Evaluation of the global DNA methylation in canine mast cell tumour samples by immunostaining of 5-methyl cytosine.

Cutaneous mast cell tumours (MCT) are the most common skin tumour in dogs, and to our knowledge, there are no previous studies regarding the global methylation of these tumours. DNA hypomethylation and hypermethylation have been described in several tumours and both mechanisms can lead to carcinogenesis. The purpose of this study was to evaluate the global DNA methylation in canine MCT. A total of 48 MCT samples were classified in grades 1, 2 and 3 or high-grade or low-grade. Monoclonal antibodies were used for the immunohistochemical detection of the 5-methylcytosine. The immunostained nuclei were classified in strong, weak or negative pattern, and these were quantified in five distinct microscopic fields (40× objective) in each slide. The results showed that global DNA hypomethylation was predominant in grade 3, high-grade, less differentiated MCT. These epigenetic changes in neoplastic mast cells warrant further detailed investigation aiming the establishment of tumour epigenetic therapies.

Liquid-based cytology and cell block immunocytochemistry in veterinary medicine: comparison with standard cytology for the evaluation of canine lymphoid samples.

Liquid-based Cytology (LBC) consists of immediate wet cell fixation with automated slide preparation. We applied LBC, cell block (CB) and immunocytochemistry to diagnose canine lymphoma and compare results with conventional cytology. Samples from enlarged lymph nodes of 18 dogs were collected and fixed in preservative solution for automated slide preparation (LBC), CB inclusion and immunophenotyping. Two CB techniques were tested: fixed sediment method (FSM) and agar method (AM). Anti-CD79a, anti-Pax5, anti-CD3 and anti-Ki67 were used in immunocytochemistry. LBC smears showed better nuclear and nucleolar definition, without cell superposition, but presented smaller cell size and worse cytoplasmic definition. FSM showed consistent cellular groups and were employed for immunocytochemistry, while AM CBs presented sparse groups of lymphocytes, with compromised analysis. Anti-Pax-5 allowed B-cell identification, both in reactive and neoplastic lymph nodes. Our preliminary report suggests that LBC and FSM together may be promising tools to improve lymphoma diagnosis through fine-needle aspiration.

Expression of NR1I3 in mouse lung tumors induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanone.

Nuclear receptor subfamily 1, group I, member 3 (NR1I3) is reported to be a possible novel therapeutic target for some cancers, including lung, brain and hematopoietic tumors. Here, we characterized expression of NR1I3 in a mouse model of lung carcinogenesis induced by 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanone (NNK), the most potent tobacco carcinogen. Lung tumors were collected from mice treated with NNK (400 mg/kg) and euthanized after 52 weeks. Benign and malignant lesions were formalin-fixed and paraffin-embedded for histology and immunohistochemistry, with samples snap-frozen for mRNA analysis. Immunohistochemically, we found that most macrophages and type I and II pneumocytes expressed NR1I3, whereas fibroblasts and endothelial cells were NR1I3-. Compared with benign lesions, malignant lesions had less NR1I3+ tumor cells. Gene expression analysis also showed an inverse correlation between NR1I3 mRNA expression and tumor size (P=0.0061), suggesting that bigger tumors expressed less NR1I3 transcripts, in accordance with our immunohistochemical NR1I3 tests. Our results indicate that NR1I3 expression decreased during progression of malignant lung tumors induced by NNK in mice.

The value of molecular expression of KIT and KIT ligand analysed using real-time polymerase chain reaction and immunohistochemistry as a prognostic indicator for canine cutaneous mast cell tumours.

This study investigated the correlation between KIT gene expression determined by immunohistochemistry and real-time polymerase chain reaction (RT-PCR) and the rate of tumour recurrence and tumour-related deaths in dogs affected with mast cell tumour (MCT). Kaplan-Meier curves were constructed to compare tumour recurrence and tumour-related death between patients. The log-rank test was used to check for significant differences between curves. KIT-I, KIT-II and KIT-III staining patterns were observed in 9 (11.11%), 50 (61.73%) and 22 (27.16%) tumours, respectively. Tumour recurrence rates and tumour-related deaths were not associated with KIT staining patterns (P = 0278, P > 0.05), KIT (P = 0.289, P > 0.05) or KIT ligand (P = 0.106, P > 0.05) gene expression. Despite the lack of association between KIT staining pattern and patient survival time, the results suggest a correlation between aberrant KIT localization and increased proliferative activity of MCTs. RT-PCR seems to be a sensible method for quantitative detection of KIT gene expression in canine MCT, although expressions levels are not correlated with prognosis.

