Lipid storage and autophagy in melanoma cancer cells

Cancer stem cells (CSC) represent a key cellular subpopulation controlling biological features such as cancer progression in all cancer types. By using melanospheres established from human melanoma patients, we compared less differentiated melanosphere-derived CSC to differentiating melanosphere-derived cells. Increased lipid uptake was found in melanosphere-derived CSC vs. differentiating melanosphere-derived cells, paralleled by strong expression of lipogenic factors Sterol Regulatory Element-Binding Protein-1 (SREBP-1) and Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ). An inverse relation between lipid-storing phenotype and autophagy was also found, since microtubule-associated protein 1A/1B-Light Chain 3 (LC3) lipidation is reduced in melanosphere-derived CSC. To investigate upstream autophagy regulators, Phospho-AMP activated Protein Kinase (P-AMPK) and Phospho-mammalian Target of Rapamycin (P-mTOR) were analyzed; lower P-AMPK and higher P-mTOR expression in melanosphere-derived CSC were found, thus explaining, at least in part, their lower autophagic activity. In addition, co-localization of LC3-stained autophagosome spots and perilipin-stained lipid droplets was demonstrated mainly in differentiating melanosphere-derived cells, further supporting the role of autophagy in lipid droplets clearance. The present manuscript demonstrates an inverse relationship between lipid-storing phenotype and melanoma stem cells differentiation, providing novel indications involving autophagy in melanoma stem cells biology

Nicotinamide inhibits melanoma in vitro and in vivo

Background: Even though new therapies are available against melanoma, novel approaches are needed to overcome resistance and high-toxicity issues. In the present study the anti-melanoma activity of Nicotinamide (NAM), the amide form of Niacin, was assessed in vitro and in vivo. Methods: Human (A375, SK-MEL-28) and mouse (B16-F10) melanoma cell lines were used for in vitro investigations. Viability, cell-death, cell-cycle distribution, apoptosis, Nicotinamide Adenine Dinucleotide+ (NAD+), Adenosine Triphosphate (ATP), and Reactive Oxygen Species (ROS) levels were measured after NAM treatment. NAM anti-SIRT2 activity was tested in vitro; SIRT2 expression level was investigated by in silico transcriptomic analyses. Melanoma growth in vivo was measured in thirty-five C57BL/6 mice injected subcutaneously with B16-F10 melanoma cells and treated intraperitoneally with NAM. Interferon (IFN)-γ-secreting murine cells were counted with ELISPOT assay. Cytokine/chemokine plasmatic levels were measured by xMAP technology. Niacin receptors expression in human melanoma samples was also investigated by in silico transcriptomic analyses. Results: NAM reduced up to 90% melanoma cell number and induced: I) accumulation in G1-phase (40% increase), ii) reduction in S- A nd G2-phase (about 50% decrease), iii) a 10-fold increase of cell-death and 2.5-fold increase of apoptosis in sub-G1 phase, iv) a significant increase of NAD+, ATP, and ROS levels, v) a strong inhibition of SIRT2 activity in vitro. NAM significantly delayed tumor growth in vivo (p ≤ 0.0005) and improved survival of melanoma-bearing mice (p ≤ 0.0001). About 3-fold increase (p ≤ 0.05) of Interferon-gamma (IFN-γ) producing cells was observed in NAM treated mice. The plasmatic expression levels of 6 cytokines (namely: Interleukin 5 (IL-5), Eotaxin, Interleukin 12 (p40) (IL12(p40)), Interleukin 3 (IL-3), Interleukin 10 (IL-10) and Regulated on Activation Normal T Expressed and Secreted (RANTES) were significantly changed in the blood of NAM treated mice, suggesting a key role of the immune response. The observed inhibitory effect of NAM on SIRT2 enzymatic activity confirmed previous evidence; we show here that SIRT2 expression is significantly increased in melanoma and inversely related to melanoma-patients survival. Finally, we show for the first time that the expression levels of Niacin receptors HCAR2 and HCAR3 is almost abolished in human melanoma samples. Conclusion: NAM shows a relevant anti-melanoma activity in vitro and in vivo and is a suitable candidate for further clinical investigations

Which is the best algorithm for evaluating a patient’s candidate to sleeve with suspected reflux or hiatal hernia: is manometry or reflux assessment always necessary

