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Oxidation of carcinogenic benzo[a]pyrene by human and rat CYP1A1 and its influencing by cytochrome b5—A comparative study
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Abstracts / Toxicology Letters 221S (2013) S59–S256
P03-08 Human breast cancer cell metastasis is attenuated by lysyl oxidase inhibitors through down-regulation of hydrogen-peroxide mediated focal adhesion kinase and the paxillin signaling pathway Li-Ching Chen 1 , Chih-Hsiung Wu 2,3,4,∗ , Wen-Sen Lee 1,∗ , Yuan-Soon Ho 4,5,6,∗ 1
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan, 2 Department of Surgery, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan, 3 Department of Surgery, Taipei Medical University-Shuang Ho Hospital, Taipei, Taiwan, 4 Center of Excellence for Cancer Research, Taipei Medical University, Taipei, Taiwan, 5 School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan, 6 Department of Laboratory Medicine, Taipei Medical University Hospital, Taipei, Taiwan The extracellular matrix (ECM) plays a critical role in the development and invasion of primary breast tumors. Lysyl oxidase (LOX), which is an ECM remodeling enzyme, appears to play roles in promoting cancer cell motility and invasion. To ascertain whether LOX overexpression in breast tumor tissues from Asian patients is associated with decreases in metastasis-free and overall survival in breast cancer patients, the mRNA levels of LOX were examined in paired tumor/normal tissue samples using real-time RT-PCR analysis (n = 246 pair-matched samples). To test whether specifically targeting LOX by inhibiting its activity (using beta-aminopropionitrile (-APN), a LOX inhibitor), mRNA expression (using siRNA), or protein expression (using 25 M magnolol) attenuates the invasion of MDA-MB-231 breast cancer cells, a cancer cell migration assay was performed. Interestingly, only 78.5% (n = 193) of the breast cancer tumors displayed detectable LOX expression. Nearly 60% (n = 120) of the cases fell into Group 1 (tumor > normal, T > N); in this group, the mean LOX expression in the tumor cells was 20.2-fold greater than in normal cells. However, in Group 2 (normal > tumor, N > T), the LOX expression level in most of the normal tissues examined (80%, 59/73) was less than 5-fold greater than in the tumor tissues. The increased level of active LOX in the invasive breast cancer cell line MDA-MB-231 was accompanied by increased phosphorylation of FAK at Tyr-576 and of paxillin at Tyr-118. We further found that LOX facilitates migration in invasive breast cancer cells through a hydrogen peroxide-mediated mechanism that involves the FAK/Src signaling pathway. Moreover, the results indicate that the inhibition of LOX using magnolol may represent a more desirable strategy for breast cancer therapy than the use of -APN. http://dx.doi.org/10.1016/j.toxlet.2013.05.055
P03-09 Inhibition of canonical Wnt signaling deregulates expression of CYP1A1 in colon cancer cells Marketa Kabatkova 1,∗ , Miroslav Machala 2 , Jan Topinka 3 , Alois Kozubik 1 , Jan Vondrácek 1 1
Institute of Biophysics AS CR, v.v.i, Brno, Czech Republic, Veterinary Research Institute, v.v.i., Brno, Czech Republic, 3 Institute of Experimental Medicine AS CR, v.v.i., Prague, Czech Republic
2
Dietary carcinogens, such as benzo[a]pyrene (BaP), a potent carcinogenic polycyclic aromatic hydrocarbon present in various
processed food, have been suggested to contribute to the development of sporadic colon carcinomas. The expression of cytochrome P450 1A1 (CYP1A1), one of the principal enzymes contributing to bioactivation of BaP, has been recently shown to be partly under control of canonical Wnt/beta-catenin signaling, which is frequently over-activated in colon cancer. Using cellular models derived from human colon epithelial cells at various stages of carcinogenesis, we inhibited canonical Wnt signaling using small synthetic inhibitor of beta-catenin or siRNA targeting betacatenin, and then we analyzed inducibility of CYP1A1 by BaP, and formation of stable BaP-7,8-diol-9,10-epoxide – DNA adducts. We found that, paradoxically, inhibition of canonical Wnt signaling by synthetic inhibitor enhanced formation of DNA adducts, while it suppressed CYP1A1 expression at both mRNA and protein level. Nevertheless, contrasting results were obtained with siRNAmediated beta-catenin knock-down. While siRNA increased the BaP-induced expression of CYP1A1 mRNA, it substantially reduced levels of CYP1A1 protein and enhanced formation of DNA adducts in a cell dependent manner. Our data suggest that inhibition of betacatenin by either synthetic inhibitor or by RNA interference may induce complex changes in CYP1A1 expression or regulation of BaP metabolism, leading to treatment- and cell-dependent enhancement or suppression of its genotoxic effects. http://dx.doi.org/10.1016/j.toxlet.2013.05.056
