NF-κB targeting by way of IKK inhibition sensitizes lung cancer cells to adenovirus delivery of TRAIL

<p>Abstract</p> <p>Background</p> <p>Lung cancer causes the highest rate of cancer-related deaths both in men and women. As many current treatment modalities are inadequate in increasing patient survival, new therapeutic strategies are required. TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in tumor cells but not in normal cells, prompting its current evaluation in a number of clinical trials. The successful therapeutic employment of TRAIL is restricted by the fact that many tumor cells are resistant to TRAIL. The goal of the present study was to test a novel combinatorial gene therapy modality involving adenoviral delivery of TRAIL (Ad5hTRAIL) and IKK inhibition (AdIKKβKA) to overcome TRAIL resistance in lung cancer cells.</p> <p>Methods</p> <p>Fluorescent microscopy and flow cytometry were used to detect optimum doses of adenovirus vectors to transduce lung cancer cells. Cell viability was assessed via a live/dead cell viability assay. Luciferase assays were employed to monitor cellular NF-κB activity. Apoptosis was confirmed using Annexin V binding.</p> <p>Results</p> <p>Neither Ad5hTRAIL nor AdIKKβKA infection alone induced apoptosis in A549 lung cancer cells, but the combined use of Ad5hTRAIL and AdIKKβKA significantly increased the amount of A549 apoptosis. Luciferase assays demonstrated that both endogenous and TRAIL-induced NF-κB activity was down-regulated by AdIKKβKA expression.</p> <p>Conclusions</p> <p>Combination treatment with Ad5hTRAIL and AdIKKβKA induced significant apoptosis of TRAIL-resistant A549 cells, suggesting that dual gene therapy strategy involving exogenous TRAIL gene expression with concurrent IKK inhibition may be a promising novel gene therapy modality to treat lung cancer.</p

TRAIL and DcR1 Expressions Are Differentially Regulated in the Pancreatic Islets of STZ- versus CY-Applied NOD Mice

TNF-related apoptosis-inducing ligand (TRAIL) is an important component of the immune system. Although it is well acknowledged that it also has an important role in Type 1 Diabetes (T1D) development, this presumed role has not yet been clearly revealed. Streptozotocin (STZ) and Cyclophosphamide (CY) are frequently used agents for establishment or acceleration of T1D disease in experimental models, including the non-obese diabetic (NOD) mice. Although such disease models are very suitable for diabetes research, different expression patterns for various T1D-related molecules may be expected, depending on the action mechanism of the applied agent. We accelerated diabetes in female NOD mice using STZ or CY and analyzed the expression profiles of TRAIL ligand and receptors throughout disease development. TRAIL ligand expression followed a completely different pattern in STZ- versus CY-accelerated disease, displaying a prominent increase in the former, while appearing at reduced levels in the latter. Decoy receptor 1 (DcR1) expression also increased significantly in the pancreatic islets in STZ-induced disease. Specific increases observed in TRAIL ligand and DcR1 expressions may be part of a defensive strategy of the beta islets against the infiltrating leukocytes, while the immune-suppressive agent CY may partly hold down this defense, contributing further to diabetes development

Bestimmung der "Organic anion transporting polypeptides" in Ovarialkarzinomen mittels Immunfluoreszenz-Mikroskopie und Immunhistochemie