Laparoscopic sleeve gastrectomy (SG) has reached wide popularity during the last 15 years, owing to limited morbidity and mortality rates, very successful weight loss results, and impact on comorbidities. However, the postoperative development or worsening of gastroesophageal reflux disease (GERD) is one of the most important drawbacks of this surgical procedure. To date, there is great heterogeneity concerning the definition of GERD, the indication for SG in patients with GERD, and the standardization of pre and postoperative diagnostic pathways. In patients with severe obesity, a strictly symptom-based diagnosis of GERD is unreliable. In fact, a high rate of silent GERD (s-GERD, asymptomatic patients despite objective evidence of GERD) has been reported. Moreover, patients with preoperative s-GERD have a significantly higher risk of experiencing GERD symptoms after SG. For these reasons, the reflux burden and the competence of the anti-reflux barrier should be carefully assessed during the preoperative work-up of patients undergoing SG. Ambulatory pH monitoring (APM) and high-resolution manometry (HRM) are useful diagnostic tools that could provide valuable evidence in the guidance of surgical strategy. In this review, we evaluate the current literature concerning the use of APM and HRM in the diagnostic pathway before SG, as well as their predictive value for the evolution of GERD in the postoperative course. Moreover, we propose a diagnostic algorithm for preoperative GERD assessment, which includes validated symptom questionnaires, upper gastrointestinal endoscopy, APM, and HRM

Evaluation of the Efficacy and Safety of a Compound of Micronized Flavonoids in Combination With Vitamin C and Extracts of Centella asiatica, Vaccinium myrtillus, and Vitis vinifera for the Reduction of Hemorrhoidal Symptoms in Patients With Grade II and III Hemorrhoidal Disease: A Retrospective Real-Life Study

Background and Aim: Several evidences have shown how, in hemorrhoidal disease, phlebotonic flavonoid agents such as quercetin reduce capillary permeability by increasing vascular walls resistance, how rutin and vitamin C have antioxidant properties, and that Centella asiatica has reparative properties towards the connective tissue. A retrospective study was designed in order to evaluate the efficacy and safety of a compound consisting of micronized flavonoids in combination with vitamin C and extracts of C. asiatica, Vaccinium myrtillus, and Vitis vinifera for grade II and III hemorrhoidal disease. Patients and Methods: Data of 49 patients, over 18, who were following a free diet regimen, not on therapy with other anti-hemorrhoid agents, treated with a compound consisting of 450 mg of micronized diosmin, 300 mg of C. asiatica, 270 mg of micronized hesperidin, 200 mg of V. vinifera, 160 mg of vitamin C, 160 mg of V. myrtillus, 140 mg of micronized quercetin, and 130 mg of micronized rutin (1 sachet or 2 tablets a day) for 7 days were collected. Hemorrhoid grade according to Goligher’s scale together with anorectal symptoms (edema, prolapse, itching, thrombosis, burning, pain, tenesmus, and bleeding) both before treatment (T0) and after 7 days of therapy (T7) were collected. Primary outcomes were the reduction of at least one degree of hemorrhoids according to Goligher’s scale assessed by proctological examination and compound safety. The secondary outcome was the reduction of anorectal symptoms assessed by questionnaires administered to patients. Results: Forty-four patients (89.8%) presented a reduction in hemorrhoidal grade of at least one grade (p < 0.001). No adverse events with the use of the compound were noted. A significant reduction was observed in all anorectal symptoms evaluated (p < 0.05). No predictors of response to the compound were identified among the clinical and demographic variables collected. Conclusion: The compound analyzed was effective and safe for patients with grade II and III hemorrhoidal disease according to Goligher’s scale

Investigating serum and tissue expression identified a cytokine/chemokine signature as a highly effective melanoma marker

