Lars Ivo Partecke

Chronic stress increases experimental pancreatic cancer growth, reduces survival and can be antagonised by beta-adrenergic receptor blockade

BACKGROUND/OBJECTIVESnChronic stress could promote tumour growth and reduce survival of pancreatic cancer patients via beta-adrenergic receptors of tumour cells. We have tested the impact of chronic acoustic and restraint stress on tumour development in an orthotopic syngeneic murine model of pancreatic cancer.nnnMETHODS AND RESULTSnTumour-bearing C57BL/6 mice exposed to chronic stress had 45% (pxa0=xa00.0138) higher circulating steroid and 111% (pxa0=xa00.0052) higher adrenal tyrosine hydroxylase levels. Their immune response was significantly suppressed: The inxa0vitro LPS response of splenocytes was significantly reduced regarding Th1- and Th2-cytokines including IFN-gamma, IL-6, IL-10 and MCP-1 (0.0011xa0<xa0pxa0<xa00.043). Also, tumours of stressed mice showed a tendency towards fewer total CD4 cells, more regulatory T cells (Treg), less T cell/tumour cell contacts and a reduction of CTLA-4 in CD4 cells (pxa0>xa00.05). TGF-beta inxa0vitro was increased by 23.4% using catecholamines (pxa0<xa00.012) and inxa0vivo employing chronic stress (pxa0<xa00.001). After 5 weeks tumour volumes were 130% (pxa0=xa00.0061) larger and median survival reduced by 13.5% (pxa0=xa00.0058). Tumours expressed more VEGF (pxa0=xa00.0334), had greater microvessel densities (pxa0=xa00.047), and an increased MMP-9 expression (pxa0=xa00.0456). Beta-catecholamines increased proliferation in tumour cells by 18% (pxa0<xa00.0001) and migration by 78% (pxa0=xa00.0348) whereas the beta-blocker propranolol reduced these effects by 25% (pxa0<xa00.0001) and 53% (pxa0=xa00.045), respectively. When stressed tumour-bearing animals were treated with propranolol tumour volumes were reduced by 69% (pxa0=xa00.0088) and survival improved by 14% (pxa0<xa00.0058).nnnCONCLUSIONSnThe potential treatment with beta-blockers of patients with pancreatic cancer or other malignancies should be further evaluated as an adjuvant anti-neoplastic agent in clinical trials.

Non-thermal plasma-treated solution demonstrates antitumor activity against pancreatic cancer cells in vitro and in vivo

Pancreatic cancer is associated with a high mortality rate. In advanced stage, patients often experience peritoneal carcinomatosis. Using a syngeneic murine pancreatic cancer cell tumor model, the effect of non-thermal plasma (NTP) on peritoneal metastatic lesions was studied. NTP generates reactive species of several kinds which have been proven to be of relevance in cancer. In vitro, exposure to both plasma and plasma-treated solution significantly decreased cell viability and proliferation of 6606PDA cancer cells, whereas mouse fibroblasts were less affected. Repeated intraperitoneal treatment of NTP-conditioned medium decreased tumor growth in vivo as determined by magnetic resonance imaging, leading to reduced tumor mass and improved median survival (61 vs 52 days; pu2009<u20090.024). Tumor nodes treated by NTP-conditioned medium demonstrated large areas of apoptosis with strongly inhibited cell proliferation. Contemporaneously, no systemic effects were found. Apoptosis was neither present in the liver nor in the gut. Also, the concentration of different cytokines in splenocytes or blood plasma as well as the distribution of various hematological parameters remained unchanged following treatment with NTP-conditioned medium. These results suggest an anticancer role of NTP-treated solutions with little to no systemic side effects being present, making NTP-treated solutions a potential complementary therapeutic option for advanced tumors.