Etwa 90% der bösartigen Ovarialtumoren entstehen aus dem Ovaroberflächenepithel. Die Behandlung des Ovarialkarzinoms hat noch immer trotz modernster Therapieverfahren und Einsatz neuester Chemotherapeutika einen begrenzten Erfolg. Prognostisch stellt das Ovarialkarzinom das ungünstigste Genitalkarzinom dar, das aufgrund der Symptomlosigkeit oder eines Symptommangels sowie schlechter Screeningmaßnahmen erst im fortgeschrittenen späten Stadium erkannt wird und daher die Haupttodesursache unter den gynäkologischen Tumoren ist. Darüber hinaus kommt es unter einer Chemotherapie häufig zu einer Chemoresistenz. Die Standardtherapie für eine Patientin in einem fortgeschrittenen Stadium mit einer platinbasierten Kombinationstherapie als Kombinationstherapie führt häufig zu einem Rezidiv. Oberstes Ziel ist es daher, potentielle Wirkstoffe für die Chemotherapie zu entwickeln, um den Tumor zu beseitigen. Die Transporterfamilie der „Organic Anion Transporting Polypeptides“ (OATPs), die zur Familie der „Solute Carriers“ (SLC) gehört und auch als SLC21A-Subfamilie bezeichnet wird, ist an der Aufnahme von verschiedenen Chemotherapeutika beteiligt. Untersuchungen von OATPs scheinen daher möglicherweise einen wichtigen Therapieansatz für die Entdeckung neuer Zytosatika zu liefern. Ziel dieser vorliegenden Diplomarbeit war es, die Lokalisation von OATP 1B1/1B3, 2A1, 2B1, 3A1, 4A1, 5A1 und 6A1 in Paraffin eingebetteten Gewebeproben von Patientinnen mit einem Ovarialkarzinom mittels Immunfluoreszenz und immunhistochemischer Färbemethoden zu untersuchen. Doppelimmunfluoreszenzfärbungen wurden durchgeführt, um eine Kolokalisation der OATPs mit Markern für epitheliale und endotheliale Zellen darzustellen. CK19 (epitheliale Zellen) oder CD34 (vaskuläre endotheliale Zellen) dienten als zelluläre Marker, um die Lokalisation der OATPs zu bestimmen. In den Überlappungen der Bilder der jeweiligen OATPs mit CK19 oder CD34 konnten die OATPs durch eine Gelb-Orangefärbung in den zellulären Membranen nachgewiesen werden. Ergebnisse zeigten, dass OATP 1B1/1B3, OATP 2A1, OATP 3A1, OATP 4A1 und OATP 5A1 tatsächlich in den CK19-positiven epithelialen Zellen lokalisiert sind. Mit dem Gefäßmarker CD34 erfolgten Doppelfärbungen zum Nachweis von OATP 2A1, OATP 2B1 und OATP 4A1. Außerdem wurden immunhistochemische Studien mit OATP 1B1/1B3 und OATP 4A1, die durch eine Braunfärbung der epithelialen Zellmembranen dargestellt wurden, durchgeführt. Zusammenfassend kann festgestellt werden, dass sowohl „Leber-spezifische“ als auch ubiquitär vorkommende OATPs in den Ovarialkarzinompräparaten nachzuweisen sind. Daher könnten diese Transportproteine für die Wirksamkeit von Arzneistoffen und Hormonen bei diesen Tumoren von Bedeutung sein.Epithelial ovarian carcinomas, which arise in the ovarian surface epithelium, constitute 90% of ovarian cancers. Treatment of ovarian cancer often has a limited success despite the application of modern therapy methods. The tumor has the worst prognosis of all gynecological malignancies, because due to the lack of sensitive diagnostic tools and non-specific clinical symptoms, ovarian cancers are not diagnosed mostly after the cancer has spread beyond the ovaries. Additionally, these malignancies are often resistant to anticancer chemotherapy. The standard first-line treatment for advanced-staged ovarian cancer is a platinum-based chemotherapy. However many patients experience a relapse following platinum-containing chemotherapy treatment. Therefore, the aim is to find novel targets for anticancer chemotherapy. The transporters from the family of organic anion transporting polypeptides (OATPs), classified as subfamily SLC21A within the superfamily of solute carriers (SLC), are mainly involved in the uptake of various anticancer drugs. Investigations of this transporter family seem to be suitable as an important therapeutic approach for the discovery of anticancer agents. The purpose of the present diploma thesis was to investigate localization of OATP 1B1/1B3, 2A1, 2B1, 3A1, 4A1, 5A1 and 6A1 in paraffin embedded tissue sections from ovarian cancer patients via immunofluorescence and immunohistochemical staining methods. Double immunofluorescence staining was done to show a possible co-localization of OATPs with marker for epithelial and endothelial cells. Therefore, cellular markers like CK19 (epithelial) or CD34 (vascular endothelial cells) were used to determine the localization of the OATPs. In the overlay picture of specific OATPs and CK19 or CD 34, respectively, OATPs were demonstrated at the cell surface by a yellow to orange color of cellular membranes. The results indicated that OATP 1B1/1B3, OATP 2A1, OATP 2B1, OATP 3A1, OATP 4A1 and OATP 5A1 are indeed located in epithelial cells that give a positive staining for CK19. Double staining images were detected for CD34 and OATP 2A1, OATP 2B1 and OATP4A1. Additionally, in immunohistochemical studies, OATP 1B1/1B3, and OATP 4A1 were visualized as clear brown staining of epithelial cell membranes. Taken together, these studies show that OATPs are widely present in ovarian tissue. Therefore, in subsequent studies, the importance of different OATPs for the cellular uptake of anticancer drugs should be investigated in ovarian cancer specimens

