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

Evolução da esquistossomose em uma zona hiperendêmica do estado de Minas Gerais: dois estudos seccionais

Two cross-sectional studies on schistosomiasis mansoni were done in Comercinho, Minas Gerais (Brazil), at an interval of 7 years. In 1974 and 1981 feces examinations (KATO-KATZ method) were done in 89 and 90% of the population (about 1,500 inhabitants) and clinical examinations were done in 78 and 92% of the patients who excreted Schistosoma mansoni eggs in the feces, respectively. The rate of infection by S. mansoni did not change (69.9% in 1974 and 70.4% in 1981), but the geometrical mean of eggs per gram of feces (431 ± 4 and 334 ± 4, respectively) and the rate of splenomegaly (11 and 7%, respectively) decreased significantly in 1981, when compared to 1974. This reduction was observed only in the central zones of the town (zones 1-2) where the rate of dwellings with piped water increased from 17 to 44%. In the surroundings (zones 3-4), where the proportion of houses with piped water did not change significantly between 1974 (10%) and 1981 (7%), the geometrical mean of S. mansoni eggs and the rate of splenomegaly did not change either.Dois estudos seccionais da esquistossomose mansoni foram desenvolvidos na cidade de Comercinho, Estado de Minas Gerais (Brasil), com intervalo de sete anos. Em 1974 e em 1981 foram feitos exames de fezes em, respectivamente, 89 e 90% da população da cidade (cerca de 1.500 habitantes) e exame clínico em, respectivamente, 78 e 92% dos pacientes que apresentavam ovos de S. mansoni nas fezes. O índice de infecção pelo S. mansoni não se modificou durante o período analisado (69,9% em 1974 e 70,4% em 1981), mas a média geométrica de ovos por grama de fezes (431 ± 4 e 334 ± 4, respectivamente) e o índice de esplenomegalia (11 e 7%, respectivamente) diminuíram significativamente em 1981, quando comparado ao observado em 1974. Esta redução ocorreu exclusivamente nas zonas centrais da cidade (zonas 1-2), onde a percentagem de domicílios com água encanada aumentou de 17 para 44%. Na periferia (zonas 3-4), onde a porcentagem de domicílios com água encanada não mudou significativamente entre 1974 (10%) e 1981 (7%), a contagem de ovos de S. mansoni e o índice de esplenomegalia também não sofreram modificações

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

O6-methylguanine (O6meG) 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 O6 position of guanine to a cysteine residue in its active center. Because O6meG repair by MGMT is a stoichiometric reaction that irreversibly inactivates MGMT, which is subsequently degraded, the repair capacity of O6meG lesions is dependent on existing active MGMT molecules. In the absence of active MGMT, O6meG is not repaired, and during replication, O6meG:T mispairs are formed. The MMR system recognizes these mispairs and introduces a gap into the strand. If O6meG remains in one of the template strands the futile MMR repair process will be repeated, generating more strand breaks (SBs). The toxicity of O6meG is, therefore, dependent on MMR and DNA SB induction of cell death. MGMT, on the other hand, protects against O6meG toxicity by removing the methyl residue from the guanine. Although removal of O6meG 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 O6meG lesions. Here, we report on a modification of the comet assay (CoMeth) that allows the qualitative assessment of O6meG 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 O6meG 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 O6meG, 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.We thank the COST Action TD0905 “Epigenetics: From Bench to Bedside” for financial support. A.A. Ramos and D. Pedro are supported by the Foundation for Science and Technology, Portugal, Grant SFRH/BD/35672/2007 and SFRH/BD/64817/2009, respectively. The work was supported by FCT research grant PEst-C/BIA/UI4050/2011, which is co-funded by the program COMPETE from QREN with co-participation from the European Community fund FEDER

Single nucleotide polymorphisms from Theobroma cacao expressed sequence tags associated with witches' broom disease in cacao

In order to increase the efficiency of cacao tree resistance to witches¿ broom disease, which is caused by Moniliophthora perniciosa (Tricholomataceae), we looked for molecular markers that could help in the selection of resistant cacao genotypes. Among the different markers useful for developing marker-assisted selection, single nucleotide polymorphisms (SNPs) constitute the most common type of sequence difference between alleles and can be easily detected by in silico analysis from expressed sequence tag libraries. We report the first detection and analysis of SNPs from cacao-M. perniciosa interaction expressed sequence tags, using bioinformatics. Selection based on analysis of these SNPs should be useful for developing cacao varieties resistant to this devastating disease. (Résumé d'auteur

Bioactivity and phenolic characterization of different medicinal and aromatic plants

Introduction: Plants are widely used to treat various diseases and have been widely recognized as a rich source of phytochemicals with antimicrobial potential. In fact, plants have received considerable attention by researchers being their biological properties widely explored. Hypothesis and aims: Medicinal and aromatic plants are known to have a wide range of uses and health benefits, and should be exploited concerning their bioactivity. Therefore, the antimicrobial activity of Satureja montana L., Origanum majorana L., Allium schoenoprasum L. and Anethum graveolens L. were evaluated and its phytochemical composition was profiled. Methodology: The antimicrobial susceptibility of Gram-positive and Gram-negative bacteria to four decoction and hydroethanolic (80:20, v/v) extracts, obtained from medicinal and aromatic plants (S. montana , O. majorana , A. schoenoprasum and A. graveolens ), was assessed aiming to identify the active extracts and the most effective were then tested against biofilms. Furthermore, the decoctions were characterized in terms of phenolic compounds by HPLC-DAD-ESI/MSn. Results: Overall, S. montana and O. majorana extracts were the most effective against Gram-positive (Staphylococcus aureus , Enterococcus faecalis and Streptococcus dysgalactiae ) and Gram-negative (Klebsiella pneumonia and Pseudomonas aeruginosa ) bacteria, with decoction presenting the most pronounced effects. O. majorana and S. montana decoction, at minimum inhibitory concentrations, were significantly effective against planktonic cells of S. aureus ATCC 25923. Concerning biofilm cells, S. montana promoted a slight antimicrobial activity against S. aureus ATCC 25923. A total of twentyfour phenolic compounds (9 phenolic acids and 15 flavonoids glycosides) were identified in S. montana and O. majorana decoctions, being rosmarinic acid the main molecule in the extracts. Conclusion: This study confirmed the bioactive potential of the medicinal and aromatic herbs, being S. montana and O. majorana decoction extracts those that showed the most promising applicability for the development of novel formulations with antimicrobial properties.info:eu-repo/semantics/publishedVersio

