EFFECTS OF CELLULAR SHORT-TERM STARVATION ON CONVENTIONAL CHEMOTHERAPY RESPONSE IN HUMAN CANCER: UNDERSTANDING OF MOLECULAR MECHANISMS AND MICRORNAS INVOLVEMENT

Background: Short Term Starvation (STS) is a type of dietary restriction able to reduce tumorigenesis and cancer progression but molecular bases of this effect are still unclear. Aim: In vitro analysis of STS effects in presence of chemotherapy and evaluation of microRNAs (miRNAs) involvement. Results: STS affects the expression profiles of miRNAs involved in chemotherapy response leading to cancer cells sensitization and to healthy cells protection

Dietary restriction: could it be considered as speed bump on tumor progression road?

Dietary restrictions, including fasting (or long-term starvation), calorie restriction (CR), and short-term starvation (STS), are considered a strong rationale that may protect against various diseases, including age-related diseases and cancer. Among dietary approaches, STS, in which food is not consumed during designed fasting periods but is typically not restricted during designated feeding periods, seems to be more suitable, because other dietary regimens involving prolonged fasting periods could worsen the health conditions of cancer patients, being they already naturally prone to weight loss. Until now, the limited amount of available data does not point to a single gene, pathway, or molecular mechanism underlying the benefits to the different dietary approaches. It is well known that the healthy effect is mediated in part by the reduction of nutrient-related pathways. The calorie restriction and starvation (long- and short-term) also suppress the inflammatory response reducing the expression, for example, of IL-10 and TNF-α, mitigating pro-inflammatory gene expression and increasing anti-inflammatory gene expression. The dietary restriction may regulate both genes involved in cellular proliferation and factors associated to apoptosis in normal and cancer cells. Finally, dietary restriction is an important tool that may influence the response to chemotherapy in preclinical models. However, further data are needed to correlate dietary approaches with chemotherapeutic treatments in human models. The aim of this review is to discuss the effects of various dietary approaches on the cancer progression and therapy response, mainly in preclinical models, describing some signaling pathways involved in these processes

A headlight on liquid biopsies: a challenging tool for breast cancer management

Breast cancer is the most frequent carcinoma and second most common cause of cancer-related mortality in postmenopausal women. The acquisition of somatic mutations represents the main mechanism through which cancer cells overcome physiological cellular signaling pathways (e.g., PI3K/Akt/mTOR, PTEN, TP53). To date, diagnosis and metastasis monitoring is mainly carried out through tissue biopsy and/or re-biopsy, a very invasive procedure limited only to certain locations and not always feasible in clinical practice. In order to improve disease monitoring over time and to avoid painful procedure such as tissue biopsy, liquid biopsy may represent a new precious tool. Indeed, it represents a basin of “new generation” biomarkers that are spread into the bloodstream from both primary and metastatic sites. Moreover, elevated concentrations of circulating tumor DNA (ctDNA) as well as circulating tumor cells (CTCs) have been found in blood plasma of patients with various tumor types. Nowadays, several new approaches have been introduced for the detection and characterization of CTCs and ctDNA, allowing a real-time monitoring of tumor evolution. This review is focused on the clinical relevance of liquid biopsy in breast cancer and will provide an update concerning CTCs and ctDNA utility as a tool for breast cancer patient monitoring during the course of disease

Analysis of tissue and circulating microRNA expression during metaplastic transformation of the esophagus

Genetic changes involved in the metaplastic progression from squamous esophageal mucosa toward Barrett's metaplasia and adenocarcinoma are almost unknown. Several evidences suggest that some miRNAs are differentially expressed in Barrett's esophagus (BE) and esophageal adenocarcinoma. Among these, miR-143, miR-145, miR-194, miR-203, miR-205, miR-215 appear to have a key role in metaplasia and neoplastic progression. The aim of this study was to analyze deregulated miRNAs in serum and esophageal mucosal tissue biopsies to identify new biomarkers that could be associated with different stages of esophageal disease. Esophageal mucosal tissue biopsies and blood samples were collected and analyzed for BE diagnosis. Quantitative Real-time PCR was used to compare miRNA expression levels in serum and 60 disease/ normal-paired tissues from 30 patients diagnosed with esophagitis, columnar-lined oesophagus (CLO) or BE. MiRNA expression analysis showed that miR-143, miR-145, miR-194 and miR-215 levels were significantly higher, while miR-203 and miR-205 were lower in BE tissues compared with their corresponding normal tissues. Esophageal mucosa analysis of patients with CLO and esophagitis showed that these miRNAs were similarly deregulated but to a lesser extent keeping the same trend and CLO appeared as intermediate step between esophagitis and BE. Analysis on circulating miRNA levels confirmed that miR-194 and miR-215 were significantly upregulated in both BE and CLO compared to esophagitis, while miR-143 was significantly upregulated only in the Barrett group. These findings suggest that miRNAs may be involved in neoplastic/ metaplastic progression and miRNA analysis might be useful for progression risk prediction as well as for monitoring of BE/CLO patients

