Annamária Szabolcs

Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

The transforming growth factor-beta (TGF-β) superfamily is one of the most diversified cell signaling pathways and regulates many physiological and pathological processes. Recently, neuropilin-1 (NRP-1) was reported to bind and activate the latent form of TGF-β1 (LAP-TGF-β1). We investigated the role of NRP-1 on Smad signaling in stromal fibroblasts upon TGF-β stimulation. Elimination of NRP-1 in stromal fibroblast cell lines increases Smad1/5 phosphorylation and downstream responses as evidenced by up-regulation of inhibitor of differentiation (Id-1). Conversely, NRP-1 loss decreases Smad2/3 phosphorylation and its responses as shown by down-regulation of α-smooth muscle actin (α-SMA) and also cells exhibit more quiescent phenotypes and growth arrest. Moreover, we also observed that NRP-1 expression is increased during the culture activation of hepatic stellate cells (HSCs), a liver resident fibroblast. Taken together, our data suggest that NRP-1 functions as a key determinant of the diverse responses downstream of TGF-β1 that are mediated by distinct Smad proteins and promotes myofibroblast phenotype.

Enhancing chemotherapy response with Bmi-1 silencing in ovarian cancer

Undoubtedly ovarian cancer is a vexing, incurable disease for patients with recurrent cancer and therapeutic options are limited. Although the polycomb group gene, Bmi-1 that regulates the self-renewal of normal stem and progenitor cells has been implicated in the pathogenesis of many human malignancies, yet a role for Bmi-1 in influencing chemotherapy response has not been addressed before. Here we demonstrate that silencing Bmi-1 reduces intracellular GSH levels and thereby sensitizes chemoresistant ovarian cancer cells to chemotherapeutics such as cisplatin. By exacerbating ROS production in response to cisplatin, Bmi-1 silencing activates the DNA damage response pathway, caspases and cleaves PARP resulting in the induction apoptosis in ovarian cancer cells. In an in vivo orthotopic mouse model of chemoresistant ovarian cancer, knockdown of Bmi-1 by nanoliposomal delivery significantly inhibits tumor growth. While cisplatin monotherapy was inactive, combination of Bmi-1 silencing along with cisplatin almost completely abrogated ovarian tumor growth. Collectively these findings establish Bmi-1 as an important new target for therapy in chemoresistant ovarian cancer.

Plasma Concentrations of High-Mobility Group Box Protein 1, Soluble Receptor for Advanced Glycation End-Products and Circulating DNA in Patients with Acute Pancreatitis

Aims: High-mobility group box protein 1 (HMGB1), a late-acting proinflammatory cytokine, is secreted actively by inflammatory cells, and released passively from necrotic cells. From the aspect that both inflammation and necrosis are involved in the pathogenesis in acute pancreatitis, the aim of the study was a joint investigation of the plasma concentrations of HMGB1, its soluble receptor for advanced glycation end-products (sRAGE), and the circulating DNA as a marker of cell death. Methods: 62 patients with acute pancreatitis (30 mild, 32 severe), 20 patients with sepsis, and 20 healthy controls were enrolled in the study. HMGB1 and sRAGE plasma levels were measured by means of ELISA. Plasma DNA concentrations were estimated by real-time quantitative PCR for the β-globin gene. Results: The circulating HMGB1 level was significantly higher in patients with severe acute pancreatitis (13.33 ± 2.11 ng/ml) than in healthy controls (0.161 ± 0.03 ng/ml) or than in patients with mild pancreatitis (2.64 ± 0.185 ng/ml). The plasma concentration of sRAGE was highest in patients with sepsis (2,210 ± 252 pg/ml), while the levels of sRAGE correlated inversely with that of HMGB1 in patients with acute pancreatitis. The plasma DNA level was significantly elevated in patients with severe acute pancreatitis (2,206 ± 452 ng/ml). Conclusion: A complex study of the plasma levels of HMGB1, sRAGE and circulating DNA can be informative in evaluations of acute pancreatitis with different levels of severity.

Designing Nanoconjugates to Effectively Target Pancreatic Cancer Cells In Vitro and In Vivo

Background Pancreatic cancer is the fourth leading cause of cancer related deaths in America. Monoclonal antibodies are a viable treatment option for inhibiting cancer growth. Tumor specific drug delivery could be achieved utilizing these monoclonal antibodies as targeting agents. This type of designer therapeutic is evolving and with the use of gold nanoparticles it is a promising approach to selectively deliver chemotherapeutics to malignant cells. Gold nanoparticles (GNPs) are showing extreme promise in current medicinal research. GNPs have been shown to non-invasively kill tumor cells by hyperthermia using radiofrequency. They have also been implemented as early detection agents due to their unique X-ray contrast properties; success was revealed with clear delineation of blood capillaries in a preclinical model by CT (computer tomography). The fundamental parameters for intelligent design of nanoconjugates are on the forefront. The goal of this study is to define the necessary design parameters to successfully target pancreatic cancer cells. Methodology/Principal Findings The nanoconjugates described in this study were characterized with various physico-chemical techniques. We demonstrate that the number of cetuximab molecules (targeting agent) on a GNP, the hydrodynamic size of the nanoconjugates, available reactive surface area and the ability of the nanoconjugates to sequester EGFR (epidermal growth factor receptor), all play critical roles in effectively targeting tumor cells in vitro and in vivo in an orthotopic model of pancreatic cancer. Conclusion Our results suggest the specific targeting of tumor cells depends on a number of crucial components 1) targeting agent to nanoparticle ratio 2) availability of reactive surface area on the nanoparticle 3) ability of the nanoconjugate to bind the target and 4) hydrodynamic diameter of the nanoconjugate. We believe this study will help define the design parameters for formulating better strategies for specifically targeting tumors with nanoparticle conjugates.

