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Industrial hemp is a multiuse crop whose phytocomplex includes terpenophenolics and flavonoids. In the present study, the phenolic and terpenophenolic compounds were assayed in the water extract of the hemp variety Futura 75. Protective effects were also investigated in human fibroblast and keratinocytes and isolate mouse skin specimens, which were exposed to hydrogen peroxide and/or to the extract (1&ndash, 500 &micro, g/mL). The results of phytochemical analysis suggested the cannabidiol, cannabidiolic acid and rutin as the prominent phytocompounds. In the in vitro system represented by human keratinocytes and fibroblasts, the hemp extract was found to be able to protect cells from cytotoxicity and apoptosis induced by oxidative stress. Moreover, modulatory effects on IL-6, a key mediator in skin proliferation, were found. In isolated rat skin, the extract reduced hydrogen peroxide-induced l-dopa turnover, prostaglandin-E2 production and the ratio kynurenine/tryptpophan, thus corroborating anti-inflammatory/antioxidant effects. The in silico docking studies also highlighted the putative interactions between cannabidiol, cannabidiolic acid and rutin with tyrosinase and indoleamine-2,3-dioxygenase, involved in l-dopa turnover and tryptophan conversion in kynurenine, respectively. In conclusion, the present findings showed the efficacy of hemp water extract as a skin protective agent. This could be partly related to the extract content in cannabidiol, cannabidiolic acid and rutin.

Tanacetum parthenium (feverfew) has traditionally been employed as a phytotherapeutic remedy in the treatment of migraine. In this study, a commercial T. parthenium water extract was investigated to explore its anti-inflammatory and neuromodulatory effects. Isolated mouse cortexes were exposed to a K+ 60 mM Krebs-Ringer buffer and treated with T. parthenium water extract. The prostaglandin E2 (PGE2) level, brain-derived neurotrophic factor (BDNF), interleukin-10 (IL-10), and IL-1&beta, gene expression were evaluated in the cortex. The effects on dopamine (DA) release and dopamine transporter (DAT) gene expression were assayed in hypothalamic HypoE22 cells. A bioinformatics analysis was conducted to further investigate the mechanism of action. The extract was effective in reducing cortex PGE2 release and IL-1&beta, gene expression. In the same experimental system, IL-10 and BDNF gene expressions increased, and in HypoE22 cells, the extract decreased the extracellular dopamine level and increased the DAT gene expression due to the direct interaction of parthenolide with the DAT. Overall, the present findings highlight the efficacy of T. parthenium water extract in controlling the inflammatory pathways that occur during cortical-spreading depression. Additionally, the inhibition of the hypothalamic DA release observed in this study further supports the role of dopaminergic pathways as key targets for novel pharmacological approaches in the management of migraine attacks.

Matrix metalloproteinases (MMPs) play a crucial role in tumor angiogenesis, and metastasis. 4&prime, geranyloxyferulic acid (GOFA) has anti-tumor and anti-inflammatory proprieties. Herein, we aimed to determine whether this compound affects cell survival, invasion, and migration through reactive oxygen species (ROS)-mediated MMPs activation of extracellular signal-regulated kinases (ERKs) and p38 signaling in lymphocytic histiocytoma (U937) and colorectal cancer (HCT116) cells. We observed that lipopolysaccharide (LPS) stimulated U937 and HCT116 cells presented abnormal cell proliferation and increased metalloproteinase (MMP-9) activity and expression. Non-cytotoxic doses of GOFA blunted matrix invasive potential by reducing LPS-induced MMP-9 expression and cell migration via inhibiting ROS/ ERK pathway. GOFA also attenuated apoptosis and cell senescence. Our findings indicate that GOFA, inhibiting cancer cell proliferation and migration, could be therapeutically beneficial to prevent tumor metastasis.

Titanium surface modification is critical for dental implant success. Our aim was to determine surfaces influence on dental pulp stem cells (DPSCs) viability and differentiation. Implants were divided into sandblasted/acid-etched (control) and sandblasted/acid-etched coated with calcium and magnesium ions (CaMg), supplied as composite (test). Proliferation was evaluated by MTT, differentiation checking osteoblastic gene expression, PGE2 secretion and matrix formation, inflammation by Interleukin 6 (IL-6) detection. MTT and IL-6 do not modify on test. A PGE2 increase on test is recorded. BMP2 is higher on test at early experimental points, Osterix and RUNX2 augment later. Alizarin-red S reveals higher matrix production on test. These results suggest that test surface is more osteoinductive, representing a start point for in vivo studies aiming at the construction of more biocompatible dental implants, whose integration and clinical performance are improved and some undesired effects, such as implant stability loss and further surgical procedures, are reduced.

