Fatty Acid Synthase as Interacting Anticancer Target of the Terpenoid Myrianthic Acid Disclosed by MS-Based Proteomics Approaches.

This research focuses on the target deconvolution of the natural compound myrianthic acid, a triterpenoid characterized by an ursane skeleton isolated from the roots of Myrianthus arboreus and from Oenothera maritima Nutt. (Onagraceae), using MS-based chemical proteomic techniques. Application of drug affinity responsive target stability (DARTS) and targeted-limited proteolysis coupled to mass spectrometry (t-LiP-MS) led to the identification of the enzyme fatty acid synthase (FAS) as an interesting macromolecular counterpart of myrianthic acid. This result, confirmed by comparison with the natural ursolic acid, was thoroughly investigated and validated in silico by molecular docking, which gave a precise picture of the interactions in the MA/FAS complex. Moreover, biological assays showcased the inhibitory activity of myrianthic acid against the FAS enzyme, most likely related to its antiproliferative activity towards tumor cells. Given the significance of FAS in specific pathologies, especially cancer, the myrianthic acid structural moieties could serve as a promising reference point to start the potential development of innovative approaches in therapy.

New biologic (Ab-IPL-IL-17) for IL-17-mediated diseases: identification of the bioactive sequence (nIL-17) for IL-17A/F function.

OBJECTIVES: Interleukin (IL) 17s cytokines are key drivers of inflammation that are functionally dysregulated in several human immune-mediated inflammatory diseases (IMIDs), such as rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD). Targeting these cytokines has some therapeutic benefits, but issues associated with low therapeutic efficacy and immunogenicity for subgroups of patients or IMIDs reduce their clinical use. Therefore, there is an urgent need to improve the coverage and efficacy of antibodies targeting IL-17A and/or IL-17F and IL-17A/F heterodimer. METHODS AND RESULTS: Here, we initially identified a bioactive 20 amino acid IL-17A/F-derived peptide (nIL-17) that mimics the pro-inflammatory actions of the full-length proteins. Subsequently, we generated a novel anti-IL-17 neutralising monoclonal antibody (Ab-IPL-IL-17) capable of effectively reversing the pro-inflammatory, pro-migratory actions of both nIL-17 and IL-17A/F. Importantly, we demonstrated that Ab-IPL-IL-17 has less off-target effects than the current gold-standard biologic, secukinumab. Finally, we compared the therapeutic efficacy of Ab-IPL-IL-17 with reference anti-IL-17 antibodies in preclinical murine models and samples from patients with RA and IBD. We found that Ab-IPL-IL-17 could effectively reduce clinical signs of arthritis and neutralise elevated IL-17 levels in IBD patient serum. CONCLUSIONS: Collectively, our preclinical and in vitro clinical evidence indicates high efficacy and therapeutic potency of Ab-IPL-IL-17, supporting the rationale for large-scale clinical evaluation of Ab-IPL-IL-17 in patients with IMIDs.

Exploring the chemical space of functionalized [1,2,4]triazolo[4,3-a]quinoxaline-based compounds targeting the bromodomain of BRD9.

