The role of STAT3 signaling in carcinogenesis

The protein STAT3 (Signal Transducer and Activator of Transcription 3) plays a central role in a range of physiologic process, and when subverted in cancer, can be a central mediator of malignant cellular function. This protein resides at the critical junction between intracellular signaling events and the regulation of genes involved in apoptosis, differentiation, and cell proliferation. The present research has intended to investigate the role of STAT3 in the nuclear compartment 1) as transcription factor in the regulation of a new promoter sequence; 2) as protein hub in modulation of enhanceosomes STAT3-specific in response to different pathways; 3) as protein involved in onset and progression of human prostate cancer. STAT3 acts as transcription factor in the regulatory region of TPX2 gene coding for a protein involved in the complex process of mitosis, and was identified as one of the microtubule-associated proteins. An inspection of the 5'-flanking region of the human BCL2L1 gene, coding for Bcl-xL, has many potentially binding sites for STAT proteins, but none of these correspond closely to the consensus sequence of Stat3. We identified a high-affinity binding sites for Stat3 located at -4305/-4297 base pairs from the transcription start of TPX2 and had a TTCCCGGAA sequence, which is identical to the sequence bound by activated Stat3 in the promoter of gene CDKN1A, coding for the protein p21WAF1/CIP1. By reporter gene assay, conducted in M14 cells treated with specific phosphopeptidic inhibitor of STAT3, we showed that this protein is recruited on the TPX2 gene promoter and regulated in vivo TPX2 expression. Whereas STAT3 is tyrosine phosphorylated by three types of kinases, it uses a precise sequence of functional actions by multiple coactivator complexes and post-translational modifications (PTMs) for mediating gene activation. Considering that we found a different set of STAT3-associated proteins in tumoral cell lines in which the activation of STAT3 can be mediated by different pathways, we could speculate not only a tumor-specific, but also a signal-dependent composition of enhanceosome STAT3-specific. Following CoIP, ChIP and RT2-PCR assays, we hypotized a functional interplay between PARP-1 STAT3-associated proteins when the transcription factor was phosphorylated by Src-kinases or after EGF stimulation, instead the association with CBP/P300 is IL-6 inducted by JAK kinases. In the study of STAT3 partners that affect its function, we demonstrated the importance of PTMs of STAT3 in prostate cancer. In this work we evaluated in parallel, by immunoblotting analysis, the variation of phosphorylation, acetylation and gluthationylation of STAT3 in cell lines and in human prostate tumor (FFPE). To investigate how differences in PTMs of STAT3 can influence gene expression and interactions with coactivators, immunoblottig analysis, Co-IP and ChIP experiments have been conducted in LNCaP and PC3 cells treated with IL-6 and H2O2, to simulate a physiological response to citokines or to oxidative stress. It has been shown that S-gluthationylation of STAT3 and the recruitment of coactivators such ERp57 and Ref-1, two protein involved in redox modification, is a response to oxidative stress, associated with a more advanced state of disease

Effects of nutrients, mainly from mediterranean dietary foods, on mesenchymal stem derived cells: growth or differentiation

During the last decade the interest for the mesenchymal cells is growing due to their possible uses in therapies to treat certain degenerative pathologies. Mesenchymal stem cells have been found in the bone marrow and they have been shown to be responsible for bone repair and fat cells production. Mesenchymal stromal cells can be obtained from a wide variety of tissues in addition to bone marrow and can differentiate into many other cell types. The study of cell differentiation and programming provides new models for drug discovery and cell therapy that now overcomes gene therapy. Senescence, cancer development and degenerative diseases depend on mesenchymal cells contribution to tissue homeostasis. On the other hand, diet and life style are included among risk factors, which can contribute to the success of pharmacological treatments. This review focuses on nutrients from Mediterranean diet and supplements, which have been shown to influence mesenchymal stem cells and cells derived from them. Dietary intake of nutrients impairs both in vitro and in vivo observations, this review aims to gather the results about the effects of food compounds on mesenchymal cells from which adipocytes and osteoblasts derive. Amino acids and proteins, carbohydrates, lipids, fatty acids and vegetable secondary metabolites, differently act on mesenchymal cells bearing on modulation of gene expression and controlling the fate of cell lineages. Remarkable, the analysis of literature shows that the main effect of nutrients on mesenchymal cells is the stimulation of transcription factors which address the cells toward proliferation or differentiation. For instance, carbohydrates, simple or complex, and lipids appear to stimulate the PPAR receptors, whereas proteins and amino acids result to act on the mTOR system and they can also stimulate the MyoD-1 transcription factor and cooperating proteins. In conclusion, nutrients can promote cell growth and differentiation of mesenchymal cells

