title: Topotecan is a substrate for multidrug resistance associated protein 4.
abstract: Topotecan (TPT) is a semisynthetic water-soluble derivative of camptothecin (CPT) used as second-line therapy in patients with metastatic ovarian carcinoma, small cell lung cancer, and other malignancies. However, both dose-limiting toxicity and tumor resistance hinder the clinical use of TPT. The mechanisms for resistance to TPT are not fully defined, but increased efflux of the drug by multiple drug transporters including P-glycoprotein (PgP), multidrug resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) from tumor cells has been highly implicated. This study aimed to investigate whether overexpression of human MRP4 rendered resistance to TPT by examining the cytotoxicity profiles using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay and cellular accumulation of TPT in HepG2 cells stably overexpressing MRP4. Two kinds of cell lines, HepG2 with insertion of an empty vector plasmid (V/HepG2), HepG2 cells stably expressing MRP4 (MRP4/HepG2), were exposed to TPT for 4 or 48 hr in the absence or presence of various MRP4 inhibitors including DL-buthionine-(S,R)-sulphoximine (BSO), diclofenac, celecoxib, or MK-571. The intracellular accumulation of TPT and paclitaxel (a PgP substrate) by V/HepG2 and MRP4/HepG2 cells was determined by incubation of TPT with the cells and the amounts of the drug in cells were determined by validated HPLC methods. The study demonstrated that MRP4 conferred a 12.03- and 6.86-fold resistance to TPT in the 4- and 48-hr drug-exposure MTT assay, respectively. BSO, MK-571, celecoxib, or diclofenac sensitised MRP4/HepG2 cells to TPT cytotoxicity and partially reversed MRP4-mediated resistance to TPT. In addition, the accumulation of TPT was significantly reduced in MRP4/HepG2 cells compared to V/HepG2 cells, and one-binding site model was found the best fit for the MRP4-mediated efflux of TPT, with an estimated K(m) of 1.66 microM and V(max) of 0.341 ng/min/106 cells. Preincubation of MRP4/HepG2 cells with BSO (200 microM) for 24 hr, celecoxib (50 microM), or MK-571 (100 microM) for 2 hr significantly increased the accumulation of TPT over 10 min in MRP4/HepG2 cells by 28.0%, 37.3% and 32.5% (P < 0.05), respectively. By contrast, there was no significant difference in intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells over 120 min. MRP4 also rendered resistance to adefovir dipivoxil (bis-POM-PMEA) and methotrexate, two reported MRP4 substrates. MRP4 did not exhibit any significant resistance to other model drugs including vinblastine, vincristine, etoposide, carboplatin, cyclosporine and paclitaxel in both long (48 hr) and short (4 hr) drug-exposure MTT assays. These findings indicate that MRP4 confers resistance to TPT and TPT is the substrate for MRP4. Further studies are needed to explore the role of MRP4 in resistance to, toxicity and pharmacokinetics of TPT in cancer patients.
authors.affiliation: Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore 117543.
authors.forename: Quan
authors.initials: Q
authors.lastname: Tian
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Sui Yung
authors.initials: SY
authors.lastname: Chan
authors.forename: Theresa May Chin
authors.initials: TM
authors.lastname: Tan
authors.forename: Wei
authors.initials: W
authors.lastname: Duan
authors.forename: Min
authors.initials: M
authors.lastname: Huang
authors.forename: Yi-Zhun
authors.initials: YZ
authors.lastname: Zhu
authors.forename: Eli
authors.initials: E
authors.lastname: Chan
authors.forename: Qiang
authors.initials: Q
authors.lastname: Yu
authors.forename: Yu-Qiang
authors.initials: YQ
authors.lastname: Nie
authors.forename: Paul Chi-Liu
authors.initials: PC
authors.lastname: Ho
authors.forename: Qi
authors.initials: Q
authors.lastname: Li
authors.forename: Ka-Yun
authors.initials: KY
authors.lastname: Ng
authors.forename: Hong-Yuan
authors.initials: HY
authors.lastname: Yang
authors.forename: Hong
authors.initials: H
authors.lastname: Wei
authors.forename: Jin-Song
authors.initials: JS
authors.lastname: Bian
authors.forename: Shu-Feng
authors.initials: SF
authors.lastname: Zhou
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000235:genetics; D019008:Drug Resistance, Neoplasm; D006801:Humans; D007202:Indicators and Reagents; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017239:Paclitaxel / Q000378:metabolism; D019772:Topotecan / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; D000972:Antineoplastic Agents, Phytogenic; D002352:Carrier Proteins; D018076:DNA, Complementary; D007202:Indicators and Reagents; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D017239:Paclitaxel; D019772:Topotecan; C073492:ABCC4 protein, human