CYP2C8及CYP3A4细胞表达体系的构建及其在小分子激酶药物对紫衫醇代谢途径抑制研究中的应用
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摘要
构建CYP2C8及其3种突变体细胞表达体系,以紫杉醇为底物研究CYP2C8基因多态性对其酶活性的影响,以及构建CYP2C8和CYP3A4共转染细胞体系研究小分子激酶抑制剂对紫杉醇代谢途径的抑制。根据基因文库分别合成CYP3A4以及CYP2C8及其3种突变体CYP2C8*2(805A>T)、CYP2C8*3(416G>A,1196A>G)、CYP2C8*4(792C>G)的基因编码片段,将其连接到PEGFP-N1表达质粒,测序验证。将CYP2C8野生型及其突变体分别转染HepG2细胞,24 h后加入紫杉醇进行孵育,通过建立好的LC-MS/MS方法对代谢物进行定量分析。同时,也将野生型CYP2C8和CYP3A4质粒按一定的浓度比转入Hep G2细胞构建共表达体系。并筛选出合适的质粒浓度比转染细胞,在加入紫杉醇孵育时,同时加入小分子激酶抑制剂,考察小分子激酶抑制剂对紫杉醇代谢途径的抑制作用。结果表明,CYP2C8*4代谢酶对紫杉醇的代谢能力存在明显差异,其中CYP2C8*2和CYP2C8*3代谢活性分别是野生型的81%(P<0.05)和87%(P<0.05),而CYP2C8*4则是野生型的65%(P<0.01)。尼洛替尼完全抑制了紫杉醇的代谢,阿法替尼对紫杉醇的两条代谢途径抑制达30%,而伊马替尼选择性抑制了CYPD3A4的活性。不同基因型CYP2C8对紫杉醇的代谢存在差异,可能是导致临床疗效不同的原因。小分子激酶抑制剂在与紫杉醇联合使用时,对紫杉醇代谢的抑制各不相同。
The aims of this work are to establish the cell expression system of CYP2C8 and its 3 mutants,to study the effects of genepolymorphisms on its enzymatic activities,and to investigate the effects of small molecular kinase inhibitors on paclitaxel metabolism withthe established CYP2C8 and CYP3A4 co-transfected cell lines.The gene fragments of CYP3A4,CYP2C8 and its three mutants(CYP2C8*2(805A>T),CYP2C8*3(416G>A,1196A>G),and CYP2C8*4(792C>G))were synthesized based on gene bank.Then those fragmentswere ligated to expression plasmid PEGFP-N1 for sequencing evaluation.Twenty-four hours after the wild and mutant plasmids were transfectedinto Hep G2 cells,paclitaxel was added and incubation proceeded,and then the metabolites were quantitatively detected by well-constructedLC-MS/MS.Concurrently,the wild CYP2C8 and CYP3A4 plasmids in certain concentration ratios were transfected into Hep G2 cells,toestablish a co-expression system.Screening the plasmids with proper concentration ratio and transfecting them to the cells,adding smallmolecule kinase inhibitor while adding paclitaxel for incubation,the inhibition of paclitaxel metabolism by small molecule kinase inhibitorswas investigated.The results showed that the metabolic enzyme of different CYP2C8 caused the significant difference of paclitaxel metabolicactivity,of which the metabolic activity of CYP2C8*2,CYP2C8*3,CYP2C8*4 remained at about 80%(P < 0.05),87%(P < 0.05),and65%(P < 0.01),respectively,as compared to the wild type.Nilotinib completely inhibited the metabolism of paclitaxel and axitinib showeda 30% inhibition,while imatinib didn't have any inhibitory effect.As conclusion,different genotypes of CYP2C8 differentially affect theoverall paclitaxel metabolism,which might be the reason that results in the varied treatment effect in clinic.Small molecule kinase inhibitorswhile combined use with paclitaxel may inhibit the metabolism of paclitaxel to different extents.
