title: Identification of cisplatin-binding proteins using agarose conjugates of platinum compounds.
abstract: Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94), heat shock protein 90 (HSP90), calreticulin, valosin containing protein (VCP), and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment.
authors.affiliation: Oregon Hearing Research Center, Oregon Health & Science University, Portland, Oregon, United States of America. karasawa@ohsu.edu
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Karasawa
authors.forename: Martha
authors.initials: M
authors.lastname: Sibrian-Vazquez
authors.forename: Robert M
authors.initials: RM
authors.lastname: Strongin
authors.forename: Peter S
authors.initials: PS
authors.lastname: Steyger
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000302:isolation & purification; D000818:Animals; D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000737:chemistry*; D037282:Calreticulin / Q000737:chemistry / Q000302:isolation & purification; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000302:isolation & purification; D002457:Cell Extracts / Q000737:chemistry*; D002460:Cell Line; D002945:Cisplatin / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry*; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology; D006001:Glycoconjugates / Q000138:chemical synthesis / Q000737:chemistry*; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification; D007687:Kidney Tubules, Proximal / Q000737:chemistry / Q000166:cytology; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D051379:Mice; D024507:Nonmuscle Myosin Type IIA / Q000737:chemistry / Q000302:isolation & purification; D009925:Organ of Corti / Q000737:chemistry / Q000166:cytology; D011485:Protein Binding; D012269:Ribosomal Proteins / Q000737:chemistry / Q000302:isolation & purification; D012685:Sepharose / Q000737:chemistry*; D000074405:Valosin Containing Protein
chemical_list: D000970:Antineoplastic Agents; D037282:Calreticulin; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D006001:Glycoconjugates; D018841:HSP90 Heat-Shock Proteins; D008562:Membrane Glycoproteins; D012269:Ribosomal Proteins; C053387:endoplasmin; C029742:ribosomal protein L5; D012685:Sepharose; D000251:Adenosine Triphosphatases; D024507:Nonmuscle Myosin Type IIA; D000074405:Valosin Containing Protein; C000614179:Vcp protein, mouse; D002945:Cisplatin