Pfaffia paniculata (Brazilian ginseng) roots decrease proliferation and increase apoptosis but do not affect cell communication in murine hepatocarcinogenesis

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Experimental and Toxicologic Pathology 62 (2010) 145–155 www.elsevier.de/etp

Pfaffia paniculata (Brazilian ginseng) roots decrease proliferation and increase apoptosis but do not affect cell communication in murine hepatocarcinogenesis Tereza Cristina da Silvaa,, Bruno Cogliatia, Ana Paula da Silvaa, Heidge Fukumasua, Gokithi Akisueb, Ma´rcia Kazumi Nagaminea, Patrı´cia Matsuzakia, Mitsue Haraguchic, Silvana Lima Go´rniaka, Maria Lu´cia Zaidan Daglia a

Department of Pathology, School of Veterinary Medicine and Animal Sciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil School of Pindamonhangaba, Pindamonhangaba, SP, Brazil c Section of Pharmacology, Division of Animal Biology, Biological Institute of Sa˜o Paulo, Sa˜o Paulo, Brazil b

Received 2 December 2008; accepted 17 March 2009

Abstract Pfaffia paniculata (Brazilian ginseng) roots and/or its extracts have shown anti-neoplastic, chemopreventive, and anti-angiogenic properties. The aim of this work was to investigate the chemopreventive mechanisms of this root in mice submitted to the infant model of hepatocarcinogenesis, evaluating the effects on cellular proliferation, apoptosis, and intercellular communication. Fifteen-day-old BALB/c male mice were given, i.p., 10 mg/g of the carcinogen N-nitrosodiethylamine (DEN). Animals were separated into three groups at weaning and were given different concentrations of powdered P. paniculata root (0%, 2%, or 10%) added to commercial food for 27 weeks. Control group (CT) was not exposed to the carcinogen and was given ration without the root. After euthanasia, the animals’ liver and body weight were measured. Liver fragments were sampled to study intercellular communication, molecular biology, and histopathological analysis. Cellular proliferation was evaluated by immunohistochemistry for PCNA, apoptosis was evaluated by apoptotic bodies count and alkaline comet technique, and intercellular communication by diffusion of lucifer yellow dye, immunofluorescence, western blot and real-time PCR for connexins 26 and 32. Chronic treatment with powdered P. paniculata root reduced cellular proliferation and increased apoptosis in the 2% group. Animals in the 10% group had an increase in apoptosis with chronic inflammatory process. Intercellular communication showed no alterations in any of the groups analyzed. These results indicate that chemopreventive effects of P. paniculata are related to the control of cellular proliferation and apoptosis, but not to cell communication and/or connexin expression, and are directly influenced by the root concentration. r 2009 Elsevier GmbH. All rights reserved. Keywords: Cancer; Hepatocarcinogenesis; Chemoprevention; Proliferation; Apoptosis; Saponin; Phytochemicals; Medicinal plants; Brazilian ginseng; Pfaffia paniculata

Corresponding author at: Departamento de Patologia, Faculdade de Medicina Veterina´ria e Zootecnia-USP, Av. Prof. Dr. Orlando Marques de Paiva, 87, CEP 05508-900 Sa˜o Paulo, SP, Brazil. Tel.: +55 11 3091 7705; fax: +55 11 3091 7829. E-mail address: [email protected] (T.C. da Silva).

