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- Estrogens and the G protein-coupled estrogen receptor in prostate cancer: angels or demons?Publication . Figueira, Marília Isabel Neto; Socorro, Sílvia Cristina da Cruz MarquesProstate cancer (PCa) is the second most frequent type of cancer among men, with an increasing incidence worldwide. PCa is highly regulated by hormones, with androgens playing a crucial role in its development and progression. However, also estrogens are accepted to influence PCa. In fact, estrogens were used in PCa treatment for decades, after the pioneer work of Huggins and Hodges demonstrating that PCa is a hormone-sensitive cancer, stimulated by androgenic activity and inhibited by suppression of androgens levels or estrogens administration. Nevertheless, despite the effectiveness of estrogens administration in delaying the progression of metastatic PCa, this therapy was discontinued because of the adverse side effects. Over the years, a vast number of studies were performed with the aim of deciphering the role of estrogens in PCa and find new estrogen-based alternatives for PCa treatment without the adverse effects. However, a contradiction in the estrogens actions remains evident in the literature until now. Many studies have pointed out estrogens as causative agents of PCa, contributing to its development and progression. Though, also a panoply of reports defended estrogens as protective against PCa, suppressing tumor growth, inducing apoptosis and inhibiting metastization, in line with their efficacy as therapeutic agents. This dual activity was associated with the diversity of receptors activated by estrogens, with the pro-carcinogenic actions associated with the activation of the estrogen receptor α (ERα), whereas anti-carcinogenic effects are linked to ERβ activation. Moreover, the discovery of G protein-coupled estrogen receptor (GPER) increased the complexity of mechanisms orchestrating estrogens actions. GPER is a membrane-bound ER that mediates the rapid, non-genomic effects of estrogens by mobilizing intracellular calcium and activating several signaling pathways. A substantial amount of evidence in different cancer types has been describing the anti-tumorigenic effects of GPER suppressing tumor growth and progression. A research area with increasing interest is the determination of the anti-carcinogenic actions of phytoestrogens, strengthened by the evidence of the lower incidence of PCa in Eastern countries, which typically have high consumption of these compounds in a consequence of plant-enriched diets. Furthermore, the mechanism of action of some of these compounds in PCa cells has been shown to require GPER activation. The present thesis aimed to answer some of the existent questions on the estrogens and GPER actions in PCa cells, which would lay the foundations for the development of new and safe estrogen-based therapies. First, it was investigated the action of estrogens as regulators of apoptosis and proliferation in prostate cells in the interplay with the SCF/c-KIT system, a set of ligand and receptor highly involved in carcinogenesis and a target of steroid hormones. Herein, it was demonstrated that E2 down-regulated the expression of the SCF/c-KIT system both in human PCa cell lines and rat prostate in vivo, which was accompanied by the effects of E2 suppressing proliferation and inducing apoptosis in rat prostates. These results supported the protective role of estrogens against PCa. Considering the promising results in other types of cancer, it was determined the effect of GPER activation in PCa cells covering several cancer hallmarks, from cell fate to metabolic reprogramming. It was found that GPER is highly expressed in PCa compared to benign prostatic hyperplasia (BPH) cases. Furthermore, GPER immunoreactivity was inversely correlated with PSA levels. GPER protein levels also were higher in neoplastic LNCaP cells than in the non-neoplastic PNT1A cells. Nevertheless, castrate-resistant PCa (CRPC) cells displayed reduced expression of GPER compared to the androgen-responsive cell line, supporting that GPER protein levels could be modulated by androgens. GPER was found to be localized at the cell membrane, endoplasmic reticulum and, residually, in the nucleus of prostate cells and this subcellular localization was altered by the GPER-specific agonist G1. G1-treatment showed a multiplicity of beneficial effects in PCa, targeting a panoply of cancer hallmarks. It reduced PCa cells viability and proliferation, arresting cell cycle and increasing apoptosis. It was also able to reduced migration and invasion, concomitantly with epithelial-mesenchymal transition (EMT) attenuation, depending on the cell line. Moreover, GPER activation by G1 increased energy metabolism and oxidative stress in PCa cells. Finally, it was evaluated how natural compounds, such as diosgenin, can influence the behavior of PCa in the interplay with GPER. Diosgenin diminished the viability of androgen-responsive and CRPC cells, without affecting the viability of non-neoplastic PNT1A cells. Moreover, the compound induced apoptosis and modulated glycolytic metabolism of PCa cells. The effects of diosgenin were enhanced when combined with G1, supported by the ability of diosgenin to augment the expression levels of GPER. Furthermore, GPER knockdown by a siRNA approach abrogated diosgenin effects inducing apoptosis of PCa cells, which implicates this receptor in the diosgenin mechanism of action. Globally, the findings of this thesis support the protective role of estrogens in PCa, disclosing new molecular targets and cellular processes. Moreover, it provides valuable information to find new therapeutic options, namely, estrogen-like, to deaccelerate the progression of disease and improve the survival of PCa patients.
