Trilobolide-6-O-isobutyrate from Sphagneticola trilobata acts by inducing oxidative stress, metabolic changes and apoptosis-like processes by caspase 3/7 activation of human lung cancer cell lines.

BACKGROUND: Lung cancer, a chronic and heterogeneous disease, is the leading cause of cancer-related death on a global scale. Presently, despite a variety of available treatments, their effectiveness is limited, often resulting in considerable toxicity and adverse effects. Additionally, the development of chemoresistance in cancer cells poses a challenge. Trilobolide-6-O-isobutyrate (TBB), a natural sesquiterpene lactone extracted from Sphagneticola trilobata, has exhibited antitumor effects. Its pharmacological properties in NSCLC lung cancer, however, have not been explored. PURPOSE: This study evaluated the impact of TBB on the A549 and NCI-H460 tumor cell lines in vitro, examining its antiproliferative properties and initial mechanisms of cell death. METHODS: TBB, obtained at 98 % purity from S. trilobata leaves, was characterized using chromatographic techniques. Subsequently, its impact on inhibiting tumor cell proliferation in vitro, TBB-induced cytotoxicity in LLC-MK2, THP-1, AMJ2-C11 cells, as well as its effects on sheep erythrocytes, and the underlying mechanisms of cell death, were assessed. RESULTS: In silico predictions have shown promising drug-likeness potential for TBB, indicating high oral bioavailability and intestinal absorption. Treatment of A549 and NCI-H460 human tumor cells with TBB demonstrated a direct impact, inducing significant morphological and structural alterations. TBB also reduced migratory capacity without causing toxicity at lower concentrations to LLC-MK2, THP-1 and AMJ2-C11 cell lines. This antiproliferative effect correlated with elevated oxidative stress, characterized by increased levels of ROS, superoxide anion radicals and NO, accompanied by a decrease in antioxidant markers: SOD and GSH. TBB-stress-induced led to changes in cell metabolism, fostering the accumulation of lipid droplets and autophagic vacuoles. Stress also resulted in compromised mitochondrial integrity, a crucial aspect of cellular function. Additionally, TBB prompted apoptosis-like cell death through activation of caspase 3/7 stressors. CONCLUSION: These findings underscore the potential of TBB as a promising candidate for future studies and suggest its viability as an additional component in the development of novel anticancer drugs prototypes.

Cytogenotoxic evaluations of leaves and stems extracts of Rubus rosifolius in primary metabolically noncompetent cells.

Plants with medicinal potential may also produce adverse effects in humans. This seems to be the case for the species Rubus rosifolius, where preliminary studies demonstrated genotoxic effects attributed to extracts obtained from leaves and stems of this plant using on HepG2/C3A human hepatoma cells as a model. Considering the beneficial properties of this plant as an antidiarrheal, analgesic, antimicrobial, and antihypertensive and its effects in the treatment of gastrointestinal diseases, the present study was developed with the aim of determining the cytotoxic and genotoxic potential of extracts of leaves and stems of R. rosifolius in primary without metabolic competence in human peripheral blood mononuclear cells (PBMC). Cell viability analyses at concentrations of between 0.01 and 100 µg/ml of both extracts did not markedly affect cell viability. In contrast, assessment of the genotoxic potential using the comet assay demonstrated significant damage to DNA within PBMC from a concentration of 10 µg/ml in the stem extract, and a clastogenic/aneugenic response without cytokinesis-block proliferation index (CBPI) alterations at concentrations of 10, 20, or 100 µg/ml for both extracts. Under our experimental conditions, the data obtained demonstrated genotoxic and mutagenic effects attributed to extracts from leaves and stems of R. rosifolius in cells in the absence of hepatic metabolism.

Melatonin modulates the Warburg effect and alters the morphology of hepatocellular carcinoma cell line resulting in reduced viability and migratory potential.

