Endogenous Coactivator ARA70 Interacts with Estrogen Receptor α (ERα) and Modulates the Functional ERα/Androgen Receptor Interplay in MCF-7 Cells

Overexpression of androgen receptor (AR) decreases estrogen receptor alpha (ERalpha) transactivation, which plays a basic role in hormone-dependent breast cancer. This transcriptional interference can be due to shared coactivators. Here we demonstrated that in MCF-7 cells ARA70, an AR-specific coactivator, interacted with endogenous ERalpha, increasing its transcriptional activity, and it was recruited to the pS2 gene promoter. Moreover, a dominant negative ARA70 down-regulated ERalpha transcriptional activity as well as pS2 mRNA. ARA70 overexpression reversed the AR down-regulatory effect on ERalpha signaling. However, in the presence of a progressive increase of transfected AR, ARA70 switched into enhancing the inhibitory effect of AR on ERalpha signaling. These opposite effects of ARA70 were further evidenced by coimmunoprecipitation assay in MCF-7wt, MCF-7-overexpressing AR, and HeLa cells, exogenously expressing an excess of ERalpha with respect to AR or an excess of AR with respect to ERalpha. Thus, ARA70 is a coactivator for ERalpha and may represent a functional link between ERalpha/AR modulating their cross-talk in models of estrogen signaling in MCF-7 and HeLa cells

Leptin Enhances, via AP-1, Expression of Aromatase in the MCF-7 Cell Line *

Leptin, a product of adipocytes, is involved in the regulation of body weight and results strongly correlated to body fat content. An excess of fat mass represents a breast cancer risk factor particularly in postmenopausal women, where estrogen production by adipose tissue through its own aromatase activity stimulates tumor progression. Leptin stimulates estrogen production through the increase of aromatase expression and activity in human luteinized granulosa cells and adipose stromal cells. In the present study, we have examined the possible link that exists between leptin and breast cancer, focusing our attention on the direct effect of leptin on aromatase activity, which may enhance estrogen production and induce tumor cell growth stimulation. We have shown that leptin enhances aromatase mRNA expression, aromatase content, and its enzymatic activity in MCF-7. Aromatase expression appears to be regulated by tissue-specific promoter. It has been demonstrated that promoters II and 1.3 are the major promoters that drive aromatase expression in MCF-7. Transient transfection experiments using vector containing human aromatase promoters II and 1.3 sequence fused with luciferase reporter gene demonstrated that leptin is able to activate this promoter. In the presence of either mitogen-activated protein kinase inhibitor PD 98059 or ERK2 dominant negative as well as in the presence of STAT3 dominant negative, the stimulatory effects of leptin on aromatase promoter, enzymatic activity, and aromatase protein content were inhibited. Functional studies of mutagenesis and electrophoretic mobility shift assay revealed that the AP-1 motif is important in determining the up-regulatory effects induced by leptin on aromatase expression in MCF-7

Ischemic Preconditioning Modulates the Peripheral Innate Immune System to Promote Anti-Inflammatory and Protective Responses in Mice Subjected to Focal Cerebral Ischemia

The development of tolerance triggered by a sublethal ischemic episode (preconditioning, PC) involves a complex crosstalk between neurons, astrocytes and microglia, although the role of the peripheral immune system in this context is largely unexplored. Here, we report that severe cerebral ischemia caused by transient middle cerebral artery occlusion (MCAo) in adult male mice elevates blood counts of inflammatory neutrophils and monocytes, and plasma levels of miRNA-329-5p. These inflammatory responses are prevented by ischemic PC induced by 15 min MCAo, 72h before the severe insult (1h MCAo). As compared with sham-operated animals, mice subjected to either ischemic PC, MCAo or a combination of both (PC+MCAo) display spleen contraction. However, protein levels of Ym1 (a marker of polarization of myeloid cells towards M2/N2 protective phenotypes) are elevated only in spleen from the experimental groups PC and PC+MCAo, but not MCAo. Conversely, Ym1 protein levels only increase in circulating leukocytes from mice subjected to 1h MCAo, but not in preconditioned animals, which is coincident with a dramatic elevation of Ym1 expression in the ipsilateral cortex. By immunofluorescence analysis, we observe that expression of Ym1 occurs in amoeboid-shaped myeloid cells, mainly representing inflammatory monocytes/macrophages and neutrophils. As a result of its immune-regulatory functions, ischemic PC prevents elevation of mRNA levels of the pro-inflammatory cytokine interleukin (IL)-1β in the ipsilateral cortex, while not affecting IL-10 mRNA increase induced by MCAo. Overall, the elevated anti-inflammatory/pro-inflammatory ratio observed in the brain of mice pre-exposed to PC is associated with reduced brain infarct volume and ischemic edema, and with amelioration of functional outcome. These findings reaffirm the crucial and dualistic role of the innate immune system in ischemic stroke pathobiology, extending these concepts to the context of ischemic tolerance and underscoring their relevance for the identification of novel therapeutic targets for effective stroke treatment