0940-2993/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2009.03.003

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Introduction Anti-cancer drugs act by various mechanisms, among them inhibition of proliferation, modulation of apoptosis, and protection of intercellular communication. These mechanisms could act as factors in the primary prevention of cancer, allowing drugs with multiple antitumor activities to be more effective in its control (De Flora, 1998; Colic and Pavelic, 2000; De Flora and Ferguson, 2005). In this context, Kelloff et al. (2000) related several studies demonstrating that anti-cancer agents from the diet can prevent the emergence of a tumor by distinct mechanisms. For instance, triterpenoid saponins from different species of plants can fight the development of neoplasms by inducing apoptosis, control of cellular proliferation (Mujoo et al., 2001; Li et al., 2005; Lemeshko et al., 2006), and positive regulation of intercellular communication (Kang et al., 2000). Apoptosis has a crucial role in cellular development, controlling the number of cells, eliminating abnormal and non-functional cells, and preventing tumor development (Thompson, 1995; Jacobson et al., 1997). Intercellular communications by gap junctions allow maintenance of tissue homeostasis. These junctions are channels formed by protein subunits known as connexins, which belong to a family of at least 20 members that is distributed among different cell types (Willecke et al., 2002). Most neoplastic cells present altered levels of connexin expression and reduction of communication by gap junctions (Yamasaki et al., 1999). These alterations are related with the increase of cellular proliferation (Budunova and Williams, 1994; Guan et al., 1995). Pfaffia paniculata (Brazilian ginseng) belongs to the Amaranthaceae family and studies have shown it has several properties, among them, antitumoral. The main isolated components of its roots are stigmasterol, sitosterol, allantoin, pfaffic acid, and its triterpenoid saponins denominated pfaffosides A, B, C, D, E, and F (Nishimoto et al., 1984). Matsuzaki et al. (2003) showed that administration of powdered root to mice by gavage reduced growth of ascitic Ehrlich tumor. da Silva et al. (2005) observed that chronic treatment with P. paniculata root added to ration of mice induced to hepatocarcinogenesis model reduced the incidence, number and area of preneoplastic lesions, indicating an inhibitory effect on steps of promotion and progression of hepatocarcinogenesis. Other studies in mice showed that administration by gavage of methanolic extract reduced neovascularization in mice cornea (Carneiro et al., 2007) and increased phagocytic activity of macrophages on ascitic form of Ehrlich tumor (Pinello et al., 2006). Also, administration of butanolic extract had anti-neoplastic action in Ehrlich tumor (Matsuzaki et al., 2006) and showed cytotoxic activity

on MCF-7 cells (Nagamine et al., 2009). Thus, the aim of this work was to investigate the chemopreventive mechanisms of powdered P. paniculata root in the diet of mice induced to hepatocarcinogenesis model, evaluating the effects on cellular proliferation, apoptosis and intercellular communication.

Materials and methods Animals Seventy male BALB/c mice from the Animal Facility colony of the Department of Pathology of the University of Sa˜o Paulo, School of Veterinary Medicine and Animal Science, Brazil, were employed throughout the experiment. Animals were housed in polycarbonate cages (4  3  5), in a room with 12 h day–night cycle, temperature of 2272 1C and relative humidity of 45–60%. All animals were cared for according to the international criteria for use of laboratory animals; the experimental procedure was approved by Bioethics Committee of our institution (protocol no. 83/02).

Plant material The powdered root of P. paniculata was kindly provided by Dr. Gokithi Akisue. A specimen of this plant was confirmed by the identification of its characteristic flowers and leaves deposited in the Goro Hashimoto (Sa˜o Paulo, Brazil) herbarium (no. 37.411).

Experimental diet The experimental diet was prepared in the Research Center for Veterinary Toxicology, CEPTOX, of School of Veterinary Medicine and Animal Science of the University of Sa˜o Paulo, Pirassununga, Brazil. The standard commercial diet (NUVILAB-CR1, Nuvital Nutrientes LTDA, Brazil) was ground, mixed with P. paniculata powdered root at 0%, 2%, and 10% of weight, re-pelletized and moistened, drying at room temperature. The control group (CT) diet received the same treatment without the powered root. Drinking water and food were supplied ad libitum.

Experimental protocol The modified infant mouse hepatocarcinogenesis model of Vesselinovitch and Mihailovich (1983) was used according to research previously reported in da Silva et al. (2005). The male 15-day-old BALB/c mice received, i.p., 10 mg/g of body weight of N-nitrosodiethylamine (DEN) (Sigma Chemical Co., St. Louis, MO, N-0756) carcinogen in physiological saline (0.9%).

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After weaning, animals were separated into three groups (n ¼ 20 animals/group) and received different concentrations of the powdered P. paniculata root (0%, 2%, or 10%) added to their diet, during 27 weeks. The CT group (n ¼ 10) was not induced to the carcinogen and received a diet without the root. After euthanasia, animals and livers were measured for relative weight calculation, and a part of the left lobe was removed for estimation of intercellular communication. Mouse livers were sampled according to Nolte et al. (2005). Representative slices from right, left, median, and caudate lobes were fixed in methacarn solution (60% methanol, 30% chloroform, and 10% acetic acid) for 24 h and routinely processed and embedded in paraffin wax. One histological section of each liver lobe of each mouse was stained with hematoxylin–eosin (H&E) for histopathological examination and immunohistochemical staining. The remaining liver fragments were frozen in liquid nitrogen and stocked at 80 1C for posterior analysis with western blot, real-time PCR and comet techniques.

