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Medical College of Georgia, Vascular Biology Center, CB 3207, Augusta, GA 30912
3 To whom correspondence and reprint requests should be addressed. E-mail: rvenema{at}mcg.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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24 h, chronic QRN exposures (24–48 h) led to early M-phase arrest and disruption of mitotic microtubule polymerization. In vivo, QRN administered i.p. to female Balb/C mice bearing both syngeneic mammary tumors and Matrigel implants suppressed angiogenesis as measured by endothelial cell immunohistochemistry and hemoglobin concentration. Taken together, these findings suggest a dual mechanism by which QRN suppresses endothelial cell proliferation, both acutely via inhibition of eNOS Ser 617 phosphorylation, and chronically via perturbation of mitotic microtubule polymerization. This novel mechanism of QRN in endothelial cells may in part explain its inhibitory action on angiogenesis and further discern a potential role of QRN as a chemopreventive agent.
KEY WORDS: • quercetin • eNOS phosphorylation • microtubules • mitotic arrest
Quercetin (3,3',4',5,7-pentahydroxyflavone) occurs naturally as a component of plant foods such as onions, apples, and green and black tea, and is one of the most abundant flavonoids in the human diet (1–6). In the United States, of an estimated daily intake of 1 g total flavonoids, on average 25 mg is thought to be quercetin (QRN)4 (1,7). QRN has received much attention in the area of nutrition and breast cancer research for several reasons. First, it exists as a component of commonly consumed fruits and vegetables, and is especially abundant in onion (6,8). In epidemiologic studies, vegetable intake is inversely associated with breast cancer risk (9–11), suggesting that breast cancer recurrence also might be modified by dietary factors (12). Second, QRN was shown to possess potent antioxidant capacity (13) and to act as an inhibitor of the phase I enzyme cytochrome P450 isozyme 1A2 (CYP1A2), an enzyme participating in carcinogen activation (14); thus, QRN is thought to confer protection against mutation and the initiation of carcinogenesis. QRN was also reported to induce cell cycle arrest (15–19), perturb microtubule polymerization (20), and trigger apoptosis of transformed cells (18,19,21), suggesting that it may also be an effective inhibitor of neoplastic cell proliferation and cancer promotion and progression. Finally, limited reports recently suggested that QRN may also affect tumor-associated angiogenesis (22,23), and thereby indirectly inhibit postinitiation carcinogenesis via suppression of endothelial cell and vascular growth necessary for tumor cell maintenance and proliferation.
Nitric oxide, a product of endothelial nitric oxide synthase (eNOS), is thought to function as the downstream effector of vascular endothelial growth factor (VEGF) signaling in endothelial cells that modulates new blood vessel formation (24–36). As an endothelium-derived substance, nitric oxide acts as an autocrine/paracrine signaling molecule and facilitates a change in endothelial cell phenotype from one that is stationary to one that displays mobility (24–26), a required first step in the angiogenic process (27,29). Nitric oxide is also a mitogen and stimulates endothelial cell proliferation via the formation of cGMP and activation of mitogen-activated protein kinase (30,35,37). These roles of nitric oxide, along with its classical function as an inducer of vasodilitation, are thought to contribute to the migration and growth of endothelial cells necessary for initiation of angiogenesis in vivo (29). Moreover, compared with quiescent cells, proliferating endothelial cells display elevated levels of eNOS expression and activity (25), whereas both basal and exogenous VEGF-stimulated vascularization is significantly reduced in eNOS knockout mice compared with wild-type controls (26). The liberation of eNOS-derived nitric oxide, therefore, is thought to be essential to the process of angiogenesis (38), leading to the suggestion that agents that limit eNOS activity might be efficacious as part of chemopreventive or chemotherapeutic regimens.
Aside from targeting the enzymatic activity of eNOS, limiting endothelial cell cycle progression is an additional mechanism by which tumor-associated angiogenesis may be suppressed in response to treatment with either natural or synthetic agents. Within the M-phase of the cell cycle, highly dynamic mitotic microtubules are essential to the proper orchestration of chromosomal segregation prior to cytokinesis. Indeed, the polymerization and dynamics of these mitotic microtubules are known to be perturbed by a variety of diverse naturally occurring compounds that arrest cells within the M-phase. Although the structures of these antimitotic compounds are heterogeneous, they have in common dose-dependent mechanisms involving microtubule disruption, transient M-phase arrest, and subsequent aberrant cell cycle progression, leading to cell death (39). This mechanism of mitotic arrest followed by ultimate cell death (or "mitotic catastrophe") was demonstrated extensively in cancerous cells treated with various antimitotic agents that bind with microtubules at 1 of 3 well-characterized tubulin protein binding sites (39–43). Although there are numerous reports of the tubulin-disrupting activities of naturally occurring antimitotic agents in cancer cells (39,43,44), relatively few studies support such a mechanism in the endothelial cell population responsible for the vascularization required for tumor progression. Moreover, to our knowledge, only 1 previous study reported inhibition of endothelial cell proliferation involving microtubule disruption in response to the action of a naturally occurring compound derived from a food (45). In light of QRN's known actions in inhibiting cell cycle progression and inducing the cell death of transformed cells (15–19,21), as well as the recent report of its ability to bind with purified tubulin proteins (20), we hypothesized that the mechanism underlying observations of angiogenesis suppression in response to QRN treatment may in part result from disruption of microtubule polymerization in endothelial cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The EMT6 cell line, established from a transplantable mouse mammary carcinoma and available from the American Type Culture Collection, was used for implantation in syngeneic female Balb/C mice to investigate the in vivo efficacy of QRN to inhibit progression of breast tumor–associated angiogenesis. Stock EMT6 cells were grown in MEM Eagle (with 2 mmol/L L-glutamine and Earle's BSS adjusted to contain 2.2 g/L sodium bicarbonate and 0.1 mmol/L nonessential amino acids) containing 10% heat-inactivated fetal bovine serum at 37°C under a 5% CO2 atmosphere. Prior to s.c. injection in mice, cells were washed and their concentration adjusted in 0.9% sterile saline.
