Valentina Tesori

Mammalian life-span determinant p66shcA mediates obesity-induced insulin resistance

Obesity and metabolic syndrome result from excess calorie intake and genetic predisposition and are mechanistically linked to type II diabetes and accelerated body aging; abnormal nutrient and insulin signaling participate in this pathologic process, yet the underlying molecular mechanisms are incompletely understood. Mice lacking the p66 kDa isoform of the Shc adaptor molecule live longer and are leaner than wild-type animals, suggesting that this molecule may have a role in metabolic derangement and premature senescence by overnutrition. We found that p66 deficiency exerts a modest but significant protective effect on fat accumulation and premature death in lepOb/Ob mice, an established genetic model of obesity and insulin resistance; strikingly, however, p66 inactivation improved glucose tolerance in these animals, without affecting (hyper)insulinaemia and independent of body weight. Protection from insulin resistance was cell autonomous, because isolated p66KO preadipocytes were relatively resistant to insulin desensitization by free fatty acids in vitro. Biochemical studies revealed that p66shc promotes the signal-inhibitory phosphorylation of the major insulin transducer IRS-1, by bridging IRS-1 and the mTOR effector p70S6 kinase, a molecule previously linked to obesity-induced insulin resistance. Importantly, IRS-1 was strongly up-regulated in the adipose tissue of p66KO lepOb/Ob mice, confirming that effects of p66 on tissue responsiveness to insulin are largely mediated by this molecule. Taken together, these findings identify p66shc as a major mediator of insulin resistance by excess nutrients, and by extension, as a potential molecular target against the spreading epidemic of obesity and type II diabetes.

Therapeutic Implications of Mesenchymal Stem Cells in Liver Injury

Mesenchymal stem cells (MSCs), represent an attractive tool for the establishment of a successful stem-cell-based therapy of liver diseases. A number of different mechanisms contribute to the therapeutic effects exerted by MSCs, since these cells can differentiate into functional hepatic cells and can also produce a series of growth factors and cytokines able to suppress inflammatory responses, reduce hepatocyte apoptosis, regress liver fibrosis, and enhance hepatocyte functionality. To date, the infusion of MSCs or MSC-conditioned medium has shown encouraging results in the treatment of fulminant hepatic failure and in end-stage liver disease in experimental settings. However, some issues under debate hamper the use of MSCs in clinical trials. This paper summarizes the biological relevance of MSCs and the potential benefits and risks that can result from translating the MSC research to the treatment of liver diseases.

Colon cancer stem cells: controversies and perspectives

Tumors have long been viewed as a population in which all cells have the equal propensity to form new tumors, the so called conventional stochastic model. The cutting-edge theory on tumor origin and progression, tends to consider cancer as a stem cell disease. Stem cells are actively involved in the onset and maintenance of colon cancer. This review is intended to examine the state of the art on colon cancer stem cells (CSCs), with regard to the recent achievements of basic research and to the corresponding translational consequences. Specific prominence is given to the hypothesized origin of CSCs and to the methods for their identification. The growing understanding of CSC biology is driving the optimization of novel anti-cancer targeted drugs.

The multikinase inhibitor Sorafenib enhances glycolysis and synergizes with glycolysis blockade for cancer cell killing

Although the only effective drug against primary hepatocarcinoma, the multikinase inhibitor Sorafenib (SFB) usually fails to eradicate liver cancer. Since SFB targets mitochondria, cell metabolic reprogramming may underlie intrinsic tumor resistance. To characterize cancer cell metabolic response to SFB, we measured oxygen consumption, generation of reactive oxygen species (ROS) and ATP content in rat LCSC (Liver Cancer Stem Cells) -2 cells exposed to the drug. Genome wide analysis of gene expression was performed by Affymetrix technology. SFB cytotoxicity was evaluated by multiple assays in the presence or absence of metabolic inhibitors, or in cells genetically depleted of mitochondria. We found that low concentrations (2.5–5 μM) of SFB had a relatively modest effect on LCSC-2 or 293 T cell growth, but damaged mitochondria and increased intracellular ROS. Gene expression profiling of SFB-treated cells was consistent with a shift toward aerobic glycolysis and, accordingly, SFB cytotoxicity was dramatically increased by glucose withdrawal or the glycolytic inhibitor 2-DG. Under metabolic stress, activation of the AMP dependent Protein Kinase (AMPK), but not ROS blockade, protected cells from death. We conclude that mitochondrial damage and ROS drive cell killing by SFB, while glycolytic cell reprogramming may represent a resistance strategy potentially targetable by combination therapies.