TRAIL Death Receptor-4, Decoy Receptor-1 and Decoy Receptor-2 Expression on CD8+ T Cells Correlate with the Disease Severity in Patients with Rheumatoid Arthritis

BACKGROUND: Rheumatoid Arthritis (RA) is a chronic autoimmune inflammatory disorder. Although the pathogenesis of disease is unclear, it is well known that T cells play a major role in both development and perpetuation of RA through activating macrophages and B cells. Since the lack of TNF-Related Apoptosis Inducing Ligand (TRAIL) expression resulted in defective thymocyte apoptosis leading to an autoimmune disease, we explored evidence for alterations in TRAIL/TRAIL receptor expression on peripheral T lymphocytes in the molecular mechanism of RA development. METHODS: The expression of TRAIL/TRAIL receptors on T cells in 20 RA patients and 12 control individuals were analyzed using flow cytometry. The correlation of TRAIL and its receptor expression profile was compared with clinical RA parameters (RA activity scored as per DAS28) using Spearman Rho Analysis. RESULTS: While no change was detected in the ratio of CD4+ to CD8+ T cells between controls and RA patient groups, upregulation of TRAIL and its receptors (both death and decoy) was detected on both CD4+ and CD8+ T cells in RA patients compared to control individuals. Death Receptor-4 (DR4) and the decoy receptors DcR1 and DcR2 on CD8+ T cells, but not on CD4+ T cells, were positively correlated with patients' DAS scores. CONCLUSIONS: Our data suggest that TRAIL/TRAIL receptor expression profiles on T cells might be important in revelation of RA pathogenesis

Surface TRAIL decoy receptor-4 expression is correlated with TRAIL resistance in MCF7 breast cancer cells

BACKGROUND: Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells. Despite this promising feature, TRAIL resistance observed in cancer cells seriously challenged the use of TRAIL as a death ligand in gene therapy. The current dispute concerns whether or not TRAIL receptor expression pattern is the primary determinant of TRAIL sensitivity in cancer cells. This study investigates TRAIL receptor expression pattern and its connection to TRAIL resistance in breast cancer cells. In addition, a DcR2 siRNA approach and a complementary gene therapy modality involving IKK inhibition (AdIKKβKA) were also tested to verify if these approaches could sensitize MCF7 breast cancer cells to adenovirus delivery of TRAIL (Ad5hTRAIL). METHODS: TRAIL sensitivity assays were conducted using Molecular Probe's Live/Dead Cellular Viability/Cytotoxicity Kit following the infection of breast cancer cells with Ad5hTRAIL. The molecular mechanism of TRAIL induced cell death under the setting of IKK inhibition was revealed by Annexin V binding. Novel quantitative Real Time RT-PCR and flow cytometry analysis were performed to disclose TRAIL receptor composition in breast cancer cells. RESULTS: MCF7 but not MDA-MB-231 breast cancer cells displayed strong resistance to adenovirus delivery of TRAIL. Only the combinatorial use of Ad5hTRAIL and AdIKKβKA infection sensitized MCF7 breast cancer cells to TRAIL induced cell death. Moreover, novel quantitative Real Time RT-PCR assays suggested that while the level of TRAIL Decoy Receptor-4 (TRAIL-R4) expression was the highest in MCF7 cells, it was the lowest TRAIL receptor expressed in MDA-MB-231 cells. In addition, conventional flow cytometry analysis demonstrated that TRAIL resistant MCF7 cells exhibited substantial levels of TRAIL-R4 expression but not TRAIL decoy receptor-3 (TRAIL-R3) on surface. On the contrary, TRAIL sensitive MDA-MB-231 cells displayed very low levels of surface TRAIL-R4 expression. Furthermore, a DcR2 siRNA approach lowered TRAIL-R4 expression on surface and this sensitized MCF7 cells to TRAIL. CONCLUSION: The expression of TRAIL-R4 decoy receptor appeared to be well correlated with TRAIL resistance encountered in breast cancer cells. Both adenovirus mediated IKKβKA expression and a DcR2 siRNA approach sensitized MCF7 breast cancer cells to TRAIL

