Browsing by Author "Pereira, Joana"
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- Adverse Outcome Pathways Associated with the Ingestion of Titanium Dioxide Nanoparticles - A Systematic ReviewPublication . Rolo, Dora; Assunção, Ricardo; Ventura, Célia; Alvito, Paula; Gonçalves, Lídia; Martins, Carla; Bettencourt, Ana; Jordan, Peter; Vital, Nádia; Pereira, Joana; Pinto, Fátima; Matos, Paulo; Silva, Maria João; Louro, HenriquetaTitanium dioxide nanoparticles (TiO2-NPs) are widely used, and humans are exposed through food (E171), cosmetics (e.g., toothpaste), and pharmaceuticals. The oral and gastrointestinal (GIT) tract are the first contact sites, but it may be systemically distributed. However, a robust adverse outcome pathway (AOP) has not been developed upon GIT exposure to TiO2-NPs. The aim of this review was to provide an integrative analysis of the published data on cellular and molecular mechanisms triggered after the ingestion of TiO2-NPs, proposing plausible AOPs that may drive policy decisions. A systematic review according to Prisma Methodology was performed in three databases of peer-reviewed literature: Pubmed, Scopus, and Web of Science. A total of 787 records were identified, screened in title/abstract, being 185 used for data extraction. The main endpoints identified were oxidative stress, cytotoxicity/apoptosis/cell death, inflammation, cellular and systemic uptake, genotoxicity, and carcinogenicity. From the results, AOPs were proposed where colorectal cancer, liver injury, reproductive toxicity, cardiac and kidney damage, as well as hematological effects stand out as possible adverse outcomes. The recent transgenerational studies also point to concerns with regard to population effects. Overall, the findings further support a limitation of the use of TiO2-NPs in food, announced by the European Food Safety Authority (EFSA).
- Alternative splicing of tumour-related Rac1b in colorectal cells is regulated by protein phosphorylation of splicing factor SRSF1Publication . Gonçalves, Vânia; Henriques, Andreia; Pereira, Joana; Neves-Costa, Ana; Moyer, Pat; Ferreira Moita, Luís; Gama-Carvalho, Margarida; Matos, Paulo; Jordan, PeterThe pre-messenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications such as protein phosphorylation, which are determined by signal transduction pathways. Here we analyzed the contribution of protein kinases to the regulation of alternative splicing variant Rac1b that is overexpressed in certain tumor types. In colorectal cells we found that depletion of AKT2, AKT3, GSK3β and SRPK1 significantly decreased endogenous Rac1b levels. Whereas knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the depletion of GSK3β or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of Rac1b expression in colorectal cells and provide further mechanistic insights into how the regulation of alternative splicing events by protein kinases can contribute to sustain tumor cell survival.
- Biological effects of ingested nanomaterials and potential adverse outcomes for human healthPublication . Vieira, Adriana; Gramacho, Ana Catarina; Rolo, Dora; Vital, Nádia; Pereira, Joana; Matos, Paulo; Jordan, Peter; Martins, Carla; Assunção, Ricardo; Alvito, Paula; Gonçalves, Lídia; Bettencourt, Ana F.; Silva, Maria João; Louro, HenriquetaThe technology based on manufactured nanomaterials (NMs) has been pointed as key enabling technology, due to its potential to improve many products and processes, namely in agriculture, food and feed industry, leading to an exponential growth. Many products, already available, have NMs, such titanium dioxide NMs used as food additives, and many others are in development. Oral exposure may occur either directly, through the consumption of products/pharmaceuticals containing NMs, or indirectly, through the ingestion of foods contaminated with NMs released from food-contact materials or even through concentration in the food chain due to environmental accumulation. Therefore, the gastrointestinal tract appears to be a probable route of exposure to NMs and may lead to systemic exposure if the body barriers are surpassed. One major concern for public health is that NMs may produce biological effects, such as genotoxicity that are associated with increased risk of cancer. Although NMs have been extensively investigated in recent years, the studies have generated contradictory results, possibly due to differences in the physicochemical properties of the NMs studied and to other variables in the test systems. This work aimed to investigate the nano-bio interactions of titanium dioxide NMs, at cellular and molecular level, in the context of intestinal tract and digestion processes, to better understand their potential adverse impacts on human health. The results of the NMs uptake by intestinal cells, as well as their cytotoxic and genotoxic effects will be presented. This nanotoxicology approach may be incorporated at early-stage in the development of new NMs for food industry, in a “safe-by-design” approach that will enable safety to keep pace with innovation.
