Anita Balázs

Curcumin and Its Analogue Induce Apoptosis in Leukemia Cells and Have Additive Effects with Bortezomib in Cellular and Xenograft Models

Combination therapy of bortezomib with other chemotherapeutics is an emerging treatment strategy. Since both curcumin and bortezomib inhibit NF-κB, we tested the effects of their combination on leukemia cells. To improve potency, a novel Mannich-type curcumin derivative, C-150, was synthesized. Curcumin and its analogue showed potent antiproliferative and apoptotic effects on the human leukemia cell line, HL60, with different potency but similar additive properties with bortezomib. Additive antiproliferative effects were correlated well with LPS-induced NF-κB inhibition results. Gene expression data on cell cycle and apoptosis related genes, obtained by high-throughput QPCR, showed that curcumin and its analogue act through similar signaling pathways. In correlation with in vitro results similar additive effect could be obsereved in SCID mice inoculated systemically with HL60 cells. C-150 in a liposomal formulation given intravenously in combination with bortezomib was more efficient than either of the drugs alone. As our novel curcumin analogue exerted anticancer effects in leukemic cells at submicromolar concentration in vitro and at 3 mg/kg dose in vivo, which was potentiated by bortezomib, it holds a great promise as a future therapeutic agent in the treatment of leukemia alone or in combination.

Prospective, Multicentre, Nationwide Clinical Data from 600 Cases of Acute Pancreatitis

Objective The aim of this study was to analyse the clinical characteristics of acute pancreatitis (AP) in a prospectively collected, large, multicentre cohort and to validate the major recommendations in the IAP/APA evidence-based guidelines for the management of AP. Design Eighty-six different clinical parameters were collected using an electronic clinical research form designed by the Hungarian Pancreatic Study Group. Patients 600 adult patients diagnosed with AP were prospectively enrolled from 17 Hungarian centres over a two-year period from 1 January 2013. Main Results With respect to aetiology, biliary and alcoholic pancreatitis represented the two most common forms of AP. The prevalence of biliary AP was higher in women, whereas alcoholic AP was more common in men. Hyperlipidaemia was a risk factor for severity, lack of serum enzyme elevation posed a risk for severe AP, and lack of abdominal pain at admission demonstrated a risk for mortality. Abdominal tenderness developed in all the patients with severe AP, while lack of abdominal tenderness was a favourable sign for mortality. Importantly, lung injury at admission was associated with mortality. With regard to laboratory parameters, white blood cell count and CRP were the two most sensitive indicators for severe AP. The most common local complication was peripancreatic fluid, whereas the most common distant organ failure in severe AP was lung injury. Deviation from the recommendations in the IAP/APA evidence-based guidelines on fluid replacement, enteral nutrition and timing of interventions increased severity and mortality. Conclusions Analysis of a large, nationwide, prospective cohort of AP cases allowed for the identification of important determinants of severity and mortality. Evidence-based guidelines should be observed rigorously to improve outcomes in AP.

SPINK1 Promoter Variants in Chronic Pancreatitis.

Objectives Serine protease inhibitor Kazal type 1 (SPINK1) provides an important line of defense against premature trypsinogen activation within the pancreas. Our aim was to identify pathogenic SPINK1 promoter variants associated with chronic pancreatitis (CP). Methods One hundred CP patients (cases) and 100 controls with no pancreatic disease from the Hungarian National Pancreas Registry were enrolled. Direct sequencing of SPINK1 promoter region was performed. Functional characterization of variants was carried out using luciferase reporter gene assay. Results Two common polymorphisms (c.-253T>C and c.-807C>T) were found in both cases and controls. Variant c.253T>C was enriched in cases relative to controls (odds ratio, 2.1; 95% confidence interval, 1.2-3.8; P = 0.015). Variant c.-215G>A was detected in 3 of 100 cases; always linked with the pathogenic variant c.194+2T>C. Novel promoter variants c.-14G>A, c.-108G>T, and c.-246A>G were identified in 1 case each. Functional analysis showed decreased promoter activity for variants c.-14G>A (80%), c.-108G>T (31%), and c.-246A>G (47%) whereas activity of variant c.-215G>A was increased (201%) and variant c.-253T>C was unchanged compared with wild type. Conclusions The common promoter variant c.-253T>C was associated with CP in this cohort. Two of 3 newly identified SPINK1 promoter variants seem to exhibit significant functional defects and should be considered potential risk factors for CP.