Cirrhosis in rats does not resolve in the long-term after induction by thioacetamide model

Introductions: Hepatic cirrhosis is a final common pathway of all chronic liver diseases, characterized by deposit of fibrillar collagen and liver failure. Materials and Methods: In this experiment, hepatic cirrhosis was induced in 15 female Wistar rats by a 14-week period, with thioacetamide solution in a 200 mg/kg dosage, via intraperitoneal. Animals were submitted to liver biopsy, and euthanized after a 80-day post-induction period. Serum biochemical analysis was performed, in addition to histopathology by H.E., Picrosirius, Alcian Blue and P.A.S. stainings, following analysis of histological activity index and staging of fibrosis. Morphometric analysis of collagen on Picrosirius slides was also performed. Results: Mortality during experimental period was low (13.33%), and after 80-day period, liver function improved, cellular changes did not altered, and deposition of acidic mucopolysaccharides and glycogen were increased. Liver histological activity did not change significantly (7.25 ± 1.30 to 6.41 ± 1.32), but staging of fibrosis was altered (3.91 ± 0.76 to 4.70 ± 1.11). Interlobular collagen showed a significant decrease (5.14 ± 2.00 to 4.00 ± 1.20), while intralobular collagen was increased (0.23 ± 0.06 to 0.36 ± 0.08). Conclusions: These findings characterize thioacetamide as a safe experimental model for induction cirrhosis, which may be used for future therapy studies.(AU)

Primary intraocular chondrosarcoma in a dog / Condrossarcoma intraocular primário em cão

A five-year-old male Cocker Spaniel was presented for evaluation of the right eye due to discomfort, abundant purulent discharge and progressive enlargement of the eyeball. The owner revealed that the right eye has appeared to be inflamed and smaller then the left eye for years. Ophthalmic examination revealed corneal perforation, buphthalmia and conjuctival hyperemia. Enucleating was performed due to signs of endophthalmitis and ocular discomfort. Histopathology revealed a multilobulated proliferation of chondrocytes producing hyaline cartilage with occasional pleomorphism and binucleate cells. A diagnosis of primary intraocular chondrosarcoma was done.

Foi atendido um cão, da raça Cocker Spaniel, de cinco anos de idade, com desconforto ocular, secreção purulenta abundante e aumento progressivo do bulbo ocular. Ao exame oftálmico, evidenciaram-se perfuração corneana, buftalmia e hiperemia conjuntival. Foi realizada enucleação em decorrência do desconforto ocular intenso e dos sinais de endoftalmite. Exame histopatológico revelou proliferação multilobulada de condrócitos produzindo cartilagem hialina com pleomorfismo ocasional e células binucleadas. Foi diagnosticado condrossarcoma intraocular primário.

Efficacy of the dietary histone deacetylase inhibitor butyrate alone or in combination with vitamin A against proliferation of MCF-7 human breast cancer cells

The combined treatment with histone deacetylase inhibitors (HDACi) and retinoids has been suggested as a potential epigenetic strategy for the control of cancer. In the present study, we investigated the effects of treatment with butyrate, a dietary HDACi, combined with vitamin A on MCF-7 human breast cancer cells. Cell proliferation was evaluated by the crystal violet staining method. MCF-7 cells were plated at 5 x 10(4) cells/mL and treated with butyrate (1 mM) alone or combined with vitamin A (10 µM) for 24 to 120 h. Cell proliferation inhibition was 34, 10 and 46% following treatment with butyrate, vitamin A and their combination, respectively, suggesting that vitamin A potentiated the inhibitory activities of butyrate. Furthermore, exposure to this short-chain fatty acid increased the level of histone H3K9 acetylation by 9.5-fold (Western blot), but not of H4K16, and increased the expression levels of p21WAF1 by 2.7-fold (Western blot) and of RARβ by 2.0-fold (quantitative real-time PCR). Our data show that RARβ may represent a molecular target for butyrate in breast cancer cells. Due to its effectiveness as a dietary HDACi, butyrate should be considered for use in combinatorial strategies with more active retinoids, especially in breast cancers in which RARβ is epigenetically altered.

E-cadherin in canine mast cell tumors: decreased expression and altered subcellular localization in Grade 3 tumors.

