BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition

Abstract

Beyond its classical role as apoptosis inhibitor, bcl-2 protein promotes tumor angiogenesis and the removal of N-terminal bcl-2 homology (BH4) domain abrogates bcl-2-induced hypoxia-inducible factor 1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in hypoxic cancer cells. Using M14 human melanoma cell line and its derivative clones stably overexpressing bcl-2 wild-type or deleted of its BH4 domain, we found that conditioned media (CM) from cells expressing BH4-deleted bcl-2 protein showed a reduced capability to increase in vitro human endothelial cells proliferation and differentiation, and in vivo neovascularization compared with CM from cells overexpressing wild-type bcl-2. Moreover, xenografts derived from cells expressing bcl-2 lacking BH4 domain showed a reduction of metastatic potential compared with tumors derived from wild-type bcl-2 transfectants injection. Stably expressing the Flag-tagged N-terminal sequence of bcl-2 protein, encompassing BH4 domain, we found that this domain is sufficient to enhance the proangiogenic HIF-1/VEGF axis under hypoxic condition. Indeed, lacking of BH4 domain abolishes the interaction between bcl-2 and HIF-1α proteins and the capability of exogenous bcl-2 protein to localize in the nucleus. Moreover, when endoplasmic reticulum-targeted bcl-2 protein is overexpressed in cells, this protein lost the capability to synergize with hypoxia to induce the proangiogenic HIF-1/VEGF axis as shown by wild-type bcl-2 protein. These results demonstrate that BH4 domain of bcl-2 is required for the ability of this protein to increase tumor angiogenesis and progression and indicate that bcl-2 nuclear localization may be required for bcl-2-mediated induction of HIF-1/VEGF axis.

Caspase-8 contributes to angiogenesis and chemotherapy resistance in glioblastoma

“…Human umbilical endothelial cells (HUVEC; PromoCell GmbH, Heidelberg, Germany) were cultured as previously reported (Gabellini et al, 2013). …”

“…The negative and positive controls contained heparin alone or heparin plus VEGF (60 ng/mice; R&D Systems, Minneapolis, MN), respectively. After 5 days, the angiogenic response was evaluated by macroscopic analysis at autopsy, and by measurement of the hemoglobin (Hb) content in the pellet of matrigel as previously reported (Gabellini et al, 2013). The values were expressed as optical density (OD at 540 nm)/100 mg of matrigel.…”

“…In vitro HUVEC cell proliferation was evaluated by a colorimetric assay at the end of treatment as described previously (Gabellini et al, 2013). Endothelial capillary tube-like network formation was assessed using Matrigel as described previously (Gabellini et al, 2013).…”

BH4 domain of Bcl-2 as a novel target for cancer therapy

“…Bcl-2 promotes tumor angiogenesis and BH4 deletion abrogates hypoxia-inducible factor (HIF)-1-mediated vascular endothelial growth factor (VEGF) expression in hypoxic tumor cells [27]. Xenografts derived from cells expressing ΔBH4 Bcl-2 showed a reduction of metastatic potential compared with those derived from cells expressing wild-type Bcl-2 [26]. A summary of proteins including those important ones highlighted above associated with the Bcl-2 BH4 domain and their functions is provided in Table 1.…”

Affinity purification-mass spectrometry analysis of bcl-2 interactome identified SLIRP as a novel interacting protein

“…In this context, we demonstrated that removal of or mutations at the BH4 domain abrogate the ability of bcl-2 to induce Vascular Endothelial Growth Factor expression and transcriptional activity, 12 reduce the interaction between bcl-2 and Hypoxia Inducible Factor-1α proteins and the capability of exogenous bcl-2 protein to localize in the nucleus 13 and mediate inhibition of autophagy.…”