AIMS: Hepatocellular Carcinoma (HCC) is a primary neoplasm derived from hepatocytes with low responsiveness and recurrent chemoresistance. Melatonin is an alternative agent that may be helpful in treating HCC. We aimed to study in HuH 7.5 cells whether melatonin treatment exerts antitumor effects and, if so, what cellular responses are induced and involved. MAIN METHODS: We evaluated the effects of melatonin on cell cytotoxicity and proliferation, colony formation, morphological and immunohistochemical aspects, and on glucose consumption and lactate release. KEY FINDINGS: Melatonin reduced cell motility and caused lamellar breakdown, membrane damage, and reduction in microvillus. Immunofluorescence analysis revealed that melatonin reduced TGF and N-cadherin expression, which was further associated with inhibition of epithelial-mesenchymal transition process. In relation to the Warburg-type metabolism, melatonin reduced glucose uptake and lactate production by modulating intracellular lactate dehydrogenase activity. SIGNIFICANCE: Our results indicate that melatonin can act upon pyruvate/lactate metabolism, preventing the Warburg effect, which may reflect in the cell architecture. We demonstrated the direct cytotoxic and antiproliferative effect of melatonin on the HuH 7.5 cell line, and suggest that melatonin is a promising candidate to be further tested as an adjuvant to antitumor drugs for HCC treatment.

Rubus rosifolius (Rosaceae) stem extract induces cell injury and apoptosis in human hepatoma cell line.

Rubus rosifolius, popularly known as "red mulberry", is a common medicinal plant in southern Brazil and is used as an antidiarrheal, analgesic, antimicrobial and antihypertensive, and to treat stomach diseases. The aim of this study was to analyze the R. rosifolius stem extract (RrSE) for possible in vitro cytotoxic and genotoxic effects, using the comet assay and the micronucleus test to assess genotoxicity, and flow cytometry to assess the impact on the cell cycle and apoptosis in HepG2/C3A cells, in addition to evaluating the expression of genes linked to the induction of DNA damage, cell cycle, apoptosis and metabolism of xenobiotics. The MTT assay observed no cytotoxic effects at concentrations between 0.01 and 100 µg/mL of the extract. However, genotoxic effects occurred in treatments with the extract from a 1 µg/mL concentration. Flow cytometry analysis revealed a significant increase in cells in the G2/M phase after treatment with 10 µg/mL, a decrease in cells in the G0/G1 phase in the treatment with 100 µg/mL, and a significant increase in total apoptotic cells. In the gene expression analysis, an increase in the CYP1A2 xenobiotics metabolizing gene expression was observed. Despite the promising pharmacological effects of R. rosifolius, the results revealed that the RrSE has genotoxic effect and induces apoptosis in HepG2/C3A cells, indicating danger in using this plant extract by humans.

Effect of probiotic supplementation on plasma metabolite profile after Roux-Y gastric bypass: a prospective, randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND/OBJECTIVE: There is evidence that metabolic profile changes after Roux-Y gastric bypass (RYGB), especially due to modifications in the gastrointestinal tract. In addition, previous studies have suggested that probiotics can modify the microbiome and produce metabolites important for metabolic health maintenance. In this sense, the aim of this study was to verify the influence of probiotic supplementation on the plasma metabolite profile after RYGB. METHODS: This was a randomized, double-blind, placebo-controlled clinical trial conducted with 31 patients subjected to RYGB surgery, randomized in probiotic group that was supplemented with a probiotic supplement (FloraVantage®) for 3 months after surgery or a placebo group. Plasma metabonomics was performed using nuclear magnetic resonance (NMR) at the preoperative period (T0) and at 45-50 days (T1) and 90-95 days (T2) during the postoperative period/intervention. RESULTS: Reductions in trimethylamine-N-oxide (TMAO) and alanine were observed in both groups, however this reduction was greater in the probiotic group (TMAO 13.82%, p = 0.01 and alanine 14.03%, p = 0.03) at T2. Additionally, ß-hydroxybutyrate (BHB) levels increased 10.77% in the probiotic group (p = 0.03) compared to the placebo group at T2. CONCLUSION: Supplementation with Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 was able to associate with significant differences in relevant plasma metabolites associated with improved metabolic health.

Trans-chalcone induces death by autophagy mediated by p53 up-regulation and ß-catenin down-regulation on human hepatocellular carcinoma HuH7.5 cell line.

