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Su1211 The Autophagy Produced by Azathioprine in HEPG2 Cells Leads to Apoptotic Cell Death Due to Inhibition With Bafilomycin A1
AGA Abstracts
analyses where there were greater than 3 studies. Combined odds ratios (OR) and 95% confidence intervals (CI) were calculated using a random effects model and the DerSimonian and Laird method, or using a fixed effect model and the Mantel-Haenszel method where there was no heterogeneity. Results: Eleven studies with combined total sample sizes ranging from 294,828 to 400,167 for each of the six risk factors were identified. Studies were performed in regions of high as well as low prevalence of IH-CCA. All studies except for those evaluating diabetes or obesity exhibited significant heterogeneity with an I2>65%. The combined OR for each of these risk factors were as follows: hepatitis B virus infection: 5.54 (CI = 3.19-9.63), hepatitis C: 4.84 (CI = 2.41-9.71), obesity: 1.56 (CI = 1.26-1.94), diabetes mellitus type II: 1.89 (CI = 1.74-2.07), smoking: 1.31 (CI = 0.95-1.82), and alcohol use: 2.81 (CI = 1.52-5.21). Sensitivity analysis with the exclusion of individual studies did not result in any significant differences in the OR for any of the risk factors with the exception of smoking. No evidence of publication bias was noted. Conclusions: Established risk factors for hepatocellular cancer such as chronic viral hepatitis B and C, alcohol use, diabetes mellitus type II, and obesity all have a strong predisposition to IH-CCA. However, there is insufficient data to support an increased risk of IH-CCA with smoking. These data suggest a common pathogenesis of primary intrahepatic epithelial cancers. Moreover, these findings are likely to have a meaningful impact by supporting new avenues for early detection and preventive strategies for IH-CCA.
(SNP) or mutations in miRNA sequence may alter miRNA expression and/or maturation. Only a few studies have been published about the association between miRNA polymorphism and malignant tumor. Hu et al reported rs11614913 T/C SNP in mir-196a2 is associated with shortened survival time of non-small cell lung cancer. We evaluated that the association between genetic variants of miRNA and survival of patients with cholangiocarcinoma Patients and Methods We enrolled 94 patients, who were diagnosed with cholangiocarcinoma between Jan. 1999 to Nov. 2006 in CHA Bundang Medical Center, CHA University and obtained blood sample. Using DNA extract from white blood cell, we analyzed mir-196a2, mir149, mir-499 and mir-146a expressions and the genotypes of four SNPs (rs2910164 C/G, rs2292832 C/T, rs11614913 T/C, and rs3746444 A/G) by PCR-RFLP assay. Patients' survival according to the genotype of four miRNAs was evaluated . Results Mean age of patients was 66.9 ± 10.7yrs old and fifty five was male and thirty nine was female. Only 13 of 92 patients had rescued with curative surgery. Median survival was 210 days (18 - 1290 days) after diagnosis of chlangiocarcinoma. According to genetic variant of miR-196a2, wild type (TT) showed significantly longer survival than in variant type (CT, CC) (Fig. 1, P = 0.018), especially in male patients (Fig. 2, p=0.011). Conclusion These findings suggest that genetic variants of microRNA 196a2 sequence could be important to predict survival of cholangiocarcinoma patients, but these findings need to be validated in larger prospective study. Su1214
Su1211 Activation of JNK in the Non-Cancerous Liver Tissue Predicts a High Risk of Recurrence After Hepatic Resection for Hepatocellular Carcinoma Toshiharu Sakurai, Satoru Hagiwara, Tatsuo Inoue, Kazuomi Ueshima, Shigenaga Matsui, Naoshi Nishida, Hiroshi Kashida, Masatoshi Kudo
The Autophagy Produced by Azathioprine in HEPG2 Cells Leads to Apoptotic Cell Death Due to Inhibition With Bafilomycin A1 Borja Hernandez-Breijo, I. D. Román, Dolores Fernández-Moreno, Jorge Monserrat, Javier P. Gisbert, Luis G. Guijarro
Aim; Hepatocellular carcinoma (HCC) ranks as the third leading cause of cancer deaths worldwide. Hepatic resection is the mainstay of curative treatment for early stage HCC. Although c-Jun N-terminal kinase (JNK) activation contributes to hepatocyte proliferation and HCC development in mice, the extent of involvement of JNK in human HCC development is unknown. The aim of this study is to assess the predictive value of JNK for post-operative recurrence in HCC. Methods; From April 2005 to March 2008, 159 patients underwent curative resection for HCC. From the 159 patients, 20 patients each matched for age, gender and etiology were registered as three groups: (i) without recurrence (no recurrence group), (ii) with recurrence within one year after surgery (early recurrence group), and (iii) with recurrence at one year or more after surgery (late recurrence group) (a cross-sectional control study). We investigated factors contributing to post-operative early and late phase recurrence. Results; Multivariate analysis using a Logistic regression model showed that JNK activation in non-cancerous liver tissue was correlated with post-operative late recurrence. (p=0.02, odds ratio; 5.79, 95%CI; 1.33-25.36). Conclusions: JNK activity in non-cancerous liver tissue is considered as a reliable predictive biomarker for post-operative recurrence in HCC.
