBA; Group II: 0.5% DMBA + 1.1 mg/15 ml potassium api-
Cocoa cathechin
genin; Group III: 05% DMBA + 2.5 mg/15 ml cocoa catechins; Group IV: 0.5% DMBA + 6 mg/
Eriocitrin and rosmarinic acid
15 ml eriocitrin; Group V: 0.5% DMBA + 1.3 mg/15 ml rosmarinic acid. The flavonoids were
DMBA
administered orally. All the animals were sacrificed after 12 weeks. Macroscopic, micro-
Hamster
scopic and immunohistochemical (PCNA and p53) analyses of the lesions were performed. Results: All the groups treated with phenolic compounds showed lower incidences of tumour, greater differentiation and lower scores in the tumour invasion front grading system in comparison with the control group. Potassium apigenin and rosmarinic acid achieved the best results, the former considerably reduced the carcinoma tumour volumes developed and both significantly reduced the intensity and aggression of the tumours. Immunoexpression of PCNA and p53 were significantly altered during DMBA-induced oral carcinogenesis. Conclusions: Animals treated with phenolic compounds, particularly potassium apigenin and rosmarinic acid, showed a lower incidence of tumours. # 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
Oral squamous cell carcinoma is the most frequently occurring cancer of the head and neck and is characterized by poor prognosis and a low patient survival rate.1,2
Recent efforts to control the incidence of oral squamous cell carcinoma, have focused on developing effective chemopreventive strategies. Chemoprevention by natural products and dietary and lifestyle changes have evolved as promising strategies for the management of cancer. In this way, dietary phytochemicals have gained significant recognition in recent
* Corresponding author at: Clı´nica Odontolo´gica Universitaria, Hospital Morales Meseguer, Avda. Marque´s de los Ve´lez s/n, Murcia 30008, Spain Tel.: +34 968 398588; fax: +34 968 398576. E-mail address: [email protected] (P. Lo´pez-Jornet). http://dx.doi.org/10.1016/j.archoralbio.2014.06.007 0003–9969/# 2014 Elsevier Ltd. All rights reserved.
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years as potential candidates for cancer chemoprevention.1–9 In vitro studies have shown that flavonoids significantly increase p53, E-cadherin and b-catenin protein expression and caspase 3, fas ligand and Fas/Apo-1 receptor activation which are strongly associated with the signal transduction pathways involved in apoptosis and cellular adhesion that affect the chemosensitivity of tumour cells. The biological behaviour of flavonoids depends on chemical structure and epidemiological studies support their role in cancer prevention.10–14 Apigenin, a flavonoid compound, has the capacity to inhibit the proliferation and cell viability in numerous cancer cell lines.14–16 Apigenin has shown chemoprotective effects in various in vivo mouse models of cancer (lung, skin, neck of the uterus, ovarian, prostate, leukaemia, and colon), in which it has been seen to reduce the size of primary tumors.17 It is also able to inhibit the mobility of tumour cells (inhibition of FAK expression)18 and to reduce the number and size of metastatic nodes in ovarian cancer, breast cancer, and melanoma.19 Initial observations have shown that procyanidin-rich fractions prepared from the seeds of Theobroma cacao, which are used to prepare cocoa, inhibit in vitro growth of several human breast cancer cell lines.20 This research has revealed oligomeric procyanidins, such as the pentameric procyanidin, to be cytotoxic to breast cancer cells. Rosmarinic acid (RA) ((2R)-2-[[(2E)-3-(3,4-dihydroxyphenyl)-1oxo-2-propenyl]]oxy-3-(3,4-dihydroxyphenyl) propanoic acid) has a number of interesting biological activities, including antiviral, antibacterial, anti-inflammatory and antioxidant effects.21–23 RA prevents mutagenic activity, which has been observed by means of micronuclear tests in Swiss mice. In addition, the reduction in tumorigenesis in a murine two-stage skin carcinogenesis model with topical application of Perilla Frutescens extract has also been attributed to the presence of RA. It has been shown to delay colorectal carcinogenesis in rodent models. The pharmacological effect of RA has been seen to act through the inhibition of several complement-dependent inflammatory processes. Recent studies confirm that caffeoyl esters such as RA show high specific antioxidant activity, delay vitamin E depletion, decrease pro-inflammatory lysophosphatidyl choline production and prevent the oxidation of low-density lipoprotein (LDL), which is compatible with its anti-inflammatory and antiatherosclerotic role in pathophysiological conditions.21–23 Among the glycoside flavonoids present in lemons, eriocitrin shows the highest antioxidant activity and has been shown to inhibit proliferation and apoptosis in different cancer cell lines.24–26 Ogata et al.27 demonstrated that lemon flavonoids and their metabolites induce apoptosis in leukaemia cell lines HL-60 dose- and time-dependently. The aim of this study was to evaluate the chemopreventive efficacy of phenolic compounds potassium apigenin, cocoa catechins, eriocitrin and rosmarinic acid on oral carcinogenesis induced in hamsters by means of topical application of DMBA.
