title: Reversal of P-glycoprotein-mediated paclitaxel resistance by new synthetic isoprenoids in human bladder cancer cell line.
abstract: We isolated a paclitaxel-resistant cell line (KK47/TX30) from a human bladder cancer cell line (KK47/WT) in order to investigate the mechanism of and reversal agents for paclitaxel resistance. KK47/TX30 cells exhibited 700-fold resistance to paclitaxel and cross-resistance to vinca alkaloids and topoisomerase II inhibitors. Tubulin polymerization assay showed no significant difference in the ratio of polymerized alpha- and beta-tubulin between KK47/WT and KK47/TX30 cells. Western blot analysis demonstrated overexpression of P-glycoprotein (P-gp) and lung resistance-related protein (LRP) in KK47/TX30 cells. Drug accumulation and efflux studies showed that the decreased paclitaxel accumulation in KK47/TX30 cells was due to enhanced paclitaxel efflux. Cell survival assay revealed that verapamil and cepharanthine, conventional P-gp modulators, could completely overcome paclitaxel resistance. To investigate whether new synthetic isoprenoids could overcome paclitaxel resistance, we synthesized 31 isoprenoids based on the structure of N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine (SDB), which could reverse multidrug resistance (MDR), as shown previously. Among those examined, trans-N,N'-bis(3,4-dimethoxybenzyl)-N-solanesyl-1,2-diaminocyclohexane (N-5228) could completely reverse paclitaxel resistance in KK47/TX30 cells. N-5228 inhibited photoaffinity labeling of P-gp by [(3)H]azidopine, suggesting that N-5228 could bind to P-gp directly and could be a substrate of P-gp. Next, we investigated structural features of these 31 isoprenoids in order to determine the structural requirements for the reversal of P-gp-mediated paclitaxel resistance, suggesting that the following structural features are important for overcoming paclitaxel resistance: (1) a basic structure of 8 to 10 isoprene units, (2) a cyclohexane ring or benzene ring within the framework, (3) two cationic sites in close proximity to each other, and (4) a benzyl group with 3,4-dimethoxy functionalities, which have moderate electron-donating ability. These findings may provide valuable information for the development of P-gp-mediated MDR-reversing agents.
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis / Q000502:physiology*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D006801:Humans; D015151:Immunoblotting; D009363:Neoplasm Proteins / Q000032:analysis; D017239:Paclitaxel / Q000378:metabolism / Q000494:pharmacology*; D011105:Polyisoprenyl Phosphate Sugars / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013729:Terpenes / Q000494:pharmacology*; D014404:Tubulin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000473:pathology; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000972:Antineoplastic Agents, Phytogenic; D009363:Neoplasm Proteins; D017239:Paclitaxel; D011105:Polyisoprenyl Phosphate Sugars; D013729:Terpenes; D014404:Tubulin; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein