Counteracting the effect of leukemia exosomes by antiangiogenic gold nanoparticles

FCT/MCTES - UID/Multi/04378/2019. FCT/MCTES - SFRH/BPD/124612/2016.Purpose: Progression of chronic myeloid leukemia (CML) is frequently associated with increased angiogenesis at the bone marrow mediated by exosomes. The capability of gold nanoparticles (AuNPs) functionalized with antiangiogenic peptides to hinder the formation of new blood vessels has been demonstrated in a chorioallantoic membrane (CAM) model. Methods: Exosomes of K562 CML cell line were isolated and their angiogenic effect assessed in a CAM model. AuNPs functionalized with antiangiogenic peptides were used to block the angiogenic effect of CML-derived exosomes, assessed by evaluation of expression levels of key modulators involved in angiogenic pathways - VEGFA, VEGFR1 (also known as FLT1) and IL8. Results: Exosomes isolated from K562 cells promoted the doubling of newly formed vessels associated with the increase of VEGFR1 expression. This is a concentration and timedependent effect. The AuNPs functionalized with antiangiogenic peptides were capable to block the angiogenic effect by modulating VEGFR1 associated pathway. Conclusion: Exosomes derived from blast cells are capable to trigger (neo)-angiogenesis, a key factor for the progression and spreading of cancer, in particular in CML. AuNPs functionalized with specific antiangiogenic peptides are capable to block the effect of the exosomes produced by malignant cells via modulation of the intrinsic VEGFR pathway. Together, these data highlight the potential of nanomedicine-based strategies against cancer proliferation.publishersversionpublishe

Exploring RAB11A Pathway to Hinder Chronic Myeloid Leukemia-Induced Angiogenesis In Vivo

project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB. This work is also funded by Fundação para a Ciência e Tecnologia in the scope of the project 2022.04315.PTDC. Publisher Copyright: © 2023 by the authors.Neoangiogenesis is generally correlated with poor prognosis, due to the promotion of cancer cell growth, invasion and metastasis. The progression of chronic myeloid leukemia (CML) is frequently associated with an increased vascular density in bone marrow. From a molecular point of view, the small GTP-binding protein Rab11a, involved in the endosomal slow recycling pathway, has been shown to play a crucial role for the neoangiogenic process at the bone marrow of CML patients, by controlling the secretion of exosomes by CML cells, and by regulating the recycling of vascular endothelial factor receptors. The angiogenic potential of exosomes secreted by the CML cell line K562 has been previously observed using the chorioallantoic membrane (CAM) model. Herein, gold nanoparticles (AuNPs) were functionalized with an anti-RAB11A oligonucleotide (AuNP@RAB11A) to downregulate RAB11A mRNA in K562 cell line which showed a 40% silencing of the mRNA after 6 h and 14% silencing of the protein after 12 h. Then, using the in vivo CAM model, these exosomes secreted by AuNP@RAB11A incubated K562 did not present the angiogenic potential of those secreted from untreated K562 cells. These results demonstrate the relevance of Rab11 for the neoangiogenesis mediated by tumor exosomes, whose deleterious effect may be counteracted via targeted silencing of these crucial genes; thus, decreasing the number of pro-tumoral exosomes at the tumor microenvironment.publishersversionpublishe

Os subprocessos do processo de escrita

O presente estudo pretende demonstrar a importância do ensino explícito e sistemático do processo de escrita e dos seus subprocessos: a planificação, a textualização, a revisão e a edição, alunos do 4.º ano do 1.º Ciclo do Ensino Básico, como forma de contribuir para a melhoria da sua competência textual. Pretende-se que os alunos do grupo experimental interiorizem a dimensão processual da escrita, levando-os a planificar, textualizar, rever, e editar, de forma a escrever melhores textos: expositivos e descritivos. Nesse sentido, foi traçado um desenho investigativo assente no seguinte dispositivo pedagógico: pré-teste, plano de intervenção e ação e pós-teste. O plano de intervenção e ação consistiu na implementação de um Percurso de Ensino/Aprendizagem para a Compreensão do Processo de Escrita na Produção de um Texto Escrito, constituído por nove sequências didáticas. Constata-se, a partir de uma análise qualitativa dos pré e pós-testes, que houve uma progressão do grupo sujeito ao plano de intervenção e ação, quanto à interiorização da dimensão processual da escrita, produzindo, deste modo, textos com maior coesão e coerência, em comparação com o grupo de controlo, comprovando, assim, os objetivos e as questões orientadoras do estudo

Hyperthermia induced by gold nanoparticles and visible light photothermy combined with chemotherapy to tackle doxorubicin sensitive and resistant colorectal tumor 3D spheroids