PCNA immunohistochemistry The effects of the treatment on cell proliferation were evaluated by PCNA immunohistochemistry. Silanized slides from liver tissue were submitted to antigen retrieval with borate buffer 0.2 M, pH 7.2, in microwave for 15 min; endogenous peroxidase was blocked with 3% hydrogen peroxide solution in methanol for 30 min. Slides were immersed in 5% skimmed milk for 10 min at 37 1C to block non-specific reactions and incubated overnight with anti-PCNA antibody (DAKO A/S, Denmark), 1:800. Then the slides were incubated with LSAB+System-HRP (Dako, Carpinteria, CA 93013, USA) kit, diaminobenzidine (DAB+Chromogen, Dako Carpinteria, CA 93013, USA) developed and counterstained with hematoxylin. Evaluation consisted in measuring the area of the three lobes and counting all PCNA-positive nuclei of hepatocytes. The areas of the three lobes were totaled and the PCNA-positive cell numbers was calculated per mm2 of tissue. The image analysis system Image Pro-Plus – version 4.5 was used for this analysis.

Apoptosis evaluation Hepatic apoptotic bodies (AB) were quantified by fluorescence microscopy as described by Stinchcombe et al. (1995) using a Nikon Eclipse E-800 microscope (Tokyo, Japan) equipped with an epifluorescence unit. This methodology is based on the property of strong eosin fluorescence of AB in H&E-stained liver tissues submitted to blue light (450–490 nm) (Stinchcombe et al., 1995; Wood et al., 1999; Ong et al., 2006).

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Identification of AB was confirmed by switching the microscope system to transmitted light and using morphological criteria established by Goldsworthy et al. (1996). AB were represented by acidophilic bodies with fragmentation or lack of chromatin accompanied by cytoplasmatic condensation and/or fragmentation. The evaluation was similar to PCNA immunohistochemistry, the three lobes area was measured and all apoptotic bodies counting. If single cells or clusters of directly neighboring cells contained multiple AB, these were assumed to be derived from the same apoptotic cell and were counted as only one event. The areas of three lobes were added and the number of apoptotic bodies was expressed per mm2 of tissue area (Stinchcombe et al., 1995). This analysis was assisted by the image analysis system Image Pro-Plus – version 4.5.

Alkaline comet assay The comet assay (or microgel electrophoresis technique) aims to estimate and detect damage in DNA single bands and is a sensitive method for identification of cellular apoptosis (Wada et al., 2003). The method described by Singh et al. (1988) with modifications (Hartmann et al., 2003) was employed. Approximately, 50 mg of frozen liver samples were homogenized in PBS (pH 7.4, 4 1C) with a tissue grinder (Dounce Tissue Grinder-Wheaton Millville, NJ, USA). Cell nuclei were preserved in the suspension and were diluted in 0.8% low melting point agarose (LMP – Sigma Chemical Company, St. Louis, USA). This mixture was added to microscope slides pre-coated with 0.5% of LMP agarose, and covered with a coverslip for 10 min at 4 1C. The coverslips were removed and the slides were then immersed in a lyses solution (2.5 M NaCl; 100 mM EDTA; 10 mM Trizma Base; 1% n-lauryl sarcosine, 1 mL Triton X-100, pH 10), for 1 h at 4 1C. The slides were washed in distilled water three times for 15 min each at 4 1C, and transferred to electrophoresis cube for DNA unwinding, for 20 min. The electrophoresis was performed in 25 V 350 mA, for 40 min at 4 1C, in a dark room in the electrophoresis alkaline buffer (300 mM NaOH, 1 mM EDTA, pH413, 74 1C). Slides were neutralized in a 0.4 M pH 5.0 Tris solution and dried vertically overnight at room temperature. Slides were then fixed in a specific solution (trichloroacetic acid, 15%, ZnSO4, 5%, glycerine, 4%, 4 1C), for 15 min; immersed three times, 1 min each, in distilled water baths, and dried vertically at room temperature. Then slides were stained in a solution mix: 32 mL solution A (50 g Na2CO3, 1 L distilled water) and 68 mL solution B (0.2 g NH4NO3, 0.2 g AgNO3, 1 g silicotungstic acid, 500 mL formaldehyde, 1 L distilled water) for 15 min. After that, slides were washed in distilled water for 1 min, and for 5 min in finished

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solution (acetic acid 1%), again in distilled water for 1 min and then dried vertically at room temperature. The apoptosis analysis was performed according to Wada et al. (2003) with some alterations. Briefly, 500 comets per mouse were analyzed with the analysis system Image Pro-Plus, version 4.5 and the percentage corresponding to the comet formed by the apoptotic cell was determined.

