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CYP1B1 inhibits cancer cell apoptosis through sp1-mediated suppression of trail expression
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
CYP1B1 activity mediated various oncogenic events in cells. In conclusion, our data suggests that CYP1B1 promotes cell proliferation and metastasis by inducing EMT and Wnt/b-catenin signaling via Sp1 induction. P40 CYP1B1 INHIBITS CANCER CELL APOPTOSIS THROUGH SP1-MEDIATED SUPPRESSION OF TRAIL EXPRESSION Yeo-Jung Kwon, Hyoung-Seok Baek, Dong-Jin Ye, Sangyun Shin, Young-Jin Chun. College of Pharmacy, Chung-Ang University, Seoul, South Korea Cytochrome P450 1B1 (CYP1B1) belongs to the CYP1 family and shares the feature as an enzyme for drug metabolisms. It has been reported that CYP1B1 expression is higher in the tumor tissues than the normal ones, especially in hormone-related cancers such as breast, ovarian and prostate cancer cells. To explore the role of CYP1B1 on cancer cell progression, we investigated whether CYP1B1 blocks apoptotic pathways. Using Western blot and RT-PCR, the expression of anti-apoptotic factors such as Bcl-2, BclxL, XIAP, survivin and Mcl-1 were examined and showed to be promoted followed by CYP1B1 overexpression or treatment with CYP1B1 inducer, 7, 12-dimethylbenz[a]anthracene (DMBA). These promoting results were reversed when cells were transfected with siRNA for CYP1B1 or treated with a CYP1B1 inhibitor, tetramethoxystilbene (TMS). Moreover, AIF, a widely known pro-apoptotic factor, showed to be translocated to cytosol from mitochondria after treatment with TMS. The expression of TRAIL, a member of tumor necrosis factor superfamily, was also examined followed by alteration of CYP1B1 expression and it showed similar changes in expression level to pro-apoptotic factors. Sp1, a transcription factor, has been reported to suppress TRAIL expression by binding TRAIL promoter. To identify whether Sp1 mediates the CYP1B1-induced suppression of TRAIL, we examined Sp1 expression after changes of CYP1B1 expression level and it showed that Sp1 is promoted by CYP1B1. Moreover, Sp1 knockdown with specific siRNA showed promotion of TRAIL-related apoptotic factors and these results were reversed by Sp1 overexpession. Moreover, attenuation of Sp1 DNA binding activity using mithramycin A, a well-known Sp1 binding inhibitor, resulted in recovery of TRAIL expression formerly suppressed by treatment with DMBA. Taken together, these data suggest that CYP1B1 blocks apoptosis via TRAIL suppression and this is mediated by Sp1 induction. P41 DIFFERENT SUSCEPTIBILITY OF CYTOCHROME P450 2C ENZYMES TO DOWN-REGULATION BY NITRIC OXIDE Ji Won Park, Choon-myung Lee, Edward T. Morgan. Department of Pharmacology, Emory University, Atlanta, GA, USA Cytochromes P450 (P450) are key enzymes in metabolizing both endogenous and xenobiotic compounds and can be induced or inhibited by numerous drugs, environmental chemicals, diet and diseases. Such changes in P450 expression and activity can cause drug toxicity and drugdrug interactions. High levels of nitric oxide (NO) are produced during activation of the immune system and previously our lab showed that nitric oxide (NO) mediates the down-regulation of several P450 enzymes including CYP2C22 and CYP2B proteins by inflammatory cytokines. The purpose of this study was to determine if human CYP2C proteins are down-regulated by NO. HeLa and Huh7 cells were transduced with lentiviral vectors containing either human CYP2C enzymes or rat CYP2C11 tagged with v5, and the transduced cells were incubated with the nitric oxide donor, DPTA-NONOate (DPTA). Preliminary studies indicated that CYP2C8 was the most sensitive of the CYP2C enzymes. With 4 hr of DPTA treatment, CYP2C8 enzymes were significantly down-regulated to 50-55% of control. However, the level of CYP2C11 was not affected by NO. For longer exposure of cells to NO, NO synthase was induced in the cells using a doxycycline inducible pLIX304-hNOS2 vector. At 24hr after doxycycline treatment, media were collected to determine NO production and cells were harvested. The treatment resulted in a ~50% downregulation of CYP2C8 protein. This downregulation was attenuated by the NOS inhibitor, L-NG-nitroarginine methyl ester (L-NAME). At both 4 hr and 24 hr, no changes of CYP2C8 mRNA levels were observed. Remarkably, the downregulation of CYP2C8 by either DPTA or doxycycline was insensitive to the