LC-MS/MS method for the simultaneous quantification of intestinal CYP and UGT activity

HIGHLIGHTSDevelopment of an analytical method for the simultaneous quantification ofprobe substrates for human intestinal enzymesSimultaneous quantification of CYP and UGT metabolites (so far almost exclusively CYP substrates).Comprehensive method validation according to current bioanalytical guidelines.Similar/superior sensitivity compared to previously published methods.Application of the method to characterize the metabolic activity of intestinalCYP2C9 / 2C19 / 2D6 / CYP3A and UGT1A1 / 2B7 ABSTRACT Many orally administered drugs are subject to first‐pass metabolism by cytochrome P450 (CYP) enzymes and uridine 5′‐diphospho‐glucuronosyltransferases (UGT). While their hepatic activity is well characterized, respective information about the intestine are very scare due to limited availability of tissue, very low microsomal protein content and the heterogeneity of the individual segments. As a consequence, determination of enzyme kinetic parameters is challenging. It was therefore the aim of this study to develop a sensitive liquid chromatography tandem mass spectrometry method for the simultaneous quantification of CYP and UGT metabolites formed by clinically relevant intestinal biotransformation enzymes: 4‐hydroxydiclofenac (CYP2C9), 5‐hydroxyomeprazole (CYP2C19), dextrorphan (CYP2D6), 1‐hydroxymidazolam (CYP3A), ezetimibe glucuronide (UGT1A) and naloxone glucuronide (UGT2B7). After precipitation of microsomal protein with acetonitrile, analytes were chromatographically separated on a C18 column with gradient elution using acetonitrile and water, both containing 0.1% formic acid and detected with a tandem mass spectrometer operating in positive mode with electron spray ionization. The assay was validated according to current bioanalytical guidelines regarding linearity, accuracy, precision, stability, recovery and matrix effects spanning an analytical range from 1 to 200 nmol/L for each analyte. The developed method was successfully applied to a proof of concept experiment using pooled human jejunal microsomes (50 &mgr;g protein/mL) in order to determine enzyme kinetic parameters. Formation of all monitored metabolites followed Michaelis‐Menten kinetics and allowed calculation of KM and Vmax values. The developed method may be useful for characterization of enzymatic activity in the human intestine which may allow more precise insights into the intestinal contribution to first pass metabolism of drugs.

Subdiaphragmatic vagotomy promotes tumor growth and reduces survival via TNFα in a murine pancreatic cancer model

This study analyses the effects of vagotomy on tumor growth and survival in a murine, pancreatic cancer model in wild-type and TNFα-knockout (−/−) mice. Throughout many operative procedures in the upper gastrointestinal tract the partial or complete transection of the vagus nerve or its local nerve fibers is unavoidable. Thereby its anti-inflammatory effects in residual tumor tissue may get lost. This effect may be mediated by tumor-associated macrophages (TAM) secreting TNFα. In an orthotopic murine pancreatic cancer model subdiaphragmatic vagotomy versus sham surgery was performed. The impact on tumor growth was monitored in wild type and TNFα −/− mice using MRI. TAMs as well as expression levels of TNFα were analyzed using immunohistochemistry. The role of TNFα on tumor growth and migration was examined in vitro. Vagotomised mice showed increased tumor growth with macroscopic features of invasive growth and had a shorter survival time. The loss of vagal modulation led to significantly increased TNFα levels in tumors and considerably elevated numbers of TAMs. In vitro TNFα significantly stimulated growth (p < 0.05) and migration (p < 0.05) of pancreatic cancer cells. TNFα −/− mice survived significantly longer after tumor implantation (p < 0.05), with vagotomy not affecting the prognosis of these animals (p > 0.05). Vagotomy can increase tumor growth and worsen survival in a murine pancreatic cancer model mediated through TAMs and TNFα. Hence, the suppression of TAMs and the modulation of TNFα dependent pathways could offer new perspectives in immunotherapies of pancreatic cancer patients especially with remaining vital tumor cells and lost vagal modulation.

