Mixture of Uncaria and Tabebuia Extracts are Potentially Chemopreventive in CBA/Ca Mice - A Long-term Experiment Ferenc Budán, István Szabo, Timea Varjas, Ghodratollah Nowrasteh, Tamás Dávid, Péter Gergely, Zsuzsa Varga, Kornélia Molnár, Balázs Kádár, Zsuzsa Orsos, et al.

To cite this version:

Ferenc Budán, István Szabo, Timea Varjas, Ghodratollah Nowrasteh, Tamás Dávid, et al.. Mixture of Uncaria and Tabebuia Extracts are Potentially Chemopreventive in CBA/Ca Mice - A Long-term Experiment. Phytotherapy Research, Wiley, 2010, 25 (4), pp.493. ￿10.1002/ptr.3281￿. ￿hal-00599836￿

HAL Id: hal-00599836 https://hal.archives-ouvertes.fr/hal-00599836 Submitted on 11 Jun 2011

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Phytotherapy Research

Mixture of Uncaria and Tabebuia Extracts are Potentially Chemopreventive in CBA/Ca Mice - A Long-term Experiment

Journal: Phytotherapy Research ManuscriptFor ID: PTR-10-0137.R1 Peer Review Wiley - Manuscript type: Full Paper

Date Submitted by the 07-Jun-2010 Author:

Complete List of Authors: Budán, Ferenc; University of Pécs, Faculty of Medicine, Institute of Public Health Szabo, István; University of Pecs, Faculty of Medicine, Institute of Public Health Varjas, Timea; University of Pécs, Faculty of Medicine, Institut of Public Health Nowrasteh, Ghodratollah; University of Pécs, Faculty of Medicine, Institute of Public Health Dávid, Tamás; CoD Cancer Information & Research Foundation Gergely, Péter; University of Debrecen, Institute of Forensic Medicine Varga, Zsuzsa; University of Pecs, Faculty of Medicine Molnár, Kornélia; Office of the Chief Medical Officer Kádár, Balázs; University of Pecs, Faculty of Medicine Orsos, Zsuzsa; University of Pécs, Faculty of Medicine, Institute of Public Health Kiss, István; University of Pecs, Faculty of Medicine, Institute of Public Health Ember, István; University of Pecs, Faculty of Medicine, Institute of Public Health

Gene expression, carcinogenesis, chemoprevention, DMBA, Uncaria, Keyword: Tabebuia

