Alice A. Ramos

Antigenotoxic effects of quercetin, rutin and ursolic acid on HepG2 cells: Evaluation by the comet assay

In the present study, the chemoprotective effects of quercetin, rutin and ursolic acid on tert-butyl hydroperoxide (t-BHP)-induced DNA damage in a human hepatoma cell line (HepG2) were investigated by the comet assay. To determine whether protection was due to direct chemical interactions alone or to cellular-mediated responses three different types of treatments were used: simultaneous incubation of cells with individual test compounds and the toxicant; pre-treatment with test compound before addition of the toxicant followed or not by a recovery period. The expression of Hsp70 was quantified by Western blotting to test the involvement of heat shock proteins in the cellular responses to the test compounds. In addition, effects on proliferation were evaluated by the MTT assay. The results show that quercetin and ursolic acid prevented DNA damage and had antiproliferative properties in HepG2 cells suggesting an anticarcinogenic potential for these compounds. The protective effects of quercetin against t-BHP-induced DNA damage seem to be due to both direct effects on t-BHP toxicity and to cellularly mediated indirect effects which reflect the potentiation of the cellular antioxidant defenses. Ursolic acid seems to exert effects only through cellularly mediated mechanisms since it was not protective in simultaneous incubation. Quercetin and ursolic acid also showed to increase the rate of DNA repair. Rutin did not have effects at any level. These results, obtained with liver cells, emphasize and confirm the chemopreventive potential of quercetin and ursolic acid, which may help explain the lower cancer incidence in human population with high dietary intakes of fruits and vegetables. These results also demonstrate that Hsp70 is not involved in the observed effects in HepG2.

Protective effects of Ursolic acid and Luteolin against oxidative DNA damage include enhancement of DNA repair in Caco-2 cells

Consumption of fruits and vegetables is associated with a reduced risk of developing a wide range of cancers including colon cancer. In this study, we evaluated the effects of two compounds present in fruits and vegetables, ursolic acid, a triterpenoid, and luteolin, a flavonoid, on DNA protection and DNA repair in Caco-2 cells using the comet assay. Ursolic acid and luteolin showed a protective effect against H(2)O(2)-induced DNA damage. Repair rate (rejoining of strand breaks) after treatment with H(2)O(2) was increased by pre-treatment of Caco-2 cells for 24h with ursolic acid or luteolin. To evaluate effects on induction of base oxidation, we exposed cells to the photosensitizer Ro 19-8022 plus visible light to induce 8-oxoguanine. Luteolin protected against this damage in Caco-2 cells after a short period of incubation. We also measured the incision activity of a cell extract from Caco-2 cells treated for 24h with test compounds, on a DNA substrate containing specific damage (8-oxoGua), to evaluate effects on base excision repair activity. Preincubation for 24h with ursolic acid enhanced incision activity in Caco-2 cells. In conclusion, we demonstrated for the first time that ursolic acid and luteolin not only protect DNA from oxidative damage but also increase repair activity in Caco-2 cells. These effects of ursolic acid and luteolin may contribute to their anti-carcinogenic effects.

Polyphenolic Compounds from Salvia Species Protect Cellular DNA from Oxidation and Stimulate DNA Repair in Cultured Human Cells