Pathogenic cellular role of the p.L104P human cationic trypsinogen variant in chronic pancreatitis

Mutations in the PRSS1 gene encoding human cationic trypsinogen are associated with hereditary and sporadic chronic pancreatitis. High-penetrance PRSS1 mutations found in hereditary pancreatitis alter activation and/or degradation of cationic trypsinogen, thereby promoting intrapancreatic trypsinogen activation. In contrast, a number of rare PRSS1 variants identified in subjects with sporadic chronic pancreatitis cause misfolding and endoplasmic reticulum (ER) stress. Mutation p.L104P is unique among natural PRSS1 variants, since it affects the substrate binding site of trypsin. The aim of the present study was to establish the clinical significance of variant p.L104P through functional analysis. We found that p.L104P trypsin exhibited decreased activity on peptide and protein substrates; however, autoactivation was slightly accelerated. Remarkably, binding of the physiological trypsin inhibitor serine protease inhibitor Kazal type 1 (SPINK1) was decreased by 70-fold. In the presence of the trypsinogen-degrading enzyme chymotrypsin C, mutant p.L104P autoactivated to higher trypsin levels than wild-type trypsinogen. This apparent resistance to degradation was due to slower cleavage at Arg(122) rather than Leu(81) Finally, secretion of mutant p.L104P from transfected cells was markedly reduced due to intracellular retention and aggregation with concomitant elevation of ER stress markers. We conclude that PRSS1 variant p.L104P exhibits a variety of phenotypic changes that can increase risk for chronic pancreatitis. Mutation-induced misfolding and associated ER stress are the dominant effects that support a direct pathogenic role in chronic pancreatitis.

Pancreatic Cancer: Multicenter Prospective Data Collection and Analysis by the Hungarian Pancreatic Study Group.

BACKGROUND AND AIMS Pancreatic cancer is a devastating disease with poor prognosis. There is very limited information available regarding the epidemiology and treatment strategies of pancreatic cancer in Central Europe. The purpose of the study was to prospectively collect and analyze data of pancreatic cancer in the Hungarian population. METHODS The Hungarian Pancreatic Study Group (HPSG) organized prospective, uniform data collection. Altogether 354 patients were enrolled from 14 Hungarian centers. RESULTS Chronic pancreatitis was present in 3.7% of the cases, while 33.7% of the patients had diabetes. Family history for pancreatic cancer was positive in 4.8%. The most frequent presenting symptoms included pain (63.8%), weight loss (63%) and jaundice (52.5%). The reported frequency of smoking and alcohol consumption was lower than expected (28.5% and 27.4%, respectively). The majority of patients (75.6%) were diagnosed with advanced disease. Most patients (83.6%) had a primary tumor located in the pancreatic head. The histological diagnosis was ductal adenocarcinoma in 90.7% of the cases, while neuroendocrine tumor was present in 5.3%. Biliary stent implantation was performed in 166 patients, 59.2% of them received metal stents. Primary tumor resection was performed in 60 (16.9%) patients. Enteral or biliary bypass was done in 35 and 49 patients, respectively. In a multivariate Cox-regression model, smoking status and presence of gemcitabine-based chemotherapy were identified as independent predictors for overall survival. CONCLUSION We report the first data from a large cohort of Hungarian pancreatic cancer patients. We identified smoking status and chemotherapy as independent predictors in this cohort.