Ursolic acid induces cell death and modulates autophagy through JNK pathway in apoptosis-resistant colorectal cancer cells

Colorectal carcinomas (CRCs) with P53 mutations have been shown to be resistant to chemotherapy with 5-fluorouracil (5-FU), the most widely used chemotherapeutic drug for CRC treatment. Autophagy is emerging as a promising therapeutic target for drug-resistant tumors. In the present study, we tested the effects of ursolic acid (UA), a natural triterpenoid, on cell death mechanisms and its effects in combination with 5-FU in the HCT15 p53 mutant apoptosis-resistant CRC cell line. The involvement of UA in autophagy and its in vivo efficacy were evaluated. Our data show that UA induces apoptosis independent of caspases in HCT15 cells and enhances 5-FU effects associated with an activation of c-jun N-terminal kinase (JNK). In this cell line, where this compound has a more pronounced effect on the induction of cell death compared to 5-FU, apoptosis corresponds only to a small percentage of the total cell death induced by UA. UA also modulated autophagy by inducing the accumulation of LC3 and p62 levels with involvement of JNK pathway, which indicates a contribution of autophagy on JNK-dependent induction of cell death by UA. By using nude mice xenografted with HCT15 cells, we verified that UA was also active in vivo decreasing tumor growth rate. In conclusion, this study shows UA's anticancer potential both in vitro and in vivo. Induction of cell death and modulation of autophagy in CRC-resistant cells were shown to involve JNK signaling.C.P.R.X. and D.F.N.P. were supported by the Foundation for Science and Technology (FCT), Portugal, through grants SFRH/BD/27524/2006 and SFRH/BD/64817/2009, respectively. C.P.W. was guest professor at University of Copenhagen through the grant SFRH/BSAB/918/2009. The work was supported by FCT research grants PTDC/QUI-BIQ/101392/2008 (NaturAge) 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

Evaluation of antifungal activity of essential oils against potentially mycotoxigenic Aspergillus flavus and Aspergillus parasiticus

The antifungal activity of essential oils of fennel (Foeniculum vulgare Mill., Apiaceae), ginger (Zingiber officinale Roscoe, Zingiberaceae), mint (Mentha piperita L., Lamiaceae) and thyme (Thymus vulgaris L., Lamiaceae) was evaluated against mycotoxin producers Aspergillus flavus and A. parasiticus. High Resolution Gas Chromatography was applied to analyze chemical constituents of essential oils. The effect of different concentrations of essential oils was determined by solid medium diffusion assay. Mycelial growth and sporulation were determined for each essential oil at the concentrations established by solid medium diffusion assay. At the fifth, seventh and ninth days the mycelial diameter (Ø mm) and spore production were also determined. FUN-1 staining was performed to assess cell viability after broth macrodilution assay. Trans-anethole, zingiberene, menthol and thymol are the major component of essential oils of fennel, ginger, mint and thyme, respectively. The effective concentrations for fennel, ginger, mint and thyme were 50, 80, 50 and 50% (oil/DMSO; v/v), respectively. The four essential oils analysed in this study showed antifungal effect. Additionally, FUN-1 staining showed to be a suitable method to evaluate cell viability of potential mycotoxigenic fungi A. flavus and A. parasiticus after treatment with essential oils.The authors are grateful to the colleagues from Laboratory of EPAMIG and Microbiology DEB/UFLA and the Micoteca da Universidade do Minho, Center for Biological Engineering, UMINHO for their support to perform this work. A special aknowledgment is also due to the FAPEMIG and MUM-UMINHO for the financial support of bench work and to CAPES for granting the first author with a PhD scholarship

Abnormal Striatal BOLD Responses to Reward Anticipation and Reward Delivery in ADHD

Altered reward processing has been proposed to contribute to the symptoms of attention deficit hyperactivity disorder (ADHD). The neurobiological mechanism underlying this alteration remains unclear. We hypothesize that the transfer of dopamine release from reward to reward-predicting cues, as normally observed in animal studies, may be deficient in ADHD. Functional magnetic resonance imaging (fMRI) was used to investigate striatal responses to reward-predicting cues and reward delivery in a classical conditioning paradigm. Data from 14 high-functioning and stimulant-naïve young adults with elevated lifetime symptoms of ADHD (8 males, 6 females) and 15 well-matched controls (8 males, 7 females) were included in the analyses. During reward anticipation, increased blood-oxygen-level-dependent (BOLD) responses in the right ventral and left dorsal striatum were observed in controls, but not in the ADHD group. The opposite pattern was observed in response to reward delivery; the ADHD group demonstrated significantly greater BOLD responses in the ventral striatum bilaterally and the left dorsal striatum relative to controls. In the ADHD group, the number of current hyperactivity/impulsivity symptoms was inversely related to ventral striatal responses during reward anticipation and positively associated with responses to reward. The BOLD response patterns observed in the striatum are consistent with impaired predictive dopamine signaling in ADHD, which may explain altered reward-contingent behaviors and symptoms of ADHD