Regucalcin (RGN) is a calcium (Ca(2+))-binding protein that displays a characteristic downregulated expression with aging in several tissues. Besides its role in regulating intracellular Ca(2+) homeostasis, RGN has been associated with the control of oxidative stress, cell proliferation, and apoptosis. Thus, the diminished expression of RGN with aging may contribute to the age-associated deterioration of cell function. In the present study, we hypothesized that the maintenance of high expression levels of RGN may prevent age-related alterations in the processes mentioned previously. First, we confirmed that RGN expression is significantly diminished in the prostate of 8-, 9-, 12-, and 24-months wild-type rats. Then, the effect of aging on lipid peroxidation, antioxidant defenses, cell proliferation, and apoptosis in the prostate of wild-type controls and transgenic rats overexpressing RGN (Tg-RGN) was investigated. The activity of glutathione and the antioxidant capacity were increased in Tg-RGN rats in response to the age-associated increase in thiobarbituric acid reactive substances levels, an effect not seen in wild type. Overexpression of RGN also counteracted the effect of aging increasing prostate cell proliferation. In contrast to wild-type animals, the prostate weight of Tg-RGN did not change with aging and was underpinned by the diminished expression of stem cell factor and c-kit, and increased expression of p53. In addition, aged Tg-RGN animals displayed increased expression (activity) of apoptosis regulators, therefore not showing the age-induced resistance to apoptosis observed in wild type. Altogether, these findings indicate the protective role of RGN against the development of age-related pathologies, such as, for example, prostate cancer.

Regucalcin (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 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.

Regucalcin (RGN) is a calcium-binding protein, which has been shown to be underexpressed in cancer cases. This study aimed to determine the association of RGN expression with clinicopathological parameters of human breast cancer. In addition, the role of RGN in malignancy of mammary gland using transgenic rats overexpressing the protein (Tg-RGN) was investigated. Wild-type (Wt) and Tg-RGN rats were treated with 7,12-dimethylbenz[α]anthracene (DMBA). Carcinogen-induced tumors were histologically classified and the Ki67 proliferation index was estimated. Immunohistochemistry analysis showed that RGN immunoreactivity was negatively correlated with the histological grade of breast infiltrating ductal carcinoma suggesting that progression of breast cancer is associated with loss of RGN. Tg-RGN rats displayed lower incidence of carcinogen-induced mammary gland tumors, as well as lower incidence of invasive forms. Moreover, higher proliferation was observed in non-invasive tumors of Wt animals comparatively with Tg-RGN. Overexpression of RGN was associated with diminished expression of cell-cycle inhibitors and increased expression of apoptosis inducers. Augmented activity of apoptosis effector caspase-3 was found in the mammary gland of Tg-RGN. RGN overexpression protected from carcinogen-induced mammary gland tumor development and was linked with reduced proliferation and increased apoptosis. These findings indicated the protective role of RGN in the carcinogenesis of mammary gland.

Regucalcin (RGN) is a calcium (Ca(2+))-binding protein widely expressed in vertebrate and invertebrate species, which is also known as senescence marker protein 30, due to its molecular weight (33 kDa) and a characteristically diminished expression with the aging process. RGN regulates intracellular Ca(2+) homeostasis and the activity of several proteins involved in intracellular signalling pathways, namely, kinases, phosphatases, phosphodiesterase, nitric oxide synthase and proteases, which highlights its importance in cell biology. In addition, RGN has cytoprotective effects reducing intracellular levels of oxidative stress, also playing a role in the control of cell survival and apoptosis. Multiple factors have been identified regulating the cell levels of RGN transcripts and protein, and an altered expression pattern of this interesting protein has been found in cases of reproductive disorders, neurodegenerative diseases and cancer. Moreover, RGN is a serum-secreted protein, and its levels have been correlated with the stage of disease, which strongly suggests the usefulness of this protein as a potential biomarker for monitoring disease onset and progression. The present review aims to discuss the available information concerning RGN expression and function in distinct cell types and tissues, integrating cellular and molecular mechanisms in the context of normal and pathological conditions. Insight into the cellular actions of RGN will be a key step towards deepening the knowledge of the biology of several human diseases.

Regucalcin (RGN) is a calcium (Ca(2+) )-binding protein underexpressed in prostate adenocarcinoma comparatively to non-neoplastic prostate or benign prostate hyperplasia cases. Moreover, RGN expression is negatively associated with the cellular differentiation of prostate adenocarcinoma, suggesting that loss of RGN may be associated with tumor onset and progression. However, the RGN actions over the control of prostate cell growth have not been investigated.

