The effect of interferon-α on the expression of cytochrome P450 3A4 in human hepatoma cells

https://doi.org/10.1016/j.taap.2011.03.019Get rights and content

Abstract

Interferon α (IFNα) is used to treat malignancies and chronic viral infections. It has been found to decrease the rate of drug metabolism by acting on cytochrome P450 enzymes, but no studies have investigated the consequences of IFNα treatment on the CYP3A4 isoform, responsible for the metabolism of a majority of drugs. In this study, we have examined the effect of IFNα on CYP3A4 catalytic activity and expression in human hepatoma cells. We found that IFNα inhibits CYP3A4 activity and rapidly down-regulates the expression of CYP3A4, independent of de novo protein synthesis. Pharmacologic inhibitors and a dominant-negative mutant expression plasmid were used to dissect the molecular pathway required for CYP3A4 suppression, revealing roles for Jak1 and Stat1 and eliminating the involvement of the p38 mitogen-activated and extracellular regulated kinases. Treatment of hepatoma cells with IFNα did not affect the nuclear localization or relative abundance of Sp1 and Sp3 transcription factors, suggesting that the suppression of CYP3A4 by IFNα does not result from inhibitory Sp3 out-competing Sp1. To our knowledge, this is the first report that IFNα down-regulates CYP3A4 expression largely through the JAK-STAT pathway. Since IFNα suppresses CYP3A4 expression, caution is warranted when IFNα is administered in combination with CYP3A4 substrates to avoid the occurrence of adverse drug interactions.

Introduction

The cytokine interferon alpha (IFNα) binds to cell surface receptors to initiate a signaling cascade that elicits antiviral or anti-proliferative events, and is used therapeutically in the treatment of viral infections, cancers and autoimmune diseases. IFNα therapy has impacted positively on the lives of hundreds of thousands of cancer patients and the full therapeutic potential of IFNα has yet to be realized (Borden, 2005). IFNα is commonly used in combination with other antiviral or antitumor agents, exploiting complementary mechanisms of action and maximizing efficacy. However, antiviral and anti-neoplastic agents often have narrow therapeutic indices (Beijnen and Schellens, 2004); thus, combinations of therapies that affect drug metabolism may expose patients to toxic or inadequate doses. Given that IFNα is commonly used as an adjunct to other therapies, and several studies have suggested that IFNα suppresses drug metabolism (Williams and Farrell, 1986, Williams et al., 1987, Jonkman et al., 1989, Craig et al., 1993, Israel et al., 1993, Islam et al., 2002), it is imperative to precisely define how drug metabolism may be altered by IFNα to predict and avoid the occurrence of untoward drug interactions.

Harmful drug–drug interactions most commonly result from the inhibition of hepatic cytochromes P450 (CYP) (Pelkonen et al., 2008). This family of heme-containing enzymes catalyzes the oxidation of exogenous and endogenous substances, increasing solubility and facilitating their elimination (Muntane-Relat et al., 1995). Inhibition of CYP enzyme activity may result from reduction in enzyme levels by decreased synthesis or increased degradation, or by inactivation of the enzyme by covalent binding of reactive intermediates to the CYP protein or heme (Pelkonen et al., 2008). While several studies have suggested that IFNα inhibits CYP 450 activity (Okuno et al., 1990, Islam et al., 2002), the mechanism by which this occurs is unknown.

Numerous CYP enzymes exist, but the most abundant hepatic isoform, responsible for the metabolism of approximately half of human medications, is CYP3A4 (Guengerich, 1999). Because CYP3A4 has a broad substrate specificity, including many anti-cancer drugs (Beijnen and Schellens, 2004), agents that affect CYP3A4 activity can trigger unexpected drug interactions. While previous work has established that other cytokines down-regulate CYP3A isoforms (Abdel-Razzak et al., 1993, Muntane-Relat et al., 1995, Tapner et al., 1996), the influence of IFNα on the expression of CYP3A4 has not been defined. In the present study, we hypothesized that IFNα treatment decreases CYP3A4 activity by down-regulating its expression. To test this hypothesis, the effect of IFNα treatment on endogenous CYP3A4 activity, transcript levels and promoter activity was assessed in human hepatoma cells. In this paper we report that IFNα reduces the catalytic activity and transcription of CYP3A4. Further investigations to delineate the signaling pathway revealed that Jak1 and Stat1 are involved in the down-regulation of CYP3A4 transcription by IFNα.