- Cellular and molecular mechanisms of toxicity of ingested nanomaterialsPublication . Gramacho, Ana Catarina; Rolo, Dora; Martins, Carla; Assunção, Ricardo; Gonçalves, Lídia M.; Bettencourt, Ana; Alvito, Paula; Pereira, Joana; Jordan, Peter; Silva, Maria João; Louro, HenriquetaThe technology based on manufactured nanomaterials (NMs) has been pointed as key enabling technology, due to its potential to improve many products and processes, namely in agriculture, food and feed industry. Many of such products, already available, have NMs such as titanium dioxide nanomaterials (TiO2) and the oral exposure may occur either directly, through the consumption of products/pharmaceuticals containing NMs, or indirectly, through the ingestion of foods contaminated with NMs released from food-contact materials or even through concentration in the food chain due to environmental accumulation. Therefore, the gastrointestinal tract (GIT) appears to be a probable route of exposure to NMs and may lead to systemic exposure if the body barriers are surpassed. One major concern for public health is that NMs may produce adverse outcomes (AO) such as genotoxic effects that are associated with increased risk of cancer. Although NMs have been extensively investigated in recent years, the studies have generated contradictory results, possibly due to differences in the physicochemical properties of the NMs studied and to other variables in the test systems. INSA has previously shown that NMs with the same chemistry, but differing in primary properties may yield different biological effects. Conversely, the NMs properties are context-dependent, i.e. can be affected by the surrounding matrix. These secondary features may be potentially more relevant for determining toxicological outcomes. In particular, processes like digestion may modify the NMs characteristics leading to unexpected toxicity in intestine cells. INGESTnano project aims to investigate the nano-bio interactions of NMs, at cellular and molecular level, in the context of intestinal tract and digestion processes, to better understand their potential negative impacts on human health with special reference to organ-specific cells. TiO2 has been selected as case-study to setup a workflow for addressing nanosafety concerns that may be in the future applied to other NMs to which GIT may be exposed. It is expected that this project will contribute to the safety evaluation of the TiO2 ingested, by elucidating key events (KE) elicited by these NMs and linking exposure to AO.
- Efeitos da aflatoxina B1 na integridade e morfologia de células intestinaisPublication . Serrenho, Inês; Vital, Nádia; Rolo, Dora; Louro, Henriqueta; Pereira, Joana; Matos, Paulo; Jordan, Peter; Alvito, Paula
- Effect of 2D and 3D growth conditions on expression of signalling GTPase Rac1b in colorectal cellsPublication . Pereira, Joana; Matos, Paulo; Jordan, PeterRac1 is a member of the Rho-family of small GTPases, which stimulates signalling pathways involved in the control of actin filament dynamics and transcriptional activation. We previously identified that Rac1b, an alternative splicing variant containing an extra exon, is overexpressed in a subset of B-Raf-mutated colorectal tumours. Although the Rac1b protein is expressed at low levels in cells, it is mostly in its active GTP-bound state and stimulates the transcription factor NFkB without affecting other classical Rac1 signalling pathways (such as lamellipodium formation or activation of the kinases PAK or JNK). Experimental depletion of Rac1b revealed an essential role in cell cycle progression and survival of colorectal cancer cells cultured under 2D conditions. Because different cell culture conditions can affect signalling pathway organization, we determined mRNA and protein expression levels and subcellular localization of Rac1b in Caco-2 cells grown as either monolayers on plastic dishes or as polarized epithelia on filters or as 3D spheroids in Matrigel. Most significantly, co-culture experiments with the generated polarized epithelia revealed that the presence of fibroblasts promoted loss of epithelial organization and a transient increase in Rac1b protein levels, implicating stromal cells in the regulation of Rac1b production and intracellular localization.
- Effect of the microenvironment on alternative splicing in colorectal cellsPublication . Pereira, JoanaAbout effect of the microenvironment on alternative splicing in colorectal cells.
- Ibuprofen inhibits overexpression of tumor-related Rac1b through SRSF1Publication . Gonçalves, Vânia; Matos, Paulo; Pereira, Joana; Jordan, PeterIntroduction: The serrated pathway to colorectal tumor formation involves oncogenic mutations in the BRAF gene, which are sufficient for initiation of hyperplastic growth but not for tumor progression. A previous analysis of colorectal tumors revealed that overexpression of splice variant Rac1b occurs in around 80% of tumors with mutant BRAF and both events proved to cooperate in tumor cell survival. Patients with inflamed human colonic mucosa also have increased expression of Rac1b as well as mice with experimentally induced colitis. The increase of Rac1b in the mouse model was specifically prevented by the nonsteroidal anti-inflammatory drug ibuprofen.