2.
Materials and methods
2.1.
Animals
The study used 50 male Syrian hamsters (Mesocricetus auratus), with an average weight of 100 g and an average
age of five months. All the animals were supplied by the University of Murcia (Spain) Research Support Services Animal Laboratory (License No. REGAES 300305440012). The hamsters were housed in cages and received food and water ad libitum. The room in which they were housed had a controlled 12/12 light/darkness cycle and a temperature of 22 8C, in compliance with European Union norms for the protection of animals used in experimentation (2010/63/UE). The experiment was approved by the University of Murcia Bioethics Committee.
2.2.
Agents
- Polycyclic aromatic hydrocarbon 7,12-dimethyl-1,2-bezantracene (DMBA) (Sigma Aldrich Co., Madrid, Spain). The DMBA was dissolved in acetone (Sigma Aldrich Co., Madrid, Spain) at 0.5%. The anaesthetic used was ketamine (Imalgene 1000 Merial, Barcelona, Spain) and xylazine (Xylagesic 2% [20 mg/ml] Laboratorios Calier S.A, Barcelona, Spain). - Concentrated extract of apigenin (90% HPLC purity) (Nutrafur, S.A., Murcia, Spain) dissolved in distilled water at a concentration of 1.1 mg/15 ml. - Concentrated extract of cocoa catechins (40% HPLC purity) (Nutrafur, S.A., Murcia, Spain) dissolved in distilled water at a concentration of 2.5 mg/15 ml. - Concentrated extract of eriocitrin (20% HPLC purity) (Nutrafur, S.A., Murcia, Spain) dissolved in distilled water at a concentration of 6 mg/15 ml. - Concentrated extract of rosmarinic acid (15% HPLC purity) (Nutrafur, S.A., Murcia, Spain) dissolved in distilled water at a concentration of 1.3 mg/15 ml.
2.3.
Experiment procedure
Fifty animals were divided randomly into five groups of ten: Group I (control group) treated with 0.5% DMBA in acetone (n = 10); Group II treated with 0.5% DMBA + concentrated extract of potassium apigenin (n = 10); Group III treated with 05% DMBA + concentrated extract of cocoa catechins (n = 10); Group IV treated with 0.5% DMBA + concentrated extract of eriocitrin (n = 10); Group V treated with 0.5% DMBA + concentrated extract of rosmarinic acid (n = 10). All the phenolic compounds were administered via the animals’ drinking water, consumed ad libitum. The animals in the study groups treated with polyphenols received pre-treatment for two weeks prior to administering the carcinogenic agent so that the animals would have plasma and tissular concentrations of each of the compounds. When the pretreatment period came to an end, the carcinogenic agent DMBA dissolved in acetone was administered topically, for which the animals were sedated three times a week by means of a mixture of ketamine and xylazine (50%). This was applied to the left cheek mucosa, as follows: the area was dried with a cotton swab, and 40 ml (200 mg) of DMBA solution was applied over a 1-min period using a micropipette. After 12 weeks of DMBA application, the animals were euthanized using a CO2 chamber.
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2.4.
Morphological and histopathological study
washed, counter stained with haematoxylin and covered with a mounting medium. For staining quantification, 3100 mm2 area were selected per slide along the inner border of the tumours, using a Leica DM600B microscope, with Leica DFC280 camera and a Leica image analysis programme supplied by Leica Microsystems AG (Solms, Germany). A histogram of pixel intensity was generated from the image; the number of total cells and strongly possitive stained cells was counted. The data for p53 and PCNA results was expressed as percentage of positive stained cells per area.