IFNα-2b (IFNα) was kindly provided by Schering-Plough (Kirkland, QC) and dissolved in phosphate-buffered saline (PBS) (Invitrogen, Burlington, ON). Cycloheximide and pathway inhibitors, including Jak1 inhibitor, the MEK inhibitor PD98059 and the p38 MAP kinase inhibitor SB203580 were from Calbiochem (Gibbstown, NJ). Cell culture media, fetal bovine serum and media supplements were from Invitrogen. Antibodies against Stat1 were from Cell Signaling Technology (Danvers, MA). Antibodies against Sp1 (H-225), Sp3 (D-20), and histones (FL-219) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rabbit secondary antibody conjugated to horseradish peroxidase was from Thermo Fisher Scientific (Ottawa, ON).

Cycloheximide and other pharmacological inhibitors were dissolved in DMSO and DMSO concentration remained constant in all experiments. To ensure an effective dose of Jak1 inhibitor was used in subsequent studies, we confirmed that treatment of HepG2 cells for 24 h with 2 μM Jak1 inhibitor prevented IFNα-stimulated phosphorylation of Stat1 (Tyr701) (data not shown). Likewise, 150 μM PD98059 blocked phosphorylation of ERK1/2 and 25 μM SB203580 prevented phosphorylation of p38 in HepG2 cells (data not shown). Phosphorylation status of Stat1 and ERK1/2 was determined using anti-phospho antibodies (Cell Signaling Technology). Phosphorylation of p38 was measured using a CASETM Cellular Activation of Signaling ELISA for p38 phosphorylation (T180/Y182) from SABiosciences (Frederick, MD), following manufacturer's instructions.

The reporter plasmid p3A4-10466-Luc (hereafter referred to as pCYP3A4-Luc), based on pGL3-Basic (Promega, Madison WI), was generously provided by Drs. Ito and Chang (University of Toronto) (Bertilsson et al., 2001). The reporter plasmid pISRE-TA-Luc (hereafter referred to as pISRE-Luc) was from Clontech (Mountain View, CA). The empty vector pRc/CMV was from Invitrogen. Addgene plasmids 8690 (pRc/CMV-STAT1α) and 8701 (pRc/CMV-STAT1α-Y701F) were used to express Stat1α (Schindler et al., 1992) or Stat1αY701F (Wen et al., 1995), respectively.

HepG2 cells (ATCC) were grown in minimum essential medium (MEM) with 2 mM L-glutamine and Earle's balanced salts adjusted to contain 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 10% fetal bovine serum, 20 U/ml penicillin and 0.02 mg/ml streptomycin. Cells were incubated at 37 °C with 5% CO2.

HepG2 cells were seeded in antibiotic-free medium at 125,000 cells/well in 24-well plates for 24 h and were then transfected with 0.5 μg reporter plasmid (pISRE-Luc or pCYP3A4-Luc) using FuGENE 6 reagent according to manufacturer's instructions (Roche, Laval, QC). In co-transfections, 1 μg expression plasmid (pRc/CMV, pRc/CMV-STAT1α, or pRc/CMV-STAT1α-Y701F) was included. Cells were incubated for 24 h prior to treatment with IFNα. Following treatment, cells were lysed with Passive Lysis Buffer (Promega) and firefly luciferase activity was determined using a Dual Luciferase Reaction assay kit (Promega). Total protein concentrations of the cell lysates were determined using a BCA kit (Pierce, Rockford, IL). Luciferase activity for each sample was normalized to the total protein concentration and expressed as relative luciferase units (RLUs) by dividing by the average normalized luciferase activity in the untreated samples.

HepG2 cells were cultured on white-walled culture plates with clear bottoms at 20,000 cells/well for 48 h then treated with IFNα. At each time point, CYP3A4 activity was determined using the P450-Glo assay according to manufacturer's instructions (Promega). Cells were washed once with fresh media, and then incubated at 37 °C for 60 min with 50 μl media containing a luminogenic CYP substrate (Luciferin-IPA). An equal volume of Luciferin Detection Reagent was added to each well and the plate was mixed briefly. Luminescence was measured directly from the cell culture plates. Net signals from each well were calculated by subtracting background luminescence values (no-cell control).