Background: Recently, our group has described that azathioprine (AZA) produces autophagy through ERK/TSC-2/mTOR/p70S6K pathway in HepG2 cells. Autophagy is a mechanism that the cell employs to defend itself from external aggressions provoked by the stress of fasting, pathogens, toxics, hypoxia or drugs. The inhibition of this process could reduce tumor cell resistance to drugs such as AZA. Aim: To investigate whether inhibition of AZA induced autophagy decreases the resistance of HepG2 cells causing cell death. Methods: We used Bafilomycin A1 as inhibitor of autophagy, which blocks H+-ATPase from lysosome. HepG2 cells were employed. To identify the effects of Bafilomycin A1 on autophagy induced by AZA, we used the following techniques: metabolic activity assay, lactate dehydrogenase release, western blot and flow cytometry. Results and conclusions: The co-treatment with AZA and Bafilomycin A1 enhanced the apoptosis of HepG2 cells with respect to AZA or Bafilomycin A1 alone. The combined treatment with both drugs, produced an increase of the staining with annexin V in viable cells, measured by flow cytometry and the proteolysis of PARP (Poly (ADP-ribose) polymerase), which are hallmarks of early apoptosis. The addition of Bafilomycin A1 enhances the effects of AZA on hepatoblastoma cells (HepG2), which are resistant to this thiopurine. When a cell activates the autophagic mechanism, its damaged proteins and organelles are sequestered by the formed autophagosomes. These autophagosomes join lysosomes to degrade the aberrant components. For this degradation, it is necessary lysosome acidification, so when H+-ATPase is blocked by Bafilomycin A1, damaged proteins are accumulated in the autophagosomes without possibility of degradation, and cells enter in an apoptotic process. Therefore, our results suggest that the combination of AZA and Bafilomycin A1 could be used in the treatment of hepatoblastoma, as it provokes the death of HepG2 cells.
Su1215 Venous Thromboembolism (VTE) in Patients With Cholangiocarcinoma: Focus on Risk Factors and Impact on Survival Mun Ki Choi, Dong Uk Kim, Hye Kyung Jeon, Bong Eun Lee, Gwang Ha Kim, Geun Am Song, Dae Hwan Kang Background/Amis: Patients with cancer are associated with a high incidence of venous thromboembolism (VTE). However, few data are available on patients with cholangiocarcinoma. The aim of this study was to evaluate the clinical characteristics and risk factors of VTE and to investigate whether VTE would affect the survival of patients with cholangiocarcinoma. Patients/Methods: From January 2004 to December 2008, total 882 patients were diagnosed with cholangiocarcinoma based on histological, cytological, or radiological findings. We retrospectively reviewed 273 patients who were finally diagnosed with cholangiocarcinoma, and had follow-up period of more than 6 months and no evidence of other malignancy. We evaluate the incidence and clinical characteristics of VTE, and investigate the risk factors of VTE. Results: We observed 40 cases of VTE. Among them, 10 patients had VTE at diagnosis. There were 14 cases of pulmonary thromboembolism with or without deep vein thrombosis, 18 of portal vein thrombosis, 4 of inferior vena cava thrombosis, and 4 of hepatic vein thrombosis. We could find that tumor stage and chemotherapy were significant associated with the occurrence of VTE (p=0.022, p=0.014, respectively). The median survival in VTE and non-VTE groups were 13.0 months and 25.0 months, respectively (log rank test, p=0.026). VTE was an unfavorable prognostic factor of cholangiocarcinoma (HR 1.626, p=0.042). Conclusions: In our study, advanced stage and treatment with chemotherapeutic agents were related to the occurrence of VTE in patients with cholangiocarcinoma. VTE was an independent unfavorable prognostic factor for survivor of cholangiocarcinoma.
