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,1
* Department of Human Nutrition and Department of Anatomy, University of Kiel, 24098 Kiel, Germany
1 To whom correspondence should be addressed. E-Mail: rment{at}anat.uni-kiel.de.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-tocopheryl succinate (-TOS) > lovastatin > apigenin > genistein > -tocopherol 
kaempferol > 
-tocopherol; chrysin and curcumin were inactive except at a concentration of 100 µmol/L. Glioma cells were similarly sensitive, with U343 more than U118, especially for -TOS and tocopherols. Among the tocopherol derivatives, -TOS (0.1 µmol/L) was the most effective in reducing VEGF release. Overall, the glycosylated flavonoids (i.e., naringin, a constituent of citrus fruits, and rutin, a constituent of cranberries) induced the greatest response to treatment at the lowest concentration in MDA human breast cancer cells. Inhibition of VEGF release by flavonoids, tocopherols, and lovastatin in these models of neoplastic cells suggests a novel mechanism for mammary cancer prevention.
KEY WORDS: • angiogenesis • nutrition • flavonoid • tocopherol • tumor
Breast cancer is the most common malignancy among women in Western countries in both incidence (24.4%) and mortality (17.8%). Consequently, breast cancer prevention is the subject of efforts to improve the lifetime and health of women. Recently, tumor angiogenesis has become in focus for tumor therapy and prevention because the development and progression of solid neoplasms require rapid and persistent growth of new blood vessels to supply the growing tumor with nutrients and oxygen (1–3). Furthermore, increased vascular proliferation was shown to correlate with a higher incidence of metastases and a worse prognosis (4). Tumor neoangiogenesis is induced by angiogenesis-promoting growth factors produced by the tumor or nonmalignant tumor stromal cells (3).
Among angiogenesis factors, vascular endothelial growth factor-A (VEGF-A,2 termed VEGF in the text) is the most important endothelial cell–selective mitogen in vitro; it induces angiogenesis in vivo and produces a profound increase in vascular permeability. VEGF, a 45-kDa dimeric glycosylated peptide, is induced by malignant transformation in the adult, promoted by low oxygen tension and growth factors/cytokines produced by the tumor (3,5,6). Several types of human tumors, including mammary carcinomas (7) and brain tumors, especially gliomas, (3,8,9) secrete VEGF. The VEGF gene encodes differently spliced mRNAs from which pre-VEGF proteins are transcribed. These proteins are secreted directly from the cells after signal sequence cleavage in the rough endoplasmatic reticulum and glycosylation in the Golgi apparatus (3,6). Thus, VEGF is not stored in cells, and synthesis correlates with release. VEGF expression is regulated by transcription factors as well as by mechanisms that control mRNA stability.
Inhibition of growth factor gene expression or effects, including that of VEGF, are novel approaches in cancer therapy (3,5,8). Because diets rich in fruits and vegetables are protective against cancer formation, they could well act on the same principle (10–12). Such diets contain a complex intake of macro- and micronutrients, making it very important to identify specific single compounds that might account for chemoprevention. Because genistein was shown to inhibit angiogenesis (13,14), VEGF expression and release (15), other flavonoids and phytochemicals might also regulate VEGF secretion/synthesis.
Flavonoids are natural plant constituents with a polyphenol structure present in a wide variety of fruits and vegetables. Compounds in seeds, citrus fruits, olive oil, tea, and red wine are commonly consumed with the human diet. The flavonoids chosen for this study were acacetin (a constituent of citrus fruits), apigenin (apple skins, citrus fruits, celery roots), chrysin (berries), genistein (the major isoflavone in soy), kaempferol (broccoli, leek), morin (fruits, Chinese herbs), naringin (citrus fruits), naringenin (citrus fruits), and rutin (cranberries). To this list, curcumin (a constituent of curry) was added as a control because of its closely related chemical structure and because of its ability to block the expression of the VEGF transcript (16).
