Efeitos do γ-orizanol e extrato hidroalcólico de Thuya occidentalis sobre linhagens de câncer de próstata responsivas e não-responsivas a andrógenos

O câncer de próstata é a segunda causa de morte entre homens no Brasil. É tipo um câncer de crescimento lento, podendo levar anos para o tumor atingir 1 cm3, porém, em alguns casos ele pode se espalhar pelo corpo, sendo o osso o principal sítio de metástase. No estágio de desenvolvimento do câncer conhecido como metástase, o principal tratamento consiste em terapia de restrição andrógena, levando as células prostáticas a pararem de proliferar, uma vez que elas crescem em resposta a presença de hormônios andrógenos, como a diidrotestosterona e testosterona. Porém, em alguns casos, as células proliferam mesmo na ausência de andrógenos e isto se deve a diversos fatores que, em geral, estão associados a mutações no receptor andrógeno e/ou alterações no metabolismo andrógeno. Quando isto acontece, os tratamentos disponíveis são menos efetivos e costumam falhar. Porém, estudos sugerem que o γ-orizanol, um fitoesterol extraído do óleo do farelo do arroz; e extratos amplamente utilizados na medicina popular, como o extrato hidroalcólico de Thuya occidentalis, poderiam atuar inibindo o desenvolvimento e progressão do câncer de próstata. Neste estudo, com o uso de abordagens bioquímicas e de biologia molecular, foi demonstrado que o tratamento com γ-orizanol diminui a viabilidade e biomassa celular em cultura, associado ao aumento da morte celular por apoptose e/ou necrose, em linhagens celulares responsivas (LNCaP) e não-responsivas a andrógenos (PC3 e DU145), além de aumentar a pERK1/2 em células LNCaP e DU145. O γ-orizanol também foi capaz de bloquear o ciclo celular em G2/M nas células PC3 e LNCaP e em G0/G1 nas células DU145. Estes efeitos foram ainda acompanhados por uma redução da expressão do gene e proteína caveolina-1- uma importante molécula envolvida no aumento da agressividade do câncer de próstata, e também, na progressão da doença para o fenótipo andrógeno resistente - nas células não-responsivas a andrógenos, e do gene PCGEM1 - gene específico da próstata regulado por andrógeno - nas células LNCaP e DU145. Ainda, γ-orizanol também mostrou capacidade de regular vários miRNAs - pequenas moléculas de RNA não codificantes de proteínas - envolvidos no controle de funções associadas ao desenvolvimento, progressão e invasão no câncer de próstata, como o miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 e miR222. O extrato hidroalcólico de Thuya occidentalis reduziu a viabilidade e biomassa celular nas linhagens responsiva (LNCaP) e não-responsivas (DU145 e PC3) a andrógenos, além de induzir parada do ciclo celular na fase G0/G1 nas células DU145 e aumentar a morte celular por apoptose e/ou necrose em todas as linhagens. Da mesma forma que o γ-orizanol, este extrato reduziu a expressão da caveolina-1 nas linhagens não-responsivas a andrógenos. Trabalhos anteriores mostram que o monoterpeno α-tujona é o principal composto ativo do extrato de Thuya occidentalis. Por cromatografia gasosa acoplada a detector de massas foi mostrada a existência de 0,0016 μg de α-tujona na dose de extrato usada neste estudo. No entanto, o tratamento com 0,0016μg de α-tujona foi efetivo somente sobre linhagem LNCaP, não tendo efeito sobre as outras linhagens estudadas, reforçando a hipótese da diferença de sensibilidade entre as linhagens responsivas e não responsivas a andrógeno e mostrando a contribuição de outros componentes do extrato nos efeitos observados neste estudo. Concluindo, estes resultados demonstram que tanto γ-orizanol como o extrato de Thuya occidentalis podem vir a ser agentes terapêuticos promissores no tratamento de câncer de próstata, não só por inibirem o crescimento celular, mas também e principalmente pela possibilidade de induzirem a recuperação da sensibilidade a andrógenos, aumentando as possibilidades de tratamento da doença.Prostate cancer is the second cause of death among men in Brazil. It is a slowgrowing cancer and it may take years for tumor to reach 1 cm3, but in some cases it can spread throughout the body and the bone is the main site of metastasis. At this cancer stage known as metastasis, the principal treatment involves antiandrogen therapy, leading to prostate cells stop proliferating, because they grow in response to presence of androgens such as testosterone and dihydrotestosterone. However, in some cases, the cells can proliferate even in the absence of androgens and this fact occurs due to many factors and they are generally associated with mutations in the androgen receptor and/or alterations in androgen metabolism. In this stage, the treatments available are less effective and usually fail. However, studies suggest that γ-oryzanol, a phytosterol extracted of rice bran oil; and extracts widely used in folk medicine, as Thuya occidentalis hidroalcolic extract, could act inhibiting the development and progression of prostate cancer. In this study, using molecular biology and biochemical approaches we showed that γ- oryzanol treatment was able to decrease cell viability and biomass in culture, and this fact was linked to increased cell death by apoptosis and/or necrosis in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) prostate cancer cell lines, besides increasing pERK1/2 in LNCaP and DU145 cells. γ- oryzanol was also able to cause cell cycle arrest at G2/M phase in LNCaP and PC3 cells and at G0/G1 phase in DU145 cells. These effects were also accompanied by a reduction in caveolin-1 gene and protein expression - an important molecule related to high aggressiveness in prostate cancer and also in the progression of the disease to androgen resistant phenotype - in androgen unresponsive cells, and also PCGEM1 gene - a prostate specific gene regulated by androgens - in LNCaP and DU145 cells. γ-oryzanol also showed ability to regulate several miRNAs - small non-coding RNA molecules - involved in the control of many functions associated with the development, progression and invasion of prostate cancer, such as miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 and miR222. Thuya occidentalis hidroalcolic extract also reduce cell viability and biomass in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) cells, in addition to inducing cell cycle arrest at G0/G1 phase in DU145 cells and to increase apoptosis and/or necrosis cell death in all cell lines. The same way that γ-oryzanol, this extract reduced the caveolin-1 expression in androgen unresponsive prostate cancer cells. Prior studies showed that the monoterpene α-thujone is the main active compound in the T. occidentalis extract. By gas chromatography coupled to mass detector it was showed the existence of 0.0016 μg of α-thujone in extract dose used in this study. However, the treatment with 0.0016 μg of α-thujone was effective only on LNCaP cell line, having no effect on the other studied lines, supporting the hypothesis of difference in sensitivity between responsive and unresponsive cell lines and showing the contribution of other components in the effects caused by the extract, observed it this study. In conclusion, these results demonstrate that both γ-oryzanol as T. occidentalis extract may become promising therapeutic agents in treatment of prostate cancer, not only inhibit cell growth but also and manly by the possibility of inducing the recovery of androgen sensitivity, increasing the treatment chances of treatment this disease