Here we report a detailed structure-activity relationship (SAR) study related to [1,2,4]triazolo[4,3-a]quinoxaline-based compounds targeting the reader module of bromodomain containing-protein 9 (BRD9). 3D structure-based pharmacophore models, previously introduced by us, were here employed to evaluate a second generation of compounds, exploring different substitution patterns on the heterocyclic core. Starting from the promising data obtained from our previously identified [1,2,4]triazolo[4,3-a]quinoxaline-based compounds 1-4, the combination of in silico studies, chemical synthesis, biophysical and in vitro assays led to the identification of a new set of derivatives, selected for thoroughly exploring the chemical space of the bromodomain binding site. In more details, the investigation of different linkers at C-4 position highlighted the amine spacer as mandatory for the binding with the protein counterpart and the crucial role of the alkyl substituents at C-1 for increasing the selectivity toward BRD9. Additionally, the importance of a hydrogen bond donor group, critical to anchor the ZA region and required for the interaction with Ile53 residue, was inferred from the analysis of our collected results. Herein we also propose an optimization and an update of our previously reported "pharm-druglike2" 3D structure-based pharmacophore model, introducing it as "pharm-druglike2.1". Compounds 24-26, 32, 34 and 36 were identified as new valuable BRD9 binders featuring IC50 values in the low micromolar range. Among them, 24 and 36 displayed an excellent selectivity towards BRD9 and a good antiproliferative effect on a panel of leukemia models, especially toward CCRF-CEM cell line, with no cytotoxicity on healthy cells. Notably, the interaction of 24 and 36 with the bromodomain and PHD finger-containing protein 1 (BRPF1) also emerged, disclosing them as new and unexplored dual inhibitors for these two proteins highly involved in leukemia. These findings highlight the potential for the identification of new attractive dual epidrugs as well as a promising starting point for the development of chemical degraders endowed with anticancer activities.

Triple Negative Breast Cancer Preclinical Therapeutic Management by a Cationic Ruthenium-Based Nucleolipid Nanosystem.

Based on compelling preclinical evidence concerning the progress of our novel ruthenium-based metallotherapeutics, we are focusing research efforts on challenging indications for the treatment of invasive neoplasms such as the triple-negative breast cancer (TNBC). This malignancy mainly afflicts younger women, who are black, or who have a BRCA1 mutation. Because of faster growing and spreading, TNBC differs from other invasive breast cancers having fewer treatment options and worse prognosis, where existing therapies are mostly ineffective, resulting in a large unmet biomedical need. In this context, we benefited from an experimental model of TNBC both in vitro and in vivo to explore the effects of a biocompatible cationic liposomal nanoformulation, named HoThyRu/DOTAP, able to effectively deliver the antiproliferative ruthenium(III) complex AziRu, thus resulting in a prospective candidate drug. As part of the multitargeting mechanisms featuring metal-based therapeutics other than platinum-containing agents, we herein validate the potential of HoThyRu/DOTAP liposomes to act as a multimodal anticancer agent through inhibition of TNBC cell growth and proliferation, as well as migration and invasion. The here-obtained preclinical findings suggest a potential targeting of the complex pathways network controlling invasive and migratory cancer phenotypes. Overall, in the field of alternative chemotherapy to platinum-based drugs, these outcomes suggest prospective brand-new settings for the nanostructured AziRu complex to get promising goals for the treatment of metastatic TNBC.

Tetrasubstituted Pyrrole Derivative Mimetics of Protein-Protein Interaction Hot-Spot Residues: A Promising Class of Anticancer Agents Targeting Melanoma Cells.

A new series of tetrasubstituted pyrrole derivatives (TSPs) was synthesized based on a previously developed hypothesis on their ability to mimic hydrophobic protein motifs. The resulting new TSPs were endowed with a significant toxicity against human epithelial melanoma A375 cells, showing IC50 values ranging from 10 to 27 µM, consistent with the IC50 value of the reference compound nutlin-3a (IC50 = 15 µM). In particular, compound 10a (IC50 = 10 µM) resulted as both the most soluble and active among the previous and present TSPs. The biological investigation evidenced that the anticancer activity is related to the activation of apoptotic cell-death pathways, supporting our rational design based on the ability of TSPs to interfere with PPI involved in the cell cycle regulation of cancer cells and, in particular, the p53 pathway. A reinvestigation of the TSP pharmacophore by using DFT calculations showed that the three aromatic substituents on the pyrrole core are able to mimic the hydrophobic side chains of the hot-spot residues of parallel and antiparallel coiled coil structures suggesting a possible molecular mechanism of action. A structure-activity relationship (SAR) analysis which includes solubility studies allows us to rationalize the role of the different substituents on the pyrrole core.