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proteasome inhibitors bortezomib or MG132. Pretreatment with calpain inhibitors ALLN and Calpain inhibitor III did not block CYP2C8 downregulation by DPTA, but partially inhibited CYP2C8 degradation after 24hr of doxycycline treatment. Taken together, these data show different effects of NO on the downregulation of CYP2C enzymes and non-lysosomal protease are partially involved in CYP2C8 downregulation by NO. Supported by grant 2R01GM69971 from the National Institutes of Health. P42 EFFECT OF CYP2C9 GENOTYPE FOR PHARMACOKINETICS OF EFAVIRENZ IN VIVO IN CYNOMOLGUS MONKEYS Kazuhide Iwasaki 1, Yusuke Kitsugi 1, Kanami Ikeda 1, Takahiro Yoshikawa 1, Shotaro Uehara 2, Yasuhiro Uno 1, Masahiro Utoh 1, Hiroshi Yamazaki 2. 1 Shin Nippon Biomedical Laboratories (SNBL), Ltd., Kainan, Japan; 2 Showa Pharmaceutical University, Machida, Japan Cynomolgus monkeys are frequently used in nonclinical studies of new drug development due to their evolutionary closeness to humans. Efavirenz (EFV) is metabolized by CYP3A4 and CYP2B6 in humans, but is mainly metabolized by CYP2C9 in cynomolgus monkeys. Because cynomolgus CYP2C9 is polymorphic, in this study, the effect of CYP2C9 polymorphisms for the pharmacokinetics of EFV and its metabolites was assessed in the genotyped cynomolgus monkeys. EFV was administered at 1 mg/kg intravenous (IV) administration and 2 mg/kg oral (PO) administration to six cynomolgus monkeys including homozygotes (homo, n¼2), heterozygote (hetero, n¼1), and wild type animals (wild, n¼3). Plasma concentrations of EFV and its metabolites were determined by high performance liquid chromatography with tandem mass spectrometry. After IV administration, EFV exhibited biphasic elimination from the plasma. Terminal half-life values of EFV were about 3 to 6 hours in wild and hetero, and about 9 hours in homo. The area under the plasma concentration-time curve (AUC) of EFV after IV administration was similar between wild and hetero, but was 1.5-fold higher in homo than in wild. After PO administration, the AUC of EFV was similar between wild and hetero, but was 2fold higher in homo than in wild. These results indicated that CYP2C9 genotype was associated with EFV metabolism. Therefore, CYP2C9 polymorphisms might account for interindividual variations of EFV clearance in cynomolgus monkeys. P43 HORMONAL CONTEXT IN THE REGULATION OF CYTOCHROME P450 (CYP) DURING LIVER INSUFFICIENCY WITH SEROTONERGIC SYSTEM DYSFUNCTION Marta Kot. Department of Pharmacokinetics and Drug Metabolism, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland Pioneering studies in the rat have demonstrated that the serotonergic system is an unknown earlier factor that influences the activity and protein levels of CYP isoforms in the liver [1,2,3]. However, the expression of CYP isoforms depends on hepatic function and can be altered by different exogenous or endogenous factors. Because liver insufficiency is associated with predisposition to e.g., hepatocellular carcinoma, the aim of this study was to show the functional consequences of serotonergic system dysfunction in the regulation of cytochrome CYP2B and CYP3A isoforms during liver insufficiency. A rat model of liver insufficiency with dysfunction of the serotonergic system was developed. To induce abnormal liver function, animals were treated with nitrosodiethylamine (DEN) at 50 mg/ kg of body weight twice a week for 7 weeks. To induce dysfunction of the serotonergic system, the removal of tryptophan in the diet was employed. The rate of serotonin synthesis is controlled by the availability of tryptophan. The serum levels of AST (aspartate transaminase), ALT (alanine transaminase) and AFP (alpha-fetoprotein) were significantly increased during DEN-evoked liver insufficiency (determined by ELISA kits). The concentration of serum serotonin was significantly increased during liver insufficiency alone and decreased after serotonergic system dysfunction during liver insufficiency (estimated using HPLC). Dysfunction of the serotonergic system during liver insufficiency generated up-regulation of CYP2B and down-regulation CYP3A gene expression (real time RT PCR analysis) with a concomitant change in protein levels (evaluated by
