Serotonin after β-Adrenoreceptors’ Exposition: New Approaches for Personalized Data in Breast Cancer Cells

Serotonin is an important monoamine in the human body, playing crucial roles, such as a neurotransmitter in the central nervous system. Previously, our group reported that β-adrenergic drugs (ICI 118,551, isoprenaline, and propranolol) influence the proliferation of breast cancer cells (MCF-7 cells) and their inherent production of adrenaline. Thus, we aimed to investigate the production of serotonin in MCF-7 cells, clarifying if there is a relationship between this production and the viability of the cells. To address this question, briefly, we treated the MCF-7 cells with ICI 118,551, isoprenaline, and propranolol, and evaluated cellular viability and serotonin production by using MTT, Sulforhodamine B (SRB) and Neutral Red (NR) assays, and HPLC-ECD analysis, respectively. Our results demonstrate that isoprenaline promotes the most pronounced endogenous synthesis of serotonin, about 3.5-fold greater than control cells. Propranolol treatment also increased the synthesis of serotonin (when compared to control). On the other hand, treatment with the drug ICI 118,551 promoted a lower endogenous synthesis of serotonin, about 1.1-fold less than what was observed in the control. Together, these results reveal that MCF-7 cells can produce serotonin, and the drugs propranolol, isoprenaline and ICI 118,551 influence this endogenous production. For the first time, after modulation of the β-adrenergic system, a pronounced cellular growth can be related to higher consumption of serotonin by the cells, resulting in decreased levels of serotonin in cell media, indicative of the importance of serotonin in the growth of MCF-7 cells.info:eu-repo/semantics/publishedVersio

β-Adrenoceptor Activation in Breast MCF-10A Cells Induces a Pattern of Catecholamine Production Similar to that of Tumorigenic MCF-7 Cells

Adrenaline, which participates in the neuroendocrine response that occurs during stress and perimenopause, may be tumorigenic. This exploratory study aimed at investigating whether non-tumorigenic and tumorigenic human breast epithelial cell lines are able to synthesize adrenaline. The study was carried out in non-tumorigenic (MCF-10A) and tumorigenic (MCF-7) human breast cell lines. Expression of enzymes involved in adrenaline synthesis was characterized by RT-qPCR, immunocytochemistry and western blot. Catecholamines and analogue compounds were quantified by HPLC-ECD. Functional assessment of the impact of drugs on cells' tumorigenic potential was assessed by determination of cell viability and clonogenic ability. Both MCF-10A and MCF-7 cells produce catecholamines, but the capacity to produce adrenaline is lower in MCF-10A cells. β-adrenoceptor activation increases the capacity of MCF-10A cells to produce adrenaline and favor both cell viability and colony formation. It is concluded that exposure of human breast epithelial cells to β-adrenoceptor agonists increases cell proliferation and the capacity to produce adrenaline, creating an autocrine potential to spread these adrenergic effects in a feed-forward loop. It is conceivable that these effects are related to tumorigenesis, bringing a new perspective to understand the claimed anticancer effects of propranolol and the increase in breast cancer incidence caused by stress or during perimenopause.info:eu-repo/semantics/publishedVersio

