Luisa Salvatori

Expression and Cellular Localization of Follicle-Stimulating Hormone Receptor in Normal Human Prostate, Benign Prostatic Hyperplasia and Prostate Cancer

PURPOSE FSH, identified as an endogenous product of the prostate, is a glycoprotein with proliferative activity. Increasing evidence of autocrine/paracrine activities of gonadotropins at extragonadal sites led us to investigate the gene expression and cellular localization of FSH-R in normal and diseased human prostates. MATERIALS AND METHODS Prostate specimens, including normal gland, BPH, PCa and human androgen refractory (PC3) and androgen dependent (LNCaP) prostate cancer cell lines (European Collection of Cell Cultures, Salisbury, United Kingdom), were analyzed for FSH-R expression by semiquantitative and real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry. We also evaluated cyclic adenosine monophosphate production by cultured PC3 and LNCaP stimulated with human FSH. RESULTS Little FSH-R expression was seen in 9 of 13 normal and 8 of 15 BPH specimens. Of 30 PCa samples 21 were FSH-R positive with generally higher expression compared to normal prostate and BPH samples. Real-time reverse transcriptase-polymerase chain reaction of matched normal/tumor pairs confirmed higher FSH-R mRNA expression in PCa. PC3 cells expressed FSH-R, while LNCaP cells were FSH-R negative. FSH-R protein was mainly localized in the glandular epithelium and in some stromal cells in normal prostate, BPH and PCa specimens. PC3 cells expressed FSH-R protein and their treatment with FSH induced a significant increase in cyclic adenosine monophosphate production. CONCLUSIONS These results indicate that a subset of PCa expresses FSH-R mRNA and protein at levels higher than those of normal and hyperplastic tissues that express FSH-R. This suggests that FSH might contribute to some cases of PCa via a receptor mediated mechanism.

Oestrogens and selective oestrogen receptor (ER) modulators regulate EGF receptor gene expression through human ER α and β subtypes via an Sp1 site

Through the analysis of the transient expression of the luciferase reporter gene in HeLa cells, an evaluation has been made of the transcriptional activity of oestrogens and of selective oestrogen receptor (ER) modulators (SERMs), mediated by the α and β isoforms of the ER, on the epidermal growth factor receptor gene promoter. Oestrogen-activated ERβ presents a lower transcriptional activity compared with ERα, probably due to structural differences in the AF-1 regions of the receptors. Also SERMs induce different responses depending on the receptor isoform bound. Indeed, the phyto-oestrogens, genistein and daidzein, act as weak agonists of the oestrogenic activity via ERα, but as full agonists when bound to ERβ. The synthetic SERM 4OH-tamoxifen, on the other hand, displays an opposite behaviour since it exerts a full agonist action through ERα, but acts as a full antagonist via ERβ. As we have previously shown for ERα, an ERβ/Sp1 functional synergism has also been highlighted, by means of gel mobility shift assays. Moreover, our results show that the sensitivity of target tissues to oestrogens and SERMs can be affected by coexpression of ERs, depending on the formation of appropriate levels of homo- and heterodimers, thus providing a useful approach to predict the effects of hormonal treatment.

Modulators of HIF1α and NFkB in Cancer Treatment: Is it a Rational Approach for Controlling Malignant Progression?

HIF1α and NFkB are two transcription factors very frequently activated in tumors and involved in tumor growth, progression, and resistance to chemotherapy. In fact, HIF1α and NFkB together regulate transcription of over a thousand genes that, in turn, control vital cellular processes such as adaptation to the hypoxia, metabolic reprograming, inflammatory reparative response, extracellular matrix digestion, migration and invasion, adhesion, etc. Because of this wide involvement they could control in an integrated manner the origin of the malignant phenotype. Interestingly, hypoxia and inflammation have been sequentially bridged in tumors by the discovery that alarmin receptors genes such as RAGE, P2X7, and some TLRs, are activated by HIF1α; and that, in turn, alarmin receptors strongly activate NFkB and proinflammatory gene expression, evidencing all the hallmarks of the malignant phenotype. Recently, a large number of drugs have been identified that inhibit one or both transcription factors with promising results in terms of controlling tumor progression. In addition, many of these molecules are natural compounds or off-label drugs already used to cure other pathologies. Some of them are undergoing clinical trials and soon they will be used alone or in combination with standard anti-tumoral agents to achieve a better treatment of tumors with reduction of metastasis formation and, more importantly, with a net increase in survival. This review highlights the central role of HIF1α activated in hypoxic regions of the tumor, of NFkB activation and proinflammatory gene expression in transformed cells to understand their progression toward malignancy. Different molecules and strategies to inhibit these transcription factors will be reviewed. Finally, the central role of a new class of deacetylases called Sirtuins in regulating HIF1α and NFkB activity will be outlined.