DNA damage can lead to carcinogenesis if replication proceeds without proper repair. This study evaluated the effects of the water extracts of three Salvia sp., Salvia officinalis (SO), Salvia fruticosa (SF), and Salvia lavandulifolia (SL), and of the major phenolic constituents, rosmarinic acid (RA) and luteolin-7-glucoside (L-7-G), on DNA protection in Caco-2 and HeLa cells exposed to oxidative agents and on DNA repair in Caco-2 cells. The comet assay was used to measure DNA damage and repair capacity. The final concentration of each sage extract was 50 microg/mL, and concentrations of RA and L-7-G were 50 and 20 microM, respectively. After a short incubation (2 h), L-7-G protected DNA in Caco-2 cells from damage induced by H(2)O(2) (75 microM); also, after a long incubation (24 h), SF, RA, and L-7-G had protective effects in Caco-2 cells. In HeLa cells, SO, SF, and RA protected against damage induced by H(2)O(2) after 24 h of incubation. Assays of DNA repair show that SO, SF, and L-7-G increased the rate of DNA repair (rejoining of strand breaks) in Caco-2 cells treated with H(2)O(2). The incision activity of a Caco-2 cell extract on a DNA substrate containing specific damage (8-oxoGua) was also measured to evaluate effects on base excision repair (BER) activity. Preincubation for 24 h with SO and L-7-G had a BER inductive effect, increasing incision activity in Caco-2 cells. In conclusion, SO, SF, and the isolated compounds (RA and L-7-G) demonstrated chemopreventive activity by protecting cells against oxidative DNA damage and stimulating DNA repair (SO, SF, and L-7-G).

Sage Tea Drinking Improves Lipid Profile and Antioxidant Defences in Humans

Salvia officinalis (common sage) is a plant with antidiabetic properties. A pilot trial (non-randomized crossover trial) with six healthy female volunteers (aged 40–50) was designed to evaluate the beneficial properties of sage tea consumption on blood glucose regulation, lipid profile and transaminase activity in humans. Effects of sage consumption on erythrocytes’ SOD and CAT activities and on Hsp70 expression in lymphocytes were also evaluated. Four weeks sage tea treatment had no effects on plasma glucose. An improvement in lipid profile was observed with lower plasma LDL cholesterol and total cholesterol levels as well as higher plasma HDL cholesterol levels during and two weeks after treatment. Sage tea also increased lymphocyte Hsp70 expression and erythrocyte SOD and CAT activities. No hepatotoxic effects or other adverse effects were observed.

Protection by Salvia Extracts Against Oxidative and Alkylation Damage to DNA in Human HCT15 and CO115 Cells

DNA damage induced by oxidative and alkylating agents contributes to carcinogenesis, leading to possible mutations if replication proceeds without proper repair. However, some alkylating agents are used in cancer therapy due to their ability to induce DNA damage and subsequently apoptosis of tumor cells. In this study, the genotoxic effects of oxidative hydrogen peroxide (H2O2) and alkylating agents N-methyl-N-nitrosourea (MNU) and 1,3-bis-(2-chloroethyl)-1-nitosourea (BCNU) agents were examined in two colon cell lines (HCT15 and CO115). DNA damage was assessed by the comet assay with and without lesion-specific repair enzymes. Genotoxic agents were used for induction of DNA damage in both cell lines. Protective effects of extracts of three Salvia species, Salvia officinalis (SO), Salvia fruticosa (SF), and Salvia lavandulifolia (SL), against DNA damage induced by oxidative and alkylating agents were also determined. SO and SF protected against oxidative DNA damage in HCT15 cells. SO and SL decreased DNA damage induced by MNU in CO115 cells. In addition to chemopreventive effects of sage plant extracts, it was also important to know whether these plant extracts may interfere with alkylating agents such as BCNU used in cancer therapy, decreasing their efficacy. Our results showed that sage extracts tested and rosmarinic acid (RA), the main constituent, protected CO115 cells from DNA damage induced by BCNU. In HCT15 cells, only SF induced a reduction in BCNU-induced DNA damage. Sage water extracts and RA did not markedly change DNA repair protein expression in either cell line. Data showed that sage tea protected colon cells against oxidative and alkylating DNA damage and may also interfere with efficacy of alkylating agents used in cancer therapy.