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1 2 3 Mixture of Uncaria and Tabebuia Extracts are Potentially Chemopreventive in CBA/Ca 4 5 Mice - A Long-term Experiment 6 1 1 1 7 FERENC BUDÁN , ISTVÁN SZABÓ , TÍMEA VARJAS , GHODRATOLLAH 8 NOWRASTEH 1, TAMÁS DÁVID 2, PÉTER GERGELY 3, ZSUZSA VARGA 4, KORNÉLIA 9 MOLNÁR 5, BALÁZS KÁDÁR 1, ZSUZSA ORSÓS 1, ISTVÁN KISS 1 and ISTVÁN EMBER 1 10 11 1 Institute of Public Health, Medical School, University of Pécs, Szigeti str. 12, H-7643 Pécs; 12 2 13 CoD Cancer Information & Research Foundation, Vörösmarty str. 15, H- 9155 Lébény; 14 3Institute of Forensic Medicine, University of Debrecen, Nagyerdei krt. 98., H-4012, 15 Debrecen; 16 4Country Hospital of Baranya, Department of Oncology, H-7623, Pécs; 17 5 18 Office of the Chief Medical Officer, Gyáli str. 2-6, H-1097, Budapest. 19 20 For Peer Review 21 22 23 Correspondence to: Ferenc Budán 24 25 [email protected] 26 Address: Szigeti str. 12, H-7624 Pécs, Hungary 27 Phone: +36-72-536-394 28 Fax: +36-72-536-395 29 30 31 Experimental work 32 33 34 Budán et al: Uncaria and Tabebuia are Chemopreventive in Long-term Mice Experiment 35 36 37 38 Key Words: Gene expression, carcinogenesis, chemoprevention, DMBA, Uncaria, 39 Tabebuia 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Abstract. 4 5 6 A long-term experimental animal model was developed by our research group for the 7 8 evaluation of potential chemopreventive effects. The inhibitory effects of agents on 9 10 carcinogen (7,12-Dimetilbenz[a]anthracene ( DMBA)) induced molecular epidemiological 11 12 13 biomarkers, in this case the expression of key onco/suppressor genes were investigated. 14 15 Expression pattern of c-myc , Ha-ras , Bcl-2, K-ras protoonco and p53 tumoursuppressor genes 16 17 18 were studied to elucidate early carcinogenic and potential chemopreventive effects. 19 TM 20 Consumption of so calledFor "CoD Peer tea” (abbreviation Review of Claw of Dragon tea) containing bark 21 22 of Uncaria guianensis , Cat's Claw (Uncaria sp. U. tomentosa ) and Palmer trumpet-tree 23 24 25 (Tabebuia sp. T. avellanedae ) was able to decrease the DMBA-induced onco/suppressor gene 26 27 overexpression in a short-term animal experiment. In following study CBA/Ca mice were 28 29 treated with 20 mg/bwkg DMBA intraperitoneally (ip. ) and expression patterns of 30 31 32 onco/suppressor genes were examined at several time intervals. According to the examined 33 34 gene expression patterns in this long-term experiment the chemopreventive effect of CoD TM 35 36 tea consumption could be confirmed. 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 In our previous studies a unique animal test model was developed through the investigation of 4 5 6 onco/suppressor genes expression patterns, as molecular epidemiological biomarkers (Ember 7 8 et al. , 1998 ). 7,12-dimetilbenz[a]anthracene ( DMBA) is a pluripotent and complete 9 10 carcinogen, therefore it is applied to induce carcinomas in animal experiments. DMBA 11 12 13 increases the expression of c-myc , Ha-ras , Bcl-2, K-ras protoonco (Gyöngyi and Somlyai, 14 15 2002) and consequently p53 tumoursuppressor genes in the lungs and in the liver, as well 16 17 18 (Sándor et al. , 1995 ) in several animal species and also in both sex, indicating both 19 20 carcinogenic exposureFor and early stepsPeer of tumour Review formation (Ember et al. , 1998 ). The purpose 21 22 was to study the early biological effect of DMBA and the protective effect of some potential 23 24 TM 25 chemopreventive agents in so called “CoD tea" (abbreviation of Claw of Dragon tea). 26 27 Consumption of “CoD TM tea”, which contains extract of Uncaria guianensis , Cat's Claw 28 29 (Uncaria sp. U. tomentosa ) and Palmer trumpet-tree (Tabebuia sp. T. avellanedae ), was able 30 31 32 to decrease the DMBA-induced overexpression of the genes mentioned above, in a short-term 33 34 animal experiment (Orsós et al. , 2007 ). According to pharmacological studies Uncaria species 35 36 have cytotoxic, anti-inflammatory, antiviral, immunostimulative, antioxidant and antibacterial 37 38 39 properties (Heitzman et al. , 2005 ). Keplinger et al. confirmed that the pentacyclic oxindol 40 41 alkaloides (POA) are the main biological active components of Uncaria species, which in 42 43 44 vitro activate lymphocyte-proliferation regulating factor in endothelial cells (Laus et al. , 45 46 1997; Keplinger et al. , 1999 ). The bark of Palmer trumpet-tree is abundant in lapachol (2- 47 48 hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone) (2-7%), lapachone and xyloidone (de 49 50 51 Santana et al. , 1968 ), which are anticarcinogenic (Ueda et al. , 1994 ) and inhibit the growth of 52 53 the human keratinocyte cell line HaCaT culture in vitro (Müller et al. , 1999 ). Our research 54 55 group supposed that CoD TM tea consumption was able to decrease the overexpression of 56 57 58 certain key onco/suppressor genes. 59 60