CYP1B1 activity mediated various oncogenic events in cells. In conclusion, our data suggests that CYP1B1 promotes cell proliferation and metastasis by inducing EMT and Wnt/b-catenin signaling via Sp1 induction. P40 CYP1B1 INHIBITS CANCER CELL APOPTOSIS THROUGH SP1-MEDIATED SUPPRESSION OF TRAIL EXPRESSION Yeo-Jung Kwon, Hyoung-Seok Baek, Dong-Jin Ye, Sangyun Shin, Young-Jin Chun. College of Pharmacy, Chung-Ang University, Seoul, South Korea Cytochrome P450 1B1 (CYP1B1) belongs to the CYP1 family and shares the feature as an enzyme for drug metabolisms. It has been reported that CYP1B1 expression is higher in the tumor tissues than the normal ones, especially in hormone-related cancers such as breast, ovarian and prostate cancer cells. To explore the role of CYP1B1 on cancer cell progression, we investigated whether CYP1B1 blocks apoptotic pathways. Using Western blot and RT-PCR, the expression of anti-apoptotic factors such as Bcl-2, BclxL, XIAP, survivin and Mcl-1 were examined and showed to be promoted followed by CYP1B1 overexpression or treatment with CYP1B1 inducer, 7, 12-dimethylbenz[a]anthracene (DMBA). These promoting results were reversed when cells were transfected with siRNA for CYP1B1 or treated with a CYP1B1 inhibitor, tetramethoxystilbene (TMS). Moreover, AIF, a widely known pro-apoptotic factor, showed to be translocated to cytosol from mitochondria after treatment with TMS. The expression of TRAIL, a member of tumor necrosis factor superfamily, was also examined followed by alteration of CYP1B1 expression and it showed similar changes in expression level to pro-apoptotic factors. Sp1, a transcription factor, has been reported to suppress TRAIL expression by binding TRAIL promoter. To identify whether Sp1 mediates the CYP1B1-induced suppression of TRAIL, we examined Sp1 expression after changes of CYP1B1 expression level and it showed that Sp1 is promoted by CYP1B1. Moreover, Sp1 knockdown with specific siRNA showed promotion of TRAIL-related apoptotic factors and these results were reversed by Sp1 overexpession. Moreover, attenuation of Sp1 DNA binding activity using mithramycin A, a well-known Sp1 binding inhibitor, resulted in recovery of TRAIL expression formerly suppressed by treatment with DMBA. Taken together, these data suggest that CYP1B1 blocks apoptosis via TRAIL suppression and this is mediated by Sp1 induction. P41 DIFFERENT SUSCEPTIBILITY OF CYTOCHROME P450 2C ENZYMES TO DOWN-REGULATION BY NITRIC OXIDE Ji Won Park, Choon-myung Lee, Edward T. Morgan. Department of Pharmacology, Emory University, Atlanta, GA, USA Cytochromes P450 (P450) are key enzymes in metabolizing both endogenous and xenobiotic compounds and can be induced or inhibited by numerous drugs, environmental chemicals, diet and diseases. Such changes in P450 expression and activity can cause drug toxicity and drugdrug interactions. High levels of nitric oxide (NO) are produced during activation of the immune system and previously our lab showed that nitric oxide (NO) mediates the down-regulation of several P450 enzymes including CYP2C22 and CYP2B proteins by inflammatory cytokines. The purpose of this study was to determine if human CYP2C proteins are down-regulated by NO. HeLa and Huh7 cells were transduced with lentiviral vectors containing either human CYP2C enzymes or rat CYP2C11 tagged with v5, and the transduced cells were incubated with the nitric oxide donor, DPTA-NONOate (DPTA). Preliminary studies indicated that CYP2C8 was the most sensitive of the CYP2C enzymes. With 4 hr of DPTA treatment, CYP2C8 enzymes were significantly down-regulated to 50-55% of control. However, the level of CYP2C11 was not affected by NO. For longer exposure of cells to NO, NO synthase was induced in the cells using a doxycycline inducible pLIX304-hNOS2 vector. At 24hr after doxycycline treatment, media were collected to determine NO production and cells were harvested. The treatment resulted in a ~50% downregulation of CYP2C8 protein. This downregulation was attenuated by the NOS inhibitor, L-NG-nitroarginine methyl ester (L-NAME). At both 4 hr and 24 hr, no changes of CYP2C8 mRNA levels were observed. Remarkably, the downregulation of CYP2C8 by either DPTA or doxycycline was insensitive to the