Su1212 Azathioprine Produces Autophagy in Hepatoblastoma Cells by Desensitization to Insulin-Like Growth Factor 1 (IGF-1) Borja Hernandez-Breijo, Irene D. Román, Dolores Fernández-Moreno, Jorge Monserrat, Javier P. Gisbert, Luis G. Guijarro Background and aims: In children, the most common primary liver cancer type is hepatoblastoma. The growth of this tumor is sensitive to insulin-like growth factor 1 (IGF-1). Our study focuses on the blocking of IGF-1 signaling on hepatoblastoma cells by azathioprine (AZA). Methods: The cell lines HepG2, HuH7 and Chang were used as models of liver cancer. IGF-1 signaling was characterized by i) metabolic activity assay; ii) immunoprecipitation; iii) western blot; iv) and cell cycle analysis measured by flow cytometry. Results and conclusions: The HepG2 cell line was the most sensitive to IGF-1. In these cells, AZA inhibits the effects of IGF-1 on cell proliferation. Interestingly, the effect of AZA was produced by sustained activation of p70S6K and subsequent IRS-1 phosphorylation on Ser307 leading to its proteosomal degradation. Moreover, we observed the uncoupling between IRS-1 and p85 PI3K, which lead to the blocking of the IGF-1 signaling. As consequence, treatment with AZA produced the inhibition of AKT and FoxO1 even in presence of IGF-1. As previously described, dephosphorylation of the tumor supressor FoxO1 inhibits cell cycle in G2/M and cellular proliferation. Moreover, the inhibition of AKT phosphorylation was essential to explain the autophagic process provoked by the treatment with AZA. It has been shown that IGF-1 is a very important growth factor for the survival of tumors. Several drugs to treat cancer are being tested in clinical trials targeting the IGF-1 signaling. Our results suggest that AZA could be useful for the treatment of pediatric liver tumors in combination with other molecules, because this thiopurine drug inhibits IGF1-mediated signaling.
Su1216 Prediction of Carcinoma After Resection in Patients With Ampullary Adenoma on Endoscopic Biopsy Ha-na Kim, Hwan Sic Yun, Woo Ik Chang, Jong Kyun Lee, Kyu Taek Lee, Kwang Hyuck Lee, Kyoung-Mee Kim, Zee Won Park Background/Aims Endoscopic treatment of ampullary adenomas has been established. However, false-negative rate of endoscopic biopsy for carcinoma is 20-30% and it remains uncertain if identifiable features predict malignancy. The purpose of this study was to evaluate the predictable factors of malignancy in ampullary adenomas on endoscopic biopsy. Methods Between 1995 and 2011, 91 ampullary adenomas were diagnosed by endoscopic biopsy. Among them, 66 adenomas were confirmed but 25 carcinomas were revealed after endoscopic or surgical resection. Various clinical, laboratory, radiological, and endoscopic findings were compared between the adenoma and carcinoma groups after resection. Results Univariate analysis revealed that the Age>65, presence of symptoms, villous components, high grade dysplasia, papilla enlargement or duct dilatation on radiologic imaging, bilirubin>2
Su1213 Genetic Variants of Micro-RNA 196a2 Sequences Can Predict Survival of Cholangiocarcinoma Kwang Hyun Ko, Chang-Il Kwon, Harry Yoon, Jeong Guil Lee, Sang Hee Song Background Cholangiocarcinoma has shown high prevalence in East Asia and is increasing in Western Europe and USA. microRNAs (miRNA, mir) are a new class of non-proteincoding, small RNAs that has function of tumor suppressors or oncogenes. They participate in diverse biological pathways and function as gene regulators. Single nucleotide polymorphisms
AGA Abstracts
S-452
analyses where there were greater than 3 studies. Combined odds ratios (OR) and 95% confidence intervals (CI) were calculated using a random effects model and the DerSimonian and Laird method, or using a fixed effect model and the Mantel-Haenszel method where there was no heterogeneity. Results: Eleven studies with combined total sample sizes ranging from 294,828 to 400,167 for each of the six risk factors were identified. Studies were performed in regions of high as well as low prevalence of IH-CCA. All studies except for those evaluating diabetes or obesity exhibited significant heterogeneity with an I2>65%. The combined OR for each of these risk factors were as follows: hepatitis B virus infection: 5.54 (CI = 3.19-9.63), hepatitis C: 4.84 (CI = 2.41-9.71), obesity: 