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- Propolis Protects GC-1spg Spermatogonial Cells against Tert-Butyl Hydroperoxide-Induced Oxidative DamagePublication . Duarte, Filipa Maia; Feijó, Mariana; Luís, Ângelo; Socorro, Sílvia; Maia, Cláudio J.; Correia, SaraPropolis is a natural resin produced by honeybees with plenty of pharmacologic properties, including antioxidant activity. Oxidative stress disrupts germ cell development and sperm function, with demonstrated harmful effects on male reproduction. Several natural antioxidants have been shown to reduce oxidative damage and increase sperm fertility potential; however, little is known about the effects of propolis. This work evaluated the role of propolis in protecting spermatogonial cells from oxidative damage. Propolis’ phytochemical composition and antioxidant potential were determined, and mouse GC-1spg spermatogonial cells were treated with 0.1–500 µg/mL propolis (12–48 h) in the presence or absence of an oxidant stimulus (tert-butyl hydroperoxide, TBHP, 0.005–3.6 µg/mL, 12 h). Cytotoxicity was assessed by MTT assays and proliferation by Ki-67 immunocytochemistry. Apoptosis, reactive oxygen species (ROS), and antioxidant defenses were evaluated colorimetrically. Propolis presented high phenolic and flavonoid content and moderate antioxidant activity, increasing the viability of GC-1spg cells and counteracting TBHP’s effects on viability and proliferation. Additionally, propolis reduced ROS levels in GC-1spg, regardless of the presence of TBHP. Propolis decreased caspase-3 and increased glutathione peroxidase activity in TBHP-treated GC-1spg cells. The present study shows the protective action of propolis against oxidative damage in spermatogonia, opening the possibility of exploiting its benefits to male fertility.
- Effects of the endocrine disruptor vinclozolin in male reproduction: a systematic review and meta-analysisPublication . Feijó, Mariana; Martins, Roberta VL; Socorro, Sílvia; Pereira, L.; Correia, SaraEndocrine-disrupting chemicals have become an issue of scientific and public discussion. Vinclozolin (VNZ) is a fungicide that competitively antagonizes the binding of natural androgens to their receptor, disturbing the function of tissues that are sensitive to these hormones, as is the case of the male reproductive organs. A systematic review with meta-analyses of rodent studies was conducted to answer the following question: Does exposure to VNZ affect sperm parameters and testicular/epididymal weight? The methodology was prespecified according to the Cochrane Handbook for Systematic Reviews and PRISMA recommendations. Sixteen articles met the inclusion criteria, comprising a total of 1189 animals. The risk of publication bias was assessed using the Trim and Fill adjustment, funnel plot, and Egger regression test. Heterogeneity and inconsistency across the findings were tested using the Q-statistic and I2 of Higgins, respectively. Sensitivitywas also analyzed. Statistical analysiswas performed on Comprehensive Meta-Analysis software (Version 2.0), using random models and weighted mean differences along with a 95% confidence interval. Sperm motility, counts, daily sperm production (evidence of publication bias), and epididymis weight were decreased in VNZ-treated animals. Exposure length and dose, as well as the time point of exposure, influenced the obtained results. Despite the moderate/high heterogeneity observed, the sensitivity analysis overall demonstrated the robustness of the findings. The quality scores of the included studies were superior to 4 in a total of 9, then classified as good. The obtained data corroborate the capability of VNZ exposure to disrupt spermatogenic output and compromise male fertility.
- The Role of Ayahuasca in Colorectal Adenocarcinoma Cell Survival, Proliferation and Oxidative StressPublication . Gonçalves, Joana; Feijó, Mariana; Socorro, Sílvia; Luís, Ângelo; Gallardo, Eugenia; Duarte, Ana PaulaThe psychedelic beverage ayahuasca is originally obtained by Banisteriopsis caapi (B. caapi) (BC) and Psychotria viridis (P. viridis) (PV). However, sometimes these plant species are replaced by others that mimic the original effects, such as Mimosa hostilis (M. hostilis) (MH) and Peganum harmala (P. harmala) (PH). Its worldwide consumption and the number of studies on its potential therapeutic effects has increased. This study aimed to evaluate the anticancer properties of ayahuasca in human colorectal adenocarcinoma cells. Thus, the maximum inhibitory concentration (IC50) of decoctions of MH, PH, and a mixture of these (MHPH) was determined. The activities of caspases 3 and 9 were evaluated, and the cell proliferation index was determined through immunocytochemical analysis (Ki-67). Two fluorescent probes were used to evaluate the production of oxidative stress and the activity of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) was also evaluated. It was demonstrated that exposure to the extracts significantly induced apoptosis in Caco-2 cells, while decreasing cell proliferation. MH and MHPH samples significantly reduced oxidative stress and significantly increased glutathione peroxidase activity. No significant differences were found in SOD activity. Overall, it was demonstrated that the decoctions have a potential anticancer activity in Caco-2 cells.