Lucifer yellow dye transfer procedure The protocol described by Sai et al. (2000) was used with modifications. Immediately after euthanasia, a part of the left hepatic lobe was excised. Three incisions were made on the surface, and a mixture of fluorescent dyes containing 0.5% lucifer yellow and 5% rhodamine–dextran in phosphate-buffered saline was dropped into the tissue surface. The dye mixture remained for 3 min, and after incubation the tissue was washed with phosphatebuffered saline three times and fixed in 10% buffered formalin for 24 h. The tissue was routinely processed and embedded in paraffin wax. The 5 mm sections were de-paraffined and were observed under fluorescence microscope. The communication between the hepatocytes was evaluated by the ratio between lucifer yellow dye diffusion area and length of incision. The mean of three incisions was considered as the data from one animal. The lucifer yellow dye transfer was analyzed in the analysis system Image Pro-Plus, version 4.5.

Connexin 32 immunofluorescence Connexins are functional when they are located in the cytoplasmic membrane; therefore, the connexin immunofluorescence aims to identify its location within the cell. The slides were incubated with polyclonal anticonnexin 32 antibody (Zymed Laboratories, San Francisco, CA 94080, USA), 1:100, at 4 1C, overnight. Slides were then incubated with biotinylated anti-rabbit secondary antibody (Dako, Carpinteria, CA 93013, USA), and afterwards were incubated with estreptavidin–fluorescein–FITC complex (DAKO A/S, Denmark). The Vectashield (Vector Laboratories, Inc., Burlingame, CA, 94010, USA) was used to prevent lost of fluorescence. Slides were observed in florescence microscope Nikon E-800.

Connexin 26 and 32 western blot In order to evaluate the expression of hepatic connexin 26 or 32, total protein was extracted from frozen fragments (40 mg). Total protein concentration was quantified with Bio-Rad protein. The protein extracts (150 mg) were subjected to electrophoresis in a 13% polyacrylamide gel in sodium dodecyl sulfate,

100 V, for 3 h. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA), by electrophoresis in a transfer buffer (Tris base, glycine, methanol, 10% sodium dodecyl sulfate, distilled water), under semi-dry conditions for 38 min with 47 mA. Saturation of non-specific sites was performed by incubating the membrane with 5% skimmed milk for 2 h at room temperature. The membrane was then incubated overnight with rabbit polyclonal anti-connexin 26 or 32 primary antibody (1:500 in 5% skimmed milk; Zymed Laboratories, San Francisco, CA, USA). A new membrane incubation was then carried out with peroxidase-labeled secondary antibody (1:500 in 5% skimmed milk; Zymed Laboratories, San Francisco, CA, USA), for 2 h at room temperature. The membrane was developed in a solution containing diaminobenzidine (DAB, Sigma Aldrich), nickel sulfate and hydrogen peroxide; then scanned (Epson perfection 1660 Photo, Nagano Ken, Japan). The Image Master (Amersham Pharmacia Biotech, Little Chalfont, UK) system was used to analyze the scoring intensity. The connexin 26 or 32 scoring intensity was analyzed by comparing the band volume with beta-actin endogenous control.

Connexin 26 or 32 real-time PCR Total RNA was extracted from the tissues with TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturers’ protocol. Five samples were used for each group. Quality of RNA samples was determined by electrophoresis in 1.5% agarose gel and staining with ethidium bromide. The 18S and 28S bands were visualized under UV light. Total RNA was treated with DNase I (Invitrogen Life Technologies). Total RNA was added with 1 mL of oligo(dT), 1 mL of dNTPs and the mix was incubated at 65 1C for 5 min. After incubation 4 mL of Superscript II Buffer 5  , 2 mL of 1 M dithiothreitol (DTT) and 1 mL of RNase OUT (Invitrogen Life Technologies) were added and incubated at 42 1C for 2 min. An aliquot of 1 mL of Superscript II was added and incubated at 42 1C for 50 min, followed by subsequent incubation at 70 1C for 15 min. In order to remove the remaining RNAse 1 mL of RNase H was added and incubated at 37 1C for 20 min. All reagents were purchased from Invitrogen Life Technologies. Real-time PCR analysis was performed with ABI Prisms 7000 Sequence Detection Systems (Applied Biosystems, Foster City, CA, USA). In this work, the Taq Man Universal Master Mix (no. 4304437; Applied Biosystems) was used and the primers and probes: 32 connexin (Cx32) forward sequence 50 GGGTGGCCTC AAGGATAGG30 ; reverse 30 GGTGTATAGACCTG TCCAGTTCATC50 ; probe FAM-30 CTCCCCAGGTG

