Microwave-Assisted, Solid-State Procedure to Covalently Conjugate Hyaluronic Acid to Curcumin: Validation of a Green Synthetic Protocol.

A microwave-assisted esterification reaction to prepare hyaluronan-curcumin derivatives by employing a solvent-free process was developed. In particular, a solid-state strategy to react two molecules characterized by totally different solubility profiles was developed. Hyaluronic acid, a highly hydrosoluble polysaccharide, was reacted with hydrophobic and even water-unstable curcumin. Microwave (MW) irradiation was employed to activate the reaction between the two solid compounds through the direct interaction with them and to preserve the integrity of the sensitive curcumin species. This new protocol can be considered efficient, fast, and also eco-friendly, avoiding the employment of toxic organic bases and solvents. A cytotoxicity test suggested that the developed hyaluronan-curcumin conjugate (HA-CUR) could be considered a candidate for its implementation as a new material. In addition, preliminary studies revealed promising anti-inflammatory activity and open future perspectives of further investigation.

Tamoxifen Activates Transcription Factor EB and Triggers Protective Autophagy in Breast Cancer Cells by Inducing Lysosomal Calcium Release: A Gateway to the Onset of Endocrine Resistance.

Among the several mechanisms accounting for endocrine resistance in breast cancer, autophagy has emerged as an important player. Previous reports have evidenced that tamoxifen (Tam) induces autophagy and activates transcription factor EB (TFEB), which regulates the expression of genes controlling autophagy and lysosomal biogenesis. However, the mechanisms by which this occurs have not been elucidated as yet. This investigation aims at dissecting how TFEB is activated and contributes to Tam resistance in luminal A breast cancer cells. TFEB was overexpressed and prominently nuclear in Tam-resistant MCF7 cells (MCF7-TamR) compared with their parental counterpart, and this was not dependent on alterations of its nucleo-cytoplasmic shuttling. Tam promoted the release of lysosomal Ca2+ through the major transient receptor potential cation channel mucolipin subfamily member 1 (TRPML1) and two-pore channels (TPCs), which caused the nuclear translocation and activation of TFEB. Consistently, inhibiting lysosomal calcium release restored the susceptibility of MCF7-TamR cells to Tam. Our findings demonstrate that Tam drives the nuclear relocation and transcriptional activation of TFEB by triggering the release of Ca2+ from the acidic compartment, and they suggest that lysosomal Ca2+ channels may represent new druggable targets to counteract the onset of autophagy-mediated endocrine resistance in luminal A breast cancer cells.

Oxidative Stress-Related Mechanisms in Melanoma and in the Acquired Resistance to Targeted Therapies.

Melanoma is a highly aggressive cancer with the poorest prognosis, representing the deadliest form of skin cancer. Activating mutations in BRAF are the most frequent genetic alterations, present in approximately 50% of all melanoma cases. The use of specific inhibitors towards mutant BRAF variants and MEK, a downstream signaling target of BRAF in the MAPK pathway, has significantly improved progression-free and overall survival in advanced melanoma patients carrying BRAF mutations. Nevertheless, despite these improvements, resistance still develops within the first year of therapy in around 50% of patients, which is a significant problem in managing BRAF-mutated advanced melanoma. Understanding these mechanisms is one of the mainstreams of the research on BRAFi/MEKi acquired resistance. Both genetic and epigenetic mechanisms have been described. Moreover, in recent years, oxidative stress has emerged as another major force involved in all the phases of melanoma development, from initiation to progression until the onsets of the metastatic phenotype and chemoresistance, and has thus become a target for therapy. In the present review, we discuss the current knowledge on oxidative stress and its signaling in melanoma, as well as the oxidative stress-related mechanisms in the acquired resistance to targeted therapies.

Peroxisome Proliferator-Activated Receptors (PPARs) and Oxidative Stress in Physiological Conditions and in Cancer.

