Hu Dong Gui, Mackenzie Peter I, Lu Lu, Meech Robyn, McKinnon Ross A
Department of Clinical Pharmacology and Flinders Centre for Innovation in Cancer, Flinders University School of Medicine, Flinders Medical Centre, Bedford Park, Australia (D.G.H., P.I.M., L.L., R.M., R.A.M.).
Department of Clinical Pharmacology and Flinders Centre for Innovation in Cancer, Flinders University School of Medicine, Flinders Medical Centre, Bedford Park, Australia (D.G.H., P.I.M., L.L., R.M., R.A.M.)
Drug Metab Dispos. 2015 May;43(5):660-8. doi: 10.1124/dmd.114.062380. Epub 2015 Feb 23.
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.
我们最近报道，在肝癌HepG2细胞中，其底物表柔比星（一种细胞毒性蒽环类抗癌药物）通过激活p53并随后将p53募集到UGT2B7启动子，从而诱导UGT2B7的表达。本研究使用相同的HepG2模型细胞系，评估了其他几种细胞毒性药物诱导UGT2B7表达的可能性。我们首先通过逆转录定量实时聚合酶链反应证明，与表柔比星的情况一样，九种细胞毒性药物，包括三种蒽环类药物（多柔比星、柔红霉素和伊达比星）和六种非蒽环类药物（丝裂霉素C、5-氟尿嘧啶、喜树碱、7-乙基-10-羟基喜树碱、拓扑替康和依托泊苷）均显著提高了UGT2B7 mRNA水平。为了研究p53在这种诱导过程中的潜在作用，我们用九种药物中的四种（多柔比星、柔红霉素、伊达比星和丝裂霉素C）进行了进一步实验。所研究的细胞毒性药物增加了p53和UGT2B7蛋白水平。通过小干扰RNA敲低p53表达可降低细胞毒性药物诱导的UGT2B7表达。荧光素酶报告基因检测显示细胞毒性药物通过先前报道的p53位点激活UGT2B7启动子。最后，染色质免疫沉淀检测证明，在暴露于丝裂霉素C（九种受试药物中最有效的UGT2B7诱导剂）后，p53被募集到UGT2B7的p53位点。综上所述，这些结果提供了进一步的证据，支持UGT2B7作为p53的靶基因。细胞毒性药物在靶肝癌细胞或可能在正常肝细胞中诱导的UGT2B7活性，可能会影响同时给药的细胞毒性药物（如表柔比星）和非细胞毒性药物（如吗啡）（它们都是UGT2B7的底物）的治疗效果。
