Dexamethasone reduces tumor recurrence and metastasis after ...

[Cancer Biology & Therapy 7:7, 1044-1050; July 2008]; ©2008 Landes Bioscience

Research Paper

Dexamethasone reduces tumor recurrence and metastasis after pancreatic tumor resection in SCID mice Jan-Hendrik Egberts, Bodo Schniewind, Miriam Pätzold, Bastian Kettler, Jürgen Tepel, Holger Kalthoff* and Anna Trauzold Division of Molecular Oncology; Department of General Surgery and Thoracic Surgery; University Hospital of Schleswig-Holstein; Kiel, Germany

Key words: pancreatic cancer, therapy, dexamethasone, inflammation and cancer, metastasis

Introduction

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Glucosteroids (GCs) are widely used for suppression of inflammation in chronic inflammatory diseases such as asthma, rheumatoid arthritis and inflammatory bowel disease.1 The predominant effects of GCs are to switch off multiple activated inflammatory genes (such as encoding cytokines, chemokines, adhesion molecules, inflammatory enzymes, receptors and proteins).1 In the oncological setting GCs are widely used as powerful cytotoxic agents in the treatment of lymphoid malignancies and as comedication in cancer therapy of solid tumors due to their effectiveness in treating the tumor related edema, inflammation, pain and preventing nausea and emesis caused by cytotoxic drugs.2-5 However, little is known about a potential impact of GCs on growth and metastasis of solid tumors like pancreatic ductal adenocarcinoma (PDAC), especially after surgical resection of the tumors when proinflammatory cytokines are released and activated.6,7 Surgery is the only potentially curative option for patients with PDAC, a disease in which the annual mortality rates are almost equivalent to the incidence rate.8 Overall, only 10–20% of diagnosed patients are applicable for resection. In these patients the five year survival rates, even after tumor free resection margins (R0-resection), are only 6–20%.9 Operative tissue trauma and systemic inflammatory response might have an influence on the development of tumor recurrence and metastasis. There are several reports on a consecutive association between surgical trauma and the development of tumor recurrence and/or metastasis in vitro and in vivo.10-12 A possible explanation for these findings might be the fact that the inflammatory reaction provoked by the surgical trauma leads to elevated levels of proinflammatory cytokines like interleukin-1 (IL1), interleukin-6 (IL6), Interleukin-8 (IL8) and tumor necrosis factor-alpha (TNFα) in the peripheral blood.6,7,13 These cytokines, in turn, seem to have a major effect on the development of local recurrences and distant tumor growth.6,7,13 In vitro, it has been shown that proinflammatory cytokines cause an enhanced (pancreatic) tumor cell adhesion to microvascular endothelial cells and might therefore increase the risk of metastasis formation.14,15 In patients suffering from PDAC a variety of immune cells including macrophages in the tumor16,17 and increased levels of interleukins (IL1, IL6, IL8, IL10 and TNFα) in the serum are detectable.18-20 Thus, there is evidence that PDAC cells in situ are regularly exposed to either endogenous (produced in an autocrine fashion) or exogenous (produced by immune or stromal

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Background: Glucocorticoids are among the most potent anti-inflammatory agents that act by inhibiting the synthesis of almost all known cytokines and influencing multiple transduction pathways. Inflammation accompanies most solid cancers including pancreatic ductal adenocarcinoma (PDAC), one of the most fatal cancers with surgery being the only potential curative therapeutic. In the present work we investigated the influence of glucocorticoids on PDAC cells in vitro as well as in vivo in a pancreatic carcinoma resection mouse model. Methods: The influence of dexamethasone (DEX), a synthetic glucocorticoid, on proliferation and IL8 secretion in pancreatic cells (BxPC3, Colo357, PancTuI) was analyzed by cell counting and ELISA. NFκB-activity of PancTuI cells treated with DEX was determined by electrophoretic mobility shift assay (EMSA). Furthermore, effects of DEX on the invasiveness were studied by a fibroblast-based invasion assay. In the mouse resection model subtotal pancreatectomy was performed after orthotopic inoculation of human PDAC cells. DEX was administered after resection as an adjuvant treatment regime and four weeks later, local recurrent tumor sizes as well as number of liver and spleen metastases were analyzed. Results: In vitro, DEX did not have an anti-proliferative effect on PDAC cells, but strongly reduced the invasiveness well as the activation of NFκB. The secretion of IL-8 into the supernatant of the tumor cells correlated inversely with the reduced activation of NFκB. In vivo, we observed a significant reduction of the local recurrent tumor volume and the number of liver and spleen metastases. Conclusions: DEX has a profound influence on the malignant phenotype of PDAC tumor cells in vitro in terms of inhibition of invasiveness and pro-inflammatory signaling. This was approved in vivo by reduced metastasizing capacity and reduced size of local tumor recurrence. Therefore, DEX-treatment appears to be an interesting therapeutical option in an adjuvant setting after pancreatic cancer resection. *Correspondence to: Holger Kalthoff; Division of Molecular Oncology; Department of General Surgery and Thoracic Surgery; University Hospital of Schleswig-Holstein; Campus Kiel; Arnold-Heller-Strasse 7; Kiel 24105 Germany; Tel.: +49.431.5971938; Fax: +49.431.5971939; Email: [email protected] Submitted: 12/22/07; Revised: 02/28/08; Accepted: 04/14/08 Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/article/6099 1044