Zerumbone exerts a beneficial effect on inflammatory parameters of cholecystokinin octapeptide-induced experimental pancreatitis but fails to improve histology.

Objective: Our experiments were designed to investigate the effects of zerumbone pretreatment on cholecystokinin octapeptide (CCK-8)-induced acute pancreatitis in rats. Methods: Male Wistar rats weighing 240 to 280 g were divided into a control group, a group treated with CCK-8, a group receiving 20 mg/kg zerumbone before CCK-8 administration, and a group treated with zerumbone only. Results: The serum amylase and lipase activities and the pancreatic weight-body weight ratio were significantly reduced by zerumbone pretreatment, but the drug failed to influence the histological parameters of pancreatitis. The anti-inflammatory effects of the drug were manifested in decreases in the cytosolic interleukin 6 and tumor necrosis factor &agr; concentrations and an elevation in the I-&kgr;B concentration, whereas the antioxidant ability of zerumbone was demonstrated by reductions in inducible nitric oxide synthase, Mn- and Cu/Zn-superoxide dismutase activities in the zerumbone-treated rats. Conclusion: Zerumbone ameliorated the changes of several parameters of acute pancreatitis probably by interfering with I-&kgr;B degradation, but in the applied dose, it failed to influence the histology of the disease.Abbreviations: CCK-8 - cholecystokinin octapeptide, DMSO - dimethyl sulfoxide, iNOS - inducible nitric oxide synthase, cNOS - constitutive nitric oxide synthase, TNF, tumor necrosis factor, IL-6 - interleukin 6, NF-&kgr;B - nuclear factor &kgr;B, SOD - superoxide dismutase, ASAT - aspartate aminotransferase

Inhibiting the growth of pancreatic adenocarcinoma in vitro and in vivo through targeted treatment with designer gold nanotherapeutics.

Background Pancreatic cancer is one of the deadliest of all human malignancies with limited options for therapy. Here, we report the development of an optimized targeted drug delivery system to inhibit advanced stage pancreatic tumor growth in an orthotopic mouse model. Method/Principal Findings Targeting specificity in vitro was confirmed by preincubation of the pancreatic cancer cells with C225 as well as Nitrobenzylthioinosine (NBMPR - nucleoside transporter (NT) inhibitor). Upon nanoconjugation functional activity of gemcitabine was retained as tested using a thymidine incorporation assay. Significant stability of the nanoconjugates was maintained, with only 12% release of gemcitabine over a 24-hour period in mouse plasma. Finally, an in vivo study demonstrated the inhibition of tumor growth through targeted delivery of a low dose of gemcitabine in an orthotopic model of pancreatic cancer, mimicking an advanced stage of the disease. Conclusion We demonstrated in this study that the gold nanoparticle-based therapeutic containing gemcitabine inhibited tumor growth in an advanced stage of the disease in an orthotopic model of pancreatic cancer. Future work would focus on understanding the pharmacokinetics and combining active targeting with passive targeting to further improve the therapeutic efficacy and increase survival.

In Vitro and in Vivo Nuclear Factor-κB Inhibitory Effects of the Cell-Penetrating Penetratin Peptide

Penetratin is a cationic cell-penetrating peptide that has been frequently used for the intracellular delivery of polar bioactive compounds. Recent studies have just revealed the major role of polyanionic membrane proteoglycans and cholesterol-enriched lipid rafts in the uptake of the peptide. Both proteoglycans and lipid-rafts influence inflammatory processes by binding a wide array of proinflammatory mediators; thus, we decided to analyze the effect of penetratin on in vitro and in vivo inflammatory responses. Our in vitro luciferase gene assays demonstrated that penetratin decreased transcriptional activity of nuclear factor-κB (NF-κB) in tumor necrosis factor (TNF)-stimulated L929 fibroblasts and lipopolysaccharide-activated RAW 264.7 macrophages. Penetratin also inhibited TNF-induced intercellular adhesion molecule-1 expression in human endothelial HMEC-1 cells. Exogenous heparan sulfate abolished the in vitro NF-κB inhibitory effects of the peptide. Uptake experiments showed that penetratin was internalized by all of the above-mentioned cell lines in vitro and rapidly entered the cells of the lung and pancreas in vivo. In an in vivo rat model of acute pancreatitis, a disease induced by elevated activities of stress-responsive transcription factors like NF-κB, pretreatment with only 2 mg/kg penetratin attenuated the severity of pancreatic inflammation by interfering with IκB degradation and subsequent nuclear import of NF-κB, inhibiting the expression of proinflammatory genes and improving the monitored laboratory and histological parameters of pancreatitis and associated oxidative stress.