Vitis vinifera (grape) contains various compounds with acknowledged phytochemical and pharmacological properties. Among the different parts of the plant, pomace is of particular interest as a winemaking industry by-product. A characterization of the water extract from grape pomace from Montepulciano d’Abruzzo variety (Villamagna doc) was conducted, and the bioactive phenolic compounds were quantified through HPLC-DAD-MS analysis. HypoE22, a hypothalamic cell line, was challenged with an oxidative stimulus and exposed to different concentrations (1 µg/mL−1 mg/mL) of the pomace extract for 24, 48, and 72 h. In the same conditions, cells were exposed to the sole catechin, in a concentration range (5–500 ng/mL) consistent with the catechin level in the extract. Cell proliferation was investigated by MTT assay, dopamine release through HPLC-EC method, PGE2 amount by an ELISA kit, and expressions of neurotrophin brain-derived neurotrophic factor (BDNF) and of cyclooxygenase-2 (COX-2) by RT-PCR. The extract reverted the cytotoxicity exerted by the oxidative stimulus at all the experimental times in a dose-dependent manner, whereas the catechin was able to revert the oxidative stress-induced depletion of dopamine 48 h and 72 h after the stimulus. The extract and the catechin were also effective in preventing the downregulation of BDNF and the concomitant upregulation of COX-2 gene expression. In accordance, PGE2 release was augmented by the oxidative stress conditions and reverted by the administration of the water extract from grace pomace and catechin, which were equally effective. These results suggest that the neuroprotection induced by the extract could be ascribed, albeit partially, to its catechin content.

Background: Cannabidiol (CBD) and cannabigerol (CBG) are non-psychotropic terpenophenols isolated from Cannabis sativa, which, besides their anti-inflammatory/antioxidant effects, are able to inhibit, the first, and to stimulate, the second, the appetite although there are no studies elucidating their role in the hypothalamic appetite-regulating network. Consequently, the aim of the present research is to investigate the role of CBD and CBG in regulating hypothalamic neuromodulators. Comparative evaluations between oxidative stress and food intake-modulating mediators were also performed. Methods: Rat hypothalamic Hypo-E22 cells and isolated tissues were exposed to either CBD or CBG, and the gene expressions of neuropeptide (NP)Y, pro-opiomelanocortin (POMC) and fatty acid amide hydrolase were assessed. In parallel, the influence of CBD on the synthesis and release of dopamine (DA), norepinephrine (NE), and serotonin (5-HT) was evaluated. The 3-hydroxykinurenine/kinurenic acid (3-HK/KA) ratio was also determined. Results: Both CBD and CBG inhibited NPY and POMC gene expression and decreased the 3-HK/KA ratio in the hypothalamus. The same compounds also reduced hypothalamic NE synthesis and DA release, whereas the sole CBD inhibited 5-HT synthesis. Conclusion: The CBD modulates hypothalamic neuromodulators consistently with its anorexigenic role, whereas the CBG effect on the same mediators suggests alternative mechanisms, possibly involving peripheral pathways.

In the present study, we investigated the water extract of Harpagophytum procumbens DC. ex Meisn. in an experimental model of inflammatory bowel diseases (IBDs). Additionally, a microbiological investigation was carried out to discriminate the efficacy against bacterial and fungal strains involved in IBDs. Finally, an untargeted proteomic analysis was conducted on more than one hundred colon proteins involved in tissue morphology and metabolism. The extract was effective in blunting the production of oxidative stress and inflammation, including serotonin, prostaglandins, cytokines, and transcription factors. Additionally, the extract inhibited the growth of Candida albicans and C. tropicalis. The extract was also able to exert a pro-homeostatic effect on the levels of a wide plethora of colon proteins, thus corroborating a protective effect. Conversely, the supraphysiological downregulation of cytoskeletal-related proteins involved in tissue morphology and antimicrobial barrier function suggests a warning in the use of food supplements containing H. procumbens extracts. © 2020 by the authors. Licensee MDPI, Basel, Switzerland., FAR 2019, Funding: This work was supported by grants from the Italian Ministry of University (FAR 2019 granted to Claudio Ferrante; FAR 2019 granted to Annalisa Chiavaroli; FAR 2019 granted to Luigi Menghini; FAR 2019 granted to Giustino Orlando; FAR 2014 granted to Lucia Recinella).