Analysis of research activity in gastroenterology: Pancreatitis is in real danger

Objective Biomedical investment trends in 2015 show a huge decrease of investment in gastroenterology. Since academic research usually provides the basis for industrial research and development (R&D), our aim was to understand research trends in the field of gastroenterology over the last 50 years and identify the most endangered areas. Methods We searched for PubMed hits for gastrointestinal (GI) diseases for the 1965–2015 period. Overall, 1,554,325 articles were analyzed. Since pancreatology was identified as the most endangered field of research within gastroenterology, we carried out a detailed evaluation of research activity in pancreatology. Results In 1965, among the major benign GI disorders, 51.9% of the research was performed on hepatitis, 25.7% on pancreatitis, 21.7% on upper GI diseases and only 0.7% on the lower GI disorders. Half a century later, in 2015, research on hepatitis and upper GI diseases had not changed significantly; however, studies on pancreatitis had dropped to 10.7%, while work on the lower GI disorders had risen to 23.4%. With regard to the malignant disorders (including liver, gastric, colon, pancreatic and oesophageal cancer), no such large-scale changes were observed in the last 50 years. Detailed analyses revealed that besides the drop in research activity in pancreatitis, there are serious problems with the quality of the studies as well. Only 6.8% of clinical trials on pancreatitis were registered and only 5.5% of these registered trials were multicentre and multinational (more than five centres and nations), i.e., the kind that provides the highest level of impact and evidence level. Conclusions There has been a clear drop in research activity in pancreatitis. New international networks and far more academic R&D activities should be established in order to find the first therapy specifically for acute pancreatitis.

The Importance of Aquaporin 1 in Pancreatitis and Its Relation to the CFTR Cl- Channel

Aquaporins (AQPs) facilitate the transepithelial water flow involved in epithelial fluid secretion in numerous tissues; however, their function in the pancreas is less characterized. Acute pancreatitis (AP) is a serious disorder in which specific treatment is still not possible. Accumulating evidence indicate that decreased pancreatic ductal fluid secretion plays an essential role in AP; therefore, the aim of this study was to investigate the physiological and pathophysiological role of AQPs in the pancreas. Expression and localization of AQPs were investigated by real-time PCR and immunocytochemistry, whereas osmotic transmembrane water permeability was estimated by the dye dilution technique, in Capan-1 cells. The presence of AQP1 and CFTR in the mice and human pancreas were investigated by immunohistochemistry. Pancreatic ductal HCO3- and fluid secretion were studied on pancreatic ducts isolated from wild-type (WT) and AQP1 knock out (KO) mice using microfluorometry and videomicroscopy, respectively. In vivo pancreatic fluid secretion was estimated by magnetic resonance imaging. AP was induced by intraperitoneal injection of cerulein and disease severity was assessed by measuring biochemical and histological parameters. In the mice, the presence of AQP1 was detected throughout the whole plasma membrane of the ductal cells and its expression highly depends on the presence of CFTR Cl- channel. In contrast, the expression of AQP1 is mainly localized to the apical membrane of ductal cells in the human pancreas. Bile acid treatment dose- and time-dependently decreased mRNA and protein expression of AQP1 and reduced expression of this channel was also demonstrated in patients suffering from acute and chronic pancreatitis. HCO3- and fluid secretion significantly decreased in AQP1 KO versus WT mice and the absence of AQP1 also worsened the severity of pancreatitis. Our results suggest that AQP1 plays an essential role in pancreatic ductal fluid and HCO3- secretion and decreased expression of the channel alters fluid secretion which probably contribute to increased susceptibility of the pancreas to inflammation.

Role of the SLC26A9 Chloride Channel as Disease Modifier and Potential Therapeutic Target in Cystic Fibrosis.

