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.

High Resolution Quantitative Proteomics of HeLa Cells Protein Species Using Stable Isotope Labeling with Amino Acids in Cell Culture(SILAC), Two-Dimensional Gel Electrophoresis(2DE) and Nano-Liquid Chromatograpohy Coupled to an LTQ-OrbitrapMass Spectrometer

“…The cells were split four times, and the incorporation of the amino acids was checked via the acidic extraction of peptides from the cells and MALDI-MS analysis (25). Apoptosis was induced by the addition of 5 M STLC (Sigma-Aldrich) to 2 ϫ 10 6 /ml HeLa cells grown in light-arginine-and light-lysine-containing medium for 16 h at 37°C in 5.0% CO 2 (26).…”

Docetaxel-Resistant Prostate Cancer Cells Remain Sensitive toS-Trityl-l-Cysteine–Mediated Eg5 Inhibition

“…In HeLa cells, STLC suppresses cell proliferation by induction of transient arrest in the G 2 -M phase of the cell cycle, followed by subsequent apoptosis (7,8). STLC-treated PC3M cells underwent a transient G 2 -M arrest, followed by polyploidy, whereas docetaxel-treated PC3M cells proceeded to apoptosis after a transient G 2 -M arrest.…”

“…STLC (Fig. 1A) is a reversible, tight-binding inhibitor of Eg5, inducing prolonged mitotic arrest and subsequent apoptosis in cell lines derived from different tumor types (6)(7)(8). STLC is currently under further chemical optimization and the best analogues have IC 50 values in vitro and in cell-based assays in the low nanomolar range (6,9,10).…”

Mutations in the human kinesin Eg5 that confer resistance to monastrol and S-trityl-l-cysteine in tumor derived cell lines

“…Induction of aberrant mitosis in tumor cells leads to consequent mitotic arrest, which is often followed by apoptotic cell death [18,19]. The fact that Eg5 inhibition in cancer cells results in a prolonged arrest in mitosis and an increased incidence of apoptosis has validated the apoptotic properties of Eg5 inhibitors [19][20][21][22][23].…”