Constructing A Cell Model With Enhanced Expression Of CYP3A4for Drug Testing

BackgroundDrug research plays an important role on the progress of medical. In the past several decades, the research of new drugs for diseases of cardiovascular, cancer, digestive system and pain have provided more choices for the treatment of disease, and have improve the health level, quality of life and the average life expectancy of the people. Drug research and development is an expensive and time-consuming process. In spite of advances in technology and increased investments, there are still90%of the new drugs which can not successfully enter to clinical. In the past decade, there is a gradually declining trend in the number of new drugs on market in the past decade. In1996,53new drugs were approved by FDA, but in2010only15. There are a lot of reasons which can account for the failure of new drug development. One of the main reasons is the drug toxicity and deficient pharmacodynamics effectiveness. Besides, it needs to confirm the pharmacokinetics, pharmacodynamics, and toxicological characteristics of the drug candidates during the drug development process, but the traditional research system lacks in vitro models for accurately and efficiently analyzing drug metabolism and toxicity at an early stage.The majority of medicine is metabolized in hepatic cells which contain a variety of enzymes involved in the biotransformation of xenobiotics and endogenous substances. Cytochrome P450enzymes (CYPs, P450s) is a monooxygenase protein superfamily containing hemoglobin sulfhydryl structure. It is an important drug-metabolizing enzyme completing detoxification and activation of relevant substrate. CYP3A subfamily is the highest level of P450s in the liver, accounting for about30%, which is the major clinical drug-metabolizing enzymes. CYP3As includes CYP3A4, CYP3A5, CYP3A7and CYP3A43. The content of CYP3A4in liver cells can reach40%. By contrast, in other organizations, such as the small intestine, kidney, lung, brain tissue, the level of CYP3A4expression is very low. Interestingly, regarding the expression of CYP3A4in brain tissue, Ghosh C et al. found that it not only involved in drug metabolism, but also had the function of protecting neurons in their recent studies. With a very wide spectrum of drug metabolism, CYP3A4involved in the metabolic processes of about50%of the clinical drugs, including hormones, immunosuppressants, calcium channel blockers, caffeine and antibiotics. Among them, nifedipine, midazolam, and testosterone are the specific substrate of CYP3A4, and can be used to detect the activity level of CYP3A4. After metabolization by CYP3A4, Some drugs, such as troglitazone and amiodarone, can produce hepatoxicity, which limited its clinical application. Some environmental carcinogens (e.g., aflatoxin B1) also produce CYP3A4-dependent hepatoxicity. In addition, CYP3A4is also closely related to drug interaction. A large number of clinical medications have been proven to induce or inhibit CYP3A4activity, leading to reduce or elevate the plasma concentration of the other drug used in combination with these drugs, resulting in weakened efficacy or even toxic effects. Terfenadine, mibefradil, astemizole and cerivastatin have been withdrawn from the market due to the serious drug interactions mentioned above.In view of the importance of CYP3A4in the process of drug metabolism, it requires a reliable in vitro model of CYP3A4metabolism to evaluate the metabolism of drug candidates and its impact on CYP3A4activity. Nowadays, in vitro models for drug development include the in vitro metabolic model based on microsomal subcellular structures, cell metabolic model, as well as tissues and organs metabolic model. The human liver microsomes is the most commonly used in vitro model, because of its available, low expense and easy to use. Supersomes, microsomes generated by genetic engineering insect cells, contain a high activity of CYP3A4. Other in vitro models also contain human liver cells S9including microsomes. These models can neither reflect the metabolism of the drug by CYP3A4in vivo, or can be used to quantitative analysis of roughly predicting the metabolism level of drug in vivo, which limits their applications. Tissue and organ metabolic models use tissue blocks or whole organs for in vitro drug metabolism test. Although these models ensure the integrity of the main metabolism to the maximum extent, which is undoubtedly conducive to reproduce the metabolic processes in the body. Due to its rare source and difficulty in maintaining activity, it is difficult to