526 MAGNETIC FINE NEEDLES AND IRON OXIDE PARTICLES IN AN ALTERNATING MAGNETIC FIELD FOR HEPATOMA TREATMENT

03a: LIVER TUMORS − a) EXPERIMENTAL of 40 mg/kg on days 1, 3, and 5 of a 21-day cycle. LDM-CTX group received CTX i.p. injection at a dose of 20 mg/kg thrice a week. Growth modulating effects, toxicity, and overall survival of MTD-CTX and LDMCTX groups were evaluated. Vascular endothelial growth factor (VEGF) levels in plasma and tissue were also measured for evaluation of antiangiogenic effect. Immunnohistochemistry was performed using CD31 antibody endothelial marker for endothelial cell expression levels. Results: LDM-CTX group showed a more significant reduction in tumor size over time than MTD-CTX (P < 0.05). Additionally, LDM-CTX group was associated with more prolonged survival than MTD-CTX group (P < 0.05). Metronomic therapy using low-dose CTX resulted in a decrease both in the microvessel density and in the VEGF expression levels of treated tumors. There was a tendency toward decreased rate of hematological and biochemical abnormalities in the LDM-CTX group. Conclusions: Metronomic therapy using low-dose CTX offers less toxicity over conventional MTD chemotherapy and would appear to have effective anticancer properties via anti-angiogenic effect in HCC. Our results suggest that metronomic scheduling of chemotherapeutic agents should be considered for future clinical trials. 524 COORDINATE REGULATION OF THE HUMAN UDPGLUCURONOSYLTRANFERASE 1A10 GENE BY ARYL HYDROCARBON RECEPTOR (AHR) AND NUCLEAR FACTOR ERYTHROID 2-RELATED FACTOR 2 (NRF2) S. Kalthoff, U. Ehmer, N. Freiberg, M. Manns, C. Strassburg. Medical School Hannover, Hannover, Germany E-mail: [email protected] Background and Aims: UDP-glucuronosyltransferases (UGTs) catalyze an important metabolic process in which many xenobiotics and endobiotics are converted to water soluble b-D-glucopyranosiduronic acids facilitating elimination from the body. UGT1A10 is capable of glucuronidating a number of phenolic compounds, steroids and 7-hydroxy-benzo[a]pyrene, which are associated with carcinogenesis and inflammation. Aim of this study was the characterization of transcriptional regulation of the human UGT1A10 gene by the 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) inducible transcription factor AhR and the tert-butyl hydroquinone (tBHQ) inducible factor Nrf2. Methods: The UGT1A10 promoter was examined by induction experiments with TCDD and tBHQ via luciferase-assay in Kyse70 cells. DNA binding sites were characterized by site-directed mutagenesis and electrophoretic mobility shift assay (EMSA). Results: TCDD-induction of the human UGT1A10 promoter led to a 7 fold and tBHQ-induction to a 2.3 fold increased luciferase expression. A xenobiotic response element (XRE), and binding of AhR was identified at position −101 and an antioxidant response element (ARE) and binding of Nrf2, was found at position −149 in the UGT1A10 promoter. Interestingly, both TCDD- and tBHQ-inducibility were decreased by mutagenesis of the XRE-site. Similarly TCDD-inducibility as well as tBHQ-inducibility were reduced when the ARE-site was mutagenized. The administration of the AhR-inhibitor 3 ,4 -dimethoxyflavone also led to a complete abolishment of TCDD- and tBHQ-inducibility. The binding of AhR to both XRE and ARE as well as the binding of Nrf2 to ARE and XRE was shown by use of specific antibodies in EMSA super shift experiments and indicate a potential interaction between these two factors. Conclusions: This is the first demonstration of coordinate regulation of human UGT1A10 by AhR and Nrf2. The specific binding sites for AhR and Nrf2 in the UGT1A10 promoter were identified. Because UGT1A10 plays a significant role in the elimination of carcinogenic compounds and drugs it may exert a protective role for the organism in situations of oxidative stress or environmental toxin exposure. Furthermore, these data may contribute to the establishment of specific therapeutic induction strategies of human UGT1A10 to augment its potentially protective role.

