title: Proteome analysis of apoptosis signaling by S-trityl-L-cysteine, a potent reversible inhibitor of human mitotic kinesin Eg5.
abstract: Mitotic kinesins represent potential drug targets for anticancer chemotherapy. Inhibitors of different chemical classes have been identified that target human Eg5, a kinesin responsible for the establishment of the bipolar spindle. One potent Eg5 inhibitor is S-trityl-L-cysteine (STLC), which arrests cells in mitosis and exhibits tumor growth inhibition activity. However, the underlying mechanism of STLC action on the molecular level is unknown. Here, cells treated with STLC were blocked in mitosis through activation of the spindle assembly checkpoint as shown by the phosphorylated state of BubR1 and the accumulation of mitosis specific phosphorylation on histone H3 and aurora A kinase. Using live cell imaging, we observed prolonged mitotic arrest and subsequent cell death after incubation of GFP-alpha-tubulin HeLa cells with STLC. Activated caspase-9 occurred before cleavage of caspase-8 leading to the accumulation of the activated executioner caspase-3 suggesting that STLC induces apoptosis through the intrinsic apoptotic pathway. Proteome analysis following STLC treatment revealed 33 differentially regulated proteins of various cellular processes, 31 of which can be linked to apoptotic cell death. Interestingly, four identified proteins, chromobox protein homolog, RNA-binding Src associated in mitosis 68 kDa protein, stathmin, and translationally controlled tumor protein can be linked to mitotic and apoptotic processes.
authors.affiliation: The Beatson Institute for Cancer Research, Bearsden, Glasgow, UK.
authors.forename: Frank
authors.initials: F
authors.lastname: Kozielski
authors.forename: Dimitrios A
authors.initials: DA
authors.lastname: Skoufias
authors.forename: Rose-Laure
authors.initials: RL
authors.lastname: Indorato
authors.forename: Yasmina
authors.initials: Y
authors.lastname: Saoudi
authors.forename: Peter R
authors.initials: PR
authors.lastname: Jungblut
authors.forename: Hanne K
authors.initials: HK
authors.lastname: Hustoft
authors.forename: Margarita
authors.initials: M
authors.lastname: Strozynski
authors.forename: Bernd
authors.initials: B
authors.lastname: Thiede
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors*; D008938:Mitosis / Q000187:drug effects; D017239:Paclitaxel / Q000494:pharmacology; D020543:Proteome / Q000032:analysis*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C470243:KIF11 protein, human; D020543:Proteome; C003724:3-tritylthio-L-alanine; D016547:Kinesins; D003545:Cysteine; D017239:Paclitaxel