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Cytochrome-C release in hepatocellular carcinoma cell apoptosis induced by cyclooxygenase-2 inhibition: evidence for involvement of mitochondria dependent signalling
310
Posters: Liver / Pathology- Research and Practice 200 (2004) 306-311
lyzed whether Ep-CAM expression is dysregulated in chronic liver diseases and hepatocellular carcinoma (HCC). Methods: A representative panel of tissues (n = 254) with chronic hepatitis C virus (HCV), hepatitis B virus (HBV), and autoimmune hepatitis (AIH), chronic alcoholic liver disease (ALD), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and HCC was analysed semiquantitatively by immunohistology for Ep-CAM expression in correlation with the stage of liver fibrosis as well as histological and biochemical parameters of necroinflammatory activity. Results: In many cases with chronic liver diseases, hepatocytes showed d e n o v o Ep-CAM expression. Expression was highest in patients with necroinflammatory diseases (HCV- and HBV-hepatitis, ALD, AIH), almost absent in chronic biliary diseases (PSC, PBC) and correlated significantly with histological/biochemical parameters of inflammatory activity and extent of fibrosis, which was particularly striking in HBV-patients. Furthermore, 14.3% of the HCCs showed tumor cell derived Ep-Cam expression. Conclusions: Our findings establish the correlation of hepatocellular EpCAM neoexpression in chronic liver diseases with fibrosis and necroinflammatory activity. This may have implications for anti-neoplastic, anti-Ep-CAM-antibodytherapy, potentially including Ep-CAM-positive HCCs.
209 Cytochrome-C release in Hepatocellular Carcinoma Cell Apoptosis induced by Cyclooxygenase-2 Inhibition: Evidence for Involvement of Mitoehondria Dependent Signalling M.A. KERN, A.M. HAUGG, E. EITENEUER, H.P. DIENES, K. BREUHAHN, P. SCHIRMACHER Institut ftir Pathologie, Universitat zu K61n
Aims: Cyclooxygenase-2 (COX-2)-controlled prostaglandin metabolism has been implicated in the pathogenesis of hepatocellular carcinoma (HCC) recently. However, the proapoptotic molecular mechanism of COX-2-inhibitors in liver tumor cells has not been established. We have examined the direct effects of COX-2 inhibition on the apoptotic cell signalling in a liver tumor cell line. Methods: The functional effects of the COX-2 selective inhibitor celecoxib (50 mM) were examined in a COX-2 positive cell line (HUH-7) by MTT-assays and TUNEL-analysis. The fluorescent probe JC-1 was used to demonstrate changes in the mitochondrial transmembrane potential (Aapm). Mitochondrial and cytoplasmic fractions of apoptosis regulating proteins were analysed by Western immunoblot. Results: The time- and dose-dependent induction of apoptosis after COX-2 inhibition correlated with activation of caspases-9, -3 and -6 and initiator caspase-8. Celecoxib treatment led to early mitochondrial changes demonstrable as a decline of transmembranous potential (AxPm)and membrane permeabilization,leading to cytosolic release of cytochrome-C. Conclusions: These data suggest that proapoptotic effects of COX2 inhibition is at least in part due to a mitochondria-dependent pathway. Upstream induction of the mitochondria-dependentpathway may be a consequence of activation of initiator caspase-8.
210
Differential subcellular distribution of Siah-1 mediate apoptosis and nuclear maintenance in human hepatocarcinogenesis K. BREUHAHN 1, M.A. KERN 1, B. HAAB2, R. HADDAD2, R SCHIRMACHER L3 1Department of Pathology and the Center of 3Molecular Medicine, University of Cologne 2Van Andel Research Institute, Grand Rapids, USA
Aims: The E3 ubiquitin ligase Siah-1 participates in degradation of several proteins, including transcription factors, transcriptional cofactors and signalling pathway components such as ~-catenin, TIEG1, and DCC. Therefore, we have examined the potential role of Siah- 1 in human hepatocarcinogenesis. Methods: Expression of Siah-1 was analysed using cDNA Microarray- and Multi-Tissue-Array Technology in normal liver tissue, Dysplastic Nodules (DNs), and hepatocellular carcinomas (HCCs). Expression of Siah-1 in HCC cell lines (HUH-7, HepG2, Hep3B) was determined by immunocytology and Western immunoblot. Overexpression of Siah-1 was induced by transient transfection while reduction of Siah-1 expression was achieved by siRNA in HCC cells. Proliferation and apoptosis were determined by Ki-67 and M30 staining, respectively. Results: In normal liver and DNs Siah-1 shows a heterogenous cytoplasmic expression in hepatocytes. In 70% of the HCCs Siah-1 transcript levels were 2-10 fold reduced as compared to normal liver. Moreover, HCCs frequently showed nuclear accumulation of Siah-1 which correlated with tumor cell proliferation. All HCC cell lines strongly expressed Siah- 1 protein in the nucleus but not in the cytoplasm. Cytoplasmic overexpression of Siaah-1 resulted in significantly increased apoptosis, whereas siRNA-mediated inhibition of Siah- 1 caused tumor cell polyploidy. Conclusions: Our data demonstrate a dual function of Siah-1 in hepatocarcinogenesis depending on its expression levels and subcellular localization. High-level expression in the cytoplasm is linked to tumor cell apoptosis, while reduced expression and nuclear accumulation correlates with tumor cell proliferation and seems to be required for proper cell division.