- Downregulated Regucalcin Expression Induces a Cancer-like Phenotype in Non-Neoplastic Prostate Cells and Augments the Aggressiveness of Prostate Cancer Cells: Interplay with the G Protein-Coupled Oestrogen Receptor?Publication . Fonseca, Lara R. S.; Carreira, Ricardo J. P.; Feijó, Mariana; Cavaco, J. E.; Cardoso, Henrique; Vaz, C. V.; Figueira, Marília I.; Socorro, SílviaBackground/objectives: Regucalcin (RGN) is a calcium-binding protein and an oestrogen target gene, which has been shown to play essential roles beyond calcium homeostasis. Decreased RGN expression was identified in several cancers, including prostate cancer (PCa). However, it is unknown if the loss of RGN is a cause or a consequence of malignancy. Also, it needs confirmation if RGN oestrogenic regulation occurs through the G-protein-coupled oestrogen receptor (GPER). This study investigates how RGN knockdown affects prostate cell fate and metabolism and highlights the GPER/RGN interplay in PCa. Methods: Bioinformatic analysis assessed the relationship between RGN expression levels and patients' outcomes. RGN knockdown (siRNA) was performed in non-neoplastic prostate and castration-resistant PCa. Wild-type and RGN knockdown PCa cells were treated with the GPER agonist G1. Viability (MTT), proliferation (Ki-67 immunocytochemistry), apoptosis (caspase-3-like activity) and migration (Transwell assays) were evaluated. Spectrophotometric analysis was used to determine glucose consumption, lactate production and lactate dehydrogenase activity. Lipid content was assessed using the Oil Red assay. Results/conclusions: Bioinformatic analysis showed that the loss of RGN correlates with the development of metastatic PCa and poor survival outcomes. RGN knockdown induced a cancer-like phenotype in PNT1A cells, indicated by increased cell viability and proliferation and reduced apoptosis. In DU145 PCa cells, RGN knockdown augmented migration and enhanced the glycolytic profile, which indicates increased aggressiveness, in line with patients' data. GPER activation modulated RGN expression in PCa cells and RGN knockdown in DU145 cells influenced GPER actions, which highlighted an interplay between these molecular players with relevance for their potential use as biomarkers or therapeutic targets.
- Characterization and evaluation of Achillea erba-rotta subsp. moschata (Wulfen) I. Richardson and Achillea millefolium L. as potential ingredients for skin applicationsPublication . Marengo, Arianna; Cagliero, Cecilia; Sgorbini, Barbara; Menzio, Giulia ; Fusani, Pietro; Duarte, Ana Paula; Luís, Ângelo Filipe Santos ; Fonseca, Lara R. S.; Feijó, Mariana Pombal ; Socorro, Sílvia; Bertea, Cinzia Margherita; Rubiolo, PatriziaEthnopharmacological relevance: Despite their different geographical distribution Achillea millefolium L. and Achillea erba-rotta subsp. moschata (Wulfen) I. Richardson are characterized by a consolidated traditional knowledge. Although this is not their first traditional use, they have also long been used as ingredients for skin healing and skin care. Aim of the study: The aim of this work was to characterize the two Achillea species from a chemical and biomolecular point of view in order to find a simple tool for their discrimination. At the same time, the biological activity of the extracts as inhibitors of the enzymes tyrosinase and elastase and as antimicrobial agents was evaluated. Materials and methods: The biomolecular analysis was performed on the DNA region trnL-F. The hydroalcholic (EtOH50 %) extracts were quali-quantitatively characterized by HPLC-PDA-MS/MS and tested for their inhibitory effect on the enzymes tyrosinase and elastase as well as for their antimicrobial activity. Cytotoxicity towards human fibroblasts was tested to evaluate their safety for potential applications. Results: Restriction Fragment Length Polymorphism (RFLP) analysis of the trnL-F DNA region was a useful tool for species discrimination. Both extracts are rich in polyphenols and showed moderate tyrosinase (26-5 %) and elastase (22-14 %) inhibitory activity at 17 μg/mL. They were also able to inhibit the growth of the bacteria and yeasts studied, generally exhibiting cytotoxicity to human fibroblasts at concentrations of 500 μg/mL or higher. Conclusions: The hydroalcoholic extracts from the aerial parts of A. millefolium and A. erba-rotta subsp. moschata can be considered good candidates for the cosmetic and health sectors, also supporting the traditional use of these species in the treatment of skin diseases.