Cancer Biology & Therapy

2008; Vol. 7 Issue 7

Anti-inflammatory therapy as an adjuvant treatment option for the postoperative course after pancreatic carcinoma resection

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cells) cytokines, pointing to a possible involvement not only in the development and progression of PDAC but also in surgery-associated tumor recurrence and metastasis. Moreover, it is known that chronic inflammation in the pancreas, e.g., chronic pancreatitis, correlates with a higher risk of PDAC development.21 We recently observed a correlation of tumor recurrence and metastasis after resection of the primary tumor in an orthotopic xenotransplant animal model in mice.16 In this model, the orthotopically inoculated tumor cells (PancTuI cells) barely metastasize into the liver or other organs but grow locally infiltrative.17 After resection of the primary tumors we observed a three fold increase in the number of liver and spleen metastasis.16 These findings imply that at the time of diagnosis or surgical resection, subclinical Figure 1. DEX-treatment has no influence on PDAC cells growth but inhibits the secretion metastases or disseminated tumor cells are already exis- of IL8. (A) BxPC3, Colo357 and PancTuI cells were treated with different concentratent and not (yet) detectable. By the manipulation and tion of DEX and counted as described in Material and Methods at time points 0 h, 24 h, 48 h and 72 h. Data represent means ± SD from three independent experiments. the following exposition to inflammatory cytokines, the (B) The amounts of IL8 in cell culture supernatants from untreated and DEX-treated cells PDAC cells might be activated and/or change their biolog- in a concentration of 106 cells (see Fig. 1A) were determined by IL8-ELISA. Different ical behavior resulting in an increased risk of metastasis concentrations of DEX were investigated: control (black bars), 0.001 μM DEX (hatched formation and tumor recurrence.22 We hypothesized that bars), 0.01 μM DEX (grey bars) and 0.1 μM DEX (white bars). The concentrations were the suppression of the release of proinflammatory cytokines normalized to the cell numbers determined in parallel. Data represent means ± SD of three independent experiments. after surgical resection might influence the development of tumor recurrence and metastasis. As described above, a NFκB has repeatedly been shown to enhance the invasive proppotent inhibition of the release can be achieved by GC, e.g., dexamethaosne (DEX), a synthetic GC. In the present study we investigated erties of cancer cells. Because DEX strongly inhibited the NFκB the effects of DEX on pancreatic tumor cells in vitro and in a murine activity in PancTuI cells we investigated whether DEX also affects the orthotopic xenotransplantation model in vivo. We demonstrate that invasiveness of these cells. Using an in vitro invasion assay detecting tumor recurrence and metastasis after surgical resection of PDAC is the ability of tumor cells to invade and digest the monolayer of fibroblasts, we showed that treatment of PancTuI cells with different significantly inhibited by administration of DEX. concentrations of DEX indeed dose-dependently inhibited invasiveResults ness blocking it completely at the concentration of 0.1 μM. DEX reduces pancreatic tumor recurrence and metastasis. To DEX inhibits NFκB, IL8 secretion and invasiveness of PDAC cells in vitro. To determine the effects of DEX on the malignancy of investigate the impact of DEX on tumor recurrence and metastasis pancreatic tumor cells we first analyzed the proliferation of BxPC3, after subtotal pancreatectomy, mice bearing PancTuI tumors were Colo357 and PancTuI cells by daily counting the cells cultivated in treated according to the scheme shown in Figure 3. After sacrifice of medium with different DEX concentrations (0.001 μM, 0.01 μM all animals we analyzed the recurrent tumor growth and metastasis. and 0.1 μM). We found that DEX had no influence on the growth All animals developed local recurrent tumors. The mean size of locally recurrent tumor was significantly reduced of the analyzed cells (Fig. 1A). Since it has been shown that DEX may affect the pro-inflam- by DEX as well in terms of tumor weight as in terms of tumor matory pathways we further determine the levels of IL8 secreted by volume. Mean tumor weight was 524.3 ± 161.3 mg for animals in cells treated with different concentration of DEX using ELISA. As control group compared to 365.75 ± 133.2 to the DEX treated mice shown in Figure 1B DEX strongly and dose-dependently inhibited (p = 0.037). The corresponding mean tumor volumes were 588.1 ± the secretion of IL8 in all analyzed cell lines (Fig. 1B). The observed 221.3 mm3 in the control and 386.0 ± 117.2 in the DEX treated group (p = 0.032). In summary, the tumor weight and volume were effects lasted for at least 72 h. IL8 is a target gene of the transcription factors NFκB and AP1. reduced by one third by DEX. After tumor resection, in all mice of the control group hepatic Therefore, we consequently determined the influence of DEX on the constitutive activity of both factors in PancTuI cells. The results of metastases were observed, whereas two mice in the DEX treated electrophoretic mobility shift assays (EMSA) depicted in Figure 2A group did not develop hepatic metastases at all. The mean number demonstrate that 0.1 μM DEX reduced NFκB activity up to 55.56% of liver metastases was 2.6 ± 1.35 in the control group and signifiand 1 μM DEX up to 62%. Interestingly, no changes of AP1 activity cant (p = 0.03) lower in the DEX treated group (mean number of were provoked by the used DEX concentrations (Fig. 2B). To study metastases 1.13 ± 0.99). A similar observation was found in the the mechanism of DEX-mediated inhibition of NFκB in PancTuI number of spleen metastases: 2.2 ± 1.6 metastases were detected in cells, we analyzed the phosphorylation status and expression levels the control group and 0.63 ± 0.74 in the DEX group (p = 0.048). of IκBα. As depicted in Figure 2C, DEX induced phosphorylation Histological examination of resected tumors and hepatic and spleen of IκBα. Interestingly, no accompanying IκB degradation could be tissues confirmed the macroscopically diagnosed metastases, which showed a similar cellular pattern to the primary or recurrent tumors, detected using antibody against total IκBα. www.landesbioscience.com


