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Here, we report new mechanistic insight into how chronic hypoxia increases 'stemness' in cancer cells. Using chemical inhibitors, we provide direct experimental evidence that ROS production and mitochondrial biogenesis are both required for the hypoxia-induced propagation of CSCs. More specifically, we show that hypoxic CSCs can be effectively targeted with i) simple mitochondrial anti-oxidants (Mito-TEMPO) and/or ii) inhibitors of mitochondrial biogenesis (Doxycycline). In this context, we discuss the idea that mitochondrial biogenesis itself may be a primary driver of "stemness" in hypoxic cancer cells, with metabolic links to fatty acid oxidation (FAO). As Doxycycline is an FDA-approved drug, we propose that it could be repurposed to target hypoxic CSCs, either alone or in combination with chemotherapy, i.e., Paclitaxel. For example, we demonstrate that Doxycycline effectively targets the sub-population of hypoxia-induced CSCs that are Paclitaxel-resistant, overcoming hypoxia-induced drug-resistance. Finally, anti-angiogenic therapy often induces tumor hypoxia, allowing CSCs to survive and propagate, ultimately driving tumor progression. Therefore, we suggest that Doxycycline could be used in combination with anti-angiogenic agents, to actively prevent or minimize hypoxia-induced treatment failure. In direct support of this assertion, Paclitaxel is already known to behave as an angiogenesis inhibitor.

Il capitolo si propone di ricostruire le policies oggetto della ricerca, in particolare la regolazione dei rapporti di lavoro, la concessione della cittadinanza agli stranieri e l’incentivazione della produzione di energia fotovoltaica, considerandone sia gli output, ovvero, a seconda dei casi, le proposte di legge, le leggi e i decreti formulati ed adottati sui tre temi, che i processi decisionali, con particolare attenzione agli attori che vi hanno preso parte. L’obiettivo è quello di ricostruire sinteticamente l’evoluzione delle tre policies dagli anni Novanta ad oggi, sintesi che consentirà, nei capitoli successivi, di comprendere come la comunicazione mediatica abbia trattato queste issues.

The use of Doxorubicin (Dox), a frontline drug for many cancers, is often complicated by dose-limiting cardiotoxicity in approximately 20% of patients. The G-protein estrogen receptor GPER/GPR30 mediates estrogen action as the cardioprotection under certain stressful conditions. For instance, GPER activation by the selective agonist G-1 reduced myocardial inflammation, improved immunosuppression, triggered pro-survival signaling cascades, improved myocardial mechanical performance, and reduced infarct size after ischemia/reperfusion (I/R) injury. Hence, we evaluated whether ligand-activated GPER may exert cardioprotection in male rats chronically treated with Dox. 1 week of G-1 (50 μg/kg/day) intraperitoneal administration mitigated Dox (3 mg/kg/day) adverse effects, as revealed by reduced TNF-α, IL-1β, LDH, and ROS levels. Western blotting analysis of cardiac homogenates indicated that G-1 prevents the increase in p-c-jun, BAX, CTGF, iNOS, and COX2 expression induced by Dox. Moreover, the activation of GPER rescued the inhibitory action elicited by Dox on the expression of BCL2, pERK, and pAKT. TUNEL assay indicated that GPER activation may also attenuate the cardiomyocyte apoptosis upon Dox exposure. Using ex vivo Langendorff perfused heart technique, we also found an increased systolic recovery and a reduction of both infarct size and LDH levels in rats treated with G-1 in combination with Dox respect to animals treated with Dox alone. Accordingly, the beneficial effects induced by G-1 were abrogated in the presence of the GPER selective antagonist G15. These data suggest that GPER activation mitigates Dox-induced cardiotoxicity, thus proposing GPER as a novel pharmacological target to limit the detrimental cardiac effects of Dox treatment. J. Cell. Physiol. 232: 1640-1649, 2017. © 2016 Wiley Periodicals, Inc.

