Figure 5

HDACI and IFNα co-operatively inhibit endothelial cell functions, and pro-angiogenic gene expression in cancer cells under hypoxic conditions in vitro. A. Human umbilical vein endothelial cells (HUVECs) were treated with control (Cont), 0.1 μM TSA and/or 500 IU/ml IFNα for 18 hours. Cell viability was evaluated with the Alamar blue assay. B. HUVECs were plated in BD Biosciences Fluroblok chambers and treated with control, 0.1 μM TSA and/or 500 IU/ml IFNα for 22 hours. Cells were stained with Cell Tracker Green CMFDA, migrated through chamber filters toward the chemo-attractant VEGF, and then quantified and expressed as optical density (OD) absorbance units. C. HUVECs were plated into BD BioCoat growth factor-reduced matrigel invasion chambers and treated with control, 0.1 μM TSA and/or 500 IU/ml IFNα for 18 hours. Cells which invaded through the Matrigel were fixed, stained with a Diff Quick staining kit, photographed and then quantified. D. HUVECs were plated onto growth factor-reduced Matrigel in 24 well plates and treated with control, 0.1 μM TSA and/or 500 IU/ml IFNα for 18 hours. Vascular sprouting was quantified by counting the numbers of complete branches per branching point. E. Neuroblastoma BE(2)-C cells were treated with control, 0.02 μM TSA and/or 500 IU/ml IFNα for 72 hours under hypoxic (1% O₂) conditions. RNA was extracted and subjected to independent semi-competitive RT-PCR analyses using trans-intron PCR primers, together with primers for the house-keeping gene β-2 microglobulin (β2M). Representative gels for each gene at the 72 hour time point were shown, and fold induction of a target gene by treatment was calculated by ascribing the ratio between the level of expression of a target gene and that of β2M as 1.0 for control treated samples. * p < 0.05, ** p < 0.01, *** p < 0.001.