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Figure 2. DEX inhibits constitutive NFκB activity and invasiveness of PancTuI cells. PancTuI cells were treated for 24 h with DEX (0.1 μM and 1 μM) and NFκB (A) as well as AP1 activity (B) in nuclear extracts were determined by EMSA (s. material and methods). The composition of NFκB complexes was proofed by supershift assay. The binding specificity of NFκB and AP1 binding activity was determined by competition assay using 100-fold excess of wildtype or mutated unlabeled NFκB or AP1-binding sequence containing oligonucleotides, respectively. (C) Western blot showing the level of phosphorylated (P-IκBα) and total IκBα in PancTuI cells treated for 24 h with DEX (0.1 μM and 1 μM). As control of equal gel-loading, β-actin levels were determined in parallel. (D) Untreated and DEX-treated (0.1 μM and 1 μM) PancTuI cells were grown on fibroblasts monolayer for 24 h. The invasive properties of PancTuI cells were analyzed as described in Material and Methods. Shown are representative results from one of three independently performed experiments.

as described previously.17 Surrounded by normal parenchyma, the lesions proved to be veritable metastases and not just tumor deposits resulting from continuous spreading (not shown). Data are summarized and shown in Figure 3 and Table 1.

Discussion GCs inhibit the synthesis of almost all known pro-inflammatory cytokines and thus influence multiple transduction pathways.1,30 DEX and similar GCs were first introduced to tumor therapy on the basis of pro-apoptotic effects in lymphoid cells and are a cornerstone in treatment of lymphomas and leukaemia.2,3 The use of GCs in solid tumors, however, does not aim to destroy malignant cells, but rather to alleviate symptoms caused by the tumor, such as edema, 1046

inflammation, pain, lack of appetite and electrolyte imbalance.31 GCs bind to glucocorticoid receptors (GRs) in the cytoplasm that translocate to the nucleus, where they bind to glucocorticoid response elements (GRE) in the promoter region of steroid-sensitive genes activating or inhibiting their transcription.32 In addition, GRs interact directly or indirectly with several other transcription factors, e.g., NFκB, AP1, p53 and CREB, thus negatively influencing the transcription of their target genes. There is fundamental evidence that the nuclear factor NFκB promotes tumorigenesis by cellular transformation, proliferation, apoptosis suppression, invasion and angiogenesis.33 The NFκB pathway is activated by TNFα, IL1 and other pro-inflammatory cytokines and mediates many of the pro-tumoral effects. 34,35



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Interestingly, increased levels of TNFα, IL1, IL6 and IL8 were found in patients suffering from pancreatic cancer.18,19,36 Furthermore, a high constitutive activity of NFκB has been reported in various pancreatic cell lines and primary tumor samples.37 Thus, the inhibition of NFκB and pro-inflammatory pathways could be a promising treatment strategy for PDAC and various other solid tumors. Despite tremendous efforts that have been made to develop new therapeutic treatment strategies for PDAC patients, only the surgical resection is known to be the sole chance for cure or is at least associated with a substantial survival benefit. Unfortunately, even after R0 resection most of PDAC patients develop local tumor recurrence and metastases and rarely survive more than five years.38 However, surgical manipulations are unavoidably