Inhibitors of the VEGF Receptor Suppress HeLa S3 Cell Proliferation via Misalignment of Chromosomes and Rotation of the Mitotic Spindle, Causing a Delay in M-Phase Progression

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.

VEGFR2 inhibition hampers breast cancer cell proliferation <i>via</i> enhanced mitochondrial biogenesis

“…It should also been noted that VEGFR2 blockade also increased the size of the cancer cells, with larger lobular or multiple nuclei, indicating the endomitosis of Ki8751-treated cancer cells. This phenomenon was further supported by the evidence showing that Ki8751 treatment detained the cells at high aneuploid phase (G4) in cell cycle analyses, and that Ki8751 inhibition delayed M phase progression 11 .…”

“…Our observations were consistent with earlier reports that VEGF is critical in maintaining cell proliferative potential, and that VEGF receptor (VEGFR) blockade by its selective inhibitor, Ki8751, significantly increases cellular senescence in metastatic colorectal cancer cells 10 . Furthermore, Ki8751 inhibition significantly delays M phase progression, and suppresses cell proliferation of HeLa S3 cells 11 .…”

Targeting Insulin-Like Growth Factor 1 Receptor Delays M-Phase Progression and Synergizes with Aurora B Inhibition to Suppress Cell Proliferation

“…Immunofluorescence staining was performed, as described previously [42,43]. In brief, cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) at room temperature for 20 min.…”

ALK Inhibitors-Induced M Phase Delay Contributes to the Suppression of Cell Proliferation

“…Western blotting was performed as described previously [52]. Briefly, cells were lysed in SDS sample buffer containing 2 µg/mL aprotinin (Seikagaku Kogyo, Tokyo, Japan), 0.8 µg/mL pepstain A (Wako Pure Chemicals, Osaka, Japan), 2 µg/mL leupeptin (Nacalai Tesque), 2 mM PMSF (Nacalai Tesque), 20 mM β-glycerophosphate (MilliporeSigma), 50 mM NaF (Wako), and 10 mM Na 3 VO 4 (Wako) and denatured at 40 and 100 • C for 20 and 5 min, respectively.…”

“…Immunofluorescence staining was performed as described previously [52,53]. Cells were grown on coverslips and fixed with 4% formaldehyde in PBS for 20 min at room temperature.…”