In previous studies, we have demonstrated that the isoflavonoid genistein is an angiogenesis inhibitor . In the present study, we have screened a number of hitherto untested isoflavonoids using inhibition of EC proliferation as an indicator of possible anti-angiogenic activity. Only, 6-ME inhibited EC proliferation with an IC50 comparable to that of genistein or the flavonoid Luteolin (around 5 μM). Interestingly, 6-ME inhibited both VEGF- and FGF2-induced proliferation of endothelial cells, whereas it had no effect on the serum-induced proliferation of four cancer cell lines. Apparently, 6-ME exhibits certain selectivity towards inhibition of EC proliferation. 6-ME is an isoflavan metabolite that has been identified in human urine following soy or red clover supplementation [20, 21, 24, 25]. However, only trace amounts of 6-ME are excreted in human urine. 6-ME originates from glycitein; the amount of the original substance is low in soy compared to daidzein and genistein, that may explain the low amounts of the metabolite .
Though 6-ME inhibited both VEGF- and FGF2-induced proliferation of ECs, we decided to study the effects of 6-ME only on VEGF-dependent EC responses, because VEGF is the most important mediator of tumor angiogenesis. Indeed, cancer cells over-express VEGF either following hypoxia or as a consequence of the genetic changes of cancer such as mutations of oncogenes and tumor suppressor genes . In fact, endothelial cells adjacent to the tumor vessels over-express VEGFR-1 and −2  establishing an angiogenic loop.
To discriminate whether the decreased number of cells in the proliferation assay derived from a truly cytostatic effect (cell cycle inhibition) of 6-ME or was the result of cytotoxicity/apoptosis, we further investigated the effect of the compound on the VEGF-induced survival of endothelial cells. 6-ME, administered alone to endothelial cell cultures did not increase the percentage of apoptotic cells compared to solvent-treated cultures. Moreover, 6-ME administered together with VEGF did not have any influence on the VEGF-induced rescue of apoptosis. This result, in other words, indicated that 6-ME did not inhibit the EC survival signaling cascades emanating from the active VEGF/VEGFR2 complex. In confirmation, 6-ME did not inhibit VEGF-induced phosphorylation of AKT, an important component of the PI3K signaling pathway, the main anti-apoptotic cascade in most cells.
Having established that 6-ME inhibits endothelial cell proliferation, we investigated whether 6-ME could inhibit other angiogenic responses of endothelial cells. Indeed, angiogenesis is a complex process that involves many partial steps such as production of proteolytic enzymes that degrade the basement membrane, migration, proliferation, tube formation, generation of basement membrane and recruitment of mural cells . Several of these processes including tube formation can be reconstituted in vitro using 3D cultures on Matrigel, a basement membrane matrix from Engelbreth-Holm-Swarm mouse tumors . Indeed, human umbilical vein endothelial cells form capillary-like structures on Matrigel substrates. 6-ME, even at high doses, did not exhibit any effect on the Matrigel assay. Migration is a critical angiogenic response of ECs allowing them to reach the membrane breach for invasion to the extracellular space. VEGF is a prime regulator of EC migration. VEGF-induced phosphorylation of Tyr1214 of VEGFR2 activates SAPK2/p38  leading to VEGF-induced actin reorganization and migration of ECs via phosphorylation of heat-shock protein-27 (HSP27)  and LIM-kinase 1 (LIMK1) . 6-ME did not exhibit any inhibitory effect on VEGF-induced migration of ECs and did not inhibit phosphorylation of p38 by the VEGF/VEGFR2 complex.
It appeared, therefore, that the main target of 6-ME was EC proliferation. Interestingly, 6-ME inhibited both VEGF- and FGF2-induced EC proliferation. In humans, upon VEGF-A binding, phosphorylation of VEGFR2 on Tyr1175 leads to recruitment of PLCγ, which in turn, via activation of PKC, phosphorylates MEK1/2 and eventually mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase-1/2 (ERK1/2) leading to proliferation of ECs . Such activation of MAPKs by VEGF is different from classic Ras-Raf-MEK-MAPK pathway, which is used by most receptor tyrosine kinases including FGF2 [13, 14]. Nevertheless, it has been shown that PKC-dependent activation of MEK1/2 requires a Ras-Raf complex formation . This PKC/Ras-Raf functional interaction is not so well understood and might include other hitherto unidentified components. PKC and Ras-Raf are the points where the VEGF and FGF2 cascades arrive just before the first downstream common effector, MEK1/2, as far as activation of MAPK is concerned. The finding that 6-ME inhibits both the VEGF and FGF2-induced EC proliferation as well as MEK1/2 phosphorylation suggests that the PKC/Ras-Raf interaction is the only point where 6-ME could target both pathways with one activity. Otherwise, 6-ME would need two activities targeting two different components upstream to MEK1/2, one for each pathway. This is a point that requires future attention.