associated with consecutive inflammatory reactions as several studies demonstrated increased serum levels of proinflammatory cytokines after major abdominal surgery.6,7,13 In vitro, it has been shown that these cytokines enhance pancreatic and colon carcinoma tumor cell adhesion to microvascular endothelial cells and promote successful tumor cell implantation resulting in an increased risk of metastases.12,14 DEX is known to inhibit the expression of various pro-inflammatory cytokines. In the present study we determined the putative anti-tumoral effects of DEX on pancreatic tumor cells in vitro by analyzing the NFκB activity, secretion of IL8 and invasiveness of PDAC cells and in vivo by analyzing the tumor growth and metastasis after resection of the primary tumor. In vitro, we observed that DEX strongly inhibits the constitutive activity of NFκB and as a consequence the expression of the established NFκB target gene IL8. DEX displayed no effects on proliferation but completely abolished the invasive properties of pancreatic tumor cells (Fig. 2). Although the ability of GCs to inhibit the NFκB activity is very well documented, the exact mechanisms for these actions are still not fully understood. In some cellular systems activated GRs are able to induce the expression of IκB with the consecutive inhibition of the NFκB activity by forming an IκB-NFκB complex.30 Another way for GCs to inhibit NFκB might be either the inhibition of the translocation of NFκB into the nucleus or the disturbance of the interaction of p65 with the basal transcriptional machinery.39 Since we did not observe any changes in the expression of IkB in DEX treated cells (Fig. 2C) the suppression of NFκB activity might be mediated by one of the above mentioned mechanisms. Overall, it is apparent that the precise steps through which glucocorticoids mediate their suppressive effects on gene transcription needs further investigation. Our in vivo data show that DEX significantly reduced the size of the recurrent tumors and also diminished the number of metastases after surgical resection of orthotopically inoculated PDAC in SCID bg mice. The recurrent tumor volume was reduced to two thirds and the number of liver and spleen metastases was more than halved compared to the control group (Fig. 3). These data suggest that proinflammatory cytokines have a severe influence on the development of local tumor recurrence and formation of distant metastases after surgical resection. These results might therefore an explanation for our recent findings, where we observed an increased number of metastases after resection of the orthotopically inoculated PancTuI cells in SCID bg mice.16,17 After surgical resection adjuvant chemotherapy has become a major component in the treatment of pancreatic cancer patients,

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Figure 3. DEX reduces pancreatic tumor recurrence and metastasis. DEX administration after subtotal pancreatectomy inhibits tumor re-growth and metastasis. Primary tumors were resected by subtotal pancreatectomy ten days after inoculation of 106 PancTuI cells and mice were treated intraperitoneally with 0.9% saline (control, n = 12), and DEX (n = 10; 2.5 mg/kg) as shown in the scheme (A). The recurrent tumor volume (B), recurrent tumor weight (C) and the number of liver (D) and spleen (E) metastases were determined on day 28 after primary tumor resection. The significances of the data were as follows: *p = 0.033; **p = 0.037; ***p = 036; ****p = 0.048.