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1 2 3 In this long term study the effect of CoD TM tea was investigated on the expression of c-myc , 4 5 6 Ha-ras , Bcl-2, K-ras and p53 onco/suppressor genes in CBA/Ca mice test model (Varga et 7 8 al. , 1991; Ember et al. , 1992; Gyöngyi and Somlyai 2000 ). 9 10 11 12 13 Materials and methods 14 15 Animals and Treatments. 16 17 TM 18 Infuse of CoD tea (D/Eu reg. numb.: PZN Deutschland: 4226037) was prepared according 19 20 to the instruction of theFor manufacturer. Peer Review 21 22 10g of the powder was suspended in 750 ml tap water and left to soak for twelve hours, and 23 24 25 then it was heated to 80 ºC for thirty minutes. After filtration and water refill, the infuse was 26 27 kept in a closed bottle avoiding light at 4 ºC. 28 29 250 ml extract is advised to be consumed three times a day, therefore the human dose is 0.167 30 31 32 g powder/kg per os (calculated for 60 kg mean body weight). 33 34 Calculated equivalent dose for mice is 0.0033 g powder in 3 ml tap water. 35 36 Quantity of total pentacyclic oxindol alkaloides (POA) is supposed to be 0.1-1.12%, based on 37 38 39 the results of Csapi et al. ( Csapi et al. , 2008 ). 40 41 Six- to eight-week-old (20±4 g) conventionally kept CBA/Ca inbred H-2K haplotype mice (6 42 43 44 males and 6 females in each group) were used for this experiment according to the following 45 TM 46 arrangement: control groups, DMBA treated groups, CoD consuming groups and DMBA 47 48 treated groups with CoD TM consumption. These setups were measured at five time intervals 49 50 51 after the first DMBA treatment: 1 week, 1 month, 3, 6 and 12 months later. 52 53 Control groups were given tap water and treated with ip. corn oil. DMBA treated groups 54 55 received 20 mg/bwkg DMBA . (Sigma Aldrich Budapest, Hungary) dissolved in corn oil 56 57 58 (Teva Pharmaceutical Industries Ltd. Debrecen, Hungary) intraperitoneally. DMBA treatment 59 60 was applied at the beginning of the experiment and in addition two months later.

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1 2 3 Gene expression investigations. 4 5 6 One week, one, three, six and twelve months later the actual four groups were sacrified and 7 8 their liver, lungs, kidneys and spleen were removed. 100 mg tissue samples from each organ 9 10 were collected, and samples of one group were pooled. 11 12 13 After homogenization of the organs, total cellular RNA was isolated using TRIZOL reagent 14 15 (Invitrogen, Paisley, UK). The RNA quality was checked by denaturing gel-electrophoresis, 16 17 18 and absorption measurement was performed at 260/280 nm (A260/A280 was over 1.8). After 19 20 the necessary dilution,For 10 µg RNA Peer was dot-blotted Review onto Hybond N + nitrocellulose membrane 21 22 23 (ECL kit, Amersham, Little Chalfont, UK) and hybridized with chemiluminescently labelled 24 25 specific probes for c-myc , Bcl-2, K-ras , p53 and Ha-ras (Professor J. Szeberényi, University 26 27 of Pécs, Hungary) genes. Isolation of RNA, hybridization and detection were performed 28 29 30 according to the instructions of the manufacturer. The membranes were rehybridized with the 31 32 constitutively expressed beta-actin gene as a positive control. The chemiluminescent signals 33 34 35 were detected on X-ray films, scanned into a computer and evaluated by Quantiscan software 36 37 (Biosoft, Cambridge, UK). The results were expressed in the percentage of the positive 38 39 control. (Data are shown in Figure 1-8.) 40 41 42 43 44 Results 45 46 In this study we investigated the level of certain onco/suppressor gene expressions. We found, 47 48 49 that in the control group basal c-myc , Ha-ras, p53, Bcl-2 and K-ras gene expressions vary 50 51 both in time and in different organs. In the liver of male mice gene expression was low 52 53 comparing to the beta-actin reference with a slight fluctuation ( Figure 1. ), in the lungs and 54 55 56 kidneys ( Figure 3, 5. ) key onco/suppressor expression was higher with the same reference. 57 58 Compared to the results of male mice (Figure 1, 3, 5, 7.) we found, that in female mice 59 60 (Figure 2, 4, 6, 8.) gene expressions were slightly lower nearly in every organ. Gene