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor superfamily. Originally described as "orphan nuclear receptors", they can bind both natural and synthetic ligands acting as agonists or antagonists. In humans three subtypes, PPARα, ß/δ, γ, are encoded by different genes, show tissue-specific expression patterns, and contribute to the regulation of lipid and carbohydrate metabolisms, of different cell functions, including proliferation, death, differentiation, and of processes, as inflammation, angiogenesis, immune response. The PPAR ability in increasing the expression of various antioxidant genes and decreasing the synthesis of pro-inflammatory mediators, makes them be considered among the most important regulators of the cellular response to oxidative stress conditions. Based on the multiplicity of physiological effects, PPAR involvement in cancer development and progression has attracted great scientific interest with the aim to describe changes occurring in their expression in cancer cells, and to investigate the correlation with some characteristics of cancer phenotype, including increased proliferation, decreased susceptibility to apoptosis, malignancy degree and onset of resistance to anticancer drugs. This review focuses on mechanisms underlying the antioxidant and anti-inflammatory properties of PPARs in physiological conditions, and on the reported beneficial effects of PPAR activation in cancer.

Biomolecular, Histological, Clinical, and Radiological Analyses of Dental Implant Bone Sites Prepared Using Magnetic Mallet Technology: A Pilot Study in Animals.

BACKGROUND: A new instrumentation exploiting magneto-dynamic technology (mallet) proposed for implant site preparation was investigated. METHODS: In the tibias of three minipigs, two sites were prepared by mallet and two by drill technique. Primary stability (ISQ) was detected after implant positioning (T0) and at 14 days (T14). X-rays and computed tomography were performed. At T14, bone samples were utilized for histological and biomolecular analyses. RESULTS: In mallet sites, histological evaluations evidenced a significant increase in the newly formed bone, osteoblast number, and a smaller quantity of fibrous tissue. These results agree with the significant BMP-4 augmentation and the positive trend in other osteogenic factors (biological and radiological investigations). Major, albeit IL-10-controlled, inflammation was present. For both techniques, at T14 a significant ISQ increase was evidenced, but no significant difference was observed at T0 and T14 between the mallet and drill techniques. In mallet sites, lateral bone condensation was observed on computed tomography. CONCLUSIONS: Using biological, histological, clinical, and radiological analyses, this study first shows that the mallet technique is effective for implant site preparation. Based on its ability to cause osseocondensation and improve newly formed bone, mallet technology should be chosen in all clinical cases of poor bone quality.

Control of Oxidative Stress in Cancer Chemoresistance: Spotlight on Nrf2 Role.

Chemoresistance represents the main obstacle to cancer treatment with both conventional and targeted therapy. Beyond specific molecular alterations, which can lead to targeted therapy, metabolic remodeling, including the control of redox status, plays an important role in cancer cell survival following therapy. Although cancer cells generally have a high basal reactive oxygen species (ROS) level, which makes them more susceptible than normal cells to a further increase of ROS, chemoresistant cancer cells become highly adapted to intrinsic or drug-induced oxidative stress by upregulating their antioxidant systems. The antioxidant response is principally mediated by the transcription factor Nrf2, which has been considered the master regulator of antioxidant and cytoprotective genes. Nrf2 expression is often increased in several types of chemoresistant cancer cells, and its expression is mediated by diverse mechanisms. In addition to Nrf2, other transcription factors and transcriptional coactivators can participate to maintain the high antioxidant levels in chemo and radio-resistant cancer cells. The control of expression and function of these molecules has been recently deepened to identify which of these could be used as a new therapeutic target in the treatment of tumors resistant to conventional therapy. In this review, we report the more recent advances in the study of Nrf2 regulation in chemoresistant cancers and the role played by other transcription factors and transcriptional coactivators in the control of antioxidant responses in chemoresistant cancer cells.

Autophagy Triggers Tamoxifen Resistance in Human Breast Cancer Cells by Preventing Drug-Induced Lysosomal Damage.

