Induction of Human UDP-Glucuronosyltransferase 2B7 Gene Expression by Cytotoxic Anticancer Drugs in Liver Cancer HepG2 Cells

Abstract

We recently reported induction of UGT2B7 by its substrate epirubicin, a cytotoxic anthracycline anticancer drug, via activation of p53 and subsequent recruitment of p53 to the UGT2B7 promoter in hepatocellular carcinoma HepG2 cells. Using the same HepG2 model cell line, the present study assessed the possibility of a similar induction of UGT2B7 by several other cytotoxic drugs. We first demonstrated by reverse transcriptase quantitative real-time polymerase chain reaction that, as observed with epirubicin, nine cytotoxic drugs including three anthracyclines (doxorubicin, daunorubicin, and idarubicin) and six nonanthracyclines (mitomycin C, 5-fluorouracil, camptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, and etoposide) significantly increased UGT2B7 mRNA levels. To investigate a potential involvement of p53 in this induction, we conducted further experiments with four of the nine drugs (doxorubicin, daunorubicin, idarubicin, and mitomycin C). The cytotoxic drugs studied increased p53 and UGT2B7 protein levels. Knockdown of p53 expression by small interfering RNA reduced cytotoxic drug-induced UGT2B7 expression. Luciferase reporter assays showed activation of the UGT2B7 promoter by cytotoxic drugs via a previously reported p53 site. Finally, chromatin immunoprecipitation assays demonstrated p53 recruitment to the UGT2B7 p53 site upon exposure to mitomycin C, the most potent UGT2B7 inducer among the nine tested drugs. Taken together, these results provide further evidence supporting UGT2B7 as a p53 target gene. The cytotoxic drug-induced UGT2B7 activity in target liver cancer cells or possibly in normal liver cells may affect the therapeutic efficacy of co-administered cytotoxic drugs (e.g., epirubicin) and noncytotoxic drugs (e.g., morphine), which are UGT2B7 substrates.

Emerging roles for UDP-glucuronosyltransferases in drug resistance and cancer progression

“…This is the case for UGT2B7 induced by several anti-cancer agents in liver cell models, for UGT2B15 induced by tamoxifen and UGT2B17 induced by exemestane in breast cancer-cell models, as well as for UGT1A4 induced by fulvestrant in breast cancer and liver cell models. [42][43][44][45][46] The notion that induction of UGT expression may lead to acquired drug resistance is further supported by the following studies. Breast cancer-cell models that were rendered resistant to methotrexate by prolonged exposure to the drug also displayed enhanced expression of several UGT1As-particularly UGT1A6-as well as enhanced glucuronidation activity.…”

The Expression Profiles of ADME Genes in Human Cancers and Their Associations with Clinical Outcomes

“…A major limitation is that tumour samples were obtained prior to therapeutic intervention. Many ADME genes (e.g., CYPs and UGTs) are induced by common cytotoxic anticancer drugs [84][85][86]. Inter-individual differences in ADME gene induction might lead to different drug responses; however, testing this hypothesis would require tumour samples to be sampled during/after treatment.…”

Intergenic Splicing between Four AdjacentUGTGenes (2B15, 2B29P2, 2B17, 2B29P1) Gives Rise to Variant UGT Proteins That Inhibit Glucuronidation via Protein-Protein Interactions

“…Total RNA was extracted from liver tissues and cancer cell lines using TRIzol reagent according to the manufacturer's protocol (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA). Reverse transcription was carried out using Invitrogen reagents as previously reported (Hu and Mackenzie, 2009;Hu et al, 2015). In brief, total RNA (∼1 mg) was treated with DNase I at room temperature for 15 minutes and then reverse-transcribed using Superscript III (50 units) and random hexamer primers (50 ng) at 50°C for 50 minutes in a 20-ml reaction containing 50 mM Tris-HCl, pH 8.0, 75 mM KCl, and 3 mM MgCl 2 .…”