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1 2 3 expression patterns of the liver were nearly the same (Figure 1, 2.) , but in the lungs and in the 4 5 6 kidneys expression was lower in female mice (Figure 4, 6.) . 7 8 The effect of DMBA treatment: 9 10 DMBA caused the overexpression of the investigated key onco/suppressor genes. Male mice 11 12 13 showed major elevations, especially in the lungs, kidneys and spleen (Figure 3, 5, 7.) . In the 14 15 lungs and kidneys these overexpressions decayed after one month but in the spleen key gene 16 17 18 expressions remained high. 19 20 In the liver the key geneFor expression Peer elevations wereReview present - they were not as high as in other 21 22 organs (Figure 1.) . Moreover, at the 12 th month control major elevations were present. In the 23 24 25 lungs, spleen and kidneys key gene expression elevations were detected. 26 27 In male mice highly increased key gene expression levels were measured in the lungs, 28 29 kidneys and spleen (Figure 3, 5, 7.) . In female mice similar - but milder - gene expressions 30 31 32 elevations were detected in the organs mentioned above (Figure 4, 6, 8.) . 33 34 In female mice major gene expression elevations were present at the 12 th month in every 35 36 organ (Figure 2, 4, 6, 8.) . 37 38 TM 39 The effect of CoD on DMBA treatment: 40 41 We compared the results of CoD TM tea consumption on DMBA treated mice to the DMBA 42 43 44 induced overexpressed cases (positive control). In case of the DMBA we arbitrary chose more 45 TM 46 than 100% gene expression increase as overexpression, and in case of the CoD treatment 47 48 we chose more than 100% key onco/suppressor gene expression decrease as major decrease in 49 50 51 comparison to the positive control. 52 53 In Table 1. (male) and Table 2. (female) we highlighted the organs - and also the 54 55 overexpressed genes - where major decrease were present. 56 57 TM 58 Comparing to the DMBA treated group, COD tea consumption decreased onco/suppressor 59 60 gene expression in the following cases: checking the one week control in the kidneys all of

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1 2 3 the key onco/suppressor gene levels decreased (Figure 5, 6.) . In the lungs c-myc , Ha-ras , p53 , 4 5 6 K-ras showed major decrease (Figure 3, 4.) and in the spleen c-myc , Ha-ras and K-ras 7 8 declined at the same time point (Figure 7, 8.) . In case of liver, the kidneys and spleen at one 9 10 year, results showed decreasing of c-myc and p53 expression (Figure 1, 2, 5, 6, 7, 8.) . Ha-ras 11 12 13 also decreased in the lungs, the kidneys and spleen (Figure 3, 4, 5, 6, 7, 8.) . There was 14 15 decrease of Bcl-2 in liver and the kidneys (Figure 1, 2, 5, 6.) and K-ras in liver, the lungs and 16 17 18 spleen (Figure 1, 2, 3, 4, 7, 8.) . 19 20 In female mice we foundFor fewer Peer differences Review than in male mice, the definite decrease of 21 22 onco/suppressor gene expressions are shown in Table 2. 23 24 25 We highlight the kidney (Figure 6.) at the one week control, in which c-myc, Ha-ras, p53 26 27 and K-ras onco/suppressor gene expression levels were decreased. In the lungs, kidneys and 28 29 spleen almost every onco/suppressor gene expression was dropped at the 12 th month (Figure 30 31 32 4, 6, 8.) . 33 34 35 36 Discussion 37 38 39 The increase of c-myc , Bcl-2, K-ras , Ha-ras and p53 gene expression indicates early 40 41 carcinogenic effects (Gyöngyi and Somlyai, 2002 ) and their overexpression is characteristic 42 43 44 in certain manifest tumours. The amplification and overexpression of c-myc gene was found 45 46 to exert an initiator role in human and in mice breast carcinogenesis as well (Dickinson et al. , 47 48 1992 ). The activation and overexpression of Ha-ras protooncogene through point mutations 49 50 51 was detected in colorectal carcinomas, as well (Csontos et al. , 2008 ). Moreover Bcl-2 is an 52 53 antiapoptotic gene, which is capable of causing immortalisation of tumour cells, especially if 54 55 it is overexpressed (Norton and Atherton, 1998 ). The overexpression and activation of K-ras 56 57 58 contribute to the formation of metastasis (Smakman et al. , 2005 ) and enhances initiation and 59 60 progression as well.