Mitochondria as playmakers of apoptosis, autophagy and senescence.

Mitochondria are the key energy-producing organelles and cellular source of reactive species. They are responsible for managing cell life and death by a balanced homeostasis passing through a network of structures, regulated principally via fission and fusion. Herein we discuss about the most advanced findings considering mitochondria as dynamic biophysical systems playing compelling roles in the regulation of energy metabolism in both physiologic and pathologic processes controlling cell death and survival. Precisely, we focus on the mitochondrial commitment to the onset, maintenance and counteraction of apoptosis, autophagy and senescence in the bioenergetic reprogramming of cancer cells. In this context, looking for a pharmacological manipulation of cell death processes as a successful route for future targeted therapies, there is major biotechnological challenge in underlining the location, function and molecular mechanism of mitochondrial proteins. Based on the critical role of mitochondrial functions for cellular health, a better knowledge of the main molecular players in mitochondria disfunction could be decisive for the therapeutical control of degenerative diseases, including cancer.

Introducing structure-based three-dimensional pharmacophore models for accelerating the discovery of selective BRD9 binders.

A well-structured in silico workflow is here reported for disclosing structure-based pharmacophore models against bromodomain-containing protein 9 (BRD9), accelerating virtual screening campaigns and facilitating the identification of novel binders. Specifically, starting from 23 known ligands co-crystallized with BRD9, three-dimensional pharmacophore models, namely placed in a reference protein structure, were developed. Specifically, we here introduce a fragment-related pharmacophore model, useful for the identification of new promising small chemical probes targeting the protein region responsible of the acetyllysine recognition, and two further pharmacophore models useful for the selection of compounds featuring drug-like properties. A pharmacophore-driven virtual screening campaign was then performed to facilitate the selection of new selective BRD9 ligands, starting from a large library of commercially available molecules. The identification of a promising BRD9 binder (7) prompted us to re-iterate this computational workflow on a second focused in-house built library of synthesizable compounds and, eventually, three further novel BRD9 binders were disclosed (8-10). Moreover, all these compounds were tested among a panel comprising other nine bromodomains, showing a high selectivity for BRD9. Preclinical bioscreens for potential anticancer activity highlighted compound 7 as that showing the most promising biological effects, proving the reliability of this in silico pipeline and confirming the applicability of the here introduced structure-based three-dimensional (3D) pharmacophore models as straightforward tools for the selection of new BRD9 ligands.

Non-coding RNAs as a new dawn in tumor diagnosis.

The current knowledge about non-coding RNAs (ncRNAs) as important regulators of gene expression in both physiological and pathological conditions, has been the main engine for the design of innovative platforms to finalize the pharmacological application of ncRNAs as either therapeutic tools or as molecular biomarkers in cancer. Biochemical alterations of cancer cells are, in fact, largely supported by ncRNA disregulation in the tumor site, which, in turn, reflects the cancer-associated specific modification of circulating ncRNA expression pattern. The aim of this review is to describe the state of the art of pre-clinical and clinical studies that analyze the involvement of miRNAs and lncRNAs in cancer-related processes, such as proliferation, invasion and metastases, giving emphasis to their functional role. A central node of our work has been also the examination of advantages and criticisms correlated with the clinical use of ncRNAs, taking into account the pressing need to refine the profiling methods aimed at identify novel diagnostic and prognostic markers and the request to optimize the delivery of such nucleic acids for a therapeutic use in an imminent future.

IL-17A neutralizing antibody regulates monosodium urate crystal-induced gouty inflammation.