TNF-related apoptosis-inducing ligand deficiency enhances survival in murine colon ascendens stent peritonitis

Background Apart from inducing apoptosis in tumor cells, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) influences inflammatory reactions. Murine colon ascendens stent peritonitis (CASP) represents a model of diffuse peritonitis. Recently, it has been demonstrated that administration of exogenous TRAIL not only induces apoptosis in neutrophils but also enhances survival in this model. The aim of this study was to examine the impact of genetic TRAIL deficiency on the course of CASP. Methods Peritonitis was induced in 6- to 8-week-old female TRAIL−/− mice as well as in wild-type mice. The sepsis severity score and survival of mice were monitored. Bacterial loads in blood as well as in the lymphoid organs were examined. Additionally, the number of apoptotic cells within the lymphoid organs was determined. Results As early as 8 hours postinduction of CASP, TRAIL−/− mice were significantly more affected by sepsis than wild-type mice, as measured by the sepsis severity score. However, during the further course of sepsis, TRAIL deficiency led to significantly decreased sepsis severity scores, resulting in an enhanced overall survival in TRAIL−/− mice. The better survival of TRAIL−/− mice was accompanied by a decreased bacterial load within the blood. In marked contrast, the number of apoptotic cells within the lymphoid organs was highly increased in TRAIL−/− mice 20 hours after induction of CASP. Conclusion Hence, exogenous and endogenous TRAIL is protective during the early phase of sepsis, while endogenous TRAIL appears to be detrimental in the later course of this disease.

Giant hepatocellular carcinoma with bone metastasis in a young adult, emerged from pigmented adenoma with beta-Catenin activation: A case report

Highlights • Hepatocellular carcinoma (HCC) is an extremely rare finding in young adult.• Giant HCCs are larger than 10 cm in diameter and mostly develop in non-cirrhotic livers.• Pigmented hepatocellular adenoma (HCA) with beta-catenin activation have an increased risk for malignant transformation.• Surgical resection is the only curative treatment with best long-term outcome.• Molecular classification of HCAs is a useful tool for risk assessment of malignant transformation.

LPS promotes resistance to TRAIL-induced apoptosis in pancreatic cancer

BackgroundThough TRAIL has been hailed as a promising drug for tumour treatment, it has been observed that many tumour cells have developed escape mechanisms against TRAIL-induced apoptosis. As a receptor of LPS, TLR 4, which is expressed on a variety of cancer cells, can be associated with TRAIL-resistance of tumour cells and tumour progression as well as with the generation of an anti-tumour immune response.MethodsIn this study, the sensitivity to TRAIL-induced apoptosis as well as the influence of LPS-co-stimulation on the cell viability of the pancreatic cancer cell lines PANC-1, BxPC-3 and COLO 357 was examined by FACS analyses and a cell viability assay. Subsequently, the expression of TRAIL-receptors was detected via FACS analyses. Levels of osteoprotegerin (OPG) were also determined using an enzyme-linked immunosorbent assay.ResultsPANC-1 cells were shown to be resistant to TRAIL-induced apoptosis. This was accompanied by significantly increased osteoprotegerin levels and a significantly decreased expression of DR4.In contrast, TRAIL significantly induced apoptosis in COLO 357 cells and to a lesser degree in BxPC-3 cells. Co-stimulation of COLO 357 as well as BxPC-3 cells combining TRAIL and LPS resulted in a significant decrease in TRAIL-induced apoptosis. In COLO 357 cells TRAIL-stimulation decreased the levels of OPG thereby not altering the expression of the TRAIL-receptors 1–4 resulting in a high susceptibility to TRAIL-induced apoptosis. Co-stimulation with LPS and TRAIL completely reversed the effect of TRAIL on OPG levels reaching a 2-fold increase beyond the level of non-stimulated cells resulting in a lower susceptibility to apoptosis.In BxPC-3, TRAIL stimulation decreased the expression of DR4 and significantly increased the decoy receptors TRAIL-R3 and TRAIL-R4 leading to a decrease in TRAIL-induced apoptosis. OPG levels remained unchanged. Co-stimulation with TRAIL and LPS further enhanced the changes in TRAIL-receptor-expression promoting apoptosis resistance.ConclusionsHere it has been shown that TRAIL-resistance in pancreatic cancer cells can be mediated by the inflammatory molecule LPS as well as by different expression patterns of functional and non-functional TRAIL-receptors.

Perspectives in General Surgery

Non-thermal plasma (NTP) offers promising opportunities to advance and refine surgical practice. The combination of stimulating tissue proliferation and decreasing bacterial load predestines NTP as add-on tool in wound treatment. Additionally, plasma-based modification of foreign materials, such as, vascular grafts or osteosynthesis materials improves their biocompatibility. Surgery in oncology is another auspicious field of application. Micrometastasis in the margins of excised tumors often cause tumor relapse. Due to selective induction of apoptosis in tumor cells, NTP treatment of wound margins after surgical bulk removal offers great potential minimize disease recurrence and improve patients’ outcome. However, compatibility with the special setting in an operating theatre is an issue not addressed yet with most plasma sources.