Endocrine resistance is a major complication during treatment of estrogen receptor-positive breast cancer. Although autophagy has recently gained increasing consideration among the causative factors, the link between autophagy and endocrine resistance remains elusive. Here, we investigate the autophagy-based mechanisms of tamoxifen resistance in MCF7 cells. Tamoxifen (Tam) triggers autophagy and affects the lysosomal compartment of MCF7 cells, such that activated autophagy supports disposal of tamoxifen-damaged lysosomes by lysophagy. MCF7 cells resistant to 5 µM tamoxifen (MCF7-TamR) have a higher autophagic flux and an enhanced resistance to Tam-induced lysosomal alterations compared to parental cells, which suggests a correlation between the two events. MCF7-TamR cells overexpress messenger RNAs (mRNAs) for metallothionein 2A and ferritin heavy chain, and they are re-sensitized to Tam by inhibition of autophagy. Overexpressing these proteins in parental MCF7 cells protects lysosomes from Tam-induced damage and preserves viability, while inhibiting autophagy abrogates lysosome protection. Consistently, we also demonstrate that other breast cancer cells that overexpress selected mRNAs encoding iron-binding proteins are less sensitive to Tam-induced lysosomal damage when autophagy is activated. Collectively, our data demonstrate that autophagy triggers Tam resistance in breast cancer cells by favoring the lysosomal relocation of overexpressed factors that restrain tamoxifen-induced lysosomal damage.

Role of rhBMP-7, Fibronectin, And Type I Collagen in Dental Implant Osseointegration Process: An Initial Pilot Study on Minipig Animals.

BACKGROUND: The biological factors involved in dental implant osseointegration need to be investigated to improve implant success. METHODS: Twenty-four implants were inserted into the tibias of six minipigs. Bone samples were obtained at 7, 14, and 56 days. Biomolecular analyses evaluated mRNA of BMP-4, -7, Transforming Growth Factor-ß2, Interleukin-1ß, and Osteocalcin in sites treated with rhBMP-7, Type 1 Collagen, or Fibronectin (FN). Inflammation and osteogenesis were evaluated by histological analyses. RESULTS: At 7 and 14 days, BMP-4 and BMP-7 increased in the sites prepared with rhBMP-7 and FN. BMP-7 remained greater at 56 days in rhBMP-7 and FN sites. BPM-4 at 7 and 14 days increased in Type 1 Collagen sites; BMP-7 increased from day 14. FN increased the TGF-ß2 at all experimental times, whilst the rhBMP-7 only did so up to 7 days. IL-1ß increased only in collagen-treated sites from 14 days. Osteocalcin was high in FN-treated sites. Neutrophilic granulocytes characterized the inflammatory infiltrate at 7 days, and mononuclear cells at 14 and 56 days. CONCLUSIONS: This initial pilot study, in a novel way, evidenced that Type 1 Collagen induced inflammation and did not stimulate bone production; conversely FN or rhBMP-7 showed neo-osteogenetic and anti-inflammatory properties when directly added into implant bone site.

In-vitro Characterization of a Hernia Mesh Featuring a Nanostructured Coating.

Abdominal hernia repair is a frequently performed surgical procedure worldwide. Currently, the use of polypropylene (PP) surgical meshes for the repair of abdominal hernias constitutes the primary surgical approach, being widely accepted as superior to primary suture repair. Surgical meshes act as a reinforcement for the weakened or damaged tissues and support tissue restoration. However, implanted meshes could suffer from poor integration with the surrounding tissues. In this context, the present study describes the preliminary evaluation of a PCL-Gel-based nanofibrous coating as an element to develop a multicomponent hernia mesh device (meshPCL-Gel) that could overcome this limitation thanks to the presence of a nanostructured biomimetic substrate for enhanced cell attachment and new tissue formation. Through the electrospinning technique, a commercial PP hernia mesh was coated with a nanofibrous membrane from a polycaprolactone (PCL) and gelatin (Gel) blend (PCL-Gel). Resulting PCL-Gel nanofibers were homogeneous and defect-free, with an average diameter of 0.15 ± 0.04 µm. The presence of Gel decreased PCL hydrophobicity, so that membranes average water contact angle dropped from 138.9 ± 1.1° (PCL) to 99.9 ± 21.6°, while it slightly influenced mechanical properties, which remained comparable to those of PCL (E = 15.7 ± 2.7 MPa, σ R = 7.7 ± 0.6 ε R = 118.8 ± 13.2%). Hydrolytic and enzymatic degradation was conducted on PCL-Gel up to 28 days, with maximum weight losses around 20 and 40%, respectively. The meshPCL-Gel device was obtained with few simple steps, with no influences on the original mechanical properties of the bare mesh, and good stability under physiological conditions. The biocompatibility of meshPCL-Gel was assessed by culturing BJ human fibroblasts on the device, up to 7 days. After 24 h, cells adhered to the nanofibrous substrate, and after 72 h their metabolic activity was about 70% with respect to control cells. The absence of detectable lactate dehydrogenase in the culture medium indicated that no necrosis induction occurred. Hence, the developed nanostructured coating provided the meshPCL-Gel device with chemical and topographical cues similar to the native extracellular matrix ones, that could be exploited for enhancing the biological response and, consequently, mesh integration, in abdominal wall hernia repair.