Following the procedure used by Feng and Wang28 we recorded the time in weeks required for 50% of the animals in each group to develop tumours (T50). The average number of oral lesions per animal was also calculated at the end of the experiment (no. of tumours/no. of animals). The size of these tumours was measured using a digital calliper (Proinsa, Vitoria, Spain) graduated at 0.01-mm intervals, along three orthogonal axes (A, B and C), and their volume was calculated. Tissue samples were fixed in 10% neutral buffered formalin for at least 48 h before setting in paraffin, sectioned at a thickness of 4 mm and stained using haematoxylin and eosin. Mucosal lesions were diagnosed on the basis of criteria established by the World Health Organization (WHO).29 The samples were examined by an observer blinded to the effects of the study who evaluated the number of carcinomas per animal (in situ and invasive). Bryne’s Invasive Tumour Front (ITF) Grading System was used to establish the invasive carcinomas’ degree of malignancy.30,31 This grading system evaluated four morphological parameters, awarding each a score of one to four to determine the degree of malignancy of each tumour. These registered the degree of keratinization, nuclear polymorphism, pattern of invasion and inflammatory response (lymphoid infiltration). The scores for all parameters were totaled to provide a total malignancy score.
2.5.
2.6.
Statistical analysis
Data were analyzed using the SPSS version 19.0 statistical package (SPSS1 Inc., Chicago, IL, USA). A descriptive study was made for each variable. For comparing qualitative data between groups a contingency table was made. To determine the degree of statistical association, one-way variance analysis was applied (ANOVA), complemented by equality comparison for pairs of means, using the minimum significant difference method, applying Bonferroni correction. Significance was established as p < 0.05.
Immunohistological study
Immunohistochemical staining was performed using a labelled streptavidin-biotin (LSAB) method. The immunoreactivity of the p53 antigen was enhanced by autoclaving the sections for 15 min in 0.01 M citrate buffer (pH 6.0). The sections were incubated overnight at 4 8C with one of the following antibodies: wild type p53 (Dako Corporation, Glostrup, Denmark) at a 1:200 dilution and PCNA (PC-10; DakoCorporation, Glostrup, Denmark) at a 1:200 dilution. Negative controls were treated with all reagents except the primary antibody. Positive controls of each antibody were also processed simultaneously (mouse intestine). The bound primary antibody was detected by incubation with the secondary antibody conjugated with horseradish peroxidase (BioGenex, San Ramon, CA, USA) for 30 min at room temperature. After rinsing with Tris-buffered saline, the antigen–antibody complex was detected using 3,30 diamminobenzidine, the substrate of horseradish peroxidase. When acceptable colour intensity was reached, the slides were
3.
Results
3.1.
General observations
When the macroscopic characteristics of the lesions were analyzed, it was found that all groups treated with phenolic compounds showed lower incidences of visible tumours than the control group, as well as lower lesion multiplicity, although these differences were not statistically significant. However, significant differences were identified for Groups III and V (Table 1) which showed lower numbers of visible tumours than the rest of the groups (1.1 0.15) ( p = 0.002). The tumour volume presented by the group treated with apigenin (Group II) was noticeably smaller than the control group and the other groups (61.01 29.51). However, these differences did not reach statistical significance ( p > 0.05) (Table 1).
3.2.
Histological study
There were no statistically significant differences in the number of lesions between groups. The control group
Table 1 – Incidence, number of tumours and tumour volume by group (ANOVA). Group
I II III IV V *
p = 0.916. p = 0.870. *** p = 0.002. **
Treatment
Control (DMBA) Apigenin Cocoa cathechin Eriocitrin Rosmarinic acid
No. of animals
10 10 10 10 10
Visible tumours Tumour incidence %
No. of tumours*
Tumour volume (mm3)**
100 90 90 90 90
1.7 0.26 1.5 0.30 2.1 0.37 1.4 0.30 1.1 0.15***
85.35 23.50 61.01 29.51 93.76 46.93 125.63 53.47 97.95 72.12
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Table 2 – Number of carcinomas observed histologically per animal and degree of invasion of microscopic lesions. Group
Treatment
No. of lesions
Microscopic lesions Tumour masses
In situ (%) Degree of tumour invasion
Carcinoma no. Incidence (%) (mean SD) (invasive and in situ) (invasive and in situ) I II III IV V
Control (DMBA) Apigenin Cocoa Cathechin Eriocitrin Rosmarinic Acid
15 8 12 13 10
1.4 0.26 0.9 0.31 1.2 0.38 0.7 0.21 0.6 0.22
presented the highest number of oral carcinomas per animal (1.4 0.26), as well as a higher incidence of tumour. The animals in the groups treated with potassium apigenin (Group II), eriocitrin (Group IV) and rosmarinic acid (Group V) showed lower mean numbers of tumours per animal, as well as lower tumour incidence and most carcinomas were in situ. Comparing groups, the groups treated with apigenin and rosmarinic acid had the highest proportions of in situ carcinomas, 77.78% and 83.33% respectively. The number invading the underlying musculature was greater in the control group (Table 2) (Fig. 1). Applying the Bryne Invasive Tumour Front (ITF) Grading System, the group treated with rosmarinic acid showed the highest proportion of well-differentiated carcinomas (83.33%). The groups treated with apigenin and rosmarinic acid achieved the lowest scores 6.6 0.9 and 6.5 13 respectively. In immunohistochemical determination, PCNA did not identify significant differences between the groups. All carcinomas subjected to PCNA study showed high scores (+++) all presenting intense degrees of proliferation. p-53 was also found to be over-expressed in 100% of carcinomas (both in situ and infiltrative) with severe intensity (+++) (Table 3) (Fig. 2).