Pancreatic cancer (PC) is one of the most lethal, chemoresistant malignancies and it is of paramount importance to find more effective therapeutic agents. Repurposing of non-anticancer drugs may expand the repertoire of effective molecules. Studies on repurposing of benzimidazole-based anthelmintics in PC and on their interaction with agents approved for PC therapy are lacking. We analyzed the effects of four Food and Drug Administration (FDA)-approved benzimidazoles on AsPC-1 and Capan-2 pancreatic cancer cell line viability. Notably, parbendazole was the most potent benzimidazole affecting PC cell viability, with half maximal inhibitory concentration (IC50) values in the nanomolar range. The drug markedly inhibited proliferation, clonogenicity and migration of PC cell lines through mechanisms involving alteration of microtubule organization and formation of irregular mitotic spindles. Moreover, parbendazole interfered with cell cycle progression promoting G2/M arrest, followed by the emergence of enlarged, polyploid cells. These abnormalities, suggesting a mitotic catastrophe, culminated in PC cell apoptosis, are also associated with DNA damage in PC cell lines. Remarkably, combinations of parbendazole with gemcitabine, a drug employed as first-line treatment in PC, synergistically decreased PC cell viability. In conclusion, this is the first study providing evidence that parbendazole as a single agent, or in combination with gemcitabine, is a repurposing candidate in the currently dismal PC therapy.

Cannabidiol (CBD) and cannabigerol (CBG) are Cannabis sativa terpenophenols. Although CBD&rsquo, s effectiveness against neurological diseases has already been demonstrated, nothing is known about CBG. Therefore, a comparison of the effects of these compounds was performed in two experimental models mimicking the oxidative stress and neurotoxicity occurring in neurological diseases. Rat astrocytes were exposed to hydrogen peroxide and cell viability, reactive oxygen species production and apoptosis occurrence were investigated. Cortexes were exposed to K+ 60 mM depolarizing stimulus and serotonin (5-HT) turnover, 3-hydroxykinurenine and kynurenic acid levels were measured. A proteomic analysis and bioinformatics and docking studies were performed. Both compounds exerted antioxidant effects in astrocytes and restored the cortex level of 5-HT depleted by neurotoxic stimuli, whereas sole CBD restored the basal levels of 3-hydroxykinurenine and kynurenic acid. CBG was less effective than CBD in restoring the levels of proteins involved in neurotransmitter exocytosis. Docking analyses predicted the inhibitory effects of these compounds towards the neurokinin B receptor. Conclusion: The results in the in vitro system suggest brain non-neuronal cells as a target in the treatment of oxidative conditions, whereas findings in the ex vivo system and docking analyses imply the potential roles of CBD and CBG as neuroprotective agents.

Aims The worldwide increase in aging population is prevalently associated with the increase of neurodegenerative diseases. Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-modulated transcriptional factors which belong to the nuclear hormone receptor superfamily which regulates peroxisome proliferation. The PPAR-γ is the most extensively studied among the three isoforms and the neuroprotective effects of PPAR-γ agonists have been recently demonstrated in a variety of preclinical models of neurological disorders. The aim of the study is to biologically evaluate the neuroprotective effects of new PPAR-γ selective agonists in an in vitro model. Main methods CTX-TNA2 rat astrocytes were treated with G3335, a PPAR-γ antagonist, to simulate the conditions of a neurological disorder. Newly synthetized PPAR-γ selective agonists were added to the cell culture. Cytotoxicity was assessed by MTT assay, catalase activity was investigated by a colorimetric assay, Reactive Oxygen Species (ROS) production and apoptosis occurrence were measured by flow cytometry. Western blotting were performed to measure the levels of protein involved in the apoptotic pathway. Key findings Four PPAR-γ agonists were selected. Among them, the GL516, a fibrate derivative, showed low cytotoxicity and proved effective in restoring the catalase activity, reducing ROS production and decreasing the apoptosis occurrence triggered by the G3335 administration. The effects of this molecule appear to be comparable to the reference compound rosiglitazone, a potent and selective PPAR-γ agonist, mainly at prolonged exposure times (96 h). Significance Based on recent evidence, hypofunctionality of the PPAR-γ in glial cells could be present in neurodegenerative diseases and could participate in pathological mechanisms through peroxisomal damage. The fibrate derivative PPAR-γ agonist GL516 emerged as the most promising molecule of the series and could have a role in preventing the pathophysiology of neurodegenerative disorders.

The aim of the present study was to investigate the role of Cyclic-nucleotide Response Element-Binding (CREB) family members and related nuclear transcription factors in the radiation response of human B lymphoma cell lines (Daudi and Ramos). Unlike the more radiosensitive Daudi cells, Ramos cells demonstrated only a moderate increase in early apoptosis after 3-5 Gy irradiation doses, which was detected with Annexin V/PI staining. Moreover, a significant and dose-dependent G2/M phase accumulation was observed in the same cell line at 24 h after both ionizing radiation (IR) doses. Western blot analysis showed an early increase in CREB protein expression that was still present at 3 h and more evident after 3 Gy IR in Ramos cells, along with the dose-dependent upregulation of p53 and NF-κB. These findings were consistent with real-time RT-PCR analysis that showed an early- and dose-dependent upregulation of NFKB1, IKBKB and XIAP gene expression. Unexpectedly, pre-treatment with SN50 did not increase cell death, but cell viability. Taken together, these findings let us hypothesise that the early induction and activation of NF-κB1 in Ramos cells could mediate necrotic cell death and be linked to other molecules belonging to CREB family and involved in the cell cycle regulation.