1.56 (CI = 1.26-1.94), diabetes mellitus type II: 1.89 (CI = 1.74-2.07), smoking: 1.31 (CI = 0.95-1.82), and alcohol use: 2.81 (CI = 1.52-5.21). Sensitivity analysis with the exclusion of individual studies did not result in any significant differences in the OR for any of the risk factors with the exception of smoking. No evidence of publication bias was noted. Conclusions: Established risk factors for hepatocellular cancer such as chronic viral hepatitis B and C, alcohol use, diabetes mellitus type II, and obesity all have a strong predisposition to IH-CCA. However, there is insufficient data to support an increased risk of IH-CCA with smoking. These data suggest a common pathogenesis of primary intrahepatic epithelial cancers. Moreover, these findings are likely to have a meaningful impact by supporting new avenues for early detection and preventive strategies for IH-CCA.
(SNP) or mutations in miRNA sequence may alter miRNA expression and/or maturation. Only a few studies have been published about the association between miRNA polymorphism and malignant tumor. Hu et al reported rs11614913 T/C SNP in mir-196a2 is associated with shortened survival time of non-small cell lung cancer. We evaluated that the association between genetic variants of miRNA and survival of patients with cholangiocarcinoma Patients and Methods We enrolled 94 patients, who were diagnosed with cholangiocarcinoma between Jan. 1999 to Nov. 2006 in CHA Bundang Medical Center, CHA University and obtained blood sample. Using DNA extract from white blood cell, we analyzed mir-196a2, mir149, mir-499 and mir-146a expressions and the genotypes of four SNPs (rs2910164 C/G, rs2292832 C/T, rs11614913 T/C, and rs3746444 A/G) by PCR-RFLP assay. Patients' survival according to the genotype of four miRNAs was evaluated . Results Mean age of patients was 66.9 ± 10.7yrs old and fifty five was male and thirty nine was female. Only 13 of 92 patients had rescued with curative surgery. Median survival was 210 days (18 - 1290 days) after diagnosis of chlangiocarcinoma. According to genetic variant of miR-196a2, wild type (TT) showed significantly longer survival than in variant type (CT, CC) (Fig. 1, P = 0.018), especially in male patients (Fig. 2, p=0.011). Conclusion These findings suggest that genetic variants of microRNA 196a2 sequence could be important to predict survival of cholangiocarcinoma patients, but these findings need to be validated in larger prospective study. Su1214
Su1211 Activation of JNK in the Non-Cancerous Liver Tissue Predicts a High Risk of Recurrence After Hepatic Resection for Hepatocellular Carcinoma Toshiharu Sakurai, Satoru Hagiwara, Tatsuo Inoue, Kazuomi Ueshima, Shigenaga Matsui, Naoshi Nishida, Hiroshi Kashida, Masatoshi Kudo
The Autophagy Produced by Azathioprine in HEPG2 Cells Leads to Apoptotic Cell Death Due to Inhibition With Bafilomycin A1 Borja Hernandez-Breijo, I. D. Román, Dolores Fernández-Moreno, Jorge Monserrat, Javier P. Gisbert, Luis G. Guijarro
Aim; Hepatocellular carcinoma (HCC) ranks as the third leading cause of cancer deaths worldwide. Hepatic resection is the mainstay of curative treatment for early stage HCC. Although c-Jun N-terminal kinase (JNK) activation contributes to hepatocyte proliferation and HCC development in mice, the extent of involvement of JNK in human HCC development is unknown. The aim of this study is to assess the predictive value of JNK for post-operative recurrence in HCC. Methods; From April 2005 to March 2008, 159 patients underwent curative resection for HCC. From the 159 patients, 20 patients each matched for age, gender and etiology were registered as three groups: (i) without recurrence (no recurrence group), (ii) with recurrence within one year after surgery (early recurrence group), and (iii) with recurrence at one year or more after surgery (late recurrence group) (a cross-sectional control study). We investigated factors contributing to post-operative early and late phase recurrence. Results; Multivariate analysis using a Logistic regression model showed that JNK activation in non-cancerous liver tissue was correlated with post-operative late recurrence. (p=0.02, odds ratio; 5.79, 95%CI; 1.33-25.36). Conclusions: JNK activity in non-cancerous liver tissue is considered as a reliable predictive biomarker for post-operative recurrence in HCC.