IN VITRO AND IN VIVO NF-κB INHIBITORY EFFECTS OF THE CELL-PENETRATING PENETRATIN PEPTIDE

Penetratin is a cationic cell-penetrating peptide that has been frequently used for the intracellular delivery of polar bioactive compounds. Recent studies have just revealed the major role of polyanionic membrane proteoglycans and cholesterol-enriched lipid rafts in the uptake of the peptide. Both proteoglycans and lipid-rafts influence inflammatory processes by binding a wide array of proinflammatory mediators; thus, we decided to analyze the effect of penetratin on in vitro and in vivo inflammatory responses. Our in vitro luciferase gene assays demonstrated that penetratin decreased transcriptional activity of nuclear factor-κB (NF-κB) in tumor necrosis factor (TNF)-stimulated L929 fibroblasts and lipopolysaccharide-activated RAW 264.7 macrophages. Penetratin also inhibited TNF-induced intercellular adhesion molecule-1 expression in human endothelial HMEC-1 cells. Exogenous heparan sulfate abolished the in vitro NF-κB inhibitory effects of the peptide. Uptake experiments showed that penetratin was internalized by all of the above-mentioned cell lines in vitro and rapidly entered the cells of the lung and pancreas in vivo. In an in vivo rat model of acute pancreatitis, a disease induced by elevated activities of stress-responsive transcription factors like NF-κB, pretreatment with only 2 mg/kg penetratin attenuated the severity of pancreatic inflammation by interfering with IκB degradation and subsequent nuclear import of NF-κB, inhibiting the expression of proinflammatory genes and improving the monitored laboratory and histological parameters of pancreatitis and associated oxidative stress.

Polymorphisms of Beta Defensins Are Associated with the Risk of Severe Acute Pancreatitis

Aims: Bacterial translocation from the intestinal tract plays an important role in severe acute pancreatitis (AP). Human β-defensins are a family of antimicrobial peptides present at the mucosal surface. The aim of this study was to investigate the relevance of single nucleotide polymorphisms (SNPs) in the DEFB1 gene and copy number polymorphisms of the DEFB4 genes in AP. Methods: 124 AP patients (30 with mild and 94 with severe disease) and 100 healthy controls were enrolled in the study. Three SNPs of the DEFB1 gene [G–20A (c.–20G→A), C–44G (c.–44C→G) and G–52A (c.–52G→A)] were genotyped by Custom TaqMan® assay. The DEFB4 gene copy number was determined by means of a TaqMan real-time PCR assay. Results: Significantly higher frequencies of the AA genotype of G–20A (c.–20G→A) and the AA genotype of G–52A (c.–52G→A) were observed among the patients with severe AP (SAP) compared with the healthy controls (38 vs. 20 and 41 vs. 18%, respectively). The GG protective genotype of C–44G (c.–44C→G) SNP was much less frequent (1%) among the patients than among the controls (9%). A higher frequency of a lower (<4) copy number of the DEFB4 gene was observed in the patients with SAP compared with the healthy controls (62 vs. 24%, respectively). Conclusions: The variations in the genes encoding human β-defensin-1 and -2 may be associated with the risk of SAP.

Simultaneous proteosome inhibition and heat shock protein induction by bortezomib is beneficial in experimental pancreatitis

The proteosome inhibitor bortezomib is used in the treatment of patients with multiple myeloma. Proteosomes are responsible for the degradation of I-kappaB, the inhibitory protein of transcription factor nuclear factor kappa B (Nf-kappaB). The heat shock protein (HSP) inducing effect of bortezomib is also documented. The aim of our work was to test the anti-inflammatory effect of bortezomib in cholecystokinin-octapeptide (CCK-8)-induced acute pancreatitis. Male Wistar rats were divided into three groups (n=8 in each). Group P received an i.p. injection of physiological saline (p.s.) 60 min. before the induction of acute pancreatitis by three hourly s.c. injections of 100 microg/kg CCK-8. Group BP received 1 mg/kg bortezomib dissolved in p.s. 1 h previous to pancreatitis induction. Group C was treated with the vehicle (p.s.). Animals were exsanguinated 4 h after the last injection of CCK-8. Bortezomib pre-treatment significantly reduced the pancreatic weight/body weight ratio, and improved the histology by decreasing the extent of vacuolization and infiltration. Bortezomib pre-treatment inhibited I-kappaBbeta degradation, and induced the synthesis of HSP72. The results confirmed the anti-inflammatory effect of bortezomib in acute experimental pancreatitis. This effect of the drug is presumably mediated by the inhibition of Nf-kappaB activation and induction of HSP synthesis.