Table 1.
Collection of clinical drugs screened in binary combinations for synergistic inhibition of HUVEC proliferation.
Table 2.
Distribution of Drug Classes.
Figure 1.
Cyclosporin A and itraconazole are synergistic inhibitors of HUVEC proliferation.
Chemical structures of cyclosporin A (A) and itraconazole (B). The CI plot for CsA+Ita indicated CI in the synergistic range across a wide range of effect levels (C). The IC50 dose of both Ita (D) and CsA (E) is significantly reduced in combination. This is also reflected in the dose reduction index (F). Bars, standard error of the mean (SEM). n = 8; * p<0.1; ** p<0.05; # p<0.005; ## p<0.0001.
Table 3.
Combination Indices and p-values versus effect level for CsA and Ita combination treatment.
Figure 2.
The potency of cyclosporin A and itraconazole against HUVEC proliferation is independent of VEGF signaling.
(A) The proliferation of HUVEC grown in standard media in the presence of the indicated doses of CsA + Ita or sunitinib was determined. (B) HUVEC were seeded in either standard media (standard), or basal media (EBM-2+2% FBS), which was changed the next day to either fresh basal media with 100 ng/mL VEGF (basal + VEGF) or VEGF vehicle (basal), or standard media (basal + standard). The cells were then treated with drug vehicle for 24 h and the incorporation of [3H]-thymidine after a 6 h pulse was determined. The dose response curves CsA + Ita and sunitinib were determined in basal media (C), basal + standard media (D), and basal media + VEGF (E). (F) The dose-response curve for VEGF-dependent proliferation (basal media + VEGF proliferation minus basal media proliferation) was also determined. Bars = SEM, n = 3.
Figure 3.
Cyclosporin A and itraconazole synergistically inhibit endothelial cell tube formation and sprout formation.
(A) HUVEC were grown on a matrigel coated plates in the presence of vehicle only (DMSO), CsA (8 µM), Ita (800 nM), or a combination of the same CsA and Ita doses. The tube networks were stained with Calcein AM. Total tube length (B), number of junctions (C), and total network size (D) were calculated using AngioQuant (n = 3). (E) HUVEC spheres were embedded in a collagen matrix in the presence of basal media (EGM-2+2% FBS) (−GF), or complete media supplemented with VEGF165 and bFGF (+GF). The spheroids were treated with drug as in (A) (n = 4; scale bar = 50 µm). Insets are magnified views of the tube architecture. Cumulative sprout length was quantified (F). The non-interaction value is denoted by the horizontal line in B–D and F. Error bars, SEM; * p<0.1; ** p<0.05.
Figure 4.
The combination of cyclosporin A and itraconazole does not cause general toxicity.
(A) HUVEC and HFF were treated with a combination of CsA and Ita for 30 hours. Cell viability was measured by Calcein AM staining (n = 3). (B) Proliferation of HeLa and Jurkat T cells treated with a combination of CsA and Ita was compared to that of HUVEC after a 30 hour incubation (n = 3). Total combined drug dose shown (the ratio of CsA to Ita was 10∶1 as in other experiments). Bars, SEM; ** p<0.05; dashed lines indicate 95% confidence bands. (C) The viability of HeLa was determined as in (A) and Jurkat viability was determined after a 24 h drug dose followed by a 6 h incubation with Alamar blue. Bars = SEM; n = 3.