In comparison with flavonoids, vitamin E, a prominent biomembrane constituent, is also a dietary antioxidant; therefore, tocopherol derivatives were included in the study (17). Vitamin E is a generic term used for all 8 naturally occurring tocopherols and tocotrienols as well as derivatives (18). The most biologically active form of vitamin E is -tocopherol (18). Unlike the redox-active -tocopherol (-TOH), -tocopheryl succinate (-TOS) is redox-silent and has a side group that can be charged at physiological pH (19). -TOS is a succinate ester of natural RRR--TOH. It is often used as the vitamin E source in commercial supplements because -TOS is more stable in the presence of oxygen than free TOH. All vitamin E in the human body is derived from nutrition. Major dietary sources of vitamin E are vegetable oils, margarine, eggs, meat, and shortening, with nuts, seeds, whole grains, and wheat germ providing additional sources. Because tocopherols (and in part flavonoids) are lipophilic nutrients, we included some drugs (e.g., lovastatin) that interfere with lipid metabolism in the study (20).
In the present study, we investigated the potency of naturally occurring and related synthetic flavonoids, tocopherols, and other compounds on basal VEGF secretion by cultured tumor cells. As tumor cell models, we choose the well-characterized breast cancer cell line MDA-MB-231 (abbreviated simply as MDA in the text) and two glioma cell lines, U343 and U118, for comparison. To verify whether the effects are restricted to tumor cells, experiments were performed also with human chondrocytes. Chondrocytes secrete VEGF in vitro and under pathological (inflammatory) conditions in vivo (21).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cell culture, stimulation experiments, and VEGF measurements. MDA cells were obtained from American Type Culture Collection) and U343 and U118 glioma cells from the "Deutsches Krebsforschungszentrum"; immortalized human chondrocytes were a gift from Mary B. Goldring, Ph.D., Beth Israel Deaconess Medical Center, Medicine/Rheumatology, Harvard Institutes of Medicine, Boston, MA (21). Cells were cultivated in glutamine-supplemented DMEM plus 10% fetal calf serum (FCS).
For stimulation, 6 x 105 cells were seeded into 8.2-cm2 culture flasks, cultivated for 4 d to confluence, washed (3 x 15 min with DMEM plus10% FCS) and stimulated with vitamin E derivatives, flavonoids, curcumin, drugs, vehicle controls (1% ethanol and 1% DMSO) or left unstimulated for 24 h in 2 mL DMEM plus 1% FCS. Culture supernatants were removed and 100-µL aliquots were analyzed for VEGF by a sandwich ELISA (R&D Systems; sensitivity 
10 ng/L). All assays were performed at least 3 times [Block, k = 1 (1) 3]. Recombinant peptides [epidermal growth factor (EGF), VEGF standard; human sequences] were obtained from Preprotech.
Proliferation assays. Proliferation assays were performed with subconfluent cells that had been precultivated for 2 d in medium with 1% FCS. Subsequently, cells were washed and then incubated with fresh medium containing 1% FCS with or without agents added. After 24 h, DNA was measured fluorimetrically using the CyQuant-method (8), and proliferation was expressed as a percentage of the nonstimulated control. Experiments were performed with 5 individual dishes for each stimulus.
Statistical analysis.
Statistical analyses were performed with commercially available software SAS PROC GLM (SAS Institute). For either cell line, each response measurement (i.e., VEGF secretion as a percentage of control) was analyzed separately. Data were analyzed using the following general linear model:
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where Yijk is the response measurement, µ is the overall mean, i is 1 (1) 28 for MDA cells, 1 (1) 12 for U118 cells, 1 (1) 8 for U343 cells, and 1 (1) 8 for chondrocytes, j is 1 (1) 3, and k (randomized repetition) is 1 (1) 3. Based on the LSMEAN-statement, all pairs of means were compared by Student's t test. Differences within cell lines were tested with an unpaired t test. Differences were considered significant at P 
0.05).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Inhibition of VEGF secretion from breast cancer cells by phytochemicals and related compounds.
