Birgit Hotz

Beyond Epithelial to Mesenchymal Transition: A Novel Role for the Transcription Factor Snail in Inflammation and Wound Healing

IntroductionSnail, a transcription factor linked to epithelial to mesenchymal transition (EMT) during embryonic development and tumor progression, is associated with migration of cells. During inflammation and tissue injury, cell movement is also observed to provide the first line of defense against bacteria and to promote wound healing. Therefore, we studied the function of Snail in activated macrophages in a variety of inflammatory processes.Materials and MethodsIn this study, we examined the expression and localization of Snail during inflammation and tissue injury in rats and human tissue specimens, by immunohistochemistry, Western blot, and real-time PCR. We investigated Snail expression after stimulation of macrophages with TGF-β1, LPS, Interleukin-8, and MMP-3 in vitro. To further understand the role of Snail in activated macrophages, we used Stealth siRNA against Snail, transfected the human macrophage cell line THP-1, and measured migration of cells in an in vitro invasion assay.Results and DiscussionWe found a strong, transient, and time-dependent activation of Snail in migrating macrophages at the sites of injury in vivo and in vitro, as well as in patients with inflammatory bowel disease. Furthermore, we showed that induction of Snail in macrophages is dependent on TGF-β1 signaling pathway. Downregulation of Snail by Stealth siRNA led to impaired migration of THP-1 cells in an invasion assay after stimulation with TGF-β1.ConclusionWe conclude that TGF-β1 induced migration of activated macrophages during inflammation and wound healing is mediated by snail. These results give insights in a novel EMT-like mechanism present in immune cell movement during tissue injury.

Magnetic resonance imaging of experimental inflammatory bowel disease: quantitative and qualitative analyses with histopathologic correlation in a rat model using the ultrasmall iron oxide SHU 555 C.

Objectives:To quantitatively and qualitatively characterize the MR findings of inflammatory bowel disease in a rat model after i.v. injection of the reticuloendothelial system cell specific ultrasmall iron oxide SHU 555 C. Materials and Methods:Colitis was induced in 15 rats using dinitrobenzene sulfonic acid instillation. Five rats served as controls. T1- and T2-weighted spin-echo- and T2*-weighted gradient-echo-sequences were acquired at 2.4 Tesla before and immediately, 15, 45, 60, and 90 minutes, and 24 hours after i.v.-injection of SHU 555 C (0.1 mmol Fe/kg). MR images were evaluated quantitatively regarding thickness and signal-to-noise ratio (SNR) of the bowel wall and qualitatively regarding overall bowel wall signal intensity and the occurrence of bowel wall ulcerations. MR findings were correlated to histology. Results:The inflamed bowel wall was significantly thicker than the noninflamed bowel wall and 90 minutes after contrast injection it showed a significant reduction of SNR in T1- (94 ± 27 vs. 61 ± 29; P < 0.01), T2- (67 ± 26 vs. 28 ± 17; P < 0.05), and T2*- (92 ± 57 vs. 10 ± 7; P < 0.05) weighted images as compared with unenhanced images. At 24 hours, the respective SNR values remained significantly reduced. The signal loss was homogeneous in 12 and focal in 3 of the 15 rats with colitis. Nine rats showed colonic wall ulcerations. In all but one animal (missed focal ulceration) MR findings correlated to the histologic findings. Conclusions:SHU 555 C leads to a significant signal intensity loss of the inflamed bowel wall in T1-, T2- and T2*-weighted images. SHU 555 C enhanced MRI findings correlate well with histologic findings.

Suramin inhibits not only tumor growth and metastasis but also angiogenesis in experimental pancreatic cancer.

