Rolipram potentiates bevacizumab-induced cell death in human glioblastoma stem-like cells

Aims: Glioblastoma cancer stem-like cells (GCSCs) promote themselves proliferation by secreting the vascular endothelial growth factor A (VEGF_A) in an autocrine manner, positively regulated by phosphodiesterase IV (PDE4). In the current study, we investigated the putative cytotoxic effect of bevacizumab, a VEGF_A blocker, alone and in combination with a specific inhibitor of PDE4 called rolipram on GCSCs isolated from human surgical tumor specimen with a focus on PI3K/AKT pathway.

Main methods: CD133+/CD15+ GCSCs were characterized by flow cytometry and expanded in a serum-free primary culture system. The cell survival, apoptosis, and protein expression values were measured using MTT assay, TUNEL staining and western blot, successively. Intracellular cAMP and free secreted VEGF_A levels were assessed by cAMP enzyme immunoassay and ELISA, respectively.

Key findings: Bevacizumab suppressed GCSCs survival with IC₅₀~6.5μg/ml and enhanced the levels of apoptosis, p53 and cleaved-caspase3 along with a decrease in free VEGF_A levels and ERKs activation. However, there was no significant modulation of AKT phosphorylation on serine 473, the intracellular PDE4A, VEGF_A and cAMP levels. More cytotoxicity in co-treated cells coupled with a more substantial decline in the free VEGF_A levels and a greater increase in the quantities of p53 and cleaved-caspase3 compared to those treated with bevacizumab alone. Co-treatment reduced phospho-AKT, endogenous VEGF_A and PDE4A values but elevated cAMP levels.

Significance: This study highlighted a booster cytotoxic effect of combined rolipram and bevacizumab treatment on the GCSCs primary culture, suggesting that this approach is warranted in treatment of GBMs overexpressing VEGF_A and PDE4A.