Anti-angiogenic and vascular disrupting effects of C9, a new microtubule-depolymerizing agent

Abstract

Background and purpose: The critical role of blood supply in the growth of solid tumours makes blood vessels an ideal target for anti-tumour drug discovery. The anti-angiogenic and vascular disrupting activities of C9, a newly synthesized microtubule-depolymerizing agent, were investigated with several in vitro and in vivo models. Possible mechanisms involved in its activity were also assessed.

Experimental approach: Microtubule-depolymerizing actions were assessed by surface plasmon resonance binding, competitive inhibition and cytoskeleton immunofluorescence. Anti-angiogenic and vascular disrupting activities were tested on proliferation, migration, tube formation with human umbilical vein endothelial cells, and in rat aortic ring, chick chorioallantoic membrane and Matrigel plug assays. Western blots and Rho activation assays were employed to examine the role of Raf-MEK-ERK (mitogen-activated ERK kinase, extracellular signal-regulated kinase) and Rho/Rho kinase signalling.

Key results: C9 inhibited proliferation, migration and tube formation of endothelial cells and inhibited angiogenesis in aortic ring and chick chorioallantoic membrane assays. C9 induced disassembly of microtubules in endothelial cells and down-regulated Raf-MEK-ERK signalling activated by pro-angiogenic factors. In addition, C9 disrupted capillary-like networks and newly formed vessels in vitro and rapidly decreased perfusion of neovasculature in vivo. Endothelial cell contraction and membrane blebbing induced by C9 in neovasculature was dependent on the Rho/Rho kinase pathway.

Conclusions and implications: Anti-angiogenic and vascular disruption by C9 was associated with changes in morphology and function of endothelial cells, involving the Raf-MEK-ERK and Rho/Rho kinase signalling pathways. These findings strongly suggest that C9 is a new microtubule-binding agent that could effectively target tumour vasculature.

Figure 1

Chemical structure of C9.

Figure 2

C9 is a microtubule-binding agent. (A) Effects of C9 on the endothelial cell cytoskeleton. Human umbilical vein endothelial cells were seeded onto coverslips coated with fibronectin and incubated overnight prior to treatment (1, 4 and 8 h, with or without 0.5 µmol·L⁻¹ C9). Cells were then fixed and stained for microtubules (green) and F-actin (red); fluorescence images were viewed by using a Leica TCS confocal microscope (43×). (B) Effects of C9 on the colchicine-binding site. Tubulin (3 µmol·L⁻¹) was incubated with colchicine to form tubulin-colchicine binding complexes prior to treatment (37°C, 60 min) with C9 (5, 10, 15, 25 and 50 µmol·L⁻¹) or vinblastine (VLB; 5, 10, 15, 25 and 50 µmol·L⁻¹). (C) Effects of C9 on the Vinca alkaloid site. Tubulin (3 µmol·L⁻¹) was incubated (37°C, 45 min) with various concentrations of C9 (25, 50, 100 and 200 µmol·L⁻¹) or vinblastine (VLB; 25, 50, 100 and 200 µmol·L⁻¹) prior to treatment with BODIPY FL-vinblastine (20 min, 37°C). (D) C9 or colchicine tubulin-binding affinity by surface plasmon resonance assay. C9 (2.9, 4.1, 5.9, 8.4, 12.0, 17.2, 35.0 and 50.0 µmol·L⁻¹) or colchicine (4.1, 5.9, 8.4, 12.0, 17.2, 24.5 and 35.0 µmol·L⁻¹) were prepared as an analyte and then injected into a Biacore 3000 system following tubulin immobilization on the chip.

Figure 3

C9 inhibited angiogenesis. (A) C9 inhibited endothelial cell proliferation. Human umbilical vein endothelial cells were incubated overnight then exposed to different concentrations of C9 (0.03, 0.06, 0.12, 0.25, 0.5, 1.0 and 2.0 µmol·L⁻¹) and cultured for 8, 12, 24, 36 and 48 h. Inhibition of proliferation was measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazalium bromide] assay. (B) Inhibitory effect of C9 on cell migration, tube formation, aortic ring and chick chorioallantoic membrane assays. Representative images and summary data (bar graphs on right hand side) showing inhibition of migration (a and b), tube formation (c and d), angiogenesis in aortic ring (e and f) and chick chorioallantoic membrane assays (g and h). Cells were treated with vehicle (a, c, e and g) or 0.5 µmol·L⁻¹ C9 (b, d and f) or 5 nmol per egg C9 (h) (see Methods); magnification: 100× (a, b, c and d), 40× (e and f), 1.6× (g and h). Results are expressed as mean ± SD; n= 3; *P < 0.01 versus control, **P < 0.005 versus control.

Figure 4

C9 down-regulated the Raf-MEK-ERK signalling pathway induced by VEGF and bFGF. Serum-starved cells were treated with different concentrations of C9 for 2 h, followed by the addition of 50 ng·mL⁻¹ VEGF or 25 ng·mL⁻¹ bFGF (10 min), then assayed by Western blotting (n= 3). bFGF, basic fibroblast growth factor; ERK, extracellular signal-regulated kinase; FGFR1, FGF receptor 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KDR, receptor for vascular endothelial growth factor; MEK, mitogen-activated ERK kinase; VEGF, vascular endothelial growth factor.

Figure 5

Vascular targeting activity of C9. (A) Neovasculature network disruption assay: cells were plated on Matrigel and allowed to form capillary-like networks for 24 h. These networks were then exposed to C9 (0.5 µmol·L⁻¹, 12 h). C9 treatment resulted in a time-dependent inhibition. Magnification, 100×. (B) Aortic ring assay; aortic rings were cultured for 7 days and then treated with C9 (0.5 µmol·L⁻¹) for 12 h. Magnification, 200×. (C) In vivo Matrigel plug assay. Fluorescence microscopy image of FITC-dextran perfusing the neovasculature before (0 h) and 1 and 3 h following C9 treatment (200 mg·kg⁻¹, i.p.), as described in Methods. The clearly visible blood vessels at 0 h were almost totally disrupted 3 h after i.p. injection of C9. Magnification, 200×.

Figure 6

Microtubule disassembly contributed to the vascular disrupting activity of C9. (A) The change in endothelial cell morphology induced by C9 was attenuated by taxol and Y27632. Endothelial cells were pretreated with vehicle control (30 min), taxol (0.2 µmol·L⁻¹, 30 min) or Y27632 (10 µmol·L⁻¹, 30 min), then incubated with C9 (0.5 µmol·L⁻¹, 1 h). Cells were examined with an inverted microscope (40×) or stained for F-actin (red); fluorescence images were obtained by a fluorescence microscope (43×). (B) Effects of C9 on newly formed vessels were antagonized by taxol and Y27632. HUVECs were seeded onto Matrigel to form a functional network, then pretreated with or without taxol (0.2 µmol·L⁻¹, 30 min) or Y27632 (10 µmol·L⁻¹, 30 min) and then exposed to C9 (0.5 µmol·L⁻¹, 12 h); magnification, 100×. (C) C9 activated Rho GTPase and increased the phosphorylation of MLC2, which was antagonized by taxol and Y27632. Serum-starved HUVECs were treated with control or taxol (0.2 µmol·L⁻¹, 30 min) or Y27632 (10 µmol·L⁻¹, 30 min), prior to analysis of Rho activity. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HUVECs, human umbilical vein endothelial cells; MLC2, myosin light chain2.