Blood-Based Diagnosis and Risk Stratification of Patients with Pancreatic Intraductal Papillary Mucinous Neoplasm (IPMN).

PURPOSE: Intraductal papillary mucinous neoplasm (IPMN) is a precursor of pancreatic ductal adenocarcinoma. Low-grade dysplasia has a relatively good prognosis, whereas high-grade dysplasia and IPMN invasive carcinoma require surgical intervention. However, diagnostic distinction is difficult. We aimed to identify biomarkers in peripheral blood for accurate discrimination. EXPERIMENTAL DESIGN: Sera were obtained from 302 patients with IPMNs and 88 healthy donors. For protein biomarkers, serum samples were analyzed on microarrays made of 2,977 antibodies. A support vector machine (SVM) algorithm was applied to define classifiers, which were validated on a separate sample set. For microRNA biomarkers, a PCR-based screen was performed for discovery. Biomarker candidates confirmed by quantitative PCR were used to train SVM classifiers, followed by validation in a different sample set. Finally, a combined SVM classifier was established entirely independent of the earlier analyses, again using different samples for training and validation. RESULTS: Panels of 26 proteins or seven microRNAs could distinguish high- and low-risk IPMN with an AUC value of 95% and 94%, respectively. Upon combination, a panel of five proteins and three miRNAs yielded an AUC of 97%. These values were much better than those obtained in the same patient cohort by using the guideline criteria for discrimination. In addition, accurate discrimination was achieved between other patient subgroups. CONCLUSIONS: Protein and microRNA biomarkers in blood allow precise diagnosis and risk stratification of IPMN cases, which should improve patient management and thus the prognosis of IPMN patients. See related commentary by Löhr and Pantel, p. 1387.

Antifibrotic and tumor microenvironment modulating effect of date palm fruit (Phoenix dactylifera L.) extracts in pancreatic cancer.

Date palm fruit (Phoenix dactylifera L.) is an endemic functional food, with great nutritional and economic importance due to its phytochemical compositions. The microenvironment of pancreatic cancer consists of cellular and acellular components, including fibroblasts, myofibroblasts, pancreatic stellate cells (PSCs), immune cells, blood vessels, extracellular matrix (ECM) and soluble proteins, such as cytokines and growth factors. The ECM represents a physical barrier that protects the tumor cell from active therapeutic compounds. In this study, four different solvents; water, ethanol, acetone, and ethyl acetate have been used to extract natural products from date palm fruit using a maceration method. The prepared extracts were investigated for antifibrotic (expression of fibronectin-1 and alpha-smooth muscle actin) and antiproliferative activity in tumor necrosis factor (TNF) stimulated PSCs in vitro. Based on the pharmacological test results, the ethyl acetate extract was subsequently partitioned into nine fractions based on polarity using silica gel column chromatography. These nine collective fractions were further evaluated for their activity. Ethanol, ethyl acetate and acetone, but not water extract significantly reduced PSC proliferation (p < 0.05). Date fruit fractions reduced fibrosis, decreased PSC activity and reversed the PSCs' fibrotic phenotype. The findings suggest a new approach for targeting pancreatic cancer through the modulation of PSC activity, thereby possibly enhancing the effect of known anticancer drugs. Moreover, date palm fruit appears to have chemopreventive activity protecting from pancreatic and probably other types of cancer, and thereby might be useful candidate to the pharmaceutical and nutraceutical industries in the development of natural compound-based industrial anticancer product.

Impact of the secretome of activated pancreatic stellate cells on growth and differentiation of pancreatic tumour cells.

Pancreatic ductal adenocarcinoma (PDAC) exists in a complex desmoplastic microenvironment. As part of it, pancreatic stellate cells (PSCs) provide a fibrotic niche, stimulated by a dynamic communication between activated PSCs and tumour cells. Investigating how PSCs contribute to tumour development and for identifying proteins that the cells secrete during cancer progression, we studied by means of complex antibody microarrays the secretome of activated PSCs. A large number of secretome proteins were associated with cancer-related functions, such as cell apoptosis, cellular growth, proliferation and metastasis. Their effect on tumour cells could be confirmed by growing tumour cells in medium conditioned with activated PSC secretome. Analyses of the tumour cells' proteome and mRNA revealed a strong inhibition of tumour cell apoptosis, but promotion of proliferation and migration. Many cellular proteins that exhibited variations were found to be under the regulatory control of eukaryotic translation initiation factor 4E (eIF4E), whose expression was triggered in tumour cells grown in the secretome of activated PSCs. Inhibition by an eIF4E siRNA blocked the effect, inhibiting tumour cell growth in vitro. Our findings show that activated PSCs acquire a pro-inflammatory phenotype and secret proteins that stimulate pancreatic cancer growth in an eIF4E-dependent manner, providing further insight into the role of stromal cells in pancreatic carcinogenesis and cancer progression.