- Estrogens down-regulate the stem cell factor (SCF)/c-KIT system in prostate cells: Evidence of antiproliferative and proapoptotic effectsPublication . Figueira, Marília I; Correia, Sara; Vaz, Cátia; Cardoso, HJ; Gomes, Inês; Marques, Ricardo; Baptista, Cláudio; Socorro, SílviaThe development of prostate cancer (PCa) is intimately associated with the hormonal environment, and the sex steroids estrogens have been implicated in prostate malignancy. However, if some studies identified estrogens as causative agents of PCa, others indicated that these steroids have a protective role counteracting prostate overgrowth. The tyrosine kinase receptor c-KIT and its ligand, the stem cell factor (SCF), have been associated with the control of cell proliferation/apoptosis and prostate carcinogenesis, and studies show that estrogens regulate their expression in different tissues, though, in the case of prostate this remains unknown. The present study aims to evaluate the role of 17β-estradiol (E2) in regulating the expression of SCF/c-KIT in human prostate cell lines and rat prostate, and to investigate the consequent effects on prostate cell proliferation and apoptosis. qPCR, Western Blot, and immuno(cito)histochemistry analysis showed that E2-treatment decreased the expression of SCF and c-KIT both in human prostate cells and rat prostate. Furthermore, the diminished expression of SCF/c-KIT was underpinned by the diminished prostate weight and reduced proliferation index. On the other hand, the results of TUNEL labelling, the increased activity of caspase-3, and the augmented expression of caspase-8 and Fas system in the prostate of E2-treated animals indicated augmented apoptosis in response to E2. The obtained results demonstrated that E2 down-regulated the expression of SCF/c-KIT system in prostate cells, which was associated with antiproliferative and proapoptotic effects. Moreover, these findings support the protective role of estrogens in PCa and open new perspectives on the application of estrogen-based therapies.
- The Pros and Cons of Estrogens in Prostate Cancer: An Update with a Focus on PhytoestrogensPublication . Figueira, Marília I; Carvalho, Tiago; Monteiro, Joana; Cardoso, Henrique J.; Correia, Sara; Vaz, CV; Duarte, Ana Paula; Socorro, SílviaThe role of estrogens in prostate cancer (PCa) is shrouded in mystery, with its actions going from angelic to devilish. The findings by Huggins and Hodges establishing PCa as a hormone-sensitive cancer have provided the basis for using estrogens in therapy. However, despite the clinical efficacy in suppressing tumor growth and the panoply of experimental evidence describing its anticarcinogenic effects, estrogens were abolished from PCa treatment because of the adverse secondary effects. Notwithstanding, research work over the years has continued investigating the effects of estrogens, reporting their pros and cons in prostate carcinogenesis. In contrast with the beneficial therapeutic effects, many reports have implicated estrogens in the disruption of prostate cell fate and tissue homeostasis. On the other hand, epidemiological data demonstrating the lower incidence of PCa in Eastern countries associated with a higher consumption of phytoestrogens support the beneficial role of estrogens in counteracting cancer development. Many studies have investigated the effects of phytoestrogens and the underlying mechanisms of action, which may contribute to developing safe estrogen-based anti-PCa therapies. This review compiles the existing data on the anti- and protumorigenic actions of estrogens and summarizes the anticancer effects of several phytoestrogens, highlighting their promising features in PCa treatment.