Gout is a paradigm of acute, self-limiting inflammation caused by the deposition of monosodium urate (MSU) crystals within intra-and/or peri-articular areas, leading to excruciating pain, joint swelling and stiffness. The infiltration of leukocytes drives the inflammatory response and remains an attractive target for therapeutic intervention. In this context, emerging evidence supports the view that systemic differentiation of Th17 cells and their in situ infiltration as one of the potential mechanisms by which these cells, and their main product IL-17, causes damage to target tissues. To test if IL-17 was having a detrimental role in gouty onset and progression we targeted this cytokine, using a neutralizing antibody strategy, in an experimental model of gout. Joint inflammation was induced in CD-1 mice by the intra-articular (i.a.) administration of MSU crystals (200 µg/20 µl). Animals from IL-17Ab-treated groups received 1, 3 and 10 µg (i.a.) in 20 µl of neutralizing antibody after MSU crystals administration. Thereafter, joints were scored macroscopically, and knee joint oedema determined with a caliper. Histological analysis, myeloperoxidase assay and western blots analysis for COX-2/mPGEs-1/IL-17R pathway were conducted at 18 h (peak of inflammation) to evaluate leukocytes infiltration and activation, followed by the analysis, in situ, of pro/anti-inflammatory cytokines and chemokines. Flow cytometry was also used to evaluate the modulation of infiltrated inflammatory monocytes and systemic Th17 and Treg profile. Treatment with IL-17Ab revealed a dose-dependent reduction of joint inflammation scores with maximal inhibition at 10 µg. The neutralizing antibody was also able to significantly reduce leukocytes infiltration and MPO activity as well the expression of JE, IL-1α, IL-1ß, IL-16, IL-17, C5a, BLC and, with a less extent IP-10, Rantes, KC, TIMP-1, SDF-1 and metalloproteinases in inflamed tissues. Biochemical analysis also revealed that IL-17Ab treatment modulated COX-2/mPGEs-1 pathway (and related PGE2 production) without interfering with IL-17R expression. Furthermore, flow cytometry analysis highlighted a selective modulation of infiltrating inflammatory monocytes (B220-/GR1hi-F480hi/CD115+) and circulating Th17, but not Treg, cells after IL-17Ab treatment. Collectively the results of this study report for the first time, that i.a. injection of MSU crystals stimulates in vivo production of Th17 cells and Th17-related inflammatory cyto-chemokines. In addition, we have demonstrated that the administration of a neutralizing antibody against IL-17 attenuates joint symptoms, swelling and leukocytes infiltration to the inflamed tissue, possibly providing a new strategy for the treatment of gouty inflammation and/or arthritis.

Down regulation of pro-inflammatory pathways by tanshinone IIA and cryptotanshinone in a non-genetic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a common form of dementia mainly characterized by the deposition of neurofibrillary tangles and ß-amyloid (Aß) peptides in the brain. Additionally, increasing evidence demonstrates that a neuro-inflammatory state plays a key role in the development of this disease. Beside synthetic drugs, the use of natural compounds represents an alternative for the development of new potential drugs for the treatment of AD. Among these, the root of Salvia miltiorhiza Bunge (also known as Danshen) used for the treatment of cardiovascular, cerebrovascular disease and CNS functional decline in Chinese traditional medicine is one of the most representative examples. We therefore evaluated the effects of tanshinone IIA (TIIA) and cryptotanshinone (CRY) (the two major lipophilic compounds of Danshen) in a non-genetic mouse model of ß-amyloid (Aß)-induced AD, which is mainly characterized by reactive gliosis and neuro-inflammation in the brain. To this aim, mice were injected intracerebroventricularly (i.c.v.) with Aß1-42 peptide (3µg/3µl) and after with TIIA and CRY (1, 3, or 10mg/kg) intraperitoneally (i.p.) 3 times weekly for 21days following the induction of experimental AD. Spatial working memory was assessed as a measure of short-term memory in mice, whereas the level of GFAP, S100ß, COX-2, iNOS and NF-kBp65 monitored by western blot and ELISA assay, were selected as markers of reactive gliosis and neuro-inflammation. Finally, by docking studies, the modulation of key pro-inflammatory enzymes and pathways involved in the AD-related neuro-inflammation were also investigated. Results indicate that TIIA and CRY alleviate memory decline in Aß1-42-injected mice, in a dose dependent manner. Moreover, the analysis of gliosis-related and neuro-inflammatory markers in the hippocampal tissues reveal a remarkable reduction in the expression of GFAP, S100ß, COX-2, iNOS and NF-kBp65 after CRY (10mg/kg) treatment. These effects were less evident, but still significant, after TIIA (10mg/kg). Finally, in silico analysis also revealed that both compounds were able to interact with the binding sites of NF-kBp65 endorsing the data from biochemical analysis. We conclude that TIIA and CRY display anti-inflammatory and neuroprotective effect in a non-genetic mouse model of AD, thus playing a role in slowing down the course and onset of AD.