4.
Discussion
The present study investigated the chemopreventive effects of phenolic compounds, potassium apigenin, cocoa catechins, eriocitrin and rosmarinic acid, using doses recommended by other authors.8,14,21–26 Apigenin has been shown to possess anticarcinogenic properties against various cancer cell lines in animal models. The choice of potassium apigenin used in this study was the result of its known chemopreventive properties against oral carcinogenesis.14 The data obtained shows that macroscopically the carcinomas developed in the hamsters treated with this flavonoid were of smaller volume than in the other study groups (61.01 29.51). Furthermore, microscope examination revealed that among this low incidence of squamous cell carcinoma, 77.78% of these were in situ carcinomas. These data agree with earlier findings made by the same research team.32 However, the present study did identify considerably different data in that 60% of samples developed tumours; this may be due to the route of administration. Immunohistochemical protein expression related to cell genesis and oncogenesis appears to be associated with the prognosis of some oral tumours. In this way, oral squamous cell carcinoma has been found to be related to the overexpression of different immunohistochemical markers such
90 60 70 60 50
Invasive Lamina Muscle (%) Propria (%) (%) 50 77.78 50 57.14 83.33
50 22.22 50 42.86 16.67
28.57 11.11 41.67 28.57 12.30
21.43 11.11 8.33 14.28 4.37
as Ki67 (nuclear proliferation antigen), Bcl-2 oncoprotein, P-16 and, as examined in the present study, PCNA and p53. The maximum expression of PCNA occurs in late G1 and S phases of the cell cycle. PCNA expression has been shown to correlate to the proliferative activity and prognosis of cancer patients.33,34 Abnormal expression of PCNA has been shown in precancerous and cancerous lesions of the oral cavity. p53 is a phosphoprotein involved in the cell cycle control, DNA repair, apoptosis and the conservation of genomic integrity. Mutations of p53, the most frequent alterations in human oral squamous cells carcinomas and HBP carcinomas, induce conformational changes that prolong the half-life of the p53 protein enabling immunolocalisation in the nuclei of malignant cells. Overexpression of PCNA and p53 in HBP carcinomas observed in the current study is indicative of increased cell proliferation and is consistent with similar findings in both human malignancies and animal tumour models. Silvan et al.8 observed that oral pretreatment with apigenin (2.5 mg kg1 body weight per day) significantly prevented tumour incidence and load in DMBA-treated hamsters and reduced the frequency of micronucleated polychromatic erythrocytes and abnormalities in the chromosome structure. Choi and Kim12 reported that apigenin-induced apoptosis through a p53-dependent pathway might have played a role in cell cycle arrest in human breast cancer SK-BR-3 cells. They also report that apigenin induced a reversible G2/M and G0/G1 cell cycle arrest by increasing p53 protein stability in a wide array of malignant cells. Recently, Silvan and Manoharan34 have found that the oral administration of apigenin regulates the expression of p53, bcl-2, PCNA, VEGF, c-fos, COX-2, NFkB,
Fig. 1 – DMBA well-defined exophytic tumours.