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TGAATG50 -NFQ and 26 connexin (Cx26) forward sequence: CAT GCA ACG TCT GGT GAA ATG; reverse: GGG CCT GGA AAT GAA GCA; Probe: 6FAM AAC GCT TGG CCC TGC CCC AAT AMGBNFQ; and the beta-actin gene (Assay ID Hs00242273_mL) was used as endogenous control. Analysis of relative gene expression data was performed according to the 24-DDCT method (Livak et al., 1995).

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groups showed similar higher increased of cellular proliferation in comparison to CT group (Fig. 3A and I). The treatment reduced cellular proliferation in the 2% group (Fig. 3C and I), which presented an inferior number of PCNA-positive nuclei, when compared with other experimental groups. The cellular proliferation of 2% group was similar to CT group one.

Cellular apoptosis Statistical analysis

Results

The groups 2% (Table 1 and Fig. 3G and I) and 10% (Table 1 and Fig. 3(H and I) showed increase in apoptosis when compared with other groups not treated (Table 1 and Fig. 3E, F, and I). Between the treated groups, the 10% group showed more AB than the 2% group. The effects observed for the 0% group (Table 1 and Fig. 3F and I) were not significant in comparison to the CT.

Effects of P. paniculata on relative liver weight

Comet assay

Mice treated with 0% or 10% groups showed significant increase in liver relative weight when compared with the CT group (Table 1 and Fig. 2). The 2% group mice did not show any alterations in relative weight.

Table 1 and Fig. 4 shows that the 10% group presented a higher percentage of comets formed by apoptotic cells when compared with other groups, corroborating the results obtained with apoptotic bodies count. The 0% or 2% groups do not show significant alterations.

The data were analyzed by one-way analysis of variance according to the most compatible Tukey– Kramer’s or Dunn’s multiple comparison tests. p-Values of o0.05 were considered significant.

Histopathological examination Effects on intercellular communication In the histopathological examination, the groups treated with P. paniculata root showed a higher incidence of diffuse mononuclear inflammatory infiltrate and areas of coagulation necrosis (Fig. 1A and B). The 10% group was more affected when compared with 2% group (Table 2). The other groups did not show these alterations.

Cell proliferation Table 1 and Fig. 3 (A–D and I) show the effects of treatment with P. paniculata root on cellular proliferation. The 0% (Fig. 3B and I) and 10% (Fig. 3D and I)

None of the groups treated with P. paniculata root presented alterations in intercellular communications of hepatocytes by gap junctions. All animals had diffusion of lucifer yellow dye inferior (Fig. 5B and C) than the one of control group (Fig. 5A and C) (CT: 116.40719.81; 0%: 88.35714.25; 2%: 85.00712.24; 10%: 94.25714.03; po0.05) but this reduction could not be identified by immunofluorescence (Fig. 7A and B). Treatment also did not affect protein expression of connexins 26 and 32 analyzed by western blot (Fig. 6A and B), also as its gene expression (Fig. 7C) analyzed by the real-time PCR.

Table 1. Relative weight, PCNA-positive cell numbers, apoptotic body numbers, and percentage of comet of apoptotic cells in mice hepatic tissue. Groups

Relative weight

PCNA+ cell number per mm2

Apoptotic bodies number per mm2

Comet (%)

CT 0% 2% 10%

4.5070.36 5.1870.40a 5.0370.35 5.4970.90a

0.1670.28 9.2675.08b 0.6970.24 9.0874.80b

0.0670.05 1.0070.31 2.9871.32a 5.6072.65a

6.4871.79 8.1472.68 10.3574.54 18.3178.06a,c

a

po0.05 vs. CT group. po0.05 vs. 2% group. c po0.05 vs. 0% group. b

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Fig. 1. Coagulation necrosis and mononuclear inflammatory infiltrate in mice induced to hepatocarcinogenesis model and treated with 10% of powdered root of P. paniculata: (A) Diffuse mononuclear infiltration and coagulation necrosis. Scale bar ¼ 100 mm. (B) Mononuclear cells in detail. Scale bar ¼ 10 mm.