Paragangliomas (PGLs) are infiltrating autonomic nervous system tumors that cause important morbidity. At present, surgery is the only effective therapeutic option for this rare tumor. Thus, new agents for PGL treatment should be identified. Using unique PGL cell models established in our laboratory, we evaluated the effect of dichloroacetate (DCA) as single agent or in a novel combination with other metabolic drugs, including GW6471 and metformin. DCA and metformin had not been tested before in PGL. DCA reduced PGL cell viability and growth through mechanisms involving reactivation of PDH complex leading to promotion of oxidative metabolism, with lowering of lactate and enhanced ROS production. This resulted in cell cycle inhibition and induction of apoptosis in PGL cells, as shown by flow cytometry and immunoblot analyses. Moreover, DCA drastically impaired clonogenic activity and migration of PGL cells. Also metformin reduced PGL cell viability as single agent and the combinations of DCA, GW6471 and metformin had strong effects on cell viability. Furthermore, combined treatments had drastic and synergistic effects on clonogenic ability. In conclusion, DCA, GW6471 and metformin as single agents and in combination appear to have promising antitumor effects in unique cell models of PGL.

The extracellular matrix (ECM) is the non-cellular component of any tissues and organs. It provides not only support, tensile strength, and scaffolding for tissues and cells, but also biochemical signals and specialized proteins. The destabilization or alteration of the ECM structural and chemical composition affects growth, morphogenesis, differentiation, migration, communication, survival of all cells as well as inflammation and immune response. Inflammation is a complex defense mechanism characterized by leukocyte migration from the vasculature to control tissue damage induced by pathogenic (bacterial or viral), traumatic, or toxic injury with subsequent deposition of extracellular matrix resulting in tissue repair. At sites of injury, phagocytic cells, namely macrophages and neutrophils, provide innate cell-mediated immunity, and immune cells are influenced in their migration by the topography and composition of the matrix architecture. The physical and biochemical ECM properties are also able to modulate a number of processes in immune cells, especially lymphocytes that can ultimately lead to inefficient immune response. Among the large number of molecules responsible for ECM homeostasis, matrix metalloproteinases, versican, hyaluronan, and thrombospondins are the most involved in inflammation and immunity.

In a previous work we reported the antiproliferative effects of (±)-MRJF4, a novel haloperidol metabolite II (HP-mII) (a sigma-1 antagonist and sigma-2 agonist) prodrug, obtained through conjugation to 4-phenylbutyric acid (PhBA) [a histone deacetylase inhibitor (HDACi)] via an ester bond. As a continuation of this work, here we report the asymmetric synthesis of compounds (R)-(+)-MRJF4 and (S)-(-)-MRJF4 and the evaluation of their biological activity on rat C6 glioma cells, derived from glioblastoma multiforme (GBM), which is the most common and deadliest central nervous system (CNS) invasive malignancy. Favourable physicochemical properties, high permeability in the parallel artificial membrane permeability assay (PAMPA), good enzymatic and chemical stability, in vivo anticancer activity, associated with the capacity to reduce cell viability and to increase cell death by apoptosis, render compound (R)-(+)-MRJF4 a promising candidate for the development of a useful therapeutic for gliomas therapy.

Many smokers have recently turned to electronic cigarettes (e-cigarettes) because they have been marketed as a cheaper, safer smokeless alternative to traditional cigarettes and a possible smoking cessation tool. Although the safety of these electronic devices is still not fully known, there is evidence of their cytotoxicity on cells belonging to the oral cavity. In a previous study by the authors, the increase of reactive oxygen production and Bax expression, followed by the occurrence of apoptosis, was demonstrated in human gingival fibroblasts (HGFs). The aim of this paper is to further investigate the effects of the e-cigarette liquids (with and without nicotine) on the same experimental model.HGFs were treated with e-cigarette fluids containing nicotine (final concentration 1 mg/mL) and the equivalent volume of a fluid without nicotine, for periods ≤48 hours. Lactate dehydrogenase assay (LDH), electronic microscopy analysis, collagen I production, flow cytometry lysosome compartment evaluation, and western blotting light chain 3 (microtubule-associated protein 1A/1B-LC3) expression were performed.Fluids containing nicotine exerted cytotoxicity as demonstrated by increased levels of LDH, in parallel to the presence of numerous vacuoles in the cytoplasm, a decrease in collagen I production, and augmented LC3 II expression. Autophagic vesicles and more procollagen I molecules were present in the cytoplasm of fibroblasts exposed to nicotine-free fluids. In the same samples, time-dependent activation of the lysosomal compartment with no changes in LC3 expression was detected.E-cigarette fluids (with and without nicotine) trigger molecular and morphologic responses in oral fibroblasts, raising concerns about their role in the pathogenesis of oral diseases.