Antioxidant activity of blackberry (Rubus sp.) genotypes from the Southern Region of Brazil

The antioxidant activity and bioactive compounds of different blackberry fruit genotypes from the major Brazilian producer region (three cultivars and four selections) were evaluated and compared to the Cherokee cultivar. Phenolic and anthocyanic extracts were obtained and evaluated for each fruit genotype. The phenolic extracts of selections 02/96 and 07/001 presented higher antioxidant activity than those of cultivars in most assays. This activity was partially correlated to the higher amount of total phenolics in these samples. Thus, the phenolic compounds are probably the major responsible for the antioxidant activity in the diphenyl-2-picrylhydrazyl radical scavenging assay (DPPH), ferric reducing antioxidant power (FRAP) assay and thiobarbituric acid reactive substances (TBARS) assay. Quercetin seems to be responsible for the antioxidant activity of blackberry phenolic extracts in the β-carotene bleaching assay. Concerning anthocyanic extracts, the selection 02/96 and Cherokee cultivar from harvest 2007 had higher antioxidant activity than the other genotypes in most assays. Anthocyanins appear to be the major responsible for the antioxidant activity of anthocyanic extracts in the DPPH and FRAP assays, although ascorbic acid also contributed to the DPPH antioxidant activity. Selection 02/96 appears to have higher antioxidant activity than the commercial cultivars cultivated in the southern Brazil and appears to be promising for nutritional and health purposes

Caracterização fisico-química e capacidade antioxidante de pitangas (Eugenia uniflora L.)