In MDA cells, 9 of the 21 compounds tested showed inhibitory activity at 0.1 µmol/L (Fig. 2). The rank order of potency for inhibition was naringin (flavanone) > rutin (flavonol) > -TOS > lovastatin > apigenin (flavone) > genistein (isoflavone) > -TOH kaempferol (flavonol) > -TOH. Moreover, the inhibitory potency of naringin, rutin, -TOS, and lovastatin was much greater than that of the other compounds (P < 0.05). Interestingly, naringin (naringenin 7-rhamnoglucoside) and rutin (quercetin 3-rhamnoglucoside), the most potent flavonoids tested, are characterized structurally by a sugar moiety. With the exception of apigenin, the flavones acacetin and chrysin were ineffective at 0.1 µmol/L in suppression of VEGF secretion. However, when tested at 100 µmol/L, acacetin and chrysin behaved quite differently. Acacetin again was ineffective, whereas chrysin inhibited VEGF release by 80% (Fig. 2). Among the flavonols used in this study, rutin and kaempferol but not morin were active as inhibitors of VEGF release. Curcumin (curcuminoid) and succinic acid were effective only at 100 and 1 µmol/L, respectively.
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-TOS, -TOH, and -TOH, also suppressed the level of secreted VEGF from human breast cancer cells (Fig. 2). The most active of these compounds was -TOS (P < 0.05), a redox-silent analog of vitamin E. The inhibitory effect of -TOS on VEGF release was maximal for the intact molecule because a reduced effect was noted when -TOH was administered alone. The redox-influencing compounds BHT and citral had only minor effects.
Inhibition of VEGF secretion from glioma cells by phytochemicals.
As with MDA cells, marked -TOS–induced inhibition could also be demonstrated with U118 (Fig. 3A) and U343 glioma cells (Fig. 3B). In U343 cells, the effect was even 1.4-times higher than that in human breast cancer cells (P < 0.05). Thus, -TOS is the only TOH derivative used that is inhibitory in all human tumor cell lines tested in this study. Although incubation with 0.1 µmol/L -TOH suppressed the secretion of VEGF in the MDA and U343 cells (P < 0.05) (Fig. 2 and Fig. 3B), it did not affect the U118 cells (Fig. 3A) when treated with 0.1 µmol/L -TOH. On the other hand, 
-TOH had no effect on the VEGF level in MDA and U118 cells but reduced VEGF secretion in U343 cells (P < 0.05).
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-TOS, -TOH, or -TOH for comparison. Here, the inhibitory effects of -TOS and -TOH tended to be lower than in MDA and U343 tumor cells (P = 0.1), but were comparable to those in U118 cells (Fig. 4). Another property that discriminates chondrocytes from tumor cells is their relative resistance to genistein. Genistein showed a small inhibitory effect only at 1 µmol/L (P < 0.05; Fig. 4A). Co-incubation of chondrocytes with 10 µg/L EGF (resulting in increased VEGF secretion) tended (P = 0.1) to reduce the inhibitory effects of -TOS, -TOH, or apigenin (Fig. 4B). In particular, EGF-exposed chondrocytes were very insensitive to apigenin, i.e., 18% inhibition required 10 µmol/L of the flavone. Thus, the concentration of apigenin required to inhibit VEGF secretion in EGF-dosed chondrocytes was 100 times higher than that required to inhibit its secretion by 17% in human breast cancer cells (P < 0.05).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Among the flavonoids, genistein and 4 other compounds exerted a pronounced inhibitory effect on VEGF secretion in MDA cells at 0.1 µmol/L. This concentration is nontoxic and can be reached in humans at least for nonglycosylated polyphenols, e.g., serum genistein concentrations are normally <1 µmol/L and reach <10 µmol/L postprandially (23–25). Compared with curcumin, an activator protein-1 inhibitor with reported antiangiogenic properties (16,26), the effects of some flavonoids occur at physiological concentrations of 0.1 µmol/L, whereas curcumin effects were observed at 100 µmol/L. Of all of the flavonoids tested, the 2 glycosylated forms, naringin (rhamnoglucoside of naringenin flavone) and rutin (rhamnoglucoside of quercetin), were the most potent inhibitors of VEGF release. The flavonoid aglycone, naringenin, does not have inhibitory activity, implying that the sugar moiety of the flavonoids may be important for blocking VEGF secretion. In addition, this finding provides an insight into the structural demands for inhibition of VEGF release by apigenin. Naringenin has been described as a breakdown product of apigenin. It differs in structure in the C-ring with apigenin lacking an additional C 2-3 double bond (Fig. 1). This double bond, however, may be essential for apigenin's action in that naringenin does not seem to have inhibitory activity. Although the water-soluble flavonoids naringin and rutin may offer promise as chemopreventive agents, their usefulness as oral antiangiogenic compounds may be limited by the restricted systemic availability of these glycosides.