Tumor Initiating Cells and Chemoresistance: Which Is the Best Strategy to Target Colon Cancer Stem Cells?

There is an emerging body of evidence that chemoresistance and minimal residual disease result from selective resistance of a cell subpopulation from the original tumor that is molecularly and phenotypically distinct. These cells are called “cancer stem cells” (CSCs). In this review, we analyze the potential targeting strategies for eradicating CSCs specifically in order to develop more effective therapeutic strategies for metastatic colon cancer. These include induction of terminal epithelial differentiation of CSCs or targeting some genes expressed only in CSCs and involved in self-renewal and chemoresistance. Ideal targets could be cell regulators that simultaneously control the stemness and the resistance of CSCs. Another important aspect of cancer biology, which can also be harnessed to create novel broad-spectrum anticancer agents, is the Warburg effect, also known as aerobic glycolysis. Actually, little is yet known with regard to the metabolism of CSCs population, leaving an exciting unstudied avenue in the dawn of the emerging field of metabolomics.

High nitric oxide production, secondary to inducible nitric oxide synthase expression, is essential for regulation of the tumour-initiating properties of colon cancer stem cells

Chronic inflammation is a leading cause of neoplastic transformation in many human cancers and especially in colon cancer (CC), in part due to tumour promotion by nitric oxide (NO) generated at inflammatory sites. It has also been suggested that high NO synthesis, secondary to inducible NO synthase (iNOS) expression, is a distinctive feature of cancer stem cells (CSCs), a small subset of tumour cells with self‐renewal capacity. In this study we explored the contribution of NO to the development of colon CSC features and evaluated potential strategies to treat CC by modulating NO production. Our data show an integral role for endogenous NO and iNOS activity in the biology of colon CSCs. Indeed, colon CSCs with high endogenous NO production (NOhigh) displayed higher tumourigenic abilities than NOlow fractions. The blockade of endogenous NO availability, using either a specific iNOS inhibitor or a genetic knock‐down of iNOS, resulted in a significant reduction of colon CSC tumourigenic capacities in vitro and in vivo. Interestingly, analysis of genes altered by iNOS‐directed shRNA showed that the knockdown of iNOS expression was associated with a significant down‐regulation of signalling pathways involved in stemness and tumour progression in colon CSCs. These findings confirm that endogenous NO plays an important role in defining the stemness properties of colon CSCs through cross‐regulation of several cellular signalling pathways. This discovery could shed light on the mechanisms by which NO induces the growth and invasiveness of CC, providing new insights into the link between inflammation and colon tumourigenesis. Copyright

Establishment of cancer cell lines from rat hepatocholangiocarcinoma and assessment of the role of granulocyte-colony stimulating factor and hepatocyte growth factor in their growth, motility and survival

BACKGROUND/AIMS Oval cells (OCs), putative hepatic stem cells, may give rise to liver cancers. We developed a carcinogenesis regimen, based upon induction of OC proliferation prior to carcinogen exposure. In our model, rats subjected to 2-acetylaminofluorene/ partial-hepatectomy followed by aflatoxin injection (APA regimen) developed well-differentiated hepatocholangiocarcinomas. The aim of this study was to establish and characterize cancer cell lines from this animal model. METHODS Cancer cells were cultured from animals sacrificed eight months after treatment, and single clones were selected. The established cell lines, named LCSCs, were characterized, and their tumorigenicity was assessed in vivo. The roles of granulocyte-colony stimulating factor (G-CSF) and hepatocyte growth factor (HGF) in LCSC growth, survival and motility were also investigated. RESULTS From primary tumors, six cell lines were developed. LCSCs shared with the primary tumors the expression of various OC-associated markers, including cMet and G-CSF receptor. In vitro, HGF conferred protection from death by serum withdrawal. Stimulation with G-CSF increased LCSC growth and motility, while the blockage of its receptor inhibited LCSC proliferation and migration. CONCLUSIONS Six cancer cell lines were established from our model of hepatocholangiocarcinoma. HGF modulated LCSC resistance to apoptosis, while G-CSF acted on LCSCs as a proliferative and chemotactic agent.