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Table 1 Incisional metastases, organ metastases (liver or spleen) metastases, pancreatic and recurrent tumor weight on day 28 after resection of PancTuI bearing mice without additional treatment (control) and DEX treatment, all groups n = 12 SCID beige mice

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Materials and Methods

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which is often combined with DEX preventing nausea and emesis caused by the cytotoxic drugs.5,40 However, controlled randomized clinical trials evaluating a potential impact of GCs on growth of solid tumors and patient survival have, to the best of our knowledge, never been performed. In vitro, some investigators observed an enhancement of the therapeutic benefits of cisplatin by DEX via regulation of tumor angiogenesis and cell cycle kinetics in a murine tumor model.41-43 But there are also concerns about the widespread use of GCs during therapy of solid tumors in combination with cytotoxic therapy describing the induction of resistance to treatment modalities in pancreatic and other solid tumor cells.28,44 A possible explanation for this effect might be the upregulation of pro survival-factors and anti-apoptotic genes as well as the induction of cell cycle arrest. The long term use of GCs might therefore be critical, especially in combination with cytotoxic therapy. However, it must be mentioned that many of these studies are in vitro studies or in vivo studies in which the tumor cells or human tumor specimens were inoculated subcutaneously without surgical manipulation. During the acute postoperative phase with an increased inflammatory response of the organism GCs might therefore have a potential benefit, until further cytotoxic treatment is started. Carefully designed clinical studies concerning the benefit of GCs after resection of pancreatic cancer would be mandatory. This underlines the importance of preclinical models that mimic the clinical situation as closely as possible and allow new therapeutic options to be tested in a systematic fashion like the one presented here.

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Incisional metastases Liver # mg metastases 12/12 428.3 11/12 5/12 115.3 6/12

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the instructions of the manufacturer. The samples were separated on native 6% polyacrylamide gels in low-ionic strength buffer (0.25 x Tris-borate-EDTA) and visualized by autoradiography. For competition assays, an 100-fold excess of unlabeled specific NFκB or AP1 oligonucleotides or 100-fold excess of unlabeled unspecific, mutated oligonucleotides were added to cell extracts ten minutes prior to the incubation with labeled NFκB binding oligonucleotides. For supershift assay, 0.4 μg of anti-p65 or 0.4 μg of anti-p50 antibodies (both from Santa-Cruz) were added to the sample and incubated for the additional one hour at 4°C. The calculation of reduction of NF activity was performed on a Macintosh computer using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http:// rsb.info.nih.gov/nih-image/). Western blot. Cells were lysed in SDS-Sample Buffer (62.5 mM Tris-HCl pH 6.8; 2% SDS; 10% glycerol; 50 mM DTT; 0.01% bromophenol blue), sonicated for ten seconds, heated for five minutes at 95°C and centrifuged for five minutes. Western Blot analysis was performed as described.24 Primary antibodies used were purchased from: Cell Signalling, Frankfurt, Germany (anti-phospho-IkappaBα), Santa Cruz Biotechnology, Heidelberg, Germany (IκBα) and Sigma, Taufkirchen, Germany (anti β-actin). Invasion assay. The invasive potential of tumor cells was determined using a trypan-blue dye-based model for cell invasion.25 Briefly, KiF-5 fibroblasts were seeded in a 24-well plate (2.5 x 105 cells/well). After four days cells were rinsed with PBS, permeabilized with 500 μl DMSO for one hour at room temperature, washed twice with PBS and overlayed with tumor cells (2 x 104 cells/well) and resuspended in culture medium with or without DEX. After additional 24 h cells were washed with PBS, stained for 15 min with 0.2% trypan-blue (Invitrogen), washed twice with PBS and photographed. Since trypan-blue stained the dead permeabilized cells, the fibroblasts layer could be distinguished from the living carcinoma cells. The observed areas of unstained cells represent regions where fibroblasts were displaced or digested by invasive tumor cells. Laboratory animals. Four-week-old female SCID beige mice weighing 14–19 g were obtained from Harlan-Winkelmann (Borchen, Germany). The mice were allowed to become acclimatized for ten days, and housed in a sterile environment, in which bedding, food and water were autoclaved (Scantainer, Denmark). Animal experiments and care were in accordance with the guidelines of institutional authorities and approved by local authorities [number: V 362-72241.121 (16-1/06)]. Orthotopic xenotransplantation of human PDAC cells and tumor resection. After median laparotomy, 30 μl of tumor cell suspension were injected into the pancreatic body to the left of the organ midline. The technique of the orthotopical tumor cell injection was performed as described in detail before.16,17 Thirty microliters of the tumor cell-MatrigelTM suspension (1 x 106 cells) were slowly injected into the pancreas. After waiting for approximately 30 s, the tumor-cell-Matrigel solution was solid and a spillage of tumor cells could be avoided. The pancreas was replaced and the abdominal wall closed using Vicryl 6/0 (Ethicon, Norderstedt, Germany). The recovery was carefully supervised with red light re-warming and volume substitution (0.5 ml of 5% glucose). Buprenorphine at 2.5 mg/kg was given intraperitoneally (i.p.) twice daily for two postoperative days for postoperative analgesia. The animals were examined