Cell-to-cell signaling influences the fate of prostate cancer stem cells and their potential to generate more aggressive tumors

An increasing number of malignancies has been shown to be initiated and propelled by small subpopulations of cancer stem cells (CSC). However, whether tumor aggressiveness is driven by CSC and by what extent this property may be relevant within the tumor mass is still unsettled. To address this issue, we isolated a rare tumor cell population on the basis of its CD44+CD24− phenotype from the human androgen-independent prostate carcinoma cell line DU145 and established its CSC properties. The behavior of selected CSC was investigated with respect to the bulk DU145 cells. The injection of CSC in nude mice generated highly vascularized tumors infiltrating the adjacent tissues, showing high density of neuroendocrine cells and expressing low levels of E-cadherin and β-catenin as well as high levels of vimentin. On the contrary, when a comparable number of unsorted DU145 cells were injected the resulting tumors were less aggressive. To investigate the different features of tumors in vivo, the influence of differentiated tumor cells on CSC was examined in vitro by growing CSC in the absence or presence of conditioned medium from DU145 cells. CSC grown in permissive conditions differentiated into cell populations with features similar to those of cells held in aggressive tumors generated from CSC injection. Differently, conditioned medium induced CSC to differentiate into a cell phenotype comparable to cells of scarcely aggressive tumors originated from bulk DU145 cell injection. These findings show for the first time that CSC are able to generate differentiated cells expressing either highly or scarcely aggressive phenotype, thus influencing prostate cancer progression. The fate of CSC was determined by signals released from tumor environment. Moreover, using microarray analysis we selected some molecules which could be involved in this cell-to-cell signaling, hypothesizing their potential value for prognostic or therapeutic applications.

Effects of the lipidosterolic extract of Serenoa repens (Permixon®) on human prostatic cell lines

Permixon® is a drug used in the treatment of benign prostatic hyperplasia. We studied its androgenic and antiandrogenic effects in the prostatic cell lines LNCaP and PC3, respectively responsive and unresponsive to androgen stimulation.

Stemness and osteogenic and adipogenic potential are differently impaired in subcutaneous and visceral adipose derived stem cells (ASCs) isolated from obese donors.

Today adipose tissue is not just considered as the primary energy storage organ, but it is also recognized as an important endocrine tissue and an abundant source of mesenchymal stem cells (adipose-derived stem cells, ASCs). During the last decade, several studies have provided preclinical data on the safety and efficacy of ASCs, supporting their use in cell-based therapy for regenerative medicine purposes. Little is known about the effect of obesity on ASCs properties. Since ASCs differentiation and proliferation are determined by their niche, the differences in body fat distribution and the obesity-related co-morbidities may have several consequences. In this study we compared ASCs of subcutaneous adipose tissue from obese (obS-ASCs) and non-obese (nS-ASCs) donors in order to compare their immunophenotype and osteogenic and adipogenic potential. Moreover, in order to evaluate the possible difference between subcutaneous and visceral fat, obS-ASCs were also compared to ASCs derived from visceral adipose tissue of the same obese donors (obV-ASCs). Our results show that subcutaneous and visceral ASCs derived from obese donors have an impaired cell proliferation, clonogenic ability and immunophenotype. Nevertheless, obS-ASCs are able to differentiate toward osteogenic and adipogenic lineages, although to a small extent with respect to non-obese donors, whereas obV-ASCs lose most of their stem cell characteristics, including multi-differentiation potential. Taken together our findings confirm that not all ASCs present the same behavior, most likely due to their biological microenvironment in vivo. The specific stimuli which can play a key role in ASCs impairment, including the effects of the obesity-related inflammation, should be further investigated to have a complete picture of the phenomenon.

Down-regulation of epidermal growth factor receptor induced by estrogens and phytoestrogens promotes the differentiation of U2OS human osteosarcoma cells

In previous studies on HeLa cells we demonstrated estrogen‐responsiveness of the epidermal growth factor receptor (EGFR) gene, as 17β‐estradiol (E2) and selective estrogen receptor modulators (SERMs) genistein (G), daidzein (D), and 4‐hydroxytamoxifen (4OH‐T) modulated its transcription in a ligand‐ and estrogen receptor (ER) isoform‐specific way. This study describes further investigations into the role of ERs in mediating the effects induced by E2 and SERMs on EGFR expression, and the relationship between the actions of ERs and EGFR in U2OS osteosarcoma cells stably expressing ERα or ERβ. Cell number and DNA content determination revealed that E2, G, and D inhibited proliferation and cell cycle progression and promoted apoptosis in both cell lines. In parallel, changes in cell morphology typical of osteoblast maturation were observed via optical microscopy. Consistently, quantitative PCR and Western blot analysis showed an up‐regulation of markers of osteoblast differentiation and bone repair, and a decrease in EGFR expression. The transfection of specific antisense (AS) oligonucleotides strengthened our hypothesis that EGFR reduction caused changes in the proliferation/differentiation pattern comparable to those induced by ER ligands. The link between the ER and EGFR pathways was confirmed by treatment with 4OH‐T, which decreased the EGFR level and produced differentiation effects via ERα, but induced both EGFR expression and proliferation effects via ERβ. In conclusion, we show that also in U2OS cells, E2 and SERMs are able to modulate the expression of the EGFR gene and can affect events strictly controlled by its signaling pathway, such as the maturation of osteoblasts. J. Cell. Physiol. 220: 35–44, 2009.