- Ibuprofen Inhibits Overexpression of Tumor-Related RAC1B through SRSF1Publication . Gonçalves, Vânia; Matos, Paulo; Pereira, Joana; Jordan, PeterThe serrated pathway to colorectal tumor formation involves oncogenic mutations in the BRAF gene, which are sufficient for initiation of hyperplastic growth but not for tumor progression [1]. A previous analysis of colorectal tumors revealed that overexpression of splice variant RAC1B occurs in around 80% of tumors with mutant BRAF and both events proved to cooperate in tumor cell survival [2]. Patients with inflamed human colonic mucosa also have increased expression of RAC1B as well as mice with experimentally induced colitis [3]. The increase of RAC1B in the mouse model was specifically prevented by the nonsteroidal anti-inflammatory drug ibuprofen [3]. The objective of our study is to understand the molecular regulation of RAC1B alternative splicing event and how it contributes to tumorigenesis. HT29 colorectal cell line was used as model to test several signaling pathways after 48h of treatment with ibuprofen. For this we analyzed the proteins of interest by Western Blot and the transcript levels by RT-PCR. Mechanistic studies in cultured HT29 colorectal tumor cells revealed that ibuprofen inhibited RAC1B expression in a cyclooxygenase inhibition–independent manner and targets directly the alternative splicing event [3]. Here, we provide evidence that ibuprofen leads to a protein kinase dependent decrease in expression of SRSF1, a splicing factor that we previously identified to promote RAC1B alternative splicing. Together, our results suggest that stromal cues, namely, inflammation, can trigger changes in RAC1B expression in the colon and identify ibuprofen as a highly specific and efficient inhibitor of RAC1B overexpression in colorectal tumors. Our data identify an additional cyclooxygenase–independent action of ibuprofen and suggest it may be beneficial in the treatment of patients with the subtype of BRAF-mutated serrated colorectal tumors. References [1] Velho S, Moutinho C, Cirnes L, Albuquerque C, Hamelin R, Schmitt F, Carneiro F, Oliveira C, and Seruca R (2008). BRAF, KRAS and PIK3CA mutations in colorectal serrated polyps and cancer: primary or secondary genetic events in colorectal carcinogenesis? BMC Cancer 8, 255. [2] Matos P, Oliveira C, Velho S, Gonçalves V, da Costa LT, Moyer MP, Seruca R, and Jordan P (2008). B-Raf V600E cooperates with alternative spliced Rac1b to sustain colorectal cancer cell survival. Gastroenterology 135, 899–906. [3] Matos P, Kotelevets L, Goncalves V, Henriques AF, Henriques A, Zerbib P, Moyer MP, Chastre E, Jordan P (2013). Ibuprofen inhibits colitis-induced overexpression of tumor-related Rac1b. Neoplasia 15(1):102-11. Acknowledgements Funding Support: grant UID/MULTI/04046/2019, SFRH/BD/109162/2015 and PTDC/BIA-MOL/28386/2017 from FCT
- Ibuprofen Inhibits Overexpression of Tumor-Related Rac1b through SRSF1Publication . Gonçalves, Vânia; Matos, Paulo; Pereira, Joana; Henriques, Andreia; Jordan, PeterAbstract: The serrated pathway to colorectal tumor formation involves oncogenic mutations in the BRAF gene, which are sufficient for initiation of hyperplastic growth but not for tumor progression. A previous analysis of colorectal tumors revealed that overexpression of splice variant Rac1b occurs in around 80% of tumors with mutant BRAF and both events proved to cooperate in tumor cell survival. Patients with inflamed human colonic mucosa also have increased expression of Rac1b as well as mice with experimentally induced colitis. The increase of Rac1b in the mouse model was specifically prevented by the nonsteroidal anti-inflammatory drug ibuprofen. Purpose: The objective of our study is to understand the molecular regulation of Rac1b alternative splicing event and how it contributes to tumorigenesis. Experimental description: HT29 colorectal cell line was used as model to test several signaling pathways after 48h of treatment with ibuprofen. For this we analyzed the proteins of interest by Western Blot and the transcript levels by RT-PCR. Results: Mechanistic studies in cultured HT29 colorectal tumor cells revealed that ibuprofen inhibited Rac1b expression in a cyclooxygenase inhibition–independent manner and targets directly the alternative splicing event. Here, we provide evidence that ibuprofen leads to a decrease in expression of SRSF1, a splicing factor that we previously identified to promote Rac1b alternative splicing. Together, our results suggest that stromal cues, namely, inflammation, can trigger changes in Rac1b expression in the colon and identify ibuprofen as a highly specific and efficient inhibitor of Rac1b overexpression in colorectal tumors. Conclusions: Our data identify an additional cyclooxygenase–independent action of ibuprofen and suggest it may be beneficial in the treatment of patients with the subtype of BRAF-mutated serrated colorectal tumors.
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