UIDB/04378/2020 SFRH/BPD/124612/2016Current cancer therapies are frequently ineffective and associated with severe side effects and with acquired cancer drug resistance. The development of effective therapies has been hampered by poor correlations between pre-clinical and clinical outcomes. Cancer cell-derived spheroids are three-dimensional (3D) structures that mimic layers of tumors in terms of oxygen and nutrient and drug resistance gradients. Gold nanoparticles (AuNP) are promising therapeutic agents which permit diminishing the emergence of secondary effects and increase therapeutic efficacy. In this work, 3D spheroids of Doxorubicin (Dox)-sensitive and -resistant colorectal carcinoma cell lines (HCT116 and HCT116-DoxR, respectively) were used to infer the potential of the combination of chemotherapy and Au-nanoparticle photothermy in the visible (green laser of 532 nm) to tackle drug resistance in cancer cells. Cell viability analysis of 3D tumor spheroids suggested that AuNPs induce cell death in the deeper layers of spheroids, further potentiated by laser irradiation. The penetration of Dox and earlier spheroid disaggregation is potentiated in combinatorial therapy with Dox, AuNP functionalized with polyethylene glycol (AuNP@PEG) and irradiation. The time point of Dox administration and irradiation showed to be important for spheroids destabilization. In HCT116-sensitive spheroids, pre-irradiation induced earlier disintegration of the 3D structure, while in HCT116 Dox-resistant spheroids, the loss of spheroid stability occurred almost instantly in post-irradiated spheroids, even with lower Dox concentrations. These results point towards the application of new strategies for cancer therapeutics, reducing side effects and resistance acquisition.publishersversionpublishe

Response of pseudomonas putida KT2440 to phenol at the level of membrane proteome

This study led to the extension and refinement of our current model for the global response of Pseudomonas putida KT2440 to phenol by getting insights into the adaptive response mechanisms involving the membrane proteome. A two-dimensional gel electrophoresis based protocol was optimized to allow the quantitative comparison of membrane proteins, by combining inner and outer membrane fractionation with membrane protein solubilization using the detergent dodecylmaltoside. Following phenol exposure, a coordinate increased content of protein subunits of known or putative solvent efflux pump systems (e.g. TtgA, TtgC, Ttg2A, Ttg2C, and PP_1516-7) and a decreased content of porins OprB, OprF, OprG and OprQ was registered, consistent with an adaptive response to reduce phenol intracellular concentration. This adaptive response may in part be mediated by post-translational modifications, as suggested by the relative content of the multiple forms identified for a few porins and efflux pump subunits. Results also suggest the important role of protein chaperones, of cell envelope and cell surface and of a more active respiratory chain in the response to phenol. All these mechanistic insights may be extended to Pseudomonas adaptation to solvents, of possible impact in biodegradation, bioremediation and biocatalysis.PhD scholarship SFRH/BD/38805/200

Liquid biopsies in myeloid malignancies

Funding Information: This work was supported by the Applied Molecular Biosciences Unit-UCIBIO which is financed by national funds from FCT/MCTES (UID/Multi/04378/2019).Hematologic malignancies are the most common type of cancer affecting children and young adults, and encompass diseases, such as leukemia, lymphoma, and myeloma, all of which impact blood associated tissues such as the bone marrow, lymphatic system, and blood cells. Clinical diagnostics of these malignancies relies heavily on the use of bone marrow samples, which is painful, debilitating, and not free from risks for leukemia patients. Liquid biopsies are based on minimally invasive assessment of markers in the blood (and other fluids) and have the potential to improve the efficacy of diagnostic/therapeutic strategies in leukemia patients, providing a useful tool for the real time molecular profiling of patients. The most promising noninvasive biomarkers are circulating tumor cells, circulating tumor DNA, microRNAs, and exosomes. Herein, we discuss the role of assessing these circulating biomarkers for the understanding of tumor progression and metastasis, tumor progression dynamics through treatment and for follow-up.publishersversionpublishe

Gene Therapy in Cancer Treatment: Why Go Nano?

The proposal of gene therapy to tackle cancer development has been instrumental for the development of novel approaches and strategies to fight this disease, but the efficacy of the proposed strategies has still fallen short of delivering the full potential of gene therapy in the clinic. Despite the plethora of gene modulation approaches, e.g., gene silencing, antisense therapy, RNA interference, gene and genome editing, finding a way to efficiently deliver these effectors to the desired cell and tissue has been a challenge. Nanomedicine has put forward several innovative platforms to overcome this obstacle. Most of these platforms rely on the application of nanoscale structures, with particular focus on nanoparticles. Herein, we review the current trends on the use of nanoparticles designed for cancer gene therapy, including inorganic, organic, or biological (e.g., exosomes) variants, in clinical development and their progress towards clinical applications.This work was supported by the Applied Molecular Biosciences Unit - UCIBIO which is financed by national funds from FCT (UIDB/04378/2020), CRR (SFRH/BPD/124612/2016) and LRG (Inn-Indigo 00002/2015 RA Detect)

Genetic biomarkers in chronic myeloid leukemia: What have we learned so far?

