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736 The Deleted in Colorectal Cancer (DCC) Dependence Receptor Constrains Tumor Progression in Colorectal Cancer Via Induction of Apoptosis
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
physiological levels of Bid and the cell’s sensitivity to apoptosis. Extending this study to the role of NS5A and Bid in inflammatory signalling, we have shown an increase of NOD1 mRNA expression in livers of HCV+ transgenic mice. This result was confirmed in primary human hepatocytes infected with HCV as well as in a cell line harboring an HCV replicon. Moreover, we were able to demonstrate for the first time that NOD1 pathway is active in human hepatocytes. Conclusion: HCV viral proteins regulate the expression of NOD1 in hepatocytes and impact on NOD signalling. Our results suggest that the HCV protein NS5A by affecting Bid impacts both apoptosis and inflammation. Bid might be an interesting target for an adjuvant anti-viral therapy important for prevention of HCC. 735 Multidrug Resistance Transporter Pgp as a Pathogenetic Factor of Non-small Cell Lung Cancer T.A. Bogush1 , E.A. Dudko1 , M.V. Tikchomirov1 , A.B. Ravcheeva1 , B.E. Polotsky2 , M.I. Davydov2 . 1 N.N. Blokhin Russian Cancer Research Center, Laboratory of Medical Chemistry, Moscow, Russian Federation, 2 N.N. Blokhin Russian Cancer Research Center, Surgical Department, Moscow, Russian Federation Introduction: Non small-cell lung cancer (NSCLC) is the most common malignancy in man. Identification of pathogenetically significant molecular determinants of NSCLC apparently is a key to understanding lung carcinogenesis processes. ABC transporter family, in particular P-glycoprotein (Pgp), are cell transporters reducing intracellular levels of xenobiotics thus protecting normal tissues from toxic and carcinogenic effects. To answer the question if Pgp is a substantial factor taking part in lung carcinogenesis we have compared level of Pgp expression in tumor tissues of NSCLC patients (pts) with normal lung tissue adjacent to NSCLC or to lung metastases of other tumors. In the latter case lung tissue is the only a niche for metastatic cells that does not involved in lung carcinogenesis and could be the most appropriate ’normal’ control for the comparison. Material and Methods: Surgical biopsy specimens of NSCLC (100), adjacent normal lung tissue (92) and adjacent normal lung tissue of pts with lung metastasis (33) were analyzed by flow cytometry. Antibodies: FITC-conjugated anti-Pgp (17F9, BD) and isotypic control mouse IgG2b (BD). Jurkat cell culture was used as a reference for the antibody activity control. Number of stained cells (level of Pgp expression) was analyzed with FlowJo software and Kolmogorov-Smirnov statistical approach. Three indexes of Pgp expression level were compared: high − Pgp revealed more than in 50% of the cells; moderate − in 30−50%; low − less than in 30% of the cells. Results: 1. Increase Pgp expression level was revealed in tumor as compared to NSCLC adjacent normal lung tissue: 39.6 and 29.7% (p = 0.001). In NSCLC low Pgp level was shown in 34.0% and high − in 25.0% of tumors. In normal lung tissue of NSCLC pts low Pgp level was in about 1.6 times more frequently (53.3%) but high − in about 3.3 times more rarely (7.6%). 2. It was not shown any difference in Pgp expression level between NSCLC and normal tissue adjacent to lung metastases (in 39.6 and 36.0% of cells, p = 0.3). In these groups percentage of pts with various levels of Pgp expression was also similar. 3. Level of Pgp expression was lower in NSCLC adjacent normal lung tissue as compared to normal lung tissue of lung metastases pts (in 29.7 and 41.2%, of cells, p = 0.003). In normal lung tissue of lung metastases pts low Pgp level was in about 2.5 times more rarely (21.2%), moderate in 1.3 times more frequently (51.5%), and high − in about 3.5 times more frequently (27.3%). Conclusion: 1. Decrease Pgp expression level in adjacent normal lung tissue as compared to NSCLC tissue, the close values of the index of NSCLC and normal tissue adjacent to lung metastases as well as decrease Pgp expression in normal tissue adjacent to NSCLC compared to normal tissue adjacent to lung metastases show that Pgp expression in normal lung tissue is an important pathogenetic factor of NSCLC. 