BACKGROUND: Hepatocellular Carcinoma (HCC) is extremely aggressive and presents low rates of response to the available chemotherapeutic agents. Many studies have focused on the search for alternative low-cost natural compounds with antiproliferative potential that selectively respond to liver cancer cells. PURPOSE: This study assessed the in vitro direct action of trans-chalcone (TC) on cells of the human HCC HuH7.5 cell line. METHODS: We subjected the HuH7.5 tumor cells to TC treatment at increasing concentrations (12.5-100 µM) for 24 and 48 h. Cell viability was verified through MTT and 50% inhibitory concentration of cells (IC50 23.66 µM) was determined within 48 h. We quantified trypan blue proliferation and light microscopy, ROS production, mitochondrial depolarization and autophagy, cell cycle analysis, and apoptosis using Muse® cell analyzer and immunocytochemical markings of p53 and ß-catenin. RESULTS: Data showed an effective dose- and time-dependent TC-cytotoxic action at low micromolar concentrations without causing toxicity to non-cancerous cells, such as erythrocytes. TC-treatment caused mitochondrial membrane damage and cell cycle G0/G1 phase arrest, increasing the presence of the p53 protein and decreasing ß-catenin, in addition, to inducing cell death by autophagy. Additionally, TC decreased the metastatic capacity of HuH7.5, which affected the migration/invasion of this type of cell. CONCLUSION: In vitro TC activity in the human HCC HuH7.5 tumor cell line is shown to be a potential molecule to develop new therapies to repair the p53 pathway and prevent the overexpression of Wnt/ß-catenin tumor development inducing autophagy cell death and decreasing metastatic capacity of HuH7.5 cell line.

Risk assessment via genotoxicity, metabolism, apoptosis, and cell growth effects in a HepG2/C3A cell line upon treatment with Rubus rosifolius (Rosaceae) leaves extract.

RUBUS ROSIFOLIUS: Sm. (Rosaceae) is a plant traditionally used in Brazil and some other countries to treat diarrhea, stomach diseases, and as an analgesic, antimicrobial, antihypertensive, and as well as other pharmacological properties. The aim of this study was to examine cytotoxic and genotoxic effects of R. rosifolius leaves extract on HepG2/C3A cells and correlate these findings with the expression of mRNA to underlying mechanisms of action. At concentrations between 0.01 and 100 µg/ml, cytotoxic effects were not detected by the MTT assay. This was confirmed by mRNA induction of the CYP3A4 gene (by RT-qPCR assay). However, genotoxic effects occurred at treatments from 1 µg/ml extract (comet and micronucleus test). An increase in the number of cells in S phase was observed at 100 µg/ml, and an elevation in apoptotic cell number was found for all tested concentrations (10, 20, or 100 µg/ml) (cell cycle and apoptosis analysis by flow cytometry). The genotoxicity induced by the extract was the main cause of the rise in the number of cells undergoing apoptosis, as indicated by rise in mRNA of CASP7 gene, and elevation of cells in the S phase of the cell cycle at the higher tested concentrations, as an attempt to repair genetic damage that occurred. These observations suggest that, despite its pharmacological potential, the use of R. rosifolius leaves extract may pose a risk to the integrity of the genetic material of human cells.

In vitro evaluation of the protective effects of plant extracts against amyloid-beta peptide-induced toxicity in human neuroblastoma SH-SY5Y cells.

