Depletion of Ras Suppressor-1 (RSU-1) promotes cell invasion of breast cancer cells through a compensatory upregulation of a truncated isoform.

Extracellular matrix (ECM)-adhesion proteins and actin cytoskeleton are pivotal in cancer cell invasion. Ras Suppressor-1 (RSU-1), a cell-ECM adhesion protein that interacts with PINCH-1, thus being connected to Integrin Linked Kinase (ILK), alpha-parvin (PARVA), and actin cytoskeleton, is up-regulated in metastatic breast cancer (BC) samples. Apart from the originally-identified gene (RSU-1L), an alternatively-spliced isoform (RSU-1-X1) has been reported. We used non-invasive MCF-7 cells, expressing only RSU-1L, and highly invasive MDA-MB-231-LM2 expressing both isoforms and generated stable shRNA-transduced cells lacking RSU-1L, while the truncated RSU-1-X1 isoform was depleted by siRNA-mediated silencing. RSU-1L depletion in MCF-7 cells resulted in complete abrogation of tumor spheroid invasion in three-dimensional collagen gels, whereas it promoted MDA-MB-231-LM2 invasion, through a compensatory upregulation of RSU-1-X1. When RSU-1-X1 was also eliminated, RSU-1L-depletion-induced migration and invasion were drastically reduced being accompanied by reduced urokinase plasminogen activator expression. Protein expression analysis in 23 human BC samples corroborated our findings showing RSU-1L to be upregulated and RSU-1-X1 downregulated in metastatic samples. We demonstrate for the first time, that both RSU-1 isoforms promote invasion in vitro while RSU-1L elimination induces RSU-1-X1 upregulation to compensate for the loss. Hence, we propose that both isoforms should be blocked to effectively eliminate metastasis.

The Role of Exercise in the Quality of Life in Patients After Pancreatectomy: A Prospective Randomized Controlled Trial.

BACKGROUND: Pancreatic resection is still a challenging operation characterised by high morbidity. The quality of life in patients after pancreatectomy is a critical outcome. The aim of our trial is to prove whether or not exercise has any benefit to the life of these patients. METHODS: The study was an open-label, randomized clinical trial. The patients were selected according to the Consolidated Standard of Reporting Trials criteria. The study was registered at the International Standard Randomized Controlled Trial registry (ISRCTN) with the study ID ISRCTN1087174. The study was approved by the Bioethics and Deontology Committee, Medical School of Aristotle University, Thessaloniki (ref: 166/29.10.2015). RESULTS: Once the allocation and the follow-up were completed, 21 patients in the exercise group and 22 in the control group were analyzed. There was no statistical difference between the two groups regarding co-morbidities and disease characteristics; however, the quality of life and the total status of health were superior in the exercise group. CONCLUSIONS: Exercise can improve the quality of life in patients after complex operations like pancreatectomy.

Effects of Wnt-1 blockade in DEN-induced hepatocellular adenomas of mice.

Recent evidence has suggested that downregulation of the Wnt/ß-catenin signaling pathway may contribute to the development and growth of HCC. Consequently, elements of this pathway have begun to emerge as potential targets for improving outcomes of anti-HCC. Thus, the present study sought to examine the effects of Wnt-1 blockade using the classical diethylnitrosamine (DEN)-induced chemical carcinogenesis mouse model of HCC. The depletion of Wnt-1 using neutralizing antisera was done for ten consecutive days at the age of 9 months and mice were examined for the following 20 days. At that time, DEN-treated mice had multiple variably-sized hepatic cell adenomas. Anti-Wnt-1 was particularly potent in suppressing the expression of critical elements of the Wnt/ß-catenin signaling pathway, such as ß-catenin and Frizzled-1 receptor, however, not Dickkopf-related protein 1. This effect co-existed with the suppression of Cyclin D1, FOXM1, NF-κΒ and c-Jun commensurate with proliferation and apoptosis blockade in hepatocellular adenomas, and reduced Bcl-2 and c-Met in the serum of mice. Nonetheless, tumor size and multiplicity were found to be unaffected, suggesting that apoptosis may be equally important to proliferation in the context of counteracting DEN induced hepatocellular adenomas of mice.

