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- Sweet Cherries as Anti-Cancer Agents: From Bioactive Compounds to FunctionPublication . Fonseca, Lara R. S.; Silva, Gonçalo R.; Luís, Ângelo; Cardoso, Henrique J.; Correia, Sara; Vaz, CV; Duarte, Ana Paula; Socorro, SílviaSweet cherries (Prunus avium L.) are among the most appreciated fruits worldwide because of their organoleptic properties and nutritional value. The accurate phytochemical composition and nutritional value of sweet cherries depends on the climatic region, cultivar, and bioaccessibility and bioavailability of specific compounds. Nevertheless, sweet cherry extracts are highly enriched in several phenolic compounds with relevant bioactivity. Over the years, technological advances in chemical analysis and fields as varied as proteomics, genomics and bioinformatics, have allowed the detailed characterization of the sweet cherry bioactive phytonutrients and their biological function. In this context, the effect of sweet cherries on suppressing important events in the carcinogenic process, such as oxidative stress and inflammation, was widely documented. Interestingly, results from our research group and others have widened the action of sweet cherries to many hallmarks of cancer, namely metabolic reprogramming. The present review discusses the anticarcinogenic potential of sweet cherries by addressing their phytochemical composition, the bioaccessibility and bioavailability of specific bioactive compounds, and the existing knowledge concerning the effects against oxidative stress, chronic inflammation, deregulated cell proliferation and apoptosis, invasion and metastization, and metabolic alterations. Globally, this review highlights the prospective use of sweet cherries as a dietary supplement or in cancer treatment.
- 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.
- Suppressed glycolytic metabolism in the prostate of transgenic rats overexpressing calcium-binding protein regucalcin underpins reduced cell proliferationPublication . Vaz, CV; Marques, Ricardo; Cardoso, HJ; Baptista, Cláudio; Socorro, SílviaRegucalcin (RGN) is a calcium-binding protein underexpressed in human prostate cancer cases, and it has been associated with the suppression of cell proliferation and the regulation of several metabolic pathways. On the other hand, it is known that the metabolic reprogramming with augmented glycolytic metabolism and enhanced proliferative capability is a characteristic of prostate cancer cells. The present study investigated the influence of RGN on the glycolytic metabolism of rat prostate by comparing transgenic adult animals overexpressing RGN (Tg-RGN) with their wild-type counterparts. Glucose consumption was significantly decreased in the prostate of Tg-RGN animals relatively to wild-type, and accompanied by the diminished expression of glucose transporter 3 and glycolytic enzyme phosphofructokinase. Also, prostates of Tg-RGN animals displayed lower lactate levels, which resulted from the diminished expression/activity of lactate dehydrogenase. The expression of the monocarboxylate transporter 4 responsible for the export of lactate to the extracellular space was also diminished with RGN overexpression. These results showed the effect of RGN in inhibiting the glycolytic metabolism in rat prostate, which was underpinned by a reduced cell proliferation index. The present findings also suggest that the loss of RGN may predispose to a hyper glycolytic profile and fostered proliferation of prostate cells.
- 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.
- The SCF/c-KIT system and imatinib actions in prostate cancer: a cross-talk with RGN?Publication . Cardoso, Henrique José Matos Morão Mingote; Batista, Cláudio Jorge Maia; Socorro, Sílvia Cristina da Cruz MarquesThe progression of prostate cancer (PCa), from an early stage confined to prostate to a more aggressive form, is associated with loss of androgen responsiveness. At this stage, PCa cells proliferate independently of androgens actions (the so-called hormone refractory prostate cancer, HRPC), which cause the failure of classical androgen ablation therapies and restricts the therapeutic options for this usually lethal form of disease. Imatinib mesylate is a chemotherapeutic drug that inhibits the tyrosine kinase activity of c-KIT receptors among others, and has been successfully used to treat leukemias and gastrointestinal stromal tumors. However, its application for treatment of PCa has not been totally effective with preclinical models and clinical experimentation producing discordant results. On the other hand, regucalcin (RGN), a calcium (Ca2+)-binding protein that regulates intracellular Ca2+ homeostasis and the activity of several proteins involved in intracellular signaling pathways, namely, kinases and phosphatases, has been associated with suppression of cell proliferation in rat prostate. These raised the question whether RGN may regulate the expression of c-KIT and its ligand, the stem cell