- Email: [email protected]
527 ROLE OF TGFβ2 IN HEPATOCELLULAR CARCINOMA PROGRESSION
S196
Poster Session − Friday, April 24
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
Poster Session − Friday, April 24
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