P03-10 Oxidation of carcinogenic benzo[a]pyrene by human and rat CYP1A1 and its influencing by cytochrome b5 —A comparative study Radek Indra ∗ , Michaela Moserova, Miroslav Sulc, Marie Stiborova Faculty of Science, Charles University, Prague, Czech Republic Benzo[a]pyrene (BaP) is a carcinogen that covalently binds to DNA after its activation with cytochrome P450 (CYP). CYP1A1 is widely accepted to be the most important enzyme in BaP activation. Using several in vitro systems, oxidation of BaP by CYP1A1 was investigated. Rat liver microsomes, in which CYP1A was induced by Sudan I, generated BaP-9,10-dihydrodiol, BaP-4,5dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol and BaP-3-ol, which were separated with HPLC and identified by mass- an NMR-spectrometry. The same metabolites were generated by human hepatic microsomes (BaP-9,10-dihydrodiol, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP3,6-dione and BaP-3-ol), but no BaP-9-ol was detectable in human microsomes. Rat recombinant CYP1A1 expressed with NADPH:CYP reductase (POR) in SupersomesTM oxidized BaP also to BaP-9,10-dihydrodiol, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol and BaP-3-ol, but additional metabolite (Mx) that was not found in rat and human hepatic microsomes was eluted by HPLC as a polar compound. Human CYP1A1 expressed with POR in this microsomal system oxidized BaP to BaP-9,10-dihydrodiol, a metabolite Mx, BaP-7,8dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol and BaP-3-ol. BaP-4,5-dihydrodiol has, however, not been detected with human CYP1A1. Addition of cytochrome b5 to the rat and human recombinant CYP1A1 systems led to a more than 2-fold increase in BaP oxidation; its effect on an electron transfer to CYP1A1 seems to be the predominant mechanism responsible for this phenomenon. Because of the similar efficiencies of rat and human CYP1A1 to oxidize BaP and their influencing by cytochrome b5 , rats seems to be a suitable model that mimic BaP oxidation in human. http://dx.doi.org/10.1016/j.toxlet.2013.05.057
Abstracts / Toxicology Letters 221S (2013) S59–S256
P03-08 Human breast cancer cell metastasis is attenuated by lysyl oxidase inhibitors through down-regulation of hydrogen-peroxide mediated focal adhesion kinase and the paxillin signaling pathway Li-Ching Chen 1 , Chih-Hsiung Wu 2,3,4,∗ , Wen-Sen Lee 1,∗ , Yuan-Soon Ho 4,5,6,∗ 1
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan, 2 Department of Surgery, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan, 3 Department of Surgery, Taipei Medical University-Shuang Ho Hospital, Taipei, Taiwan, 4 Center of Excellence for Cancer Research, Taipei Medical University, Taipei, Taiwan, 5 School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan, 6 Department of Laboratory Medicine, Taipei Medical University Hospital, Taipei, Taiwan The extracellular matrix (ECM) plays a critical role in the development and invasion of primary breast tumors. Lysyl oxidase (LOX), which is an ECM remodeling enzyme, appears to play roles in promoting cancer cell motility and invasion. To ascertain whether LOX overexpression in breast tumor tissues from Asian patients is associated with decreases in metastasis-free and overall survival in breast cancer patients, the mRNA levels of LOX were examined in paired tumor/normal tissue samples using real-time RT-PCR analysis (n = 246 pair-matched samples). To test whether specifically targeting LOX by inhibiting its activity (using beta-aminopropionitrile (-APN), a LOX inhibitor), mRNA expression (using siRNA), or protein expression (using 25 M magnolol) attenuates the invasion of MDA-MB-231 breast cancer cells, a cancer cell migration assay was performed. Interestingly, only 78.5% (n = 193) of the breast cancer tumors displayed detectable LOX expression. Nearly 60% (n = 120) of the cases fell into Group 1 (tumor > normal, T > N); in this group, the mean LOX expression in the tumor cells was 20.2-fold greater than in normal cells. However, in Group 2 (normal > tumor, N > T), the LOX expression level in most of the normal tissues examined (80%, 59/73) was less than 5-fold greater than in the tumor tissues. The increased level of active LOX in the invasive breast cancer cell line MDA-MB-231 was accompanied by increased phosphorylation of FAK at Tyr-576 and of paxillin at Tyr-118. We further found that LOX facilitates migration in invasive breast cancer cells through a hydrogen peroxide-mediated mechanism that involves the FAK/Src signaling pathway. Moreover, the results indicate that the inhibition of LOX using magnolol may represent a more desirable strategy for breast cancer therapy than the use of -APN. http://dx.doi.org/10.1016/j.toxlet.2013.05.055