Cell culture. Human pancreatic adenocarcinoma cells PancTuI, Colo357 and BxPC3 were cultured in RPMI-1640 medium, supplemented with 10% FCS, 2 mM glutamine and 1 mM sodium pyruvate.For stimulation experiments cells were treated with dexamethasone (Merck Pharma GmbH, Darmstadt, Germany). For animal experiments, cells were trypsinized, resuspended in MatrigelTM (BD Bioscience, Heidelberg, Germany) at a concentration of 106 cells/ml and stored on ice until injection. IL8 ELISA. For determination of IL8 concentrations in culture supernatants IL8-Immunoassays (R&D Systems, Wiesbaden, Germany) were used according to the provided protocols. Electrophoretic mobility shift assay (EMSA). For detection of the AP1 and NFκB activity, nuclear extracts were prepared as described.23 Electrophoretic mobility shift assays were performed by analyzing 5 μg of nuclear extract with the Gelshift AP1 or NFκB family (Carcinoma) kits (Active Motif, Rixensart, Belgium) following 1048




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10. van Rossen ME, Hofland LJ, van den Tol MP, van Koetsveld PM, Jeekel J, Marquet RL, et al. Effect of inflammatory cytokines and growth factors on tumour cell adhesion to the peritoneum. J Pathol 2001; 193:530-7. 11. Eggermont AM, Steller EP, Sugarbaker PH. Laparotomy enhances intraperitoneal tumor growth and abrogates the antitumor effects of interleukin-2 and lymphokine-activated killer cells. Surgery 1987; 102:71-8. 12. ten Kate M, Hofland LJ, van Grevenstein WM, van Koetsveld PV, Jeekel J, van Eijck CH. Influence of proinflammatory cytokines on the adhesion of human colon carcinoma cells to lung microvascular endothelium. Int J Cancer 2004; 112:943-50. 13. Baigrie RJ, Lamont PM, Kwiatkowski D, Dallman MJ, Morris PJ. Systemic cytokine response after major surgery. Br J Surg 1992; 79:757-60. 14. ten Kate M, Hofland LJ, van Koetsveld PM, Jeekel J, van Eijck CH. Pro-inflammatory cytokines affect pancreatic carcinoma cell. Endothelial cell interactions. Jop 2006; 7:454-64. 15. Nozawa F, Hirota M, Okabe A, Shibata M, Iwamura T, Haga Y, et al. Tumor necrosis factor alpha acts on cultured human vascular endothelial cells to increase the adhesion of pancreatic cancer cells. Pancreas 2000; 21:392-8. 16. Egberts JH, Schniewind B, Sipos B, Hinz S, Kalthoff H, Tepel J. Superiority of extended neoadjuvant chemotherapy with gemcitabine in pancreatic cancer: A comparative analysis in a clinically adapted orthotopic xenotransplantation model in SCID beige mice. Cancer Biol Ther 2007; 6:1227-32. 17. Tepel J, Kruse ML, Kapischke M, Haye S, Sipos B, Kremer B, et al. Adjuvant treatment of pancreatic carcinoma in a clinically adapted mouse resection model. Pancreatology 2006; 6:240-7. 18. Karayiannakis AJ, Syrigos KN, Polychronidis A, Pitiakoudis M, Bounovas A, Simopoulos K. Serum levels of tumor necrosis factor-alpha and nutritional status in pancreatic cancer patients. Anticancer Res 2001; 21:1355-8. 19. Sclabas GM, Fujioka S, Schmidt C, Evans DB, Chiao PJ. NFkB in pancreatic cancer. Int J Gastrointest Cancer 2003; 33:15-26. 20. Ebrahimi B, Tucker SL, Li D, Abbruzzese JL, Kurzrock R. Cytokines in pancreatic carcinoma: correlation with phenotypic characteristics and prognosis. Cancer 2004; 101:2727-36. 21. Jura N, Archer H, Bar-Sagi D. Chronic pancreatitis, pancreatic adenocarcinoma and the black box in-between. Cell Res 2005; 15:72-7. 