In this study, we found that acetyl-11-keto-β-boswellic acid (AKBA) was a potent angiogenesis inhibitor and inhibited multiple steps of VEGF-mediated tumor angiogenesis, including cell proliferation, migration, invasion, and tube formation. As evidenced in our xenograft human prostate tumor mouse model experiments, tumor growth was significantly inhibited when AKBA antagonized tumor angiogenesis.
VEGF receptor inhibitors are a promising class of cancer treatment drugs (6
). VEGF is the primary and the most potent inducer of angiogenesis. VEGF activates cellular signaling pathways by binding to its receptor tyrosine kinase, which promotes several events required for angiogenesis, including endothelial cell survival, proliferation, and migration and vascular permeability (2
). Currently, over 20 agents with antiangiogenic properties, including a VEGF-neutralizing antibody, soluble receptors, receptor antagonists, and tyrosine kinase inhibitors, are either already approved for cancer treatment or undergoing clinical (phase I–III) studies (2
). VEGF signaling events relevant to tumor growth and angiogenesis are mainly mediated by VEGFR2 (8
). In the present study, we found that a half-maximum inhibitory concentration of 1.68 μMol/L AKBA significantly blocked VEGFR2’s kinase activity, making AKBA a potent VEGFR2 inhibitor. Previous study also reported that AKBA inhibited basic fibroblast growth factor-induced signaling responses (32
), suggesting that AKBA might be a broad receptor tyrosine kinase inhibitor.
In clinical trials, antiangiogenic therapy with VEGF antagonists has so far produced disappointing results (33
). Successful antiangiogenic therapy may require the simultaneous blockade of signaling pathways downstream from multiple proangiogenic factor receptors (34
). Previous studies have shown that the Src family kinase is substantially involved in VEGF-induced angiogenesis in vitro
and in vivo
). By interacting with its downstream molecule, FAK, Src regulates cell motility (37
) and vascular permeability (9
). In the current study, we found that a low concentration of AKBA (5 μMol/L) effectively inhibited VEGF-triggered Src and FAK activation in endothelial cells.
Recently, AKT/mTOR/p70S6K signaling has been identified as a novel, functional mediator in angiogenesis (10
). In the current study, we found that AKBA significantly decreased the phosphorylation of mTOR and p70S6K, and its upstream kinase, AKT and ERK, indicating that AKBA suppresses tumor angiogenesis by inhibiting VEGFR2 and blocking its multiple downstream signaling components, thereby suppressing endothelial cell migration, proliferation, and survival (). Interestingly, the mTOR/p70S6K pathway has been found to regulate the expression of proangiogenic factors such as interleukin-8 and VEGF in various human carcinomas by regulating hypoxia-inducible factor-1 α expression at the translational level (38
), making the pathway a potential target for anticancer therapy. Therefore, AKBA may suppress tumor angiogenesis by inducing tumor cells to produce fewer proangiogenic molecules.
In the present studies, we demonstrate that 5 μMol/L AKBA was sufficient to inhibit VEGF-induced angiogenic responses in in vitro
and ex vivo
angiogenesis assays while 10 μMol/L AKBA completely blocked capillary-like structure formation and mircovessel sprouting. Our data indicate that AKBA’s anti-angiogenic activity inhibits tumor growth in vivo
much earlier than its cytotoxic effects on tumor cells. Furthermore, we found that AKBA induced endothelial cell apoptosis through a caspase-dependent pathway, which suggests that AKBA inhibits endothelial cell survival not only by blocking VEGFR2 but also by blocking certain extrinsic death receptors on the cell surface or triggering the apoptotic cascade of the intrinsic mitochondrial pathway, as suggested by previous studies that higher concentrations of AKBA (30–50 μMol/L) directly interact with IκB kinase (13
) to suppress nuclear factor-κB–regulated gene expression (14
In conclusion, we found that AKBA potently inhibited tumor growth and angiogenesis by targeting VEGFR2 activation and mTOR signaling pathways, suggesting that AKBA has a potential role in cancer therapy.