Fig 1.
The structure and bioactivity of LXY18 and 11i.
(A, B) The chemical structures of LXY18 and 11i. (C) Concentration-response curves for Calu-6 and Hela cells as determined by the MTT assay. Cell numbers are relative to vehicle control (0.1% DMSO). Each point represents the mean ± SD from three independent experiments with measurements done triplicate. The IC50 was calculated with GraphPad Prism 8.0.2. (D) The localization of AURKB. RPE-MYCBcl2 cells were treated for 6 hours with the indicated compounds before immunofluorescent staining for AURKB and kinetochores with human CREST serum. DNA staining was with DAPI. Cells treated with vehicle (0.1% of DMSO) were used as a negative control. The numbers of early anaphase cells scored and those with AURKB mislocation under each treatment are indicated. Bar: 10μm.
Fig 2.
LXY18 exerts broad-spectrum activity in suppressing the growth of cancer cell lines.
(A) The IC50, Emax, and AOC of LXY18 in 18 cancer cell lines treated over a wide concentration range for 3 days before viable cell number was quantified by MTT assay. The IC50, Emax, and AOC were calculated with GraphPad Prism 8.0.2. Histograms show the combined data from three independent experiments presented as mean ± SD. (B) The reverse correlation between IC50 and either Emax or AOC.
Fig 3.
LXY18 elicits apoptosis in cancer lines but not in nontransformed cell lines.
(A) Cell morphology in response to LXY18. The indicated cell lines were exposed to 40 nM of LXY18 for 48 hours. Images were acquired using an inverted tissue culture microscope. Scale bar, 10 μm. (B) LXY18 elicits apoptosis in cancer cell lines. The indicated cell lines were exposed to 40 or 80 nM of LXY18 for 48 hours prior to lysis and Western analysis of protein. (C) Cell death could be rescued by inhibiting caspases. NCI-H23 and NCI-H460 were treated for 48 hours with 40 nM LXY18 either alone or in combination with 100 μM of Z-VAD fmk for 24 hours. RPE A19 and IMR90 cell lines were also treated with 40 nM of LXY18 for 48 hours, while NIH 3T3 and Rat1A cell lines were exposed to 40 nM of LXY18 for 72 hours. Vehicle control was 0.1% DMSO. At the endpoint, the Trypan blue exclusion assay was used to detect dead/living cells and the percent dead cells in the population was calculated. Data from three independent experiments with triplicate measurements are presented as mean ± SD. *p<0.0001.
Fig 4.
LXY18 elicits a prolonged mitotic arrest, cytokinetic failure, and cell death.
Cells were subjected to a time-lapse analysis following exposure to 40nM LXY18 with a duration of 70 hours (RPE A19) or 75 hours (NCI-H23 and Hela). (A) The histogram shows the mitotic index at 24, 48, and 72 hours. A total of 200–600 cells were scored for each data point. (B) The duration of mitotic arrest was quantified for each cell line with LXY18 treatment (n is the number of mitotic cells scored in each group). (C) Endpoint analysis showing the fate of cells followed throughout the entire time course of the experiment. (D) Histogram showing the mitotic index as mean ± SD (*p<0.0001). Cells were treated with 40 nM LXY18 for 48 hours or with vehicle control (0.1% DMSO).
Fig 5.
Time-lapse images of cells treated with LXY18.
Cells were treated with or without 40 nM of LXY18 and then subjected to time-lapse video filming. Representative images are presented. (A) NCI-H23. (B) Hela. (C) RPE A19. Bars: 10 μm.
Fig 6.
Aphidicolin protects cells from cell death elicited by LXY18.
NCI-H23 cells were treated as indicated. (A) The morphology of NCl-H23 cells. Scale bars, 100 μm. (a) Vehicle control (0.1% DMSO) for 48 hours. (b) LXY18, 40 nM for 48 hours. (c) Results of assay with 1st LXY18 (40nM) + 2nd Aphidicolin (3 μg/mL). LXY18 was for 48 hours with addition of 3 μg/mL aphidicolin for the last 24 hours. (d) Results of assay with 1st Aphidicolin (3 μg/mL) + 2nd LXY18 (40nM). LXY18 was for 48 hours, with aphidicolin given 24 hours before the addition of LXY18. Cell viability was determined by Trypan blue assays. (B) The percentage of dead cells. The percentage of dead cells in 6A was quantified. Combined data from three independent experiments with triplicate measurements are presented as mean ± SD. *p<0.0001.
Fig 7.
LXY18 fails to inhibit the catalytic activities of MKLP2 and NEK6, NEK7, and NEK 9.
(A) Kinome-wide selectivity profiling of 430 kinases with LXY18. The scatter plot shows the percentage of inhibition of each of 430 kinases by 2 μM of LXY18. (B) The impact of LXY18 on the MKLP2’s ATPase activity. (C) Western blot for the expression of AURKB, INCENP, and MKLP2 after treatment of NCI-H23 cells for the indicated time with 200 nM of LXY18.