Nucleos(t)ide analogues for the prevention of hepatitis B recurrence after liver transplantation do not affect serum phosphorus levels.

BACKGROUND: Nucleos(t)ide analogues (NAs) constitute the backbone of treatment for the prevention of hepatitis B virus recurrence after liver transplantation (LT). Decline in serum phosphorus levels is a common side effect of nucleotide therapy. Our aim was to assess the impact of nucleotide treatment on the occurrence of hypophosphatemia after LT and determine possible predictors. METHODS: We retrospectively analyzed data from liver transplant recipients who had been transplanted for various indications. All patients were evaluated every 3 months. Each patient was considered to be having hypophosphatemia when at least one value of serum phosphorus ≤2.5 mg/dL was detected. RESULTS: In total, 109 patients [83 males (76%)] with a mean age of 55±10 years were included. 46/67 (67%) patients with hepatitis B received a nucleotide. The rate of hypophosphatemia (55%) was not different between patients with hepatitis B and those transplanted for other indications (62%). Patients receiving a nucleotide did not run a greater risk of hypophosphatemia than patients receiving only nucleosides (59% vs. 48%, P=0.39). Male gender and everolimus use were associated with the occurrence of hypophosphatemia in patients with hepatitis B. In multivariate analysis only gender was associated with hypophosphatemia (odds ratio 11.43, 95%CI -2.11 to -0.49; P=0.0025). CONCLUSIONS: Hypophosphatemia occurs in more than half of liver transplant recipients regardless of the indication for LT. Male gender and everolimus use seem to predispose to hypophosphatemia, whereas the type of antiviral agent does not.

Telomere Length in Peripheral Blood Mononuclear Cells of Patients on Chronic Hemodialysis Is Related With Telomerase Activity and Treatment Duration.

Telomere shortening to a critical limit is associated with replicative senescence. This process is prevented by the enzyme telomerase. Oxidative stress and chronic inflammation are factors accelerating telomere loss. Chronic hemodialysis, typically accompanied by oxidative stress and inflammation, may be also associated with replicative senescence. To test this hypothesis, we determined telomere length and telomerase activity in peripheral blood mononuclear cells (PBMCs) in a cross-sectional study. Hemodialysis patients at the University Hospital Larissa and healthy controls were studied. Telomere length was determined by the TeloTAGGG Telomere Length Assay and telomerase activity by Telomerase PCR-ELISA (Roche Diagnostics GmbH, Mannheim, Germany). We enrolled 43 hemodialysis patients (17 females; age 65.0 ± 12.7 years) and 23 controls (six females; age 62.1 ± 15.7 years). Between the two groups, there was no difference in telomere length (6.95 ± 3.25 vs. 7.31 ± 1.96 kb; P = 0.244) or in telomerase activity (1.82 ± 2.91 vs. 2.71 ± 3.0; P = 0.085). Telomere length correlated inversely with vintage of hemodialysis (r = -0.332, P = 0.030). In hemodialysis patients, positive telomerase activity correlated with telomere length (r = 0.443, P = 0.030). Only age, and neither telomere length nor telomerase activity, was an independent survival predictor (hazard ratio 1.116, 95% confidence interval 1.009-1.234, P = 0.033). In this study, telomere length and telomerase activity in PBMCs are not altered in hemodialysis patients compared with healthy controls. Long duration of hemodialysis treatment is associated with telomere shortening and positive telomerase activity with an increased telomere length in PBMCs of hemodialysis patients. The underlying mechanism and clinical implications of our findings require further investigation.

Ras suppressor-1 promotes apoptosis in breast cancer cells by inhibiting PINCH-1 and activating p53-upregulated-modulator of apoptosis (PUMA); verification from metastatic breast cancer human samples.