2,3,7,8-tetrachlorodibenzo-p-dioxin and the viral infection.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a widespread highly toxic environmental contaminant, suppresses immune response and leads to an increased susceptibility to infectious agents. In particular, several studies have provided evidence that TCDD decreases resistance to numerous viruses. Indeed, in vivo and in vitro investigations showed that the presence of TCDD is able to interfere with the replication of both human and animal viruses, such as influenza A viruses, coxsackie virus B3, immunodeficiency virus type-1 (HIV-1), cytomegalovirus (CMV), herpes simplex II, and bovine herpesvirus 1. Moreover, TCDD could induce an exacerbation of latent infection produced by HIV-1, CMV or Epstein-Barr virus. In this review, we first describe the general effects of TCDD exposure on mammalian cells, then we focus on its influence on the viral infections. Overall, the available data support the concept that TCDD exposure may act as an additional risk factor in promoting of viral diseases.

Site-specific replacement of the thymine methyl group by fluorine in thrombin binding aptamer significantly improves structural stability and anticoagulant activity.

Here we report investigations, based on circular dichroism, nuclear magnetic resonance spectroscopy, molecular modelling, differential scanning calorimetry and prothrombin time assay, on analogues of the thrombin binding aptamer (TBA) in which individual thymidines were replaced by 5-fluoro-2'-deoxyuridine residues. The whole of the data clearly indicate that all derivatives are able to fold in a G-quadruplex structure very similar to the 'chair-like' conformation typical of the TBA. However, only ODNs TBA-F4: and TBA-F13: have shown a remarkable improvement both in the melting temperature (ΔTm ≈ +10) and in the anticoagulant activity in comparison with the original TBA. These findings are unusual, particularly considering previously reported studies in which modifications of T4 and T13 residues in TBA sequence have clearly proven to be always detrimental for the structural stability and biological activity of the aptamer. Our results strongly suggest the possibility to enhance TBA properties through tiny straightforward modifications.

Cysteine Prevents the Reduction in Keratin Synthesis Induced by Iron Deficiency in Human Keratinocytes.

L-cysteine is currently recognized as a conditionally essential sulphur amino acid. Besides contributing to many biological pathways, cysteine is a key component of the keratin protein by its ability to form disulfide bridges that confer strength and rigidity to the protein. In addition to cysteine, iron represents another critical factor in regulating keratins expression in epidermal tissues, as well as in hair follicle growth and maturation. By focusing on human keratinocytes, the aim of this study was to evaluate the effect of cysteine supplementation as nutraceutical on keratin biosynthesis, as well as to get an insight on the interplay of cysteine availability and cellular iron status in regulating keratins expression in vitro. Herein we demonstrate that cysteine promotes a significant up-regulation of keratins expression as a result of de novo protein synthesis, while the lack of iron impairs keratin expression. Interestingly, cysteine supplementation counteracts the adverse effect of iron deficiency on cellular keratin expression. This effect was likely mediated by the up-regulation of transferrin receptor and ferritin, the main cellular proteins involved in iron homeostasis, at last affecting the labile iron pool. In this manner, cysteine may also enhance the metabolic iron availability for DNA synthesis without creating a detrimental condition of iron overload. To the best of our knowledge, this is one of the first study in an in vitro keratinocyte model providing evidence that cysteine and iron cooperate for keratins expression, indicative of their central role in maintaining healthy epithelia.