factor (SCF). Therefore, the present dissertation firstly aimed to analyze the cytotoxic effects of imatinib in two cell line models of HRPC, DU145 and PC3 cells. Moreover, the effect of RGN on the expression of SCF/c-KIT in rat prostate was evaluated by means of a transgenic animal model overexpressing RGN (Tg-RGN). Finally, the subcellular localization of RGN in HRPC cell lines and its association with a-tubulin was investigated. DU145 and PC3 cells were incubated with 20 µM imatinib for 48 and 72 hours. The MTS assay was used to assess cell viability in response to imatinib and the colorimetric measurement of the enzymatic activity of caspase-3 was included as an end-point of apoptosis. The expression of cell-cycle and apoptosis regulators in response to imatinib, and the expression of SCF/c-KIT in Tg-RGN vs. wild-type rats were determined by real-time PCR and Western Blot. The expression of RGN in HRPC cells lines in its association with a-tubulin were evaluated through fluorescent immunocytochemistry. Treatment with imatinib decreased the viability of DU145 cells at 48 and 72 hours. Although imatinib decreased the viability of PC3 cells upon 6 hours of treatment, thereafter cell viability significantly increased in relation to control. Accordingly, the enzymatic activity of caspase-3 was increased in DU145 cells whereas diminished activity of caspase-3 was observed in PC3 cells treated with imatinib for 48 and 72 hours. Moreover, DU145 cells displayed reduced expression of anti-apoptotic protein Bcl-2 and increased levels of the executioners of apoptosis caspase-8 and caspase-9. No differences were observed on the expression levels of these apoptosis related proteins in PC3 cells. The mRNA expression of cell cycle inhibitor p21 was increased in both DU145 and PC3 cells. Also, the mRNA levels of VEGF were decreased in DU145 cells in response to Imatinib but the opposite effect was seen in PC3 cells. To start explaining the differential response of DU145 and PC3 cells to imatinib, the expression of c- KIT receptor in these cell lines was characterized. Fluorescent immunocytochemistry and Western Blot analysis showed that the expression of the active membrane-bound c-KIT is decreased in PC3 cells relatively to DU145. In addition, PC3 cells presented increased expression of truncated isoforms of c-KIT. Relatively to RGN the results obtained showed that the expression of SCF/c-KIT system is diminished in the prostate of Tg-RGN animals, which is in accordance with the antiproliferative effects of RGN, and indicates that regulation of SCF/c-KIT system may be a mechanism by which RGN restricts proliferation. Moreover, it was confirmed the expression of RGN in HRPC cells and its co-localization with a-tubulin, a fundamental component of microtubules. The results presented in this dissertation indicated that Imatinib was effective inducing apoptosis of DU145 cells likely through the inactivation of c-KIT. On the other hand, the paradoxical effects of imatinib in PC3 cells may be associated with the presence of truncated isoforms of c-KIT for which no definitive role has been established. These findings also contributed to understand the inefficacy of imatinib as therapeutic option in PCa. Moreover, the role of RGN as an antiproliferative molecule controlling cell cycle was further highlighted by the observed decreased expression of SCF/c- KIT system with overexpression of RGN, as well as, by the association of RGN with components of the cell division machinery.
- 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.
- Sweet Cherry Extract Targets the Hallmarks of Cancer in Prostate Cells: Diminished Viability, Increased Apoptosis and Suppressed Glycolytic MetabolismPublication . Silva, Gonçalo; Vaz, CV; Catalão, Beatriz; Ferreira, Susana; Cardoso, HJ; Duarte, A. P.; Socorro, SílviaThe present work evaluated the anticancer properties of sweet cherry (Prunus avium) extract on human prostate cells. Several sweet cherry cultivars from Fundão (Portugal) were methanol-extracted and their phytochemical composition characterized. The Saco "late harvest" extract was highly-enriched in anthocyanins and selected for use in biological assays. Non-neoplastic (PNT1A) and neoplastic (LNCaP and PC3) human prostate cells were treated with 0-2,000 μg/ml of extract for 48-96 h. Cell viability was evaluated by the MTT assay. Apoptosis, oxidative stress, and glycolytic metabolism were assessed by Western blotting and enzymatic assays. Glucose consumption and lactate production were measured spectrophotometrically. Saco cherry extract diminished the viability of neoplastic and non-neoplastic cells, whereas enhancing apoptosis in LNCaP. Cherry extract-treatment also diminished oxidative damage and suppressed glycolytic metabolism in LNCaP cells. These findings widened the knowledge on the mechanisms by which cherry extract modulate cell physiology, demonstrating their broad action over the hallmarks of cancer.
- 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.