The aims of this work are to establish the cell expression system of CYP2C8 and its 3 mutants,to study the effects of genepolymorphisms on its enzymatic activities,and to investigate the effects of small molecular kinase inhibitors on paclitaxel metabolism withthe established CYP2C8 and CYP3A4 co-transfected cell lines.The gene fragments of CYP3A4,CYP2C8 and its three mutants(CYP2C8*2(805A>T),CYP2C8*3(416G>A,1196A>G),and CYP2C8*4(792C>G))were synthesized based on gene bank.Then those fragmentswere ligated to expression plasmid PEGFP-N1 for sequencing evaluation.Twenty-four hours after the wild and mutant plasmids were transfectedinto Hep G2 cells,paclitaxel was added and incubation proceeded,and then the metabolites were quantitatively detected by well-constructedLC-MS/MS.Concurrently,the wild CYP2C8 and CYP3A4 plasmids in certain concentration ratios were transfected into Hep G2 cells,toestablish a co-expression system.Screening the plasmids with proper concentration ratio and transfecting them to the cells,adding smallmolecule kinase inhibitor while adding paclitaxel for incubation,the inhibition of paclitaxel metabolism by small molecule kinase inhibitorswas investigated.The results showed that the metabolic enzyme of different CYP2C8 caused the significant difference of paclitaxel metabolicactivity,of which the metabolic activity of CYP2C8*2,CYP2C8*3,CYP2C8*4 remained at about 80%(P < 0.05),87%(P < 0.05),and65%(P < 0.01),respectively,as compared to the wild type.Nilotinib completely inhibited the metabolism of paclitaxel and axitinib showeda 30% inhibition,while imatinib didn't have any inhibitory effect.As conclusion,different genotypes of CYP2C8 differentially affect theoverall paclitaxel metabolism,which might be the reason that results in the varied treatment effect in clinic.Small molecule kinase inhibitorswhile combined use with paclitaxel may inhibit the metabolism of paclitaxel to different extents.
引文
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[8]Ramalingam SS,Mlfrankel M.Carboplatin and Paclitaxel in combination with either vorinostat or placebo for first-line therapy of advanced non-small-cell lung cancer[J].J Clin Oncol,2010,28(1):56-62.
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[10]Monsarrat B,Mariel E,Cros S,et al.Taxol metabolism.Isolationand identification of three major metabolites of taxol in ratbile[J].Drug Metabolism&Disposition,1990,18(6):895-901.
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[13]Wang Y,Wang M,Qi H,et al.Pathway-dependent inhibition ofpaclitaxel hydroxylation by kinase inhibitors and assessment ofdrug-drug interaction potentials[J].Drug Metabolism&Dispos,2014,42(4):782-795.
[14]Leskel?S,Jara C,Leandro-García LJ,et al.Polymorphisms incytochromes P450 2C8 and 3A5 are associated with paclitaxelneurotoxicity[J].Pharmacogenomics Journal,2011,11(2):121-129.
[15]Hertz DL,Motsinger-Reif AA,Amy Drobish,et al.CYP2C8*3predicts benefit/risk profile in breast cancer patients receivingneoadjuvant paclitaxel[J].Breast Cancer Research&Treatment,2012,134(1):401-410.
[16]Hertz DL,Roy S,Motsinger-Reif AA,et al.CYP2C8*3 increasesrisk of neuropathy in breast cancer patients treated withpaclitaxel[J].Ann Oncol,2013,24(6):1472-1478.
[17]Graan AJMD,Laure E,Sprowl JA,et al.CYP3A4*22 genotype andsystemic exposure affect paclitaxel-induced neurotoxicity[J].Clin Cancer Res,2013,19(12):3316-3324.
[18]Behrendorff JB,Moore CD,Kim KH,et al.Directed evolutionreveals requisite sequence elements in the functional expressionof P450 2F1 in Escherichia coli[J].Chemical Research inToxicology,2012,25(9):1964-1974.
[19]Imaoka S,Yamada T,Hiroi T,et al.Multiple forms of humanP450 expressed in Saccharomyces cerevisiae[J].BiochemicalPharmacology,1996,51(8):1041-1050.
[20]路珂,曾苏,姚形炜.利用细菌/杆状病毒系统在昆虫细胞中表达人CYP2E1[J].浙江大学学报:医学版,2008,37(2):118-125.