Suramin inhibits the proliferation of several human tumors in vivo and in vitro. In this study, the effects of Suramin on proliferation and angiogenesis were investigated in human pancreatic cancer cell lines and in an orthotopic nude mouse model of human pancreatic cancer. The effects of Suramin on proliferation, viability, cell cycle, and apoptosis were studied in five human pancreatic cancer cell lines. Suramin inhibited the proliferation of pancreatic cancer cells in a dose-dependent manner and reduced viability at high concentrations. Cell cycle analysis revealed a decreased S-phase fraction in most cell lines, whereas the apoptotic fraction was not notably different. In vivo treatment with Suramin significantly reduced pancreatic tumor size (MiaPaCa-2, −74%; AsPC-1, −41%; and Capan-1, −49%) and metastatic spread (MiaPaCa-2, −79%; AsPC-1, −34%; and Capan, −38%). As a parameter for angiogenic activity, vascular endothelial growth factor (VEGF) secretion was measured, revealing reduced VEGF concentrations under Suramin treatment in both cell culture medium and ascites. Also, microvessel density quantified in primary tumors was reduced in animals treated with Suramin. Therefore, Suramin inhibits the proliferation of human pancreatic cancer in vitro and in vivo. The therapeutic effects seem to involve cell cycle kinetics and may be in part related to the antiangiogenic action of the drug.

Selective inhibition of endothelin receptor A as an anti-angiogenic and anti-proliferative strategy for human pancreatic cancer

Endothelin-1 (ET-1) plays a major role in tumor proliferation and angiogenesis of various types of cancer acting through endothelin receptors A and B (ETRA and ETRB). The aim of this study was to analyze theET-1/ETRsystem inhumanpancreatic cancer cell linesandto evaluate the effect of a selective endothelin A inhibitor in vitro and in vivo in an orthotopic mouse model. Three different human pancreatic cancer cell lines, MiaPaCa-2, AsPC-1, and Panc-1, were studied. We found that proliferation of human pancreatic carcinoma cells expressing ETRA was significantly reduced with a selective antagonist. Hypoxic conditions led to improved results compared to a normoxic environment (MiaPaCa-2: -53% vs. -18%; AsPC-1: -54% vs. -46%). Proliferation of ETRA negative Panc-1 cells was not decreased. In vivo, the selective ETRA inhibition resulted in reduced angiogenesis as measured by lower microvessel densities (MiaPaCa-2: -47%; AsPC-1: -55%). The blockade of ETRA decreased the volume (MiaPaCa-2: -87%; AsPC-1: -28%) and metastatic spread (MiaPaCa-2: -95.5%; AsPC-1: -27%) of receptorpositive tumors, thereby increasing survival in experimental pancreatic cancer. ETRA blockade did not show an effect on ETRA negative Panc-1 tumors. Therefore, targeting ETRA with a selective antagonist might provide a new approach to reducing proliferation and angiogenesis in human pancreatic cancer.

Fluid resuscitation with human albumin or hydroxyethyl starch--are there differences in the healing of experimental intestinal anastomoses?

Abstract Objectives. Restoration of the macro- and microcirculation is important for the healing of gastrointestinal anastomoses. Colloids and crystalloids are widely used for blood volume therapy. We evaluated the effects of human albumin, hydroxyethyl starch (HES) 130/0.4 and saline on the microcirculation and on wound healing in colon anastomoses in rats. Material and methods. Male Wistar rats received a colonic end-to-end anastomosis. The animals were randomized into three groups and a single 3-ml dose of either 20% human albumin, 6% HES 130/0.4 or 0.9% saline was applied intravenously. Six, 24, 48, 96 h and 2 weeks after the procedure, 10 animals per group were reanesthetized. Measurements of capillary blood flow, vessel permeability and anastomosis bursting pressure were performed. The amounts of vascular endothelial growth factor (VEGF) and IL-6 in the plasma were determined by enzyme-linked immunosorbent assays, and the mRNA levels of VEGF and collagen types I and III were measured by real-time polymerase chain reaction. Results. No significant differences were found between albumin, HES 130/0.4 and saline in capillary blood flow, vessel permeability and anastomotic bursting pressure in this rat model. Concentrations of collagen I and III mRNA were significantly elevated after 96 h in animals that had received HES 130/0.4 or albumin. RNA and protein levels of VEGF and interleukin-6 were unaffected by therapy. Conclusions. Human albumin, which is still widely used in the clinical setting, had no advantage over HES 130/0.4 and saline with regard to anastomotic healing in this animal model. Nevertheless, we prefer HES 130/0.4 because it is more effective for volume therapy than saline and has a better availability and is less expensive than human albumin.