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525 PROTEASOME INHIBITION BY BORTEZOMIB DURING ONCOLYTIC VIROTHERAPY OF HCC LEADS TO IMPROVED THERAPEUTIC EFFICACY MEDIATED BY MOLECULAR AND IMMUNOLOGICAL MECHANISMS B. Boozari1 , N. Woller1 , B. Fleischmann-Mundt1 , T.C. Wirth2 , M.P. Manns1 , S. Kubicka1 , F. K¨uhnel1 . 1 Gastroenterologie, Hepatologie und Endokrinologie, Medizinische Hochschule Hannover, Hannover, Germany; 2 Department of Microbiology, University of Iowa, Iowa City, Iowa, USA E-mail: kuehnel.fl[email protected] Background and Aims: Conditionally replicating adenoviral vectors provide a challenging tool for tumor treatment but potential interference with Bortezomib, a novel proteasome inhibitor and anti-cancer-agent, is unkown. Aim of this work was to investigate potential interference or exploitable synergisms if applied as combination treatment in HCC. Methods: Huh-7 cells were treated with Bortezomib (10 nM) and/or hTERT-Ad virotherapy (MOI 25) and cell death mechanisms were investigated by different methods with a focus on ER-stress and subsequent unfolded protein response (UPR). In vivo, therapeutic efficacy was determined in xenotransplant models. Immune-mediated therapeutic effects were investigated in a syngeneic murine model (BNL-cells/Balb/c-mice). Results: Bortezomib-dependent apoptosis was accompagnied by ERstress as suggested by dose-dependent Caspase-3 activation and XIAPdownregulation, and increase of UPR-markers like CHOP and BIP. The UPR was predominantly activated via the IRE1a/XBP-1 but not the PERK/ATF-4 pathway. Additional hTERT-Ad infection strongly shifted the UPR towards the activation of ATF-6 and the PERK/ATF-4 pathway and activation of downstream targets like SAPK/Jun-Kinase and phosphorylation of c-Jun. Importantly, viral infection lead to elimination of the antiapoptotic mediator Mcl-1 which was only partially rescued by Bortezomib, but a complete decay of the protective chaperone BIP could be observed. These molecular alterations increased caspase-3 activity and oncolytic efficacy in vitro. Low-dose Bortezomib neither influenced adenoviral protein expression nor viral DNA replication, and only slightly reduced viral particle numbers. Improved therapeutic efficacy was confirmed in vivo in s.c. Huh7-xenografts on nude mice. We then addressed Bortezomib/hTERT-Ad-dependent modulation of immunogenic cell surface proteins and could show that combination treatment lead to a significant increase of Hsp70 presented on the cell surface. To investigate therapeutic efficacy of combination treatment in an immunocompetent environment in vivo we applied a murine syngeneic model of subcutaneously engrafted liver tumors (BNL/Balb/c) and systemically inoculated BNLlung metastasis. Intratumoral application of hTERT-Ad in combination with systemic administration of Bortezomib lead to improved growth reduction of the primary tumor and of distant lung metastasis. Conclusion: Proteasome inhibition by Bortezomib in combination with adenoviral oncolytic therapy could provides a promising approach for the treatment of a primary HCC and distant metastases. 526 MAGNETIC FINE NEEDLES AND IRON OXIDE PARTICLES IN AN ALTERNATING MAGNETIC FIELD FOR HEPATOMA TREATMENT J.R. Zuchini1 , C.H. Huang1 , S.C. Huang2 , Y.H. Shih1 , H.W. Tsai3 , S.J. Hwang2 , C.F. Huang4 , G.B. Lee2 , X.-Z. Lin1 . 1 Department of Internal Medicine, National Cheng Kung University Hospital, 2 Department of Engineering Science, National Cheng Kung University, 3 Department of Pathology, National Cheng Kung University Hospital, Tainan, 4 Graduate Institute of Communication Engineering, Tatung University, Taipei, Taiwan E-mail: [email protected] Background and Aims: Focal hepatoma is often treated by radiofrequency ablation currently, but this method is painful, limited in effective area and considerably expensive. We designed a new system using fine needles and iron oxide nanoparticles in an alternating magnetic field (AMF) to generate hyperthermia for treating hepatoma in an animal model.