Comparison of the tumor cell secretome and patient sera for an accurate serum-based diagnosis of pancreatic ductal adenocarcinoma.

Pancreatic cancer is the currently most lethal malignancy. Toward an accurate diagnosis of the disease in body liquids, we studied the protein composition of the secretomes of 16 primary and established cell lines of pancreatic ductal adenocarcinoma (PDAC). Compared to the secretome of non-tumorous cells, 112 proteins exhibited significantly different abundances. Functionally, the proteins were associated with PDAC features, such as decreased apoptosis, better cell survival and immune cell regulation. The result was compared to profiles obtained from 164 serum samples from two independent cohorts - a training and a test set - of patients with PDAC or chronic pancreatitis and healthy donors. Eight of the 112 secretome proteins exhibited similar variations in their abundance in the serum profile specific for PDAC patients, which was composed of altogether 189 proteins. The 8 markers shared by secretome and serum yielded a 95.1% accuracy of distinguishing PDAC from healthy in a Receiver Operating Characteristic curve analysis, while any number of serum-only markers produced substantially less accurate results. Utility of the identified markers was confirmed by classical enzyme linked immunosorbent assays (ELISAs). The study highlights the value of cell secretome analysis as a means of defining reliable serum biomarkers.

Clinical proteomics: promises, challenges and limitations of affinity arrays.

After the establishment of DNA/RNA sequencing as a means of clinical diagnosis, the analysis of the proteome is next in line. As a matter of fact, proteome-based diagnostics is bound to be even more informative, since proteins are directly involved in the actual cellular processes that are responsible for disease. However, the structural variation and the biochemical differences between proteins, the much wider range in concentration and their spatial distribution as well as the fact that protein activity frequently relies on interaction increase the methodological complexity enormously, particularly if an accuracy and robustness is required that is sufficient for clinical utility. Here, we discuss the contribution that protein microarray formats could play towards proteome-based diagnostics.

Prediction of recurrence of non muscle-invasive bladder cancer by means of a protein signature identified by antibody microarray analyses.

About 70% of newly diagnosed cases of bladder cancer are low-stage, low-grade, non muscle-invasive. Standard treatment is transurethral resection. About 60% of the tumors will recur, however, and in part progress to become invasive. Therefore, surveillance cystoscopy is performed after resection. However, in the USA and Europe alone, about 54 000 new patients per year undergo repeated cystoscopies over several years, who do not experience recurrence. Analysing in a pilot study resected tumors from patients with (n = 19) and without local recurrence (n = 6) after a period of 5 years by means of an antibody microarray that targeted 724 cancer-related proteins, we identified 255 proteins with significantly differential abundance. Most are involved in the regulation and execution of apoptosis and cell proliferation. A multivariate classifier was constructed based on 20 proteins. It facilitates the prediction of recurrence with a sensitivity of 80% and a specificity of 100%. As a measure of overall accuracy, the area under the curve value was found to be 91%. After validation in additional sample cohorts with a similarly long follow-up, such a signature could support decision making about the stringency of surveillance or even different treatment options.

Proteome variations in pancreatic stellate cells upon stimulation with proinflammatory factors.

Pancreatic stellate cells are key mediators in chronic pancreatitis and play a central role in the development of pancreatic fibrosis, stromal formation, and progression of pancreatic cancer. This study was aimed at investigating molecular changes at the level of the proteome that are associated with the activation of pancreatic stellate cells by proinflammatory factors, namely TNF-α, FGF2, IL6, and chemokine (C-C motif) ligand 4 (CCL4). They were added individually to cells growing in serum-free medium next to controls in medium supplemented with serum, thus containing a mixture of them all, or in serum-free medium alone. Variations were detected by means of a microarray of 810 antibodies targeting relevant proteins. All tested factors triggered increased proliferation and migration. Further analysis showed that TNF-α is the prime factor responsible for the activation of pancreatic stellate cells. CCL4 is associated with cellular neovascularization, whereas FGF2 and IL6 induction led to better cellular survival and decreased apoptotic activity of the stellate cells. The identified direct effects of individual cytokines on human pancreatic stellate cells provide new insights about their contribution to pancreatic cancer promotion.

Immunoassay-based proteome profiling of 24 pancreatic cancer cell lines.