Water extracts of tree Hypericum sps. protect DNA from oxidative and alkylating damage and enhance DNA repair in colon cells

Diet may induce colon carcinogenesis through oxidative or alkylating DNA damage. However, diet may also contain anticarcinogenic compounds that contribute to cancer prevention. DNA damage prevention and/or induction of repair are two important mechanisms involved in cancer chemoprevention by dietary compounds. Hypericum sps. are widely used in traditional medicine to prepare infusions due to their beneficial digestive and neurologic effects. In this study, we investigated the potential of water extracts from three Hypericum sps. and some of their main phenolic compounds to prevent and repair oxidative and alkylating DNA damage in colon cells. The results showed that water extracts of Hypericum perforatum, Hypericum androsaemum, Hypericum undulatum, quercetin and rutin have protective effect against oxidative DNA damage in HT29 cells. Protective effect was also observed against alkylating DNA damage induced by methyl-methanesulfonate, except for H. androsaemum. With regard to alkylating damage repair H. perforatum, H. androsaemum and chlorogenic acid increased repair of alkylating DNA damage by base excision repair pathway. No effect was observed on nucleotide excision repair pathway. Antigenotoxic effects of Hypericum sps. may contribute to colon cancer prevention and the high amount of phenolic compounds present in Hypericum sps. play an important role in DNA protective effects.

DNA Damage Protection and Induction of Repair by Dietary Phytochemicals and Cancer Prevention: What Do We Know?

DNA damage accumulates in cells over time as a result of exposure to a variety of exogenous and endogenous agents. These damages, if not repaired properly, can generate mutations in somatic or germline cells, which are involved in the pathogenesis of many diseases, such as cancer. To maintain genomic integrity generation after generation, organisms possess multiple mechanisms such as inhibition of carcinogen uptake into the cells, induction of detoxification enzymes, increased cellular defenses that prevent DNA damage, enhancement of DNA repair, increased anti-inflammatory activity, inhibition of cell proliferation, and modulation of apoptosis through effects on signal transduction pathways (de Kok et al., 2008); (Pan et al., 2008). In the last decades, several dietary constituents have been shown to modulate all these processes. Epidemiological studies as well as laboratory data suggest that consumption of fruits and vegetables is associated with a reduced risk of developing a wide range of cancers. It has been estimated that 3040% of all tumours can be prevented with a correct lifestyle and diet, in particular colon cancer (Rajamanickam and Agarwal, 2008). Multiple mechanisms have been proposed to explain the chemopreventive effects of phytochemicals. Protection of DNA from damage and modulation of DNA repair assume an important role on prevention of mutations and consequently of the carcinogenic process. The comet assay or single cell gel electrophoresis (SCGE) assay is a rapid, sensitive and relatively simple method for assessing DNA damage and its repair in individual cells. The standard comet assay measures DNA breaks and alkali-labile sites that are converted to strand breaks. With its widespread use, several modifications on the comet assay have been made that allow the quantification of other types of DNA damage as well as DNA repair rates. With the inclusion of an extra step on the comet assay by using specific DNA repair enzymes, different base lesions can be identified by the introduction of breaks at sites of base damage. In this regard, Endonuclease III, FPG and AlkA have been used to detect oxidized pyrimidines, modified purines and alkylpurines bases, respectively. With these

Modulation of DNA damage prevention and signaling pathways in diet induced colon cancer prevention

Colorectal cancer (CRC) is a common malignancy and significant cause of mortality in Western societies. It develops through an accumulation of genetic and epigenetic alterations, transforming normal colon cells and giving them growth advantage. Epigenetic alterations are reversible and studies have shown that dietary compounds can alter the epigenetic status and reactivate epigenetically-silenced genes. Many food plants are rich in bioactive compounds and have shown to posses anticancer properties. We proposed to explore the effects of sage (Salvia officinalis (SO)) water extract (herbal tea) drinking on colon cancer prevention and modulation of epigenetic events. F344 rats were used to study the effects of sage tea drinking on pre-initiation (SO treatment before AOM exposure) and post-initiation (SO after AOM exposure) phases of carcinogenesis. We found a chemopreventive effect of SO in the pre-initiation group, but not in the post-initiation. We then investigated if SO affected AOM metabolism, searching for effects on CYP2E1 expression and activity. We found that AOM decreased CYP2E1 activity when compared with control, but SO treatment before AOM prevented this effect. The capacity of SO in vivo treatment to protect colonocytes from H2O2 damage induced in vitro was also investigated. SO decreased significantly the oxidative H2O2-induced DNA damage. We also are searching for alterations in expression of key proteins involved in signalling pathways important for cell proliferation or apoptosis and proteins involved in DNA repair. Sage water extract seems to have the ability to prevent CRC and studies to further explore this potential are ongoing.