This study aimed to check the effect of zoledronic acid (ZA) at subtoxic dose on human osteoblasts (HOs) in terms of cell viability, apoptosis occurrence, and differentiation induction. ZA belongs to the family of bisphosphonates (BPs), largely used in the clinical practice for the treatment of bone diseases, often associated with jaw osteonecrosis onset. Their pharmacological action consists in the direct block of the osteoclast-mediated bone resorption along with indirect action on osteoblasts. HOs were treated choosing the highest limit concentration (10−5 M) which does not induce toxic effects. Live/dead staining, flow cytometry, mitochondrial membrane potential assay, osteocalcin western blotting, gp38 RT-PCR, collagen type I, PGE2, and IL-6 ELISA assays were performed. Similar viability level between control and ZA-treated samples is found along with no significant increase of apoptotic and necrotic cells in ZA-treated sample. To establish if an early apoptotic pathway was triggered, Bax expression and mitochondrial membrane potential were evaluated finding a higher protein expression in control sample and a good integrity of mitochondrial membrane in both experimental points. Type I collagen secretion and alkaline phosphatase (ALP) activity appear increased in ZA-treated sample, osteocalcin expression level is reduced in ZA-treated cells, whereas no modifications of gp38 mRNA level are evidenced. No statistical differences are identified in PGE2 secretion level whereas IL-6 secretion is lower in ZA-treated HOs with respect to control ones. These results highlight that ZA, delaying the osteoblastic differentiation process versus the osteocytic lineage, strengthens its pharmacological activity enhancing bone density. The knowledge of ZA effects on osteoblasts at subtoxic dose allows to improve therapeutic protocols in order to strengthen drug pharmacological activity through a combined action on both osteoclastic and osteoblastic cells.

Migraine is one of the most common neurological disorder, which has long been related to brain serotonin (5-HT) depletion and neuro-inflammation. Despite many treatment options are available, the frequent occurrence of unacceptable adverse effects further supports the research toward nutraceuticals and herbal preparations, among which Tanacetum parthenium and Salix alba showed promising anti-inflammatory and neuro-modulatory activities. The impact of extract treatment on astrocyte viability, spontaneous migration and apoptosis was evaluated. Anti-inflammatory/anti-oxidant effects were investigated on isolated rat cortexes exposed to a neurotoxic stimulus. The lactate dehydrogenase (LDH) release, nitrite levels and 5-HT turnover were evaluated, as well. A proteomic analysis was focused on specific neuronal proteins and a fingerprint analysis was carried out on selected phenolic compounds. Both extracts appeared able to exert in vitro anti-oxidant and anti-apoptotic effects. S. alba and T. parthenium extracts reduced LDH release, nitrite levels and 5-HT turnover induced by neurotoxic stimulus. The downregulation of selected proteins suggest a neurotoxicity, which could be ascribed to an elevated content of gallic acid in both S. alba and T. parthenium extracts. Concluding, both extracts exert neuroprotective effects, although the downregulation of key proteins involved in neuron physiology suggest caution in their use as food supplements.

H9c2 are rat heart embryonic myoblasts, with skeletal muscle properties, which terminally differentiate by fusing and forming multinucleated myotubes. Here we investigated the possible involvement of Protein Kinases C (PKCs) in H9c2 cell differentiation and explored the interplay of these enzymes both with reactive oxygen species (ROS), upstream physiological mediators of cell differentiation, and with nitric oxide (NO), downstream target of PKC activation, known for being involved in apoptosis induction in differentiated myoblasts. Cells were induced to differentiate (6 days) under low serum culture conditions and assayed for the expression of cell cycle (cyclin A) and differentiation markers (morphology and myogenin). Both ROS and in vivo production of NO were found increased after 6 days of differentiation, when the activation of PKC-δ isoform was 14-fold increased compared with the undifferentiated control cells. The parallel analysis of apoptotic features demonstrated a small increase in Annexin-V+ cells and a concomitant increase in PARP cleavage and Bax expression. Interestingly, a reduced percentage of differentiated cells was obtained both in the presence of Rottlerin, a highly selective PKC-δ pharmacologic inhibitor, and, moreover, with the use of PKC-δ siRNA technology, further supporting the involvement of PKC-δ in switching on the events related to skeletal muscle myoblast differentiation.