Mast cell tumors (MCTs) are the most frequent round cell tumors in dogs and comprise approximately 21% of all canine cutaneous tumors. MCTs are highly invasive and metastatic corresponding to the histological grade. E-cadherin is an adhesion molecule expressed in epithelial cells and although it is an epithelial cellular marker, studies have shown expression of E-cadherin in canine round cell tumors. To better characterize the expression pattern of E-cadherin in several different histological grades of MCTs in dogs, the expression and localization of the adhesion molecule was investigated using immunohistochemistry. For this purpose, 18 cutaneous MCTs were classified into three histological grades, 1, 2 or 3. Clinical history and follow-up data were available for all of the dogs. Cytoplasmic and nuclear expressions of E-cadherin in all three types of tumors were verified by immunostaining using two different antibodies. There was decreased E-cadherin expression in the more aggressive MCTs (Grade 3), suggesting an association between E-cadherin and tumor aggressiveness. Additionally, the loss of E-cadherin expression in either the cytoplasm or nucleus in more aggressive and undifferentiated tumor types confirmed the importance of cellular adhesion in tumor behavior.

Efficacy of the dietary histone deacetylase inhibitor butyrate alone or in combination with vitamin A against proliferation of MCF-7 human breast cancer cells.

The combined treatment with histone deacetylase inhibitors (HDACi) and retinoids has been suggested as a potential epigenetic strategy for the control of cancer. In the present study, we investigated the effects of treatment with butyrate, a dietary HDACi, combined with vitamin A on MCF-7 human breast cancer cells. Cell proliferation was evaluated by the crystal violet staining method. MCF-7 cells were plated at 5 x 10(4) cells/mL and treated with butyrate (1 mM) alone or combined with vitamin A (10 µM) for 24 to 120 h. Cell proliferation inhibition was 34, 10 and 46% following treatment with butyrate, vitamin A and their combination, respectively, suggesting that vitamin A potentiated the inhibitory activities of butyrate. Furthermore, exposure to this short-chain fatty acid increased the level of histone H3K9 acetylation by 9.5-fold (Western blot), but not of H4K16, and increased the expression levels of p21WAF1 by 2.7-fold (Western blot) and of RARß by 2.0-fold (quantitative real-time PCR). Our data show that RARß may represent a molecular target for butyrate in breast cancer cells. Due to its effectiveness as a dietary HDACi, butyrate should be considered for use in combinatorial strategies with more active retinoids, especially in breast cancers in which RARß is epigenetically altered.

Modelo de suplementação nutricional com fatores hepatotróficos aumenta proliferação celular em fígado de ratos sadios / Model of nutritional supplementation with hepatotrophic factors increases cell proliferation in the liver of healthy rats

Foram avaliados dois protocolos de administração, em ratos sadios, de uma solução de fatores hepatotróficos (FH), composta por aminoácidos, vitaminas, sais minerais, glicose, insulina, glucagon e triiodotironina (T3). A solução foi administrada durante 10 dias, 40mg/kg/dia, i.p., em duas, grupo 2xFH (n=15), ou três doses, grupo 3xFH (n=15), diárias. Foram observados os efeitos na proliferação celular dos hepatócitos, na angiogênese e na matriz extracelular hepática, assim como as possíveis reações adversas. Os animais dos grupos 2xFH e 3xFH apresentaram aumento da massa hepática de 30,1 por cento e 22,5 por cento, respectivamente, em relação ao grupo-controle (CT; n=15). O índice de proliferação hepatocelular foi maior nos grupos 2xFH (1,4 por cento) e 3xFH (1,2 por cento) em relação ao grupo CT (0,53 por cento), e a densitometria relativa do fator de crescimento do endotélio vascular pelo imunoblot não revelou diferença estatística entre os três grupos. Nos grupos 2xFH e 3xFH, houve redução do colágeno intersticial em relação ao grupo CT. A solução de FH estimulou o crescimento hepático e reduziu o volume de colágeno perissinusoidal. A administração em três doses diárias resultou em mortalidade de 26,7 por cento, possivelmente pelo excessivo estresse da manipulação e pela menor adaptação fisiológica dos ratos, o que não ocorreu nos grupos 2xFH e CT. Para esse tipo de abordagem em ratos, o procedimento experimental mais apropriado, seguro, com melhor chance de adaptação dos animais e com resultados significativos é a aplicação dos FH em duas doses diárias.