Serum Albumin Is Inversely Associated With Portal Vein Thrombosis in Cirrhosis

We analyzed whether serum albumin is independently associated with portal vein thrombosis (PVT) in liver cirrhosis (LC) and if a biologic plausibility exists. This study was divided into three parts. In part 1 (retrospective analysis), 753 consecutive patients with LC with ultrasound-detected PVT were retrospectively analyzed. In part 2, 112 patients with LC and 56 matched controls were entered in the cross-sectional study. In part 3, 5 patients with cirrhosis were entered in the in vivo study and 4 healthy subjects (HSs) were entered in the in vitro study to explore if albumin may affect platelet activation by modulating oxidative stress. In the 753 patients with LC, the prevalence of PVT was 16.7%; logistic analysis showed that only age (odds ratio [OR], 1.024; P = 0.012) and serum albumin (OR, -0.422; P = 0.0001) significantly predicted patients with PVT. Analyzing the 112 patients with LC and controls, soluble clusters of differentiation (CD)40-ligand (P = 0.0238), soluble Nox2-derived peptide (sNox2-dp; P < 0.0001), and urinary excretion of isoprostanes (P = 0.0078) were higher in patients with LC. In LC, albumin was correlated with sCD4OL (Spearman's rank correlation coefficient [r(s)], -0.33; P < 0.001), sNox2-dp (r(s), -0.57; P < 0.0001), and urinary excretion of isoprostanes (r(s), -0.48; P < 0.0001) levels. The in vivo study showed a progressive decrease in platelet aggregation, sNox2-dp, and urinary 8-iso prostaglandin F2 alpha-III formation 2 hours and 3 days after albumin infusion. Finally, platelet aggregation, sNox2-dp, and isoprostane formation significantly decreased in platelets from HSs incubated with scalar concentrations of albumin. Conclusion: Low serum albumin in LC is associated with PVT, suggesting that albumin could be a modulator of the hemostatic system through interference with mechanisms regulating platelet activation

Analysis of miRNA expression profile induced by short term starvation in breast cancer cells treated with doxorubicin

Recent studies showed that dietary approaches restricting food intake can be helpful to hinder tumor progression. To date, the molecular mechanisms are unclear and a key role seems to be exerted by nutrient-related signaling pathways. Since several evidences showed that non-coding small RNAs, including microRNAs, are correlated to cancer progression and antiblastic treatment response, our work aims to study their involvement in a triple negative breast cancer (TNBC) cell line treated with doxorubicin under Short Term Starvation (STS) condition. Human TNBC cell line MDA-MB-231 and healthy breast cell line MCF10A were treated with 1 μM doxorubicin for 24 h under STS condition for 48 h and miRNA expression profiles were analyzed using Taqman® Low Density Array A human microRNA microfluidic cards. In addition, the expression of specific mRNAs and miRNAs differentially expressed under STS was analyzed using Real-time PCR analyses. MiRNA expression profile analysis in MDA-MB-231 and MCF10A cells treated with doxorubicin under STS for 48 h could explain the molecular mechanisms underlying anticancer effects associated to STS. Among deregulated miRNAs, a subset, including miR-15b, miR-23a, miR-26a, miR-29a, miR-106b, miR-128, miR-149, miR-181a, miR- 192, miR-193b, miR-195, miR-324-3p and miR-494, has been shown to be involved in pathways related to drug sensitivity/resistance. The obtained data from our study suggest a potential involvement of some miRNAs in molecular pathways mediating the anticancer effects of STS in doxorubicin-treated breast cancer cells. Preliminary results seem to be encouraging and, in future, could allow the discovery of new potential targets useful for the development of new therapeutic approaches

Local permeability changes, passive degassing and related gas hazard at the Baia di Levante area (Vulcano island, Italy)