22. Soeth E, Grigoleit U, Moellmann B, Roder C, Schniewind B, Kremer B, et al. Detection of tumor cell dissemination in pancreatic ductal carcinoma patients by CK 20 RT-PCR indicates poor survival. J Cancer Res Clin Oncol 2005; 131:669-76. 23. Schafer H, Diebel J, Arlt A, Trauzold A, Schmidt WE. The promoter of human p22/PACAP response gene 1 (PRG1) contains functional binding sites for the p53 tumor suppressor and for NFkappaB. FEBS Lett 1998; 436:139-43. 24. Trauzold A, Wermann H, Arlt A, Schutze S, Schafer H, Oestern S, et al. CD95 and TRAIL receptor-mediated activation of protein kinase C and NFkappaB contributes to apoptosis resistance in ductal pancreatic adenocarcinoma cells. Oncogene 2001; 20:4258-69. 25. Trauzold A, Roder C, Sipos B, Karsten K, Arlt A, Jiang P, et al. CD95 and TRAF2 promote invasiveness of pancreatic cancer cells. Faseb J 2005; 19:620-2. 26. Tepel J, Kruse ML, March C, Fiedler A, Kapischke M, Ketterer T, et al. Terminally modified oligodeoxynucleotides directed against p53 in an orthotopic xenograft model: a novel adjuvant treatment strategy for pancreatic ductal carcinoma. Pancreas 2004; 28:1-12. 27. Hunneyball IM, Crossley MJ, Spowage M. Pharmacological studies of antigen-induced arthritis in BALB/c mice. I. Characterization of the arthritis and the effects of steroidal and non-steroidal anti-inflammatory agents. Agents Actions 1986; 18:384-93. 28. Sui M, Chen F, Chen Z, Fan W. Glucocorticoids interfere with therapeutic efficacy of paclitaxel against human breast and ovarian xenograft tumors. Int J Cancer 2006; 119:712-7. 29. Buckwell A. Limiting clinical signs appendices. Lab Anim Sci Assoc Winter Newsl 1992:16-7. 30. Auphan N, DiDonato JA, Rosette C, Helmberg A, Karin M. Immunosuppression by glucocorticoids: inhibition of NFkB activity through induction of IkappaB synthesis. Science 1995; 270:286-90. 31. Rutz HP. Effects of corticosteroid use on treatment of solid tumours. Lancet 2002; 360:1969-70. 32. Barnes PJ. Corticosteroid effects on cell signalling. Eur Respir J 2006; 27:413-26. 33. Pacifico F, Leonardi A. NFkB in solid tumors. Biochem Pharmacol 2006; 72:1142-52. 34. Nakashima H, Nakamura M, Yamaguchi H, Yamanaka N, Akiyoshi T, Koga K, et al. Nuclear factorkappaB contributes to hedgehog signaling pathway activation through sonic hedgehog induction in pancreatic cancer. Cancer Res 2006; 66:7041-9. 35. Aggarwal BB. Nuclear factorkappaB: the enemy within. Cancer Cell 2004; 6:203-8. 36. Ariapart P, Bergstedt-Lindqvist S, van Harmelen V, Permert J, Wang F, Lundkvist I. Resection of pancreatic cancer normalizes the preoperative increase of tumor necrosis factor alpha gene expression. Pancreatology 2002; 2:491-4. 37. Sebens S, Arlt A, Schafer H. NFkappaB as a molecular target in the therapy of pancreatic carcinoma. Recent Results Cancer Res 2008; 177:151-64. 38. Smeenk HG, Tran TC, Erdmann J, van Eijck CH, Jeekel J. Survival after surgical management of pancreatic adenocarcinoma: does curative and radical surgery truly exist? Langenbecks Arch Surg 2005; 390:94-103. 39. De Bosscher K, Vanden Berghe W, Vermeulen L, Plaisance S, Boone E, Haegeman G. Glucocorticoids repress NFkappaB-driven genes by disturbing the interaction of p65 with the basal transcription machinery, irrespective of coactivator levels in the cell. Proc Natl Acad Sci USA 2000; 97:3919-24.