Alzheimer's disease (AD) is the most common form of dementia and has no cure. Therapeutic strategies focusing on the reduction of oxidative stress, modulation of amyloid-beta (Aß) toxicity and inhibition of tau protein hyperphosphorylation are warranted to avoid the development and progression of AD. The aim of this study was to screen the crude extracts (CEs) and ethyl-acetate fractions (EAFs) of Guazuma ulmifolia, Limonium brasiliense, Paullinia cupana, Poincianella pluviosa, Stryphnodendron adstringens and Trichilia catigua using preliminary in vitro bioassays (acetylcholinesterase inhibition, antioxidant activity and total polyphenol content) to select extracts/fractions and assess their protective effects against Aß25-35 toxicity in SH-SY5Y cells. The effect of the EAF of S. adstringens on mitochondrial membrane potential, lipid peroxidation, superoxide production and mRNA expression of 10 genes related to AD was also evaluated and the electropherogram fingerprints of EAFs were established by capillary electrophoresis. Chemometric tools were used to correlate the in vitro activities of the samples with their potential to be evaluated against AD and to divide extracts/fractions into four clusters. Pretreatment with the EAFs grouped in cluster 1 (S. adstringens, P. pluviosa and L. brasiliense) protected SH-SY5Y cells from Aß25-35-induced toxicity. The EAF of S. adstringens at 15.62 µg/mL was able completely to inhibit the mitochondrial depolarization (69%), superoxide production (49%) and Aß25-35-induced lipid peroxidation (35%). With respect to mRNA expression, the EAF of S. adstringens also prevented the MAPT mRNA overexpression (expression ratio of 2.387x) induced by Aß25-35, which may be related to tau protein hyperphosphorylation. This is the first time that the neuroprotective effects of these fractions have been demonstrated and that the electropherogram fingerprints for the EAFs of G. ulmifolia, L. brasiliense, P. cupana, P. pluviosa and S. adstringens have been established. The study expands knowledge of the in vitro protective effects and quality control of the evaluated fractions.

Effects of folic acid on the antiproliferative efficiency of doxorubicin, camptothecin and methyl methanesulfonate in MCF-7 cells by mRNA endpoints.

There is a lack of consensus on whether the role of folate in cancer cells is protective or harmful. The use of folates in combination with cancer-targeting therapeutic regimens requires detailed information to ensure their safe and proper use. Therefore, we evaluated the effects of folic acid (FA) in combination with the chemotherapeutic compounds doxorubicin (DXR), camptothecin (CPT) and methyl methanesulfonate (MMS) on the growth of MCF-7 cells. The data generated from the RTCA assays demonstrated that FA did not affect proliferation in MCF-7 cells treated with DXR and CPT; however, FA reduced the efficacy of MMS treatment. RTCA data also confirmed that DXR and CPT exert their cytotoxic effects in a time-dependent manner and that CPT induced a significantly greater decrease in MCF-7 cell proliferation compared with DXR. The MTT assay failed to detect a reduction in cell proliferation in cells treated with MMS. We quantified the mRNA expression levels of genes associated with cellular stress response, cell cycle and apoptosis pathways using RT-qPCR. The addition of FA to DXR or CPT promoted a similar shift in the gene expression profile of MCF-7 cells compared with cells treated with DXR or CPT without FA; however, this shift did not alter the bioactivity of these drugs. Rather, it indicated that these drugs promoted cell death by alternative mechanisms. In contrast, the addition of FA to MMS reduced the efficacy of the drug without changing the gene expression profile. None of the genes encoding folate receptors that were analyzed were differentially expressed in cells treated with or without FA. In conclusion, supplementation with 450 µM FA was not cytotoxic to MCF-7 cells. However, the addition of FA to anti-cancer drugs must be performed cautiously as the properties of FA might lead to a reduction in drug efficacy.

Role of 1α,25-Dihydroxyvitamin D3 in Adipogenesis of SGBS Cells: New Insights into Human Preadipocyte Proliferation.

