Anti-Angiogenic Activity of Bortezomib Is blocked by a 50–60 KDa Protein Secreted by Various tumor Cell Lines

Background: Bortezomib is a reversible proteasome inhibitor displaying significant clinical activity in the treatment of multiple myeloma. Apart from direct antitumor cell activity via inhibition of the nuclear factor kappa B pathway in myeloma cells additional anti-angiogenic effects have been reported.

Aims: This study aimed to analyze the cellular effects and molecular targets of bortezomib in endothelial cells. In particular, we wanted to assess the its effects on proliferating as well as quiescent endothelial cells in vitro. Anti-angiogenic activity in vivo was studied in the chicken chorioallantoic membrane (CAM) model using tumor xenografts.

Methods: Cell viablility of human umbilical vein endothelial cells (HUVEC) and endothelial colony forming cells (ECFC) was determined by flow cytometry; DNA synthesis was measured by BrDU-incorporation. All experiments were performed on mitotic and growth-arrested cells, respectively. Proteins involved in cell cycle arrest and apoptosis were analysed by Western Blots and antibody microarrays. Anti-angiogenic effects in vivo were studied in the chorioallantoic membrane (CAM) assay and the B16F10 xenograft model. Secretome analysis of bortezomib-resistant cells was performed by size exclusion chromatography (SEC), 2D gel electrophoresis (2D-PAGE) and mass spectroscopy (MS, Maldi-TOF).

Results: In proliferating endothelial cells, bortezomib significantly reduced cell viability and proliferation in a dose-dependent fashion with an EC₅₀ of 5 ng/mL. This is about ten times lower than the EC₅₀ determined for established multiple myeloma cell lines (OPM-2, LP1 and RPMI 8226). A dose-dependent increase of cell cycle arrest was accompanied by upregulation of the cycline-dependent kinase inhibitors p21^CIP1, p27 ^KIP1 and the tumor suppressor p53. Bortezomib at higher concentrations induced apoptosis only in proliferating endothelial cells by induction of the pro-apoptotic Bok and Noxa proteins. Vessel formation in the CAM-assay was strongly inhibited by bortezomib. In contrast,, growth and vascularization of melanoma (B16F10) xenografts in the CAM assay was not inhibited by bortezomib. A protein secreted spontaneously by melanoma cells neutralized the antiangiogenic action of bortezomib. By size exclusion chromatography (SEC) and proteasome activity assays a 50–60 KDa protein could be isolated and identified to cause this activity. Besides melanoma cells a variety of other cell lines derived from soldi tumors, such as the colorectal carcinoma cells HRT18 and prostate carcinoma cells PC3 were found to release this protein constitutively at high concentrations. Mass spectrometry data on this protein will be presented at the meeting.

Conclusions: Besides its direct anti-myeloma activity by suppressing the nuclear factor kappa B pathway in myeloma cells bortezomib induces apoptosis in proliferating endothelial cells via the p53 pathway and its downstream targets Noxa and Bok, and inhibits angiogenesis in vivo.. Unexpetedly, we observed many solid tumor cell lines to release a soluble protein that can effectively block the antiangiogenic activity of bortezomib in vivo..