The solute carrier family 26, member 9 (SLC26A9) is an epithelial chloride channel that is expressed in several organs affected in patients with cystic fibrosis (CF) including the lungs, the pancreas, and the intestine. Emerging evidence suggests SLC26A9 as a modulator of wild-type and mutant CFTR function, and as a potential alternative target to circumvent the basic ion transport defect caused by deficient CFTR-mediated chloride transport in CF. In this review, we summarize in vitro studies that revealed multifaceted molecular and functional interactions between SLC26A9 and CFTR that may be implicated in normal transepithelial chloride secretion in health, as well as impaired chloride/fluid transport in CF. Further, we focus on recent genetic association studies and investigations utilizing genetically modified mouse models that identified SLC26A9 as a disease modifier and supported an important role of this alternative chloride channel in the pathophysiology of several organ manifestations in CF, as well as other chronic lung diseases such as asthma and non-CF bronchiectasis. Collectively, these findings and the overlapping endogenous expression with CFTR suggest SLC26A9 an attractive novel therapeutic target that may be exploited to restore epithelial chloride secretion in patients with CF irrespective of their CFTR genotype. In addition, pharmacological activation of SLC26A9 may help to augment the effect of CFTR modulator therapies in patients with CF carrying responsive mutations such as the most common disease-causing mutation F508del-CFTR. However, future research and development including the identification of compounds that activate SLC26A9-mediated chloride transport are needed to explore this alternative chloride channel as a therapeutic target in CF and potentially other muco-obstructive lung diseases.

A Common CCK-B Receptor Intronic Variant in Pancreatic Adenocarcinoma in a Hungarian Cohort:

Objectives Variant c.811+32C>A in intron 4 of the cholecystokinin-B receptor gene (CCKBR) was reported to correlate with higher pancreatic cancer risk and poorer survival. The variant was suggested to induce retention of intron 4, resulting in a new splice form with enhanced receptor activity. Our objective was to validate the c.811+32C>A variant as an emerging biomarker for pancreatic cancer risk and prognosis. Methods We genotyped variant c.811+32C>A in 122 pancreatic adenocarcinoma case patients and 106 control subjects by sequencing and examined its association with cancer risk and patient survival. We tested the functional effect of variant c.811+32C>A on pre–messenger RNA splicing in human embryonic kidney 293T and Capan-1 cells transfected with CCKBR minigenes. Results The allele frequency of the variant was similar between patients and control subjects (18.4% and 17.9%, respectively). Survival analysis showed no significant difference between median survival of patients with the C/C genotype (266 days) and patients with the A/C or A/A genotypes (257 days). CCKBR minigenes with or without variant c.811+32C>A exhibited no difference in expression of the intron-retaining splice variant. Conclusion These data indicate that variant c.811+32C>A in CCKBR does not have a significant impact on pancreatic cancer risk or survival in a Hungarian cohort.

Pancreatic epithelial fluid and bicarbonate secretion is significantly elevated in the absence of peripheral serotonin

We read the manuscript by Sonda et al 1 recently published in Gut with great interest. The authors elegantly demonstrated that lack of peripheral serotonin (5-HT) in tryptophan hydroxylase 1 knockout (TPH1−/−) mice remarkably limited pancreatic damage and leucocyte infiltration during the early phase of cerulein-induced acute pancreatitis (AP) and identified 5-HT as an important regulator of zymogen secretion in acinar cells. Although the study was very comprehensive, the ductal function of TPH1−/− mice was not investigated, which might be another key player in this protection. Notably, 5-HT was shown to inhibit fluid and HCO3− secretion of pancreatic ductal epithelial cells,2 which play a pivotal role in pancreatic physiology and can influence the severity of AP.3 Thus, we investigated the possible alterations of pancreatic ductal secretion in TPH1−/− mice. To achieve our aim, intralobular/interlobular pancreatic ducts were isolated from the pancreas of wild-type (TPH1+/+) and TPH1−/− mice. HCO3− secretion was measured by three different, but complementary methods using …