Cachrys libanotis L. Extracts: Photocytotoxic Effects on UVA-Irradiated Human Melanoma Cells

Melanoma is the most aggressive form of skin cancer. Photochemotherapy, combining the action of a light source and a chemical photosensitizer, is one of the most interesting current therapeutic approaches. Plants represent a rich source of photoactive compounds, and furanocoumarins are some of the most important naturally occurring phytoconstituents. The aim of this study was to evaluate the photocytotoxic potential of Cachrys libanotis L. (Apiaceae) from Southern Italy. This species belongs to a genus rich in furanocoumarins and widely distributed in Europe. The aerial parts were extracted through both traditional maceration and pressurized cyclic solid-liquid (PCSL) extraction using Naviglio extractor®. Qualitative and quantitative analyses were performed to detect the coumarins content using GC-MS, and the photocytotoxic effects of the extracts were assessed on UVA-irradiated C32 melanoma cells. The apoptotic responses were also evaluated. Furthermore, phenolic content and the in vitro antioxidant potential were also estimated. Xanthotoxin, bergapten and isopimpinellin were identified and quantified. Both extracts affected cell viability in a concentration-dependent manner after irradiation for 1 hour at a dose of 1.08 J/cm2. Sample obtained through PCSL extraction was the most effective, with an IC50 equal to 3.16 μg/mL, a very interesting value if compared with the positive control bergapten. This extract induced up-regulation of apoptotic signals such as BAX and PARP cleavage and, in the presence of UVA radiation, it caused a greater upregulation of p21 protein. Obtained results suggest that investigated species could be a good candidate for further studies aimed to find new drugs with photocytotoxic potential

Il Recettore Estrogenico alfa e IRS-1 (Insulin Receptor Substate-1) come target dell’azione del Recettore Estrogenico beta