stract Silver-based products have been proven to be effective in retarding and preventing bacterial growth since ancient times. In the field of restorative dentistry, the use of silver ions/nanoparticles has been explored to counteract bacterial infections, as silver can destroy bacterial cell walls by reacting with membrane proteins. However, it is also cytotoxic towards eukaryotic cells, which are capable of internalizing nanoparticles. In this work, we investigated the biological effects of Chitlac-nAg, a colloidal system based on a modified chitosan (Chitlac), administered for 24-48 h to a co-culture of primary human gingival fibroblasts and Streptococcus mitis in the presence of saliva, developed to mimic the microenvironment of the oral cavity. We sought to determine its efficiency to combat oral hygiene-related diseases without affecting eukaryotic cells. Cytotoxicity, reactive oxygen species production, apoptosis induction, nanoparticles uptake, and lysosome and autophagosome metabolism were evaluated. In vitro results show that Chitlac-nAg does not exert cytotoxic effects on human gingival fibroblasts, which seem to survive through a homoeostasis mechanism involving autophagy. That suggests that the novel biomaterial Chitlac-nAg could be a promising tool in the field of dentistry.

Hyaluronic Acid (HA) has been already approved by Food and Drug Administration (FDA) for osteoarthritis (OA), while its use in the treatment of tendinopathy is still debated. The aim of this study was to evaluate in human rotator cuff tendon derived cells the effects of four different HA on cell viability, proliferation, apoptosis and the expression of collagen type I and collagen type III.An in vitro model was developed on human tendon derived cells from rotator cuff tears to study the effects of four different HA preparations (Ps) (sodium hyaluronate MW: 500-730 KDa - Hyalgan®, 1000 kDa Artrosulfur HA®, 1600 KDa Hyalubrix® and 2200 KDa Synolis-VA®) at various concentrations. Tendon derived cells morphology were evaluated after 0, 7 and 14 d of culture. Viability, proliferation, apoptosis were evaluated after 0, 24 and 48 h of culture. The expression and deposition of collagen type I and collagen type III were evaluated after 1, 7 and 14 d of culture.All HAPs tested increased viability and proliferation, in dose dependent manner. HAPs already reduce apoptosis at 24 h compared to control cells (without HAPs). Furthermore, HAPs stimulated the synthesis of collagen type I in a dose dependent fashion over 14 d, without increase in collagen type III; moreover, in the presence of Synolis-VA® the expression and deposition of collagen type I was significantly higher as compare with the other HAPs.HAPs enhanced viability, proliferation and expression of collagen type I in tendon derived cells.

Background Hyaluronic Acid (HA) has been already approved by Food and Drug Administration (FDA) for osteoarthritis (OA), while its use in the treatment of tendinopathy is still debated. The aim of this study was to evaluate in human rotator cuff tendon derived cells the effects of four different HA on cell viability, proliferation, apoptosis and the expression of collagen type I and collagen type III. Methods An in vitro model was developed on human tendon derived cells from rotator cuff tears to study the effects of four different HA preparations (Ps) (sodium hyaluronate MW: 500-730 KDa - Hyalgan®, 1000 kDa Artrosulfur HA®, 1600 KDa Hyalubrix® and 2200 KDa Synolis-VA®) at various concentrations. Tendon derived cells morphology were evaluated after 0, 7 and 14 d of culture. Viability, proliferation, apoptosis were evaluated after 0, 24 and 48 h of culture. The expression and deposition of collagen type I and collagen type III were evaluated after 1, 7 and 14 d of culture. Results All HAPs tested increased viability and proliferation, in dose dependent manner. HAPs already reduce apoptosis at 24 h compared to control cells (without HAPs). Furthermore, HAPs stimulated the synthesis of collagen type I in a dose dependent fashion over 14 d, without increase in collagen type III; moreover, in the presence of Synolis-VA® the expression and deposition of collagen type I was significantly higher as compare with the other HAPs. Conclusions HAPs enhanced viability, proliferation and expression of collagen type I in tendon derived cells.

Anticancer therapy addresses the destruction of tumour cells which try to counteract the effect of drugs and/or ionising radiation. Thus the knowledge of the threshold over which the cells do not resist such agents could help in the setting up of therapy protocols. Since a key role was assigned to Cyclic nucleotide Response Element Binding protein (CREB) multigenic family (which is composed of several nuclear transcription factors involved in c-AMP signalling in cell differentiation, proliferation, apoptosis, survival and adaptive response and in hematopoiesis and acute leukemias), attention was paid to the activation of Erk cascade and of the downstream kinases and transcription factors such as p90RSK and CREB. K562 erythroleukemia cell survival to 1.5 Gy ionising radiation with or without etoposide treatment seemed to involve Erk phosphorylation which, regulating p90 RSK, should activate CREB. In parallel, p38 MAP kinase activity down-modulation, along with low caspase-3 activity, and no modification of Bax and Bcl2 levels, supported such evidence. Thus, endogenous CREB activation, triggering a potent survival signal in K562 cells exposed to 1.5 Gy with or without etoposide, led us to suggest that using specific inhibitors against CREB, such as modified phosphorothionate oligodeoxynucleotides (ODN) corresponding to CREB-1 sequence, anticancer therapy efficacy could be improved.