P03-09 Inhibition of canonical Wnt signaling deregulates expression of CYP1A1 in colon cancer cells Marketa Kabatkova 1,∗ , Miroslav Machala 2 , Jan Topinka 3 , Alois Kozubik 1 , Jan Vondrácek 1 1
Institute of Biophysics AS CR, v.v.i, Brno, Czech Republic, Veterinary Research Institute, v.v.i., Brno, Czech Republic, 3 Institute of Experimental Medicine AS CR, v.v.i., Prague, Czech Republic
2
Dietary carcinogens, such as benzo[a]pyrene (BaP), a potent carcinogenic polycyclic aromatic hydrocarbon present in various
processed food, have been suggested to contribute to the development of sporadic colon carcinomas. The expression of cytochrome P450 1A1 (CYP1A1), one of the principal enzymes contributing to bioactivation of BaP, has been recently shown to be partly under control of canonical Wnt/beta-catenin signaling, which is frequently over-activated in colon cancer. Using cellular models derived from human colon epithelial cells at various stages of carcinogenesis, we inhibited canonical Wnt signaling using small synthetic inhibitor of beta-catenin or siRNA targeting betacatenin, and then we analyzed inducibility of CYP1A1 by BaP, and formation of stable BaP-7,8-diol-9,10-epoxide – DNA adducts. We found that, paradoxically, inhibition of canonical Wnt signaling by synthetic inhibitor enhanced formation of DNA adducts, while it suppressed CYP1A1 expression at both mRNA and protein level. Nevertheless, contrasting results were obtained with siRNAmediated beta-catenin knock-down. While siRNA increased the BaP-induced expression of CYP1A1 mRNA, it substantially reduced levels of CYP1A1 protein and enhanced formation of DNA adducts in a cell dependent manner. Our data suggest that inhibition of betacatenin by either synthetic inhibitor or by RNA interference may induce complex changes in CYP1A1 expression or regulation of BaP metabolism, leading to treatment- and cell-dependent enhancement or suppression of its genotoxic effects. http://dx.doi.org/10.1016/j.toxlet.2013.05.056
P03-10 Oxidation of carcinogenic benzo[a]pyrene by human and rat CYP1A1 and its influencing by cytochrome b5 —A comparative study Radek Indra ∗ , Michaela Moserova, Miroslav Sulc, Marie Stiborova Faculty of Science, Charles University, Prague, Czech Republic Benzo[a]pyrene (BaP) is a carcinogen that covalently binds to DNA after its activation with cytochrome P450 (CYP). CYP1A1 is widely accepted to be the most important enzyme in BaP activation. Using several in vitro systems, oxidation of BaP by CYP1A1 was investigated. Rat liver microsomes, in which CYP1A was induced by Sudan I, generated BaP-9,10-dihydrodiol, BaP-4,5dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol and BaP-3-ol, which were separated with HPLC and identified by mass- an NMR-spectrometry. The same metabolites were generated by human hepatic microsomes (BaP-9,10-dihydrodiol, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP3,6-dione and BaP-3-ol), but no BaP-9-ol was detectable in human microsomes. Rat recombinant CYP1A1 expressed with NADPH:CYP reductase (POR) in SupersomesTM oxidized BaP also to BaP-9,10-dihydrodiol, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol and BaP-3-ol, but additional metabolite (Mx) that was not found in rat and human hepatic microsomes was eluted by HPLC as a polar compound. Human CYP1A1 expressed with POR in this microsomal system oxidized BaP to BaP-9,10-dihydrodiol, a metabolite Mx, BaP-7,8dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol and BaP-3-ol. BaP-4,5-dihydrodiol has, however, not been detected with human CYP1A1. Addition of cytochrome b5 to the rat and human recombinant CYP1A1 systems led to a more than 2-fold increase in BaP oxidation; its effect on an electron transfer to CYP1A1 seems to be the predominant mechanism responsible for this phenomenon. Because of the similar efficiencies of rat and human CYP1A1 to oxidize BaP and their influencing by cytochrome b5 , rats seems to be a suitable model that mimic BaP oxidation in human. http://dx.doi.org/10.1016/j.toxlet.2013.05.057