Efeitos antigenotóxicos dos compostos naturais na linha celular HepG2

O consumo de compostos de origem vegetal tem sido inversamente relacionado com o risco de doenças associadas ao stress oxidativo assim como o cancro. Compostos naturais que diminuam o stress oxidativo e consequente extensão dos danos sobre o DNA reduzem potencialmente o risco de desenvolvimento de cancro. Assim, justifica-se o estudo dos efeitos de compostos naturais de origem vegetal na protecção do DNA. O objectivo do presente trabalho foi investigar o potencial efeito quimopreventivo da quercetina (Q), rutina (R) e ácido ursólico (AU) sobre os danos oxidativos no DNA induzidos pelo tert-butil hidroperoxido (t-BHP) numa linha celular do hepatoma humano (HepG2) pelo método “comet assay”. Para compreender que possíveis mecanismos estavam envolvidos na protecção do DNA foram avaliados os efeitos de diferentes tipos de tratamento com os compostos naturais: incubação simultânea; pré-tratamento e pré-tratamento com período de recuperação. Um outro objectivo foi avaliar o efeito dos compostos naturais sobre a proliferação celular através do método da redução do brometo de 3- (4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio (MTT). Os nossos resultados mostraram que a Q protege o DNA dos danos induzidos pelo t-BHP em células HepG2 através de diferentes mecanismos. A forma glicosilada, R, não teve efeitos a nenhum dos níveis. O AU protegeu o DNA dos danos induzidos pelo t-BHP nos ensaios de pré-incubação com ou sem período de recuperação. Os efeitos antiproliferativos da Q e do AU aumentaram com o tempo de incubação das células, com os respectivos compostos. A rutina não teve efeitos sobre a proliferação celular. O potencial anticarcinogénico da Q e do AU no fígado parece ser devido a diferentes mecanismos: prevenção dos danos no DNA através da modulação do sistema antioxidante endógeno, das enzimas envolvidas no metabolismo dos xenobióticos e reparação dos danos através da modulação do sistema de reparação e inibição da proliferação celular. No caso da Q um outro mecanismo envolvido na prevenção dos danos é actividade antioxidante. A quimoprevenção com agentes naturais eficazes pode ser usada como uma estratégia para diminuir a incidência de diversos tipos de cancro através da inclusão na alimentação de factores protectores que fortifiquem os mecanismos de defesa do organismo.The consumption of natural some compounds is inversely correlated with the risk of diseases associated to oxidative stress such as cancer. Natural compounds that decrease oxidative stress and consequent DNA damage reduce the potentially risk of cancer development. Thus, the study of effects of natural compounds of vegetable origin in protection of DNA is justified. The aim of the present study was to investigate the potential chemoprotective effects of quercetin (Q), rutin (R) and ursolic acid (UA) on tert-butyl hydroperoxide (t-BHP)-induced oxidative DNA damage in a human hepatoma cell line (HepG2) by the comet assay. To determine whether any effects were due to direct chemical interactions or to cellular mediated responses three different types of treatments were used: simultaneous treatment; pre-treatment and pre-treatment with recovery period. In addition, we performed the MTT (3- (4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide) assay to evaluate effects on cell proliferation. Our results show that Q is protective against HepG2 DNA damage induced by t-BHP through mechanisms at three levels. The glycosylated form, R showed no effects at any levels. UA was effective in protecting DNA against damage induced by t-BHP in pre-incubation experiments with or without a recovery period. Antiproliferative effects of Q and UA increased with incubation time of cells. R showed no effects on cellular proliferation. The anticarcinogenic potential of Q and UA on liver seems to be due to different mechanisms: prevention of DNA damage through a modulation of endogenous antioxidant systems and enhancement of xenobiotic metabolising enzymes and DNA repair by modulation of repair system and inhibition of cell proliferation. In the case of Q another mechanism involved in prevention of DNA damage is activity antioxidant of this compound. Quemoprevention with effective natural agents can be used as a strategy to decrease the incidence of several cancer types through their inclusion in the diet as protective agents that fortify the defence mechanisms of the organism.POCTI/AGR/62040/200