Table 2. Incidence of areas of coagulation necrosis and mononuclear inflammatory infiltrate in mice induced to hepatocarcinogenesis model and treated with powdered root of P. paniculata. Groups (%)

2 10

n

20 21

Incidence (%) Infiltrate

Necrosis

20 52.38

15 23.81

Fig. 2. Relative liver weight of mice treated or not with powdered root of Pfaffia paniculata for 27 weeks: asignificant difference in relation to CT group (po0.01).

Discussion In a previous study, we demonstrated that treatment with powdered P. paniculata root added to ration reduced the incidence, number, and area of preneoplastic lesions in mice submitted to hepatocarcinogenesis (da Silva et al., 2005). From this initial action, we evaluated

in this work the possible mechanisms that could be involved in chemoprevention, by analysis of cellular proliferation, apoptosis, and intercellular communication by gap junctions. Immunohistochemistry for PCNA was used in the evaluation of cell proliferation. PCNA is an auxiliary protein for DNA polymerase d that may be involved not only in DNA replication but also in DNA repair mechanisms (Xue et al., 2003). The PCNA expression increases at the end of the G1 phase immediately preceding DNA synthesis, reaches a maximum during the phase S, and declines through G2 phase (Cox, 1997). PCNA has been used in many other studies involving the evaluation of cell proliferation (Avanzo et al., 2004; Torres et al., 2005; Fukumasu et al., 2006; Oloris et al., 2007; Fukumasu et al., 2008). According to our experience, it is a suitable cell proliferation marker because there is a comparison of cell proliferation indexes in mice submitted to different doses of P. paniculata. Our results point towards different mechanisms involved in chemoprevention of hepatocarcinogenesis in mice, which are directly linked to concentrations of P. paniculata in ration. Similar effects were also reported by da Silva et al. (2005), in relation to quantitative aspects of preneoplastic lesions. In the present work, cellular proliferation was significantly higher in both the mice that received 10% of root in ration and the 0% group that had not received the treatment. As a consequence, this result promoted an increase in liver relative weight, which was observed in both groups. This hyperplasic process is directly related to liver response to injury of any nature, especially toxic. These data are confirmed by the presence of diffuse mononuclear inflammatory infiltrate and coagulation necrosis in

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Fig. 3. Evaluation of treatment with Pfaffia paniculata on cellular proliferation (scale bar ¼ 40 mm) or apoptosis induction (scale bar ¼ 20 mm) per mm2 of liver: (A, E) CT group; (B, D) 0% group; (C, E) 2% group; and (D, F) 10%. (I) aSignificant difference in relation to CT group and 0% group (po0.05); bsignificant difference in relation to CT group and 2% group (po0.05).

Fig. 4. Percentage of comet formed by apoptotic cells observed in liver cells of mice treated or not with powdered root of Pfaffia paniculata for 27 weeks. aSignificant difference in relation to CT group (po0.05); bsignificant difference in relation to 0% group (po0.05).

hepatic parenchyma. Thus, analysis of histopathologycal and cellular findings allow us to infer that these animals had a regenerative liver response to chronic

injury that in this case was represented by administration of 10% of P. paniculata in the diet. Moreover, the 0% group mice presented a result of carcinogen administrated. The 2% group mice showed less cellular proliferation in comparison with other groups. In addition, the fact that this group presented a low incidence of hepatic inflammatory process might be the result of a lower toxicity of this concentration and an effective proliferation reduction due to treatment with the P. paniculata roots. The 2% or 10% treatments showed an increase of apoptotic bodies in comparison with other groups, which was confirmed by comet assay results. This result can explain the reduction in area and number of neoplastic lesions reported by da Silva et al. (2005) in both concentrations. These results can be explained by the triterpenoid saponins present in P. paniculata roots and are in accordance with studies that show that induction of apoptosis (Mujoo et al., 2001; Li et al., 2005; Lemeshko et al., 2006; Wakabayashi et al., 1998; Haridas et al., 2001)

