Effects of Salvia officinalis in the liver : relevance of glutathione levels

Tese de Doutoramento em Ciências BiológicasSalvia officinalis L (Lamiaceae, salva comum) é uma planta aromática que cresce espontaneamente em Portugal, bastante conhecida pelas suas propriedades medicinais. Duas das principais propriedades medicinais atribuídas a esta planta são como antioxidante e antidiabética. No entanto, ainda não existem suficientes evidências experimentais que demonstrem estes efeitos e em particular os mecanismos inerentes em sistemas biológicos. No presente trabalho, os estudos dos efeitos antioxidantes e antidiabéticos foram realizados no I fígado e em hepatócitos. Uma vez que os compostos da salva passam primeiro pelo fígado após a absorção gastrointestinal, estes podem atingir concentrações consideráveis neste órgão possibilitando a indução de efeitos biológicos. Além disso, o fígado tem uma grande capacidade de biotransformação, é bastante susceptível a efeitos tóxicos de xenobióticos e é também afectado por bastantes doenças onde se reconhece o envolvimento de stresse oxidativo. Deste modo, para além dos prováveis benefícios do consumo de salva para o fígado, estes estudos permitiram-nos observar possíveis efeitos tóxicos desta planta e interacções com outros compostos. Três tipos de extractos foram estudados: o óleo essencial (OE), um extracto metanólico e a extracto aquoso de salva preparado sob a forma de um chá. Numa primeira série de experiências (capítulo 2), usando ensaios com suspensões de hepatócitos de rato, observou-se que o OE de salva não teve efeitos antioxidantes. Pelo contrário, a altas concentrações (maiores que 2 ~1/ml), o óleo de salva induziu significativamente morte nas células, a qual foi acompanhada por uma depleção nos níveis de GSH (forma reduzida da glutationa) mas não por uma indução da peroxidação lipídica. A diminuição dos níveis de GSH foi provavelmente um dos mecanismos responsáveis pelos efeitos tóxicos do OE. Em seguida, após uma experiência in vivo com ratinhos onde se verificou que o chá de salva melhorava a resposta antioxidante do fígado (capítulo 3), no c.apítulo 4 foram estudados os efeitos directos de salva em células HepG2 (linha celular de hepatoma humano). A salva protegeu contra a morte celular induzida pelo tert-butil hidroperóxido - um modelo de stresse oxidativo - revelando o seu potencial antioxidante directo em células. A sua acção nos níveis de GSH foi muito importante, prevenindo significativamente a sua depleção. O efeito citoprotector dos extractos da salva foi dependente da composição dos extractos ym compostos fenólicos bem como da sua actividade antiradicalar. Também no capítulo 4, verificou-se que os mesmos extractos eram capazes de elevar os níveis basais de GSH em células HepG2, indicando uma capacidade em melhorar as defesas antioxidantes. o aumento de GSH poderá ter acontecido através da indução da síntese de novo da glutationa, o que é também corroborado por uma experiência anterior com hepatócitos de rato em cultura (capítulo 3). Nessa experiência, o chá de salva dado in vivo fez recuperar os níveis de GSH dos hepatócitos, após o isolamento com colagenase, para valores mais altos do que na situação controlo. Adicionalmente, o chá de salva induziu significativamente as actividades das enzimas GST (capítulo 3), GPox e NADPH citocromo P450 redutase bem como, em alguma extensão, algumas enzimas citocromo P450 em fígado de ratinho (capítulo 5). Estes efeitos antioxidantes indirectos parecem deixar o fígado mais bem preparado para combater uma situação de stresse oxidativo e o efeito tóxico de xenobióticos. No entanto, contrariamente ao que era esperado, o tratamento prévio com chá de salva não protegeu a hepatotoxicidade induzida pelo tetracloreto de carbono (CC4) em ratinhos (capítulo 5). Em vez disso, ocorreu uma potenciação da toxicidade do CC4. Esta interacção pode ser explicada, pelo menos em