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THE ROLE OF STAT3 SIGNALING IN CARCINOGENESIS
Full text linkThe 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.This work was sponsored by "Istituto Pasteur-Fondazione Cenci Bolognetti
STAT3/ERP57/TPX2 axis and process of “androgen escape” in prostate cancer
No full textThe mechanisms of Prostate Cancer (PCa) progression through hormone-dependent to hormone refractory form is still unclear. Many data indicate that JAK/STAT signaling contributes to tumor resistance and STAT3 hyperactivation is observed in a variety of human cancers. Moreover, several authors suggested that ERp57 (GRP58/PDIA3), a disulfide isomerases protein, is associated with modulation of STAT3 activity. We investigate the role of STAT3-ERp57-TPX2 axis in the ormone-responsive and androgen-refractory tumor using human PCa cell lines, LNCaP (androgen-sensitive) and PC3 (androgen-refractory), untreated and stimulated with IL-6 and EGF. Immunoblotting and CoIP analysis were performed to confirm STAT3 activation and ERp57-STAT3 interaction. To investigate the physiological relevance of STAT3-ERp57-TPX2 axis, we inhibited STAT3 or ERp57 activity and the expression levels of TPX2 was monitored by qRT-PCR. The results showed that increased STAT3-ERp57 complex association determines an TPX2 overexpression. In conclusion, this study showed that STAT3-ERp57-TPX2 axis is correlated with tumor progression and it suggests a functional role of STAT3/ERp57 complex in the Androgen Escape
Androgen-Escape": the role of STAT3
No full textThe pathophysiological process ‘‘Androgen Escape’’ is a clinical phase in which the tumoral prostatic cells obtain the ability to survive and proliferate without the required signals delivered normally by circulating androgens. Androgen insensitivity reflects the ability to grow without (or with very low) circulating androgens, but it should not be confused with an absence of the intracellular signaling activated downstream of androgen binding to AR. This event occurs when the tumoral prostatic cell can over activates the androgenic pathway by several mechanisms. A convergence point of some of these pathways is the protein STAT3 (Signal Transducer and Activator of Transcription). The phosphorylation of STAT-3 of tyrosine 705 in the cytoplasm, by IL-6 and EGF, leads to its dimerization, translocation into the nucleus, DNA binding, and then, expression of genes that regulate cell proliferation, differentiation, and apoptosis. In prostate cancer (CaP) AR and STAT3 pathway are constitutively activated; they coexist in the hormone-responsive tumor and crosstalk in androgen-refractory tumor. The aim of our study was to investigate the role of STAT3 and its post-translational modifications (PTMs), like phosphorilation, acetilation, glutathionylation, responsible of a functional variability of the protein as transcription factor. To evaluate STAT3 activation and its PTMs we performed Western Blotting analysis on two human prostate cancer cell lines LNCaP, androgen-sensitive, and PC3, androgen-refractory, untreated and stimulated with IL-6 (25 ng/ml), EGF (100 ng/ml) and H2O2 (100 mM). The results showed differences between the profiles of PTMs in the two cell lines and in the different conditions. After, we examined by RT2-PCR, the expression levels of STAT3 target genes, under the same conditions: the data obtained showed a relation between PTMs of STAT3 and P21 – Signal transduction Abstracts FEBS Journal 278 (Suppl. 1) 74–445 (2011) a 2011 The Authors Journal compilation a 2011 Federation of European Biochemical Societies 35
β-Caryophellene inhibits DNA-damage induced by tobacco smoke in mammalian cells
Full text linkExposure 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
Chemopreventive effects of β-caryophyllene against cigarette smoke damage in upper airway cells
Full text linkExposure to cigarette smoke induces damages in different organs and tissues, and high incidence of precancerous lesions and malignancies particularly at upper respiratory tract.1 Increasing levels of reactive oxygen species (ROS) and the activation of STAT3 pathway seems to be involved in smoke injury and oncogenic proliferation of damaged cells.1 In order to find new strategies for preventing cancer development in smokers, in present study we evaluated the ability of the natural sesquiterpene β-caryophyllene (CRY) to inhibit smoke damage in human epithelial bronchial upper airway (BEAS-2B) cells, by studying the inhibition of STAT3-phosphorylation, pro-oxidant damage and oncogenic proliferation induced by a condensed sample of cigarette smoke (CSC), according to previous methods.2,3 In our experiments, CSC (25-150 g/ml) strongly increased the levels of both phosphorylated STAT3 and intracellular ROS, and the cell migration capacity. CRY (1-10 g/ml) significantly reduced the activation of STAT3 pathway and the pro-oxidant effects of CSC (75 μg/ml). Furthermore, a remarkable inhibition of CSC-induced cell migration was highlighted, so suggesting a possible interference of CRY with metastatic ability of damaged cells. Data obtained highlight interesting protective properties of CRY and encourage further studies in order to evaluate its possible use as a chemopreventive agent against smoke damage.
References
1. Wu et al. (2014). Free Rad. Biol. Med. 69, 208–218.
2. Chichiarelli et al. (2010). Arch Biochem Biophys 494, 178-193.
3. Duan et al. (2013). Plos One 8, e57941
The induction of Maspin expression by a glucosamine-derivative has an antiproliferative activity in prostate cancer cell lines
Full text linkMammary 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
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