PPARs are mediators of anti-cancer properties of superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with conjugated linoleic acid.

Breast cancer chemotherapy can cause side effects due to nonspecific drug delivery, low solubility and fast metabolism of drugs used in conventional therapy. Moreover, the therapeutic effect of the drugs is often reduced by the strengthening of chemoresistance, which occurs via a variety of mechanisms. Different strategies have been developed to reduce multidrug resistance (MDR)-associated gene expressions including the use of surfactants and polymers. In this study superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with conjugated linoleic acid (CLA) reduced the number and viability of cells in comparison with both untreated cells or cells treated with SPIONs alone. This cytostatic effect correlated with the increase of peroxisome proliferator-activated receptors γ (PPARγ). The necrotic cell death induced, as a consequence, an inflammatory process, as evidenced by the decrease of the anti-inflammatory PPARα and increase of pro-inflammatory TNFα and IL-1ß. PPARs were examined because CLA is one of their natural ligands. The antitumor effect observed was accompanied by a down-regulation of p-glycoprotein (P-gp), which was the first important discovered efflux transporter belonging to MDR, and of ALDH3A1, an enzyme able to metabolize some drugs, reducing their effects. The down-regulation of P-gp correlated with the increase of cytokines. The ALDH3A1 decrease correlated with the increase of PPARγ. Based on these results, PPARs are molecular mediators of anti-cancer effect of SPIONs functionalized with CLA, being changes in these nuclear receptors correlated with induction of cytotoxicity and inflammation, and decreased ability of cancer cells in blocking anti-cancer drug effect.

Innovative superparamagnetic iron-oxide nanoparticles coated with silica and conjugated with linoleic acid: Effect on tumor cell growth and viability.

One of the goals for the development of more effective cancer therapies with reduced toxic side effects is the optimization of innovative treatments to selectively kill tumor cells. The use of nanovectors loaded with targeted therapeutic payloads is one of the most investigated strategies. In this paper superparamagnetic iron oxide nanoparticles (SPIONs) coated by a silica shell or uncoated, were functionalized with single-layer and bi-layer conjugated linoleic acid (CLA). Silica was used to protect the magnetic core from oxidation, improve the stability of SPIONs and tailor their surface reactivity. CLA was used as novel grafting biomolecule for its anti-tumor activity and to improve particle dispersibility. Mouse breast cancer 4T1 cells were treated with these different SPIONs. SPIONs functionalized with the highest quantity of CLA and coated with silica shell were the most dispersed. Cell viability was reduced by SPIONs functionalized with CLA in comparison with cells which were untreated or treated with SPIONs without CLA. As regards the types of SPIONs functionalized with CLA, the lowest viability was observed in cells treated with uncoated SPIONs with the highest quantity of CLA. In conclusion, the silica shell free SPIONs functionalized with the highest amount of CLA can be suggested as therapeutic carriers because they have the best dispersion and ability to decrease 4T1 cell viability.

Biocompatibility versus peritoneal mesothelial cells of polypropylene prostheses for hernia repair, coated with a thin silica/silver layer.