parte, pelo aumento significativo induzido pelo chá da proteína CYP 2EI. Estes resultados chamam a atenção para possíveis interacções entre a salva e fármacos ou outros xenobióticos metabolizados pelo fígado. No entanto, isso provavelmente não acontecerá, a menos que uma elevada quantidade de salva for consumi da por um longo período de tempo. Em relação aos estudos das actividades antidiabéticas da salva, descritos no capítulo 6, verificou-se que o chá desta planta reduziu a glucose plasmática em jejum em ratinhos, indicando efeitos ao nível da gluconeogénese. Isto foi confirmado com experiências utilizando hepatócitos em cultura isolados de ratos, nos quais o chá de salva reduziu a indução da gluconeogénese pelo glucagon. Além disso, verificou-se que o chá de salva aumentou o consumo de glucose pelas células e que o OE aumentou a sensibilidade. das células à insulina e inibiu a gluconeogénese. No geral, estes efeitos assemelham-se aos obtidos com o fármaco metformina, usado no tratamento e prevenção da diabetes. No entanto, em hepatócitos isolados de ratos diabéticos induzidos pela estreptozotocina, estes efeitos não foram observados. Os resultados sugerem contudo, que a salva poderá ser utilizada na prevenção da diabetes tipo 2, principalmente em pessoas em risco de a desenvolver. Em conclusão, os resultados aqui apresentados confirmam os efeitos antioxidantes e hipoglicémicos de salva em sistemas biológicos, embora o seu uso terapêutico contra doenças do fígado e a diabetes necessite de mais investigação. No entanto, neste momento a salva pode ser encarada como um suplemento alimentar que poderá ter efeitos benéficos na prevenção, a baixo custo, da diabetes tipo 2 e de doenças do fígado. Para além disso, a prevenção de outro tipo de doenças também poderá ser considerada, principalmente aquelas onde é conhecido o envolvimento de stresse oxidativo, tal como o cancro e doenças neurodegenerativas. Por último, toma-se relevante relembrar que o consumo de salva, por períodos prolongados de tempo, em conjunto com certos fármacos poderá levar a efeitos indesejados devido a interacções planta-fármaco.Salvia officinalis L. (Lamiaceae, common sage) is an aromatic plant that grows in Portugal well known for its medical properties. Antioxidant and antidiabetic effects are among the medicinal properties attributed to this plant. However, both still lack of biological experimental confrnnation. Since sage compounds, after gastrointestinal absorption, first pass through the liver, givingan opportunity to accumulate to considerable concentrations, the antioxidant and antidiabetic studies reported here focused on the liver. In addition, the liver is a biotransforming organ, susceptible to toxic effects of xenobiotics and affected by several tiver ., diseases where oxidative stress is known to be involved. Therefore, these studies allowed us to observe, besides the benefits of sage consumption to the tiver, the possible toxic effects of sage and interactions with other drugs. Three different extracts of sage were studied: the essential oi! (EO), a methanolic extract and a water extract (prepared as a tea). In chapter 2, sage EO was tested in freshly isolated rat hepatocytes. No direct antioxidant effects were observed in tiver cells. On the contrary, at higher concentrations (more than 2 ~l/ml), sage EO induced significant cell death, which was accompanied by GSH. (glutathione - reduced form) depletion but not by tipid peroxidation. The GSH depletion induced by sage EO was probably an important mechanism that explains EO toxic effects. Then, after an in vivo experiment with mice, where sage tea drinking (for 14 days) improved the tiver antioxidant status (chapter 3), the direct effects of sage were studied on HepG2 cells (a human hepatoma cell tine) in chapter 4. Sage extracts protected against cell death induced by tert-butyl hydroperoxide - a model of oxidative stress - revealing their direct antioxidant effects at cellular leveI. Sage was able to act at a cell critical parameter, the GSH levels, preventing GSH depletion. The cytoprotective effect of sage was found to be dependent on its composition in phenotic compounds and their antiradical activity. AIso in chapter 4, not only did sage extracts prevent GSH depletion in a situation of oxidative stress, they also increased basal GSH levels in HepG2 cells. This fact indicated a capacity of sage extracts to improve basal cell antioxidant defences. The increase in basal GSH levels may possibly have happened by induction of de novo glutathione synthesis, which was corroborated by a different set of experiments using rat hepatocytes in culture (chapter 3). In that study, sage tea given in vivo restored hepatocyte GSH levels after collagenase isolation to higher levels when compared with controls. Sage tea was, in addi~ion, able to induce significantly the activities of GST (chapter 3), GPox and NADPH cytochrome P450 reductase and also, slightly, some CYP enzymes (chapter 5) in mice liver. These indirect antioxidant effects may leave liver cells better prepared to face oxidative stress and toxicants. However, contrarily to what was expected, sage tea drinking did not protect carbon tetrachloride (CCI4)-induced hepatotoxicity in mice (chapter 5). lnstead, sage tea potentiated the , toxicity of CCl4 in mice liver of both genders. This herb-toxicant interaction may be explained, at least in part, by the significant induction of CYP 2El protein by sage tea. Since the dose of sage tea used in this study was much higher than what is usually taken by humans, sage tea-drug interactions are not likely to happen in humans. Nevertheless, these results draw attention to possible herb-drug interactions between sage and drugs metabolised by the liver. Regarding the antidiabetic effects (chapter 6), sage tea was found to lower fasting plasma glucose in mice indicating effects on gluconeogenesis. This was confmned in the experiments using normal rat hepatocytes in culture, where sage tea drinking induced a decrease in gluconeogenesis in response to glucagon. ln addition, sage tea drinking increased glucose uptake capacity and, sage EO further increased hepatocyte sensitivity to insulin and inhibited gluconeogenesis. Overall, these effects resemble those obtained with the pharmaceutical drug metformin used in the treatment and prevention oftype 2 diabetes mellitus. ln primary cultures of hepatocytes isolated from streptozotocin-induced diabetic rats none of these activities, however, was observed. Nevertheless, the metformin-like effect observed here suggests a possible type 2 diabetes preventive potential of sage tea, mainly in people at risk of developing it. ln conclusion, the antioxidant effects of sage, at cellular and liver levels, were demonstrated by these results as well as its hypoglycaemic effects. ln order to apply sage products as a therapeutical tool for diabetes and liver diseases more research has, however, to be done. Nevertheless, sage products may now be considered as a functional food or a food supplements that could have a beneficial impact in low cost prevention strategies of diabetes and liver diseases. ln addition, the treatment and/or the prevention of other kind of diseases where oxidative stress is known to be involved, such as cancer and neurodegenerative diseases, may also benefit from the regular consumption of sage. Care should be taken, however, not to use high doses of sage products, over extended periods of time, in combination with conventional pharmaceutical drugs, since undesired herb-drugs interaction may take place.Ministério da Ciência e do Ensino Superior - Programa Operacional Ciência, Tecnologia e Inovação, Programa Operacional Ciência e Inovação 2010Fundação para a Ciência e a Tecnologia (FCT) - POCTI/AGR/43482/2001, POCI/AGR/62040/2004Fundo Social Europe

