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Cited by 17Pharmacogenetics and Drug MetabolismDrug Transport and Resistance MechanismsDrug-Induced Hepatotoxicity and Protection

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Enhancement of CYP3A4 Activity in Hep G2 Cells by Lentiviral Transfection of Hepatocyte Nuclear Factor-1 Alpha

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Chiang, Tsai-Shin, Yang, Kai-Chiang, Chiou, Ling-Ling, Huang, Guan-Tarn, Lee, Hsuan-Shu·2014·PLoS ONE
DOI 10.1371/journal.pone.0094885PMC PMC3986372
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Abstract

Human hepatoma cell lines are commonly used as alternatives to primary hepatocytes for the study of drug metabolism in vitro. However, the phase I cytochrome P450 (CYP) enzyme activities in these cell lines occur at a much lower level than their corresponding activities in primary hepatocytes, and thus these cell lines may not accurately predict drug metabolism. In the present study, we selected hepatocyte nuclear factor-1 alpha (HNF1α) from six transcriptional regulators for lentiviral transfection into Hep G2 cells to optimally increase their expression of the CYP3A4 enzyme, which is the major CYP enzyme in the human body. We subsequently found that HNF1α-transfected Hep G2 enhanced the CYP3A4 expression in a time- and dose-dependent manner and the activity was noted to increase with time and peaked 7 days. With a multiplicity of infection (MOI) of 100, CYP3A4 expression increased 19-fold and enzyme activity more than doubled at day 7. With higher MOI (1,000 to 3,000), the activity increased 8- to 10-fold; however, it was noted the higher MOI, the higher cell death rate and lower cell survival. Furthermore, the CYP3A4 activity in the HNF1α-transfected cells could be induced by CYP3A4-specific inducer, rifampicin, and metabolized nifedipine in a dose-dependent manner. With an MOI of 3,000, nifedipine-metabolizing activity was 6-fold of control and as high as 66% of primary hepatocytes. In conclusion, forceful delivery of selected transcriptional regulators into human hepatoma cells might be a valuable method to enhance the CYP activity for a more accurate determination of drug metabolism in vitro.