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

Nutrition and chemoprevention of colorectal cancer

Tese de doutoramento em Ciências especialidade em Biologiaexogenous and endogenous agents. These damages, when not repaired properly, can generate mutations in somatic or germline cells, which are involved in the pathogenesis of many diseases, including cancer. In the last decades, epidemiological studies as well as laboratory data suggest that consumption of some fruits and vegetables and a correct lifestyle is associated with a reduced risk of developing CRC. 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 global aim of this work was to clarify the role of the consumption of some plants of genus Salvia and their individual phytochemicals on DNA damage prevention and induction of DNA repair and consequent effect on colon cancer prevention. Their potential interaction with alkylating agents used as chemotherapeutic drugs was also evaluated. In the first study (Chapter II) we evaluate the effects of three Salvia sp. water extracts, Salvia officinalis (SO), Salvia fruticosa (SF), and Salvia lavandulifolia (SL), and the main phenolic constituents rosmarinic acid (RA) and luteolin-7-glucoside (L-7-G) on DNA protection against oxidative damage in two human cell lines, Caco-2 and HeLa cells. SF, RA, and L-7-G significantly decreased DNA strand breaks induced by H2O2 in Caco-2 cells. The protective effects were more pronounced at a short period (2h) than at a long period of incubation (24h). In contrast, in HeLa cells, SO, SF, and RA had protective effects against damage induced by H2O2 after a long period of incubation. Protection against 8-oxoGua, a specific base oxidation, was conferred by RA in Caco-2 cells after a short period of incubation, while no protective effects were observed in HeLa cells. Effects of the three Salvia sp. water extracts and phenolic compounds on DNA repair in Caco-2 cells were also assessed (Chapter II). Pre-incubation for 24h with SO, SF and L-7-G increased the extent of rejoining of strand breaks in Caco-2 cells after treatment with H2O2. SO and L-7-G also have a BER inductive effect in Caco-2 cells. In the second study (Chapter II) the effects of two common compounds, ursolic acid (UA) and luteolin (Lut), present in Sage plants and also in fruits, vegetables and spices, on DNA protection against oxidative DNA damage and modulation of DNA repair in human colon cells (Caco-2) were evaluated. We showed that both compounds, Lut and UA, protected DNA of Caco-2 cells from strand breaks induced by H2O2. However, only Lut protected DNA against 8-oxoGua in Caco-2 after a short period of incubation. On DNA repair, pre-incubation for 24h with UA or Lut increased the extent of rejoining of strand breaks. UA also had a BER inductive effect, increasing incision activity of a Caco-2 cell extract. In the third study (Chapter III) effects of the three Salvia sp. water extracts and the main phenolic constituent, RA, on DNA protection and repair of DNA damage induced by the oxidative and alkylating agents in two colon cancer cell lines, HCT15 and CO115 cells were evaluated. Salvia extracts protected against H2O2-induced DNA damage in HCT15 cells (SO and SF) and also protected CO115 cells from DNA damage induced by MNU (an alkylating agent) (SO and SL). This protection may be beneficial as a chemopreventive mechanism; however may decrease therapeutic efficacy of alkylating anticancer drugs. So, another aim of this study was to evaluate if consumption of Salvia sp. and RA might interfere with alkylating agents used in cancer therapy such as BCNU. We showed that the Salvia extracts tested and RA decreased DNA damage induced by BCNU, in CO115 (SO, SF, SL and RA) and HCT15 cells (SF). No effect was observed on MPG protein expression, a DNA repair enzyme involved in Nalkylations repair. In the fourth study (Chapter IV), we develop a new application of the comet assay to assess mutagenic alkylation DNA damages, specifically, O6meG levels in proliferative and MMR proficient cells by the use of a specific inhibitor of MGMT. With this new application we expect to contribute to the widespread use of the comet assay to test potential chemopreventive and chemotherapeutic drugs that act by inducing the activity of MGMT and the repair of O6meG base damage. In conclusion our results showed that Salvia extracts and some isolated compounds tested have effect on DNA damage protection not only against oxidative but also against alkylating DNA damage. Enhancement of DNA repair is another important mechanism that is involved in the chemopreventive effect of Salvia extracts and the phytochemicals tested.Os danos no ADN acumulam-se nas células ao longo do tempo como resultado da exposição a uma grande variedade de agentes endógenos e exogenos. Estes danos, se não forem devidamente reparados, podem gerar mutações em células somáticos ou germinais, os quais estão