Validation of an LC-MS/MS Method for Urinary Lactulose and Mannitol Quantification: Results in Patients with Irritable Bowel Syndrome

Aim. Lactulose/mannitol ratio is used to assess intestinal barrier function. Aim of this work was to develop a robust and rapid method for the analysis of lactulose and mannitol in urine by liquid chromatography coupled to tandem mass spectrometry. Lactulose/mannitol ratio has been measured in pediatric patients suffering from irritable bowel syndrome. Methods. Calibration curves and raffinose, used as internal standard, were prepared in water : acetonitrile 20 : 80. Fifty μL of urine sample was added to 450 μL of internal standard solution. The chromatographic separation was performed using a Luna NH2 column operating at a flow rate of 200 μL/min and eluted with a linear gradient from 20% to 80% water in acetonitrile. Total run time is 9 minutes. The mass spectrometry operates in electrospray negative mode. Method was fully validated according to European Medicine Agency guidelines. Results and Conclusions. Linearity ranged from 10 to 1000 mg/L for mannitol and 2.5 to 1000 mg/L for lactulose. Imprecision in intra- and interassay was lower than 15% for both analytes. Accuracy was higher than 85%. Lactulose/mannitol ratio in pediatric patients is significantly higher than that measured in controls. The presented method, rapid and sensitive, is suitable in a clinical laboratory.

May Gender or Ethnicity Affect Delta Over Baseline Values Obtained by 13-C Urea Breath Test?

INTRODUCTION 13C-urea breath test (UBT) is a non-invasive test for detecting active H. pylori infection. Previous studies showed a correlation of delta over baseline (DOB) values with bacterial load, mucosal inflammation and successful eradication. Gender has been shown to affect DOB in children. Aim of our study was to verify whether gender or ethnicity affects DOB in adults. PATIENTS AND METHODS We retrospectively analyzed data of 2922 patients (1024M/1898F mean age 47±15 years) that underwent UBT in our outpatient unit, from October 2015 to October 2016. Patients were divided based on gender and ethnicity; mean DOB values were then compared. RESULTS 686 pts (23.4%, 258M/428F, mean age 45±17 years) of 2922 pts showed a positive UBT. Prevalence of H. pylori infection was significantly higher in males compared to females (29% vs 22%; p=0,03). Females showed a significant higher mean DOB (34±25 vs 27,6±22; p=0,008). A total of 2922 UBT were performed during the study period (F:1898, 65%; M: 1024 35%). The prevalence of H. pylori infection is 32% in those from Eastern Countries, 28% in those from South America and 40% in both those coming from Africa and Asia. We found significantly lower DOB values in Italians compared to non-Italian (mean DOB 36±27 vs 69±32; p<0.0001). CONCLUSION Our study showed that in our geographic area, prevalence H. pylori infection is higher in males. Moreover, it demonstrates for the first time in our geographic area that adult females show a significantly higher DOB compared to males (p=0,008). Whether this effect may be due to hormonal differences, able to influence gastric emptying, bacterial load, or even the production of urease by H. pylori, merits further investigation.

O0009 FUNCTIONAL SIGNATURE UNDERLYING LIVER CANCER PROGRESSION IN CELL LINES ESTABLISHED FROM A STEM CELL DERIVED HEPATOCHOLANGIOCARCINOMA

Anna Chiara Piscaglia 1, Nathalie Saulnier 1, Valentina Tesori 2, Marta Barba 1, Mariachiara Campanale 1, Flaminia Purchiaroni 1, Emanuela D’angelo 1, Giovanni Cammarota 1, Giovanni Pani 2, Antonio Gasbarrini 1. 1Department of Internal Medicine; Catholic University of Rome – College of Medicine, “a. Gemelli” Hospital, Italy; 2Inst. of General Pathology – Catholic University of Rome – College of Medicine, “a. Gemelli” Hospital, Italy