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Methods: Sprague-Dawley (SD) rats were inoculated with N1-S1 cell line into the liver orthotopically. After 7 days, the SD-rats were randomized into four groups that received local treatment: group 1 received a normal saline injection; group 2 an injection of magnetic nanoparticles of Ferucarbotran; in group 3, fine-needles were placed; and group 4, received fine-needles and nanoparticles combined. The temperature remained constant at 37ºC before and after treatment. The average tumor size was 1848±1722 mm3 , 1831±1556 mm3 , 1863±1055 mm3 and 1678±826 mm3 for each group respectively. After receiving treatment, the rats were placed on an alternating magnetic field with a power of 20kw during 5−20 minutes per day for 3 days. The temperature of the tumor was maintained between 55−60ºC. At day 30 after treatment, tumor size and mortality were assessed and pathology samples were studied. Results: On day 30 after treatment, average tumor size for each group was 32643±38566 mm3 , 8715±16172 mm3 , 30±93 mm3 and 39±116 mm3 and the survival rates were: 35%, 45%, 92% and 100%, the tumor regression rate was 0%, 28%, 91% and 89% respectively. There were no changes of temperature in tumor and anus during treatment for group 1 and 2; while the intratumor temperature elevated to 55- 60ºC during treatment but did not change at the anus for group 3 and 4. Tumor size was significantly smaller and survival rate was prolonged in groups 3 and 4 (p < 0.05). Groups 3 and 4 presented obvious calcifications, necrosis, apoptosis and few inflammatory changes from pathology studies. Conclusions: Our study demonstrates that using magnetic fine-needles with or without iron oxide particles in an AMF for hyperthermia can effectively inhibit hepatoma growth in rats and prolong their survival. We anticipate that this approach can be applied clinically in the near future for patients suffering from hepatoma.

527 ROLE OF TGFb2 IN HEPATOCELLULAR CARCINOMA PROGRESSION M.V. Makarova1 , N.E. Donner1 , D.I. Fleishman1 , O.V. Morozova2 , N.L. Lazarevich1 . 1 Immunochemistry, 2 Laboratory of Carcinogenic Substances, Russian Cancer Research Center, Moscow, Russia E-mail: [email protected] Background and Aims: Transforming growth factor (TGFb) 1 is a crucial cytokine in hepatocellular carcinoma (HCC) tumorigenesis. Its increased levels in patients’ serum and urine are associated with disease progression. While the role of TGFb1 in the control of hepatic proliferation, cell death and epithelial plasticity is a subject of extensive investigations, the impact of TGFb2 in these processes is still underestimated. In order to investigate the role of overproduction of TGFb2 in the development of malignant phenotype of HCC we used an experimental model of mouse HCC progression in which a highly differentiated slow-growing transplantable mouse HCC (sgHCC) rapidly gave rise in vivo to a highly invasive fast-growing variant (fgHCC). This model is characterized by overproduction of TGFb2 which is accompanied with downregulation of hepatocyte nuclear factor (HNF) 4a that is a key regulator for maintenance of epithelial morphology, cell adhesion and regulation of liver-specific gene expression. Methods: We used siRNA technique to knockdown TGFb2 in H33 cell culture obtained from fgHCC. The amount of TGFb2 was determined by ELISA. To analyze gene expression we used RT-PCR. The effect of TGFb2 inactivation in dedifferentiated HCC cells was analyzed using proliferation, colony formation, cell motility tests and tumorigenic assay. Results: We obtained H33-siTGFb2 culture in which the amount of TGFb2 was decreased 3.5 folds relative to control H33. Inhibition of TGFb2 induced HNF4a re-expression in H33. Together with HNF4a induction, expression of C/EBPa, transcription factor essential for the maintenance of hepatic differentiation, was upregulated in H33-siTGFb2. Inactivation of TGFb2 in dedifferentiated HCC cells led to alterations in several TGFb responsive genes expression, cell growth retardation, decrease of cell motility, reduction of colony formation properties in vitro, and reduction of tumorigenic potential of H33 cells after subcutaneous injection into syngenic recipient mice.