BACKGROUND/AIMS: Compared with non-obese individuals, obese individuals commonly store more vitamin D in adipose tissue. VDR expression in adipose tissue can influence adipogenesis and is therefore a target pathway deserving further study. This study aims to assess the role of 1,25(OH)2D3 in human preadipocyte proliferation and differentiation. METHODS: RTCA, MTT, and trypan blue assays were used to assess the effects of 1,25(OH)2D3 on the viability, proliferation, and adipogenic differentiation of SGBS cells. Cell cycle and apoptosis analyses were performed with flow cytometry, triglycerides were quantified, and RT-qPCR was used to assess gene expression. RESULTS: We confirmed that the SGBS cell model is suitable for studying adipogenesis and demonstrated that the differentiation protocol induces cell maturation, thereby increasing the lipid content of cells independently of treatment. 1,25(OH)2D3 treatment had different effects according to the cell stage, indicating different modes of action driving proliferation and differentiation. In preadipocytes, 1,25(OH)2D3 induced G1 growth arrest at both tested concentrations without altering CDKN1A gene expression. Treatment with 100 nM 1,25(OH)2D3 also decreased MTT absorbance and the lipid concentration. Moreover, increased normalized cell index values and decreased metabolic activity were not induced by proliferation or apoptosis. Exposure to 100 nM 1,25(OH)2D3 induced VDR, CEBPA, and CEBPB expression, even in the preadipocyte stage. During adipogenesis, 1,25(OH)2D3 had limited effects on processes such as VDR and PPARG gene expression, but it upregulated CEBPA expression. CONCLUSIONS: We demonstrated for the first time that 1,25(OH)2D3 induces changes in preadipocytes, including VDR expression and growth arrest, and increases the lipid content in adipocytes treated for 16 days. Preadipocytes are important cells in adipose tissue homeostasis, and understanding the role of 1,25(OH)2D3 in adipogenesis is a crucial step in ensuring adequate vitamin D supplementation, especially for obese individuals.

Risk Assessment via Metabolism and Cell Growth Inhibition in a HepG2/C3A Cell Line Upon Treatment with Arpadol and its Active Component Harpagoside.

Harpagophytum procumbens (Hp) has been used as antiinflammatory and analgesic agent for the treatment of rheumatic diseases. The principal active component of Hp is harpagoside (HA). We tested the toxicity of this new therapeutic agent in a hepatic cell line (HepG2/C3A). Hp was found to be cytotoxic, and HA was found to decrease the number of cells in S phase, increase the number of cells in G2/M phase and induce apoptosis. Neither Hp nor HA was genotoxic. The expression of CDK6 and CTP3A4 was reduced by Hp, and both HA and Hp caused a significant reduction of CYP1A2 and CYP3A4 expression. It is possible that the cytotoxicity caused by HA and Hp does not involve transcriptional regulation of the cyclins and CDKs tested but is instead related to the inhibition of metabolism. This is evidenced by the results of an MTT assay and changes in the expression of genes related to drug metabolism, leading to cell death. Indeed, the cells exhibited decreased proliferation upon exposure to Hp and HA. The data show that treatment with either Hp or HA can be cytotoxic, and this should be taken into consideration when balancing the risks and benefits of treatments for rheumatic diseases. Copyright © 2016 John Wiley & Sons, Ltd.

Analysis of the genotoxic potential of low concentrations of Malathion on the Allium cepa cells and rat hepatoma tissue culture.

Based on the concentration of Malathion used in the field, we evaluated the genotoxic potential of low concentrations of this insecticide on meristematic and F1 cells of Allium cepa and on rat hepatoma tissue culture (HTC cells). In the A. cepa, chromosomal aberrations (CAs), micronuclei (MN), and mitotic index (MI) were evaluated by exposing the cells at 1.5, 0.75, 0.37, and 0.18mg/mL of Malathion for 24 and 48hr of exposure and 48hr of recovery time. The results showed that all concentrations were genotoxic to A. cepa cells. However, the analysis of the MI has showed non-relevant effects. Chromosomal bridges were the CA more frequently induced, indicating the clastogenic action of Malathion. After the recovery period, the higher concentrations continued to induce genotoxic effects, unlike the observed for the lowest concentrations tested. In HTC cells, the genotoxicity of Malathion was evaluated by the MN test and the comet assay by exposing the cells at 0.09, 0.009, and 0.0009mg/5mL culture medium, for 24hr of exposure. In the comet assay, all the concentrations induced genotoxicity in the HTC cells. In the MN test, no significant induction of MN was observed. The genotoxicity induced by the low concentrations of Malathion presented in this work highlights the importance of studying the effects of low concentrations of this pesticide and demonstrates the efficiency of these two test systems for the detection of genetic damage promoted by Malathion.

Effects of genistein and daidzein on cell proliferation kinetics in HT29 colon cancer cells: the expression of CTNNBIP1 (ß-catenin), APC (adenomatous polyposis coli) and BIRC5 (survivin).