Two protocols of hepatotrophic factors (HF) administration, in solution composed by aminoacids, vitamins, mineral salts, glucose, insulin, glucagon, and triiodothyronine were evaluated in healthy rats. This solution was administered for 10 days, (40mg/kg/day) i.p., in two (group 2xFH; n=15) or three daily doses (group 3xFH n=15). The effects on hepatocytes cell proliferation, angiogenesis, and hepatic extracellular matrix, and also possible adverse reactions were analyzed. Animals of groups 2xFH and 3xFH presented an increase in hepatic mass of 30.1 percent and 22.5 percent, respectively, when compared rats of control group (CT; n=15). Hepatocellular proliferation index was higher in rats of groups 2xFH (1.4 percent) and 3xFH (1.2 percent) when compared to CT group animals (0.53 percent), and the relative densitometry of the vascular endothelial growth factor analyzed with immunoblot did not show a significant difference among the three groups. Rats of groups 2xFH and 3xFH showed a reduction of interstitial collagen when compared to CT rats. HF solution stimulated hepatic growth and reduced the volume of perisinusoidal collagen. Administration in three daily doses resulted in 26.7 percent mortality, possibly due to excessive stress from manipulation and lower physiological adaptation of rats, which did not occur in rats of groups 2xFH and CT. The more appropriate and safer experimental procedure for this approach in rats with higher chance of animal adaptation and significant results is the application of HF in two daily doses.

Identification of hepatic stem/progenitor cells in canine hepatocellular and cholangiocellular carcinoma.

Hepatic progenitor cells (HPCs) are bipotential stem cells residing in human and animal livers that are able to differentiate towards the hepatocytic or cholangiocytic lineages. HPCs are present in both hepatocellular (HCC) and cholangiocellular carcinoma (CC) in humans; and a small percentage of HCC can originate from cancer stem cells. However, its distribution in canine liver tumour has not been studied. Herein, we searched for stem/progenitor cells in 13 HCC and 7 CC archived samples by immunohistochemical analysis. We found that both liver tumours presented a higher amount of K19-positive HPCs. Besides, 61.6% of HCC cases presented immature CD44-positive hepatocytes. Nevertheless, only two cases presented CD133-positive cells. As observed in humans, hepatic canine tumours presented activated HPCs, with important differentiation onto hepatocytes-like cells and minimal role of cancer stem cells on HCC. These findings reiterate the applicability of canine model in the search for new therapies before application in humans.

Translocator protein (18 kDa) mediates the pro-growth effects of diazepam on Ehrlich tumor cells in vivo.

The Translocator Protein (TSPO), previously known as the peripheral-type benzodiazepine receptor, is a ubiquitous drug- and cholesterol-binding protein that is up regulated in several types of cancer cells. TSPO drug ligands (e.g., diazepam) induce or inhibit tumor cell proliferation, depending on the dose and tissue origin. We have previously shown that TSPO is expressed in Ehrlich tumor cells and that diazepam increases proliferation of these cells in vitro. Here, we investigated the in vivo effects of diazepam on Ehrlich tumor growth and the role of TSPO in mediating this process. Oral administration of diazepam to mice (3.0mg/kg/day for 7 days) produced plasma and ascitic fluid drug concentrations of 83.83 and 54.12 nM, respectively. Diazepam increased Ehrlich tumor growth, likely due to its ability to increase tumor cell proliferation and Reactive Oxygen Species production. Radioligand binding assays and nucleotide sequencing revealed that Ehrlich tumor cell TSPO had the same pharmacological and biochemical properties as TSPO described in other tumor cells. The estimated K(d) for PK 11195 in Ehrlich tumor cells was 0.44 nM and 8.70 nM (low and high binding site, respectively). Structurally diverse TSPO drug ligands with exclusive affinity for TSPO (i.e., 4-chlordiazepam, Ro5-4864, and isoquinoline-carboxamide PK 11195) also increased Ehrlich tumor growth. However, clonazepam, a GABA(A)-specific ligand with no affinity for TSPO, failed to do so. Taken together, these data suggest that diazepam induces in vivo Ehrlich tumor growth in a TSPO-dependent manner.

Immunohistochemical characterization of canine prostatic intraepithelial neoplasia.

