Additive/synergistic efficacy of zoledronic acid and LBH589 in the spheroid-based cellular angiogenesis assay

Abstract

e14627 Background: The compounds Zoledronic acid (ZA) and LBH589 (histon deactetylase (HDAC) inhibitor) have shown in previous experiments antitumoral and antiangiogenic effects. Aim of this study was to determine their ability to inhibit VEGF-A induced endothelial cell (EC) sprouting and to observe potential additive/synergistic effects of the combination ZA and LBH589 in the spheroid-based cellular 3D angiogenesis assay. Methods: HUVEC spheroids were embedded in a 3D collagen gel, stimulated with VEGF-A [25 ng/ml] and treated for 24h with different concentrations of ZA alone or in combination with LBH589. Sprouting intensity of EC spheroids was quantitated by an image analysis system determining the cumulative sprout length per spheroid using an inverted microscope and digital imaging software. The mean of the cumulative sprout length of 10 randomly selected spheroids was analyzed as an individual data point. Results: Before testing the compounds in combination the single IC50 values for ZA (42 μM) and LBH589 (2.9 μM) were determined. The IC50 of ZA has to be considered with caution because of the low inhibitory effect which did not reach basal sprouting levels. In the first combination experiment a dilution series of ZA was tested in combination with a constant LBH589 concentration [2.9 μM, IC50 value as determined previously]. The combination of ZA with LBH589 was ∼330 fold more effective in the inhibition of EC sprouting than ZA alone [0.58 μM compared to 190 μM]. However, the sprouting intensity in the combination of LBH589 and ZA was very low. Therefore the experiment was repeated with a dilution series of ZA in combination with a constant LBH589 concentration that showed no inhibition of EC sprouting previously (1 μM). The second combination experiment revealed IC50 values of 1300 μM for ZA alone and 1.5 μM in combination with LBH589 [1 μM]. The combination LBH589 and ZA is again ∼870 fold more effective in the inhibition of EC sprouting than ZA alone. Conclusions: Our results indicate a very strong synergistic effect of the combination ZA and LBH589 in the spheroid-based angiogenesis assay. No significant financial relationships to disclose.