Dysregulation of mucosal membrane transporters and drug metabolizing enzymes in ulcerative colitis

Intestinal transporters and metabolizing enzymes are the important factors of the intestinal absorption barrier. Because there is evidence that their expression and function may be affected during inflammatory conditions, we investigated gene expression, protein abundance, and regulation of relevant intestinal transporters and metabolizing enzymes in the intestinal mucosa of patients with ulcerative colitis (UC). Specimens from inflamed and noninflamed tissues of 10 patients with UC as well as colonic control tissues of 10 patients without inflammation were subjected to gene (9 enzymes, 15 transporters, 9 cytokines) and microRNA (Nxa0= 54) expression analysis. Protein abundance was quantified by liquid chromatography-tandem mass spectrometry-based targeted proteomics. Gene expression of several metabolizing enzymes (e.g., CYP2C9, UGT1A1) and transporters such as ABCB1 (ABCB1), ABCG2 (ABCG2), and monocarboxylate transporter 1 (MCT1, SLC16A1) were significantly decreased during inflammation and negatively correlated to microRNAs. On contrary, multidrug resistance-protein 4 (MRP4, ABCC4), organic anion-transporting polypeptide 2B1 (OATP2B1, SLCO2B1), and organic cation transporter-like 2 (ORCTL2, SLC22A18) were significantly elevated in inflamed tissue. However, at protein level, these findings could only be confirmed for MCT1. UC is associated with complex changes in the intestinal expression of enzymes, transporters, cytokines, and microRNAs, which may affect efficacy of anti-inflammatory drug therapy or the disease state itself.

The MRI Sepsis Score: An Innovative Tool for the Evaluation of Septic Peritonitis in Mice Using 7-Tesla Small Animal MRI

Background: Magnetic resonance imaging (MRI) techniques are rarely used in the context of abdominal sepsis and in sepsis research. This study investigates the impact of MRI for monitoring septic peritonitis in an animal model (colon ascendens stent-induced peritonitis, CASP). The CASP model closely mimics that of human disease and is highly standardized. The most frequently employed readout parameter in mouse CASP studies is prolonged or decreased rate of survival. Monitoring the progression of peritonitis via MRI could provide a helpful tool in the evaluation of severity. The use of alternative readout systems could very well reduce the number of research animals. Perspectively, clinical improvement after certain treatment could be classified. Methods: This study describes for the first time MRI findings following the induction of septic peritonitis in mice using the CASP model. Two sublethal groups of mice with septic peritonitis were investigated. Each had received one of two differing stent diameters in order to control the leakage of feces into the abdominal cavity. Each mouse served as its own control. Imaging and analyses were performed blinded. Gut diameters, stomach volume, abdominal organ wall diameters, and volume of the adrenal glands were measured. Serum corticosterone levels were detected using ELISA. Serum IL-6, TNF-α, IL-1β, and IL-10 levels were screened by cytometric bead array. Statistical analysis was performed using the Mann-Whitney U test for nonparametric probes and the Kruskal-Wallis and t tests. Results: Using a 7-tesla MRI scanner 24 and 48 h after induction of septic peritonitis, interenteric fluid, organ swelling of spleen and adrenal glands, as well as dilatation of the stomach were compared to nonseptic conditions. Swelling of adrenal glands resulted in an increased serum corticosterone level. In addition, the wall of the intestine bowel was thickened. Based upon these findings, an MRI score (MRI sepsis score, MSS) for abdominal sepsis in mice was established. Reduced stent sizes led to reduced severity of the abdominal sepsis, which could be reproduced in the MSS, which is described here for the first time. Conclusions: Intraabdominal variations during septic peritonitis are detectable by MRI techniques. MRI methods should become a more important tool for the evaluation of abdominal peritonitis. MSS could provide an interesting tool for the evaluation of therapeutic strategies.