Thus, inhibition of MEK1/2 and consequently ERK1/2 phophorylation was the sole cardinal effect of 6-ME on the signaling cascade of VEGF in HUVECs; activation of AKT and P38 were unaffected. This mechanism is strikingly different compared to the effects of the flavonoid luteolin on VEGF signaling in HUVECs . Luteolin, inhibited the PI3K/AKT pathway abolishing downstream survival signals, but also enhanced the pro-apoptotic MKK3/MKK6/p38 pathway of VEGF eliciting a strong apoptotic effect in ECs. Regarding the anti-mitotic activity, luteolin inhibited VEGF-induced phosphorylation of p70 S6K, a downstream effector of PI3K responsible for G1 progression. Surprisingly, luteolin did not affect VEGF-induced phosphorylation of ERK1/2 MAP kinases. Thus, two representatives (luteolin and 6-ME) of closely related isomeric compound classes (flavonoids and Isoflavonoids) exhibited entirely different molecular targets concerning the VEGF-dependent signaling cascades in HUVECs. Perhaps, the fact that these compounds are competitive inhibitors of ATP binding  allows them to target a variety of tyrosine and serine kinases [31, 32].
6-ME was eventually tested in animal models. For this purpose, we used a murine tumor xenograft model utilizing A-431 cells, a human epidermoid carcinoma cell line that produces VEGF . 6-ME administered orally in this model was devoid of any effect. The experimental and control tumors did not show any difference in their average volumes). We postulated that low bioavailability is the reason for the lack of effect. Indeed, estimation of the free, conjugated and total amounts of 6-ME in the plasma of the mice revealed that the maximum concentration achieved was 1.23 μM, a value below the in vitro IC50 of the compound (around 5-10μM). Several factors contribute to the bioavailability including absorption, distribution, metabolism and elimination. There are no extensive studies on these issues concerning isoflavonoids. However, the studies so far [20, 21] anticipate that isoflavones are rather poorly bioavailable. In a study in human adults, consumption of 50 mg of isoflavones per day yielded plasma concentrations ranging from 0.2-3.2 μmol/L. Indeed, following consumption of food rich in soy or red clover only traces of 6-ME were detected in soy human urine . The low biovailability excludes any significant contribution of 6-ME to the protective function of plant-based diets on cancer incidence.
However, biovailable analogs of 6-ME could be used therapeutically to target tumor angiogenesis. Alternatively, 6-ME could be loaded in nanoparticles targeted to ECs, where they could be endocytosed and eventually release their cargo. Indeed, when injected directly to the xenograft tumors, to bypass its low biovailability, 6-ME suppressed tumor vascularization resulting to a statistically significant decrease in the volumes of murine A-431 xenograft tumors. Thus, 6-ME acquires the potential to be developed into a therapeutic anti-cancer agent. In this capacity, 6-ME or 6-ME analogs have two very important and unique properties. 6-ME inhibits only VEGF-induced MEK1/2 activation inhibiting exclusively EC proliferation without influencing VEGF-induced survival. Thus, one can anticipate that it targets only dividing ECs in the vicinity of tumors, without affecting the survival of the quiescent normal endothelium. Moreover, it inhibits also FGF2, which an alternative angiogenic factor expressed when ECs develop resistance (Angiogenic Redundancy)  against current anti-VEGF treatments . This is a very important issue in the anti-VEGF treatments.
In conclusion, 6-ME, a natural isoflavone found also in humans, inhibits VEGF- and FGF2-induced proliferation of ECs. The molecular target of 6-ME is upstream of MEK1/2 inhibiting phosphorylation of MEK1/2 and ERK1/2 kinases that are important components of the mitogenic MAPK pathway. 6-ME does not affect the PI3K/AKt pathway, thereby not affecting VEGF-dependent survival of ECs. Oral administration in mice fails to achieve sufficient plasma concentrations to inhibit neovascularization and growth of xenograft tumors in mice. However, direct injections of 6-ME to the xenograft tumors, to bypass its low biovailability, suppress tumor vascularization resulting to a statistically significant decrease in the volumes of murine A-431 xenograft tumors. Concomitant inhibition of VEGF- and FGF2-induced EC proliferation and targeting only dividing ECs without affecting the survival of ECs are two properties rendering 6-ME as an attractive molecule for the development of a novel anti-angiogenic intervention in cancer treatment.