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Table 3 – Histological grading of invasive carcinomas: invasive tumour front graduation system and immunohistochemical scores. Group
Treatment
Animals (n = 10)
Microscopic lesions Degree of tumour differentiation (% of masses)
Immunohistological detection
PCNA Invasive tumour p53 accumulation front graduation (%) accumulation (%) system
Well Moderately Poorly I II III IV V
Control (DMBA) Apigenin Cocoa cathechin Eriocitrin Rosmarinic acid
10 10 10 10 10
50 66.67 41.67 71.43 83.33
35.71 33.33 50 0 0
cyclin D1 and up regulated the expression of Bax, caspase-3 and 9 in hamsters treated with DMBA. These findings demonstrate the modulating effect of apigenin on the expression pattern of molecular markers of apoptosis, cell proliferation, angiogenesis, and inflammation in DMBA induced buccal pouch carcinogenesis in hamsters. The over-expression of cyclooxygenase-2 (COX-2) has been demonstrated in many malignant and premalignant lesions in humans, including oral lesions. COX-2 is related with more aggressive tumours of worse prognosis and appears to be
14.29 0 8.33 28.57 16.67
8 0.646 6.6 0.98 7 0.707 8.1 1.487 6.5 1.310
81 77 79 77 82
90 91 84 92 89
involved in the maintenance of tumour growth and angiogenesis.35 Feng and Wang28 observed a reduction in the number and volume of tumours developed in animals treated with celecoxib (COX-2 inhibitor) compared with the DMBA-treated animals. COX-2 Inhibition resulting from the administration of the flavones used in this study is probably, at least partly, responsible for the results obtained. Osakabe et al.36 conclude that part of the anticarcinogenic effects of these extracts is due to rosmarinic acid acting via two independent mechanisms: the inhibition of the inflammatory
Fig. 2 – (a) Muscular invasion in an invasive carcinoma of control group, haematoxylin and eosin, original magnification 40T; (b) cords of epithelial cells and keratin pearl detail, haematoxylin and eosin, original magnification 100T; (c) nuclear PCNA and (d) p-53 immunoexpression, 40T and 10T respectively (original magnification).
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response and the scavenging of reactive oxygen radicals. Results of the present study show that rosmarinic acid has an inhibitory effect on oral carcinogenesis. The flavone eriocitrin is abundant in lemon and lime but not in all Citrus fruits.37 It is obtained from the Citrus peel and is used in numerous multivitaminic complexes, in which the antioxidant activity of the ‘‘bioflavonoids’’, for maintaining capillary integrity and peripheral circulation, is of note. Citrus peels and their extracts have been reported to have potent pharmacological activities and health benefits due to the abundance of flavonoids in citrus fruits, particularly in the peels. Previous studies demonstrated that oral administration of Gold Lotion (GL), an extract of multiple varieties of citrus peels containing abundant flavonoids, effectively suppressed azoxymethane (AOM)-induced colonic tumorigenesis.38 Lai et al.39 explored the anti-tumour effects of GL using a human prostate tumour xenograft mouse model and they reported that treatment with GL by both intraperitoneal (i.p.) injection and oral administration dramatically reduced both the weights (57–100% inhibition) and volumes (78–94% inhibition) of the tumours without any observed toxicity. These inhibitory effects were accompanied by mechanistic down-regulation of the protein levels of inflammatory enzymes (inducible nitric oxide synthase, iNOS and cyclooxygenase-2, COX-2), metastasis (matrix metallopeptidase-2, MMP-2 and MMP-9), angiogenesis (vascular endothelial growth factor, VEGF), and proliferative molecules, as well as by the induction of apoptosis in prostate tumours. The effects of cocoa catechins are related to their capacity for improving endothelial function and to increased active nitric oxide. It has been seen that they inhibit the proliferation of colon, breast and prostate cancer and reduce tumour marker enzyme activity during hepatocarcinogenesis.40 In the literature review performed in preparation for the present study, no research into cocoa catechins and oral carcinogenesis was identified. For this reason, further research is required into the action of this flavonoid in cases of oral cancer. Eriocitrin has anti-inflammatory and anticarcinogenic properties and appears to play an important role in cancer; several studies have looked at its anticarcinogenic activity in citric fruits and mint, in which it is an important metabolite.25,26 The present study found that groups treated with cocoa catechins and eriocitrin did not undergo such clearly beneficial effects. The tumours developed were of greater volume than the control group.
5.
Conclusions
All the phenolic compounds tested in this study were found to reduce the incidence of tumour. Potassium apigenin and rosmarinic acid showed the best results, the former considerably reducing tumour volume of the carcinomas developed in the group, and both compounds significantly reduced the incidence and aggression of the tumours developed.
Funding None.
Conflict of interest None declared.
Ethical approval All the animals were supplied by the University of Murcia (Spain) Research Support Services Animal Laboratory (License No. REGAES 300305440012) compliance with European Union norms for the protection of animals used in experimentation (86/609/EEC). The experiment was approved by the University of Murcia Bioethics Committee.
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