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

Abstract

Anaplastic lymphoma kinase (ALK), a receptor-type tyrosine kinase, is involved in the pathogenesis of several cancers. ALK has been targeted with small molecule inhibitors for the treatment of different cancers, but absolute success remains elusive. In the present study, the effects of ALK inhibitors on M phase progression were evaluated. Crizotinib, ceritinib, and TAE684 suppressed proliferation of neuroblastoma SH-SY5Y cells in a concentration-dependent manner. At approximate IC50 concentrations, these inhibitors caused misorientation of spindles, misalignment of chromosomes and reduction in autophosphorylation. Similarly, knockdown of ALK caused M phase delay, which was rescued by re-expression of ALK. Time-lapse imaging revealed that anaphase onset was delayed. The monopolar spindle 1 (MPS1) inhibitor, AZ3146, and MAD2 knockdown led to a release from inhibitor-induced M phase delay, suggesting that spindle assembly checkpoint may be activated in ALK-inhibited cells. H2228 human lung carcinoma cells that express EML4-ALK fusion showed M phase delay in the presence of TAE684 at about IC50 concentrations. These results suggest that ALK plays a role in M phase regulation and ALK inhibition may contribute to the suppression of cell proliferation in ALK-expressing cancer cells.

Combination Treatment of OSI-906 with Aurora B Inhibitor Reduces Cell Viability via Cyclin B1 Degradation-Induced Mitotic Slippage

“…Cell viability was assessed using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) based on the manufacturer’s instructions, as described previously [ 14 ]. A549 cells seeded in 96-well plates (3000–4000 cells per well) were cultured with either OSI-906, ZM447439, or their combination for 3 days.…”

“…This is supported by the fact that increases in plasma IGF1 levels are associated with cancer risk [ 9 , 10 , 11 , 12 ]. Recently, we reported an abnormal cell division upon inhibition of receptor-type tyrosine kinases, including EphA2 [ 13 ], ALK [ 14 ], VEGF receptor [ 15 ], and IGF1R [ 16 ].…”

The tyrosine kinase v‐Src modifies cytotoxicities of anticancer drugs targeting cell division

“…Immunofluorescence staining was performed as described previously 14 . In brief, cells were cultured on coverslips and fixed with 100% methanol at −30ºC for 5 minutes.…”

“…The number of live cells per well was estimated using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions as described previously. 14 In brief, HeLa S3/v-Src cells at 3.0 or 8.0 × 10 3 cells/well in a 96-well plate were cultured with or without inhibitors/anticancer drugs in the presence or absence of 2 ng/mL Dox for 24 hours. After washing these drugs out, the cells were further cultured for 2 days.…”

“…Immunofluorescence staining was performed as described previously. 14 In brief, cells were cultured on coverslips and fixed with 100% methanol at −30ºC for 5 minutes. After permeabilizing and blocking with PBS(−) containing 0.1% saponin and 3% bovine serum albumin for 30 minutes, the cells were incubated with the primary antibody for 1 hour and subsequently with the secondary antibody for 1 hour along with 1 µM Hoechst 33342 for DNA staining.…”