Abstract
BackgroundIncreased consumption of plant-based diets has been linked to the presence of certain phytochemicals, including polyphenols such as flavonoids. Several of these compounds exert their protective effect via inhibition of tumor angiogenesis. Identification of additional phytochemicals with potential antiangiogenic activity is important not only for understanding the mechanism of the preventive effect, but also for developing novel therapeutic interventions.ResultsIn an attempt to identify phytochemicals contributing to the well-documented preventive effect of plant-based diets on cancer incidence and mortality, we have screened a set of hitherto untested phytoestrogen metabolites concerning their anti-angiogenic effect, using endothelial cell proliferation as an end point. Here, we show that a novel phytoestrogen, 6-methoxyequol (6-ME), inhibited VEGF-induced proliferation of human umbilical vein endothelial cells (HUVE) cells, whereas VEGF-induced migration and survival of HUVE cells remained unaffected. In addition, 6-ME inhibited FGF-2-induced proliferation of bovine brain capillary endothelial (BBCE) cells. In line with its role in cell proliferation, 6-ME inhibited VEGF-induced phosphorylation of ERK1/2 MAPK, the key cascade responsible for VEGF-induced proliferation of endothelial cells. In this context, 6-ME inhibited in a dose dependent manner the phosphorylation of MEK1/2, the only known upstream activator of ERK1/2. 6-ME did not alter VEGF-induced phosphorylation of p38 MAPK or AKT, compatible with the lack of effect on VEGF-induced migration and survival of endothelial cells. Peri-tumor injection of 6-ME in A-431 xenograft tumors resulted in reduced tumor growth with suppressed neovasularization compared to vehicle controls (P < 0.01).Conclusions6-ME inhibits VEGF- and FGF2-induced proliferation of ECs by targeting the phosphorylation of MEK1/2 and it downstream substrate ERK1/2, both key components of the mitogenic MAPK pathway. Injection of 6-ME in mouse A-431 xenograft tumors results to tumors with decreased neovascularization and reduced tumor volume suggesting that 6-ME may be developed to a novel anti-angiogenic agent in cancer treatment.
Highlights
Physiological angiogenesis is a strictly regulated finetuned process
Vascular endothelial growth factor A (VEGFA), referred to as VEGF, represents a critical inducer of tumor angiogenesis and is the first-choice target of antiangiogenic therapies tested in clinical trials [1]
Endothelial cell proliferation is regulated by activation of PLCγ, a SH2-domain-containing molecule that interacts directly with activated VEGFR-2 and mediates the phosphorylation of mitogen-activated protein kinase (MAPK)/ extracellular – signal-regulated kinase 1/2 (ERK1/2) cascade [5]
Summary
Screening of flavonoids revealed that 6-methoxyequol is a specific inhibitor of endothelial cell proliferation exhibiting minor anti-mitotic effect on tumor cells We screened a selection of isoflavonoids on endothelial cell proliferation seeking to identify additional structures with antiangiogenic activity compared to that of genistein. 6-methoxyequol inhibits activation of the MEK1/2-ERK1/2 pathway by VEGF Having established that 6-ME inhibits only endothelial cell proliferation without affecting survival, migration and tube formation, we sought mechanistic confirmation of these findings. 6-ME did not affect VEGFinduced phosphorylation of p38 MAPK (Figure 2D, lower panel), a signaling cascade that mediates the induction of endothelial cell migration by VEGF [12]. These results, together with the fact that 6-ME does not inhibit PLC-γ activation, as VEGF-induced calcium release in not affected (Additional file: Figure 4), exclude the kinase activity of VEGFR2/KDR of being the target of 6-ME. At day 8 mice survival was 78% in the 6- methoxyequol group and 44% in the vehicle group
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