UIDP/04378/2020 UIDB/04378/2020 LA/P/0140/2020Chronic Myeloid Leukemia (CML) is a rare malignant proliferative disease of the hematopoietic system, whose molecular hallmark is the Philadelphia chromosome (Ph). The Ph chromosome originates an aberrant fusion gene with abnormal kinase activity, leading to the buildup of reactive oxygen species and genetic instability of relevance in disease progression. Several genetic abnormalities have been correlated with CML in the blast phase, including chromosomal aberrations and common altered genes. Some of these genes are involved in the regulation of cell apoptosis and proliferation, such as the epidermal growth factor receptor (EGFR), tumor protein p53 (TP53), or Schmidt-Ruppin A-2 proto-oncogene (SRC); cell adhesion, e.g., catenin beta 1(CTNNB1); or genes associated to TGF-β, such as SKI like proto-oncogene (SKIL), transforming growth factor beta 1 (TGFB1) or transforming growth factor beta 2 (TGFB2); and TNF-α pathways, such as Tumor necrosis factor (TNFA) or Nuclear factor kappa B subunit 1 (NFKB1). The involvement of miRNAs in CML is also gaining momentum, where dysregulation of some critical miRNAs, such as miRNA-451 and miRNA-21, which have been associated to the molecular modulation of pathogenesis, progression of disease states, and response to therapeutics. In this review, the most relevant genomic alterations found in CML will be addressed.publishersversionpublishe

Peptide-coated gold nanoparticles for modulation of angiogenesis in vivo

In this work, peptides designed to selectively interact with cellular receptors involved in the regulation of angiogenesis were anchored to oligo-ethylene glycol-capped gold nanoparticles (AuNPs) and used to evaluate the modulation of vascular development using an ex ovo chick chorioallantoic membrane assay. These nanoparticles alter the balance between naturally secreted pro- and antiangiogenic factors, under various biological conditions, without causing toxicity. Exposure of chorioallantoic membranes to AuNP–peptide activators of angiogenesis accelerated the formation of new arterioles when compared to scrambled peptide-coated nanoparticles. On the other hand, antiangiogenic AuNP–peptide conjugates were able to selectively inhibit angiogenesis in vivo. We demonstrated that AuNP vectorization is crucial for enhancing the effect of active peptides. Our data showed for the first time the effective control of activation or inhibition of blood vessel formation in chick embryo via AuNP-based formulations suitable for the selective modulation of angiogenesis, which is of paramount importance in applications where promotion of vascular growth is desirable (eg, wound healing) or ought to be contravened, as in cancer development

Programa de intervenção para aprendizagem do texto expositivo

UIDB/04647/2020 UIDP/04647/2020Neste texto, apresenta-se um estudo sobre o impacto que o ensino explícito e sistemático do processo de escrita e dos seus subprocessos: a planificação, a textualização, a revisão e a edição tem na qualidade de textos expositivos produzidos por alunos de 4.º ano de escolaridade, em Portugal. Foi desenvolvida uma intervenção no sentido de ensinar explicitamente estes subprocessos, aplicados aos textos expositivos. O desenho investigativo contemplou um diagnóstico da situação através da realização de um pré-teste; uma intervenção didática, visando implementar um conjunto de sequências didáticas em que a dimensão processual da escrita era objeto de ensino explícito; e, por último, a realização de um pós-teste para avaliar o impacto da intervenção na qualidade dos textos. Constata-se que, a partir de uma análise mista dos pré e pós-testes, houve uma progressão do grupo experimental, no que se refere à interiorização da dimensão processual da escrita e à qualidade dos textos. In this text, we present a study focused on the impact of explicit and systematic teaching of the writing process and its subprocesses have in the quality of expository texts produced: Planning, textualization, revision and editing, by students of 4th grade in Portugal. An intervention was developed to explicitly teach these subprocesses, applied to expository texts. The research design contemplated a diagnosis of the situation by a pre-test; a didactic intervention, aiming to implement a set of didactic sequences in which the procedural dimension of writing was the object of explicit teaching; and, finally, a post-test to evaluate the impact of intervention on the quality of texts. In conclusion, from a mixed analysis of the pre- and post-tests, we can observe there was a progression of the experimental group, regarding the internalization of the procedural dimension of writing and the quality of texts.publishersversionpublishe