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

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

Colon cancer chemoprevention by sage tea drinking: decreased DNA damage and cell proliferation

Salvia officinalis and some of its isolated compounds have been found to be preventive of DNA damage and increased proliferation in vitro in colon cells. In the present study, we used the azoxymethane model to test effects of S. officinalis on colon cancer prevention in vivo. The results showed that sage treatment reduced the number of ACF formed only if administered before azoxymethane injection, demonstrating that sage tea drinking has a chemopreventive effect on colorectal cancer. A decrease in the proliferation marker Ki67 and in H2O2-induced and azoxymethane-induced DNA damage to colonocytes and lymphocytes were found with sage treatment. This confirms in vivo the chemopreventive effects of S. officinalis. Taken together, our results show that sage treatment prevented initiation phases of colon carcinogenesis, an effect due, at least in part, to DNA protection, and reduced proliferation rates of colon epithelial cell that prevent mutations and their fixation through cell replication. These chemopreventive effects of S. officinalis on colon cancer add to the many health benefits attributed to sage and encourage its consumption.Fundação para a Ciência e a Tecnologia (FCT) - grant SFRH/BD/35672/2007, SFRH/BD/64817/200

A deep residual architecture for skin lesion segmentation

In this paper, we propose an automatic approach to skin lesion region segmentation based on a deep learning architecture with multi-scale residual connections. The architecture of the proposed model is based on UNet [22] with residual connections to maximise the learning capability and performance of the network. The information lost in the encoder stages due to the max-pooling layer at each level is preserved through the multi-scale residual connections. To corroborate the efficacy of the proposed model, extensive experiments are conducted on the ISIC 2017 challenge dataset without using any external dermatologic image set. An extensive comparative analysis is presented with contemporary methodologies to highlight the promising performance of the proposed methodology

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

Oral health-related quality of life in elderly: an umbrella review of systematic reviews from a multidisciplinary rehabilitation point-of-view

Background. Poor oral health is highly prevalent among elderlies and may impact quality of life of elderly people. In this scenario, oral health has been often linked to general health and chronic disorders, including distinct features of frailty. The aim of the present umbrella review of systematic reviews was to assess the scientific literature on the correlation between oral health related quality of life (OHRQoL) and elderly to present a multidisciplinary approach to these complex patients. Methods. We performed a literature search of the databases PubMed/Medline, Scopus, Web of Science, and Physiotherapy Evidence Database electronic databases. Two independent reviewers performed the literature research from the inception to 25th November 2023 and screened the studies for eligibility. Results. The search resulted in a total of 676 results eligible articles. After removal of duplicates and full-text screening, a total of 3 systematic reviews were considered to meet the inclusion criteria and were included for this review. Conclusions. Frailty is very common in elderly such as a poor oral health. In this scenario, malnutrition and bad lifestyle habits may affect not only the determinism of many systemic non-communicable diseases but also oral health quality. Taken together, the findings of this umbrella review of systematic reviews showed a strict correlation between the frailty, typical condition of ageing people, and a poor OHRQoL. Therefore, it is mandatory to implement the oral health prevention with specific protocols of oral rehabilitation to improve the OHRQoL in elderly

Determination of the radiochemical purity of 99mTc-Tetrofosmin: comparision between five methods

GOAL: The manufacturing and distribution of strips of instant thin - layer chromatography with silica gel (ITLC - SG) (reference method) is currently discontinued so there is a need for an alternative method f or the determination of radiochemical purity (RCP) of 99m Tc - tetrofosmin. This study aims to compare five alternative methods proposed by the producer to determine the RCP of 99m Tc - tetrofosmin. METHODS: Nineteen vials of tetrofosmin were radiolabelled with 99m Tc and the percentages of the RCP were determined. Five different methods were compared with the standard RCP testing method (ITLC - SG, 2x20 cm): Whatman 3MM (1x10 cm) with acetone and dichloro - methane (method 1); Whatman 3MM (1x1 0 cm) with ethyl acetate (method 2); aluminum oxide - coated plastic thin - layer chromatography (TLC) plate (1x10 cm) and ethanol (method 3); Whatman 3MM (2x20 cm) with acetone and dichloro - methane (method 4); solid - phase extraction method C18 cartridge (meth od 5). RESULTS: The average values of RCP were 95,30% ± 1,28% (method 1), 93,95 ± 0,61% (method 2), 96,85% ± 0,93% (method 3), 92,94% ± 0,99% (method 4) and 96,25% ± 2,57% (method 5) (n=12 each), and 93,15% ± 1,13% for the standard method (n=19). There we re statistical significant differences in the values obtained for methods 1 (P=0,001), 3 (P=0,000) and 5 (P=0,004), and there were no statistical significant differences in the values obtained for methods 2 (P=0,113) and 4 (P=0,327). CONCLUSION: From the results obtained, methods 2 and 4 showed a higher correlation with the standard method. Unlike method 4, method 2 is less time - consuming than the reference method and can overcome the problems associated with the solvent toxicity. The remaining methods (1, 3 and 5) tended to overestimate RCP value compared to the standard method

OC33 Chromatographic approaches to study pine nut skin: exploitation of its composition and bioactivities

Pine nut skin (PNS) is an unexploited and uncharacterized by-product recovered during pine nut processing. The exploitation of by-products as sources of valuable compounds agrees with the current demand for the reduction of waste, and a transition to more sustainable production and consumption1. Therefore, PNS characterization and bioactive potentialities were assessed. The utilization of several chromatographic techniques allowed the characterization of PNS phenolic compounds (HPLC-DAD-UV and HPLC-DAD-ESI-MSn), and the carbohydrates quantification and structural characterization, after specific derivatization (GC-FID and GC-MS). PNS subcritical water extraction using microwave was optimized and the obtained extracts, separated into low-molecularweight (rich in phenolic compounds) and high-molecular-weight (rich in carbohydrates), were evaluated regarding their digestibility and prebiotic activity. The prebiotic potential was assessed by quantifying the short-chain fatty acids (HPLC-UV) produced after the in vitro faecal fermentation. HPLC-DAD-ESI-MSn allowed to identify PNS phenolic compounds, namely protocatechuic, p-coumaric, and caffeic acids, while HPLC-DAD-UV enabled the monomers identification of proanthocyanidins ((epi)catechins) and hydrolysable tannins (protocatechuic acid), after acid methanolysis. GC techniques allowed to disclose the polysaccharides structures (xyloglucans and pectic polysaccharides) and their degradation by microbiota. The fermentation of both extracts rich in phenolic compounds and rich in polysaccharides resulted in an increased production of acetic, propionic, and butyric acids when compared to the commercial prebiotic inulin, proposing these PNS extracts as prebiotic agents.The work was supported through the projects UIDB/50006/2020 and UIDP/50006/2020, funded by FCT/MCTES through national funds. Soraia P. Silva, Alondra González and Dalila Roupar thank FCT/MCTES and ESF through NORTE 2020 for their PhD grants (ref. SFRH/BD/136471/2018, SFRH/BD/06268/2021 and SFRH/DB/139884/2018 respectively). Elisabete Coelho thanks the research contract (CDL-CTTRI-88-ARH/2018 – REF. 049-88-ARH/2018) funded by national funds (OE), through FCT, in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. Clarisse Nobre acknowledges FCT for the assistant research contract 2021.01234.CEECIND.info:eu-repo/semantics/publishedVersio