Metastasis, responsible for most deaths from breast cancer (BC), is a multistep process leading to cancer cell spread. Extracellular matrix (ECM)-related adhesion and apoptosis resistance play pivotal role in metastasis. Ras suppressor-1 (RSU-1) localizes to cell-ECM adhesions and binds to pro-survival adhesion protein PINCH-1. Little is known about the role of RSU-1 in BC. In the present study, we investigated the role of RSU-1 in BC metastasis using two BC cell lines that differ in terms of their metastatic potential and a set of 32 human BC samples from patients with or without lymph node metastasis. We show that RSU-1 is upregulated in the aggressive MDA-MB-231 cells compared to MCF-7 and that its silencing by siRNA leads to upregulation of PINCH-1, induction of proliferation and reduction of apoptosis through downregulation of the pro-apoptotic gene p53-upregulated-modulator-of-apoptosis (PUMA). Our findings in the cell lines were further validated in the human BC tissues where normal adjacent tissues were used as controls. We demonstrate for the first time, that RSU-1 expression is upregulated in metastatic BC samples and downregulated in non-metastatic while it is negatively correlated with PINCH-1 and positively correlated with PUMA expression, suggesting that a pro-apoptotic mechanism is in place in metastatic BC samples and identifying RSU-1 as a potentially interesting molecule that needs to be evaluated further as a novel BC metastasis biomarker.

Polymorphisms of renin-angiotensin system and natriuretic peptide receptor A genes in patients of Greek origin with a history of myocardial infarction.

We assessed the association between (CA)n repeat polymorphism of angiotensinogen (AGT), 250 base pair (bp) insertion/deletion (I/D) of angiotensin-converting enzyme (ACE), tetranucleotide repeat polymorphism (TCTG)n of renin (REN), (CT)n repeat polymorphism of the natriuretic peptide receptor A (NPRA) genes, and the presence and extent of coronary artery disease (CAD) in Greek patients with a history of myocardial infarction (MI). A total of 158 post-MI patients referred for coronary angiography were compared with 144 controls. The SS genotype of the AGT gene was related with an increased risk for 3-vessel CAD (odds ratio [OR], 1.94; 95% confidence interval [CI], 1.05-3.61; P = .041), whereas the SL genotype was related with a decreased risk (OR, 0.44; 95% CI, 0.22-0.87; P = .019). Moreover, there was a trend for the SL genotype of the REN gene toward increased risk for CAD. There was a significant association between (CA)n polymorphism of the AGT gene and the extent of CAD in Greek patients with a history of MI.

Spur cells and spur cell anemia in hospitalized patients with advanced liver disease: Incidence and correlation with disease severity and survival.

AIM: Spur cell anemia (SCA) is a form of acquired hemolytic anemia seen in patients with advanced cirrhosis and particularly in patients with alcoholic cirrhosis. The aim of the present study was to evaluate the incidence of spur cells and spur cell anemia in patients with advanced liver disease and to correlate the presence of spur cell anemia with survival. METHODS: During a 33-month period, all patients with advanced cirrhosis (Child-Pugh-Turcott score [CPT]>/=7] who were hospitalized in our department for various reasons were included in this study. RESULTS: A total of 54 patients were included in the study; 26 patients had spur cells on peripheral blood smear (median 4, range 1-14). Patients with spur cells had more advanced liver disease compared with those without spur cells (CPT score, P < 0.0001 and MELD score, P < 0.0001), lower hemoglobin levels (P < 0.0001), higher bilirubin levels (total/unconjugated, P < 0.0001), higher reticulocyte count (P < 0.0001) and more prolonged international normalized ratio (INR; P < 0.0001). Patients with 5% spur cells or more had more advanced disease compared with patients with 1-4% spur cells (CPT score, P = 0.004 and MELD score, P = 0.003), lower hemoglobin levels (P = 0.033), more elevated bilirubin levels (total/unconjugated, P = 0.006) and more prolonged INR (P = 0.04). Three-month survival was lower in patients with spur cells compared with patients without spur cells (P = 0.017 and P = 0.104, respectively). Patients with 5% spur cells or more had lower 3-month survival compared with those with 1-4% spur cells (P = 0.014). CONCLUSION: Presence of spur cells in patients with advanced cirrhosis is not always accompanied by spur cell anemia. The presence of 5% spur cells or more and/or hemolytic anemia is associated with poor prognosis and these patients might have to be given priority for liver transplantation.

Association of renin-angiotensin system and natriuretic peptide receptor A gene polymorphisms with hypertension in a Hellenic population.