Development of a new application of the comet assay to assess levels of O6-methylguanine in genomic DNA (CoMeth)

O(6)-methylguanine (O(6)meG) is one of the most premutagenic, precarcinogenic, and precytotoxic DNA lesions formed by alkylating agents. Repair of this DNA damage is achieved by the protein MGMT, which transfers the alkyl groups from the O(6) position of guanine to a cysteine residue in its active center. Because O(6)meG repair by MGMT is a stoichiometric reaction that irreversibly inactivates MGMT, which is subsequently degraded, the repair capacity of O(6)meG lesions is dependent on existing active MGMT molecules. In the absence of active MGMT, O(6)meG is not repaired, and during replication, O(6)meG:T mispairs are formed. The MMR system recognizes these mispairs and introduces a gap into the strand. If O(6)meG remains in one of the template strands the futile MMR repair process will be repeated, generating more strand breaks (SBs). The toxicity of O(6)meG is, therefore, dependent on MMR and DNA SB induction of cell death. MGMT, on the other hand, protects against O(6)meG toxicity by removing the methyl residue from the guanine. Although removal of O(6)meG makes MGMT an important anticarcinogenic mechanism of DNA repair, its activity significantly decreases the efficacy of cancer chemotherapeutic drugs that aim at achieving cell death through the action of the MMR system on unrepaired O(6)meG lesions. Here, we report on a modification of the comet assay (CoMeth) that allows the qualitative assessment of O(6)meG lesions after their conversion to strand breaks in proliferating MMR-proficient cells after MGMT inhibition. This functional assay allows the testing of compounds with effects on O(6)meG levels, as well as on MGMT or MMR activity, in a proliferating cell system. The expression of MGMT and MMR genes is often altered by promoter methylation, and new epigenetically active compounds are being designed to increase chemotherapeutic efficacy. The CoMeth assay allows the testing of compounds with effects on O(6)meG, MGMT, or MMR activity. This proliferating cell system complements other methodologies that look at effects on these parameters individually through analytical chemistry or in vitro assays with recombinant proteins.

Drug resistance in glioblastoma and cytotoxicity of seaweed compounds, alone and in combination with anticancer drugs: A mini review

BACKGROUND Glioblastomas (GBM) are one of the most aggressive tumor of the central nervous system with an average life expectancy of only 1-2 years after diagnosis, even with the use of advanced treatments with surgery, radiation, and chemotherapy. There are several anticancer drugs with alkylating properties that have been used in the therapy of malignant gliomas. Temozolomide (TMZ) is one of them, widely used even in combination with ionizing radiation. However, the main disadvantage of using these types of drugs in the treatment of GBM is the development of cancer drug resistance. Research of bioactive compounds with anticancer activity has been heavily explored. PURPOSE This review focuses on a carotenoid and a phlorotannin present in seaweed, namely fucoxanthin and phloroglucinol, and their anticancer activity against glioblastoma. The combination of natural compounds with conventional drugs is also discussed. CONCLUSION Several natural compounds existing in seaweeds, such as fucoxanthin and phoroglucinol, have shown cytotoxic activity in models in vitro and in vivo, acting through different molecular mechanisms, such as antioxidant, antiproliferative, DNA damage/DNA repair, proapoptotic, antiangiogenic and antimetastic. Within the scope of interactions with conventional drugs, there are evidences that some seaweed compounds could be used to potentiate the action of anticancer drugs. However, their effects and mechanisms of action, alone or in combination with anticancer drugs, namely TMZ, in glioblastoma cell, still few explored and require more attention due to the unquestionable high potential of these marine compounds.