Pancreatic ductal adenocarcinoma is one of the most deadly forms of cancers, with a mortality that is almost identical to incidence. The inability to predict, detect or diagnose the disease early and its resistance to all current treatment modalities but surgery are the prime challenges to changing the devastating prognosis. Also, relatively little is known about pancreatic carcinogenesis. In order to better understand relevant aspects of pathophysiology, differentiation, and transformation, we analysed the cellular proteomes of 24 pancreatic cancer cell lines and two controls using an antibody microarray that targets 741 cancer-related proteins. In this analysis, 72 distinct disease marker proteins were identified that had not been described before. Additionally, categorizing cancer cells in accordance to their original location (primary tumour, liver metastases, or ascites) was made possible. A comparison of the cells' degree of differentiation (well, moderately, or poorly differentiated) resulted in unique marker sets of high relevance. Last, 187 proteins were differentially expressed in primary versus metastatic cancer cells, of which the majority is functionally related to cellular movement.

Secretome profiling with antibody microarrays.

Following the advances in human genome sequencing, attention has shifted in part toward the elucidation of the encoded biological functions. Since proteins are the driving forces behind very many biological activities, large-scale examinations of their expression variations, their functional roles and regulation have moved to the central stage. A significant fraction of the human proteome consists of secreted proteins. Exploring this set of molecules offers unique opportunities for understanding molecular interactions between cells and fosters biomarker discovery that could advance the detection and monitoring of diseases. Antibody microarrays are among the relatively new proteomic methodologies that may advance the field significantly because of their relative simplicity, robust performance and high sensitivity down to single-molecule detection. In addition, several aspects such as variations in amount, structure and activity can be assayed at a time. Antibody microarrays are therefore likely to improve the analytical capabilities in proteomics and consequently permit the production of even more informative and reliable data. This review looks at recent applications of this novel platform technology in secretome analysis and reflects on the future.

Analysis conditions for proteomic profiling of mammalian tissue and cell extracts with antibody microarrays.

Antibody microarrays are a developing tool for global proteomic profiling. A protocol was established that permits robust analyses of protein extracts from mammalian tissues and cells rather than body fluids. The factors optimized were buffer composition for surface blocking, blocking duration, protein handling and processing, labeling parameters like type of dye, molar ratio of label versus protein, and dye removal, as well as incubation parameters such as duration, temperature, buffer, and sample agitation.

Single-step procedure for the isolation of proteins at near-native conditions from mammalian tissue for proteomic analysis on antibody microarrays.

The process of extracting comprehensive proteome representations is a crucial step for many proteomic studies. While antibody microarrays are an evolving and promising methodology in proteomics, the issue of protein extraction from tissues for this kind of analysis has never been addressed. Here, we describe a single-step extraction buffer for the isolation of proteins from mammalian tissues under native conditions in an effective and reproducible manner. Protein was extracted from cell lines BxPC-3 and SU.86.86, rat organs (pancreas, liver, heart and lung) and human pancreatic cancer tissues using several buffer systems that contained individual nonionic or zwitterionic detergents in comparison to commercial extraction buffers. Also, detergent combinations were used that included at least one polymeric phenylethylene glycol, a long-chain amidosulfobetaine, cholate and a zwitterionic detergent. Extracts were analyzed for protein quantity and quality. The detergent cocktails exhibited superior extraction capacity. Additionally, they demonstrated a substantially higher recovery of membrane and compartmental proteins as well as much better preservation of protein functionality. Also, they did not interfere with subsequent analysis steps such as labeling. In Western blot and antibody microarray assays, they outperformed the other buffer systems, indicating that they should also be useful for other types of proteomic studies.

Antiglycation and antioxidant effect of carnosine against glucose degradation products in peritoneal mesothelial cells.

BACKGROUND/AIM: Toxicity with advanced glycation end products (AGEs) is a major problem in uremic patients. Treatment with peritoneal dialysis (PD) exacerbates AGE formation as a result of bioincompatibility of the conventional peritoneal dialysis fluid (PDF). The presence of glucose degradation products (GDPs) in PDF is the main cause of its bioincompatibility. Carnosine is an endogenous dipeptide with a powerful antiglycation/antioxidant activity. In an attempt to improve PDF biocompatibility, we evaluated the effect of carnosine in human peritoneal mesothelial cells (HPMC) incubated with PDF or GDPs in vitro. METHODS: HPMC were incubated for short or prolonged time with PDF in the presence or absence of carnosine. Similarly, HPMC were incubated in the same condition but with a combination of GDPs. Following the incubation, cells were tested for their viability, protein carbonyl content and reactive oxygen species (ROS) production. RESULTS: Results demonstrated a significant protective effect of carnosine to HPMC in both acute and chronic conditions with PDF or GDPs as judged by the enhancement of cell viability, preserved protein from modification and decreased ROS production. CONCLUSION: Carnosine enhanced HPMC viability against the toxic effect of GDPs probably through protection of cellular protein from modification and from ROS-mediated oxidative damage. The salutary effect of carnosine may render it a desirable candidate for improving PDF biocompatibility and reducing AGE complications in PD patients.