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Figure 1 Selection of HNF1α as the optimal regulator for induction of CYP3A4 expression in Hep G2 cells. Cells were infected by control or indicated recombinant lentiviruses and cultured for 7 days before determination of the CYP3A4 expression and activity. (A) CYP3A4 mRNA expression in normal control (NC) Hep G2 cells and cells infected (at MOI = 100) with control virus or recombinant lentiviruses respectively expressing HNF1α, HNF1β, RXR, PXR, SOX17, or XBP-1. Optimal CYP3A4 expression occurs in cells infected with the HNF1α-expressing lentivirus. (B) Co-infection of Hep G2 cells with the HNF1α lentivirus and either HNF1β, RXR, PXR, SOX17, or XBP-1 did not increase CYP3A4 mRNA expression levels. (C) Left panel: A representative bright field image of control HepG2 cells. Right panel: A representative bright field image and fluorescent image of HepG2 cells infected at MOI = 3,000 by lentiviruses containing HNF1α, showing the reporter copGFP fluorescence. Scale bar = 100 µm. (D) Flow cytometry analysis showing percentage of copGFP+ Hep G2 cells 7 days post-infection with HNF1α-expressing lentivirus at various MOI. (E) Increased CYP3A4 enzyme activity and (F) protein levels in Hep G2 cells infected with the HNF1α-expressing lentivirus (MOI = 100) compared with normal control cells (NC) or cells infected with control virus. **P<0.01, ***P<0.001 compared with NC cells.
Figure 1 Selection of HNF1α as the optimal regulator for induction of CYP3A4 expression in Hep G2 cells. Cells were infected by control or indicated recombinant lentiviruses and cultured for 7 days before determination of the CYP3A4 expression and activity. (A) CYP3A4 mRNA expression in normal control (NC) Hep G2 cells and cells infected (at MOI = 100) with control virus or recombinant lentiviruses respectively expressing HNF1α, HNF1β, RXR, PXR, SOX17, or XBP-1. Optimal CYP3A4 expression occurs in cells infected with the HNF1α-expressing lentivirus. (B) Co-infection of Hep G2 cells with the HNF1α lentivirus and either HNF1β, RXR, PXR, SOX17, or XBP-1 did not increase CYP3A4 mRNA expression levels. (C) Left panel: A representative bright field image of control HepG2 cells. Right panel: A representative bright field image and fluorescent image of HepG2 cells infected at MOI = 3,000 by lentiviruses containing HNF1α, showing the reporter copGFP fluorescence. Scale bar = 100 µm. (D) Flow cytometry analysis showing percentage of copGFP+ Hep G2 cells 7 days post-infection with HNF1α-expressing lentivirus at various MOI. (E) Increased CYP3A4 enzyme activity and (F) protein levels in Hep G2 cells infected with the HNF1α-expressing lentivirus (MOI = 100) compared with normal control cells (NC) or cells infected with control virus. **P<0.01, ***P<0.001 compared with NC cells.CC BY · PubMed Central
Figure 2 Enzyme activity of CYP3A4, CYP1A1/1B1, and CYP2C9 in HNF1α-transfected Hep G2 cells. (A) CYP3A4 activities in cells from day 1 to day 14 with various MOI are presented as the detected luminescence level normalized to the respective amount of genomic DNA. With various MOI, the activities peaked at day 7. With MOI≧500, the activities increased to 6–10 folds. At day 7, the activities of CYP1A1/1B1 (B) and CYP2C9 (C) dose-dependently increased to only around 2 folds at higher MOI. Control virus did not change the activities significantly. ***P<0.001 compared with normal control cells (MOI = 0).
Figure 2 Enzyme activity of CYP3A4, CYP1A1/1B1, and CYP2C9 in HNF1α-transfected Hep G2 cells. (A) CYP3A4 activities in cells from day 1 to day 14 with various MOI are presented as the detected luminescence level normalized to the respective amount of genomic DNA. With various MOI, the activities peaked at day 7. With MOI≧500, the activities increased to 6–10 folds. At day 7, the activities of CYP1A1/1B1 (B) and CYP2C9 (C) dose-dependently increased to only around 2 folds at higher MOI. Control virus did not change the activities significantly. ***P<0.001 compared with normal control cells (MOI = 0).CC BY · PubMed Central
Figure 3 Characterization of HNF1α-transfected Hep G2 cells with various MOI 7 days post-infection. (A) The relative survival rate decreased as the viral MOI increased. (B) Cell cycle analysis revealed the percentage of apoptotic cells increased as the MOI increased. AP: apoptotic cells; G1/G0: G1/G0 phase; S: S phase; G2/M: G2/M phase. Control virus with MOI = 3,000 did not affect cell survival or apoptosis. *P<0.05, **P<0.01.
Figure 3 Characterization of HNF1α-transfected Hep G2 cells with various MOI 7 days post-infection. (A) The relative survival rate decreased as the viral MOI increased. (B) Cell cycle analysis revealed the percentage of apoptotic cells increased as the MOI increased. AP: apoptotic cells; G1/G0: G1/G0 phase; S: S phase; G2/M: G2/M phase. Control virus with MOI = 3,000 did not affect cell survival or apoptosis. *P<0.05, **P<0.01.CC BY · PubMed Central
Figure 4 Inducibility of CYP3A4 enzyme activity by rifampicin in HNF1α-transfected Hep G2 cells at various MOI. Light gray bars: with rifampicin; dark gray bars: vehicle only; white bars: induction fold increase. *P<0.05, **P<0.01, ***P<0.001.
Figure 4 Inducibility of CYP3A4 enzyme activity by rifampicin in HNF1α-transfected Hep G2 cells at various MOI. Light gray bars: with rifampicin; dark gray bars: vehicle only; white bars: induction fold increase. *P<0.05, **P<0.01, ***P<0.001.CC BY · PubMed Central
Figure 5 Nifedipine metabolism activity in HNF1α-transfected Hep G2 cells cultured for 7 days post-infection. The metabolic activity was determined by measuring the levels of oxidized nifedipine. Oxidized nifedipine was quantified by HPLC-ESI-MS/MS. HH: human hepatocytes used as positive control. The nifedipine metabolism activities increased as viral MOI increased. The activity of the cells infected by control virus with MOI = 3,000 is similar to that in normal control cells (MOI = 0). *P<0.05, **P<0.01.
Figure 5 Nifedipine metabolism activity in HNF1α-transfected Hep G2 cells cultured for 7 days post-infection. The metabolic activity was determined by measuring the levels of oxidized nifedipine. Oxidized nifedipine was quantified by HPLC-ESI-MS/MS. HH: human hepatocytes used as positive control. The nifedipine metabolism activities increased as viral MOI increased. The activity of the cells infected by control virus with MOI = 3,000 is similar to that in normal control cells (MOI = 0). *P<0.05, **P<0.01.CC BY · PubMed Central

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Metadata

Authors
Chiang, T.-S., Yang, K.-C., Chiou, L.-L., Huang, G.-T., & Lee, H.-S.
Affiliations
  • National Taiwan University (TW)
  • Taipei Medical University (TW)
Corresponding author
Huang, Guan-Tarn
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2014-04-14
Volume
9
Issue
4
Pages
e94885
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0094885
PMC
PMC3986372
OpenAlex
W2095442432
Cited by
17
References
37
Citations by year
2026: 1 · 2025: 1 · 2024: 2 · 2022: 1 · 2021: 1 · 2020: 1 · 2019: 4 · 2018: 2 · 2016: 2 · 2015: 2
FWCI
1.53
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Science Council
  • National Taiwan University
Topics
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Pharmacogenetics and Drug Metabolism
  • Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
Keywords
CYP3A4, HNF1A, HepG2 cells, lentiviral transfection, cytochrome P450 induction, rifampicin, multiplicity of infection, HNF1B, transcription factors
MeSH terms
Cell Survival, Cytochrome P-450 Enzyme System, Humans, Nifedipine, Rifampin, Transduction, Genetic, Transfection, Lentivirus, Apoptosis, Hepatocyte Nuclear Factor 1-alpha, Cytochrome P-450 CYP3A, Hep G2 Cells, HEK293 Cells

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