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- Promoter Demethylation Upregulates STEAP1 Gene Expression in Human Prostate Cancer: In Vitro and In Silico AnalysisPublication . Rocha, Sandra; Sousa, Inês; Gomes, Inês M.; Arinto, Patrícia; Pinheiro, Pedro Costa; Coutinho, Eduarda; Santos, Cecilia; Jerónimo, Carmen; Lemos, Manuel C.; Passarinha, L A; Socorro, Sílvia; Baptista, Cláudio MaiaThe Six Transmembrane Epithelial Antigen of the Prostate (STEAP1) is an oncogene overexpressed in several human tumors, particularly in prostate cancer (PCa). However, the mechanisms involved in its overexpression remain unknown. It is well known that epigenetic modifications may result in abnormal gene expression patterns, contributing to tumor initiation and progression. Therefore, this study aimed to analyze the methylation pattern of the STEAP1 gene in PCa versus non-neoplastic cells. Bisulfite amplicon sequencing of the CpG island at the STEAP1 gene promoter showed a higher methylation level in non-neoplastic PNT1A prostate cells than in human PCa samples. Bioinformatic analysis of the GEO datasets also showed the STEAP1 gene promoter as being demethylated in human PCa, and a negative association with STEAP1 mRNA expression was observed. These results are supported by the treatment of non-neoplastic PNT1A cells with DNMT and HDAC inhibitors, which induced a significant increase in STEAP1 mRNA expression. In addition, the involvement of HDAC in the regulation of STEAP1 mRNA expression was corroborated by a negative association between STEAP1 mRNA expression and HDAC4,5,7 and 9 in human PCa. In conclusion, our work indicates that STEAP1 overexpression in PCa can be driven by the hypomethylation of STEAP1 gene promoter.
- Assessment of the Bioaccessibility and Bioavailability of the Phenolic Compounds of Prunus avium L. by in Vitro Digestion and Cell ModelPublication . Gonçalves, Joana; Ramos, Rodrigo; Luís, Ângelo; Rocha, Sandra; Rosado, Tiago; Gallardo, Eugenia; Duarte, Ana PaulaThe fruit of Prunus avium L., commonly known as sweet cherry, is an excellent source of phytochemicals, namely, phenolic compounds. This study aims to determine in vitro the bioavailability and the bioactivity of phenolic compounds present in cherries. Caco-2 cells were used, and the permeability and integrity of the monolayer formed were investigated. After incubation, the phenolic compounds that permeabilized the cell monolayer were quantified using a high-performance liquid chromatography–diode array detector, and the antioxidant activity was evaluated by the 1,1-diphenyl-2-picrylhydrazyl method. The results demonstrated that the phenolic compounds of sweet cherries after undergoing a simulated digestion were absorbed by the cellular barrier, becoming bioavailable. Contrary to what was found after incubation with the in vitro digested extract, the integrity of the cell monolayer was altered and its permeability increased upon incubation with the sweet cherry phenolic extract. Regarding the antioxidant activity, it was verified that this decreased after the absorption by the cellular monolayer. This study suggests that digestion is an indispensable process for absorption because without it the phenolic content of the food matrix does not become bioaccessible.
