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In Vitro Approaches to Study Regulation of Hepatic Cytochrome P450 (CYP) 3A Expression by Paclitaxel and Rifampicin

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Cancer Drug Resistance

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1395))

Abstract

Cancer is the second leading cause of mortality worldwide; however the response rate to chemotherapy treatment remains slow, mainly due to narrow therapeutic index and multidrug resistance. Paclitaxel (taxol) has a superior outcome in terms of response rates and progression-free survival. However, numerous cancer patients are resistant to this drug. In this investigation, we tested the hypothesis that induction of cytochrome P450 (Cyp)3a11 gene by paclitaxel is downregulated by the inflammatory mediator, lipopolysaccharide (LPS), and that the pro-inflammatory cytokine, tumor necrosis factor (TNF)-α, attenuates human CYP3A4 gene induction by rifampicin. Primary mouse hepatocytes were pretreated with LPS (1 μg/ml) for 10 min, followed by paclitaxel (20 μM) or vehicle for 24 h. RNA was extracted from the cells by trizol method followed by cDNA synthesis and analysis by real-time PCR. Paclitaxel significantly induced gene expression of Cyp3a11 (~30-fold) and this induction was attenuated in LPS-treated samples. Induction and subsequent downregulation of CYP3A enzyme can impact paclitaxel treatment in cancer patients where inflammatory mediators are activated. It has been shown that the nuclear receptor, pregnane X receptor (PXR), plays a role in the induction of CYP enzymes. In order to understand the mechanisms of regulation of human CYP3A4 gene, we co-transfected HepG2 cells (human liver cell line) with CYP3A4-luciferase construct and a PXR expression plasmid. The cells were then treated with the pro-inflammatory cytokine, TNFα, followed by the prototype CYP3A inducer rifampicin. It is well established that rifampicin activates PXR, leading to CYP3A4 induction. We found that induction of CYP3A4-luciferase activity by rifampicin was significantly attenuated by TNFα. In conclusion, we describe herein several in vitro approaches entailing primary and cultured hepatocytes, real-time PCR, and transcriptional activation (transfection) assays to investigate the molecular regulation of CYP3A, which plays a pivotal role in the metabolism of numerous chemotherapeutic drugs. Genetic or drug-induced variation in CYP3A and/or PXR expression could contribute to drug resistance to chemotherapeutic agents in cancer patients.

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Acknowledgements

This work was supported by NIH grants R21-DA035751 to RG and R01-ES-009132, R01-HL-112516, and R01-ES-019689 to BM.

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Authors and Affiliations

  1. Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, TX, 77030, USA

    Romi Ghose, Pankajini Mallick & Guncha Taneja

  2. Department of Pediatrics, Baylor College of Medicine, Houston, TX, 77030, USA

    Chun Chu & Bhagavatula Moorthy

Authors
  1. Romi Ghose
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  2. Pankajini Mallick
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  3. Guncha Taneja
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  4. Chun Chu
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  5. Bhagavatula Moorthy
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Correspondence to Bhagavatula Moorthy .

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Editors and Affiliations

  1. New University of Lisbon, Lisbon, Portugal

    Jose Rueff

  2. New University of Lisbon, Lisbon, Portugal

    António Sebastião Rodrigues

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Ghose, R., Mallick, P., Taneja, G., Chu, C., Moorthy, B. (2016). In Vitro Approaches to Study Regulation of Hepatic Cytochrome P450 (CYP) 3A Expression by Paclitaxel and Rifampicin. In: Rueff, J., Rodrigues, A. (eds) Cancer Drug Resistance. Methods in Molecular Biology, vol 1395. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3347-1_4

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  • DOI: https://doi.org/10.1007/978-1-4939-3347-1_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3345-7

  • Online ISBN: 978-1-4939-3347-1

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