- Prostate cancer cells metabolism on the interplay of androgenic regulation and metabolic environmentPublication . Cardoso, Henrique José Matos Morão Mingote; Socorro, Sílvia Cristina da Cruz Marques; Madureira, PatríciaProstate Cancer (PCa) is one of the most common cancer in men and represents the fifth leading cause of cancer deaths. In an initial phase of PCa, the so-called androgen-sensitive stage, PCa cells are extremely dependent on androgens actions to survive and proliferate. This condition allows the effectiveness of androgens deprivation therapy (ADT) that reduce the circulating levels of androgens or block their action. The continuous administration of ADT renders PCa cells resistant to treatment, becoming capable of survive and metastasize even in the absence or very low circulating levels of androgens. At this moment, it is reached the stage of castrate-resistant prostate cancer (CRPC), a condition with high mortality rates and treatment limitations. In the last years, a substantial amount of data showed that cancer cells have the ability of reprogramming metabolism to survive and metastasize. Warburg studies were pioneer showing that tumour cells predominantly use glycolysis for obtaining energy, in detriment of oxidative phosphorylation, with the production of high amounts of lactate. These findings opened the door to the metabolic adaptation being considered a hallmark of cancer. Currently, it is accepted that mitochondrial oxidative phosphorylation and glycolysis, support tumour cells survival and growth. Primary PCa cases differ from other cancer types by the fact that is less glycolytic, and the idea that predominantly use fatty acids and glutamine as energy substrates has been gaining consistency. Indeed, glycolysis is only overactivated in more advanced stages of the disease, in CRPC. However, the understanding of how each metabolic pathway sustains PCa cells survival and growth still is incomplete. Besides the recognized functions as the main drivers of PCa survival and growth, androgens have been indicated as metabolic regulators in PCa, modulating glycolysis and cell lipid handling. Nevertheless, there are several issues in the role of androgens controlling PCa metabolism that need to be clarified. Although efforts have been made in recent years to develop new drugs for PCa treatment, namely, for CRPC, they have shown limited duration of clinical and survival benefits. Following the trend observed in other cancer types, treatment approaches targeting metabolism also have been explored in the case of PCa and CRPC. However, there are important knowledge gaps in the understanding of PCa cells metabolism that should be fulfilled to improve its efficacy and to avoid resistance and bypassing metabolic pathways. The main goal of this thesis was to clarify the role androgens and the metabolic environment in shaping PCa metabolism, and how this interplay can affect PCa cell fate. The present thesis first established the PCa cells dependency on the different metabolic pathways (glycolysis, glutaminolysis and lipid metabolism). It was demonstrated that CRPC cells have higher metabolic rates being more glycolytic than the androgen-sensitive cells, especially the PC3 cells, which also showed a higher capacity to oxidize glutamine. Androgen-responsive LNCaP cells displayed a higher capacity for using fatty acids as mitochondrial fuels. These findings allowed to demonstrate a differential dependency and capacity of fuel use between androgen-sensitive and CRPC cells. Next, we determined the relevance of glutaminolysis for PCa cells survival and growth and the effect of androgens in the regulation of glutamine metabolism. Treatment of PCa cells with 5α-dihydrotestosterone (DHT, 10nM) potentiated glutamine metabolism in PCa cells, whereas the inhibition of glutaminase activity diminished cell viability and migration, and increased apoptosis, particularly in the CRPC. Moreover, cotreatment with glutaminase inhibitor BPTES and the anti-androgen bicalutamide had a synergic effect suppressing LNCaP cells viability, which highlights the benefit of co-targeting androgen receptor and glutamine metabolism in PCa treatment. Glutaminolysis inhibition also had an impact on glycolysis and lipid metabolism. The role of androgens in regulating lipid metabolism and the influence of these hormones and LDL-cholesterol modulating PCa cells fate were evaluated. DHT upregulated the expression of fatty acid synthase and carnitine palmitoyltransferase 1A in androgensensitive PCa cells. LDL-cholesterol enrichment increased PCa cells viability, proliferation, and migration dependently on DHT. This in vitro approach supports clinical and epidemiological data linking obesity and cholesterol with PCa, and first implicated androgens in this relationship. Finally, we investigated the effect of different glucose availability on the PCa cells response to therapy. For this purpose, it was used the receptor tyrosine kinase inhibitor imatinib and two cell line models of CRPC. Higher glucose availability improved the effectiveness of imatinib suppressing survival and growth of CRPC cells Moreover, imatinib treatment stimulated the glycolytic metabolism of CRPC cells. Overall, it was showed that hyperglycemia, the main serum alteration in diabetic patients, potentiated the effects of imatinib in CRPC cells, which raises the curiosity about the efficacy of this drug for treatment of castration-resistant diabetic patients. In conclusion, the main findings of this thesis confirmed the crucial actions of androgens in regulating the metabolism of PCa cells. These effects were pivotal for PCa cells obtaining energy and triggered proliferation and metastasis. Another innovative result of the present thesis was the identification of the cholesterol and androgens interplay in inducing survival and invasiveness features of PCa cells. Moreover, this dissertation demonstrated the flexibility of PCa cells using different energy sources and contributed to a better understanding of the role of lipids and glutamine in PCa. The molecular mechanism underlying the metabolic support of cancer cell survival and growth were highlighted. Overall, the information gathered in this thesis supports the metabolic environment and androgens as “co-authors” orchestrating the reprogramming of PCa and cancer development. Further research on this interplay could be a basis for the development of new treatment approaches for PCa.
- 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.