2. Decrease Pgp expression in normal lung tissue could be substantial factor promoting the process of lung carcinogenesis. Supported by Russian Foundation for Basic Research (Grant N 10−04– 00551). 736 The Deleted in Colorectal Cancer (DCC) Dependence Receptor Constrains Tumor Progression in Colorectal Cancer Via Induction of Apoptosis M. Castets1 , L. Broutier1 , Y. Molin1 , M. Brevet2 , G. Chazot1 , N. Gadot2 , J.Y. Scoazec2 , A. Bernet1 , P. Mehlen1 . 1 Centre de Recherche en ´ ´ Cancerologie de Lyon (CRCL), Apoptose Cancer et Developpement, ´ de Lyon (CRCL), Lyon, France, 2 Centre de Recherche en Cancerologie ´ Differenciation Endocrine, Lyon, France Background: Dependence receptors share the common property to be active and to trigger cell apoptosis in absence of their ligand. These receptors are then thought to act as tumor suppressors by preventing tumor progression in settings of ligand limitation. Reciprocally, two selective advantages could be
S175
acquired by tumor cells to escape dependence receptors-induced cell death: (i) loss of dependence receptors expression, or (ii) autocrine gain of ligand expression, that would free tumor cells from ligand availability in exracellular medium, and could thus favor tumor growth and metastatic spreading. DCC belongs to the functional family of dependence receptors. DCC was first characterized as a putative suppressor gene in colorectal cancers since loss of one DCC allele or decreased DCC expression occurs in more than 70% of the cases. However, the role of DCC as a tumor suppressor has been a matter of debate for the last 15 years. Indeed, DCC was also shown to play a major role in neuronal navigation as a receptor for the neuronal guidance cue netrin-1. Moreover, to date, no animal model has supported the view that the DCC loss-of-function is implicated causally in tumor escape. Material and Methods: To specifically adress the role of DCC-induced apoptosis in tumorigenesis and to bypass perinatal death of DCC knockout mice, we created a mouse model in which DCC’s pro-apoptotic activity is genetically silenced by introduction of a point-mutation in its intracellular domain. Results: While the loss of DCC-induced apoptosis in this mouse model is not associated with a major disorganisation of the intestines, it leads to a decrease of basal intestinal apoptosis and to occurrence of spontaneous intestinal neoplasia at a relatively low frequency. Loss of DCC-induced apoptosis is also associated with an increase in the number and the aggressivness of intestinal tumors in a predisposing APC mutant context, resulting in the development of highly invasive adenocarcinomas. Conclusions: These results demonstrate that DCC functions as a tumor suppressor, via its ability to trigger tumor cell apoptosis. 737 Evaluation of Anti-angiogenic Therapy on the Effects of Angiogenic and Apoptotic Pathways in Breast Cancer Cell Line F. Karaca1 , S. Inan1 , S. Muftuoglu2 , K. Ozbilgin1 , S. Vatansever1 . 1 Celal Bayar University Faculty of Medicine, Histology and Embriyology, Manisa, Turkey, 2 Hacettepe University Faculty of Medicine, Histology and Embriyology, Ankara, Turkey Breast cancer is one of the most common cancer types in the world. In various studies it has been reported that angiogenic mechanisms are important in cancer development and invasion. It has also been reported that antiangiogenic treatment usage in addition to anti-chemotherapeutic treatment is effective for the prevention on the tumor progression. Apoptosis, a genetically programmed process of autonomous cell death. Cell death in tumours is commonly attributed to the induction of apoptosis. In recent studies, it is reported that one of the anti-vomiting drugs, thalidomide, inhibits angiogenic mechanisms. At the same time it is reported that propranolol treatment, the