Soybean isoflavonoids have received significant attention due to their potential anticarcinogenic and antiproliferative effects and possible role in many signal transduction pathways. However, their mechanisms of action and their molecular targets remain to be further elucidated. In this paper, we demonstrated that two soybean isoflavones (genistein and daidzein) reduced the proliferation of the human colon adenocarcinoma grade II cell line (HT-29) at concentrations of 25 and 50-100 µM, respectively. We then investigated the effects of genistein and daidzein by RT-PCR on molecules that involved in tumor development and progression by their regulation of cell proliferation. At a concentration of 50 µM genistein, there was suppressed expression of ß-catenin (CTNNBIP1). Neither genistein nor daidzein affected APC (adenomatous polyposis coli) or survivin (BIRC5) expression when cells were treated with concentrations of 10 or 50 µM. These data suggest that the down-regulation of ß-catenin by genistein may constitute an important determinant of the suppression of HT-29 cell growth and may be exploited for the prevention and treatment of colon cancer.

Evaluation of the cytotoxicity, mutagenicity and antimutagenicity of a natural antidepressant, Hypericum perforatum L. (St. John's wort), on vegetal and animal test systems.

BACKGROUND: St. John's wort (Hypericum perforatum L.) is an herbaceous plant that is native to Europe, West Asia and North Africa and that is recognized and used worldwide for the treatment of mild and moderate depression. It also has been shown to be therapeutic for the treatment of burns, bruises and swelling and can be used for its wound healing, antiviral, antimicrobial, antioxidant, analgesic, hepato-protective and anxiolytic properties. The aim of this study was to evaluate the potential cytotoxic, mutagenic and antimutagenic action of H. Perforatum. METHODS: Meristematic cells were used as the test system for Allium cepa L., and bone marrow cells from Rattus norvegicus, ex vivo, were used to calculate the mitotic index and the percentage of chromosomal aberration. Statistical analysis was performed using the chi-square test. RESULTS: This medicinal plant had no cytotoxic potential in the vegetal test system evaluated. In the animal test system, none of the acute treatments, including intraperitoneal gavage and subchronic gavage, were cytotoxic or mutagenic. Moreover, this plant presented antimutagenic activity against the clastogenic action of cyclophosphamide, as confirmed in pre-treatment (76% reduction in damage), simultaneous treatment (95%) and post-treatment (97%). CONCLUSIONS: Thus, the results of this study suggest that the administration of H. perforatum, especially by gavage similar to oral consumption used by humans, is safe and with beneficial antimutagenic potential.

Modulação do efeito mutagênico do benzo[a]pireno e bleomicina por extratos de isoflavonas em células de hepatoma de roedor / Modulation of the mutagenic effect of benzo[a]pyrene and bleomycin by isoflavone extracts in a rat hepatoma cell line

Estudos epidemiológicos mostram que a ingestão de alimentos ricos em isoflavonas (fitoestrógenos), como a soja, confere proteção contra vários tipos de câncer, o que aumenta o interesse científico e popular sobre esses compostos. No presente estudo, os fitoestrógenos de extrato de soja foram testados quanto aos efeitos genotóxicos e modulador de benzo [a] pireno e bleomicina. Dois fitoestrogênios foram avaliados in vitro, o fitoestrógenos “A” foi fornecido pela Embrapa-Soja, Londrina - PR, e o fitoestrógenos “B” foi comprado em uma farmácia de manipulação local. Os métodos utilizados foram o teste do Cometa (genotoxicidade e antigenotoxicidade) e teste do Micronúcleo com Bloqueio Citocinese mutagenicidade) em células de hepatoma de rato (HTC celulares). As isoflavonas foram testadas em três concentrações pré-estabelecidas pelo ensaio de citotoxidade MTT. Ambos os extratos de isoflavonas não mostraram efeitos genotóxicos no ensaio do cometa, mas mostraram indução de micronúcleo. Na avaliação dos fitoestrogênios para um efeito modulador, ambos os extratos fitoestrogênios mostraram efeito antigenotóxico no ensaio do cometa.

