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.
Highlights
Hepatocytes are the main cell type in human body and play a major role in the metabolism of drugs through the activity of their abundant cytochrome P450 (CYPs) enzymes
To further our understanding of CYP enzymes and to advance the use of hepatoma cells as replacements for primary hepatocytes, we investigated the expression and enzyme activity of CYP3A4 in Hep G2 cells following the delivery of hepatocyte nuclear factor-1 alpha (HNF1a) using a lentivirus system
We selected HNF1a as a candidate transcriptional regulator to optimally increase the expression and activity of CYP3A4 in Hep G2 cells (Fig. 1). We found that it increased CYP3A4 activity in a dose-dependent manner (Fig. 2, 4, and 5) and infected cells still responded to rifampicin, a known CYP3A4 inducer (Fig. 4)
Summary
Hepatocytes are the main cell type in human body and play a major role in the metabolism of drugs through the activity of their abundant cytochrome P450 (CYPs) enzymes. They present themselves as the most reliable model cell in which to study drug metabolism in vitro [1,2]. Human hepatoma cell lines have attracted significant research attention as convenient and reliable alternatives to primary human hepatocytes These cell types can be obtained and maintained in vitro. Their CYP enzyme levels are much lower than primary human hepatocytes [6,7,8,9], which may result in the inaccurate detection of certain drug metabolism processes [10]
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