International audience; Estrogens control a wide number of aspects of human physiology and play a key role in multiple diseases, including cancer. Estrogens act by binding to and activating the cognate receptor (ER), however numerous studies have revealed that the G protein-coupled receptor named GPR30/GPER mediates also estrogen signals. As ER and GPER share the ability to bind to same compounds, the use of GPER-selective ligands has allowed a better understanding of the biological responses mediated by GPER. In the present study, we designed and synthesized two novel carbazole derivatives and then investigated their ability to interact with and activate the GPER-mediated transduction pathway in breast cancer cells. Both compounds did not activate the classical ER in MCF7 cells, whereas one of the two compounds synthesized triggered through GPER the rapid ERK activation in ER-negative SkBr3 cells, demonstrating a good affinity for GPER in docking studies. The characterization of this novel selective GPER agonist could represent a potential useful tool to provide further insights into the physiopathological role exerted by GPER.

Activation of c-Jun, a major component of the AP-1 transcription factor, represents a paradigm for transcriptional response to stress. Transactivation of c-Jun is regulated by Jun-N-terminal kinases (JNKs) through phosphorylation at serine 63 and 73 (S63/S73), as well as at threonine 91 and 93 (T91/T93). How these two groups of phosphoacceptor sites respond to different grades of genotoxic stress and whether DNA-damage pathways influence the extent of their JNK-dependent phosphorylations remain to be elucidated. Here, we show that following a short exposure to the DNA-damaging compound etoposide, c-Jun phosphorylation is restricted to S63/S73. In contrast, JNK-dependent phosphorylation of T91/T93 requires continuous exposure to the drug and is impaired by caffeine treatment or alanine substitution of the adjacent threonine 95 (T95). Conversely, c-Jun mutations switching the T95/Q96 site into a canonical site for mitogen activated protein kinase (MAPK) phosphorylation (T95/P96) rescues T91/T93 phosphorylation in presence of caffeine, suggesting that a preceding phosphorylation at T95 exposes T91/T93 to JNK-dependent phosphorylation. Moreover, we show that alanine substitution at T95

MAPK phosphorylation of various substrates is mediated by the presence of docking sites, including the D domain and the DEF motif. Depending on the number and sequences of these domains, substrates are phosphorylated by specific subsets of MAPKs. For example, a D domain targets JNK to c-Jun, whereas a DEF motif is required for ERK phosphorylation of c-Fos. JunD, in contrast, contains both D and DEF domains. Here we show that these motifs mediate JunD phosphorylation in response to either ERK or JNK activation. An intact D domain is required for phosphorylation and activation of JunD by both subtypes of MAPK. The DEF motif acts together with the D domain to elicit efficient phosphorylation of JunD in response to the epidermal growth factor (EGF) but has no function on JunD phosphorylation and activation by JNK signaling. Furthermore, we show that conversion of a c-Jun sequence to a canonical DEF domain, as it is present in JunD, elicits c-Jun activation in response to EGF. Our results suggest that evolution of a particular modular system of MAPK targeting sequences has determined a differential response of JunD and c-Jun to ERK activation

Phytoestrogens are a chemically diverse group of compounds made by plants that can have estrogenic effects in animals. Both tumorigenic and antitumorigenic effects have been reported. Although estrogens stimulate the growth of many breast tumors, there is a negative correlation between the incidence of breast cancer and the phytoestrogen-rich diet of certain Asian populations. To begin to resolve this paradox, we have analyzed the estrogenic properties of genistein and quercetin, two flavonoid phytoestrogens particularly abundant in soybeans. Trans-activation experiments with a transfected reporter gene for nuclear estrogen receptors (ER) show strong activation of the endogenous ER alpha by both phytoestrogens in two MCF7 human breast cancer cell lines. This is supported by the observation that the two phytoestrogens induce the down-regulation of ER alpha mRNA and protein levels. Using chimeric proteins consisting of the hormone binding domains of ER alpha and ER beta fused to the Gal4 DNA binding domain, we have established that genistein and quercetin are full estrogenic agonists of both ER isoforms. Ligand binding experiments with purified ER alpha and ER beta confirm that the two phytoestrogens are ER ligands. At concentrations that are sufficient to obtain substantial transcriptional activity, they stimulate the proliferation of two ER alpha-dependent breast cancer cell lines. At high concentrations, such as those reached with a soy-rich diet, genistein and quercetin are strong cytotoxic agents that even kill ER-independent HeLa cells. Thus, the mode of action of phytoestrogens and the balance between being risk or chemopreventive factors for breast cancer may depend on the dietary load.