beta blocker drug, shrinks in hemangiomas in infantil hemangioma cases. In this study our aim was to investigate the effects of anti-chemotherapeutic (Paclitaxel [PX]) and anti-angiogenic treatment (Thalidomide [TD] and Propranolol [PR]) on the distribution of the Vascular Endothelial Growth Factor (VEGF) and Caspase 3 in the invasive human breast cancer cell line (MDAMB-231-ECACC-92101203), by using immunohistochemical method. MDA-MB-231 cells were reproduced in RPMI-1640 medium with 10% Fetal bovine serum, 1% L-Glutamine, 1% antibiotic, including 5% CO2 in a 37ºC humid incubator. After drugs of IC50 were calculated, the cells were put into a 24 well plate. The study was carried out under 6 groups; control group (MDAMB-231), PX group (1.4 nM); PR group (70 mM); TD group (75 mM); PX+PR group and PX+TD group. The drug effects at the 48th hour were evaluated. After fixating the cells with paraformaldehyde, the avidin-biotin peroxidase method was employed by using the anti-VEGF and anti-Caspase 3 primary antibodies. Immunohistochemical distribution intensities of primary antibodies were scored as minimal (−/+), mild (+), moderate (++) and strong (+++) and analyzed comparatively by using the statistic ANOVA test. It was observed that, the immunoreactivity of VEGF was observed as strong, moderate, mild and minimal in the control, PX, PR and TD groups, respectively. Immunoreactivity of Caspase 3 was observed as strong in the PX group, mild in the PR and TD groups and minimal in the control group. In the PX+PR and PX+TD groups, the immunoreactivity of VEGF was decreased compared to the control and PX groups. In these groups, the immunoreactivity of Caspase 3 was similar compared to the PX group and increased compared to the control, PR and TD groups. Angiogenic drugs such as Propranolol and Thalidomide in addition to the anti-chemotherapeutic therapy might less effective on the apoptotic pathways, but might inhibit the tumor invasion and metastasis by affected angiogenic mechanisms. Thus progressive inhibition of angiogenesis may contribute to tumor initiation, growth and metastasis in the breast cancer.
european journal of cancer 48, suppl. 5 (2012) S25–S288
physiological levels of Bid and the cell’s sensitivity to apoptosis. Extending this study to the role of NS5A and Bid in inflammatory signalling, we have shown an increase of NOD1 mRNA expression in livers of HCV+ transgenic mice. This result was confirmed in primary human hepatocytes infected with HCV as well as in a cell line harboring an HCV replicon. Moreover, we were able to demonstrate for the first time that NOD1 pathway is active in human hepatocytes. Conclusion: HCV viral proteins regulate the expression of NOD1 in hepatocytes and impact on NOD signalling. Our results suggest that the HCV protein NS5A by affecting Bid impacts both apoptosis and inflammation. Bid might be an interesting target for an adjuvant anti-viral therapy important for prevention of HCC. 735 Multidrug Resistance Transporter Pgp as a Pathogenetic Factor of Non-small Cell Lung Cancer T.A. Bogush1 , E.A. Dudko1 , M.V. Tikchomirov1 , A.B. Ravcheeva1 , B.E. Polotsky2 , M.I. Davydov2 . 1 N.N. Blokhin Russian Cancer Research Center, Laboratory of Medical Chemistry, Moscow, Russian Federation, 2 N.N. Blokhin Russian Cancer Research Center, Surgical Department, Moscow, Russian Federation Introduction: Non small-cell lung cancer (NSCLC) is the most common malignancy in man. Identification of pathogenetically significant molecular determinants of NSCLC apparently is a key to understanding lung carcinogenesis processes. ABC transporter family, in particular P-glycoprotein (Pgp), are cell transporters reducing intracellular levels of xenobiotics thus protecting normal tissues from toxic and carcinogenic effects. To answer the question if Pgp is a substantial factor taking part in lung carcinogenesis we have compared level of Pgp expression in tumor tissues of NSCLC patients (pts) with normal lung tissue adjacent to NSCLC or to lung metastases of other tumors. In the latter case lung tissue is