Background: Recently, our group has described that azathioprine (AZA) produces autophagy through ERK/TSC-2/mTOR/p70S6K pathway in HepG2 cells. Autophagy is a mechanism that the cell employs to defend itself from external aggressions provoked by the stress of fasting, pathogens, toxics, hypoxia or drugs. The inhibition of this process could reduce tumor cell resistance to drugs such as AZA. Aim: To investigate whether inhibition of AZA induced autophagy decreases the resistance of HepG2 cells causing cell death. Methods: We used Bafilomycin A1 as inhibitor of autophagy, which blocks H+-ATPase from lysosome. HepG2 cells were employed. To identify the effects of Bafilomycin A1 on autophagy induced by AZA, we used the following techniques: metabolic activity assay, lactate dehydrogenase release, western blot and flow cytometry. Results and conclusions: The co-treatment with AZA and Bafilomycin A1 enhanced the apoptosis of HepG2 cells with respect to AZA or Bafilomycin A1 alone. The combined treatment with both drugs, produced an increase of the staining with annexin V in viable cells, measured by flow cytometry and the proteolysis of PARP (Poly (ADP-ribose) polymerase), which are hallmarks of early apoptosis. The addition of Bafilomycin A1 enhances the effects of AZA on hepatoblastoma cells (HepG2), which are resistant to this thiopurine. When a cell activates the autophagic mechanism, its damaged proteins and organelles are sequestered by the formed autophagosomes. These autophagosomes join lysosomes to degrade the aberrant components. For this degradation, it is necessary lysosome acidification, so when H+-ATPase is blocked by Bafilomycin A1, damaged proteins are accumulated in the autophagosomes without possibility of degradation, and cells enter in an apoptotic process. Therefore, our results suggest that the combination of AZA and Bafilomycin A1 could be used in the treatment of hepatoblastoma, as it provokes the death of HepG2 cells.
Su1215 Venous Thromboembolism (VTE) in Patients With Cholangiocarcinoma: Focus on Risk Factors and Impact on Survival Mun Ki Choi, Dong Uk Kim, Hye Kyung Jeon, Bong Eun Lee, Gwang Ha Kim, Geun Am Song, Dae Hwan Kang Background/Amis: Patients with cancer are associated with a high incidence of venous thromboembolism (VTE). However, few data are available on patients with cholangiocarcinoma. The aim of this study was to evaluate the clinical characteristics and risk factors of VTE and to investigate whether VTE would affect the survival of patients with cholangiocarcinoma. Patients/Methods: From January 2004 to December 2008, total 882 patients were diagnosed with cholangiocarcinoma based on histological, cytological, or radiological findings. We retrospectively reviewed 273 patients who were finally diagnosed with cholangiocarcinoma, and had follow-up period of more than 6 months and no evidence of other malignancy. We evaluate the incidence and clinical characteristics of VTE, and investigate the risk factors of VTE. Results: We observed 40 cases of VTE. Among them, 10 patients had VTE at diagnosis. There were 14 cases of pulmonary thromboembolism with or without deep vein thrombosis, 18 of portal vein thrombosis, 4 of inferior vena cava thrombosis, and 4 of hepatic vein thrombosis. We could find that tumor stage and chemotherapy were significant associated with the occurrence of VTE (p=0.022, p=0.014, respectively). The median survival in VTE and non-VTE groups were 13.0 months and 25.0 months, respectively (log rank test, p=0.026). VTE was an unfavorable prognostic factor of cholangiocarcinoma (HR 1.626, p=0.042). Conclusions: In our study, advanced stage and treatment with chemotherapeutic agents were related to the occurrence of VTE in patients with cholangiocarcinoma. VTE was an independent unfavorable prognostic factor for survivor of cholangiocarcinoma.
