Figure 1.
(A) Chemical structure of R1498; (B) R1498 was subjected KINOMEScan® and the most potent hits with Kd< 0.2 µM are shown; the assay was carried out in triplicate, and data were presented as mean±standard deviation; (C) R1498 was tested in Z-lyte kinase assays including VEGFR2, Aurora A and B, the dose response curve was shown and mean IC50 values from three independent experiments were indicated. (D) The in vitro anti-tumor growth activity of R498 was assayed on 16 cell lines including HCC, GC and NPC. Data from triplicated independent assays were presented as mean±standard deviation.
Figure 2.
R1498 targets Aurora kinases in cells.
(A) SGC-7901 gastric cancer cells were treated with 5 µM R1498 for 24 h, then fixed and stained for phospho-Aurora A(T288) (green), MPM2 (red) and DNA (blue). MPM2 was used to locate the mitotic cells. Bright green foci pointed by white arrow heads indicated the high level of phospho-Aurora A(T288) in centromere (upper panel). R1498 treated SGC-7901 gastric cancer cells as above were stained for phospho-Histone H3(H10) (green), MPM2 (red) and DNA (blue) (lower panel). Representative images from 2 independent assays are shown. (B) R1498 treated SGC-7901 cells as above was subjected DNA analysis by flow cytometry via propidium iodide staining. The histograms were further analyzed by Modfit 3.0 for cell cycle distribution. The cells with DNA content more than 4N (>4N) mean aneuploidy caused by endoduplication. Representative histograms from 3 independent assays are shown. (C) SGC-7901 synchronized with nocodazole (300 nM) for 12 h were treated with various concentration of R1498, then lysed and subjected for western blot with corresponding antibodies. (D) In vitro tubulin polymerization assay was performed with R1498 and docetaxel, and the OD340 was measured and recorded in a time lapsed manner. Representative histograms from 2 independent assays are shown.
Figure 3.
R1498 targets VEGF signaling in cells.
(A) HUVECs cultured in 10% FBS, or 0.5% FBS with VEGF 165 were treated with various R1498 for 72 h, and then analyzed for proliferation endpoint. The curves were fitted with Graphpad Prism5.0. The three independent assays were carried out in triplicated and IC50s were indicated as mean±standard deviation; (B) HUVECs grown on Matrigel™ were treated with 3 µM R1498 or equivalent DMSO for 8 h. Representative images from 2 independent assays are shown.
Figure 4.
In vivo efficacy of R1498 on cell line derived xenografts.
BEL-7402, SGC-7901 and CNE-2 xenograft bearing nude mice were treated with 25 mg/kg R1498 or sorafenib (or cyclophosphamide (CTX)) as indicated schedule; the tumor volume was recorded and plotted against days post inoculation (upper panel), the body weight changes compared with the day of starting treatment was plotted (lower panel). The volume and relative body weight changes were indicated as mean±standard error (n = 12 in vehicle groups, n = 10 in R1498/sorafenib/ cyclophosphamide groups). The studies were repeated for confirmation twice. The representative curves are shown here.
Figure 5.
R1498 antagonized angiogenesis and Aurora A activity in vivo.
The tumors harvested from said BEL-7402 study (n = 12 in vehicle groups, n = 10 in R1498/sorafenib/CTX groups) were sectioned and subjected for immunohistochemistry of CD31 (angiogenesis marker, A) and phosphor-Aurora A(T288) (Aurora kinase A activity marker, B) staining. The slides were reviewed and scored by two independent pathologists. The scores were presented as mean±standard error. (** p< 0.01; Log-rank test).
Figure 6.
In vivo efficacy of R1498 on patient primary tumor derived xenografts.
R1498 was administrated to nude mice bearing xenografts derived from three different gastric cancer patients as indicated schedule, the tumor volumes were recorded and plotted against days post inoculation (left panel), together with the body weight changes (right panel). The volume and relative body weight changes were indicated as mean±standard error (n = 10).