Antanas Gulbinas

Inhibition of Heme Oxygenase-1 Increases Responsiveness of Pancreatic Cancer Cells to Anticancer Treatment

Heme oxygenase-1 (HO-1) is believed to represent a key enzyme for the protection of cells against “stress.” Its overexpression in different types of human cancers supports the notion that HO-1 provides a growth advantage and contributes to cellular resistance against chemotherapy and radiotherapy. Given the poor survival rates of patients with pancreatic cancer due to its aggressive growth behavior and its exceptional resistance to all known forms of anticancer treatment, we have investigated the expression of HO-1 in human pancreatic cancer cells growth behavior and prognosis. Expression of HO-1 was analyzed in human pancreatic cancer samples in comparison with normal pancreas by quantitative PCR, Western blot, and confocal microscopy. The influence of radiotherapy and chemotherapy on HO-1 expression in pancreatic cancer cell lines was evaluated. Furthermore, HO-1 expression was specifically suppressed by small interfering RNA transfection and subsequently the alterations of growth behavior and resistance to anticancer treatment were tested. Human pancreatic cancer showed a 6-fold and 3.5-fold HO-1 up-regulation in comparison to normal pancreas based on mRNA and protein level, respectively (P < 0.05). Cancer tissues revealed marked HO-1 immunoreactivity in tumor cells and in tumor associated immunocytes. Treatment of the pancreatic cancer cell lines with gemcitabine or radiation strongly induced HO-1 expression. Targeted knockdown of HO-1 expression led to pronounced growth inhibition of the pancreatic cancer cells and made tumor cells significantly more sensitive to radiotherapy and chemotherapy. Therefore, specific inhibition of HO-1 expression may be a new option in pancreatic cancer therapy and may be used as sensitizer to chemotherapy and radiotherapy.

Value of the different prognostic systems and biological markers for predicting severity and progression of acute pancreatitis

Abstract Objective. Several tools have been developed for severity stratification in acute pancreatitis (AP). They include single biochemical markers and complex scoring systems, all of which aim at an early detection of severe AP to optimize monitoring and treatment of these patients. The aim of this study was to reassess and compare the value of some known and newly-introduced prognostic markers in the clinical context. Material and Methods. We have conducted a prospective observational study. One hundred and eight patients with a diagnosis of AP and onset of the disease within last 72 h were included in this study. Clinical data and expression results of some serum biochemical markers were used for statistical analysis. The diagnostic performance of scores predicting severity and progression of AP, cut-off values, specificity, and sensitivity were established using receiver operating characteristic curve analysis. Results. Among single biochemical markers, C-reactive protein remains the most useful. Despite its delayed increase, it is accurate, cheap, and widely available. Interleukin-6 and macrophage migration inhibitory factor seem to be new promising parameters for use in clinical routine. Pancreas specific scores (Imrie-Glasgow, pancreatitis outcome prediction) and scores assessing organ dysfunction (acute physiology and chronic health evaluation II, multiple organ dysfunction score, and Marshall score) remain of value in determining the severity, complications, and possible outcome of AP. Conclusions. Indication, timing, and consequences of the methods applied need to be carefully considered and incorporated into clinical assessments. Currently, there is no single prognostic marker that would cover the whole range of problems associated with the treatment of AP. The prediction of severity and progression of AP can be achieved using a series of accurate methods. The decision to undertake interventional or surgical treatment is the most complex task requiring both clinical judgment and meticulous monitoring of the patient.

Finite element modeling of cooled-tip probe radiofrequency ablation processes in liver tissue

Finite element model of radiofrequency ablation (RFA) with cooled-tip probe in liver has been developed by employing COMSOL Multiphysics software. It describes coupled electric, thermal and sodium chloride solution infiltration flow phenomena taking place during ablation processes. Features of hydraulic capacity, saturation of the tissue by infiltration, and dependency of electrical conductivity on the damage integral of the tissue have been supplied to the model. RFA experiments have validated the model. Physical parameters describing hydraulic capacity and hydraulic conductivity in the tissue, as well as, the relation of electrical conductivity against the value of damage integral have been determined.