Structure and Configuration of Phosphoeleganin, a Protein Tyrosine Phosphatase 1B Inhibitor from the Mediterranean Ascidian Sidnyum elegans.

A new phosphorylated polyketide, phosphoeleganin (1), has been isolated from the Mediterranean ascidian Sidnyum elegans. Its structure and configuration have been determined by extensive use of 2D NMR and microscale chemical degradation and/or derivatization. Phosphoeleganin (1) inhibited the protein tyrosine phosphatase 1B (PTP1B) activity.

Glucose deprivation promotes activation of mTOR signaling pathway and protein synthesis in rat skeletal muscle cells.

Signaling through mammalian target of rapamycin (mTOR) has been shown to play a central role in the regulation of skeletal muscle growth induced by a wide range of stimuli either mechanical or metabolic, such as growth factors and amino acids. Here, we demonstrate that mTOR and its downstream target, the ribosomal S6 kinase (p70(S6K)), are activated in L6 myocytes by a short-term glucose deprivation. Such response is specific of skeletal muscle and is likely responsible for the increased rate of protein synthesis and expression of the muscle-specific proteins during recovery from glucose deprivation. Nitric oxide and phosphatidylinositol-3-kinase (PI3K) are upstream positive regulators of mTOR since their pharmacological inhibition prevents the activation of p70(S6K) in response to glucose deprivation. We therefore propose a model of response to a brief period of glucose deprivation that may occur in skeletal muscle cells during resistance exercise and that may lead to protein accretion when blood flow recovers and all nutrients are again available.

Guanine-based amphiphiles: synthesis, ion transport properties and biological activity.

Novel amphiphilic guanine derivatives, here named Gua1 and Gua2, have been prepared through few, simple and efficient synthetic steps. In ion transport experiments through phospholipid bilayers, carried out to evaluate their ability to mediate H(+) transport, Gua2 showed high activity. When this compound was investigated for ion-selective transport activities, no major differences were observed in the behaviour with cations while, in the case of anions, selective activity was observed in the series I(-)>Br(-)>Cl(-)>F(-). The bioactivity of these guanine analogues has been evaluated on a panel of human tumour and non-tumour cell lines in preliminary in vitro cytotoxicity assays, showing a relevant antiproliferative profile for Gua2.

Expanding the potential of G-quadruplex structures: formation of a heterochiral TBA analogue.

In order to expand the potential applications of G-quadruplex structures, we explored the ability of heterochiral oligodeoxynucleotides based on the thrombin-binding aptamer (TBA) sequence to fold into similar complexes, with particular focus on their resistance in biological environments. A combination of CD and NMR techniques was used. Similarly to TBA, the ODN ggTTggtgtggTTgg (lower case letters indicate L residues) is able to fold into a chair-like antiparallel G-quadruplex structure, but has a slightly higher thermal stability. The discovery that heterochiral ODNs are able to form stable G-quadruplex structures opens up new possibilities for their development in several fields, as aptamers, sensors and, as recently shown, as catalysts for enantioselective reactions.

Insight into Iron, Oxidative Stress and Ferroptosis: Therapy Targets for Approaching Anticancer Strategies.