Androgens 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.

O cancro da mama é uma doença heterogénea que compreende uma grande variedade de alterações moleculares e diferentes tipos de resposta em termos clínicos. Esta diversidade reside nos múltiplos fatores que podem levar à transformação maligna das células, em consequência da desregulação de diferentes processos fisiológicos. A regucalcina (RGN) é uma proteína de ligação ao cálcio (Ca2+) e cuja principal função conhecida é regular a homeostase do Ca2+ intracelular, mas podendo também estar envolvida na regulação da proliferação celular, apoptose e metabolismo das células. A RGN também foi identificada como um gene regulado por hormonas, incluindo os esteroides sexuais como os androgénios. Para além disso, foi anteriormente associada a determinadas patologias e tendo mesmo sido identificada como uma proteína subexpressa em casos humanos de cancro da mama, próstata ou fígado. No fígado, a subexpressão da RGN foi detetada em lesões pré-neoplásicas, ou seja, antes da aquisição do fenótipo neoplásico, o que sugere que a sua diminuição pode estar ligada ao início do processo de transformação tumorigénico. Apesar destas evidências, os mecanismos moleculares subjacentes às funções da RGN na mama permanecem por identificar. Nesta tese, colocámos a hipótese de que a sobreexpressão da RGN poderá exercer uma ação protetora em relação à carcinogénese mamária. De modo a avaliar esta questão, o composto 7,12 dimetilbenz[α]antraceneno, o qual é conhecido por induzir carcinogénese mamária em rato, foi administrado a ratos transgénicos que sobreepressam a RGN (Tg-RGN) e aos respetivos controlos (Wt, do inglês wild-type). Os ratos Tg-RGN apresentaram, notavelmente, uma menor incidência de tumores (25.8 %) comparativamente aos animais controlo (100 %). A classificação histológica também demonstrou uma clara resistência dos ratos Tg-RGN à tumorigénese, ao serem bastante mais resistentes à progressão dos tumores para estadios mais agressivos. Verificou-se uma muito menor percentagem de tumores do tipo invasivo nos animais transgénicos (3.8 % vs 45.8 % nos Wt). Para além disso, foi observado um aumento da atividade proliferativa nos tumores não-invasivos nos Wt comparativamente aos animais TG-RGN, o que indica a menor capacidade invasiva. A avaliação metabólica dos tumores benignos da glândula demonstrou que os tumores de ratos Tg-RGN possuem uma menor expressão e atividade da lactato desidrogenase (LDH), característica que normalmente se encontra associada a uma restrição da progressão tumoral e a um decréscimo da agressividade. Contudo, em tecido mamário não-neoplásico de ratos Tg-RGN observou-se uma restrição do metabolismo glicolítico, o que é indicativo de uma redução dos níveis energéticos no tecido. Estes resultados podem ser de extrema importância para a diminuição da proliferação celular e constituir um mecanismo adicional, pelo qual a RGN previne o desenvolvimento tumoral. De facto, a sobreexpressão da RGN originou uma diminuição da expressão de genes envolvidos na regulação ciclo celular e de oncogenes na glândula mamária de ratos Tg-RGN. Mais ainda, a expressão do P53 e a atividade da caspase-3 também foi encontrada aumentadas concomitantemente com a sobreexpressão da RGN, o que sugere uma ação protetora da RGN no aparecimento do tumor, a qual pode ser mediada também pela regulação das vias apoptóticas. Em contrapartida, a expressão da RGN em células MCF-7 de cancro da mama diminui pela ação do androgénio não-aromatizável 5α-dihidrotestosterona, ao passo que a expressão do canal de Ca2+ do tipo L aumentou. Estes resultados sustentam a diminuição da viabilidade celular evidenciada nestas células e sugerem o envolvimento destes modeladores do Ca2+ no controlo da proliferação celular mamária, o que no caso do canal de Ca2+ do tipo L nunca antes tinha sido sugerido. Em conclusão, o trabalho apresentado nesta tese destaca a preponderância da RGN numa diversidade de mecanismos fisiológicos e fisiopatológicos na glândula mamária. As evidências biológicas aqui apresentadas confirmam o papel protetor da RGN na carcinogénese mamária, ao restringir processos biológicos reconhecidos como fundamentais para o desenvolvimento do cancro, nomeadamente, a proliferação celular e as alterações no metabolismo celular, ao mesmo tempo que aumenta a morte celular por apoptose.