Decreased formation of advanced glycation end-products in peritoneal fluid by carnosine and related peptides.

BACKGROUND: Formation of advanced glycation end-products (AGEs) is a major problem in uremic patients treated with peritoneal dialysis (PD). Application of additives with known anti-glycosylation properties to PD fluid may be beneficial in minimizing the formation of AGEs. This study aimed to evaluate the effect of carnosine and its related peptides homocarnosine and anserine against the formation of AGEs in PD fluid. METHODS: PD solutions (1.5% dextrose) were incubated with human serum albumin (HSA) or collagen (type IV) with or without 10 mmol/L of each of carnosine, anserine, homocarnosine, histidine, and aminoguanidine. The formation of AGEs was followed by fluorescence spectrophotometry at weekly intervals for 7 weeks. For the determination of the acute effect of carnosine and related compounds, HSA and collagen were incubated with 4.25% dextrose PD solutions for 24 hours, followed by incubation with 20 mmol/L of carnosine and related compounds for another 24 hours. The rate of AGE formation was monitored by fluorescence spectrophotometry. RESULTS: Carnosine and related compounds showed effective regression in AGE formation in both types of proteins in both long- and short-term exposure to PD fluids at a rate of effectiveness of the order of carnosine > homocarnosine > anserine, aminoguanidine > histidine in long-term exposure, and homocarnosine > carnosine > aminoguanidine > anserine > histidine in short-term exposure. CONCLUSION: Carnosine and related peptides could suppress the formation of AGEs initiated by PD fluid. This observation may provide a new therapeutic approach for the prevention and treatment of the AGE-related complications in PD patients.

Elevated levels of alkanals, alkenals and 4-HO-alkenals in plasma of hemodialysis patients.

BACKGROUND/AIMS: Several studies have implicated reactive carbonyl compounds (RCOs), especially those derived from lipid peroxidation, in the development of complications frequently associated with hemodialysis (HD) treatment. However, there is still much unknown regarding the nature and concentration of RCOs in HD patients. This study was designed to evaluate the level of toxic aldehydes in the plasma of HD patients and to determine the extent to which these aldehydes contribute to RCO toxicity among these patients. METHODS: 15 aldehydes of the alkanal, alkenal and 4-HO-alkenal type were measured in the plasma of 17 HD patients and 20 healthy controls. In addition, protein modification markers such as carbonyl content (CO), free thiol (SH) and residual free amino groups, as well as amyloid fibrils were also determined. RESULTS: 11 of the 15 aldehydes were significantly elevated in the HD group when compared with the controls. Correlation studies in the HD group revealed high relationships between total alkenals plus total 4-HO-alkenals versus CO, total alkanals versus NH2, total aldehydes versus SH, and total 4-HO-alkenals versus fibril. CONCLUSION: The increased levels of alkanals, alkenals and 4-HO-alkenals of lipid peroxidation in the plasma of HD patients may greatly contribute to the toxicity of RCOs. The pattern of modification of plasma protein by each group of aldehydes may provide new evidence on the in vivo mechanisms of toxicity triggered by these aldehydes on their target molecules.

Hypoglycemic and antioxidant effect of oleuropein in alloxan-diabetic rabbits.

Patients with diabetes mellitus are likely to develop certain complication such as retinopathy, nephropathy and neuropathy as a result of oxidative stress and overwhelming free radicals. Treatment of diabetic patients with antioxidant may be of advantage in attenuating these complications. Oleuropein, the active constituent of olive leaf (Olea europaea), has been endowed with many beneficial and health promoting properties mostly linked to its antioxidant activity. This study aimed to evaluate the significance of supplementation of oleuropein in reducing oxidative stress and hyperglycemia in alloxan-induced diabetic rabbits. After induction of diabetes, a significant rise in plasma and erythrocyte malondialdehyde (MDA) and blood glucose as well as alteration in enzymatic and non-enzymatic antioxidants was observed in all diabetic animals. During 16 weeks of treatment of diabetic rabbits with 20 mg/kg body weight of oleuropein the levels of MDA along with blood glucose and most of the enzymatic and non-enzymatic antioxidants were significantly restored to establish values that were not different from normal control rabbits. Untreated diabetic rabbits on the other hand demonstrated persistent alterations in the oxidative stress marker MDA, blood glucose and the antioxidant parameters. These results demonstrate that oleuropein may be of advantage in inhibiting hyperglycemia and oxidative stress induced by diabetes and suggest that administration of oleuropein may be helpful in the prevention of diabetic complications associated with oxidative stress.