In vitro evaluation of bovine lactoferrin potential as an anticancer agent

"Available online 2 September 2014"Bovine lactoferrin (bLF) was shown to efficiently inhibit the growth of MCF-7, T-47D, MDA-MB-231 and Hs578T breast cancer cells in a concentration-dependent manner. However, apoptosis was only induced in MCF-7 cells, which was associated with the mitochondria membrane depolarisation and a decrease of Bcl-2 levels. bLF led to the cycle arrest of MCF-7 cells at G1/G0 phase, as well as a significant decrease in the expression of CDC25c. The possibility that the observed anticancer effects could be due to the high exogenous bLF concentrations in the culture media was excluded. Moreover, bLF was shown to restrain the colony formation of MCF-7 cells, although it promoted cell migration. This later effect was unspecific and related to the presence of a high protein concentration in the culture or medium. The results gathered in this work provide valuable insights for the evaluation and further study of the potential of bLF in cancer therapy.Zhang Y. acknowledges the Erasmus Mundus External Cooperation window (Bridging the Gap) for supporting his PhD grant. The authors acknowledge the financial support from the Strategic Projects PEst-OE/EQB/LA0023/2013 and PEst-OE/AGR/UI4033/2014, as well as the project ref. RECI/BBB-EBI/0179/2012 (project number FCOMP-01-0124-FEDER-027462) funded by Fundacao para a Ciencia e a Tecnologia, Portugal

Phenolic compounds protect HepG2 cells from oxidative damage : relevance of glutathione levels

Prova tipográfica (In Press)In the present work, the potential hepatoprotective effects of five phenolic compounds were evaluated against oxidative damages induced by tert-butyl hydroperoxide (t-BHP) in HepG2 cells in order to relate in vitro antioxidant activity with cytoprotective effects. t-BHP induced considerable cell damages to HepG2 cells as shown by significant LDH leakage, increased lipid peroxidation, DNA damage as well as decreased reduced glutathione (GSH) levels. All tested phenolic compounds significantly decreased cell death induced by t-BHP (when in co-incubation). If the effects of quercetin are given the reference value 1, the compounds rank in the following order according to inhibition of cell death: luteolin (4.0) > quercetin (1.0) > rosmarinic acid (0.34) > luteolin-7-glucoside (0.30) > caffeic acid (0.21). The results underscore the importance of the compound’s lipophilicity in addition to its antioxidant potential for its biological activity. All tested phenolic compounds were found to significantly decrease lipid peroxidation and prevent GSH depletion induced by t-BHP, but only luteolin and quercetin significantly decreased DNA damage. Therefore, the lipophilicity of the natural antioxidants tested appeared to be of even higher importance for DNA protection than for cell survival. The protective potential against cell death was probably achieved mainly by preventing intracellular GSH depletion. The phenolic compounds studied here showed protective potential against oxidative damages induced in HepG2 cells that could be beneficial against liver diseases where it is known that oxidative stress plays a crucial role.Fundação para a Ciência e Tecnologia -SFRH/BD/6942/2001; POCTI/AGR/62040/2004

Drinking of Salvia officinalis tea increases CCl4-induced hepatotoxicity in mice

In a previous study, the drinking of a Salvia officinalis tea (prepared as an infusion) for 14 days improved liver antioxidant status in mice and rats where, among other factors, an enhancement of glutathione-S-transferase (GST) activity was observed. Taking in consideration these effects, in the present study the potential protective effects of sage tea drinking against a situation of hepatotoxicity due to free radical formation, such as that caused by carbon tetrachloride (CCl4), were evaluated in mice of both genders. Contrary to what was expected, sage tea drinking significantly increased the CCl4-induced liver injury, as seen by increased plasma transaminase levels and histology liver damage. In accordance with the previous study, sage tea drinking enhanced significantly GST activity. Additionally, glutathione peroxidase was also significantly increased by sage tea drinking. Since CCl4 toxicity results from its bioactivation mainly by cytochrome P450 (CYP) 2E1, the expression level of this protein was measured by Western Blot. An increase in CYP 2E1 protein was observed which may explain, at least in part, the potentiation of CCl4-induced hepatotoxicity conferred by sage tea drinking. The CCl4-induced hepatotoxicity was higher in females than males. In conclusion, our results indicate that, although sage tea did not have toxic effects of its own, herb–drug interactions are possible and may affect the efficacy and safety of concurrent medical therapy with drugs that are metabolized by phase I enzymes.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/6942/2001, POCI/AGR/62040/200

Quercetin synergistically induces sensitivity to 5-fluorouracil through p53 modulation in colorectal cancer cells