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1 2 3 The carcinogenic effect of DMBA in the lungs is well known (Stowers and Anderson, 1985 ), 4 5 6 and correlates to our finding, because due to the treatment, all of the examined genes had 7 8 increased expression in comparison to the control in both sexes. According to the gene 9 10 expression patterns, CoD TM tea consumption in the lungs of male group seems to exert 11 12 13 tumour formation inhibitory effect, usually as the results of several anticancer effects, such as 14 15 anti-inflammatory, antioxidant (Heitzman et al. , 2005 ) and DNA topoisomerase II partial 16 17 18 inhibitory (Esteves-Souza et al. , 2007 ) effects, as well. 19 20 Complete (both initiatorFor and promoter) Peer chemical Review carcinogens activate and overexpress Ha-ras 21 22 proto-oncogene (Dickinson et al. , 1992; Perjési et al. , 2006 ), so the Ha-ras overexpression 23 24 TM 25 caused by DMBA correlates to these facts. Furthermore, CoD tea consumption decreased 26 27 the overexpression of onco/suppressor genes induced by DMBA in the kidneys of male mice, 28 29 presumably due to the result of antitumour properties of lapachone (Ueda et al. , 1994 ) and 30 31 32 inhibitory effects of POA on cancer cell proliferation (Lee et al. , 2000 ). 33 34 Generally speaking, in male animals CoD TM tea consumption seemed to exert higher 35 36 chemopreventive effect on carcinogen increased onco/suppressor gene expression, than in 37 38 39 female groups. This could be explained with the noncompetitive, concentration-dependent 40 41 oestrogen receptor binding property, and therefore oestrogen inhibitory effect of U. 42 43 44 tomentosa 's. But -according to the litarature- in long-term experiment, tamoxifen like paradox 45 46 oestrogen enhancer effects were found (Salazar and Jayme, 1998 ). 47 48 Oestrogen receptor beta (ERbeha) is supposed to be effected ( Bukulmez et al. , 2008 ) by the 49 50 51 oestrogen receptor activating effect of Uncaria extracts. 52 53 Anti-inflammatory activity of Uncaria extracts (Heitzman et al. , 2005 ) is able to silence 54 55 COX-2, thereby aromatase expression as well (Sirianni et al. , 2009 ), leading to oestrogen 56 57 58 level decreasing. Oestrogen level decreasing might slow the proliferation of certain sensitive 59 60 cells ( Sirianni et al. , 2009 ). However, oestrogen increased the expression of Ha-ras - and