Vulcano, the southernmost island of the Aeolian archipelago (Italy), is presently characterized by active fumarolic fields located along the rim of La Fossa cone and the shoreline of the Baia di Levante beach, in the northern portion of the island.The Baia di Levante fumarolic vents are fed by a shallow hydrothermal aquifer heated by magmatic gases rising from the deep down, with a spatial distribution strongly affected by the local fracture network. These fractures are the expression of a deformation field, dominated by a northward motion to Lipari, abruptly decaying to the Vulcanello peninsula, immediately northward of the Baia di Levante beach. Variable rates of fluid transfer to the surface, following permeability changes affecting the fracture network are among the results of stress field variations over time which induce fluctuations in the pressure state of the hydrothermal system. Under these conditions, increments in hydrothermal gas flow, able to cause an increase of gas hazard, could be determined by a rearrangement of the shallow permeability distribution induced by changes in the deformation field. In this case not associated to any variation in the volcanic activity state. Since 2009 an huge gas flow increment has been noticed in some undersea vents of the Baia di Levante area, leading to increase of gas hazard in their immediate surroundings. On the contrary, the acquired data from the INGV volcanic surveillance program didn’t suggest any correlated increase of the magmatic fluid component in the degassing activity.In July 2015, we carried out multi-parametric geochemical surveys in this area, based on direct (thermocouple) and indirect (thermal infrared camera and pyrometer) soil temperature, soil CO2 flux, atmospheric concentration of CO2 and H2S measurements at low elevation (one meter a.s.l.). The chemical and isotopic composition of low temperature fumarole gases was determined too.The comparison of the new data with previous surveys carried out in the same area, and the general information from the INGV monitoring program exclude a possible renewal of volcanic activity as the source for the observed anomalies.The most reliable cause for the observed localized gas flow anomalies should therefore be referred to a rearrangement of the local shallow permeability field driven by geodynamic stress variations. The differential subsidence rate acting in the Baia di Levante area, as resulting from the geodetic data from literature, could be accounted as the engine able to close and open fractures, modifying the permeability distribution and, finally, conveying major amount of gases in restricted areas where an increased gas hazard is observed.PublishedVienna, Austria4V. Dinamica dei processi pre-eruttivi6V. Pericolosità vulcanica e contributi alla stima del rischio2SR. VULCANI - Servizi e ricerca per la Societ

VISCERAL FAT TISSUE ACTIVITY DOES NOT CORRELATE TO HIGH GRADE PROSTATE CANCER RISK AT BIOPSY

Introduction/Aim: High-grade prostate cancer (PCa) has been reported in association with metabolic syndrome (MS). In a previous study we found no significant correlation between body mass index (BMI) and prevalence of high Gleason score at biopsy (1). BMI could be not an accurate marker of the endocrine activity of visceral adipose tissue responsible of high plasmatic levels of adipokines promoting PCa aggressiveness. We estimated visceral adiposity dysfunction by the visceral adiposity index (VAI) considering waist circumference (WC), BMI, triglycerides (TG) and high density lipoproteins (HDL) plasma levels of each patient (2). The aim was to correlate VAI values with PCa detection rate and Gleason patterns 4 and 5 at biopsy. Patients and Methods: Patients who underwent prostate biopsy for suspicious digital rectal examination and/or elevated PSA levels were enrolled. After informed consent, a transrectal prostate biopsy, 12 cores at least (24 in case of re-biopsy), was performed. VAI was expressed as: WC/[39.68 + (1.88 × BMI)] × TG/1.03 × 1.31/HDL, assuming VAI=1 in healthy, non obese men with TG and HDL levels within normal limits. PCa detection at biopsy, Gleason score patterns, VAI and BMI were statistically analyzed using the Mann Whitney U-test. Results: Ninety-five patients were entered with a median age of 67 years (range=47-79). The median BMI was 27 kg/m2 (range=17.4-40) and the median VAI was 4.47 (range=1.3-15.6). Median PSA was 7.9 ng/ml (range=0.47- 53). A prostate nodule was palpable in 27 (28.4%) patients. Ten patients (10.5%) had a previous negative biopsy. A prostate cancer was diagnosed in 43 (45.2%) patients, Gleason patterns 4 and 5 were detected in 18 (41.8%) patients. Median BMI and VAI were 27.4 Kg/m2 and 26.3 Kg/m2 (p=0.53) and 4.25 and 4.66 (p=0.28) in patients with positive and negative biopsy, respectively. Median BMI and VAI resulted 27.7 Kg/m2 and 27.3 Kg/m2 and 4.78 and 3.98 in patients with and without Gleason patterns 4 or 5, respectively. No statistically significant difference was highlighted for VAI (p=0.37) or BMI (p=0.85). Discussion and Conclusion: The identification of patients harboring an aggressive PCa remains an important goal. To date the relation between MS and PCa remains contradictory. Moreover, an accurate marker of MS has not yet been determined (3). VAI might be a more accurate marker than BMI in indicating the activity of visceral fat. In spite of VAI adoption, our analysis does not reveal any statistically significant correlation between VAI, PCa detection rate and incidence of Gleason patterns 4 or 5 at biopsy. Diet, race and other environmental and genetic factors, playing a promoting or protective role in PCa, should be also considered in further studies. The statistical support of the GSTU Foundation is acknowledged. 1 Serretta V, Caruana G, Sommatino F, et al: Does exist A correlation between BMI and Gleason patterns 4 and 5 at prostate biopsy? J Cytol Histol 4: 182, 2013. 2 Amato MC, Giordano C, Galia M et al: Visceral Adiposity Index: a reliable indicator of visceral fat function associated with cardiometabolic risk. Diabetes Care 33: 920, 2010. 3 Bhindi B, Locke J, Alibhai SM et al: Dissecting the association between metabolic syndrome and prostate cancer risk: analysis of a large clinical cohort. Eur Urol 67(1): 64-70, 2015. Epub 2014 Feb 14