[21]Gerets HHJ,Tilmant K,Gerin B,et al.Characterization of primaryhuman hepatocytes,Hep G2 cells,and Hepa RG cells at the m RNAlevel and CYP activity in response to inducers and their predictivityfor the detection of human hepatotoxins[J].Cell Biology&Toxicology,2012,28(2):69-87.
[22]薛正楷.稳定表达人CYP3A4基因与Bama小型猪CYP3A基因的Hep G_2细胞株的建立及探针药物代谢表征的比较研究[D].重庆:重庆医科大学,2009.
[23]Westerink WMA,Schoonen WGEJ.Cytochrome P450 enzymelevels in Hepg2 cells and cryopreserved primary human hepatocytesand their induction in Hepg2 cells[J].Toxicology in Vitro,2007,21(8):1581-1591.
[24]谢章明,陈枢青.人CYP的异源表达及其在新药研发早期的作用[J].浙江大学学报:医学版,2013,1(1):109-113.
[25]Vignati L,Turlizzi E,Monaci S,et al.An in vitro approach to detectmetabolite toxicity due to CYP3A4-dependent bioactivation ofxenobiotics[J].Toxicology,2005,216:154-167.
[26]梁艳,李彦,白清清.CYP450基因多态性研究进展[J].武警医学,2014,25(5):529-532.
[27]Sonnichsen DS,Liu Q,Schuetz EG,et al.Variability in humancytochrome P450 paclitaxel metabolism[J].J Pharmacol ExpTher,1995,275(2):566-575.
[28]VáclavíkováR,HorskyS,?imek P,et al.Paclitaxel metabolismin rat and human liver microsomes is inhibited by phenolicantioxidants[J].Naunyn-Schmiedeberg’s Archives ofPharmacology,2003,368(3):200-209.
[2]Nebot N,Crettol S,D’Esposito F,et al.Participation of CYP2C8and CYP3A4 in the N-demethylation of imatinib in human hepaticmicrosomes[J].Br J Pharmacol,2010,161(5):1059-1069.
[3]Daily EB,Aquilante CL.Cytochrome P450 2C8 pharmacogenetics:a review of clinical studies[J].Pharmacogenomics,2009,10(9):1489-1510.
[4]Gertz M,Tsamandouras N,S?ll C,et al.Reduced physiologicallybased pharmacokinetic model of repaglinide:impact of OATP1B1and CYP2C8 genotype and source of in vitro data on the predictionof drug-drug interaction risk[J].Pharmaceutical Research,2014,31(9):2367-2382.
[5]Khan MS,Barratt DT,Somogyi AA.Impact of CYP2C8*3polymorphism on in vitro metabolism of imatinib to N-desmethylimatinib[J].Xenobiotica,2016,46(3):278-287.
[6]Yu L,Shi D,Ma L,et al.Influence of CYP2C8 polymorphisms on thehydroxylation metabolism of paclitaxel,repaglinide and ibuprofenenantiomers in vitro[J].Biopharmaceutics&Drug Disposition,2013,34(5):278-287.
[7]Fountzilas G,Kalofonos HP,Dafni U,et al.Paclitaxel and epirubicinversus paclitaxel and carboplatin as first-line chemotherapy inpatients with advanced breast cancer:a phase III study conductedby the Hellenic Cooperative Oncology Group[J].Annals of Oncol,2004,15(10):1517-1526.
[8]Ramalingam SS,Mlfrankel M.Carboplatin and Paclitaxel in combination with either vorinostat or placebo for first-line therapy of advanced non-small-cell lung cancer[J].J Clin Oncol,2010,28(1):56-62.
[9]郭培凤,郭云利.紫杉醇应用相关不良反应观察[J].医学信息,2014(2):643.
[10]Monsarrat B,Mariel E,Cros S,et al.Taxol metabolism.Isolationand identification of three major metabolites of taxol in ratbile[J].Drug Metabolism&Disposition,1990,18(6):895-901.