The results with flavonoid aglycones indicate that factors other than polarity participate in controlling VEGF secretion because their potency does not relate directly to the number of the hydroxyl groups. For example, morin (5 OH groups), which is more hydrophilic than genistein (3 OH groups), was ineffective in this model, whereas genistein lowered VEGF secretion by 14%. These data suggest that flavonoid glycosides with high water solubility may work through mechanisms different from those of flavonoid aglycones with greater lipid solubility.
Among the 3 flavones used, the most active compound at 0.1 µmol/L was apigenin. This flavonoid isomer of genistein, with hydroxylations at positions 5, 7, and 4', inhibits VEGF secretion with potency comparable to that of genistein. The suppression of VEGF secretion by chrysin was achieved only at a 1000-fold higher concentration. In contrast to apigenin and chrysin, acacetin was without effect, even at the highest concentration tested (100 µmol/L). Thus, it is likely that only subsets of flavones are potentially effective inhibitors of VEGF release.
For comparison, we performed some selective experiments on the susceptibility of VEGF also in nonmalignant cells. In fact, tumor cells were more sensitive to the isoflavone than chondrocytes in which genistein reduced VEGF only at higher concentrations (>1 µmol/L). In this context, we can rule out that cessation of cell proliferation in response to genistein dosing accounts for the decreased VEGF concentrations in the medium compartment that occurred with the chondrocytes.
Of all the of vitamin E derivatives tested, the succinyl analog of -TOH (-TOS) was the most potent inhibitor of VEGF release in MDA cells. -TOH and -TOH were equipotent blocking agents, whereas -TOH was without effect at vitamin E concentrations below those in the serum (27). Our data suggest that vitamin E analogs with low lipid solubilities are more potent inhibitors than the highly apolar ones.
Two different mechanisms for the inhibition of VEGF release by vitamin E derivatives seem possible: alteration of membrane fluidity and permeability or antioxidant effects influencing signaling pathways. The redox-silent -TOS may behave (in part) as a membrane-stabilizing agent, as was shown for -tocopheryl acetate (28). Like other antioxidants, the redox-active -TOH and -TOH can influence cellular redox status, which in turn modulates signaling pathways involved in the regulation of gene expression (29). Both mechanisms could also explain the effects of glycosylated flavonoids naringin and rutin on VEGF secretion. These amphiphilic phytochemicals accumulate in the cell surface and thereby interact with lipid constituents of biomembranes. The modification of membrane receptor and enzyme activities as well as antioxidant effects might both influence signal transduction pathways.
To expand our observations with flavonoids and vitamin E derivatives, we also characterized the activity of drugs affecting (among others) vitamin A metabolism (20,30–32), i.e., lovastatin, cyclophosphamide, chloral hydrate, citral, and ketoconazol. However, only lovastatin, which reduces cholesterol and inhibits retinyl ester hydrolase (20), potently inhibited VEGF secretion from MDA cells as previously reported for Ha-ras-transformed murine tumor cells (32).
In conclusion, inhibition of VEGF synthesis/secretion seems to be a potential molecular mechanism for the anticancer activities of specific secondary plant factors, probably through membrane perturbation and antioxidant actions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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-TOH, -tocopherol; -TOS, -tocopheryl succinate; -TOH, -tocopherol; -TOH, -tocopherol. 
Manuscript received 8 November 2005. Initial review completed 2 December 2005. Revision accepted 22 March 2006.
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