carry out. Cell models, the body’s smallest full-featured performer, can better reflect the physiological activities of the body than the simple biochemical models and organelle models. Compared with the tissue and organ metabolic model, cell models have greater feasibility in technology application. Cell models is the increased used technology in modern drug discovery. Therefore, cell models with a high expression of CYP3A4is very important for drug development.ObjectiveUp-regulating CYP3A4expression in C3A cells stably transfected by chimeric hPXR to enhance its potential applications in drug development.Methods1. Specific primers were designed according to sequences of gene hPXR and p53-AD included in the GenBank. Then, the target gene hPXR and p53-AD were amplified by RT-PCR from normal human liver tissue-derived cDNA pool.2. According to the sequence of CYP3A4gene included in the GenBank database, specific primers were designed. The two regulatory sequences of CYP3A45’-flanking region (-362to+53and-7838to-7208) were amplified from normal human liver tissue-derived cDNA pool using RT-PCR method.3. The hPXR and p53-AD gene was directionally cloned into the eukaryotic expression vector pCI-neo by restriction endonuclease sites. Then the recombinant eukaryotic expression vector pCI-hPXR-p53was screened by restriction enzyme digestion and identified by sequencing.4. The hPXR and p53-AD gene was directionally cloned into the eukaryotic expression vector pCI-neo by restriction endonuclease sites. Then the recombinant eukaryotic expression vector pCI-p53-hPXR was screened by restriction enzyme digestion and identified by sequencing.5. The two regulatory sequences of CYP3A45’-flanking region (-362to+53and-7838to-7208) were directionally cloned into the eukaryotic expression vector pGL3-basic by restriction endonuclease sites. Then the recombinant eukaryotic expression vector CYP3A4.XREM.luc was screened by restriction enzyme digestion and identified by sequencing.6. After pCI-hPXR-p53and pcDNA3.1(+) were digested by endonuclease Nhe I and BamH I, their products were combined to get an intermediate vector pcDNA3.1-hPXR. There was a restriction site EcoR I in the intermediate vector. Then, both the pcDNA3.1-hPXR and pCI-neo vectors were digested by Nhe I/EcoR I and their products were combined. The resulting vectors were identified by restriction enzyme digestion assay.7. HEK293T and C3A cells were transiently transfected with the target plasmid (pCI-neo, pCI-hPXR, pCI-hPXR-p53or pCI-p53-hPXR), reporter plasmid (CYP3A4.XREM.luc) and internal control plasmid (TK) in a ratio of46ng:140ng:14ng, respectively. After transfection for24hours,0.2μl of10mM rifampicin solution was added to the rifampin group,0.2μl DMSO to the control group. Dual luciferase detection was carried out48hours after the medicine.8. C3A cells were transfected with vector pCI-neo, pCI-hPXR, pCI-hPXR-p53and pCI-p53-hPXR, respectively. Twenty four hours later,2μl of10mM rifampin solution was added to the rifampin group,2μl DMSO to the control group. Another48hours later, cells were harvested for quantitative PCR detection.9. C3A cells were transfected with chimeric hPXRs, and the stable transformants were selected with G418. Proliferating colonies were expanded and had their CYP3A4mRNA expression detected. The clone with the highest expression of CYP3A4was screened out by CYP3A4mRNA level analysis in comparison with WT C3A cells.10. CYP3A4protein in WT C3A and modified cells stably expressing chimeric hPXR were detected by western blot.11. The microsomes from WT C3A and modified cells stably expressing chimeric hPXR were extracted. Testosterone was used as CYP3A4-specific substrate, and the formation of its metabolite was measured by HPLC.12. WT C3A and modified cells stably expressing chimeric hPXR were seed on6-well plates, and testosterone was added as CYP3A4-specific substrate, and the formation of its metabolite was measured by HPLC.Results1. p53-AD and hPXR gene was amplified from the normal human liver tissue-derived cDNA pool using specific primers.2. p53-AD and hPXR gene was directionally cloned into the eukaryotic expression vector pCI-neo by the restriction endonuclease. Eukaryotic expression vector pCI-hPXR-p53and pCI-p53-hPXR were successfully constructed.3. The two regulatory sequences of CYP3A45’-flanking region (-362to+53and-7838to-7208) were amplified from the normal human liver tissue-derived cDNA pool using specific primers.4. The two regulatory sequences of CYP3A45’-flanking region (-362to+53and-7838to-7208) were directionally cloned into the eukaryotic expression vector