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1 2 3 consequently other oncogenes (Sándor et al. , 1995 ) - in endometrial cancer cells via 4 5 6 increasing tyrosine kinase (TK) activity ( Fujimoto et al ., 1995 ). Aromatase synthetize 7 8 oestrogen in males dominantly, thus the inhibition of it underpins the higher efficacy of 9 10 CoD TM tea in male groups, than in observed in female groups. 11 12 13 According to the expression patterns of the examined molecular epidemiological key 14 15 onco/suppressor biomarker genes, in this long-term experiment the chemopreventive effect of 16 17 TM 18 CoD tea consumption could be confirmed. 19 20 For Peer Review 21 22 Acknowledgements 23 24 25 The authors express their special thanks to Zsuzsanna Bayer and Mónika Herczeg for their 26 27 valuable technical assistance. 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 References 5 6 7 8 Bukulmez O, Hardy DB, Carr BR, Word RA, Mendelson CR. 2008 Inflammatory status 9 influences aromatase and steroid receptor expression in endometriosis. Endocrinology. 10 149(3): 1190-204. 11 12 Csapi B, Csupor D, Veres K, Szendrei K, Hohmann J. 2008. Phythochemical analysis of 13 14 Uncaria products commercially available in Hungary. Planta Medica 74: 1100. 15 16 Csontos Z, Nádasi E, Csejtey A, Illényi L, Kassai M, Lukács L, Kelemen D, Kvarda A, 17 Zólyomi A, Horváth OP, Ember I. 2008. Oncogene and tumor suppressor gene expression 18 changes in the peripheral blood leukocytes of patients with colorectal cancer. Tumori 94(1): 19 79-82. 20 For Peer Review 21 Dickson RB, Salomon DS and Lippman ME. 1992. Tyrosine kinase receptor--nuclear 22 23 protooncogene interactions in breast cancer. Cancer Treat Res. 61: 249-273. 24 25 Ember I, Kiss I and Pusztai Z. 1998. Effect of 7,12-dimethylbenz[a]anthracene on 26 onco/suppressor gene action in vivo : a short-term experiment. Anticancer Res. 18: 445-447. 27 28 Ember I, Varga Cs, Herceg L, Raposa T. 1992. Szekunder tumorok állatkísérletes modellje. In 29 vivo citosztatikus kezelési protokollok karcinogén-leukemogén hatása. Magyar Onkológia 46: 30 31 87-97. [Article in Hungarian] 32 33 Esteves-Souza A, Figueiredo DV, Esteves A, Câmara CA, Vargas MD, Pinto AC, Echevarria 34 A. 2007. Cytotoxic and DNA-topoisomerase effects of lapachol amine derivatives and 35 interactions with DNA. Braz J Med Biol Res 40(10): 1399-1402. 36 37 Fujimoto J, Ichigo S, Hori M, Morishita S, Tamaya T. 1995. Estrogen induces c-Ha-ras 38 39 expression via activation of tyrosine kinase in uterine endometrial fibroblasts and cancer cells. 40 J Steroid Biochem Mol Biol. 55(1): 25-33. 41 42 Gyöngyi Z and Somlyai G. 2000. Deuterium depletion can decrease the expression of C-myc 43 Ha-ras and p53 genes in carcinogen-treated mice. In Vivo 3: 437-439. 44 45 Gyöngyi Z, Grama L, Nádasi E, Sándor J, Németh A, Varga C, Kiss I and Ember I. 2002. 46 47 Flow cytometric analysis of DMBA-induced early in vivo ras expression. In Vivo 16: 323- 48 326. 49 50 Heitzman ME, Neto CC, Winiarz E, Vaisberg AJ, Hammond GB. 2005. Ethnobotany, 51 phytochemistry and pharmacology of Uncaria (Rubiaceae). Phytochemistry 66(1): 5-29. 52 53 Keplinger K, Laus G, Wurm M, Dierich MP, Teppner H. 1999. Uncaria tomentosa (Willd.) 54 55 DC. – Ethnomedicinal use and new pharmacological, toxicological and botanical results. J 56 Ethnopharmacol 64: 23-34. 57 58 Laus G, Brossner D, Keplinger K. 1997. Alkaloids of Peruvian Uncaria tomentosa. 59 Phytochemistry 45: 855-860. 60

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1 2 3 Lee JS, Kim J, Kim BY, Lee HS, Ahn JS, Chang YS. 2000. Inhibition of phospholipase 4 5 cgamma1 and cancer cell proliferation by triterpene esters from Uncaria rhynchophylla. J Nat 6 Prod. 63(6): 753-756. 7 8 Müller K, Sellmer A, Wiegrebe W. 1999. Potential antipsoriatic agents: lapacho compounds 9 as potent inhibitors of HaCaT cell growth. J Nat Prod. 62(8): 1134-1136. 10 11 Norton JD, Atherton GT. 1998. Coupling of cell growth control and apoptosis functions of Id 12 proteins. Mol Cell Biol. (4): 2371-2381. 13 14 TM 15 Orsós Zs, Nádasi E, Dávid T, Ember I, Kiss I. 2007. Effect of CoD tea consumption in a 16 „short-term” test system on the expression of onco- and tumor suppressor genes. 17 Egészségtudomány 50: 195-107. [Article in Hungarian] 18 19 Perjési P, Ember I, Bozak RE, Nádasi E, Rozmer Z, Varjas T, Hicks RJ. 2006. Effect of the 20 chalcone analog For E,E-bis(2-hydroxybenzylidene) Peer Review acetone on the 7,12- 21 22 dimethylbenz[a]anthracene-induced Ha-ras gene activity in vivo. In Vivo 20(1): 141-146. 23 24 Salazar EL, Jayme V. 1998. Depletion of specific binding sites for estrogen receptor by 25 Uncaria tomentosa. Proc West Pharmacol Soc. 41: 123-124. 26 27 de Santana CF, De Lima O, D'albuquerque IL, Lacerda AL, Martins DG. 1968. Antitumoral 28 and toxicological properties of extracts of bark and various wood components of Pau d'arco 29 30 (Tabebuia avellanedae ). Rev Inst Antibiot 8: 89-94. 31 32 Sándor J, Ambrus T and Ember I. 1995. The function of the p53 gene suppressor in 33 carcinogenesis. Orv Hetil. 35: 1875-1883. [Article in Hungarian] 34 35 Sirianni R, Chimento A, De Luca A, Zolea F, Carpino A, Rago V, Maggiolini M, Andò S, 36 Pezzi V. 2009. Inhibition of cyclooxygenase-2 down-regulates aromatase activity and 37 38 decreases proliferation of Leydig tumor cells. J Biol Chem. 284(42):28905-16. 39 40 Smakman N, Borel Rinkes IH, Voest EE, Kranenburg O. 2005. Control of colorectal 41 metastasis formation by K-Ras. Biochim Biophys Acta 1756(2): 103-114. 42 43 Stowers SJ, Anderson MW. 1985. Formation and persistence of benzo(a)pyrene metabolite- 44 DNA adducts. Environ Health Perspect. 62: 31-9. 45 46 47 Ueda S, Umemura T, Dohguchi K, Matsuzaki T, Tokuda H, Nishino H, Iwashima A. 1994. 48 Production of anti-tumour-promoting furanonaphthoquinones in Tabebuia avellanedae cell 49 cultures. Phytochemistry 36: 323-325. 50 51 Varga Cs, Ember I, Raposa T. 1991. Comparative studies on genotoxic and carcinogenic 52 effects of different cytostatic protocols. I. In vivo cytogenetic analyses in CBA mice. Cancer 53 54 Letters 60: 199-203. 55 56 57 58 59 60