The artificial 4-zinc-finger protein Bagly binds human utrophin promoter A at the endogenous chromosomal site and activates transcription.

Our aim is to upregulate the expression of the dystrophin-related gene utrophin in Duchenne muscular dystrophy, in this way complementing the lack of dystrophin function. To achieve utrophin upregulation, we designed and engineered synthetic zinc-finger based transcription factors. We have previously shown that the artificial 3-zinc-finger protein Jazz, fused with the appropriate effector domain, is able to drive the transcription of a test gene from utrophin promoter A. Here we report a novel artificial 4-zinc-finger protein, Bagly, which binds with optimized affinity-specificity to a 12 bp DNA target sequence that is internal to human utrophin promoter A. Bagly was generated adding to Jazz protein an extra-fourth zinc finger, derived from transcription factor YY1. Importantly, the Bagly DNA target sequence is statistically present in the human genome only 210 times, about 60 fewer times than the 9 bp Jazz DNA target sequence. Thanks to its additional zinc-finger domain, Bagly protein shows enhanced transcriptional activity. Moreover, we demonstrated Baglys effective access and binding to active chromatin in the chromosomal context and its ability to upregulate endogenous utrophin.

Cell death, proliferation and repair in human myocarditis responding to immunosuppressive therapy

In this study, we evaluate cell death, proliferation and repair in left ventricular endomyocardial biopsies from 20 patients with active lymphocytic myocarditis worsening or recovering from cardiac dysfunction after 6-months immunosuppression. Apoptosis and necrosis were assessed by in situ ligation of hairpin probes, proliferation by Ki67 and MCM5 labelling of myocytes, repair by electron microscopy, morphometric study of percent myofibrillar area and real-time polymerase chain reaction of α-and β-Myosin Heavy Chain (MHC). Apoptosis and necrosis decreased in post- vs pretreatment biopsies by 85 and 62%, respectively in responders, while increased by 42 and 46% in nonresponders. Ki67 and MCM5-positive myocytes were higher vs controls at baseline and increased by 43 and 38% at follow-up in responders and by 75 and 63% in nonresponders. Myofibrillar area reduced in pretreatment samples, increased by 33% at follow-up in responders, correlated with percent enhancement of ejection fraction and was associated with increased α-MHC expression and α/β-MHC ratio. In follow-up biopsies of nonresponders, myofibrillar area diminished by 36% and correlated with percent decrease of ejection fraction. Our results suggest that recovery of cardiac function in myocarditis responding to immunosuppression is associated with inhibition of cell death, activation of cell proliferation and with newly synthesized contractile material.

Hypoxia Promotes the Inflammatory Response and Stemness Features in Visceral Fat Stem Cells From Obese Subjects

Low‐grade chronic inflammation is a salient feature of obesity and many associated disorders. This condition frequently occurs in central obesity and is connected to alterations of the visceral adipose tissue (AT) microenvironment. Understanding how obesity is related to inflammation may allow the development of therapeutics aimed at improving metabolic parameters in obese patients. To achieve this aim, we compared the features of two subpopulations of adipose‐derived stem cells (ASC) isolated from both subcutaneous and visceral AT of obese patients with the features of two subpopulations of ASC from the same isolation sites of non‐obese individuals. In particular, the behavior of ASC of obese versus non‐obese subjects during hypoxia, which occurs in obese AT and is an inducer of the inflammatory response, was evaluated. Obesity deeply influenced ASC from visceral AT (obV‐ASC); these cells appeared to exhibit clearly distinguishable morphology and ultrastructure as well as reduced proliferation, clonogenicity and expression of stemness, differentiation and inflammation‐related genes. These cells also exhibited a deregulated response to hypoxia, which induced strong tissue‐specific NF‐kB activation and an NF‐kB‐mediated increase in inflammatory and fibrogenic responses. Moreover, obV‐ASC, which showed a less stem‐like phenotype, recovered stemness features after hypoxia. Our findings demonstrated the peculiar behavior of obV‐ASC, their influence on the obese visceral AT microenvironment and the therapeutic potential of NF‐kB inhibitors. These novel findings suggest that the deregulated hyper‐responsiveness to hypoxic stimulus of ASC from visceral AT of obese subjects may contribute via paracrine mechanisms to low‐grade chronic inflammation, which has been implicated in obesity‐related morbidity. J. Cell. Physiol. 231: 668–679, 2016.