Whole Blood DNA Aberrant Methylation in Pancreatic Adenocarcinoma Shows Association with the Course of the Disease: A Pilot Study

Pancreatic tumors are usually diagnosed at an advanced stage in the progression of the disease, thus reducing the survival chances of the patients. Non-invasive early detection would greatly enhance therapy and survival rates. Toward this aim, we investigated in a pilot study the power of methylation changes in whole blood as predictive markers for the detection of pancreatic tumors. We investigated methylation levels at selected CpG sites in the CpG rich regions at the promoter regions of p16, RARbeta, TNFRSF10C, APC, ACIN1, DAPK1, 3OST2, BCL2 and CD44 in the blood of 30 pancreatic tumor patients and in the blood of 49 matching controls. In addition, we studied LINE-1 and Alu repeats using degenerate amplification approach as a surrogate marker for genome-wide methylation. The site-specific methylation measurements at selected CpG sites were done by the SIRPH method. Our results show that in the patient’s blood, tumor suppressor genes were slightly but significantly higher methylated at several CpG sites, while repeats were slightly less methylated compared to control blood. This was found to be significantly associated with higher risk for pancreatic ductal adenocarcinoma. Additionally, high methylation levels at TNFRSCF10C were associated with positive perineural spread of tumor cells, while higher methylation levels of TNFRSF10C and ACIN1 were significantly associated with shorter survival. This pilot study shows that methylation changes in blood could provide a promising method for early detection of pancreatic tumors. However, larger studies must be carried out to explore the clinical usefulness of a whole blood methylation based test for non-invasive early detection of pancreatic tumors.

Value of routine clinical tests in predicting the development of infected pancreatic necrosis in severe acute pancreatitis.

Objective. Fine-needle aspiration (FNA) is the procedure of choice for accurate diagnosis of infected necrosis. However, invasive procedures increase the risk of secondary pancreatic infection and the timing of FNA is still a matter for debate. Our objective was to assess the value of routine clinical tests to determine the minimal risk for infected necrosis, thereby optimizing timing and selection of patients for image-guided FNA. Material and methods. This prospective, non-randomized study comprised 90 patients with acute necrotizing pancreatitis. The data of 52 patients were used for discriminant function analysis to determine the differences between patients with infected necrosis and those with sterile necrosis. Cut-off points for variables were established using receiver operating characteristic (ROC) curve analysis and logistic regression was performed to determine the risk of infected necrosis. The clinical relevance of the defined diagnostic system was prospectively tested in a further 38 consecutive patients with acute necrotizing pancreatitis (ANP). Results. Discriminant function analysis showed that C-reactive protein (CRP) and white blood cell (WBC) values were significant discriminators between patients with sterile necrosis and those with infected necrosis. Cut-off values of 81 mg/l for CRP and 13×109/l for WBC were established. The predicted risk for infected necrosis is approx. 1.4% if both tests are below the defined cut-off values. Consequently, we found FNA unnecessary in this subset of patients, unless otherwise indicated, as this invasive procedure per se carries a certain risk of bacterial contamination. Conclusions. Routine clinical tests are helpful in diagnosing the development of infected necrosis. Based on the application of classification functions, the timing and selection of patients for image-guided FNA can be optimized.

Inter-locus as well as intra-locus heterogeneity in LINE-1 promoter methylation in common human cancers suggests selective demethylation pressure at specific CpGs