[11]Walle T,Kumar GN,Mc Millan JM,et al.Taxol metabolism in rathepatocytes[J].Biochemcal Pharmacology,1993,46(9):1661-1664.
[12]Taniguchi R,Kumai T,Matsumoto N,et al.Utilization ofhuman liver microsomes to explain individual differences inpaclitaxel metabolism by CYP2C8 and CYP3A4[J].Journal ofPharmacological Sciences,2005,97(1):83-90.
[13]Wang Y,Wang M,Qi H,et al.Pathway-dependent inhibition ofpaclitaxel hydroxylation by kinase inhibitors and assessment ofdrug-drug interaction potentials[J].Drug Metabolism&Dispos,2014,42(4):782-795.
[14]Leskel?S,Jara C,Leandro-García LJ,et al.Polymorphisms incytochromes P450 2C8 and 3A5 are associated with paclitaxelneurotoxicity[J].Pharmacogenomics Journal,2011,11(2):121-129.
[15]Hertz DL,Motsinger-Reif AA,Amy Drobish,et al.CYP2C8*3predicts benefit/risk profile in breast cancer patients receivingneoadjuvant paclitaxel[J].Breast Cancer Research&Treatment,2012,134(1):401-410.
[16]Hertz DL,Roy S,Motsinger-Reif AA,et al.CYP2C8*3 increasesrisk of neuropathy in breast cancer patients treated withpaclitaxel[J].Ann Oncol,2013,24(6):1472-1478.
[17]Graan AJMD,Laure E,Sprowl JA,et al.CYP3A4*22 genotype andsystemic exposure affect paclitaxel-induced neurotoxicity[J].Clin Cancer Res,2013,19(12):3316-3324.
[18]Behrendorff JB,Moore CD,Kim KH,et al.Directed evolutionreveals requisite sequence elements in the functional expressionof P450 2F1 in Escherichia coli[J].Chemical Research inToxicology,2012,25(9):1964-1974.
[19]Imaoka S,Yamada T,Hiroi T,et al.Multiple forms of humanP450 expressed in Saccharomyces cerevisiae[J].BiochemicalPharmacology,1996,51(8):1041-1050.
[20]路珂,曾苏,姚形炜.利用细菌/杆状病毒系统在昆虫细胞中表达人CYP2E1[J].浙江大学学报:医学版,2008,37(2):118-125.
[21]Gerets HHJ,Tilmant K,Gerin B,et al.Characterization of primaryhuman hepatocytes,Hep G2 cells,and Hepa RG cells at the m RNAlevel and CYP activity in response to inducers and their predictivityfor the detection of human hepatotoxins[J].Cell Biology&Toxicology,2012,28(2):69-87.
[22]薛正楷.稳定表达人CYP3A4基因与Bama小型猪CYP3A基因的Hep G_2细胞株的建立及探针药物代谢表征的比较研究[D].重庆:重庆医科大学,2009.
[23]Westerink WMA,Schoonen WGEJ.Cytochrome P450 enzymelevels in Hepg2 cells and cryopreserved primary human hepatocytesand their induction in Hepg2 cells[J].Toxicology in Vitro,2007,21(8):1581-1591.
[24]谢章明,陈枢青.人CYP的异源表达及其在新药研发早期的作用[J].浙江大学学报:医学版,2013,1(1):109-113.
[25]Vignati L,Turlizzi E,Monaci S,et al.An in vitro approach to detectmetabolite toxicity due to CYP3A4-dependent bioactivation ofxenobiotics[J].Toxicology,2005,216:154-167.
[26]梁艳,李彦,白清清.CYP450基因多态性研究进展[J].武警医学,2014,25(5):529-532.
[27]Sonnichsen DS,Liu Q,Schuetz EG,et al.Variability in humancytochrome P450 paclitaxel metabolism[J].J Pharmacol ExpTher,1995,275(2):566-575.
[28]VáclavíkováR,HorskyS,?imek P,et al.Paclitaxel metabolismin rat and human liver microsomes is inhibited by phenolicantioxidants[J].Naunyn-Schmiedeberg’s Archives ofPharmacology,2003,368(3):200-209.