envolvidos na patogenicidade de muitas doenças, como o cancro. Nas últimas décadas, estudos epidemiológicos, assim como resultados laboratoriais sugerem que o consumo de alguns frutos e vegetais está associado ao baixo risco de desenvolver CRC. Diversos mecanismos têm sido propostos para explicar os efeitos quimopreventivos dos fitoquímicos. A proteção dos danos no ADN e a reparação do ADN assumem um importante papel na prevenção das mutações e consequentemente prevenção do processo carcinogénico. O objectivo geral deste trabalho foi esclarecer sobre o papel do consumo de algumas plantas do género Salvia e de alguns dos seus fitoquímicos na prevenção dos danos no ADN e indução da reparação do ADN e consequente efeito na prevenção do CRC. A sua potencial interação com agentes alquilantes usados como fármacos quimioterapêuticos foi também avaliada. No primeiro estudo (capítulo II) avaliamos o efeito dos extratos aquosos de três plantas do género Salvia, Salvia officinalis (SO), Salvia fruticosa (SF) e Salvia lavandulifolia (SL) e os seus principais constituintes, ácido rosmarínico (AR) e a luteolina-7- glucosídeo (L-7-G) na proteção do ADN contra danos oxidativos em duas linhas celulares humanas, Caco-2 e HeLa. SF, RA e L-7-G diminuem significativamente as quebras no ADN induzidas pelo H2O2 em células Caco-2. O efeito protetor foi mais evidente para períodos curtos de incubação (2h) do que para períodos longos (24h). Pelo contrário, em células HeLa, SO, SF e AR tiveram um efeito protetor contra danos induzidos pelo H2O2 após um longo período de incubação. A proteção contra 8-oxoGua, que é uma base oxidada, foi conferida pelo AR em células Caco-2 após um curto período de incubação, enquanto em células HeLa não foi observado efeito protetor. O efeito dos extratos aquosos das três plantas do género Salvia e dos compostos fenólicos na reparação do ADN foi também avaliado em células Caco-2. A pré-incubação de 24h com SO, SF e L-7-G aumentou a reparação das quebras de cadeia em células Caco-2 após tratamento com H2O2. SO e L-7-G também tiveram um efeito indutivo no sistema de reparação BER em células Caco-2. No segundo estudo (capítulo II) foram avaliados os efeitos de dois compostos, o ácido ursólico (AU) e a luteolina (Lut), que estão presentes nas plantas do género Salvia e também em frutos, vegetais e especiarias, na proteção do ADN contra danos oxidativos e na reparação do ADN em células humanas do colon (Caco-2). Demonstramos que ambos os compostos, Lut e AU, protegeram o ADN das células Caco-2 das quebras de cadeia induzidas pelo H2O2. No entanto, apenas a Lut protegeu as células Caco-2 contra 8-oxoGua após um curto período de incubação. Na reparação do ADN, uma préincubação de 24h com o UA ou Lut aumentou a reparação das quebras de cadeia. O AU também teve um efeito indutor no sistema de reparação BER, aumentando a actividade de corte do extrato celular de Caco-2. No terceiro estudo (capítulo III) foram avaliados os efeitos dos extratos aquosos das três plantas do género Salvia e do principal constituinte fenólico, o AR, na proteção e reparação do ADN contra danos induzidos por agentes oxidativos e alquilantes em duas linhas celulares do cancro do cólon, HCT15 e CO115. Os extratos de salvia protegeram contra os danos no ADN induzidos pelo H2O2 nas células HCT15 (SO e SF) e também protegeram as células CO115 dos danos no ADN induzidos pelo MNU (que é um agente alquilante) (SO e SL). Esta proteção pode ser benéfica como mecanismo quimiopreventivo, no entanto pode diminuir a eficácia terapêutica dos fármacos alquilantes anticancerígenos. Um outro objectivo deste trabalho foi avaliar a potencial interação entre o consumo de plantas do género Salvia e do AR com os fármacos alquilantes usados na terapia do cancro, como por exemplo o BCNU. Demonstramos que os extratos de Salvia e o AR diminuíram os danos no ADN induzidos pelo BCNU em células CO115 (SO, SF, SL e AR) e em células HCT15 (SF). Não foram observados efeitos na expressão da proteína MPG, uma importante enzima de reparação do ADN involvida na reparação dos danos N-alquilantes. No quarto estudo (capítulo IV), desenvolvemos uma nova aplicação do ensaio cometa para avaliar danos alquilantes mutagénicos, especialmente O6meG em células proliferativas e com o sistema de reparação MMR funcional, usando um inibidor específico da MGMT. Com esta nova aplicação, nós esperamos contribuir para um maior uso do ensaio cometa para testar agentes quimiopreventivos e fármacos quimioterapêuticos que atuam através da indução da actividade da MGMT e reparação do dano O6meG. Em conclusão, os nossos resultados mostraram que os extratos de Salvia e alguns dos compostos testados tem efeito na proteção do ADN não só contra danos oxidativos mas também contra danos alquilantes. A reparação do ADN é um outro importante mecanismo que está involvido nos efeitos quimiopreventivos dos extratos de Salvia e dos fitoquímicos testados