BackgroundHypomethylation of long interspersed element (LINE)-1 has been observed in tumorigenesis when using degenerate assays, which provide an average across all repeats. However, it is unknown whether individual LINE-1 loci or different CpGs within one specific LINE-1 promoter are equally affected by methylation changes. Conceivably, studying methylation changes at specific LINE-1 may be more informative than global assays for cancer diagnostics. Therefore, with the aim of mapping methylation at individual LINE-1 loci at single-CpG resolution and exploring the diagnostic potential of individual LINE-1 locus methylation, we analyzed methylation at 11 loci by pyrosequencing, next-generation bisulfite sequencing as well as global LINE-1 methylation in bladder, colon, pancreas, prostate, and stomach cancers compared to paired normal tissues and in blood samples from some of the patients compared to healthy donors.ResultsMost (72/80) tumor samples harbored significant methylation changes at at least one locus. Notably, our data revealed not only the expected hypomethylation but also hypermethylation at some loci. Specific CpGs within the LINE-1 consensus sequence appeared preferentially hypomethylated suggesting that these could act as seeds for hypomethylation. In silico analysis revealed that these CpG sites more likely faced the histones in the nucleosome. Multivariate logistic regression analysis did not reveal a significant clinical advantage of locus-specific methylation markers over global methylation markers in distinguishing tumors from normal tissues.ConclusionsMethylation changes at individual LINE-1 loci are heterogeneous, whereas specific CpGs within the consensus sequence appear to be more prone to hypomethylation. With a broader selection of loci, locus-specific LINE-1 methylation could become a tool for tumor detection.

Tumor‐related factors and patient's age influence survival after resection for ampullary adenocarcinoma

BACKGROUND/PURPOSE The majority of surgeons agree that ampullary adenocarcinoma should be removed by partial pancreatoduodenectomy. Favoring extended resection, based on the uncertainty of the preoperative diagnosis and the higher probability of clear resection margins, we aimed to disclose the results of this surgical procedure in terms of postoperative morbidity and mortality, and to identify prognosticators of long-term survival. METHODS We documented, prospectively, 25 consecutive patients with adenocarcinoma of the papilla of Vater in whom pylorus-preserving pancreatoduodenectomy was performed. Clinical data, pathology reports, International Union Against Cancer (UICC) tumor stage, postoperative morbidity, mortality, and long-term follow-up results were evaluated. The Kaplan-Meier method and log-rank test were applied for univariate analysis. The Cox proportional hazard model was used for multivariate analysis. RESULTS Postoperative mortality was 4%, overall morbidity was 32%, and pancreas-associated morbidity was 8%. Mean survival time was 53.8 months. Tumor size, N status, UICC stage, lymphatic invasion, blood vessel infiltration, R0 resection, and age of patient at the cutoff of 70 years were independent predictors of survival on univariate analysis. Multivariate analysis, however, disclosed no independent predictors of prognosis. CONCLUSIONS Pancreatoduodenectomy for ampullary carcinoma is reasonable in terms of postoperative morbidity and mortality. Tumor-related factors, R0 resection, and advanced age appeared as the main predictors of survival.

Fatty acids of erythrocyte membrane in acute pancreatitis patients

AIM To evaluate changes in the fatty acid composition of erythrocyte membrane phospholipids during severe and mild acute pancreatitis (AP) of alcoholic and nonalcoholic etiology. METHODS All consecutive patients with a diagnosis of AP and onset of the disease within the last 72 h admitted to the Hospital of Lithuanian University of Health Sciences between June and December 2007 were included. According to the Acute Physiology and Chronic Health Evaluation (APACHE II) scale, the patients were subdivided into the mild (APACHE II score < 7, n = 22) and severe (APACHE II score ≥ 7, n = 17) AP groups. Healthy individuals (n = 26) were enrolled as controls. Blood samples were collected from patients on admission to the hospital. Fatty acids (FAs) were extracted from erythrocyte phospholipids and expressed as percentages of the total FAs present in the chromatogram. The concentrations of superoxide dismutase and glutathione peroxidase were measured in erythrocytes. RESULTS We found an increase in the percentages of saturated and monounsaturated FAs, a decrease in the percentages of total polyunsaturated FAs (PUFAs) and n-3 PUFAs in erythrocyte membrane phospholipids of AP patients compared with healthy controls. Palmitic (C16:0), palmitoleic (C16:1n7cis), arachidonic (C20:4n6), docosahexaenoic (DHA, C22:6n3), and docosapentaenoic (DPA, C22:5n3) acids were the major contributing factors. A decrease in the peroxidation and unsaturation indexes in AP patients as well as the severe and mild AP groups as compared with controls was observed. The concentrations of antioxidant enzymes in the mild AP group were lower than in the control group. In severe AP of nonalcoholic etiology, the percentages of arachidic (C20:0) and arachidonic (C20:4n6) acids were decreased as compared with the control group. The patients with mild AP of nonalcoholic etiology had the increased percentages of total saturated FAs and gama linoleic acid (C18:3n6) and the decreased percentages of elaidic (C18:1n9t), eicosapentaenoic acid (EPA, C20:5n3), DPA (C22:5n3), DHA (C22:6n3) as well as total and n-3 PUFAs in erythrocyte membrane phospholipids. CONCLUSION The composition of FAs in erythrocyte membranes is altered during AP. These changes are likely to be associated with alcohol consumption, inflammatory processes, and oxidative stress.