Colorectal tumors (CRC) with microsatellite instability (MSI) show resistance to chemotherapy with 5-fluorouracil (5-FU), the most widely used pharmacological drug for CRC treatment. The aims of this study were to identify compounds that increase sensitivity of MSI CRC cells to 5-FU and characterize their dependence on the p53 status of the cells. Two MSI human CRC derived cell lines were used: CO115 wildtype for p53 and HCT15 that harbors a p53 mutation. The sensitivity of these cells to 5-FU was evaluated by TUNEL assay and the effects on apoptosis induction of co-incubation of the flavonoids, quercetin (Q) or luteolin (L), with 5-FU were characterized. The mechanisms of apoptosis induction were assessed by western blot and p53 mediated effects confirmed by small interference RNA (siRNA) in CO115 and in HCT116 wt and p53 knockout cells. Our results demonstrate that CO115 is more sensitive to 5-FU than the p53 mutated HCT15. Additive effects on apoptosis were shown for L (in both cell lines) and Q (in HCT15). In CO115 Q synergistically induced apoptosis with 5-FU. Apoptosis induction was caspase dependent in CO115 cells but not in HCT15 cells. Both flavonoids increased p53 expression in both cell lines, an effect particularly remarkable for Q. The synergistic effect of Q and 5-FU in CO115 involved the activation of the mitochondrial pathway with an increase in the expression of cleaved caspase 9 and 3 and PARP, as well as a decrease in Bcl-2 expression. Importantly, knockdown of p53 by siRNA in CO115 cells and p53 knockout in HCT116 cells totally abrogated apoptosis induction, demonstrating the dependence on p53 modulation of apoptosis induction by Q. This study suggests the potential applicability of these phytochemicals for enhancement 5-FU efficiency in CRC therapy, especially Q in p53 wild-type tumors.Fundação para a Ciência e a Tecnologia (FCT

Hypericum androsaemum water extract inhibits proliferation in human colorectal cancer cells through effects on MAP kinases and PI3K/Akt pathway

MAP kinase and PI3K/Akt signalling pathways are commonly altered in colorectal carcinoma (CRC) leading to tumor growth due to increased cell proliferation and inhibition of apoptosis. Several species of the genus Hypericum are used in Portugal to prepare herbal teas to which digestive tract effects are attributed. In the present study, the antiproliferative and proapoptotic effects of the water extracts of H. androsaemum (HA) and H. perforatum (HP) were investigated in two human colon carcinoma-derived cell lines, HCT15 and CO115, which harbour activating mutations of KRAS and BRAF, respectively. Contrarily to HP, HA significantly inhibited cell proliferation and induced apoptosis in both cell lines. HA decreased BRAF and phospho-ERK expressions in CO115, but not in HCT15. HA also decreased Akt phosphorylation in CO115 and induced p38 and JNK in both cell lines. HA induced cell cycle arrest at S and G2/M phases as well as caspase-dependent apoptosis in both cell lines. Chlorogenic acid (CA), the main phenolic compound present in the HA extract and less represented in the HP water extract, did, however, not show any of those effects when used individually. In conclusion, water extract of HA, but not of HP, controlled CRC proliferation and specifically acted on mutant and not wild-type BRAF. The effect of HA was, however, not due to CA alone.CPRX was supported by the Foundation for Science and Technology (FCT), Portugal, through the grant SFRH/BD/27524/2006 and the work was supported by the FCT research grants PTDC/AGR-AAM/70418/2006 (HypericumBiotech) and PEst-C/BIA/UI4050/2011. All projects are co-funded by the program COMPETE from QREN with co-participation from the European Community fund FEDER

Salvia fruticosa, salvia officinalis and rosmarinic acid induce apoptosis and inhibit proliferation of human colorectal cell lines: the role in MAPK/ERK pathway