Based on the multifaceted molecular machinery that tightly controls iron cellular homeostasis, this review delves into its paradoxical, potentially dangerous role in biological systems, with a special focus on double-edged sword correlations with cancer. Indeed, though iron is a vital micronutrient and a required cofactor participating in several essential cell functions, its tendency to cause oxidative stress can be related both to cancer risk and to the activation of cancer cell death pathways. In this scenario, ferroptosis refers to an iron-dependent form of regulated cell death (RCD) powered by an overload of lethal peroxides sharing distinctive oxidized phospholipid profiles. As a unique cell death pathway, ferroptosis is both morphologically and mechanistically different from other types of programmed cell death involving executioner family proteins. The accumulation of cytotoxic lipid peroxides encompasses a cellular antagonism between ferroptosis execution and defense systems, with iron-dependent death occurring when ferroptosis-promoting activities significantly exceed the cellular antioxidant defenses. The most recent molecular breakthroughs in the execution of ferroptosis have aroused great consideration in tumor biology, as targeting ferroptosis can provide new tools for exploring therapeutic strategies for tumor suppression. Mutations and death/survival pathway alterations, as well as distinctive metabolic regulations of cancer cells, including the propensity to generate ROS, are seen as features that can render cancer cells unprotected to ferroptosis, thereby exposing vulnerabilities which deserve further attention to be regarded as targetable for cancers with limited therapeutic options.

Tuning Peptide-Based Nanofibers for Achieving Selective Doxorubicin Delivery in Triple-Negative Breast Cancer.

Introduction: The design of delivery tools that efficiently transport drugs into cells remains a major challenge in drug development for most pathological conditions. Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer with poor prognosis and limited effective therapeutic options. Purpose: In TNBC treatment, chemotherapy remains the milestone, and doxorubicin (Dox) represents the first-line systemic treatment; however, its non-selective distribution causes a cascade of side effects. To address these problems, we developed a delivery platform based on the self-assembly of amphiphilic peptides carrying several moieties on their surfaces, aimed at targeting, enhancing penetration, and therapy. Methods: Through a single-step self-assembly process, we used amphiphilic peptides to obtain nanofibers decorated on their surfaces with the selected moieties. The surface of the nanofiber was decorated with a cell-penetrating peptide (gH625), an EGFR-targeting peptide (P22), and Dox bound to the cleavage sequence selectively recognized and cleaved by MMP-9 to obtain on-demand drug release. Detailed physicochemical and cellular analyses were performed. Results: The obtained nanofiber (NF-Dox) had a length of 250 nm and a diameter of 10 nm, and it was stable under dilution, ionic strength, and different pH environments. The biological results showed that the presence of gH625 favored the complete internalization of NF-Dox after 1h in MDA-MB 231 cells, mainly through a translocation mechanism. Interestingly, we observed the absence of toxicity of the carrier (NF) on both healthy cells such as HaCaT and TNBC cancer lines, while a similar antiproliferative effect was observed on TNBC cells after the treatment with the free-Dox at 50 µM and NF-Dox carrying 7.5 µM of Dox. Discussion: We envision that this platform is extremely versatile and can be used to efficiently carry and deliver diverse moieties. The knowledge acquired from this study will provide important guidelines for applications in basic research and biomedicine.

CL316,243, a selective ß3-adrenoceptor agonist, activates protein translation through mTOR/p70S6K signaling pathway in rat skeletal muscle cells.

Functional ß3-adrenoceptors have been found in skeletal muscle where they mediate metabolic oxidation and glucose utilization. Whether ß3-adrenoceptors (ARs) also play any role in muscle protein metabolism still remains uncertain. By using rat L6 myocyte cultures, we found that CL316,243, a ß3-AR selective agonist, at the concentration of 10(-6) M for 24 h, induced a significant increase of skeletal muscle constitutive proteins such as H- and L-myosin and ß-actin. Such effect was correlated to an increased expression of phosphorylated p70(S6K) that was significantly inhibited by ß3-AR antagonist, SR 59230A, but not by ß2-AR antagonist, ICI-118,551. The CL316,243-induced activation of p70(S6K) was markedly inhibited by wortmannin, a PI3K inhibitor, and rapamycin, a specific inhibitor of mTOR, suggesting a critical involvement of the PI3K-mTOR-p70(S6K) signaling cascade in the anabolic response of L6 cells to ß3-AR agonist. Taken together, these results suggest that stimulation of ß3-AR in skeletal muscle cells activates a specific signaling pathway leading to protein synthesis and, eventually, muscle growth.