- Estrogenic Regulation of the SCF/c-KIT System in Prostate Cells: a Relationship with Prostate Cancer?Publication . Figueira, Marília Isabel Neto; Batista, Cláudio Jorge Maia; Socorro, Sílvia Cristina da Cruz MarquesProstate cancer (PCa) is the most common type of oncological disorder in men, for which an increasing incidence has been reported. Development and progression of PCa have been highly related with the circulating and intraprostatic hormonal milieu. Despite the classical role of androgens as stimulating agents in PCa growth, currently, estrogens also have been implicated in the prostate carcinogenesis. However, a duality for the possible role of estrogens in prostate cells has been gaining consistency over the last years. If some studies defend that estrogens are potential causative agents of PCa, other strong evidences indicate that these steroids may be protective against PCa. The tyrosine kinase receptor c-KIT and its ligand, the Stem Cell Factor (SCF) are powerful agents stimulating cell proliferation in a broad range of tissues, and the SCF/c-KIT interaction seems to play a crucial role in carcinogenesis. Moreover, it has been shown that estrogens modulate the expression of SCF/c-KIT system in several tissues, except the prostate. The present work aims to evaluate the role of estrogens regulating the SCF/c-KIT expression in human prostate cell lines and in rat prostate in vivo. The consequent effects on proliferation and apoptosis of prostatic cells will also be determined. Neoplastic (LNCaP, DU145 and PC3) and non-neoplastic (PNT1A) human prostate cell lines were cultured in presence or absence of 100 nM 17b-estradiol (E2) for different time periods. Adult male Wistar rats were daily injected with vehicle (control) or E2 (250 mg/day/kg) for 5 days. After treatment, animals were euthanized under anesthesia and prostates were collected, weighted and either fixed in 4 % paraformaldehyde or snap frozen in liquid nitrogen. The expression analysis of SCF and c-KIT in response to E2 was performed by means of real-time PCR, Western Blot and immunocito/histochemistry. The proliferation in rat prostate cells was estimated via fluorescent immunohistochemistry of Ki67. The protein ratio of Bax (pro-apoptotic)/Bcl-2 (anti-apoptotic), the expression of caspase-9, Fas and Fas- L, the enzymatic activity of caspase-3 and a TUNEL assay were used to evaluate apoptosis. The results obtained showed a decreased expression of both SCF and c-KIT in response to E2-treatment either in human prostate cells or rat prostate in vivo, which suggested a restricted proliferation of prostate cells in response to estrogens. This fact was confirmed in vivo by the diminished prostate weight and reduced Ki67 proliferation index observed in the E2-treated group. In addition, the enzymatic activity of caspase-3 was increased in response to E2, which indicates that estrogens induced apoptosis in rat prostate. The enhanced expression of the Fas system in the prostate of E2-treated animals suggests the involvement of the extrinsic pathway in the estrogen-induced apoptosis. In conclusion, the present work demonstrated that estrogens down-regulate the SCF/c-KIT system in neoplastic and non-neoplastic human prostate cells and in rat prostate in vivo. Moreover, estrogens have anti-proliferative and apoptosis-inducer effects in prostate exerted likely through the down-regulation of the SCF/c-KIT system. These findings also provided a body of evidence supporting the protective role of estrogens against development of PCa.
- Effect of Diosgenin in Suppressing Viability and Promoting Apoptosis of Human Prostate Cancer Cells: An Interplay with the G Protein-Coupled Oestrogen Receptor?Publication . Figueira, Marília I; Marques, Ricardo; Cardoso, Henrique J.; Fonseca, Lara R. S.; Duarte, Ana Paula; Silvestre, Samuel; Socorro, SílviaDiosgenin is a phytosteroid sapogenin with reported antitumoral activity. Despite the evidence indicating a lower incidence of prostate cancer (PCa) associated with a higher consumption of phytosteroids and the beneficial role of these compounds, only a few studies have investigated the effects of diosgenin in PCa, and its mechanisms of action remain to be disclosed. The present study investigated the effect of diosgenin in modulating PCa cell fate and glycolytic metabolism and explored its potential interplay with G protein-coupled oestrogen receptor (GPER). Non-neoplastic (PNT1A) and neoplastic (LNCaP, DU145, and PC3) human prostate cell lines were stimulated with diosgenin in the presence or absence of the GPER agonist G1 and upon GPER knockdown. Diosgenin decreased the cell viability, as indicated by the MTT assay results, which also demonstrated that castrate-resistant PCa cells were the most sensitive to treatment (PC3 > DU145 > LNCaP > PNT1A; IC50 values of 14.02, 23.21, 56.12, and 66.10 µM, respectively). Apoptosis was enhanced in diosgenin-treated cells, based on the increased caspase-3-like activity, underpinned by the altered expression of apoptosis regulators evaluated by Western blot analysis, which indicated the activation of the extrinsic pathway. Exposure to diosgenin also altered glucose metabolism. Overall, the effects of diosgenin were potentiated in the presence of G1. Moreover, diosgenin treatment augmented GPER expression, and the knockdown of the GPER gene suppressed the proapoptotic effects of diosgenin in PC3 cells. Our results support the antitumorigenic role of diosgenin and its interest in PCa therapy, alone or in combination with G1, mainly targeting the more aggressive stages of the disease.
- Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility?Publication . Figueira, Marília I; Cardoso, HJ; Correia, Sara; Baptista, Cláudio; Socorro, SílviaThe c-KIT, a tyrosine kinase receptor, and its ligand the stem cell factor (SCF) play an important role in the production of male and female gametes. The interaction of SCF with c-KIT is required for germ cell survival and growth, and abnormalities in the activity of the SCF/c-KIT system have been associated with human infertility. Recently, it was demonstrated that gonadotropic and sex steroid hormones, among others, regulate the expression of SCF and c-KIT in testicular and ovarian cells. Therefore, the hormonal (de)regulation of SCF/c-KIT system in the testis and ovary may be a cause underpinning infertility. In the present review, we will discuss the effects of hormones modulating the expression levels of SCF and c-KIT in the human gonads. In addition, the implications of hormonal regulation of SCF/c-KIT system for germ cell development and fertility will be highlighted.
- 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.
- Paradoxical and contradictory effects of imatinib in two cell line models of hormone-refractory prostate cancerPublication . Cardoso, HJ; Vaz, CV; Correia, Sara; Figueira, Marília I; Marques, Ricardo; Baptista, Cláudio; Socorro, SílviaImatinib mesylate is a chemotherapeutic drug that inhibits the tyrosine kinase activity of c-KIT and has been successfully used to treat leukemias and some solid tumors. However, its application for treatment of hormone-refractory prostate cancer (HRPC) has shown modest effectiveness and did not follow the outcomes in cultured cells or animal models. Moreover, the molecular pathways by which imatinib induces cytotoxicity in prostate cancer cells are poorly characterized.
- 5α-Dihydrotestosterone regulates the expression of L-type calcium channels and calcium-binding protein regucalcin in human breast cancer cells with suppression of cell growthPublication . Marques, Ricardo; Peres, Carina; Vaz, Cátia; Gomes, Inês; Figueira, Marília I; Cairrão, Elisa; Verde, Ignacio; Baptista, Cláudio; Socorro, SílviaAndrogens have been associated with the development of normal breast, and their role in mammary gland carcinogenesis has also been described. Several studies reported that androgens inhibit breast cancer cell growth, whereas others linked their action with the modulation of calcium (Ca(2+)) pumps, Ca(2+) channels and Ca(2+)-binding proteins. Also, it is known that deregulated Ca(2+) homeostasis has been implicated in the pathophysiology of breast. The L-type Ca(2+) channels (LTCCs) were found to be up-regulated in colon, colorectal and prostate cancer, but their presence in breast tissues remains uncharacterized. On the other hand, regucalcin (RGN) is a Ca(2+)-binding protein involved in the control of mammary gland cell proliferation, which has been identified as an androgen target gene in distinct tissues except breast. This study aimed to confirm the expression and activity of LTCCs in human breast cancer cells and investigate the effect of androgens in regulating the expression of α1C subunit (Cav1.2) of LTCCs and Ca(2+)-binding protein RGN. PCR, Western blot, immunofluorescence and electrophysiological experiments demonstrated the expression and activity of Cav1.2 subunit in MCF-7 cells. The MCF-7 cells were treated with 1, 10 or 100 nM of 5α-dihydrotestosterone (DHT) for 24-72 h. The obtained results showed that 1 nM DHT up-regulated the expression of Cav1.2 subunit while diminishing RGN protein levels, which was underpinned by reduced cell viability. These findings first confirmed the presence of LTCCs in breast cancer cells and opened new perspectives for the development of therapeutic approaches targeting Ca(2+) signaling.