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Mechanisms of the antiangiogenic activity by the hop flavonoid xanthohumol: NF-B and Akt as targets

Adriana Albini^*, Raffaella Dell’Eva^*, Roberta Vené^*, Nicoletta Ferrari^*, Donald R. Buhler, Douglas M. Noonan^,1 and Gianfranco Fassina^,||

^* Molecular Oncology Laboratory, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy;
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon, USA;
Dipartimento di Scienze Cliniche e Biologiche, Universita' degli Studi dell'Insubria, Varese, Italy;
Tumor Progression Unit, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy; and
^|| Istituto di Bioimmagini e Fisiologia Molecolare, Sezione di Genova, Consiglio Nazionale delle Ricerche, Genoa, Italy

¹Correspondence: Facoltà di Medicina e Chirurgia, Dipartimento di Scienze Cliniche e Biologiche, Universita' degli Studi dell'Insubria, Via Ottorino Rossi 9, Varese 21100, Italy. E-mail: douglas.noonan{at}uninsubria.it

SPECIFIC AIMS

Xanthohumol (XN) is a flavonoid of the chalcone chemical class isolated from hop ‘cones’, the female inflorescences of the hop plant (Humulus lupulus L.). It is largely used in the brewing industry as a preservative and flavoring agent. Several natural compounds of plant origin and dietary components, have been shown to have cancer chemoprevention and/or therapeutic activities. We have shown that inhibition of angiogenesis is a mechanism by which dietary components prevent cancer, a concept we termed "angioprevention." XN has several properties that suggest it may be an angiopreventive agent. Here we show for the first time that XN can inhibit growth of a vascular tumor in vivo; histopathology indicated that much of this effect was by blocking angiogenesis which was confirmed by in vitro and in vivo analyses. XN represses both the NF-B and Akt pathways in endothelial cells, indicating that they are major targets in the molecular mechanism of XN. Our data provide the basis for clinical tumor prevention trials with this hop extract.

PRINCIPAL FINDINGS

1. XN inhibition of angiogenesis in vivo
The matrigel sponge model is a rapid and quantitative system for measuring tumor angiogenesis in vivo. A cocktail of VEGF, TNF-, and heparin induced an intense angiogenic reaction in matrigel implants. Addition of 5 µM XN to this cocktail in the matrigel significantly inhibited the angiogenic response in vivo (P<0.01, t test) as measured by hemoglobin content (Fig. 1 A).

View larger version (20K): [in this window] [in a new window]   Figure 1. A) Inhibition of angiogenesis in vivo by XN as estimated by the hemoglobin content of the gels. Addition of XN to the gels significantly (**P<0.01, t test) prevented vascularization. Means ±SE are shown. B, C) Preventive oral administration of XN in the drinking water potently and dose-dependently inhibited angiogenesis in vivo that was macroscopically visible (B) and significant when hemoglobin content was quantified (C). Means ±SE are shown (***P<0.001 with respect to positive control, 2-way ANOVA). D) Inhibition of tumor growth in vivo after oral administration of XN. The growth rate of KS-IMM tumors in animals receiving XN was significantly reduced as compared with the tumors in control animals. Differences were statistically significant from day 20 on (**P<0.01, ***P<0.001, 2-way ANOVA, means ±SE are shown). The weights of tumors recovered were significantly smaller (**P=0.0039, t test) for the animals receiving XN than those of tumors from the control animals (inset).

2. Oral administration of XN inhibits angiogenesis in vivo
We then investigated the antiangiogenic activity of XN by oral administration, the preferred route for chemoprevention treatment in humans. We found that administration of XN in the drinking water (starting 3 days before matrigel injection) potently and dose-dependently induced a significant inhibition of angiogenesis in vivo. Doses as low as 2 µM in the drinking water induced a significant prevention of angiogenesis that was further reduced at higher doses (P<0.001 with respect to positive control (0) ANOVA) (Fig. 1B, C ). Histological examination of the pellets indicates a strong reduction of vessel formation in gels implanted in mice receiving XN, with a variable reduction of the cellular infiltrate. Strong angiogenesis inhibition by XN with no adverse effects on animal health parameters was noted at doses as high as 200 µM, indicating low or absent toxicity.

3. Oral administration of XN prevents vascular tumor growth in vivo
We also examined whether XN was able to prevent vascular tumor growth in vivo. The Kaposi's sarcoma cell line KS-IMM forms highly angiogenic tumors when injected s.c. in male nude mice. The growth of tumors in male nude mice receiving 20 µM XN in an angioprevention setting was significantly reduced (Fig. 1D ). No differences were noted in the body weight or general health parameters in the treated animals compared with controls, indicating limited or no toxicity of XN treatment. Tumors removed from the animals receiving XN were significantly smaller than controls as determined by tumor weight (Fig. 1D , inset). Histological analyses indicated that untreated tumors showed extensive areas of vascularization interspersed with occasional zones of necrosis. In tumors from XN-treated animals the areas of necrosis and fibrosis were much more prevalent, often containing vessels.