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1 2 3 4 5 Liver Lungs Spleen Kidneys 6 7 c-myc, 8 c-myc, Ha-ras, 9 1 week Ha-ras, x x p53, 10 p53, Bcl-2, 11 K-ras 12 K-ras 13 c-myc, 14 1 month c-myc K-ras K-ras p53, 15 K-ras 16 c-myc, 17 Ha-ras, c-myc, 18 c-myc x 3 months p53, Ha-ras, 19 20 For PeerBcl-2, ReviewBcl-2, 21 K-ras 22 Ha-ras, 23 6 months x x x 24 p53 25 c-myc, c-myc, c-myc, c-myc, 26 Ha-ras, 27 12 Ha-ras, Ha-ras, Ha-ras, p53, 28 p53, p53, p53, months Bcl-2, 29 K-ras K-ras Bcl-2 30 K-ras 31 32 Table 1. The effect of CoD TM on key onco/suppressor genes in male CBA/Ca mice 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 Liver Lungs Spleen Kidneys 6 7 c-myc, Ha-ras, 8 p53, Ha-ras, 1 week p53, x 9 K-ras p53, K-ras 10 K-ras 11 Ha-ras, 12 13 1 month x Bcl-2, x Bcl-2 14 K-ras 15 3 months p53 x x x 16 17 6 months x x x Bcl-2 18 19 c-myc, c-myc, c-myc, c-myc, p53, Ha-ras, Ha-ras, Ha-ras, 20 12 months For Peer Review 21 Bcl-2, p53, p53, p53, 22 K-ras K-ras Bcl-2 Bcl-2 23 24 TM 25 Table 2. The effect of CoD on key onco/suppressor genes in female CBA/Ca mice 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 100 5 control DMBA COD tea COD + DMBA 6 7 8 80 9 10 11 12 60 13 14 15 16 40 17 18 19 20 20 For Peer Review 21 22 23 0 24 p53 p53 p53 p53 p53 K-ras K-ras K-ras K-ras K-ras BCL2 BCL2 BCL2 BCL2 25 BCL2 c-myc c-myc c-myc c-myc c-myc Ha-ras Ha-ras Ha-ras Ha-ras Ha-ras 26 1 week 1 month 3 month 6 month 12 month 27

28 29 30 Figure 1. The gene expression profile of liver of male CBA/Ca mice 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3