Epidemiologic studies have shown that nutrition is a key factor in modulating sporadic colorectal carcinoma (CRC) risk. Aromatic plants of the genus Salvia (sage) have been attributed many medicinal properties, which include anticancer activity. In the present study, the antiproliferative and pro-apoptotic effects of water extracts of Salvia fruticosa (SF) and Salvia officinalis (SO) and of their main phenolic compound rosmarinic acid (RA) were evaluated in two human colon carcinoma-derived cell lines, HCT15 and CO115, which have different mutations in the MAPK/ERK and PI3K/Akt signalling pathways. These pathways are commonly altered in CRC leading to increased proliferation and inhibition of apoptosis. Our results show that SF, SO and RA induce apoptosis in both cell lines, whereas cell proliferation was inhibited by the two sage extracts only in HCT15. SO, SF and RA inhibited ERK phosphorylation in HCT15 and had no effects on Akt phosphorylation in CO115 cells. The activity of sage extracts seems to be due, at least in part, to the inhibition of MAPK/ERK pathway.POCI/AGR/62040/2004. CPRX and CFLSFRH/BD/27524/2006 and SFRH/BPD/26316/2006Foundation for Science and Technology (FCT

Keratin-based peptide: biological evaluation and strengthening properties on relaxed hair

A peptide based on a fragment of hair keratin type II cuticular protein, keratin peptide (KP), was studied as a possible strengthening agent for weakened relaxed hair. The peptide was prepared both in aqueous water formulation (WF) and organic solvent formulations (OF), to determine the effect of organic solvents on peptide interaction with hair and the differences in hair recovery. Both peptide formulations were shown to improve mechanical and thermal properties of weakened hair with peptide in OF showing the stronger effect. As a potential new hair care product, and so would necessitate contact with skin, the cytotoxicity and genotoxicity of the peptide were also evaluated through different methodologies (Alamar Blue assay, 2′-7′-dichlorofluorescein probe, cell morphology and growth and evaluation of DNA damage by an alkaline version of the comet assay) in skin fibroblasts. These tests are indicators of the potential of peptide to cause irritation on skin or to be carcinogenic, respectively. The peptide in WF did not cause cytotoxicity or genotoxicity in any of the concentrations tested. The presence of OF, however, induced a 20% decrease in cell viability in all of the range of concentrations used after 72-h incubation. Moreover, OF inhibited cell growth and was considered genotoxic at first contact with cells. The peptide was therefore considered a promising strengthening agent for hair and was shown to be innocuous when applied in WF.The work was supported by a PhD Grant Fellowship (scholarship SFRH/BD/38363/2007) from FCT "Fundacao para a Ciencia e Tecnologia", Portugal. The authors have declared no conflict of interest

Ursolic acid, a dietary phytochemical, decreases KRAS signaling and modulates cell death pathways in resistant CRC cells

Publicado em "BMC Proceedings 2012, 6(Suppl 3)"KRAS mutations are frequent in colorectal cancer (CRC) and have the potential to activate proliferation and inhibit cell death through effects on MAPK/ERK and PI3K/Akt signaling pathways. Because diet is one of the most important determinants of CRC incidence and progression, we studied the effects of the dietary triterpenoid ursolic acid (UA) on proliferation and cell death induction in human CRC derived KRAS mutated cell lines. Our results show that UA decreases cell proliferation and induces cell death while decreasing signaling through KRAS as indicated by a decrease in ERK and Akt phosphorylation (western blot). UA also induced cell death. TP53 mutated cells are known to be resistant to the chemotherapeutic drug 5-FU. Caspase independent apoptosis (Tunel assay), was increased 6 fold by co-incubation of UA with 5-FU. However, apoptosis was only a small percentage of the total cell death induced by UA. In order to explain these observations, we looked into effects on autophagy. Autophagy is emerging as a promising therapeutic target for drug resistant tumors. UA modulated autophagy by inducing the accumulation of LC3 II and p62 levels an effect dependent on JNK activation. In conclusion, this study shows UA’s anticancer potential as a modulator of KRAS signaling and cell death mechanisms increasing sensitivity to the chemotherapeutic drug 5-FU