Epidemiologic studies show that the intake of foods rich in isoflavones (phytoestrogens), such as soybeans, confers protection against various types of cancer, what increases the scientific and popular interest on these compounds. In the present study, phytoestrogens extracts from soybeans were tested for genotoxic potential and modulatory effects on benzo[a]pyrene and bleomycin. Two phytoestrogens were evaluated in vitro, phytoestrogen “A” was supplied by EMBRAPA-Soja, Londrina – PR, and phytoestrogen “B” was purchased in a local drug store. The methods used were the comet assay (genotoxicity and antigenotoxicity) and micronucleus test with cytokinesis block (mutagenicity) in rat hepatoma cells (HTC cell). The isoflavones were tested at three concentrations pre-established by the MTT cytotoxicity assay. Both isoflavone extracts showed no genotoxic effects in the comet assay, but showed induction of micronucleus. In the evaluation of the phytoestrogens for a modulatory effect, both phytoestrogens extracts showed antigenotoxicity in the comet assay.

Activity of selenium on cell proliferation, cytotoxicity, and apoptosis and on the expression of CASP9, BCL-XL and APC in intestinal adenocarcinoma cells.

Intestinal cancers are correlated with diet. Thus, determining and understanding nutrient-genome interactions is important. The present work assessed the action of the oligoelement selenium on cell proliferation, cytotoxicity, and in situ apoptosis induction and on the expression CASP9, BCL-XL and APC genes in intestinal adenocarcinoma cells (HT29). HT29 cells were cultured and treated with selenium at concentrations of 5, 50 and 500ng/mL with or without the damage-inducing agent doxorubicin. These cells were then evaluated for cytotoxicity (MTT), cell proliferation and in situ apoptosis induction. To evaluate gene expression, only the cells treated with 500ng/mL of selenium were used. RNA was extracted from these cells, and the expressions of CASP9, BCL-XL and APC were analyzed by the RT-PCR method. The GAPDH gene was used as a reference gene. The MTT assay showed that selenium was not cytotoxic at any of the concentrations tested. The cell proliferation assay showed that selenium did not interfere with cell proliferation at the three concentrations tested. In contrast, when the three concentrations were combined with doxorubicin, a significant decrease in the proliferation rate was observed. The apoptosis rate was significantly increased in the selenium (500ng/mL) and doxorubicin group. CASP9 expression was increased and BCL-XL expression decreased in the selenium (500ng/mL) and doxorubicin group. APC was significantly increased in the selenium group alone. These results show that selenium increases apoptosis, especially when it is associated with a damage-inducing agent. Also, selenium has an important role in the expression of the APC gene, which is related to cell cycle regulation.

Evaluation of Agaricus blazei in vivo for antigenotoxic, anticarcinogenic, phagocytic and immunomodulatory activities.

The development of various types of cancer results from the interaction among endogenous, environmental and hormonal factors, where the most notable of these factors is diet. The aim of the present study was to determine the antigenotoxic, anticarcinogenic, phagocytic and immunomodulatory activities of Agaricus blazei. The test antigenotoxicity (Comet Assay) and anticarcinogenic (Test of Aberrant Crypt Foci) assess changes in DNA and/or intestinal mucosa that correlate to cancer development. Tests of phagocytosis in the spleen and differential count in blood cells allow the inference of modulation of the immune system as well as to propose a way of eliminating cells with DNA damage. Supplementation with the mushroom was carried out under pre-treatment, simultaneous treatment, post-treatment and pre-treatment+continuous conditions. Statistical analysis demonstrated that the mushroom did not have genotoxic activity but showed antigenotoxic activity. Supplementation caused an increase in the number of monocytes and in phagocytic activity, suggesting that supplementation increases a proliferation of monocytes, consequently increasing phagocytic capacity especially in the groups pre-treatment, simultaneous and pre-treatment+continuous. The data suggest that A. blazei could act as a functional food capable of promoting immunomodulation which can account for the destruction of cells with DNA alterations that correlate with the development of cancer, since this mushroom was demonstrated to have a preventive effect against pre-neoplastic colorectal lesions evaluated by the aberrant crypt foci assay. According to these results and the literature, it is believed that supplementation with A. blazei can be an efficient method for the prevention of cancer as well as possibly being an important coadjuvant treatment in chemotherapy.