THE ROLE OF INTRAVESICAL GLYCOSAMINOGLYCANS IN TOXICITY INDUCED BY ADJUVANT INTRAVESICAL THERAPY: GENETIC LABORATORY EVIDENCE

Introduction and Objectives: The intravesical administration of hyaluronic acid and chondroitin sulphate solution (HA-CS) has been proven active in patients affected by interstitial cystitis (1). The gene expression of fibronectin (FN) in bladder washings has recently been correlated with local toxicity of adjuvant intravesical therapy (2). The aim of the study was to investigate the genetic evidence of the healing or protective action that HA-CS could carry out also in patients suffering from topical toxicity induced by intravesical adjuvant therapy given for non-muscle invasive bladder cancer. Materials and Methods: The study included 50 patients submitted to adjuvant intravesical therapy with mitomycin, epirubicin or bacillus Calmette–Guérin (BCG). Ten age-matched healthy patients were enrolled as control group. Before, during and after intravescical therapy, bladder washing samples were collected to investigate the gene expression of FN. In 9 more patients the samples were collected also immediately before and a week after the instillation of HA-CS. Topical toxicity was classified into 3 grades: 0-1, light (no medical therapy); 2, moderate (medical therapy); 3, severe (instillation postponed). Bladder washing samples were analyzed by isolation of cellular RNA using a miRNeasy Mini Kit (Qiagen®). RT-PCR was performed in order to analyze FN gene expression. Changes in the FN content were calculated using the ΔΔCt method after normalization with endogenous reference 18s rRNA and calibrating Ct value for each RNA obtained for triplicate reactions. Statistical analysis was performed to correlate the FN gene expression to tumor characteristics, treatment, topical toxicity and intravesical administration of HA-CS. Results: FN median value before the adjuvant treatment was 1.1-fold, with higher levels in patients with multiple tumors (median FN=1.5; mean=3.9; p=0.0003). Twenty patients (34%) showed grade 2-3 toxicity. Compared to controls (FN=1), FN increased during therapy a median of 4-fold (range=0.2-45.2; mean=7.5) in presence of grade 2-3 toxicity, remaining stable in asymptomatic patients (median FN=0.6; range=0.1-3.2), with a statistically significant difference (p=0.0005). In 9 patients, one week after single instillation of HA-CS, the median FN gene expression decreased from 3.2 to 0.33 with concomitant symptomatic relief. Discussion and Conclusion: Fibronectin is a fundamental element for the repair of urothelial damage. FN gene is probably activated by the need of fibronectin for healing process and down-regulated by the integrity of bladder urothelium. In our preliminary experience FN gene expression in bladder washings resulted strictly related to local toxicity induced by intravesical therapy. It increases after transurethral resection (TUR) of multiple tumors due to the greater urothelial damage. It increases also during intravesical therapy reaching the highest levels in case of severe toxicity due to the extensive urothelial damage. A single instillation of intravesical hyaluronic acid and chondroitin sulphate solution induces a rapid reduction of FN gene expression levels, particularly when high levels are present. The FN gene downregulation induced in patients with toxicity is due to intravesical therapy and might represent an objective and measurable indicator of the healing activity of intravesical instillation of HA-CS. 1 Van Agt S et al: Treatment of interstitial cystitis by intravesical instillation of hyaluronic acid: A prospective study on 31 patients, Prog Urol 21: 218-225, 2011. 2 Serretta V et al: Fibronectin (FN), Epidermal Growth Factor-Receptor (EGF-R) and Heparin-Binding Epidermal Growth Factor-like Growth Factor (HB- EGF) urinary expression and topical toxicity of adjuvant intravesical therapy for non muscle invasive bladder cancer (NMI-BC). 28th EAU Congress, Stockholm, 2014. Eur Urol Suppl 13: e409, 2014