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proteasome inhibitors bortezomib or MG132. Pretreatment with calpain inhibitors ALLN and Calpain inhibitor III did not block CYP2C8 downregulation by DPTA, but partially inhibited CYP2C8 degradation after 24hr of doxycycline treatment. Taken together, these data show different effects of NO on the downregulation of CYP2C enzymes and non-lysosomal protease are partially involved in CYP2C8 downregulation by NO. Supported by grant 2R01GM69971 from the National Institutes of Health. P42 EFFECT OF CYP2C9 GENOTYPE FOR PHARMACOKINETICS OF EFAVIRENZ IN VIVO IN CYNOMOLGUS MONKEYS Kazuhide Iwasaki 1, Yusuke Kitsugi 1, Kanami Ikeda 1, Takahiro Yoshikawa 1, Shotaro Uehara 2, Yasuhiro Uno 1, Masahiro Utoh 1, Hiroshi Yamazaki 2. 1 Shin Nippon Biomedical Laboratories (SNBL), Ltd., Kainan, Japan; 2 Showa Pharmaceutical University, Machida, Japan Cynomolgus monkeys are frequently used in nonclinical studies of new drug development due to their evolutionary closeness to humans. Efavirenz (EFV) is metabolized by CYP3A4 and CYP2B6 in humans, but is mainly metabolized by CYP2C9 in cynomolgus monkeys. Because cynomolgus CYP2C9 is polymorphic, in this study, the effect of CYP2C9 polymorphisms for the pharmacokinetics of EFV and its metabolites was assessed in the genotyped cynomolgus monkeys. EFV was administered at 1 mg/kg intravenous (IV) administration and 2 mg/kg oral (PO) administration to six cynomolgus monkeys including homozygotes (homo, n¼2), heterozygote (hetero, n¼1), and wild type animals (wild, n¼3). Plasma concentrations of EFV and its metabolites were determined by high performance liquid chromatography with tandem mass spectrometry. After IV administration, EFV exhibited biphasic elimination from the plasma. Terminal half-life values of EFV were about 3 to 6 hours in wild and hetero, and about 9 hours in homo. The area under the plasma concentration-time curve (AUC) of EFV after IV administration was similar between wild and hetero, but was 1.5-fold higher in homo than in wild. After PO administration, the AUC of EFV was similar between wild and hetero, but was 2fold higher in homo than in wild. These results indicated that CYP2C9 genotype was associated with EFV metabolism. Therefore, CYP2C9 polymorphisms might account for interindividual variations of EFV clearance in cynomolgus monkeys. P43 HORMONAL CONTEXT IN THE REGULATION OF CYTOCHROME P450 (CYP) DURING LIVER INSUFFICIENCY WITH SEROTONERGIC SYSTEM DYSFUNCTION Marta Kot. Department of Pharmacokinetics and Drug Metabolism, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland Pioneering studies in the rat have demonstrated that the serotonergic system is an unknown earlier factor that influences the activity and protein levels of CYP isoforms in the liver [1,2,3]. However, the expression of CYP isoforms depends on hepatic function and can be altered by different exogenous or endogenous factors. Because liver insufficiency is associated with predisposition to e.g., hepatocellular carcinoma, the aim of this study was to show the functional consequences of serotonergic system dysfunction in the regulation of cytochrome CYP2B and CYP3A isoforms during liver insufficiency. A rat model of liver insufficiency with dysfunction of the serotonergic system was developed. To induce abnormal liver function, animals were treated with nitrosodiethylamine (DEN) at 50 mg/ kg of body weight twice a week for 7 weeks. To induce dysfunction of the serotonergic system, the removal of tryptophan in the diet was employed. The rate of serotonin synthesis is controlled by the availability of tryptophan. The serum levels of AST (aspartate transaminase), ALT (alanine transaminase) and AFP (alpha-fetoprotein) were significantly increased during DEN-evoked liver insufficiency (determined by ELISA kits). The concentration of serum serotonin was significantly increased during liver insufficiency alone and decreased after serotonergic system dysfunction during liver insufficiency (estimated using HPLC). Dysfunction of the serotonergic system during liver insufficiency generated up-regulation of CYP2B and down-regulation CYP3A gene expression (real time RT PCR analysis) with a concomitant change in protein levels (evaluated by