Cytotoxicity of Seaweed Compounds, Alone or Combined to Reference Drugs, against Breast Cell Lines Cultured in 2D and 3D

Seaweed bioactive compounds have shown anticancer activities in in vitro and in vivo studies. However, tests remain limited, with conflicting results, and effects in combination with anticancer drugs are even scarcer. Here, the cytotoxic effects of five seaweed compounds (astaxanthin, fucoidan, fucosterol, laminarin, and phloroglucinol) were tested alone and in combination with anticancer drugs (cisplatin—Cis; and doxorubicin—Dox), in breast cell lines (three breast cancer (BC) subtypes and one non-tumoral). The combinations revealed situations where seaweed compounds presented potentiation or inhibition of the drugs’ cytotoxicity, without a specific pattern, varying according to the cell line, concentration used for the combination, and drug. Fucosterol was the most promising compound, since: (i) it alone had the highest cytotoxicity at low concentrations against the BC lines without affecting the non-tumoral line; and (ii) in combination (at non-cytotoxic concentration), it potentiated Dox cytotoxicity in the triple-negative BC cell line. Using a comparative approach, monolayer versus 3D cultures, further investigation assessed effects on cell viability and proliferation, morphology, and immunocytochemistry targets. The cytotoxic and antiproliferative effects in monolayer were not observed in 3D, corroborating that cells in 3D culture are more resistant to treatments, and reinforcing the use of more complex models for drug screening and a multi-approach that should include histological and ICC analysis

Fucoxanthin Holds Potential to Become a Drug Adjuvant in Breast Cancer Treatment: Evidence from 2D and 3D Cell Cultures

(This article belongs to the Special Issue Compounds from Marine Sources as Hits and Leads for Pharmaceutical, Cosmeceutical and Industrial Applications)Fucoxanthin (Fx) is a carotenoid derived from marine organisms that exhibits anticancer activities. However, its role as a potential drug adjuvant in breast cancer (BC) treatment is still poorly explored. Firstly, this study investigated the cytotoxic effects of Fx alone and combined with doxorubicin (Dox) and cisplatin (Cis) on a panel of 2D-cultured BC cell lines (MCF7, SKBR3 and MDA-MB-231) and one non-tumoral cell line (MCF12A). Fucoxanthin induced cytotoxicity against all the cell lines and potentiated Dox cytotoxic effects towards the SKBR3 and MDA-MB-231 cells. The combination triggering the highest cytotoxicity (Fx 10 µM + Dox 1 µM in MDA-MB-231) additionally showed significant induction of cell death and genotoxic effects, relative to control. In sequence, the same combination was tested on 3D cultures using a multi-endpoint approach involving bioactivity assays and microscopy techniques. Similar to 2D cultures, the combination of Fx and Dox showed higher cytotoxic effects on 3D cultures compared to the isolated compounds. Furthermore, this combination increased the number of apoptotic cells, decreased cell proliferation, and caused structural and ultrastructural damages on the 3D models. Overall, our findings suggest Fx has potential to become an adjuvant for Dox chemotherapy regimens in BC treatment.The Strategic Funding UIDB/04423/2020 and UIDP/04423/2020 partially supported this research, through national funds provided by FCT and ERDF to CIIMAR/CIMAR, in the framework of the program PT2020. The Doctoral Program in Biomedical Sciences, of the ICBAS-University of Porto, offered additional funds.info:eu-repo/semantics/publishedVersio

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 licenseBackground: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licenseBackground: Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide. Methods: A multimethods analysis was performed as part of the GlobalSurg 3 study—a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital. Findings: Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3·85 [95% CI 2·58–5·75]; p<0·0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63·0% vs 82·7%; OR 0·35 [0·23–0·53]; p<0·0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer. Interpretation: Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised. Funding: National Institute for Health and Care Research