The development of prostate cancer is believed to be a multistep process, progressing sequentially from normal epithelium, to prostatic intraepithelial neoplasia (PIN) and, finally, to invasive neoplasia. Malignant stem cells within the basal cell layer of the prostatic epithelium are believed to play an important role in the failure of androgen-ablation therapy that occurs in the most advanced form of prostate cancer. The aim of the present study was to immunohistochemically characterize the lesions of canine PIN. Prostatic tissue from five dogs with PIN was compared with normal prostate tissue from nine further dogs. There was an increase in the number of basal epithelial cells in lesions consistent with PIN as defined by expression of the nuclear protein p63. These lesions had elevated expression of proliferating cell nuclear antigen (PCNA) and heterogeneous labelling for the nuclear androgen receptor (AR). These findings suggest that the basal cells present in PIN may play a role in canine prostate carcinogenesis and that the proliferation of these cells occurs despite the heterogeneous expression of the AR.

Modulation of extracellular matrix by nutritional hepatotrophic factors in thioacetamide-induced liver cirrhosis in the rat

Nutritional substances associated to some hormones enhance liver regeneration when injected intraperitoneally, being denominated hepatotrophic factors (HF). Here we verified if a solution of HF (glucose, vitamins, salts, amino acids, glucagon, insulin, and triiodothyronine) can revert liver cirrhosis and how some extracellular matrices are affected. Cirrhosis was induced for 14 weeks in 45 female Wistar rats (200 mg) by intraperitoneal injections of thioacetamide (200 mg/kg). Twenty-five rats received intraperitoneal HF twice a day for 10 days (40 mL·kg-1·day-1) and 20 rats received physiological saline. Fifteen rats were used as control. The HF applied to cirrhotic rats significantly: a) reduced the relative mRNA expression of the genes: Col-α1 (-53 percent), TIMP-1 (-31.7 percent), TGF-β1 (-57.7 percent), and MMP-2 (-41.6 percent), whereas Plau mRNA remained unchanged; b) reduced GGT (-43.1 percent), ALT (-17.6 percent), and AST (-12.2 percent) serum levels; c) increased liver weight (11.3 percent), and reduced liver collagen (-37.1 percent), regenerative nodules size (-22.1 percent), and fibrous septum thickness. Progranulin protein (immunohistochemistry) and mRNA (in situ hybridization) were found in fibrous septa and areas of bile duct proliferation in cirrhotic livers. Concluding, HF improved the histology and serum biochemistry of liver cirrhosis, with an important reduction of interstitial collagen and increased extracelullar matrix degradation by reducing profibrotic gene expression.

Expression of connexins in normal and neoplastic canine bone tissue.

Previous studies showed that intercellular communication by gap junctions has a role in bone formation. The main connexin involved in the development, differentiation, and regulation of bone tissue is connexin (Cx) 43. In addition, Cx46 is also expressed, mostly localized within the trans-Golgi region. Alterations in the expression pattern and aberrant location of these connexins are associated with oncogenesis, demonstrating a deficient gap junctional intercellular communication (GJIC) capacity in neoplastic tissues. In this study, we evaluated normal and neoplastic bone tissues regarding the expression of Cx43 and Cx46 by immunofluorescence, gene expression of these connexins by real-time PCR, and their correlation with cell proliferation index and deposition of collagen. Fourteen neoplastic bone lesions, including 13 osteosarcomas and 1 multilobular tumor of bone, were studied. The mRNA levels of Cx43 were similar between normal and neoplastic bone tissue. In normal bone tissue, the Cx43 protein was found mainly in the intercellular membranes. However, in all bone tumors studied here, the Cx43 was present in both cell membranes and also aberrantly in the cytoplasm. Regarding only tumor samples, we determined a possible inverse correlation between Cx43 expression and cellular proliferation, although a positive correlation between Cx43 expression and collagen deposition was also noted. In contrast, Cx46 had lower levels of expression in neoplastic bone tissues when compared with normal bone and was found retained in the perinuclear region. Even though there are differences between these two connexins regarding expression in neoplastic versus normal tissues, we concluded that there are differences regarding the subcellular location of these connexins in normal and neoplastic dog bone tissues and suggest a possible correlation between these findings and some aspects of cellular proliferation and possibly differentiation.

In vitro chemosensitivity of canine mast cell tumors grades II and III to all-trans-retinoic acid (ATRA).