- Astrocyte-derived GDNF is a potent inhibitor of microglial activationPublication . Rocha, Sandra Catarina Moreira; Baltazar, Graça Maria FernandesParkinson’s disease is characterized by the selective loss of dopaminergic neurons in the substantia nigra pars compacts. The aetiology of this disease is not completely clarified; however several hypotheses have been advanced regarding the loss of dopaminergic neurons. Among them, neuroinflammation has been increasingly recognized as a major factor in the pathogenesis of Parkinson’s disease, and increasing evidence suggests that microglial cells are a predominant source of inflammation contributing for the dopaminergic neurodegeneration. Astrocytes play vital roles in the maintenance of the normal brain function and diverse studies suggest that they could act as physiological regulators preventing excessive inflammatory microglial responses. However, little is known regarding how astrocytes may modulate the microglial activation. Due to the relevance of astrocytes-microglia interactions in the regulation of brain inflammation, it is important to identify the mediators involved in this process, which could act as natural anti-inflammatory agents in the brain. In this way, the major goal of the present work was to evaluate of the effect of soluble mediators release by astrocytes on microglial activation induced by the inflammatory agent Zymosan A, as well as to identify the nature of these mediators. For the determination of the effect of these molecules in the microglial activity, ventral midbrain microglial primary cultures were previously exposed to astrocytes conditioned media (or culture medium – control), and then treated with 5 μg/mL ofZymosan. Studies previously made indicated that this concentration of Zymosan provokes an accented increase of the microglia phagocytic activity and increased ROS generation, showing no citotoxici effect to the cells. However, the pre-incubation of the microglial cells with astrocytes conditioned media was capable to prevent the characteristic increase of the phagocytic activity and ROS production induced by Zymosan A, which levels remained at control levels. To evaluate the nature of the soluble mediators release by astrocytes able to prevent the microglial activation, specific antibodies recognizing some neurotrophics factors known by its neuroprotectors properties of substantia nigra. were used to block their action on the astrocytes conditioned media. Those antibodies were: anti-GDNF, anti-CDNF and anti condicioned media, we observed that the glial cells line-derived neurotrophic factor (GDNF) seems to be a soluble mediator capable to completely prevent the microglial activation induced by Zymosan A, whereas, the remaining mediators do not exerted an effect on microglial activation. To confirm this fact, specific knockdown of GDNF was achived in astrocytes cell cultures and the resultant condicined medium from this cultures, when applied to microglia cultures before Zymosan A, were not capable to prevent its activation. Finally, to clarify if this effect was an isolated of GDNF or if other molecules were laso involved the levels of GDNF on the astrocytes conditioned media were quntidied by ELISA assay. Based on the obtained values, three concentrations of GDNF (100 pg/mL, 200 pg/mL and 400 pg/mL), diluted in culture medium, were tested to verify its capability to prevent microglia activation induced by Zymosan A. The results have shown that the three concentrations of GDNF were capable to suppress microglial activation induced by Zymosan A. However, the achieved protection was not in a dose dependent manner, as initially expected. Taken together, the results obtained in this work demonstrate that GDNF a neurotrophic factor expressed by astrócitos, has the capacity to modulate the microglial inflammatory response. In this way, GDNF could be use to develop a potencial therapy to prevent neuroinflammation, and in this way contributing to the reduction of the development of Parkinson’s Disease pathogenesis.
- Unravelling the effect of STEAP1 knockdown in prostate cancer cells: from protein expression profile to clinical applicationsPublication . Rocha, Sandra Catarina Moreira da; Socorro, Sílvia Cristina da Cruz Marques; Passarinha, Luís António PaulinoProstate cancer (PCa) is one of the most common cancers in the male population and continues to be a considerable source of morbidity and mortality for men worldwide. In an early stage of the disease, the cancer is confined to the prostate gland depending on the stimulating action of androgens. This dependence is the characteristic that allows the use of androgen-deprivation therapy (ADT), which is based on reducing circulating levels of androgens or blocking their actions. The continuous administration of this therapy makes the PCa cells resistant to treatment, being able to survive, extravasate the prostate gland and cause metastases. This stage of the disease is the so-called castrate-resistant PCa (CRPC) and involves the use of chemotherapeutic agents. The limitations of the existent therapeutic options have been stimulating the use of combined therapies targeting survival regulators and proteins involved in the regulation of prostate cell growth and its emergence as a promising option for the treatment of PCa. The development and progression of PCa results from several genetic and metabolic changes, which give tumor cells treatment resistance, exacerbated proliferation capacity and invasive features. Interestingly, many cancer-associated proteins have been reported to have a multifunctional role in controlling the biological changes acquired by PCa cells. Among them is STEAP1 (“Six-Transmembrane