Dottorato di Ricerca in Biologia Animale, XXII Ciclo,a.a.2008-2009Estrogen signalling through ERα plays a central role in many diseases such as breast and endometrial cancer, osteoporosis and cardiovascular disease. In 1996 it has been discovered a second estrogen receptor in rat prostate which has been named ERβ. Both receptors are co-expressed in many tissues and, surprisingly, ERβ was shown to sometimes mediate opposite effects to ERα. In the human, ERα is encoded at 6q25.1 and ERβ at 14q23.2, they have a high degree of homology in the DNA-binding domain (96%), but differ in the ligand-binding domain (53%). Estradiol is a natural ligand of ERα, while DPN (diarylpropionitrile) is a selective agonist of ERβ. The activation of ERα through estradiol increases cell proliferation and positively influences breast tumorogenesis and cell invasion, on the contrary, ERβ, also in the absence of the ligand, appears to oppose the action of ERα, suggesting that the estrogen receptor β could play an important role in carcinogenesis. To better define the action of ERβ, we used an estrogen receptor α positive MCF-7 breast cancer cell line overexpressing, through transient transfection, ERβ. In these cells the ectopic expression of ERβ induces a decrease of cell proliferation and a block of cell cycle in G2/M phase. Taking into account these data, we focused the attention on two proteins involved in the transduction of mitotic signallings: IRS-1 and ERα. The first one, is the major substrate of the tyrosine kinase activity of IR (Insulin Receptor) and IGF-R (Insulin- Like Growth Factor Receptors). This protein exerts a key role in the functional synergism between the growth factor/insulin and estradiol in the mammary gland. In fact, ERα is important to sustain IRS-1 protein expression. and to transactivate IRS-1 gene. On the basis of this finding, we wanted to study the possible influence of ERβ on IRS-1 by monitoring mRNA and protein contents in MCF-7 breast cancer cells. In this context, our results show that the ectopic expression of ERβ is able to negatively regulate the transactivation of IRS-1 promoter thus leading to a decrease of IRS-1 signalling. Further investigations indicate that ERβ appears to act as a dominant2 negative regulator of ERα expression in these cells. Indeed, ERβ represses the transcriptional activity of ERα gene promoter . To better investigate the molecular mechanism underlying the negative regulation of ERβ on ERα gene, we performed EMSA (Electrophoretic Mobility Shift Assay) experiments, by using ERE labelled probes present in ERα promoter. Our results show a decreased binding of the nuclear extracts, obtained from MCF-7 cells overexpressing ERβ, to the ERE labelled nucleotide. In addition, chromatin immunoprecipitation analysis reveals in MCF-7 cells overexpressing ERβ, the specific recruitment of the nuclear corepressor NCoR to these ERE sequences of ERα promoter. The recruitment of this corepressor weakens the binding between ERβ and ERE sequence, thus producing a repression of ERα transcriptional activity. This study is the first evidence to demonstrate that ERβ acts as a dominant negative regulator of ERα gene in breast cancer cells. Our data, in agreement with other findings, tend to better justify how Estrogen Receptor β overexpression in many tissues reduces ERα-regulated gene transcription and, in this way, inhibits the proliferative responses in breast cancer cells.Università della Calabri

Pathway infiammatorio del Sistema del Complemento nelle ascidie: sequenziamento e caratterizzazione funzionale del recettore dell’anafilatossina C3a di Ciona intestinalis

Dottorato di Ricerca in Biologia Animale, XIX Ciclo,a.a.2005-2006In mammals, the bioactive fragment C3a, released from C3 during complement activation, is a potent mediator of inflammatory reactions and exerts its functional activity through the specific binding to cell surface G protein-coupled seven-transmembrane receptors. Recently, a C3a-mediated chemotaxis of hemocytes has been demonstrated in the deuterostome invertebrate Ciona intestinalis and an important role for this molecule in inflammatory processes has been suggested. In this study, we have cloned and characterized the CiC3aR molecule involved in the CiC3a-mediated chemotaxis and studied its expression profile. The sequence of CiC3aR, encoding a 95,394 Da seventransmembrane domain protein, shows the highest sequence homology with mammalian C3aRs. Northern blot analysis revealed that the CiC3aR is expressed abundantly in the heart and neural complex and to a lesser extent, in the ovaries, hemocytes, and larvae. Three polyclonal antibodies raised against peptides corresponding to CiC3aR regions of the first and second extracellular loop and of the third intracellular loop, react specifically in Western blotting with a single band of 98-102 kDa in hemocyte protein extracts. Immunostaining performed on circulating hemocytes with the three specific antibodies revealed that CiC3aR is constitutively expressed only in hyaline and granular amoebocytes. In chemotaxis experiments, the antibodies against the first and second extracellular loop inhibited directional migration of hemocytes toward the synthetic peptide reproducing the CiC3a C-terminal sequence, thus providing the compelling evidence that C. intestinalis.expresses a functional C3aR homologous to the mammalian receptor. These findings further elucidate the evolutionary origin of the vertebrate complement-mediated proinflammatory processUniversità della Calabri

Estrogen receptor alpha interferes with LKBl/AMPK/mTOR signaling activation in adiponectin-treated breast cancer cells