The current status of immunotherapy for cancer is here summarized with particular attention to the new methodologies developed for ex vivo expansion of T cells from neonatal cord blood. Umbilical cord blood (UCB) mononuclear cells (MNC) generate CD45RA naive T lymphocytes when cultured under serum-deprived conditions with appropriate combinations of growth factors. These ex vivo generated T cells resemble precursors for the lymphoid lineage present in adult bone marrow in terms of active transcription of RAG-2 and pTalpha.

A novel cyclic prodrug of S-allyl-glutathione (CP11), obtained by using an acyloxy-alkoxy linker, was estimated for its pharmacokinetic and biological properties. The stability of CP11 was evaluated at pH 1.2, 7.4, in simulated fluids with different concentrations of enzymes, and in human plasma. The anti-inflammatory ability of CP11 was assessed in U937 cells, an immortalized human monocyte cell line. Results showed that CP11 is stable at acidic pH showing a possible advantage for oral delivery due to the longer permanence in the stomach. Having a permeability coefficient of 2.49 × 10(-6) cm s(-1), it was classified as discrete BBB-permeable compound. Biological studies revealed that CP11 is able to modulate inflammation mediated by LPS in U937 cells preventing the increase of ROS intracellular levels through interaction with the MAPK pathway.

Electronic cigarettes (e-cigarettes) are generally acknowledged as a safer alternative to the use of combusted tobacco products. Nevertheless, there are increasing conflicting claims concerning the effect of these novel industrial products on the health of e-cigarettes users. The aim of this work was to investigate the effects of the liquids of e-cigarettes on human gingival fibroblasts (HGFs) and to compare the effects of nicotine-containing fluid to the fluid itself. HGFs were treated with different concentrations (0–5 mg/mL) of fluids of e-cigarettes for different times (0–72 h) and cytotoxicity was analyzed by MTT assay. Fluids were administered also after being vaped (e.g., warmed into the cartomizer). Apoptosis occurrence and Bax expression were evaluated by flow cytometry; ROS production was analyzed by fluorescence optical microscopy. Both nicotine-containing and nicotine-free fluids induced an increased ROS production after 24 h, along with an increased Bax expression, followed by apoptosis occurrence after 48 h of exposure. The cytotoxicity exerted on HGFs by e-cigarettes fluids is not entirely ascribable to nicotine. Since the e-cigarettes are advertised as a safer alternative to traditional ones, especially for the possibility of “smoking” nicotine-free fluids, further studies are necessary to clarify the mechanism involved in the occurrence of cytotoxicity exerted by such compounds. Our results suggest a role for e-cigarette fluids in the pathogenesis of oral diseases, such as periodontitis.

Zoledronic acid (ZA) belongs to bisphosphonates (BPs), drugs administered to treat resorptive bone diseases. Although ZA is largely used in the clinical practice, significant adverse effects of ZA, such as osteonecrosis of the jaw (ONJ), were recorded. The aim of this work was to evaluate the role of Nitric Oxide (NO) in the in vitro response of Human Gingival Fibroblasts (HGFs) to 1, 5, 10 and 100μM ZA. HGFs morphology was evaluated through phase contrast microscopy and live/ dead staining; MTT and ELISA assays were applied to measure cell viability, Collagen Type I and IL6 secretion. ROS production and mitochondrial membrane potential were evaluated by flow cytometry; NO production and NOS activity by spectrophotometric analysis; eNOS and nNOS expression by fluorescence microscopy. Viable fibroblasts are evidenced in control sample while floating dead cells and cells close to detachment phase in ZA treated sample along with decreased level of Collagen Type I. Control sample shows higher number of viable cells respect to ZA treated one and ROS production increases when ZA is added. Released NO in ZA treated sample appears higher and NO overproduction is related to increased nNOS activity. IL 6 secretion level is higher in ZA treated sample than in control one. Our results suggest ROS involvement in NO overproduction, due to nNOS recruitment, both at low and high doses. In turn, NO release seems to be able to trigger the inflammatory response only when high doses are administered., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