4 100 5 6 control DMBA COD tea COD + DMBA 7 8 80 9 10 11 12 60 13 14 15 16 40 17 18 19 20 20 For Peer Review 21 22 23 0 24 p53 p53 p53 p53 p53 K-ras K-ras K-ras K-ras K-ras BCL2 BCL2 BCL2 BCL2 BCL2 c-myc c-myc c-myc c-myc c-myc Ha-ras Ha-ras Ha-ras Ha-ras 25 Ha-ras 26 1 week 1 month 3 month 6 month 12 month 27

28 Figure 2. The gene expression profile of liver of female CBA/Ca mice 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 100 5 control DMBA COD tea COD + DMBA 6 7 8 80 9 10 11 12 60 13 14 15 16 40 17 18 19 20 20 For Peer Review 21 22 23 0 24 p53 p53 p53 p53 p53

25 K-ras K-ras K-ras K-ras K-ras BCL2 BCL2 BCL2 BCL2 BCL2 c-myc c-myc c-myc c-myc c-myc 26 Ha-ras Ha-ras Ha-ras Ha-ras Ha-ras 1 week 1 month 3 month 6 month 12 month 27 28 29 30 Figure 3. The gene expression profile of the lungs of male CBA/Ca mice 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 100 6 7 control DMBA COD tea COD + DMBA 8 9 80 10 11 12 13 60 14 15 16 17 40 18 19 20 For Peer Review 21 20 22 23 24 25 0 p53 p53 p53 p53 26 p53 K-ras K-ras K-ras K-ras K-ras BCL2 BCL2 BCL2 BCL2 BCL2 c-myc c-myc c-myc c-myc c-myc Ha-ras Ha-ras Ha-ras Ha-ras 27 Ha-ras 28 1 week 1 month 3 month 6 month 12 month 29 30 Figure 4. The gene expression profile of the lungs of female CBA/Ca mice 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 100 6 7 control DMBA COD tea COD + DMBA 8 9 80 10 11 12 13 60 14 15 16 17 40 18 19 20 For Peer Review 21 20 22 23 24 25 0 p53 p53 p53 p53 26 p53 K-ras K-ras K-ras K-ras K-ras BCL2 BCL2 BCL2 BCL2 BCL2 c-myc c-myc c-myc c-myc c-myc Ha-ras Ha-ras Ha-ras Ha-ras 27 Ha-ras 28 1 week 1 month 3 month 6 month 12 month 29 30 Figure 5. The gene expression profile of the kidneys of male CBA/Ca mice 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 100 6 7 control DMBA COD tea COD + DMBA 8 9 80 10 11 12 13 60 14 15 16 17 40 18 19 20 For Peer Review 21 20 22 23 24 25 0 p53 p53 p53 p53 26 p53 K-ras K-ras K-ras K-ras K-ras BCL2 BCL2 BCL2 BCL2 BCL2 c-myc c-myc c-myc c-myc c-myc Ha-ras Ha-ras Ha-ras Ha-ras 27 Ha-ras 28 1 week 1 month 3 month 6 month 12 month 29 30 Figure 6. The gene expression profile of the kidneys of female CBA/Ca mice 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 100 5 control DMBA COD tea COD + DMBA 6 7 8 80 9 10 11 12 60 13 14 15 16 40 17 18 19 20 20 For Peer Review 21 22 23 24 0 p53 p53 p53 p53 p53

25 K-ras K-ras K-ras K-ras K-ras BCL2 BCL2 BCL2 BCL2 BCL2 c-myc c-myc c-myc c-myc c-myc 26 Ha-ras Ha-ras Ha-ras Ha-ras Ha-ras 1 week 1 month 3 month 6 month 12 month 27 28 29 Figure 7. The gene expression profile of spleen of male CBA/Ca mice 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 100 5 control DMBA COD tea COD + DMBA 6 7 8 80 9 10 11 12 60 13 14 15 16 40 17 18 19 20 20 For Peer Review 21 22 23 24 0 p53 p53 p53 p53 p53 K-ras K-ras K-ras K-ras 25 K-ras BCL2 BCL2 BCL2 BCL2 BCL2 c-myc c-myc c-myc c-myc c-myc Ha-ras Ha-ras Ha-ras Ha-ras 26 Ha-ras 1 week 1 month 3 month 6 month 12 month 27 28 29 30 Figure 8. The gene expression profile of spleen of female CBA/Ca mice 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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