Cell surface expression of VEGF receptors Although western blot analysis did not show any overall change in expression, to determine if receptor localization was affected by hypoxia or bevacizumab treatment, cell surface localization of VEGFR2 and Neuropilin1 AZD2171 solubility was eval uated by flow cytometry. Inhibitors,Modulators,Libraries VEGFR1 localization was not analyzed, as no suitable antibody Inhibitors,Modulators,Libraries for FACS analysis was available. Although all cell lines showed Neuropilin1 protein ex pression to varying intensities, this did not necessarily translate to cell surface expression, with no detectable expression on H522, HCT 116, HT 29 or KM12. Neuropilin1 was expressed on the cell surface at a high level in one breast tumor cell line, followed by A498. Expression was present to a lesser degree in HOP62 and HS 578 T exhibiting approximately 10 15% of cells with receptors at the cell surface.

In contrast to Neuropilin1, VEGFR2 expression was Inhibitors,Modulators,Libraries more limited on the surface of tumor cells, in line with western blot analysis. As expected endothelial cells showed expression of VEGFR2 and only one tumor cell line, MDA MB 231, with high numbers of VEGFR2 positive cells compared to the other tumor cell lines. The other tumor cell lines that had VEGFR2 pro tein expression, H522, HOP62 and HCT 116, did not show VEGFR2 on their surface with the percentages of positive cells remaining below 10%. Treatment under hypoxia or with bevacizumab did not influence any ob vious change in either Neuropilin1 or VEGFR2 mem brane expression.

Analysis of hypoxic VEGFA induction in tumor cells Activation Inhibitors,Modulators,Libraries of HIF 1 under hypoxia should lead to a var iety of gene expression changes, including induction of VEGFA, which may preferentially trigger specific chan ges in tumor cells. To Inhibitors,Modulators,Libraries this end, cells were incubated under normoxic and hypoxic conditions for 24 hours and total VEGFA mRNA levels were measured by quan titative real time PCR. Most tumor cells reacted to the hypoxic environment with the induction of either VEGFA or GLUT1 mRNA after 24 hours of hypoxia exposure, however to variable degrees within the different tumor entities. Three tumor cell lines had significant induction of VEGFA, which did not exactly match the pattern of GLUT1 mRNA where six cell lines showed significant induction. MDA MB 231 and A498 showed no transcriptional regula tion of the two classical hypoxia inducible genes whereas KM12 and H522 demonstrated induction of only GLUT1.

HS 578 T responded to the hypoxic environment with a 2. 7 fold increase of VEGFA over the normoxic control and 2. 8 fold change for GLUT1. HOP62 showed the highest induction of VEGFA with up to 3 fold along all investigated tumor cell lines. For the two colorectal tumor cell lines HCT 116 and HT 29 VEGFA was upregulated to a similar Nutlin-3 price extent under hypoxic conditions with 2. 5 fold and 2. 4 fold.