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect the skin and soft tissue of dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. While retinoids are well recognized as promising antitumor agents, there have been only a few reports about retinoids' effect on canine cancers. The aim of this study was to investigate the chemosensitivity of MCT grades II and III to all-trans retinoic acid (ATRA). Immediately after surgical resection, MCT were prepared for primary culture. Samples of MCTs were also fixed in formalin for histopathology and grading according to the classification of Patnaik et al. (Veterinary Pathology 21(5):469-474, 1984). The best results were obtained when neoplastic mast cells were co-cultivated with fibroblasts. Cultured mast cells were, then, treated with concentrations of 10(-4) to 10(-7) M of ATRA, in order to evaluate their chemosensitivity to this retinoid. MTT assay was performed to estimate cell growth and death. The highest level of mast cell chemosensivity was obtained at the dose of 10(-4) M (p < 0,002). MCT of grades II or III were equally susceptible to the treatment with ATRA. Cell death was observed on the first 24 h until 48 h. According to these results, ATRA may be a potential chemotherapeutic agent for the treatment of canine MCT.

Can established cultured papilloma cells harbor bovine papillomavirus?

Papillomaviruses have been reported to be very difficult to grow in cell culture. Also, there are no descriptions of cell cultures from lesions of bovine cutaneous papillomatosis, with identification of different bovine papilloma virus (BPV) DNA sequences. In the present report, we describe primary cell cultures from samples of cutaneous lesions (warts). We investigated the simultaneous presence of different BPV DNA sequences, comparing the original lesion to different passages of the cell cultures and to peripheral blood. BPV 1, 2 and 4 DNA sequences were found in lesion samples, and respective cell cultures and peripheral blood, supporting our previous hypothesis of the possible activity of these sequences in different samples and now also showing how they can be maintained in different passages of cell cultures.

Simultaneous presence of bovine papillomavirus and bovine leukemia virus in different bovine tissues: in situ hybridization and cytogenetic analysis.

Bovine papillomavirus (BPV) DNA sequences were detected in different tissues, in addition to epithelium. Cytogenetic abnormalities were observed in blood lymphocytes. The presence of more than one virus in a single tissue is a difficult aspect to evaluate, especially when the DNA sequences are detected in tissues that are not specifically targeted by the virus. BPV and bovine leukemia virus (BLV) are clastogenic, causing chromosome aberrations in peripheral blood lymphocytes. In the present study, we investigated the simultaneous presence of DNA sequences of both viruses and the possibility of vertical transmission and compared the types of chromosome aberrations related to viral action. BPV 1, 2, and 4 DNA sequences were found in three females of the herd and in their offspring. BLV DNA sequences were not detected in their progeny. A newborn calf that was negative for BLV infection showed specific chromosome rearrangements possibly related to the effect of infection with BPV.

Paullinia cupana Mart var. sorbilis, guaraná, reduces cell proliferation and increases apoptosis of B16/F10 melanoma lung metastases in mice.

We showed that guaraná (Paullinia cupana Mart var. sorbilis) had a chemopreventive effect on mouse hepatocarcinogenesis and reduced diethylnitrosamine-induced DNA damage. In the present experiment, we evaluated the effects of guaraná in an experimental metastasis model. Cultured B16/F10 melanoma cells (5 x 10(5) cells/animal) were injected into the tail vein of mice on the 7th day of guaraná treatment (2.0 mg P. cupana/g body weight, per gavage) and the animals were treated with guaraná daily up to 14 days until euthanasia (total treatment time: 21 days). Lung sections were obtained for morphometric analysis, apoptotic bodies were counted to calculate the apoptotic index and proliferating cell nuclear antigen-positive cells were counted to determine the proliferation index. Guaraná-treated (GUA) animals presented a 68.6% reduction in tumor burden area compared to control (CO) animals which were not treated with guaraná (CO: 0.84 +/- 0.26, N = 6; GUA: 0.27 +/- 0.24, N = 6; P = 0.0043), a 57.9% reduction in tumor proliferation index (CO: 23.75 +/- 20.54, N = 6; GUA: 9.99 +/- 3.93, N = 6; P = 0.026) and a 4.85-fold increase in apoptotic index (CO: 66.95 +/- 22.95, N = 6; GUA: 324.37 +/- 266.74 AB/mm(2), N = 6; P = 0.0152). In this mouse model, guaraná treatment decreased proliferation and increased apoptosis of tumor cells, consequently reducing the tumor burden area. We are currently investigating the molecular pathways of the effects of guaraná in cultured melanoma cells, regarding principally the cell cycle inhibitors and cyclins.