SNS-032 prevents tumor cell-induced angiogenesis by inhibiting vascular endothelial growth factor

Abstract

Cell proliferation, migration, and capillary network formation of endothelial cells are the fundamental steps for angiogenesis, which involves the formation of new blood vessels. The purpose of this study is to investigate the effect of a novel aminothiazole SNS-032 on these critical steps for in vitro angiogenesis using a coculture system consisting of human umbilical vein endothelial cells (HUVECs) and human glioblastoma cells (U87MG). SNS-032 is a potent selective inhibitor of cyclin-dependent kinases 2, 7, and 9, and inhibits both transcription and cell cycle. In this study, we examined the proliferation and viability of HUVECs and U87MG cells in the presence of SNS-032 and observed a dose-dependent inhibition of cellular proliferation in both cell lines. SNS-032 inhibited threedimensional capillary network formations of endothelial cells. In a coculture study, SNS-032 completely prevented U87MG cell-mediated capillary formation of HUVECs. This inhibitor also prevented the migration of HUVECs when cultured alone or cocultured with U87MG cells. In addition, SNS-032 significantly prevented the production of vascular endothelial growth factor (VEGF) in both cell lines, whereas SNS-032 was less effective in preventing capillary network formation and migration of endothelial cells when an active recombinant VEGF was added to the medium. In conclusion, SNS-032 prevents in vitro angiogenesis, and this action is attributable to blocking of VEGF.

Figure 1

Effect of SNS-032 on HUVEC and U87MG cellular growth and viability. (A and D) Cell growth was measured by CTG luminescent assay in 96-well plates in the presence of increasing doses of SNS-032, as indicated, for 24, 48, and 72 hours, and the results are expressed as percent growth inhibition in response to SNS-032 (A, HUVEC; D, U87MG). *P < .001 compared with untreated control (0 µM). (B and E) Cell viability was measured by TBE assay. Treatment conditions were identical as indicated in the CTG assay, and results were expressed as percent viability (B, HUVEC; E, U87MG). *P < .01 compared with untreated control (0 µM). (C and F) Immunoblot analysis of survivin. Cells were treated as indicated, and lysates were collected at the indicated time points and immunoblotted with survivin antibodies (C, HUVEC; F, U87MG). Bars = SD of six replicates from two independent experiments.

Figure 2

Effect of SNS-032 on tube formation by HUVECs. Cells were seeded on Matrigel-coated wells in the presence of different doses of SNS-032, as indicated, and incubated for 8 hours to form a capillary network, and the total number of branched tubes was then counted. The assay was run in duplicate and repeated thrice. (A) Representative images of capillary network formation. (B) Statistical analysis of three independent experiments. Bars, SD. *P < .05 and **P < .001 compared with untreated control.

Figure 3

Effect of SNS-032 on the migration of HUVECs. Cells were seeded onto PET membrane insert with or without SNS-032, as indicated. The insert was then placed into the well; after 8 hours, migrated cells were stained and photographed. The assay was run in duplicate and repeated thrice. (A) Representative images of endothelial cell migration were shown. (B) The total number of migrated cells per field (x200) was measured. Bars = SD calculated from three independent experiments. **P < .001, significantly different from untreated control.

Figure 4

SNS-032 prevents VEGF expression in both glioblastoma and endothelial cells. Expression of VEGF in U87MG and HUVECs was determined by ELISA (A and B), RT-PCR (C), and quantitation of VEGF mRNA bands (D). Cells were exposed to different doses of SNS-032, as indicated, for 24 hours. (A) VEGF protein released by U87MG cells into the conditioned medium was measured by ELISA kit. Bars = SD (n = 3). *P < .05 and **P < .001 compared with untreated control. (B) HUVECs were treated as indicated. Conditioned media were collected and then concentrated up to 10-fold using a 10-kDa molecular weight cutoff centricon concentrator (Amicon; Millipore, Billerica, MA). VEGF protein was measured by ELISA kit. Bars = SD (n = 3). **P < .001 compared with CoCl₂ treatment. (C) Effect of SNS-032 on VEGF mRNA in U87MG cells. The expression of VEGF mRNA was analyzed by RT-PCR. β-Actin expression was included as an internal control. (D) VEGF mRNA expression in U87MG cells was quantitated by densitometry and then normalized with β-actin, as shown in the bar graph. The ratios of VEGF/β-actin at 0 µM SNS-032 were assigned to be 100%. Bars = SD (n = 3). **P < .001 compared with untreated control.

Figure 5

SNS-032 inhibited the U87MG cell-induced tube formation of HUVECs. U87MG cells were treated with or without SNS-032 for 8 hours and then incubated with fresh media without SNS-032 for 24 hours, followed by the collection of conditioned media (CM). (A) Capillary network formation of HUVECs using CM. No-treatment panel indicates that a blank medium (serum-free DMEM) was added to HUVECs for network formation. In the other two panels, CM was added to HUVECs, as indicated. The tube formation assay of HUVECs was performed using CM prepared from U87MG cells instead of HUVEC medium. (B) The total number of branched tubes was counted, and results from three independent experiments were presented for statistical analysis. Error bars = SD. **P < .001 between untreated CM and SNS-032 - treated CM or blank CM. (C) VEGF level was analyzed in the conditioned medium by ELISA.

Figure 6

Effect of SNS-032 on the migration of HUVECs cocultured with U87MG cells. U87MG cells were cultured in the well (lower chamber). Cells were then treated with or without SNS-032 for 8 hours. After treatment, the medium from the lower chamber was replaced with fresh DMEM without SNS-032, and the insert (upper chamber) containing a monolayer of HUVECs in DMEM was then placed into the well. After 8 hours, migrated cells were photographed and counted. (A) Schematic diagram of coculture assay. (B) Control. HUVECs were seeded onto the upper chamber, whereas the bottom chamber contains only DMEM without U87MG cells. (C) HUVECs cocultured with U87MG cells untreated with SNS-032. (D) HUVECs cocultured with U87MG cells pretreated with 0.5 µM SNS-032. (E) Pooled data from three experiments for statistical analysis comparing the number of migrated cells in coculture with and without SNS-032 treatment. Bars = SD. **P < .001 between treated and untreated U87MG cells with SNS-032 or blank culture medium.

Figure 7

Effect of SNS-032 on exogenously added VEGF-induced capillary network formation and migration of HUVECs. (A) HUVECs in endothelial cell basal medium seeded onto a growth factor-reduced Matrigel and then treated with SNS-032 (0.5 µM) for 1 hour. Recombinant VEGF₁₆₅ (100 ng/ml) was added into the endothelial cell basal medium; after 8 hours, capillary network formations by HUVECs were analyzed. (B) Branched tubes were counted, and the numbers of branched tubes with and without VEGF and VEGF + SNS-032 treatment were compared. Bars = SD (n = 3). *P < .001, significantly different from untreated control (0 µM SNS-032) or from VEGF + SNS-032 treatment. (C) Cells were seeded onto a Biocoat (Bedford, MA) (PET) membrane in growth factor-reduced endothelial cell medium, treated with SNS-032 followed by the addition of rhVEGF (100 ng/ml). (D) After 8 hours, migrated cells were counted, and statistical analysis was performed. Bars = SD. **P < .001, compared with untreated control. *P < .05, compared with the sample treated with VEGF.