INTRODUCTION: Hypertension results from the interaction of genetic and environmental factors. Since the renin-angiotensin and the natriuretic peptide systems contribute to blood pressure regulation, variations in the relative genes are candidates for the development of hypertension. MATERIALS AND METHODS: In 194 hypertensives and 304 controls of Hellenic origin, the possible association between the (CA)n repeat polymorphism of angiotensinogen (AGT), the 250 bp insertion/deletion (I/D) of angiotensin-converting enzyme (ACE), the tetranucleotide repeat polymorphism (TCTG)n of renin, and the (CT)n repeat polymorphism of the natriuretic peptide receptor A (NPRA) and hypertension was assessed. RESULTS: No association between AGT and NPRA polymorphisms and hypertension was observed. The presence of ID or DD genotype of ACE was associated with an increased risk for hypertension compared with the II genotype (OR: 1.782 [95% CI: 1.032-3.077]), whereas the LL genotype of the renin gene was associated with a decreased risk compared with the SS genotype (OR: 0.174 [95% CI: 0.044-0.689]). However, after adjustment for confounding factors only the latter association remained. CONCLUSIONS: In the present study conducted in a homogeneous Hellenic population, no associations between AGT,ACE, and NPRA gene polymorphisms and hypertension were found. The presence of a significant negative association between the LL polymorphism of the renin gene and hypertension requires further confirmation.

Bilateral Morgagni Hernia: Primary Repair without a Mesh.

We present a case of bilateral Morgagni hernia in a 68-year-old male with an intermittent history of progressive onset of breath shortness and occasional cardiac arrhythmias. Diagnosis was made by clinical examination and the findings in a plain chest radiograph and was confirmed by computed tomography scan. The patient was operated electively and subjected to a transabdominal approach. A bilateral subcostal incision revealed a large right side anterior diaphragmatic defect with a hernia containing the ascending colon, the majority of the transverse colon and a huge amount of omentum. Also a second smaller defect was found on the left side with no hernia inside. After large bowel and omentum had been taken down to the peritoneal cavity, both defects were primarily closed using interrupted nylon sutures without the use of a mesh. The patient recovered very well, had an uneventful postoperative course and was released on the 5th postoperative day. 15-month follow-up failed to reveal any signs of recurrence.

Leptin receptor isoforms mRNA expression in peripheral blood mononuclear cells from patients with chronic viral hepatitis.

There is accumulating evidence that leptin has a pleiotropic role in hematopoiesis, immune response, fibrogenesis, and hepatocarcinogenesis. We investigated the expression of leptin and leptin receptor (OB-R) at the protein level by flow cytometry and also quantified by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) the two major leptin receptor isoforms (OB-Rl, OB-Rs) in peripheral blood mononuclear cells (PBMCs) of patients with hepatitis B (HBV; n = 31), hepatitis C (HCV; n = 34), and nonviral liver disease (n = 25), and healthy controls (n = 36), as well as in liver tissues of HBV (n = 8), HCV (n = 7), and healthy individuals (n = 6). Serum leptin levels were measured in all participants (N = 126). We observed significantly lower OB-Rl and OB-Rs mRNA levels in PBMCs of HBV and HCV patients compared with healthy controls and nonviral liver disease patients (P < 0.05). Flow cytometry analysis confirmed the real-time RT-PCR results. Expression of leptin and OB-Rl was significantly increased in viral hepatitis liver tissues compared with healthy tissues (P < 0.01). OB-Rl mRNA levels were not associated with hepatitis patients' clinical status (inactive, chronic hepatitis, or cirrhosis). We also found decreased serum leptin in HBV and HCV patients compared with healthy individuals and the nonviral liver disease group. Leptin was expressed in 3 of 34 HCV (8.8%) and 19 of 25 (76%) nonviral liver disease patients. Moreover, expression of OB-Rl and OB-Rs were associated when all individuals were grouped together (r = 0.78, P < 0.001). In conclusion, our findings may suggest the involvement of the leptin system in the immunopathology of chronic viral hepatitis.

Correlation between radiation-induced telomerase activity and human telomerase reverse transcriptase mRNA expression in HeLa cells.