Conclusion: TGFb signaling due to overexpression of TGFb2 can contribute HCC progression through the influence on the key properties such as proliferation and differentiation, particularly due to HNF4a repression. The work was supported by RFBR grant 07−04–01422.

528 TNF-a AFFECTS THE CAPACITY OF STROMAL FIBROBLASTS TO STIMULATE THE MIGRATION OF COLON CANCER CELLS L. Mueller1 , J. Schumacher1 , B. Temel1 , L. von Seggern2 , H. Kalthoff1 , D. Broering1 . 1 Department of General and Thoracic Surgery, University Hospital of Schleswig-Holstein, Campus Kiel, Kiel, 2 Hepato-Biliary Surgery, University Hospital Hamburg-Eppendorf, Hamburg, Germany E-mail: [email protected] Background: Inflammation plays key roles in invasion, angiogenesis and metastasis. Given the multifaceted roles of tumor-necrosis-factor-alpha (TNF-a) in these processes, its effects on the fibroblasts that constitute the desmoplastic stroma in colorectal metastases are of interest. Methods: Primary cultures of cancer-associated stromal fibroblasts (CAFs) were generated from human tissues harvested during hepatic resection. TNF-a expression in tissue was examined by immunohistochemistry. Activation of nuclear-factor-kappaB (NF-úB) activity was measured by gel mobility shift assay. The effect of TNF-a on migratory capacity and gene expression of CAFs was tested in presence/absence of parthenolide, an herbal inhibitor of NF-úB. Gene expression in tissues and cell cultures was examined by Northern blot analysis. Protein measurements in the cell culture supernatant were performed with cytometric capture beads. Results: The colorectal metastases display immunoreactivity for TNF-a in tumor cells and leukocyte cells, whereas stromal fibroblasts are negative. To investigate transcriptional effects of TNF-a on CAFs, we analysed the expression of potential inflammatory target genes that are involved in tumor progression. CAFs that were exposed for 24 hours to TNF-a (10 ng/ml) showed a dramatic increased expression of interleukin-6 (IL-6), monocyte-chemotactic protein-1 (MCP-1) and intercellular cell adhesion molecule-1 (ICAM-1). Increasing concentrations of parthenolide (1, 5, 10 mM) dose-dependently inhibited the activation of NF-úB by TNF-a exposure for 30 min, as well as the TNF-a effect on IL-6 and MCP-1 mRNA and protein expression. Exposure of CAFs with TNF-a significantly increased the chemotaxis of HT29 colon carcinoma cells towards these cells in a coculture migration chamber system. This migratory effect activated by paracrine TNF-a was inhibited by co-incubation with parthenolide. Conclusion: CAFs are an important target for inflammatory signaling mediated by TNF-a/NF-úB in the context of tumor-stroma interaction and may play an important role in metastasis progression. The inhibition of NF-úB activation may be an interesting strategy in antitumor therapy targeting hepatic colorectal carcinoma metastatis.

529 FGL2 PROTHROMBINASE CONTRIBUTES TO TUMOR HYPERCOAGULABILITY VIA IL-2 AND IFN-g K. Su1 , F. Chen1 , W.-M. Yan1 , Q.-L. Zeng1 , L. Xu1 , D. Xi1 , B. Pi1 , X.-P. Luo2 , Q. Ning1 . 1 Department of Infectious Disease, 2 Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, China E-mail: [email protected] Aims: Fibrinogen-like protein 2/fibroleukin (fgl2) has been reported to play a vital role in the pathogenesis in MHV-3 (mouse hepatitis virus) induced fulminant and severe hepatitis, spontaneous abortion, allo- and xeno- graft rejection by mediating “immune coagulation”. Fgl2 functions as an immune coagulant with the ability to cleave prothrombin to thrombin directly. Therefore, this study was designed to examine the role of fgl2 in tumor development. Methods: Tumor tissues from 133 patients with six types of distinct cancers and the animal tumor tissues from human hepatocellular carcinoma