MARCADORES DE STRESS OXIDATIVO NA POLINEUROPATIA AMILOIDÓTICA FAMILIAR

MARCADORES DE STRESS OXIDATIVO NA POLINEUROPATIA AMILOIDÓTICA FAMILIAR Henrique Reguengo1,2, Maria Luís Cardoso2, Teresa Coelho3, Ana Martins3, Marta Novais3, Luísa Gomes1, Isabel Fonseca3, Berta Martins4, Franklim Marques2 1Serviço de Química Clínica, HSA/CHP, 2FFUP, 3Unidade Clínica de Paramiloidose, HSA/CHP, 4Laboratório de Imunogenética, ICBAS/UP. Hospital de Santo António, Centro Hospitalar do Porto (HSA/CHP), Porto. Faculdade de Farmácia, Universidade do Porto (FF/UP), Porto. Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto (ICBAS/UP), Porto. Introdução A Polineuropatia Amilóidotica Familiar (PAF) é uma amiloidose hereditária associada a variantes de transtirretina (TTR), em que ocorre deposição sistémica de amilóide, principalmente a nível dos nervos periféricos. Alguns estudos sugerem que o stress oxidativo pode estar envolvido na formação e modificação das fibrilas de amilóide. Fisiologicamente, o organismo defende-se das agressões mediadas pelos radicais livres utilizando diversas reservas antioxidantes celulares. Estudos realizados revelam diferentes respostas ao stress oxidativo envolvendo o malondialdeído (MDA), a capacidade antioxidante total (TAS), algumas vitaminas (A e E) e enzimas: glutationa reductase (GRed) e a superóxido dismutase (SOD). Objectivos Este estudo pretendeu quantificar alguns marcadores de stress oxidativo e analisar as diferenças entre doentes com PAF e portadores assintomáticos. Material e Métodos Foram incluídos 40 doentes com diagnostico de PAF e 45 portadores assintomáticos da mutação, procedentes da Unidade Clínica de Paramiloidose. Procedeu-se à avaliação da TAS da GRed e da SOD, respectivamente através dos Kit comerciais da Randox TAS NX2332, Glutationa Reductase ref. GR 2368, RANSOD ref. SD 125 e do MDA com o kit comercial da Zeptometrix, OXItek TBARS. Ref. 0801192. A comparação entre os grupos foi efectuada pelo teste t de student para amostras independentes e a relação entre as variáveis pela correlação de Pearson. Resultados Os valores de TAS e GRed foram significativamente mais elevados no grupo de doentes com PAF comparativamente aos portadores assintomáticos (P=0.02 e P=0.03, respectivamente). Entre os dois grupos, não se verificaram diferenças significativas no MDA, SOD e vitaminas A e E. No grupo de doentes com PAF, foi encontrada uma correlação significativa entre a TAS e a função renal avaliada pela creatinina sérica (r=0.60, P=0.01) e pela cistatina C (r=0.51, P=0.01). Nos portadores assintomáticos apenas a creatinina se correlacionou com a TAS, mas não a cistatina C. Apesar do grupo dos doentes com PAF apresentar valores mais elevados de creatinina sérica, as diferenças não foram significativas e o valor máximo apresentado foi de 1,46 mg/dl nos PAF e 1,02 mg/dl nos portadores assintomáticos. Conclusão Apesar do MDA não apresentar diferenças estatisticamente significativas, a capacidade antioxidante parece ser superior nos doentes com PAF comparativamente aos portadores assintomáticos. Os resultados revelam ainda uma correlação positiva significativa entre a TAS e os níveis séricos de creatinina e de cistatina C. É possível que o aumento dos valores de TAS reflicta um mecanismo de defesa ao aumento de stress oxidativo, geralmente associado à disfunção renal, avaliada pela creatinina e pela cistatina C. Apresentador: Henrique Reguengo, Técnico Superior de Saúde, Serviço de Quimica Clínica, HSA/CHP; Aluno de Doutoramento em Ciências Farmacêuticas, FF/UP

Carbidopa Alters Tryptophan Metabolism in Breast Cancer and Melanoma Cells Leading to the Formation of Indole-3-Acetonitrile, a Pro-Proliferative Metabolite

Carbidopa is used for the treatment of Parkinson's disease (PD) as an inhibitor of DOPA decarboxylase, and PD patients taking carbidopa have a lower incidence of various tumors, except for breast cancer and melanoma. Recently, it was shown that carbidopa inhibits tryptophan-2,3-dioxygenase (TDO) and kynureninase enzymes. In the present study, the effect of carbidopa on the viability and metabolic profile of breast cancer MCF-7 and melanoma A375 cells was investigated. Carbidopa was not effective in inhibiting MCF-7 and A375 proliferation. Liquid chromatography and mass spectrometry revealed a new compound, identified as indole-3-acetonitrile (IAN), which promoted a concentration-dependent increase in the viability of both cell lines. The results suggest that treatment with carbidopa may alter tryptophan (Trp) metabolism in breast cancer and melanoma leading to the formation of a pro-proliferative Trp metabolite, which may contribute to its failure in reducing breast cancers and melanoma incidence in PD patients taking carbidopa.This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT, in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274).” NV thanks FCT for IF position, Fundação Manuel António da Mota (FMAM, Portugal) for support. The contents of this report are solely the responsibility of the authors and do not necessarily represent the official views of the FCT and FMAMinfo:eu-repo/semantics/publishedVersio