Hernias are generally repaired using synthetic prostheses. Infection may already be present or develop during implantation. Based on the increasing resistance to antibiotics, and the well-known antimicrobial properties of silver (Ag), the possibility of coating hernia prostheses with a nanostructured layer containing Ag was explored. Prostheses (Clear Mesh Composite [CMC]) made up of two polypropylene layers (macroporous light mesh and thin transparent film) were tested with human mesothelial cells from omentum biopsies. Mesotheliocytes modulate abdominal wall healing producing cytokines, growth factors, and adhesion molecules. Evaluating the growth of these cells on CMC or film alone showed that cell numbers on CMC increased over time, and were higher than those on film alone. Vimentin immunostaining confirmed the cells to be mesotheliocytes. Subsequently, the biocompatibility of mesh layer, coated or not with a thin layer of Ag/SiO2 -nanoclusters, was analyzed, showing no difference in absence or presence of Ag/SiO2 . Differently, TGF-ß2 production, involved in tissue repair and fibrosis, increased in the presence of Ag/SiO2 . Moreover, Ag/SiO2 -coated mesh showed antibacterial properties. In conclusion, the mesh layer coated with Ag/SiO2 afforded cell growth, and showed antibacterial activity. Coating only the mesh layer did not decrease film transparency, and did not favor the formation of adhesions on the visceral side. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1586-1593, 2017.

Mitochondrial Dysfunction in Cancer and Neurodegenerative Diseases: Spotlight on Fatty Acid Oxidation and Lipoperoxidation Products.

In several human diseases, such as cancer and neurodegenerative diseases, the levels of reactive oxygen species (ROS), produced mainly by mitochondrial oxidative phosphorylation, is increased. In cancer cells, the increase of ROS production has been associated with mtDNA mutations that, in turn, seem to be functional in the alterations of the bioenergetics and the biosynthetic state of cancer cells. Moreover, ROS overproduction can enhance the peroxidation of fatty acids in mitochondrial membranes. In particular, the peroxidation of mitochondrial phospholipid cardiolipin leads to the formation of reactive aldehydes, such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA), which are able to react with proteins and DNA. Covalent modifications of mitochondrial proteins by the products of lipid peroxidation (LPO) in the course of oxidative cell stress are involved in the mitochondrial dysfunctions observed in cancer and neurodegenerative diseases. Such modifications appear to affect negatively mitochondrial integrity and function, in particular energy metabolism, adenosine triphosphate (ATP) production, antioxidant defenses and stress responses. In neurodegenerative diseases, indirect confirmation for the pathogenetic relevance of LPO-dependent modifications of mitochondrial proteins comes from the disease phenotypes associated with their genetic alterations.

The Omega-3 Fatty Acid Docosahexaenoic Acid Modulates Inflammatory Mediator Release in Human Alveolar Cells Exposed to Bronchoalveolar Lavage Fluid of ARDS Patients.

BACKGROUND: This study investigated whether the 1 : 2 ω-3/ω-6 ratio may reduce proinflammatory response in human alveolar cells (A549) exposed to an ex vivo inflammatory stimulus (bronchoalveolar lavage fluid (BALF) of acute respiratory distress syndrome (ARDS) patients). Methods. We exposed A549 cells to the BALF collected from 12 ARDS patients. After 18 hours, fatty acids (FA) were added as docosahexaenoic acid (DHA, ω-3) and arachidonic acid (AA, ω-6) in two ratios (1 : 2 or 1 : 7). 24 hours later, in culture supernatants were evaluated cytokines (TNF-α, IL-6, IL-8, and IL-10) and prostaglandins (PGE2 and PGE3) release. The FA percentage content in A549 membrane phospholipids, content of COX-2, level of PPARγ, and NF-κB binding activity were determined. RESULTS: The 1 : 2 DHA/AA ratio reversed the baseline predominance of ω-6 over ω-3 in the cell membranes (P < 0.001). The proinflammatory cytokine release was reduced by the 1 : 2 ratio (P < 0.01 to <0.001) but was increased by the 1 : 7 ratio (P < 0.01). The 1 : 2 ratio reduced COX-2 and PGE2 (P < 0.001) as well as NF-κB translocation into the nucleus (P < 0.01), while it increased activation of PPARγ and IL-10 release (P < 0.001). Conclusion. This study demonstrated that shifting the FA supply from ω-6 to ω-3 decreased proinflammatory mediator release in human alveolar cells exposed to BALF of ARDS patients.