211
Analyses of central mediators indicating structural integrity of TGFlA-signaling in human HCCs T. LONGERICH 1, K. BREUHAHN 1, K. PETMECKY 1, R SCHIRMACHER 1'2 lInstitut ftir Pathologie 2Zentmm flit Molekulare Medizin, Universit~t zu K61n
Aims: TGF[5 is a central mitoinhibitory factor for hepatocytes and alterations of TGF[~-signaling have been demonstrated in many human cancers. In contrast, alterations of TGF(-signalingin human hepatocellular carcinomas (HCCs) have been documented only infrequently. In order to detect possible alterations in TGF~-signaling, we analysed the intracellular signal transducer Smad4, the transcriptional co-repressors Ski and SnoN, and the TGF[3-regulated target gene MMP2 at the mRNA- and protein-level. Methods: For analyses of mRNA-expression we performed realtime-RT-PCR of Smad4, Ski, SnoN, and MMP2 of 30 HCCs, 4 peritumorous liver tissues, three HCC cell lines, and one normal control liver tissue. Additionaly, we performed mutational analyses of exons 1 of the Ski and SnoN genes, that are known to govern transforming capacity. Protein-analyses were performed either by immunohistology or by Western-immunoblotting. Results: Smad4-mRNA was detected in all HCCs, but was downregulated in 85% of HCCs. Immunohistologicalloss of Smad4-expression was found in 10% of HCCs, preferentially in poorly differenciated HCCs. Ski and SnoN were not found to be overexpressed neither at the mRNA nor the protein-level with the exception of one HCC with moderately increased Ski/SnoN-mRNA levels. Mutational analyses of exon 1 of Ski and SnoN revealed no mutations. MMP2 was expressed in all HCCs and expression levels correlated with Smad4, Ski, and SnoN-levels, respectively. Conclusions: In summary, Smad4 was only deficient in 10% of HCCs and Ski/SnoN did not show significant overexpression or mutations. Together with the good correlation of the expressionlevels of all mediators analysed, this suggests, that TGF~-signaling is intact in the majority of human HCCs.
Posters: Liver / Pathology- Research and Practice 200 (2004) 306-311
lyzed whether Ep-CAM expression is dysregulated in chronic liver diseases and hepatocellular carcinoma (HCC). Methods: A representative panel of tissues (n = 254) with chronic hepatitis C virus (HCV), hepatitis B virus (HBV), and autoimmune hepatitis (AIH), chronic alcoholic liver disease (ALD), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and HCC was analysed semiquantitatively by immunohistology for Ep-CAM expression in correlation with the stage of liver fibrosis as well as histological and biochemical parameters of necroinflammatory activity. Results: In many cases with chronic liver diseases, hepatocytes showed d e n o v o Ep-CAM expression. Expression was highest in patients with necroinflammatory diseases (HCV- and HBV-hepatitis, ALD, AIH), almost absent in chronic biliary diseases (PSC, PBC) and correlated significantly with histological/biochemical parameters of inflammatory activity and extent of fibrosis, which was particularly striking in HBV-patients. Furthermore, 14.3% of the HCCs showed tumor cell derived Ep-Cam expression. Conclusions: Our findings establish the correlation of hepatocellular EpCAM neoexpression in chronic liver diseases with fibrosis and necroinflammatory activity. This may have implications for anti-neoplastic, anti-Ep-CAM-antibodytherapy, potentially including Ep-CAM-positive HCCs.
209 Cytochrome-C release in Hepatocellular Carcinoma Cell Apoptosis induced by Cyclooxygenase-2 Inhibition: Evidence for Involvement of Mitoehondria Dependent Signalling M.A. KERN, A.M. HAUGG, E. EITENEUER, H.P. DIENES, K. BREUHAHN, P. SCHIRMACHER Institut ftir Pathologie, Universitat zu K61n
Aims: Cyclooxygenase-2 (COX-2)-controlled prostaglandin metabolism has been implicated in the pathogenesis of hepatocellular carcinoma (HCC) recently. However, the proapoptotic molecular mechanism of COX-2-inhibitors in liver tumor cells has not been established. We have examined the direct effects of COX-2 inhibition on the apoptotic cell signalling in a liver tumor cell line. Methods: The functional effects of the COX-2 selective inhibitor celecoxib (50 mM) were examined in a COX-2 positive cell line (HUH-7) by MTT-assays and TUNEL-analysis. The fluorescent probe JC-1 was used to demonstrate changes in the mitochondrial transmembrane potential (Aapm). Mitochondrial and cytoplasmic fractions of apoptosis regulating proteins were analysed by Western immunoblot. Results: The time- and dose-dependent induction of apoptosis after COX-2 inhibition correlated with activation of caspases-9, -3 and -6 and initiator caspase-8. Celecoxib treatment led to early mitochondrial changes demonstrable as a decline of transmembranous potential (AxPm)and membrane permeabilization,leading to cytosolic release of cytochrome-C. Conclusions: These data suggest that proapoptotic effects of COX2 inhibition is at least in part due to a mitochondria-dependent pathway. Upstream induction of the mitochondria-dependentpathway may be a consequence of activation of initiator caspase-8.