NIVEIS DE SUPERÓXIDO DISMUTASE REVELAM STRESS OXIDATIVO AUMENTADO NA POLINEUROPATIA AMILOIDÓTICA FAMILIAR

NIVEIS DE SUPERÓXIDO DISMUTASE REVELAM STRESS OXIDATIVO AUMENTADO NA POLINEUROPATIA AMILOIDÓTICA FAMILIAR Henrique Reguengo1,2, Maria Luís Cardoso2, Teresa Coelho3, Ana Martins3, Marta Novais3, Madalena Cruz1, Isabel Fonseca3, Berta Martins4, Franklim Marques2 1Serviço de Química Clínica, HSA/CHP, 2FFUP, 3Unidade Clínica de Paramiloidose, HSA/CHP, 4Laboratório de Imunogenética, ICBAS/UP. Hospital de Santo António, Centro Hospitalar do Porto (HSA/CHP), Porto. Faculdade de Farmácia, Universidade do Porto (FF/UP), Porto. Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto (ICBAS/UP), Porto. Introdução A polineuropatia amiloidótica familiar (PAF) é provocada por mutações no gene da transtirretina, principalmente em resultado da mutação TTRv30M no caso de Portugal. Estudos recentes em vários tipos de amiloidoses revelam que o stress oxidativo pode estar envolvido quer na produção das fibrilas de proteína amilóide, quer na modificações pós formação das fibrilas. A enzima superóxido dismutase (SOD) desempenha um importante papel antioxidante que protege as células expostas aos radicais superóxido. O presente estudo pretendeu avaliar o stress oxidativo nesta patologia. Material e Métodos A amostra em estudo incluiu 40 doentes com PAF e 45 portadores assintomáticos da mutação, seguidos habitualmente na Unidade Clínica de Paramiloidose do CHP. Foi ainda considerado um grupo controlo de 26 indivíduos saudáveis. Avaliou-se a concentração de SOD intra-eritocitária com um método colorimétrico disponível no KIT RANSOD ref SD 125 da Randox. A análise estatística foi efectuada utilizando o software SPSS, versão 19. Resultados Os valores de SOD obtidos foram os seguintes: Controlos: (1208±254 U/g Hb), Portadores assintomáticos (1436±62 U/g Hb), Doentes com PAF (1455±350 U/g Hb). Os valores resultaram da média de três réplicas. Os valores de SOD foram significativamente mais elevados no grupo de doentes com PAF e nos portadores assintomáticos, comparativamente ao grupo controlo (respectivamente P=0.003 e P=0.013). Não foram encontradas diferenças estatisticamente significativas entre os doentes com PAF e os portadores assintomáticos. No grupo de doentes com PAF verificou-se uma correlação positiva significativa entre os valores de SOD e Proteína C reactiva (r=0.45, P =0.013). Conclusão A alteração da concentração da SOD nos doentes com PAF sugere uma maior exposição dos mesmos a fenómenos de stress oxidativo comparativamente ao grupo controlo. Dado que este fenómeno pode ter influência quer no despoletar, quer no curso da patologia da doença, justifica-se uma maior atenção e estudo deste fenómeno nestes doentes. Apresentador: Henrique Reguengo, Técnico Superior de Saúde, Serviço de Quimica Clínica, HSA/CHP; Aluno de Doutoramento em Ciências Farmacêuticas, FF/UP

Quantitative Proteomic Analysis of Skeletal Muscle Detergent- Resistant Membranes in a Smith-Lemli-Opitz Syndrome Mouse

Smith-Lemli-Opitz syndrome (SLOS) is an inborn error of metabolism affecting the last step of cholesterol biosynthesis. It is characterized by a deficiency of the enzyme 7-dehydrocholesterol reductase and accumulation of 7-dehydrocholesterol (7DHC) in cells and body fluids. Given the similarities between 7DHC and cholesterol, 7DHC can be incorporated into cell membranes in lieu of cholesterol. Nevertheless, due to their structural differences and distinct affinity to other membrane components, this substitution alters membrane properties and one can expect to find abnormalities in membrane protein composition. In order to identify differences in membrane proteins that could facilitate our understanding of SLOS physiopathology, we isolated detergent-resistant membranes (DRMs) from the skeletal muscle of Dhcr7T93M/T93M mice and C57/BL6 controls and performed comparative proteomic analysis using iTRAQ for peptide quantification. A total of 133 proteins were identified in the DRM fraction: 17 (13%) proteins demonstrated increased expression in SLOS mice, whereas, 21 (16%) showed decreased expression. Characterization of functional point of view and bioenergetics pathway and transmembrane transport responded to the major differences between the two groups of animals

Pharmacological and Non-Pharmacological Agents versus Bovine Colostrum Supplementation for the Management of Bone Health Using an Osteoporosis-Induced Rat Model

Osteoporosis is defined by loss of bone mass and deteriorated bone microarchitecture. The present study compared the effects of available pharmacological and non-pharmacological agents for osteoporosis [alendronate (ALE) and concomitant supplementation of vitamin D (VD) and calcium (Ca)] with the effects of bovine colostrum (BC) supplementation in ovariectomized (OVX) and orchidectomized (ORX) rats. Seven-month-old rats were randomly allocated to: (1) placebo-control, (2) ALE group (7.5 μg/kg of body weight/day/5 times per week), (3) VD/Ca group (VD: 35 μg/kg of body weight/day/5 times per week; Ca: 13 mg/kg of body weight/day/3 times per week), and (4) BC supplementation (OVX: 1.5 g/day/5 times per week; ORX: 2 g/day/5 times per week). Following four months of supplementation, bone microarchitecture, strength and bone markers were evaluated. ALE group demonstrated significantly higher Ct.OV, Ct.BMC, Tb.Th, Tb.OV and Tb.BMC and significantly lower Ct.Pr, Tb.Pr, Tb.Sp, Ct.BMD and Tb.BMD, compared to placebo (p < 0.05). BC presented significantly higher Ct.Pr, Ct.BMD, Tb.Pr, Tb.Sp, and Tb.BMD and significantly lower Ct.OV, Ct.BMC, Tb.Th, Tb.OV and Tb.BMC compared to ALE in OVX rats (p < 0.05). OVX rats receiving BC experienced a significant increase in serum ALP and OC levels post-supplementation (p < 0.05). BC supplementation may induce positive effects on bone metabolism by stimulating bone formation, but appear not to be as effective as ALE