the only a niche for metastatic cells that does not involved in lung carcinogenesis and could be the most appropriate ’normal’ control for the comparison. Material and Methods: Surgical biopsy specimens of NSCLC (100), adjacent normal lung tissue (92) and adjacent normal lung tissue of pts with lung metastasis (33) were analyzed by flow cytometry. Antibodies: FITC-conjugated anti-Pgp (17F9, BD) and isotypic control mouse IgG2b (BD). Jurkat cell culture was used as a reference for the antibody activity control. Number of stained cells (level of Pgp expression) was analyzed with FlowJo software and Kolmogorov-Smirnov statistical approach. Three indexes of Pgp expression level were compared: high − Pgp revealed more than in 50% of the cells; moderate − in 30−50%; low − less than in 30% of the cells. Results: 1. Increase Pgp expression level was revealed in tumor as compared to NSCLC adjacent normal lung tissue: 39.6 and 29.7% (p = 0.001). In NSCLC low Pgp level was shown in 34.0% and high − in 25.0% of tumors. In normal lung tissue of NSCLC pts low Pgp level was in about 1.6 times more frequently (53.3%) but high − in about 3.3 times more rarely (7.6%). 2. It was not shown any difference in Pgp expression level between NSCLC and normal tissue adjacent to lung metastases (in 39.6 and 36.0% of cells, p = 0.3). In these groups percentage of pts with various levels of Pgp expression was also similar. 3. Level of Pgp expression was lower in NSCLC adjacent normal lung tissue as compared to normal lung tissue of lung metastases pts (in 29.7 and 41.2%, of cells, p = 0.003). In normal lung tissue of lung metastases pts low Pgp level was in about 2.5 times more rarely (21.2%), moderate in 1.3 times more frequently (51.5%), and high − in about 3.5 times more frequently (27.3%). Conclusion: 1. Decrease Pgp expression level in adjacent normal lung tissue as compared to NSCLC tissue, the close values of the index of NSCLC and normal tissue adjacent to lung metastases as well as decrease Pgp expression in normal tissue adjacent to NSCLC compared to normal tissue adjacent to lung metastases show that Pgp expression in normal lung tissue is an important pathogenetic factor of NSCLC. 2. Decrease Pgp expression in normal lung tissue could be substantial factor promoting the process of lung carcinogenesis. Supported by Russian Foundation for Basic Research (Grant N 10−04– 00551). 736 The Deleted in Colorectal Cancer (DCC) Dependence Receptor Constrains Tumor Progression in Colorectal Cancer Via Induction of Apoptosis M. Castets1 , L. Broutier1 , Y. Molin1 , M. Brevet2 , G. Chazot1 , N. Gadot2 , J.Y. Scoazec2 , A. Bernet1 , P. Mehlen1 . 1 Centre de Recherche en ´ ´ Cancerologie de Lyon (CRCL), Apoptose Cancer et Developpement, ´ de Lyon (CRCL), Lyon, France, 2 Centre de Recherche en Cancerologie ´ Differenciation Endocrine, Lyon, France Background: Dependence receptors share the common property to be active and to trigger cell apoptosis in absence of their ligand. These receptors are then thought to act as tumor suppressors by preventing tumor progression in settings of ligand limitation. Reciprocally, two selective advantages could be
S175
acquired by tumor cells to escape dependence receptors-induced cell death: (i) loss of dependence receptors expression, or (ii) autocrine gain of ligand expression, that would free tumor cells from ligand availability in exracellular medium, and could thus favor tumor growth and metastatic spreading. DCC belongs to the functional family of dependence receptors. DCC was first characterized as a putative suppressor gene in colorectal cancers since loss of one DCC allele or decreased DCC expression occurs in more than 70% of the cases. However, the role of DCC as a tumor suppressor has been a matter of debate for the last 15 years. Indeed, DCC was also shown to play a major role in neuronal navigation as a receptor for the neuronal guidance cue netrin-1. Moreover, to date, no animal model has supported the view that the DCC loss-of-function is implicated causally in tumor escape. Material and Methods: To specifically adress the role of DCC-induced apoptosis in tumorigenesis and to bypass perinatal death of DCC knockout mice, we created a mouse model in which DCC’s pro-apoptotic activity is genetically silenced by introduction of a point-mutation in its intracellular domain. Results: While the loss of DCC-induced apoptosis in this mouse model is not associated with a major disorganisation of the intestines, it leads to a decrease of basal intestinal apoptosis and to occurrence of spontaneous intestinal neoplasia at a relatively low frequency. Loss of DCC-induced apoptosis is also associated with an increase in the number and the aggressivness of intestinal tumors in a predisposing APC mutant context, resulting in the development of highly invasive adenocarcinomas. Conclusions: These results demonstrate that DCC functions as a tumor suppressor, via its ability to trigger tumor cell apoptosis. 737 Evaluation of Anti-angiogenic Therapy on the Effects of Angiogenic and Apoptotic Pathways in Breast Cancer Cell Line F. Karaca1 , S. Inan1 , S. Muftuoglu2 , K. Ozbilgin1 , S. Vatansever1 . 1 Celal Bayar University Faculty of Medicine, Histology and Embriyology, Manisa, Turkey, 2 Hacettepe University Faculty of Medicine, Histology and Embriyology, Ankara, Turkey Breast cancer is one of the most common cancer types in the world. In various studies it has been reported that angiogenic mechanisms are important in cancer development and invasion. It has also been reported that antiangiogenic treatment usage in addition to anti-chemotherapeutic treatment is effective for the prevention on the tumor progression. Apoptosis, a genetically programmed process of autonomous cell death. Cell death in tumours is commonly attributed to the induction of apoptosis. In recent studies, it is reported that one of the anti-vomiting drugs, thalidomide, inhibits angiogenic mechanisms. At the same time it is reported that propranolol treatment, the beta blocker drug, shrinks in hemangiomas in infantil hemangioma cases. In this study our aim was to investigate the effects of anti-chemotherapeutic (Paclitaxel [PX]) and anti-angiogenic treatment (Thalidomide [TD] and Propranolol [PR]) on the distribution of the Vascular Endothelial Growth Factor (VEGF) and Caspase 3 in the invasive human breast cancer cell line (MDAMB-231-ECACC-92101203), by using immunohistochemical method. MDA-MB-231 cells were reproduced in RPMI-1640 medium with 10% Fetal bovine serum, 1% L-Glutamine, 1% antibiotic, including 5% CO2 in a 37ºC humid incubator. After drugs of IC50 were calculated, the cells were put into a 24 well plate. The study was carried out under 6 groups; control group (MDAMB-231), PX group (1.4 nM); PR group (70 mM); TD group (75 mM); PX+PR group and PX+TD group. The drug effects at the 48th hour were evaluated. After fixating the cells with paraformaldehyde, the avidin-biotin peroxidase method was employed by using the anti-VEGF and anti-Caspase 3 primary antibodies. Immunohistochemical distribution intensities of primary antibodies were scored as minimal (−/+), mild (+), moderate (++) and strong (+++) and analyzed comparatively by using the statistic ANOVA test. It was observed that, the immunoreactivity of VEGF was observed as strong, moderate, mild and minimal in the control, PX, PR and TD groups, respectively. Immunoreactivity of Caspase 3 was observed as strong in the PX group, mild in the PR and TD groups and minimal in the control group. In the PX+PR and PX+TD groups, the immunoreactivity of VEGF was decreased compared to the control and PX groups. In these groups, the immunoreactivity of Caspase 3 was similar compared to the PX group and increased compared to the control, PR and TD groups. Angiogenic drugs such as Propranolol and Thalidomide in addition to the anti-chemotherapeutic therapy might less effective on the apoptotic pathways, but might inhibit the tumor invasion and metastasis by affected angiogenic mechanisms. Thus progressive inhibition of angiogenesis may contribute to tumor initiation, growth and metastasis in the breast cancer.