PURPOSE: To quantify and correlate human telomerase reverse transcriptase (hTERT) mRNA expression with telomerase activity (TA) after ionizing irradiation of HeLa cells. MATERIALS AND METHODS: TA and hTERT mRNA expression were evaluated, at 24-h intervals, in HeLa cells cultured for up to 144 h, before and after treatment with increasing doses of 6 MV photon ionizing radiation (5 - 20 Gy), using the telomeric repeat amplification protocol (TRAP) assay and real-time reverse transcriptase polymerase chain reaction (RT-PCR), respectively. Cell viability was determined using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. A prototype phantom was constructed for accurate irradiation of HeLa cells. RESULTS: Treated cells showed a decrease in viability with increasing radiation dose, and a correlation was observed with post-treatment period. TA and hTERT mRNA expression of HeLa cells increased for the first 24 h after irradiation. The maximal increases were approximately two times the un-irradiated cell levels at 24 h post-irradiation, followed by a decrease and a return to the control levels 72 h post-irradiation. The time-course of telomerase activation after 24 h, differed among radiation doses. A dose-dependent G2/M arrest was observed 24 h post-irradiation, along with an increase in polyploidy 48 h post-irradiation and afterwards. CONCLUSION: A correlation between TA and hTERT mRNA expression and a radiation induced cell cycle dependent modification of hTERT mRNA expression was established for the first 24 h post-irradiation.

Ductal plate malformation and congenital hepatic fibrosis Clinical and histological findings in four patients.

Congenital hepatic fibrosis belongs to the fibrocystic diseases of the liver and represents ductal plate malformation of interlobular bile ducts, along with a destructive cholangiopathy associated with fibrosis. Four patients with congenital hepatic fibrosis are described. Their median age at presentation was 25 years; none of them had a family history of liver or renal disease. Variceal bleeding was the initial manifestation in three patients. All of them required frequent endoscopic variceal ligation sessions and distal splenorenal shunting was also performed in two, almost obviating the need for further variceal ligation. Variceal bleeding did not recur during follow-up. One of these three patients rarely exhibited acute cholangitis; administration of ursodeoxycholic acid resulted in complete remission. In contrast, the fourth patient showed frequent severe episodes of acute cholangitis but normal cholangiographic findings. He underwent liver transplantation but died 2 months later. Laboratory findings disclosed pancytopenia in all patients whereas hepatic synthetic capacity was well preserved. Renal function was unaffected despite the presence of polycystic kidneys in two patients. In summary, congenital hepatic fibrosis can also be diagnosed in older ages, might have strikingly different manifestations and is associated with prominent portal hypertension necessitating aggressive management in order to prevent variceal bleeding.

Improvement of rat liver graft function after storage in University of Wisconsin solution containing testicular hyaluronidase.

Hyaluronan accumulates at sites of inflammation, which affects the organization of matrix and thereby the proliferation, migration, and adherence of cells. In this study we investigated possible beneficial effects of the hyaluronan-degrading enzyme hyaluronidase on rat liver graft viability. Orthotopic rat liver transplantation was performed using a cuff technique in Wistar AL Bacharach Glaxo (WAG) rats grafted with WAG livers, which had been stored in the University of Wisconsin (UW) solution or in UW solution enriched with testicular hyaluronidase. Liver tissue architecture, as well as tissue and serum hyaluronan levels, were determined using immunohistochemistry and biochemical assays. Addition of testicular hyaluronidase (0.4 mg/mL) to livers preserved for 24 hours in cold UW solution followed by brief exposure to Ringer's lactate both prolonged the function of the grafted livers and improved their viability (4 of 10 grafts survived, compared with 0 of 10 in the control group). Hyaluronidase treatment did not damage the liver tissue architecture, and a reduced edema was observed in the survivors. Furthermore, 10 minutes after restoration of circulation, higher serum hyaluronan levels were observed in nonsuccessful compared with successful transplantations, whereas no differences in the levels of other serum viability markers were detected. We conclude that addition of testicular hyaluronidase to storage UW solution limits liver cell damage and considerably improves graft function. Furthermore, our data suggest that serum hyaluronan level is a better marker than other serum markers for early evaluation of postoperative graft function.