The induction of Maspin expression by a glucosamine-derivative has an antiproliferative activity in prostate cancer cell lines

Mammary serine protease inhibitor or Maspin has been characterized as a class II tumor suppressor gene in several cancer types, among them prostate cancer (CaP). Androgen ablation is an effective therapy for CaP, but with short-term effectiveness, thus new therapeutic strategies are actively sought. The present study is aimed to explore the effects of a glucosamine derivative, 2-(N-Carbobenzyloxy)L-phenylalanylamido-2-deoxy-β-D-glucose (NCPA), on two CaP cell lines, PC3 and LNCaP. In particular we analyzed the impact of NCPA on Maspin production, cell viability and cell cycle progression and apoptosis/necrosis pathway activation has been determined in PC3 and LNCaP cell lines. NCPA is able to stimulate Maspin production in PC3 and not in LNCaP cell lines. NCPA blocks the PC3 cell cycle in G1 phase, by inhibiting Cyclin D1 production and induces the apoptosis, therefore interfering with aggressiveness of this androgen-insensitive cell line. Moreover, NCPA is able to induce the expression of Maspin in LNCaP cell line treated with androgen receptor inhibitor, Bicalutamide, and in turn to stimulate the apoptosis of these cells. These findings suggest that NCPA, stimulating the endogenous production of a tumor suppressor protein, could be useful in the design of new therapeutic strategies for treatment of CaP

Biochemical and computational studies of the interaction between a glucosamine derivative, NAPA, and the IKKα kinase

The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose (NAPA), was shown to inhibit the kinase activity of IKKα, one of the two catalytic subunits of IKK complex, decreasing the inflammatory status in osteoarthritis chondrocytes. In the present work we have investigated the inhibition mechanism of IKKα by NAPA by combining computational simulations, in vitro assays and Mass Spectrometry (MS) technique. The kinase in vitro assay was conducted using a recombinant IKKα and IKKtide, a 20 amino acid peptide substrate derived from IkBα kinase protein and containing the serine residues Ser32 and Ser36. Phosphorylated peptide production was measured by Ultra Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS), and the atomic interaction between IKKα and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKKα kinase activity with an IC50 of 0.5 mM, to decrease the Km value from 0.337 mM to 0.402 mM and the Vmax from 0.0257 mM·min-1 to 0.0076 mM·min-1. The computational analyses indicate the region between the KD, ULD and SDD domains of IKKα as the optimal binding site explored by NAPA. Biochemical data indicate that there is a non-significant difference between Km and Ki whereas there is a statistically significant difference between the two Vmax values. This evidence, combined with computational results, consistently indicates that the inhibition is non-competitive, and that the NAPA binding site is different than that of ATP or IKKtide

β-Caryophellene inhibits DNA-damage induced by tobacco smoke in mammalian cells

Exposure to smoke induces damages in different organs and tissues, being the upper respiratory tract the first and continuously exposed target (Huang and Chen, 2011). A high incidence of precancerous lesions and malignancies has been also highlighted in smokers. In this context, inhibiting the smoke-induced damages by using chemopreventive agents could represent an effective approach for human health care. In the present study, the natural sesquiterpene β-caryophyllene (CRY) was studied for its ability to inhibit the DNA-damage induced by condensed smoke (CSC; obtained from standard 3R4F cigarettes) in human epithelial bronchial upper airway cells (.BEAS-2B and HepG2) The cytokinesis-block micronucleus (CBMN) assay (Di Sotto et al., 2010) was carried out in order to detect the presence of micronuclei (MN) in the cytoplasm of the cell exposed to the CSC, as markers of genetic damage. Furthermore, taking into account that an increase in the intracellular reactive oxygen species (ROS) has been associated with the acute toxic effects of smoke, the ability of the test substance to inhibit the CSC-induced oxidative stress was evaluated by the 2,7-dichlorofluorescein diacetate (DCFH-DA) assay (Duan et al., 2013). Finally, as cigarette smoke is reputed able to increase the activation of STAT3 protein in bronchial HBECs cells, so resulting in survival of damaged cells (Wu et al., 2014), we have evaluated the ability of CRY to inhibit the STAT3-phosphorylation induced by CSC, according to Chichiarelli et al. (2010). In our experiments, CSC (1-100 g/ml) significantly increased the MN frequency in both cell lines already at the lower concentrations. In DCFH-DA assay, CSC induced an increase of the DCFH fluorescence with respect to the vehicle, so suggesting the presence of higher intracellular levels of ROS. Furthermore, an increase of the CSC-induced STAT-3 phosphorylation was found in both cell lines at different incubation times. When the smoke sample was tested in the presence of CRY (1-25 g/ml), a significant reduction in the CSC-induced MN-frequency was found (maximum inhibition of about 60%). These results suggest a possible preventive role of the substance against CSC and agree with previous data obtained in bacteria (Di Sotto et al., 2013). The effect of CRY could be due to its lipophilic structure and to its high affinity for the phospholipid bilayers (Sarpietro et al., 2015). On the basis of these features, CRY could be able to induce a change in the cell membrane permeability so hindering the uptake into cells of the genotoxic species contained in CSC. CRY was also able to reduce the oxidative stress and to inhibit the STAT3 phosphorylation induced by CSC. Taking into account that the STAT3 protein has been found activated by an increased intracellular oxidative stress, the reduction of its phosphorylation induced by CRY could be strictly connected to the inhibition of the CSC-mediated pro-oxidant effects. STAT3-activation is involved in the progression of pre-neoplastic lesions induced by cigarette smoking: its inhibition could represent a possible mechanism for the protective properties of CRY against the precancerous events associated to the smoke exposure. On the whole, data obtained in the present research highlight protective properties of CRY and encourage further studies in order to evaluate its possible use as a chemopreventive agent against smoke damage