HuR mediated post-transcriptional regulation as a new potential adjuvant therapeutic target in chemotherapy for pancreatic cancer

AIM To investigate the expression of HuR in pancreatic ductal adenocarcinoma (PDA) and to assess the effects of HuR silencing on the expression of cyclooxygenase-2 (COX-2) and heme oxygenase-1 (HO-1) and the in vitro response to gemcitabine (GEM) treatment in pancreatic cell lines. METHODS We compared the expression of HuR, COX-2, and HO-1 in PDA and normal pancreatic tissue using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. In addition, the HuR, COX-2 and HO-1 were analyzed in four types of cancer cell lines (MiaPaca2, Su.86.86, Capan-1, and Capan-2) with and without GEM treatment. Immunocytofluorescence analysis was used to investigate HuR localization in cells. Cell viability and response to GEM after HuR silencing were determined with the 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide test and the crystal violet clonogenic assay, respectively. To measure apoptosis, activation of caspases 3/7 was evaluated using immunofluorescence. RESULTS In PDA tissue obtained from patients not treated with GEM, HuR mRNA expression was 3.2 times lower (P < 0.05) and COX-2 and HO-1 mRNA expression was 2.3-fold and 7.2-fold higher (P < 0.05), respectively, than normal pancreatic tissue (from organ donor). qRT-PCR analysis showed that HuR, COX-2, and HO-1 mRNA were overexpressed in all cancer cell lines treated with the half maximal inhibitory concentration (IC50) dose of GEM compared with control cells (P < 0.05). Western blot analysis revealed that COX-2 and HO-1 levels were significantly decreased in cancer cells after HuR silencing. Furthermore, HuR silencing increased the response to GEM treatment and decreased cell viability by 11.6%-53.7% compared to control cell lines. Caspases 3 and 7 were activated after HuR silencing and GEM treatment in all pancreatic cancer cell lines. In comparison, treatment with GEM alone did not activate caspases 3 and 7 in the same cell lines. CONCLUSION HuR mediated post-transcriptional upregulation of COX-2 and HO-1 expression after GEM treatment in pancreatic cancer cells. HuR silencing significantly increased the effectiveness of GEM treatment in vitro.

Heme oxygenase-1 gene promoter polymorphism is associated with the development of necrotizing acute pancreatitis.

Objectives Acute pancreatitis is a severe and frequently life-threatening disease, which can lead to pancreatic necrosis, acute lung injury, systemic inflammatory response syndrome, and other complications. In this study, we hypothesized that the expression of heme oxygenase-1 determined by the number of guanidinium thiocyanate (GT) repeats can influence the occurrence of acute pancreatitis. Methods Patients with acute pancreatitis (n = 131) and age- and sex-matched healthy controls (n = 108) were studied. The polymerase chain reaction products were analyzed by ABI 3130 genetic analyzer and the exact size of the polymerase chain reaction products was determined by GeneMapper software. A short allele was defined as containing 27 GT repeats or fewer, whereas a long allele was more than 27 repeats. Results The subjects were categorized into 3 groups on the basis of the genotype results: 1 short and 1 long, 2 short, and 2 long alleles (L/L). Patients with necrotizing disease more frequently were carriers of LL genotype compared with those who had edematous acute pancreatitis. Furthermore, logistic regression analysis revealed that the presence of L/L allele type doubles the risk for developing pancreatic necrosis in patients with acute pancreatitis. Conclusions The polymorphism of the GT repeats in the heme oxygenase-1 promoter region may be a risk factor for developing severe and necrotizing acute pancreatitis.