The high number (>10(8-10)) of primary human pro-erythroblasts (CD36high/CD235alow) obtainable in HEMA culture (Migliaccio et al., 2002) is exploited here to analyse the expression of proteins implicated in erythropoietin (EPO)-signalling (STATs, PI-3K, and PLCs) during the process of erythroid maturation. Human pro-erythroblasts progressed in 4 days of culture with EPO into basophilic- (CD36high/CD235amedium, 24 h), polychromatic-(CD36high/CD235ahigh, 48 h), and, finally, orthochromatic-(CD36low/CD235ahigh, 72-96 h) erythroblasts. During this maturation, STAT-1 was expressed up to the orthochromatic stage, expression of STAT-5, as well as of its target proteins BclxL and IRF1, remained constant up to 48 h (polychromatic-erythroblasts) but decreased by 96 h (orthochromatic-erythroblasts), while that of STAT-3 decreased constantly from 24 h on and became undetectable by 96 h. Expression of PI-3K rapidly decreased with differentiation since only 50% of original protein levels were detected by 48 h. On the other hand, among the members of PLC families investigated, PLC beta4 was not expressed, PLC beta2, delta1, and gamma2 were expressed at constant levels throughout the maturation process, while expression of PLC beta3 and of PLC gamma1 decreased, as PI-3K, by 24 h and that of PLC beta1 was induced by 6 h and became undetectable by 24 h. In conclusion, these data depict the dynamic signalling scenario associated with the maturation of erythroid cells and provide the first indication that members of PLC families (PLC beta1, beta3, and gamma1) might be involved in the control of erythroid differentiation in humans.

Hypoxia (reduced oxygen tension) is a critical stimulus which switches on a cell rapid response, determining damage and death in some cells, and adaptation and survival in others. Here we report that K562 erythroleukemia cells exposed to hypoxia, proliferated more slowly and the percentage of dead cells increased after 22 h. In parallel HIF (Hypoxia Inducible Factor)-1alpha and Bax level increased, as well as the PKC (Protein Kinase C) delta/Erk (Extracellular Signal Regulated Kinase) pathways being activated. The low level of ROS after 5h of hypoxia did not modify cell cycle progression or affect cell death, whereas HIF-1alpha/CBP (CREB Binding Protein) co-immunoprecipitation and MAPK (Mitogen Activated Protein Kinase)/CREB (c-AMP Response Element Binding) protein signalling pathway activation determined the adaptive survival response. We suggest a dual role for HIF-1alpha in providing a survival or death signal, based on hypoxia duration, and consider the nuclear transcription factor, CREB, to be a possible target for hypoxic therapy against leukemia disease.

In order to discover molecular biomarkers in radiation response we investigated the effects of X-radiation on radioresistant K562 cells by using a comparative proteomic analysis. In treated cells 29 up-regulated and 10 down-regulated proteins were detected by image analysis and identified by mass spectrometry. Elongation factor 1 alpha 1 and stress-70 protein showed a 6.2 and 5.4 fold increase respectively in treated cells. Additional proteins such us pi and omega classes glutathione transferases, ATP synthase D chain, were also found to be up-regulated, suggesting that the enzyme belonging to the cellular detoxification system against oxidative stress and energetic metabolism may have a key role in the cellular response to radiation injury. This data set may provide a useful tool to design a combined chemo- and radiotherapic strategy against leukemia disease.

The study of the ability of chemotherapeutic agents and/or ionizing radiation (IR) to induce cell death in tumor cells is essential for setting up new and more efficient therapies against human cancer. Since drug and ionizing radiation resistance is an impediment to successful chemotherapy against cancer, we wanted to check if etoposide/ionizing radiation combined treatment could have a synergic effect to improve cell death in K562, a well-known human erythroleukemia ionizing radiation resistant cell line. In this study, we examined the role played by JNK/SAPK, p53, and mitochondrial pathways in cell death response of K562 cells to etoposide and IR treatment. Our results let us suppose that the induction of cell death, already evident in 15 Gy exposed cells, mainly in 15 Gy plus etoposide, may be mediated by JNK/SAPK pathway. Moreover, p53 is a potential substrate for JNK and may act as a JNK target for etoposide and ionizing radiation. Thus further investigation on these and other molecular mechanisms underlying the cell death response following etoposide and ionizing radiation exposure could be useful to overcome resistance mechanisms in tumor cells.

We describe a new two-step culture method for mass production in vitro of erythroid cells from either CD34+ (10(5) cells/mL) or light-density (10(6) cells/mL) cells purified from the blood of normal donors and thalassemic patients. The method includes (i) culture of the cells in the presence of dexamethasone and estradiol (10(-6) M each) and (ii) the growth factors SCF (50 ng/mL), IL-3 (1 ng/mL), and EPO (1 U/mL). In their proliferative phase, these cultures generated approximately 1.2 x 10(7) erythroblasts for each milliliter of blood collected from normal donors or thalassemic patients. They were composed mostly (90%) of CD45(low)/glycophorin (GPA)(neg)/CD71(1ow) cells at day 7, 50-60\% of which became CD45(neg)/GPA+/CD71high by days 15-20. However, when cells from days 7 to 12 of the proliferative phase were transferred in differentiation medium containing EPO and insulin, they progressed to mature erythroblasts (g90% benzidine(pos) and CD45(neg)/GPA+/CD71medium) in 4 days. Because of the high number of erythroid cells that are generated from modest volumes of blood, this method will prove useful in donor-specific studies of erythroid differentiation.