Dottorato di Ricerca in Medicina Traslazionale. Ciclo XXXBreast cancer is the most common type of tumor and the leading cause of cancer-related deaths in women, worldwide. The cause of breast cancer is multifactorial and includes hormonal, genetic and environmental cues. Obesity is now an accepted risk factor for breast cancer in postmenopausal women, particularly for the hormone-dependent subtype of mammary tumor. Obesity has regarded as a multifactorial disorder characterized by an increased number and size of adipocytes. Adipose tissue is an active metabolic and endocrine organ that secretes many adipocytokines, which act as key mediators in several obesity-associated diseases. Among these, adiponectin represents the most abundant adipose tissue-excreted protein, which exhibits insulin sensitizing, antiinflammatory, and antiatherogenic properties Adiponectin has been proposed as having a key role in the pathogenesis of cardiovascular disease and type 2 diabetes along with obesity-associated malignancies, such as breast cancer. An inverse correlation is reported between obesity and adiponectin, for which low levels of adiponectin represent a risk factor for breast cancer. The role of adiponectin on breast tumorigenesis seems to be dependent on cell phenotypes. Indeed, several in vitro and in vivo studies demonstrated that low adiponectin levels repressed growth in ER-negative breast cancer cells whereas increased proliferation in ER- positive cells. Adiponectin interacts with specific receptors and exerts its effects, including regulation of cell survival, apoptosis and metastasis, via a plethora of signaling pathways. The key molecule of adiponectin action is AMP-activated protein kinase (AMPK), which is mainly activated by liver kinase B1 (LKB1). On the basis of this observations, the aim of the present study was to investigate the effect of adiponectin on LKB1/AMPK signaling in ER-negative (MDA-MB-231) and positive (MCF-7) breast cancer cells. In MCF-7 cells, upon low adiponectin levels, ER impaired LKB1/AMPK interaction by recruiting LKB1 as coactivator at nuclear level, sustaining breast tumor growth. In this condition, AMPK signaling was not working, letting fatty acid synthesis still active. In contrast, in MDA-MB-231 cells the phosphorylated status of AMPK and ACC appeared enhanced, with consequent inhibition of both lipogenesis and cell growth. Thus, in the presence of adiponectin, ERα signaling switched energy balance of breast cancer cells towards a lipogenic phenotype. The same results on tumor growth were reproduced in a xenograft model. These results emphasize how adiponectin action in obese patients is tightly dependent on ERα, addressing that adiponectin may work as growth factor in ERα- positive breast cancer cells.Università della Calabri

L'assunzione del genotipo neuroendocrino da parte di cellule epiteliali neoplastiche quale fattore di resistenza all'aggressione terapeutica nell'ademacarcinoma prostatico

Scuola di Dottorato "Life Sciences", Ciclo XXVII, a.a. 2014Università della Calabri

L'uso di biomateriali innovativi per la veicolazione di farmaci antitumorali

Dottorato di Ricerca in Biochimica Cellulare ed Attività dei Farmaci in Oncologia, XXVII Ciclo, a.a. 2014Università della Calabri

Cachrys libanotis L. Extracts: Photocytotoxic Effects on UVA-Irradiated Human Melanoma Cells

Melanoma is the most aggressive form of skin cancer. Photochemotherapy, combining the action of a light source and a chemical photosensitizer, is one of the most interesting current therapeutic approaches. Plants represent a rich source of photoactive compounds, and furanocoumarins are some of the most important naturally occurring phytoconstituents. The aim of this study was to evaluate the photocytotoxic potential of Cachrys libanotis L. (Apiaceae) from Southern Italy. This species belongs to a genus rich in furanocoumarins and widely distributed in Europe. The aerial parts of the plant were extracted through both traditional maceration and pressurized cyclic solid-liquid (PCSL) extraction using a Naviglio extractor®. Qualitative and quantitative analyses were performed to detect the coumarin content using GC-MS, and the photocytotoxic effects of the extracts were assessed on UVA-irradiated C32 melanoma cells. The apoptotic responses were also evaluated. Furthermore, the phenolic content and in vitro antioxidant potential were estimated. Xanthotoxin, bergapten and isopimpinellin were identified and quantified. Both extracts affected the cell viability in a concentration-dependent manner after irradiation for 1 h at a dose of 1.08 J/cm2. The sample obtained through PCSL extraction was the most effective, with an IC50 equal to 3.16 μg/mL, a very interesting value if compared with the positive control bergapten. This extract induced upregulation of apoptotic signals such as BAX and PARP cleavage, and in the presence of UVA radiation, it caused a greater upregulation of the p21 protein. The obtained results suggest that the investigated species could be a good candidate for further studies aiming to find new drugs with photocytotoxic potential