Diabetes-Resistant NOR Mice Are More Severely Affected by Streptozotocin Compared to the Diabetes-Prone NOD Mice: Correlations with Liver and Kidney GLUT2 Expressions

Nonobese Diabetic (NOD) mice are susceptible strains for Type 1 diabetes development, and Nonobese Diabetes-Resistant (NOR) mice are defined as suitable controls for NOD mice in non-MHC-related research. Diabetes is often accelerated in NOD mice via Streptozotocin (STZ). STZ is taken inside cells via GLUT2 transmembrane carrier proteins, the major glucose transporter isoforms in pancreatic beta cells, liver, kidneys, and the small intestine. We observed severe adverse effects in NOR mice treated with STZ compared to NOD mice that were made diabetic with a similar dose. We suggested that the underlying mechanism could be differential GLUT2 expressions in pancreatic beta cells, yet immunofluorescent and immunohistochemical studies revealed similar GLUT2 expression levels. We also detected GLUT2 expression profiles in NOD and NOR hepatic and renal tissues by western blot analysis and observed considerably higher GLUT2 expression levels in liver and kidney tissues of NOR mice. Although beta cell GLUT2 expression levels are frequently evaluated as a marker predicting STZ sensitivity in animal models, we report here very different diabetic responses to STZ in two different animal strains, in spite of similar initial GLUT2 expressions in beta cells. Furthermore, use of NOR mice in STZ-mediated experimental diabetes settings should be considered accordingly

Treatment With Recombinant Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Alleviates the Severity of Streptozotocin-Induced Diabetes

OBJECTIVE: To evaluate the potential therapeutic effect of recombinant human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) treatment in a model of type 1 diabetes. RESEARCH DESIGN AND METHODS: Recombinant TRAIL was added in vitro to primary human and mouse peripheral blood mononuclear cells (PBMCs) and isolated human islets to evaluate the expression of the immunoregulatory gene SOCS1. Diabetes was induced by five consecutive daily injections of low-concentration (50 mg/kg) streptozotocin (STZ) in C57 black mice (n = 24). A group of these mice (n = 12) was co-injected with recombinant TRAIL (20 microg/day) for 5 days, and the diabetic status (glycemia and body weight) was followed over time. After 6 weeks, circulating levels of insulin, TNF-alpha, and osteoprotegerin (OPG) were measured, and animals were killed to perform the histological analysis of the pancreas. RESULTS: The in vitro exposure of both PBMCs and human islets to recombinant TRAIL significantly upregulated the expression of SOCS1. With respect to STZ-treated animals, mice co-injected with STZ+TRAIL were characterized by 1) lower levels of hyperglycemia, 2) higher levels of body weight and insulinemia, 3) a partial preservation of pancreatic islets with normal morphology, and 4) a lower expression of both systemic (TNF-alpha and OPG) and pancreatic (vascular cell adhesion molecule [VCAM]-1) inflammatory markers. CONCLUSIONS: Overall, these data demonstrate that the administration of recombinant TRAIL ameliorates the severity of STZ-induced type 1 diabetes, and this effect was accompanied by the upregulation of SOCS1 expressio

Discovery and Validation of a New Class of Small Molecule Toll-Like Receptor 4 (TLR4) Inhibitors

Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C17H27NO9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the β-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases. © 2013 Neal et al