Association Between Activation of Atypical NF-kappa B1 p105 Signaling Pathway and Nuclear beta-Catenin Accumulation in Colorectal Carcinoma

Recent studies have demonstrated that increased expression of coding region determinant‐binding protein (CRD‐BP) in response to β‐catenin signaling leads to the stabilization of β‐TrCP1, a substrate‐specific component of SCF E3 ubiquitin ligase complex, resulting in an accelerated degradation of IκBα and activation of canonical nuclear factor‐κB (NF‐κB) pathway. Here, we show that the noncanonical NF‐κB1 p105 pathway is constitutively activated in colorectal carcinoma specimens, being particularly associated with β‐catenin‐mediated increased expression of CRD‐BP and β‐TrCP1. In the carcinoma tissues exhibiting high levels of nuclear β‐catenin the phospho‐p105 levels were increased and total p105 amounts were decreased in comparison to that of normal tissue indicating an activation of this NF‐κB pathway. Knockdown of CRD‐BP in colorectal cancer cell line SW620 resulted in significantly higher basal levels of both NF‐κB inhibitory proteins, p105 and IκBα. Furthermore decreased NF‐κB binding activity was observed in CRD‐BP siRNA‐transfected SW620 cells as compared with those transfected with control siRNA. Altogether, our findings suggest that activation of NF‐κB1 p105 signaling in colorectal carcinoma might be attributed to β‐catenin‐mediated induction of CRD‐BP and β‐TrCP1.

Spezifisches Endothelzell-Targeting mit einem Shiga-like-Toxin-VEGF Fusionsprotein als neue Therapiestrategie für das Pankreaskarzinom

SLT-VEGF Fusionsprotein reduziert Primartumorwachstum und Disseminierung des experimentellen Pankreaskarzinoms, was in einem verbesserten 14-Wochen-Uberleben der Versuchstiere resultiert. Die reduzierte mikrovaskulare Gefasdichte deutet darauf hin, das dieser Effekt in erster Linie durch einen toxischen Effekt von SLT-VEGF auf Tumorendothelzellen vermittelt wird. Die eingesetzten Therapien waren nicht mit systemischen Nebenwirkungen wie Gewichtsverlust assoziiert.

Der Angiogenese-Inhibitor IM862 verbessert das Überleben beim experimentellen Pankreaskarzinom auch nach verzögertem Therapiebeginn

IM862 is a dipeptide of L-glutamyl-L-tryptophan with antiangiogenic properties and potential antitumor activity. This study evaluated the effect of IM862 on human pancreatic cancer in vitro and in vivo. Proliferation of three pancreatic cancer cell lines (MIAPaCa-2, AsPC-1, HPAF-2) was significantly inhibited only at high concentrations of IM862 (> 100 µg/ml). In vivo, IM862 (100 mg/kg) reduced tumor size and metastasis in an orthotopic nude mouse model, thereby increasing survival, even after a delayed onset of therapy (6 weeks after tumor induction). In vitro data, reduced microvessel density in tumors, and diminished plasma concentration of the proangiogenic mediator VEGF indicate that IM862 inhibits tumor angiogenesis rather than proliferation of pancreatic cancer cells.

Non-invasive quantification of anti-angiogenic therapy by contrast-enhanced MRI in experimental pancreatic cancer.