- Obesogens-induced deregulation of periprostatic adipose tissue: a driven force in the onset and progression of prostate cancer?Publication . Feijó, Mariana Pombal ; Socorro, Sílvia Cristina da Cruz Marques; Correia, Sara Carina de Lima; Kiss-Tóth, EndreProstate cancer (PCa) is a hormone-dependent cancer whose development and progression are strongly influenced by the tumour microenvironment and exogenous factors, such as environmental influences. The periprostatic adipose tissue (PPAT), by its anatomical proximity and functional crosstalk with prostate cells, emerged as a key driver of tumour growth, particularly in obesity, with the secretome of “obese” PPAT being associated with enhanced tumour aggressiveness. On the other hand, epidemiological and experimental studies have implicated endocrine-disrupting chemicals (EDCs) as environmental risk factors for PCa. Given the hormone dependency of PCa, it is predictable that it is a cancer highly susceptible to the influence of environmental exposures, namely EDCs and specifically those with obesogenic properties (i.e. obesogens), which are capable of disrupting both endocrine and metabolic pathways. Notably, based on their mechanisms of action and the cellular and molecular alterations they induce, obesogens may promote tumorigenesis either directly by acting on prostate cells or indirectly by inducing adipose tissue dysfunction. However, the extent to which obesogenic compounds drive these alterations and the consequent impact on prostate tumorigenesis remain largely unknown. Moreover, despite the well-established effects of obesogens on adipose tissue, no study has characterised their actions on PPAT. Addressing these gaps is critical to understanding how environmental factors intersect with adipose tissue biology, influencing interorgan communication between the prostate and adipose tissue and PCa development. Based on this scientific rationale, this doctoral thesis hypothesises that obesogen-induced PPAT dysfunction represents a driving force in the initiation and progression of PCa. Tributyltin (TBT) is a well-characterised obesogenic EDC and a potent regulator of adipogenesis, widely used in experimental settings to investigate the effects of obesogens. Therefore, using the obesogen model TBT this thesis aimed to: (i) characterise the morphological and secretory alterations of PPAT following TBT exposure; (ii) assess the impact of TBT-induced PPAT dysregulation on prostate cell fate, metabolism, oxidative and inflammatory status, and response to chemotherapeutic drugs; and (iii) identify the molecular targets and signalling pathways mediating the crosstalk between dysregulated PPAT and prostate cells. First, it was demonstrated that in vivo exposure to TBT (50 μg/kg) besides increasing rat body weight, enhanced PPAT somatic index and altered its functional phenotype. TBT treatment promoted a shift in rat prostate cells toward a glycolytic and lipogenic metabolic profile and stimulated oncogenic signalling pathways, including increased phosphorylated/total protein kinase B (pAKT/AKT) ratio and androgen receptor expression. Moreover, macrophage infiltration and a shift in macrophage polarisation towards a pro-inflammatory phenotype were observed both in the prostate and PPAT of TBT-exposed animals, suggesting that TBT can perturb the local prostate-PPAT immune status, contributing to an environment permissive to prostate carcinogenesis. These findings confirmed the effects of TBT on prostate cells, supporting the hypothesis and the investigation into the contribution of PPAT-mediated effects in altering prostate cell behaviour. Culture of the PPAT from rats exposed to TBT clearly demonstrated that TBT induced a dysregulation of the PPAT secretome. TBT-treated PPAT (TBT-PPAT) displayed increased leptin/adiponectin ratio and C-C motif chemokine ligand 7 (CCL7) levels. This adipokine/chemokine profile induced by TBT mimics that observed in obesity and is concurrent with a metabolic reprogramming associated with enhanced glucose, free fatty acids, and lipid peroxidation. Importantly, ex vivo exposure of rat PPAT to TBT (100 nM) recapitulated the findings obtained in vivo concerning the features of its secretome, which are capable of having an impact on prostate cell fate. The results obtained in the subsequent preclinical approaches using co-cultures and conditioned media (CM) assays confirmed the ability of TBT-PPAT enhance the viability, proliferation and migration, as well as apoptosis resistance, in all the studied prostate cell line models, namely non-neoplastic prostate epithelial cells (PNT1A), and androgen-sensitive (22Rv1) and androgen-insensitive (DU145 and PC3) PCa cells. Notably, the TBT-PPAT secretome increased the expression of CCL7 receptor, the C-C motif receptor 3 (CCR3), in prostate cells, which, together with the enhanced CCL7 secretion observed in our experimental setting, raised curiosity about the role of the CCL7-CCR3 axis underlying the pro-tumorigenic effects of TBT-PPAT. The use of a CCR3 antagonist significantly reduced TBT-PPAT induced migration across all cell lines, allowing to implicate the CCL7 and CCR3 in the observed responses of prostate cells. Other molecular targets beyond the CCL7-CCR3 axis were also highlighted. It is the case of tribbles homolog 1 (TRIB1), a pseudokinase involved in tumorigenesis and lipid homeostasis that was overexpressed across all studied cell models exposed to TBT-PPAT-CM. Cell fate alterations observed upon TBT treatment were accompanied by metabolic changes with distinct outcomes in non-neoplastic and neoplastic cell lines: in PNT1A, enhanced fatty acid β-oxidation and synthesis indicate a plausible shift toward a cancer-like metabolic profile; in 22Rv1, the unaltered metabolic and oxidative status suggests the activation of alternative signalling pathways sustaining TBT-PPAT effects; in DU145 and PC3, the distinct metabolic responses observed underscore the differential responsiveness of androgen-insensitive PCa cell subtypes to adipose-derived cues. The investigation in the present thesis was extended to a clinically relevant setting. Human PPAT obtained from patients submitted to radical prostatectomy or prostatic adenomectomy (Millin’s procedure) was treated ex vivo with TBT (100 nM) to confirm if the human tissues resemble the pro-tumorigenic cues identified in controlled experimental models. This approach demonstrated that human PPAT is a target of obesogenic dysregulation and showed that the secretome of obesogen-dysregulated PPAT can significantly enhance the viability of prostate cells. Moreover, the presence of human TBT-PPAT reduced the sensitivity of PCa cells to docetaxel and cabazitaxel, suggesting that obesogenic dysregulation contributes to PCa resistance to taxane-based chemotherapy. Overall, the scientific evidence gathered in this thesis identifies PPAT as a key target of obesogenic EDCs, which disrupt PPAT function and its crosstalk with prostate cells, thereby contributing to the initiation and progression of PCa. These findings open new avenues for developing interventions aimed at modulating PPAT activity to counteract its tumour-promoting effects and emphasise obesity as a critical modulator of PCa aggressiveness.
- Effects of 17ß-Estradiol and Endocrine Disruptor Methoxychlor in Spermatogonial Stem Cells: a Protective Effect of Regucalcin?Publication . Feijó, Mariana Pombal ; Correia, Sara Carina de Lima; Socorro, Sílvia Cristina da Cruz MarquesEndocrine disrupting chemicals (EDCs) are a set of compounds, either natural or produced by man, that interfere with the endocrine function by altering hormone metabolism, synthesis, and mechanism of action. In the last years, estrogens have emerged as important regulators of germ cell fate, although, the beneficial or detrimental effects of these hormones in spermatogenesis remains controversial, which raised the concern about the EDCs with estrogenic behavior. Methoxychlor (MXC) is an insecticide extensively used in the agricultural sector, which displays endocrine disrupting activity by mimicking estrogens actions (xenoestrogenic). Although it has been proved that MXC can affect the male reproductive function, little is known regarding the impact of this EDC in the spermatogonial stem cell (SSCs) population. SSCs are the adult stem cell population in the testis, having self-renewal capability and high differentiation rates, and its biological activity is the foundation of spermatogenesis. Therefore, any threat disturbing SSCs population can have a detrimental impact on the spermatogenic output and male fertility. Regucalcin (RGN) is a calcium (Ca2+)-binding protein that has been associated with the control of cell proliferation, oxidative stress, apoptosis, and metabolism. Furthermore, the protective role of RGN for the germ cell population upon exposure to damaging factors, such as oxidative stress, apoptosis inducers, freezing, and radiation has been suggested. So, it is highly likely to hypothesize that RGN may have a similar behavior against EDCs actions in SSCs. In the present dissertation, the impact of 17?