Evaluation of extracts from Coccoloba mollis using the Salmonella/microsome system and in vivo tests.

The common everyday use of medicinal plants is an ancient, and still very widespread practice, whereby the need for studies on their possible toxicity and mutagenic properties. The species Coccoloba mollis has been much used in phytotherapy, mainly in cases involving loss of memory and stress. In order to investigate its genotoxic and mutagenic potential, ethanolic extracts from the leaves and roots underwent Salmonella/microsome assaying (TA98 and TA100 strains, with and without exogenous metabolism - S9), besides comet and micronucleus tests in vivo.There was no significant increase in the number of revertants/plate of Salmonella strains in any of the analyzed root-extract concentrations, although the extract itself was extremely toxic to the Salmonella TA98 strain in the tests carried out with S9 (doses varying from 0.005 to 0.5 µg/plate). On the other hand, the leaf-extract induced mutations in the TA98 strain in the absence of S9 in the highest concentration evaluated, although at very low mutagenic potency (0.004 rev/ µg). Furthermore, there was no statistically significant increase in the number of comets and micronuclei, in treatments involving Swiss mice. It was obvious that extracts of Coccoloba mollis, under the described experimental conditions, are not mutagenic.

Evaluation of the genotoxic and anti-genotoxic activities of silybin in human hepatoma cells (HepG2).

Silybin (SB), a constituent of the medicinal plant Silybum marianum, is reported to be a potent hepatoprotective agent, but little is currently known regarding its genotoxicity, mutagenicity and potential chemopreventive properties. In this study, we evaluated the ability of SB to induce DNA migration and micronuclei (MN) formation in human hepatoma cells (HepG2). Also, possible preventive effects of SB on MN formation induced by three different mutagens, bleomycin (BLEO), benzo[a]pyrene (B[a]P) and aflatoxin B(1) (AFB(1)), were studied. To clarify the possible mechanism of SB antimutagenicity, three treatment protocols were applied: pretreatment, in which SB was added before the application of the mutagens; simultaneous treatment, in which SB was added during treatment and post-treatment, in which SB was added after the application of the mutagens. At concentrations up to 100 microM, SB was non-genotoxic, while at a concentration of 200 microM, SB induced DNA migration, generated oxidized DNA bases, reduced cell viability, decreased the replicative index of the cells and induced oxidative stress. It is noteworthy that SB was able to reduce the genotoxic effect induced by B[a]P, BLEO and AFB(1) in pretreatment and simultaneous treatments but had no significant effect on DNA damage induction in post-treatment. Taken together, our findings indicate that SB presents anti-genotoxic activity in vitro, which suggests potential use as a chemopreventive agent.

Evaluation of extracts from Coccoloba mollis using the Salmonella/microsome system and in vivo tests

The common everyday use of medicinal plants is an ancient, and still very widespread practice, whereby the need for studies on their possible toxicity and mutagenic properties. The species Coccoloba mollis has been much used in phytotherapy, mainly in cases involving loss of memory and stress. In order to investigate its genotoxic and mutagenic potential, ethanolic extracts from the leaves and roots underwent Salmonella/microsome assaying (TA98 and TA100 strains, with and without exogenous metabolism - S9), besides comet and micronucleus tests in vivo.There was no significant increase in the number of revertants/plate of Salmonella strains in any of the analyzed root-extract concentrations, although the extract itself was extremely toxic to the Salmonella TA98 strain in the tests carried out with S9 (doses varying from 0.005 to 0.5 µg/plate). On the other hand, the leaf-extract induced mutations in the TA98 strain in the absence of S9 in the highest concentration evaluated, although at very low mutagenic potency (0.004 rev/µg). Furthermore, there was no statistically significant increase in the number of comets and micronuclei, in treatments involving Swiss mice. It was obvious that extracts of Coccoloba mollis, under the described experimental conditions, are not mutagenic.