Background Currently, early changes of tumor vasculature after angiogenesis inhibition can only be evaluated by histopathology, a method not suitable in a clinical setting. Purpose To quantify effects of different angiogenesis inhibitors on the microvasculature of orthotopically implanted pancreatic cancers by contrast-enhanced magnetic resonance imaging (MRI) in order to establish a non-invasive technique for monitoring antiangiogenic cancer treatment. Material and Methods DSL-6A/C1 pancreatic cancers were implanted in the pancreas of 109 Lewis rats. Three weeks later, antiangiogenic treatment was initiated by administration of Bevacizumab (n = 38) or Suramin (n = 27) while the control group (n = 44) remained untreated. Dynamic MRI was performed 24 h, 1 week, and 4 weeks after treatment initiation. Fractional tumor plasma volume (fPV, %) and vascular permeability (KPS, mL/min/100 cc) were calculated based on the MRI data by using a pharmacokinetic model. Results Twenty-four hours after the initial dose, a significant decline in KPS was observed in the Bevacizumab group compared to the control and Suramin group (0.002 ± 0.008; 0.057 ± 0.046 and 0.064 ± 0.062 (mean ± SD); P < 0.05). At 1 week, fPV was significantly smaller in Bevacizumab and Suramin treated tumors compared to control tumors (6.25 ± 2.74, 7.47 ± 3.44, and 15.10 ± 9.97, respectively; P < 0.05). Differences in tumor volumes were first observed after 4 weeks of treatment with significantly larger control tumors (4380.3 ± 1590.6 vs. 869.6 ± 717.2 and 1676.5 ± 2524.1 mm3; P < 0.05). Conclusion Dynamic MRI can quantify antiangiogenic effects on tumor microvasculature before changes in tumor volumes are detectable. Thus, this technique is a reasonable addition to morphological MRI and may be applied as an alternative to histopathology.

Ein inaktiver Rezeptor verhindert ein effektives EGF-Rezeptor-Targeting beim experimentellen Magenkarzinom

Overexpression of epidermal growth factor receptor 1 (EGFR1) plays a central role in malignant transformation and tumor progression. EGFR1 inhibition by specific antibodies or tyrosine-kinase inhibitors yielded therapeutic effects in colon and lung cancer. This study evaluated the effect of EGFR1 inhibition alone or in combination with chemotherapy on human gastric cancer cell lines in vitro and in an orthotopic nude mouse model. 3 human gastric cancer cell lines (MKN-45, AGS, NCI-N87) were exposed to Carboplatin (0–3000 μg/ml), Irinotecan (0–1000 μg/ml), Docetaxel (0–300 μg/ml), or to the EGFR1-antibody Cetuximab (0–100 μg/ml) and the EGFR-tyrosine-kinase inhibitor Erlotinib (0–10 μM). Cell proliferation was assessed after 24 hours by MTT-assay. 1 cmm fragments from subcutaneous MKN-45 donor tumors were implanted into the gastric corpus of 48 nude mice. The application of Carboplatin (20 mg/kg, weekly ip.), Cetuximab (1 mg, weekly ip.) or the combination of both substances started 4 weeks after tumor induction and was continued for 14 weeks or until death. Primary tumor volume, local infiltration and metastatic spread (dissemination score) were determined at autopsy. All cell lines were evaluated for receptor status, K-Ras and Raf mutations. Carboplatin was most effective among the evaluated chemotherapeutics and reduced proliferation of gastric cancer cells in a dose dependent manner (MKN-45: –81 %; AGS: –57 %; NCI-N87: –67 %). High concentrations of the EGFR1 inhibitors Cetuximab and Erlotinib only reduced proliferation of MKN-45 cells (–16 % and –17 %, respectively). Carboplatin, Cetuximab or the combination of both substances did not exert an effect in vivo, when therapy was started 4 weeks after tumor induction. An inactive EGFR1 in MKN-45 cells may be a possible explanation for lacking effects of the EGFR1 inhibitors.