Su1212 Azathioprine Produces Autophagy in Hepatoblastoma Cells by Desensitization to Insulin-Like Growth Factor 1 (IGF-1) Borja Hernandez-Breijo, Irene D. Román, Dolores Fernández-Moreno, Jorge Monserrat, Javier P. Gisbert, Luis G. Guijarro Background and aims: In children, the most common primary liver cancer type is hepatoblastoma. The growth of this tumor is sensitive to insulin-like growth factor 1 (IGF-1). Our study focuses on the blocking of IGF-1 signaling on hepatoblastoma cells by azathioprine (AZA). Methods: The cell lines HepG2, HuH7 and Chang were used as models of liver cancer. IGF-1 signaling was characterized by i) metabolic activity assay; ii) immunoprecipitation; iii) western blot; iv) and cell cycle analysis measured by flow cytometry. Results and conclusions: The HepG2 cell line was the most sensitive to IGF-1. In these cells, AZA inhibits the effects of IGF-1 on cell proliferation. Interestingly, the effect of AZA was produced by sustained activation of p70S6K and subsequent IRS-1 phosphorylation on Ser307 leading to its proteosomal degradation. Moreover, we observed the uncoupling between IRS-1 and p85 PI3K, which lead to the blocking of the IGF-1 signaling. As consequence, treatment with AZA produced the inhibition of AKT and FoxO1 even in presence of IGF-1. As previously described, dephosphorylation of the tumor supressor FoxO1 inhibits cell cycle in G2/M and cellular proliferation. Moreover, the inhibition of AKT phosphorylation was essential to explain the autophagic process provoked by the treatment with AZA. It has been shown that IGF-1 is a very important growth factor for the survival of tumors. Several drugs to treat cancer are being tested in clinical trials targeting the IGF-1 signaling. Our results suggest that AZA could be useful for the treatment of pediatric liver tumors in combination with other molecules, because this thiopurine drug inhibits IGF1-mediated signaling.
Su1216 Prediction of Carcinoma After Resection in Patients With Ampullary Adenoma on Endoscopic Biopsy Ha-na Kim, Hwan Sic Yun, Woo Ik Chang, Jong Kyun Lee, Kyu Taek Lee, Kwang Hyuck Lee, Kyoung-Mee Kim, Zee Won Park Background/Aims Endoscopic treatment of ampullary adenomas has been established. However, false-negative rate of endoscopic biopsy for carcinoma is 20-30% and it remains uncertain if identifiable features predict malignancy. The purpose of this study was to evaluate the predictable factors of malignancy in ampullary adenomas on endoscopic biopsy. Methods Between 1995 and 2011, 91 ampullary adenomas were diagnosed by endoscopic biopsy. Among them, 66 adenomas were confirmed but 25 carcinomas were revealed after endoscopic or surgical resection. Various clinical, laboratory, radiological, and endoscopic findings were compared between the adenoma and carcinoma groups after resection. Results Univariate analysis revealed that the Age>65, presence of symptoms, villous components, high grade dysplasia, papilla enlargement or duct dilatation on radiologic imaging, bilirubin>2
Su1213 Genetic Variants of Micro-RNA 196a2 Sequences Can Predict Survival of Cholangiocarcinoma Kwang Hyun Ko, Chang-Il Kwon, Harry Yoon, Jeong Guil Lee, Sang Hee Song Background Cholangiocarcinoma has shown high prevalence in East Asia and is increasing in Western Europe and USA. microRNAs (miRNA, mir) are a new class of non-proteincoding, small RNAs that has function of tumor suppressors or oncogenes. They participate in diverse biological pathways and function as gene regulators. Single nucleotide polymorphisms
AGA Abstracts
S-452