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daily for general signs of distress and particularly for inoculationrelated problems (e.g., jaundice). Ten days after tumor cell inoculation, a median re-laparotomy was performed and the tumor-bearing pancreas was carefully mobilized. Subtotal pancreatectomy was performed as follows: general anesthesia was induced using Fentanyl/Midazolam/Medetomidin (dosage 0.05 mg/kg; 5 mg/kg; 0.5 mg/kg). The supplying blood vessels were ligated with Vicryl 6/0. The tail, body and parts of the head of the pancreas were removed by a transsection close to the duodenum. A narrowing of the stomach or duodenum was carefully avoided. In all phases of the operation meticulous care was taken to ensure complete hemostasis, which was achieved above all by suture ligation of surrounding connective tissue. Closure of the abdominal wall and postoperative care were identical to the procedure described earlier.16,26 In vivo treatment. All mice survived the resection procedure and were randomly assigned into two treatment groups: DEX group (n = 12) with application of dexamethasone at a dose of 2.5 mg/kg and control group (n = 12) with administration 200 μl saline i.p. The administered doses of DEX at a body weight of 20 g turned out to be 50 μg/day. This dosage has been administered before and turned out to be well tolerated in mice.27,28 Treatment was started on day after subtotal pancreatectomy and lasted for 28 consecutive until sacrifice of the animals. To avoid unnecessary suffering, it was planned to sacrifice all animals in each group, if one animal met Buckwell’s criteria indicating severe distress.29 Regimes are summarized in Figure 3A. Statistical analysis. In vivo data were analysed using SPSS 11.0 (SPSS Inc., Chicago, IL, USA). Owing to skewed data distribution (tested by Shapiro-Wilk test) different groups were analysed non-parametrically by Mann-Whitney U-test. Differences were considered statistically significant at a level of p < 0.05. Acknowledgements

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We thank Birgit Fricke and Angelika Duttmann for excellent technical assistance. The support by Birte Summa is highly appreciated. Some of the data are part of the doctoral thesis of M.P. This work was supported by the Deutsche Forschungsgemeinschaft SFB 415 (project A3) and a Gerok-Fellowship plus intramural funding given to J.H.E.; MOIN-SH (Molecular Imaging North). References 1. Barnes PJ. Anti-inflammatory actions of glucocorticoids: molecular mechanisms. Clin Sci (Lond) 1998; 94:557-72. 2. Frei E, 3rd, Karon M, Levin RH, Freireich EJ, Taylor RJ, Hananian J, et al. The effectiveness of combinations of antileukemic agents in inducing and maintaining remission in children with acute leukemia. Blood 1965; 26:642-56. 3. Richardson PG, Mitsiades C, Schlossman R, Munshi N, Anderson K. New drugs for myeloma. Oncologist 2007; 12:664-89. 4. Raslan A, Bhardwaj A. Medical management of cerebral edema. Neurosurg Focus 2007; 22:12. 5. Dexamethasone alone or in combination with ondansetron for the prevention of delayed nausea and vomiting induced by chemotherapy. The Italian Group for Antiemetic Research. N Engl J Med 2000; 342:1554-9. 6. Aosasa S, Ono S, Mochizuki H, Tsujimoto H, Osada S, Takayama E, et al. Activation of monocytes and endothelial cells depends on the severity of surgical stress. World J Surg 2000; 24:10-6. 7. Badia JM, Whawell SA, Scott-Coombes DM, Abel PD, Williamson RC, Thompson JN. Peritoneal and systemic cytokine response to laparotomy. Br J Surg 1996; 83:347-8. 8. Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, et al. Cancer statistics 2007. CA Cancer J Clin 2007; 56:106-30. 9. Bilimoria KY, Bentrem DJ, Ko CY, Tomlinson JS, Stewart AK, Winchester DP, et al. Multimodality therapy for pancreatic cancer in the U.S.: utilization, outcomes and the effect of hospital volume. Cancer 2007; 110:1227-34.

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