title: In Vitro Approaches to Study Regulation of Hepatic Cytochrome P450 (CYP) 3A Expression by Paclitaxel and Rifampicin.
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.
authors.affiliation: Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, TX, 77030, USA.
authors.forename: Romi
authors.initials: R
authors.lastname: Ghose
authors.affiliation: Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, TX, 77030, USA.
authors.forename: Pankajini
authors.initials: P
authors.lastname: Mallick
authors.affiliation: Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, TX, 77030, USA.
authors.forename: Guncha
authors.initials: G
authors.lastname: Taneja
authors.affiliation: Department of Pediatrics, Baylor College of Medicine, Houston, TX, 77030, USA.
authors.forename: Chun
authors.initials: C
authors.lastname: Chu
authors.affiliation: Department of Pediatrics, Baylor College of Medicine, Houston, TX, 77030, USA. bmoorthy@bcm.tmc.edu.
authors.forename: Bhagavatula
authors.initials: B
authors.lastname: Moorthy
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis / Q000235:genetics*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D017930:Genes, Reporter / Q000235:genetics; D056945:Hep G2 Cells; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008099:Liver / Q000187:drug effects* / Q000201:enzymology* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D051379:Mice; D017239:Paclitaxel / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction; D012293:Rifampin / Q000494:pharmacology*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D018076:DNA, Complementary; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases; D051544:Cytochrome P-450 CYP3A; D017239:Paclitaxel; D012293:Rifampin
keywords: CYP3A; Chemotherapy; HepG2; Hepatocytes; Inflammation