This study was carried out to obtain more information about the physicochemical properties, composition, and antioxidant activity of pitanga fruits (Eugenia uniflora L.), particularly fruits from the State of Rio Grande do Sul, Brazil. Pitanga with different flesh colors (purple, red, and orange) from tree selections cultivated at Embrapa Clima Temperado (RS-Brazil) were analyzed. Only slight differences were observed in the quality parameters and in the proximate and fatty acid compositions among the fruits studied. The extracts from purple-fleshed pitanga had the highest total phenolic and anthocyanin contents along with the highest antioxidant capacity. The antioxidant capacity (DPPH and FRAP assays) of methanolic pitanga extracts was highly correlated with the total phenolic content, but in ethanolic extracts, the anthocyanin content was correlated only with the FRAP antioxidant capacity. Orange fleshed pitanga had higher β-cryptoxanthin and β-carotene levels than those of the red fruit, which had higher lycopene content. The results indicate that the purple-fleshed pitanga, cultivated in Rio Grande do Sul, is a rich source of phenolic compounds and has high antioxidant capacity. The red and orange-fleshed pitanga, on the other hand, are rich sources of carotenoids.Este estudo foi realizado para obter mais informações sobre as propriedades físico-químicas, composição e atividade antioxidante de frutos de pitanga (Eugenia uniflora L.), especialmente os do Rio Grande do Sul (Brasil). Foram comparadas pitangas com diferentes colorações de polpa (roxa, vermelha e laranja) de seleções cultivadas na Embrapa Clima Temperado (RS-Brasil). Foram observadas pequenas diferenças nos parâmetros de qualidade e na composição centesimal e de ácidos graxos entre as frutas com diferentes colorações de polpa. Os extratos de pitanga roxa apresentaram maiores conteúdos totais de fenólicos e de antocianinas, bem como, a maior capacidade antioxidante. A capacidade antioxidante (valores de DPPH e FRAP) dos extratos metanólicos de pitanga apresentou alta correlação com o conteúdo de fenólicos totais, mas nos extratos etanólicos, o conteúdo de antocianinas correlacionou-se apenas com a capacidade antioxidante avaliada pelo método de FRAP. A pitanga de cor laranja apresentou maiores teores de β-criptoxantina e β-caroteno, enquanto que a de cor vermelha continha alto teor de licopeno. Os resultados indicam que a pitanga de cor roxa, cultivada no Rio Grande do Sul, é uma fonte rica de compostos fenólicos e possui alta capacidade antioxidante. As de cor vermelha e laranja, por outro lado, são fontes ricas de carotenoides.147154Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Physicochemical characterization and antioxidant capacity of pitanga fruits (Eugenia uniflora L.)

This study was carried out to obtain more information about the physicochemical properties, composition, and antioxidant activity of pitanga fruits (Eugenia uniflora L.), particularly fruits from the State of Rio Grande do Sul, Brazil. Pitanga with different flesh colors (purple, red, and orange) from tree selections cultivated at Embrapa Clima Temperado (RS-Brazil) were analyzed. Only slight differences were observed in the quality parameters and in the proximate and fatty acid compositions among the fruits studied. The extracts from purple-fleshed pitanga had the highest total phenolic and anthocyanin contents along with the highest antioxidant capacity. The antioxidant capacity (DPPH and FRAP assays) of methanolic pitanga extracts was highly correlated with the total phenolic content, but in ethanolic extracts, the anthocyanin content was correlated only with the FRAP antioxidant capacity. Orange fleshed pitanga had higher β-cryptoxanthin and β-carotene levels than those of the red fruit, which had higher lycopene content. The results indicate that the purple-fleshed pitanga, cultivated in Rio Grande do Sul, is a rich source of phenolic compounds and has high antioxidant capacity. The red and orange-fleshed pitanga, on the other hand, are rich sources of carotenoids