210
Differential subcellular distribution of Siah-1 mediate apoptosis and nuclear maintenance in human hepatocarcinogenesis K. BREUHAHN 1, M.A. KERN 1, B. HAAB2, R. HADDAD2, R SCHIRMACHER L3 1Department of Pathology and the Center of 3Molecular Medicine, University of Cologne 2Van Andel Research Institute, Grand Rapids, USA
Aims: The E3 ubiquitin ligase Siah-1 participates in degradation of several proteins, including transcription factors, transcriptional cofactors and signalling pathway components such as ~-catenin, TIEG1, and DCC. Therefore, we have examined the potential role of Siah- 1 in human hepatocarcinogenesis. Methods: Expression of Siah-1 was analysed using cDNA Microarray- and Multi-Tissue-Array Technology in normal liver tissue, Dysplastic Nodules (DNs), and hepatocellular carcinomas (HCCs). Expression of Siah-1 in HCC cell lines (HUH-7, HepG2, Hep3B) was determined by immunocytology and Western immunoblot. Overexpression of Siah-1 was induced by transient transfection while reduction of Siah-1 expression was achieved by siRNA in HCC cells. Proliferation and apoptosis were determined by Ki-67 and M30 staining, respectively. Results: In normal liver and DNs Siah-1 shows a heterogenous cytoplasmic expression in hepatocytes. In 70% of the HCCs Siah-1 transcript levels were 2-10 fold reduced as compared to normal liver. Moreover, HCCs frequently showed nuclear accumulation of Siah-1 which correlated with tumor cell proliferation. All HCC cell lines strongly expressed Siah- 1 protein in the nucleus but not in the cytoplasm. Cytoplasmic overexpression of Siaah-1 resulted in significantly increased apoptosis, whereas siRNA-mediated inhibition of Siah- 1 caused tumor cell polyploidy. Conclusions: Our data demonstrate a dual function of Siah-1 in hepatocarcinogenesis depending on its expression levels and subcellular localization. High-level expression in the cytoplasm is linked to tumor cell apoptosis, while reduced expression and nuclear accumulation correlates with tumor cell proliferation and seems to be required for proper cell division.
211
Analyses of central mediators indicating structural integrity of TGFlA-signaling in human HCCs T. LONGERICH 1, K. BREUHAHN 1, K. PETMECKY 1, R SCHIRMACHER 1'2 lInstitut ftir Pathologie 2Zentmm flit Molekulare Medizin, Universit~t zu K61n
Aims: TGF[5 is a central mitoinhibitory factor for hepatocytes and alterations of TGF[~-signaling have been demonstrated in many human cancers. In contrast, alterations of TGF(-signalingin human hepatocellular carcinomas (HCCs) have been documented only infrequently. In order to detect possible alterations in TGF~-signaling, we analysed the intracellular signal transducer Smad4, the transcriptional co-repressors Ski and SnoN, and the TGF[3-regulated target gene MMP2 at the mRNA- and protein-level. Methods: For analyses of mRNA-expression we performed realtime-RT-PCR of Smad4, Ski, SnoN, and MMP2 of 30 HCCs, 4 peritumorous liver tissues, three HCC cell lines, and one normal control liver tissue. Additionaly, we performed mutational analyses of exons 1 of the Ski and SnoN genes, that are known to govern transforming capacity. Protein-analyses were performed either by immunohistology or by Western-immunoblotting. Results: Smad4-mRNA was detected in all HCCs, but was downregulated in 85% of HCCs. Immunohistologicalloss of Smad4-expression was found in 10% of HCCs, preferentially in poorly differenciated HCCs. Ski and SnoN were not found to be overexpressed neither at the mRNA nor the protein-level with the exception of one HCC with moderately increased Ski/SnoN-mRNA levels. Mutational analyses of exon 1 of Ski and SnoN revealed no mutations. MMP2 was expressed in all HCCs and expression levels correlated with Smad4, Ski, and SnoN-levels, respectively. Conclusions: In summary, Smad4 was only deficient in 10% of HCCs and Ski/SnoN did not show significant overexpression or mutations. Together with the good correlation of the expressionlevels of all mediators analysed, this suggests, that TGF~-signaling is intact in the majority of human HCCs.