Abstract
Cell division is the process by which replicated chromosomes are separated into two daughter cells. Although regulation of M phase has been extensively investigated, not all regulating factors have been identified. Over the course of our research, small molecules were screened to identify those that regulate M phase. In the present study, the vascular endothelial growth factor receptor (VEGFR) inhibitors A83-01, SU4312, and Ki8751 were examined to determine their effects on M phase. Treatment of HeLa S3 cells with these inhibitors suppressed cell proliferation in a concentration-dependent manner, and also suppressed Akt phosphorylation at Ser473, a marker of Akt activation. Interestingly, cleaved caspase-3 was detected in Adriamycin-treated cells but not in inhibitor-treated cells, suggesting that these inhibitors do not suppress cell proliferation by causing apoptosis. A cell cycle synchronization experiment showed that these inhibitors delayed M phase progression, whereas immunofluorescence staining and time-lapse imaging revealed that the M phase delay was accompanied by misalignment of chromosomes and rotation of the mitotic spindle. Treatment with the Mps1 inhibitor AZ3146 prevented the SU4312-induced M phase delay. In conclusion, the VEGFR inhibitors investigated here suppress cell proliferation by spindle assembly checkpoint-induced M phase delay, via misalignment of chromosomes and rotation of the mitotic spindle.
Highlights
Cell division is the process by which organelles, cytoplasm, and replicated chromosomes are separated into two daughter cells
We found that three vascular endothelial growth factor receptor (VEGFR) inhibitors, A83-01, SU4312, and Ki8751, delayed M phase progression, accompanied by misalignment of chromosomes and rotation of the mitotic spindle, at concentrations around the IC50
Because cleaved caspase-3 was not detected by immunofluorescence and Western blot analyses when cells were treated with these inhibitors, the resulting suppression of cell proliferation may have been caused not by apoptosis, but by induction of M phase delay
Summary
Cell division is the process by which organelles, cytoplasm, and replicated chromosomes are separated into two daughter cells. VEGFR is mainly expressed in endothelial cells, and is one of the more extensively investigated RTKs. VEGF/VEGFR signaling is the most important regulatory factor of angiogenesis and mediates various cellular responses, including vascular permeability and cell survival, migration, and proliferation in blood vascular endothelial cells [5]. The phosphorylated tyrosine residues in the intracellular domain are recognized by the Src homology 2 domain of the proteins, which stimulate downstream signals including the phospholipase Cγ (PLCγ)–ERK1/2 pathway, the PI3K–AKT pathway, and Src. Phosphorylated tyrosine residue of VEGFR at 1173 recruits PLCγ; PLCγ is activated and catalyzes generation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). Phosphorylated tyrosine residue of VEGFR at 1173 recruits PLCγ; PLCγ is activated and catalyzes generation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) This regulates the RAF1–MEK–ERK1/2 cascade via activation of PKCβ2. In addition to endothelial cells, VEGFR is expressed in breast [6], bladder [7], colorectal [8,9], and gastric [10] cancers; it is an attractive target for cancer chemotherapy
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