Epithelial Antigen of the Prostate”). STEAP1 is a transmembrane protein identified as overexpressed in PCa and several other types of cancer. In non-tumor tissues, STEAP1 expression is low and only restricted to the prostate gland, suggesting that the increase in its expression may favor and accompany tumor development. STEAP1 has been suggested to act as an oncogene, as this protein appears to have the ability to regulate cell proliferation, apoptosis, invasion and metastasis leading to PCa progression. Furthermore, several studies have shown that STEAP1 is involved in intercellular communication and can function as an ion channel or transporter protein. Thus, taking into account the various biological processes that may be controlled by STEAP1, its location and overexpression at the cell surface, and its low expression in non-tumor tissues, it is predictable that this protein plays a crucial role in the maintenance of cell homeostasis and might be used as a possible therapeutic target for PCa. However, the clinical significance of STEAP1 expression in PCa and its usefulness as a biomarker still needs clarification. Furthermore, there are few studies on the regulation of STEAP1 expression in PCa and on cellular mechanisms underlying biological functions in PCa. The present thesis aimed to first establish the clinical significance of the STEAP1 protein in PCa and explore its usefulness as a biomarker. Its relationship with the other proteins of the STEAP family (STEAP2, STEAP3 and STEAP4) was also investigated. Using databases and bioinformatics tools, it was found that the STEAP1 gene is differentially expressed in PCa, from benign lesions to metastatic adenocarcinoma, suggesting that deregulation of this gene may be involved in the early stages of the development of the disease. Concerning to the other STEAP family proteins, our results indicate that STEAP2 and STEAP4 are also overexpressed in PCa, but STEAP3 is underexpressed. However, only the high expression of STEAP1 was associated with a poor prognosis of patients with PCa, compared to patients without alterations in the expression of STEAP family proteins. Furthermore, comparing only patients with alterations in the expression of these proteins, we found that the overexpression of STEAP1 continues to be associated with a worse prognosis, while the overexpression of STEAP4 indicates a good prognosis for patients with PCa, meaning a longer survival time. The dysregulation of STEAP1 can have a high impact on the development of PCa, and the work developed in this thesis demonstrated that STEAP1 can be regulated by epigenetic mechanisms. In addition to confirming the high expression of STEAP1 in neoplastic prostate cell lines (LNCaP) and the low expression of this gene in non-neoplastic prostate cell lines (PNT1A), it was found that the CpG island located at near the transcription start site is methylated in PNT1A cells and demethylated in LNCaP cells. This result suggested that demethylation of the STEAP1 gene promoter may contribute to its overexpression in PCa. The recruitment of enzymes, such as DNA methyltransferases and histone deacetylases, are essential for epigenetic modifications to occur. The treatment of PNT1A cells with inhibitors of these enzymes increased the expression of the STEAP1 gene, suggesting once again that the hypomethylation of the STEAP1 gene promoter may be associated with its overexpression in PCa. The global characterization of the proteome of LNCaP cells silenced for STEAP1 in order to unravel new molecular and cellular functions associated with STEAP1 was another focus of this dissertation. For this, LNCaP cells silenced, or not, for STEAP1 were subjected to protein identification and quantification techniques. Comparing the two experimental groups (STEAP1 siRNA LNCaP cells versus scramble siRNA-LNCaP cells), 526 proteins were found to be differentially expressed. The cellular processes of endocytosis, RNA transport, apoptosis, amino acid biosynthesis and metabolic pathways were the most significant, suggesting that STEAP1 can modulate these biological processes. In fact, STEAP1 silencing promotes apoptosis of PCa cells, attenuating cell proliferation and metabolism, stimulates endocytosis and diminishes intercellular communication by vesicles. These results emphasized the oncogenic role of STEAP1 in PCa. Finally, it was found that the action of antiandrogenic drugs on LNCaP cells, namely bicalutamide, enzalutamide and apalutamide, appears to be independent of STEAP1 expression levels. On the other hand, treatment of LNCaP cells with taxane-based chemotherapeutic drugs, paclitaxel, docetaxel and cabazitaxel, appears to depend on STEAP1 levels. High STEAP1 expression levels seem to promote a more efficient action of chemotherapeutic drugs based on taxanes, with stimulation of apoptosis and a decrease in uncontrolled proliferation of prostate cells. In conclusion, the main results of this thesis confirmed the crucial action of STEAP1 in development and progression of PCa. This dissertation showed that STEAP1 overexpression may be used as a prognostic biomarker, indicating that patients with PCa overexpressing this gene have a shorter survival time; also it showed that the reduction of STEAP1 expression might be a therapeutic strategy to decrease the development and progression of PCa; and yet, in an innovative way, this thesis demonstrated that the action of chemotherapeutic drugs based on taxanes are more effective in the treatment of PCa overexpressing STEAP1. Additional investigation into the relationship between STEAP1 and PCa will be a fundamental basis for the development of new therapeutic approaches for PCa, as well as its usefulness as biomarker.