HepG2 cells grown to 50% confluency in 100 mm dishes were exposed to IFNα and/or pharmacological inhibitors. Treated and untreated cells were lysed with RLT buffer (Qiagen Inc., Mississauga, ON) and homogenized using QIAshredder columns (Qiagen). Total cellular RNA was extracted with the Qiagen RNeasy Kit (Qiagen). Thirty microgram RNA was reverse-transcribed to cDNA using the High Capacity cDNA Reverse Transcription kit from Applied Biosystems (Foster City, CA), then diluted 1:2 with water and used as template. Real-time PCR quantification was performed using a LightCycler Instrument (Roche). Reactions were carried out in a final volume of 20 μl, using 5 μl template, 4 μl FastStart SYBR Green Plus mastermix (Roche), 500 nM of the CYP3A4 primers (forward: 5′-CCT TAC ACA TAC ACA CCC TTT GGA AGT-3′ and reverse: 5′-AGC TCA ATG CAT GTA CAG AAT CCC CGG TTA-3′) (Jover et al., 2002) and 300 nM of the glucose-6-phosphate dehydrogenase primers (forward: 5′-GCC CCT CGC TGC TGC TAC TA-3′ and reverse: 5′-CGC CCT CCT CCT TCC TTC TGT-3′) (Jover et al., 2002). After an initial 10 min denaturation at 95 °C, amplification proceeded with 50 cycles of 10 s at 95 °C, 5 s at 58 °C and 20 s at 72 °C. Ct values were determined using the LightCycler Relative Quantification software. CYP3A4 mRNA levels were corrected for glucose-6-phosphate dehydrogenase levels (reference gene) and ΔΔCt was determined by normalizing to a calibrator (untreated) sample. To compare the results of independent experiments, the relative fold change was determined to be 2 ΔΔCt (Livak and Schmittgen, 2001) and converted to % change by multiplying by 100. Both PCR reactions gave rise to a single peak when melting curves were analyzed.

To examine the toxicity of IFNα and to ensure that non-cytotoxic concentrations of pharmacological inhibitors were selected for this study, a CytoTox 96 Nonradioactive Cytotoxicity Assay Kit (Promega) was used according to manufacturer's instructions. This kit measures the activity of lactose dehydrogenase, a cytosolic enzyme that is released upon cell lysis, in the culture medium.

HepG2 cells in 100 mm dishes were washed twice with cold PBS and nuclear proteins were extracted using the NE-PER nuclear and cytoplasmic extraction reagent kit (Pierce). Equal amounts of proteins were separated by SDS-PAGE and transferred to Immobilon-P PVDF membranes (Millipore, Bedford, MA). Antibodies were diluted in Tris-buffered saline (TBS) containing 5% BSA and 0.1% Tween-20 at the following ratios: anti-Sp1 (H-225), 1:2000; anti-Sp3 (D-20), 1:5000; anti-Stat1 and anti-histones H1 (FL-219), 1:1000. Membranes incubated with diluted anti-Sp1, anti-Sp3, or anti-histones antibodies for 1 h at room temperature while membranes incubated with diluted anti-Stat1 antibody overnight at 4 °C. The Pierce SuperSignal West Dura Extended Duration Substrate chemiluminescence kit was used to detect horseradish peroxidase-labeled secondary antibodies.

Data from each experimental condition were summarized using means and standard deviations. Overall difference between treatment and non-treatment groups were assessed using two-factor Analysis of Variance. If indicated, individual differences at each time point were performed using two sample t-tests with Bonferroni adjustment for multiple comparisons. Proportional differences between matched observations were performed using the Wilcoxon Signed Rank test. All tests were two-sided and considered to be statistically significant at p < 0.05.

Section snippets

IFNα decreases CYP3A4 activity in human hepatoma cells

Several in vivo studies have suggested that IFNα treatment inhibits drug metabolism by CYP enzymes. Administration of IFNα impaired antipyrine clearance in hepatitis patients (Williams and Farrell, 1986), and decreased theophylline metabolism in cancer patients (Israel et al., 1993), as well as in hepatitis patients and healthy volunteers (Williams et al., 1987). Melanoma patients treated with high-dose IFNα exhibited differential effects on CYP isozyme activity, with significant inhibition of

Discussion

Given that IFNα is frequently used in combination with other therapies to treat malignancies and chronic hepatitis B and C, it is of great importance to establish the impact of IFNα treatment on CYP3A4 activity to avoid unexpected adverse drug interactions. Drug metabolism is known to be altered during infection or inflammation, therefore previous in vitro studies using human and rat hepatocytes focused on the effects of inflammatory cytokines, including IFNγ, tumor necrosis factor-α,

Conflict of interest statement

The authors declare that they have no conflict of interest in this work.

Acknowledgments

We thank Michèle Lemieux and Jennifer Whitteker for their technical assistance and Drs. Aaron Farnsworth and Michael Rosu-Myles for critical review of this manuscript. We would also like to acknowledge Dr. Rémy Aubin for helpful discussions and Dr. Rodney Breau for statistical analysis. This work is funded by the Canadian Regulatory Strategy for Biotechnology.

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    This work was funded by Genomic Research Development Initiative (GRDI) from the Government of Canada. AMH is supported by a Scholarship from King Abdulaziz University, through the Saudi Arabian Cultural Bureau in Canada.

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