Key role of the expression of bone morphogenetic proteins in increasing the osteogenic activity of osteoblast-like cells exposed to shock waves and seeded on bioactive glass-ceramic scaffolds for bone tissue engineering.

In this work, the role of shock wave-induced increase of bone morphogenetic proteins in modulating the osteogenic properties of osteoblast-like cells seeded on a bioactive scaffold was investigated using gremlin as a bone morphogenetic protein antagonist. Bone-like glass-ceramic scaffolds, based on a silicate experimental bioactive glass developed at the Politecnico di Torino, were produced by the sponge replication method and used as porous substrates for cell culture. Human MG-63 cells, exposed to shock waves and seeded on the scaffolds, were treated with gremlin every two days and analysed after 20 days for the expression of osteoblast differentiation markers. Shock waves have been shown to induce osteogenic activity mediated by increased expression of alkaline phosphatase, osteocalcin, type I collagen, BMP-4 and BMP-7. Cells exposed to shock waves plus gremlin showed increased growth in comparison with cells treated with shock waves alone and, conversely, mRNA contents of alkaline phosphatase and osteocalcin were significantly lower. Therefore, the shock wave-mediated increased expression of bone morphogenetic protein in MG-63 cells seeded on the scaffolds is essential in improving osteogenic activity; blocking bone morphogenetic protein via gremlin completely prevents the increase of alkaline phosphatase and osteocalcin. The results confirmed that the combination of glass-ceramic scaffolds and shock waves exposure could be used to significantly improve osteogenesis opening new perspectives for bone regenerative medicine.

In vitro study of manganese-doped bioactive glasses for bone regeneration.

A glass belonging to the system SiO2-P2O5-CaO-MgO-Na2O-K2O was modified by introducing two different amounts of manganese oxide (MnO). Mn-doped glasses were prepared by melt and quenching technique and characterized by means of X-ray diffraction (XRD), scanning electron microscopy (SEM) observation and energy dispersion spectrometry (EDS) analysis. In vitro bioactivity test in simulated body fluid (SBF) showed a slight decrease in the reactivity kinetics of Mn-doped glasses compared to the glass used as control; however the glasses maintained a good degree of bioactivity. Mn-leaching test in SBF and minimum essential medium (MEM) revealed fluctuating trends probably due to a re-precipitation of Mn compounds during the bioactivity process. Cellular tests showed that all the Mn-doped glasses, up to a concentration of 50 µg/cm(2) (µg of glass powders/cm(2) of cell monolayer), did not produce cytotoxic effects on human MG-63 osteoblasts cultured for up to 5 days. Finally, biocompatibility tests demonstrated a good osteoblast proliferation and spreading on Mn-doped glasses and most of all that the Mn-doping can promote the expression of alkaline phosphatase (ALP) and some bone morphogenetic proteins (BMPs).

CLA reduces inflammatory mediators from A427 human lung cancer cells and A427 conditioned medium promotes differentiation of C2C12 murine muscle cells.

Conjugated linoleic acid (CLA) is thought to have anti-proliferative and anti-inflammatory properties, but its effect on cancer cachexia is unknown. Two effects were here investigated: that of CLA on inflammatory mediator production in human lung cancer cells, and that of reduced mediators on the myogenic differentiation of murine muscle C2C12 cells. The latter cells were grown in medium conditioned by human lung cancer A427 cells, with or without CLA, to mimic only the effect of molecules released from the tumor "in vivo", excluding the effect of host-produced cachectic factors. The results obtained show that CLA was found to reduce the production of tumor necrosis factor-α, interleukin (IL)-1ß and prostaglandin E2 (PGE2), but had no effect on IL-6 production. The mechanisms underlying the effect of CLA on cytokine or PGE2 release in A427 cells are probably mediated by activation of peroxisome proliferator-activated receptor (PPAR)α, which increased at 24 h CLA treatment. In turn, the reduced content of inflammatory mediators in medium conditioned by A427 cells, in the presence of CLA, allowed muscle cells to proliferate, again by inducing PPAR. The involvement of PPARα was demonstrated by treatment with the antagonist MK-886. The findings demonstrate the anti-inflammatory and myogenic action of CLA and point to its possible application as a novel dietary supplement and therapeutic agent in inflammatory disease states, such as cachexia.