pGL3-basic by the restriction endonuclease. Eukaryotic expression vector CYP3A4.XREM.luc with the two regulatory sequences inserted was successfully constructed.5. Both pCI-hPXR-p53and pcDNA3.1(+) were digested by restriction endonucleases Nhe I/BamH I and their products were combined to gain an intermediate vector pcDNA3.1-hPXR. There was a restriction site EcoR I in the intermediate vector. Then, both pcDNA3.1-hPXR and pCI-neo were digested by Nhe I/EcoR I and their products were combined to construct subclone vector pCI-hPXR.6. The effects of pCI-neo, pPCI-hPXR, pCI-hPXR-p53and pCI-p53-hPXR expression vectors on CYP3A4.XREM.luc reporter gene in HEK293T cells were detected by dual fluorescence detection. The results showed in the RIF group, the activity of luciferase in cell transfected with chimeric hPXR constructs is stronger than that in cell transfected with hPXR or pCI-neo construct; compared with hPXR, the activity of luciferase activated by pCI-hPXR-p53and pCI-p53-hPXR increased1.5and9.5folds respectively, and the difference was statistically significant (p<0.05). So it is in RIF(-) group (p<0.05).7. The effects of pCI-neo, pPCI-hPXR, pCI-hPXR-p53and pCI-p53-hPXR expression vectors on CYP3A4.XREM.luc reporter gene in C3A cells were detected by dual fluorescence detection. The results showed in the RIF group, the activity of luciferase in cell transfected with chimeric hPXR constructs is stronger than that in cell transfected with hPXR or pCI-neo construct; compared with native hPXR, the activity of luciferase activated by pCI-hPXR-p53and pCI-p53-hPXR increased5.4and9.1folds respectively, and the difference was statistically significant (p<0.05). So it is in RIF(-) group (p<0.05).8. CYP3A4mRNA expression in C3A cells transiently transfected pCI-neo, pCI-hPXR, pCI-hPXR-p53and pCI-p53-hPXR respectively were quantified by Q-PCR. The results showed in the RIF (+) group, the level of CYP3A4expression in cell transfected with chimeric hPXR constructs is stronger than that in cell transfected with hPXR or pCI-neo construct; compared with native hPXR, the level of CYP3A4expression activated by pCI-hPXR-p53and pCI-p53-hPXR increased2.3and1.65folds respectively, and the difference was statistically significant (p<0.05). So it is in RIF(-) group (p<0.05).9. C3A cells were transfected with chimeric hPXRs, and the stable transformants were selected with G418. And13proliferating colonies were observed, including7cell lines stably expressing pCI-hPXR-p53(A1-A7) and6cell lines stably expressing pCI-p53-hPXR(B1-B6).10. CYP3A4mRNA expressions in13cell lines were detected by Q-PCR. Our results showed that the CYP3A4mRNA expression in A5cell was the highest in all the cell lines. Compared with WT C3A, its CYP3A4mRNA expression enhanced2.5 folds(p<0.05). Therefore, A5cell line was selected out for further study.11. CYP3A4protein expression in WT C3A and A5cells was detected by western blot. The results showed that the CYP3A4protein levels in A5cell line increased1.5folds as compared to WT C3A, and were relatively comparable with those observed in human liver.12. We detected the formation of6β-hydroxytestosterone, the metabolite of CYP3A4-specific substrate testosterone, using microsomal fractions isolated from A5and WT C3A cells. Although the concentration of6β-hydroxytestosterone was beyond the lowest limit of detection in microsomes from WT C3A cells, there was a dramatic increase in the formation of6β-hydroxytestosterone (714pmol/mg protein/min) in the microsomes from A5cells. Then, using a cell-based assay for the same substrate drug, we found no testosterone metabolite could be detected in WT C3A cells; but a significant increase of metabolite6β-hydroxytestosterone formation (55pmol/mg protein/min) was observed in A5cells. Moreover, the CYP3A4activity was significantly induced when the cells were treated with RIF.ConclusionIn this study, we successfully constructed4eukaryotic expression vectors (pCI-hPXR-p53, pCI-p53-hPXR, pCI-hPXR, CYP3A4.XREM.luc). The results of dual fluorescence detection showed that chimeric hPXRs (pCI-hPXR-p53and pCI-p53-hPXR) had stronger activation of the CYP3A4promoter than pCI-hPXR. The fact that chimeric hPXRs had more potent activation of CYP3A4transcription was further confirmed by Q-PCR.After C3A cell were stably transfected with chimeric hPXRs, the A5cell line was selected out with the highest expression of CPY3A4mRNA. The expression levels of CYP3A4protein and CYP3A4-mediated drug metabolism in A5cells were enhanced accordingly. Undoubtably, it provides a new in vitro cell model for drug metabolism and toxicity studies.