4. Effects of XN on tumor and endothelial cell growth, survival, and apoptosis
Regarding the mechanism of XN anti-angiogenesis, in vitro XN inhibited the proliferation of both endothelial (HUVEC, human umbilical vascular endothelial cells) and KS-IMM tumor cells at micromolar concentrations in a dose-dependent manner that was maximal at 10 µM or higher but did not cause increased apoptosis. XN had no additive effect on the apoptosis induced by vincristine; on the contrary, 5–15 µM XN was associated with a slight reduction in apoptosis. Using measurement of LDH release, which permits estimation of both cell growth and death, cytostasis with low levels of cytotoxicity were found for 10 µM XN in HUVEC. In contrast, the growth inhibitory effects at 25 µM XN were associated with a high rate (>80%) of cell death, apparently in the absence of DNA fragmentation.

5. XN inhibits migration and invasion of endothelial and tumor cells and the formation of endothelial networks
Migration, invasion, and matrix remodeling are required for genesis of new vessels. XN inhibited endothelial cell chemotaxis and invasion in vitro at concentrations as low as 5 µM, with complete blockage at 10 µM or greater. HUVE cells plated on a matrigel layer spontaneously organized into networks in a process that reflects the final morphogenesis phase of the angiogenic process. XN at 5–10 µM concentrations reduced the formation of these networks. We examined the effects of XN on metalloprotease activities by gelatin zymography and found that MMP-2 (matrix metalloprotease-2) release by HUVEC was inhibited at 25 µM with a reduction evident at 5–10 µM. Addition of XN directly to the digestion buffer had no effect, indicating that XN does not interact directly with MMP-2 but acts on the cells, modulating its production.

6. XN inhibits translocation and activation of NF-B and growth factor-induced Akt phosphorylation
Tumor invasion is influenced by numerous gene products that are regulated by NF-B. This nuclear factor also regulates aspects of inflammation and the expression of a number of chemokines and angiogenic growth factors. Proinflammatory stimulation leads to rapid phosphorylation, ubiquitinylation, and degradation of the inhibitory protein IB, freeing NF-B for translocation to the nucleus to activate transcription of its target genes. We hypothesized that XN could affect the NF-B pathway and we found that in endothelial cells, the antiangiogenic effects of XN correlated with a block of NF-B activation and IB phosphorylation, with a consequent arrest of NF-B translocation to the nucleus (Fig. 2 A, B).

View larger version (32K): [in this window] [in a new window]   Figure 2. The effects of XN on NF-B activation, and IB and Akt phosphorylation. A) ELISA analysis demonstrated treatment with XN reduced the amount of active NF-B both in resting and TNF--stimulated cells. Means ±SE are shown (*P<0.05, t test). B) Western blot analysis shows that XN strongly repressed phosphorylated IB levels in HUVE cells after stimulation with TNF- evident soon after simulation. C) Western blot analysis demonstrated that XN also blocked IGF-induced Akt phosphorylation in HUVE cells.

XN also repressed phosphorylation of endothelial Akt in response to stimulation by IGF-1 (Fig. 2C) , suggesting that XN interferes with the molecular mechanisms of cell migration and survival.

CONCLUSIONS AND SIGNIFICANCE

In parallel with the evolution of prevention in cardiovascular medicine, interest in cancer chemoprevention recently received great attention. Among chemopreventive compounds, natural nontoxic products (or their synthetic analogs), in particular dietary components, have been under scrutiny for their potential to halt or reverse the development or the progression of cancer. Several flavonoids, including chalcones, have been shown to inhibit the proliferation of cancer cells and tumor growth in preclinical studies and are now tested in phase II trials of prevention. Previous studies based on in vitro analyses as a good candidate for chemoprevention.

Our data show that XN is a potent orally available antiangiogenic, antitumoral, chemopreventive agent whose collective properties place it in the realm of angioprevention agents. XN appears to affect several different pathways that lead to efficient inhibition of angiogenesis in vivo.

In vitro, XN inhibited endothelial cell chemotaxis and invasion at concentrations as low as 5 µM. This was associated with reduced formation of endothelial cell networks on matrigel in an in vitro morphogenesis assay. XN inhibited HUVE cell proliferation, and to an even greater extent KS-IMM tumor cell proliferation. The XN doses effective on migration did not induce HUVE or KS cell apoptosis, suggesting that migration and growth are the principal targets for the reduction of angiogenesis and tumor growth observed in vivo. The inhibitory effects on NF-B and Akt pathways could be involved in the effects of XN on endothelial cell mobility (Fig. 3 ).

View larger version (41K): [in this window] [in a new window]   Figure 3. Summary of the effects of XN on endothelial cells. XN is readily absorbed from the diet and has systemic activity. In endothelial cells, XN is able to repress NF-B activation at or upstream of the phosphorylation of IB by IKK, thus preventing IB degradation and NF-B release. XN also represses Akt activation in response to growth factor stimulation of endothelial cells, preventing direct effects of Akt promoting cell survival and migration while enhancing the inhibition of NF-B. These activities lead to endothelial cell quiescence and angiogenesis inhibition, but not to systemic toxicity.

Taken together, our data show that XN is a potent orally available antiangiogenic chemoprevention agent whose mechanism targets endothelial cell migration, invasion, and proliferation. We also provide the first evidence of an antitumor activity of XN and show that this appears to be due to its angiopreventive properties. The levels of XN in beer indicate, however, that a daily intake of numerous liters might be necessary to attain significant chemoprevention effects. Therefore, enrichment of XN in the beer extraction in brewing and/or the isolation of XN administered as a dietary supplement will be needed for future trials. This work is the first complete demonstration of preclinical cancer prevention in vivo by this agent and should open the way to human investigation.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5128fje;

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