Oral mucosa produces cytokines and factors influencing osteoclast activity and endothelial cell proliferation, in patients with osteonecrosis of jaw after treatment with zoledronic acid.

OBJECTIVES: The intravenous injection of bisphosphonates, currently used as treatment for osteoporosis, bone Paget's disease, multiple myeloma, or bone metastases, can cause jaw bone necrosis especially in consequence of trauma. The present research aimed to clarify the mechanisms underlying bone necrosis, exploring involvement of the oral mucosa "in vivo." PATIENTS AND METHODS: Specimens of oral mucosa were removed from bisphosphonate-treated patients with or without jaw bone necrosis. In mucosa specimens, expression was evaluated of: cytokines involved in the inflammatory process, factors involved in osteoclast activity, i.e., receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin, a factor involved in cell proliferation, namely hydroxymethylglutaryl coenzyme A reductase, and a factor involved in angiogenesis, namely vascular endothelial growth factor (VEGF). RESULTS: Interleukin (IL)-6 and the RANK/osteoprotegerin ratio were significantly elevated in mucosa from patients with versus without jaw necrosis, whereas hydroxymethylglutaryl coenzyme A reductase and VEGF were significantly decreased. CONCLUSIONS: Our results suggest that mucosa, stimulated by bisphosphonate released from the bone, can contribute to the development of jaw necrosis, reducing VEGF, and producing IL-6 in consequence of hydroxymethylglutaryl coenzyme A reductase reduction. In turn, IL-6 stimulates osteoclast activity, as shown by the increased RANKL/osteoprotegerin ratio. CLINICAL RELEVANCE: The results of this study suggest the importance of evaluating during bisphosphonate treatment the production of IL-6, RANKL, osteoprotegerin, and VEGF, in order to monitor the jaw osteonecrosis onset. To avoid repeated mucosa excisions, the determination of these factors could be carried out in crevicular fluid.

Effect of n-3 fatty acids on patients with advanced lung cancer: a double-blind, placebo-controlled study.

PUFA from fish oil appear to have anti-inflammatory and anti-oxidative effects and improve nutritional status in cancer patients. With this as background, the aim of the present study was to investigate the effect of EPA plus DHA on inflammatory condition, and oxidative and nutritional status in patients with lung cancer. In our multicentre, randomised, double-blind trial, thirty-three patients with a diagnosis of advanced inoperable non-small-cell lung cancer and undergoing chemotherapy were divided into two groups, receiving four capsules/d containing 510 mg of EPA and 340 mg of DHA, or 850 mg of placebo, for 66 d. At the start of chemotherapy (T0), after 8 d (T1), 22 d (T2) and 66 d (T3), biochemical (inflammatory and oxidative status parameters) and anthropometric parameters were measured in both groups. A significant increase of body weight in the n-3 group at T3 v. T0 was observed. Concerning inflammation, C-reactive protein and IL-6 levels differed significantly between the n-3 and placebo groups at T3, and progressively decreased during chemotherapy in the n-3 group, evidencing n-3 PUFA anti-inflammatory action. Concerning oxidative status, plasma reactive oxygen species levels increased in the placebo group v. the n-3 group at the later treatment times. Hydroxynonenal levels increased in the placebo group during the study, while they stabilised in the n-3 group. Our data confirm that the continual assumption of EPA plus DHA determined an anti-inflammatory and anti-oxidative action which could be considered a preliminary goal in anti-cachectic therapy.