The identification of reliable and quantitative melanoma biomarkers may help an early diagnosis and may directly affect melanoma mortality and morbidity. The aim of the present study was to identify effective biomarkers by investigating the expression of 27 cytokines/chemokines in melanoma compared to healthy controls, both in serum and in tissue samples. Serum samples were from 232 patients recruited at the IDI-IRCCS hospital. Expression was quantified by xMAP technology, on 27 cytokines/chemokines, compared to the control sera. RNA expression data of the same 27 molecules were obtained from 511 melanoma-and healthy-tissue samples, from the GENT2 database. Statistical analysis involved a 3-step approach: analysis of the single-molecules by Mann–Whitney analysis; analysis of paired-molecules by Pearson correlation; and profile analysis by the machine learning algorithm Support Vector Machine (SVM). Single-molecule analysis of serum expression identified IL-1b, IL-6, IP-10, PDGF-BB, and RANTES differently expressed in melanoma (p < 0.05). Expression of IL-8, GM-CSF, MCP-1, and TNF-α was found to be significantly correlated with Breslow thickness. Eotaxin and MCP-1 were found differentially expressed in male vs. female patients. Tissue expression analysis identified very effective marker/predictor genes, namely, IL-1Ra, IL-7, MIP-1a, and MIP-1b, with individual AUC values of 0.88, 0.86, 0.93, 0.87, respectively. SVM analysis of the tissue expression data identified the combination of these four molecules as the most effective signature to discriminate melanoma patients (AUC = 0.98). Validation, using the GEPIA2 database on an additional 1019 independent samples, fully confirmed these observations. The present study demonstrates, for the first time, that the IL-1Ra, IL-7, MIP-1a, and MIP-1b gene signature discriminates melanoma from control tissues with extremely high efficacy. We therefore propose this 4-molecule combination as an effective melanoma marker

c-FLIP regulates autophagy by interacting with Beclin-1 and influencing its stability

c-FLIP (cellular FLICE-like inhibitory protein) protein is mostly known as an apoptosis modulator. However, increasing data underline that c-FLIP plays multiple roles in cellular homoeostasis, influencing differently the same pathways depending on its expression level and isoform predominance. Few and controversial data are available regarding c-FLIP function in autophagy. Here we show that autophagic flux is less effective in c-FLIP−/− than in WT MEFs (mouse embryonic fibroblasts). Indeed, we show that the absence of c-FLIP compromises the expression levels of pivotal factors in the generation of autophagosomes. In line with the role of c-FLIP as a scaffold protein, we found that c-FLIPL interacts with Beclin-1 (BECN1: coiled-coil, moesin-like BCL2-interacting protein), which is required for autophagosome nucleation. By a combination of bioinformatics tools and biochemistry assays, we demonstrate that c-FLIPL interaction with Beclin-1 is important to prevent Beclin-1 ubiquitination and degradation through the proteasomal pathway. Taken together, our data describe a novel molecular mechanism through which c-FLIPL positively regulates autophagy, by enhancing Beclin-1 protein stability

Bioinformatics in Italy : BITS 2012, the ninth annual meeting of the Italian Society of Bioinformatics

The BITS2012 meeting, held in Catania on May 2-4, 2012, brought together almost 100 Italian researchers working in the field of Bioinformatics, as well as students in the same or related disciplines. About 90 original research works were presented either as oral communication or as posters, representing a landscape of Italian current research in bioinformatics.This preface provides a brief overview of the meeting and introduces the manuscripts that were accepted for publication in this supplement, after a strict and careful peer-review by an International board of referees

P-rex1 cooperates with PDGFRβ to drive cellular migration in 3D microenvironments

Expression of the Rac-guanine nucleotide exchange factor (RacGEF), P-Rex1 is a key determinant of progression to metastasis in a number of human cancers. In accordance with this proposed role in cancer cell invasion and metastasis, we find that ectopic expression of P-Rex1 in an immortalised human fibroblast cell line is sufficient to drive multiple migratory and invasive phenotypes. The invasive phenotype is greatly enhanced by the presence of a gradient of serum or platelet-derived growth factor, and is dependent upon the expression of functional PDGF receptor β. Consistently, the invasiveness of WM852 melanoma cells, which endogenously express P-Rex1 and PDGFRβ, is opposed by siRNA of either of these proteins. Furthermore, the current model of P-Rex1 activation is advanced through demonstration of P-Rex1 and PDGFRβ as components of the same macromolecular complex. These data suggest that P-Rex1 has an influence on physiological migratory processes, such as invasion of cancer cells, both through effects upon classical Rac1-driven motility and a novel association with RTK signalling complexes