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Fig. 5. Diffusion of lucifer yellow dye in mice liver tissue treated with powdered root of Pfaffia paniculata for 27 weeks. (A) CT group; (B) 10% group, diffusion representative of treated groups: asignificant difference in relation to CT (po0.05). Scale bar ¼ 40 mm.

and cellular proliferation (Mujoo et al., 2001; Koratkar and Rao, 1997; Watanabe et al., 2000; Lin et al., 2003) are some of the mechanisms of antitumoral action of saponins. Thus, chemopreventive effects in these animals could be related to a joint action of these mechanisms. Although we did not evaluate the molecular mechanisms responsible by the results obtained, studies showed that saponins from different sources have antitumoral properties similar to the ones of P. paniculata, which could explain our results. Popovich and Kitts (2002) studied saponins from Panax ginseng and observed that the induction of apoptosis occurred by DNA fragmentation. According to Park et al. (1997), induction of apoptosis by saponins from P. ginseng occurs by activation of caspase by Bcl2. Lemeshko et al. (2006) showed that saponins from Acacia victoriae induce apoptosis by increasing sensibility to oxidative stress by releasing cytochrome c, activating the apoptotic cascade. The increase in

permeability caused by A. victoriae would result from interaction between saponins and cellular membranes forming cholesterol-depending channels (Li et al., 2005). Control of cellular proliferation in tumor cells by saponins occurs by inhibition of proteins that are responsible for cellular division as cyclin-dependent kinases (Mujoo et al., 2001; Livak et al., 1995). Liu et al. (2000) observed that saponins from P. ginseng reduce cellular proliferation by activating expression of p21 and p27, which are genes that inhibit cyclin kinases. In compensation, molecular studies would be extremely interesting in order to evaluate the influence of active substances present in P. paniculata roots on proliferation and/or apoptosis of tumor cells. Gap junctions are responsible for intercellular flow of molecules with molecular weight inferior to 1000– 2000 Da and are related to maintenance of tissue homeostasis (Yamasaki, 1990). Loss of these junctions causes a decrease in intercellular communication, relating to promotion and progression of carcinogenic

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process. Dagli et al. (2004) demonstrated that mice bearing connexin 32 mutation and submitted to a hepatocarcinogenesis model had a higher susceptibility

Fig. 6. (A) Density of connexins 32 and 26 in relation to betaactin, analyzed by western blot in liver of mice treated or not with powdered root of Pfaffia paniculata for 27 weeks. (B) Imunoblots corresponding to connexins 32, 26 and beta-actin. There is no significant difference between groups.

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to lesions caused by DEN. Studies related to effects of saponins on intercellular communication by gap junctions are limited to P. ginseng, which could indicate that it is a particular property of active substances of this plant. Kang et al. (2000) showed, in vitro, that administration of different fractions of saponins from P. ginseng-protected cells from inhibition of intercellular communication by TPA or H2O2, and according to Zhang et al. (2001), several molecular mechanisms could be involved in these effects. However, in this study, none of the animals evaluated present significant alterations in cellular communication, evaluated by functional and molecular methods. Thus, it is suggested that active substances present in powdered root from P. paniculata do not exert modulation in gap junctions and, consequently, this mechanism is not involved in its chemopreventive potential. In conclusion, our results demonstrated that the chemopreventive effects presented in hepatocarcinogenesis in mice originated by two main mechanisms: inhibition of cellular proliferation and increase in apoptosis, but were not related to cell communication and/or connexin expression. These mechanisms were directly related to concentration of P. paniculata in food, where animals that received 2% had a more effective mechanism of tumor control and lower hepatic toxicity.

Fig. 7. Connexin 32 imunofluorescence in mice liver tissue. (A) CT group. (B) mice treated with powdered root of Pfaffia paniculata. Scale bar ¼ 20 mm. (C) Expression of genes of connexin proteins 26 and 32, analyzed by real-time PCR in liver of mice treated or not with powdered root of Pfaffia paniculata for 27 weeks. There is no significant difference between groups.

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Acknowledgements This work was part of the thesis presented by Tereza Cristina da Silva to the Comparative and Experimental Pathology Program of the Department of Pathology, School of Veterinary Medicine and Animal Science, University of Sa˜o Paulo. Tereza Cristina da Silva was a recipient of a fellowship from CAPES, Brazil. This work benefited of grants from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP, Proc. number 06/51678-3) and was also supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, CNPq.

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