-estradiol (E2) and MXC on SSCs glycolytic metabolism and survival/apoptosis and the influence of RGN in attenuating their effects were evaluated. For this purpose, a rat spermatogonial stem cell line (GC-6spg) transfected to overexpress RGN (GC6-spg/RGN) was cultured. After confirming RGN overexpression by means of Western blot analysis and immunofluorescence, GC6-spg/RGN cells and mock-transfectants (GC-6spg/Mock) were exposed either to 100 nM of E2 or 25 µM of MXC for 48 hours. Glucose consumption and lactate production, as well as, the expression and activity of glycolytic metabolism and apoptosis regulators were evaluated by spectrophotometric assays and Western blot analysis. The results obtained showed an increased glycolytic activity in GC-6spg cells overexpressing RGN (GC-6spg/RGN) compared to the mock-transfectants, regardless of E2 or MXC treatments, as indicated be the augmented glucose consumption and lactate production. E2 treatment did not affect the glycolytic metabolism of GC-6spg cells, though, in the case of MXC exposure, an enhanced glycolytic metabolism was shown. Nevertheless, RGN overexpression diminished the effect of MXC. Concerning apoptosis, it was found that GC-6spg/RGN cells displayed diminished apoptosis compared with mock-transfectants, namely, by the observed diminution of Bax (proapoptotic)/Bcl-2 (antiapoptotic) protein ratio, p53 expression and caspase-3 activity. E2 also seems to decrease the apoptotic rate of GC-6spg cells whereas upon MXC treatment apoptosis was increased. Interestingly, overall, RGN overexpression tended to counteract E2 and MXC effects over apoptosis. The present study is the first evidence that SSCs metabolism and apoptosis can be modulated by hormonal factors, namely E2 and the EDC with xenoestrogenic properties, MXC. Indeed, MXC was shown to greatly change the apoptotic status and metabolism of GC-6spg cells, with E2-treatment displaying mild effects. Furthermore, RGN was identified as a possible protective mechanism against the damaging effects of MXC in GC-6spg cells. Although preliminary, the obtained findings also highlight for the impact that MXC exposure might have disrupting the SSCs population and compromising male fertility.
- Revisão e Atualização da Imunofenotipagem do Lavado Broncoalveolar no Centro Hospitalar Universitário da Cova da BeiraPublication . Feijó, Mariana Pombal ; Monteiro, Andreia Sofia dos Reis; Correia, Sara Carina de LimaA lavagem broncoalveolar é uma técnica amplamente aceite como método de diagnóstico complementar em várias patologias do trato respiratório inferior, especialmente no que diz respeito ao grupo das doenças do interstício pulmonar. O Serviço de Patologia Clínica do Centro Hospitalar Universitário da Cova da Beira conta com uma experiência de aproximadamente 13 anos no estudo de lavados broncoalveolares (LBA) por citometria de fluxo (CMF) e análise morfológica. Assim, é de extrema importância efetuar um estudo retrospetivo, com o objetivo de rever a bibliografia recente, atualizar o conhecimento relativo aos diferentes perfis celulares encontrados no LBA em condições normais e patológicas e verificar a necessidade de atualização dos protocolos vigentes. Com a finalidade anteriormente referida, os resultados da análise morfológica e por CMF dos 763 LBA realizados entre 2006 e 2018 foram recolhidos, organizados e estudados tendo em conta outros dados importantes como o diagnóstico final, idade, sexo e hábitos tabágicos do paciente. Além disso, os resultados de CMF dos LBA realizados entre 2016 e 2018 foram reanalisados, de forma a identificar novas populações celulares. Os grupos estudados foram: doenças do interstício pulmonar (sarcoidose, fibrose pulmonar idiopática (FPI), pneumonite por hipersensibilidade (PH)), doenças obstrutivas (doença pulmonar obstrutiva crónica (DPOC) e asma brônquica (AB)), infeciosas (pneumonia) e indivíduos sem patologia pulmonar. Quanto ao padrão celular dos LBA estudados tem-se como principais resultados: (1) a linfocitose verificada na sarcoidose e na PH, (2) a razão CD4/CD8 aumentada na sarcoidose, (3) o aumento de neutrófilos na pneumonia e na PH, (4) o aumento de linfócitos natural killer (NK) na DPOC, (5) a inespecificidade do padrão celular do LBA na FPI e (6) a diminuição da razão CD4/CD8, assim como o aumento de macrófagos nos fumadores em comparação com os não fumadores. Quanto à identificação de novas populações celulares com relevância clínica no LBA, verificou-se a presença de células NKT, demonstrando-se significativamente elevadas nas doenças obstrutivas estudadas. Após revisão bibliográfica foi possível verificar que os resultados apresentados neste estudo estão de acordo com o descrito na literatura. Sendo que a atualização dos protocolos vigentes torna-se imprescindível para a melhoria da abordagem analítica deste tipo de líquido biológico tendo em vista a identificação de novas populações celulares com interesse clínico e o registo de alguns pormenores que podem influenciar os resultados e a sua interpretação, tais como hábitos tabágicos, número de células epiteliais, seringas utilizadas, aspeto macroscópico e volume instilado e recolhido. Esta atualização tornará a análise do LBA mais completa e útil para o clínico nas decisões de diagnóstico.