Glucosamine and its peptidyl-derivative NAPA: novel therapeutic strategy for chondrocytes matrix remodeling

Cartilage degradation, due to an imbalance between anabolic and catabolic rate of chondrocyte metabolism, is the main feature of Osteoarthritis (OA). To date, OA is mainly treated with Non Steroidal Anti-Inflammatory Drugs (NSAIDs), in order to reduce arthritis-related symptoms. In the last decades an increasing number of patients have started to use supplements, such as Glucosamine (GlcN) and chondroitin sulfate, as potential chondroprotective agents. Several in vivo clinical trials as well as in vitro experiments have been performed reporting inconsistent outcomes. Previously, in our lab we analyzed the anabolic effects of GlcN and its N-acetyl-phenylalanine derivative (NAPA) in a rabbit OA model, finding that intra-articular administration of GlcN and NAPA was very effective in reducing cartilage changes in injured rabbit knee. GlcN and NAPA intra-articular administration allows higher concentrations to be reached in the joints compared to oral administration, thus providing an explanation for the ability of both molecules to interfere with OA progression. We also studied the effects of GlcN and NAPA on inflammatory pathways, finding that both molecules can interfere with MAP kinase and NF-kB pathways, by interfering with IKK activity. Finally, we studied the effectiveness of GlcN and NAPA one the biosynthetic activity and hence the matrix production of human primary chondrocytes cultured in micromasses, which represent a good tridimensional culture model. We explored the ability of GlcN and NAPA to stimulate the synthesis of collagen type II (Coll II), Aggrecan (ACAN) and Small Leucine-Rich Proteoglycans (SLRPs). After 6 weeks, micromasses stimulated with GlcN + NAPA still showed a large amount of ECM compared to untreated cells. Moreover, Collagen type II was more abundant and better organized compared to that produced by untreated cells. Finally, cells resulted viable in both treated and untreated micromasses, even if in the middle of untreated micromasses, few dead cells were observed, whereas in the treated micromasses only viable cells and cells completely surrounded by ECM were detected

Effects of a glucosamine-derivative on prostate cancer cell line PC3

Prostate Cancer (CaP) is the most common form of male tumor and is the second leading cause of cancer death. Androgen ablation has proved to be an effective therapy for metastatic prostate cancer, but the regression of metastatic lesions lasts only 18 to 24 months. Mammary serine protease inhibitor or Maspin is a 42 kDa, a non-inhibitory member of the serine protease inhibitor superfamily, and has been characterized as a class II tumor suppressor gene in several cancer types, among them prostate cancer (CaP), due to its ability to inhibit metastasis. In normal prostate epithelial cells, Maspin is highly expressed whereas in prostate cancer cell lines its expression is almost completely suppressed. Previously, it has been demonstrated that NCPA, a glucosamine-derivative synthetized in our laboratory, was able to inhibit IKKα nuclear translocation and to stimulate the production and nuclear localization of Maspin in an osteosarcoma cell line: 143B. IKKα, one of two catalytic subunits of NF-B transcriptional factors, enhances tumor promotion by repressing, among other mechanisms, maspin promoter. Tumor-suppressing and anti-metastatic activities of Maspin have been attributed to its ability to inhibit both invasiveness and cell cycle progression and to stimulate apoptosis of tumor cells. Aim of this presentation is to analyze the ability of NCPA to affect metastatic and proliferation activity of PC3, which is an androgen-insensitive prostate cancer cell line