Cell growth assay. BAE cells were seeded at a density of 1.25 x 104 cells/cm2 in 25-cm2 Corning tissue culture flasks. Cultures (n = 4/group) were exposed to increasing doses of QRN or DMSO for 48 h, with fresh medium and treatment replenishment after the initial 24 h, prior to harvesting (via trypsinization) and cell counting on a hemocytometer.
[3H]thymidine incorporation assay of DNA synthesis. BAE cells were seeded at a density of 1.25 x 104 cells/cm2 in Costar 48-well plates. Cultures (n = 5/group) were exposed to increasing doses of QRN or DMSO for 48 h, with replenishment of fresh medium containing treatments and [3H]thymidine (1 µCi) following the initial 24 h. The medium was then removed and the cells assayed for tritium incorporation as previously described (44).
L-Arginine to L-citrulline conversion assay of eNOS activity. Following QRN or DMSO treatment of BAE cell cultures, assay of eNOS activity was carried out as described previously (46).
Western blot analysis.
Following QRN or DMSO treatment of BAE cell cultures, the samples were washed twice with Tris-buffered saline, harvested in ice-cold lysis buffer (1% Triton X-100, 50 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaF, 15 mmol/L NaPPi, 1 mmol/L Na3VO4, 1.6 mg/L aprotinin, 10 mg/L Leupeptin, 1 µmol/L pepstatin), centrifuged (15,000 x g for 15 min at 4°C), and assayed for total protein content. Aliquots of equal protein concentration were fractionated by SDS-PAGE (7.5%) and transferred to nitrocellulose membranes. The membranes were blocked and then incubated with primary antibody to phosphorylated eNOS (Ser 116, Ser 617, Ser 635, Ser 1179, Thr 497; Upstate USA) or phosphorylated Akt (Ser 473, Cell Signaling Technology). After incubation with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) from the appropriate species, immunodetection was carried out using enhanced chemiluminescence (34080, Pierce Biotechnology). Exposed X-ray film was then scanned into Adobe Photoshop, and densitometric analysis performed on an Apple Macintosh G4 computer using the public domain NIH Image program [developed at the U.S. National Institutes of Health and available on the Internet (47)]. Equal loading of total protein was confirmed by reprobing membranes for 
-tubulin (CP06, EMD Biosciences).
Cell cycle analysis and quantification of the mitotic index. BAE cells were treated with either QRN or DMSO and harvested via trypsinization at the times indicated. The cells were then prepared for analysis using a Becton Dickinson FACSCalibur Flow Cytometer as previously described (44).
To quantify cells in mitosis, BAE cells were seeded at a density of 1.25 x 104 cells/cm2 on 12-mm round glass coverslips; 24 h later, the cells were treated with increasing doses of QRN or DMSO for an additional 24 h. After being washed once with PBS, cells were fixed with glutaraldehyde (1% in PBS, for 10 min at room temperature) followed by sodium borohydride (1 g/L in PBS, twice for 10 min each). Cells were then rinsed 3 times with PBS, permeabilized with wash buffer (0.1% Triton X-100, 1% bovine serum albumin in Tris-buffered saline) for 10 min, and incubated with 4',6-diamidino-2-phenylindole (268298, 10 mg/L, EMD Biosciences) for 30 min at room temperature. Samples were washed again 5 times with wash buffer, washed once with dH2O, and mounted on microscope slides with mounting medium (622701, MP Biomedicals). A total of 1000 cells per dose group were counted, and cells displaying either mitotic nuclei or aberrant micronuclei were scored under a fluorescence microscope.
Immunofluorescence tubulin staining. BAE cells were sparsely plated on 12-mm round glass coverslips, and 24 h later, they were exposed to 50 or 100 µmol/L QRN or DMSO vehicle for 24 h. After being washed once with PBS, cells were fixed and stained as previously described (44) prior to analysis by fluorescence microscopy.
Intraperitoneal QRN and angiogenesis progression in vivo. Female Balb/C mice (Harlan) at 10 wk of age were housed individually in polycarbonate cages and maintained on a 12-h light:dark cycle (lights on at 0700) with AIN-93G diet [(48), without tert-butylhydroquinone] and water available ad libitum. All procedures were carried out in accordance with a protocol approved by the Institutional Animal Care Committee of the Medical College of Georgia. EMT6 cells were administered to the right flank region of mice via s.c. injection, at a threshold dose determined in pilot experiments to yield tumor development in all mice within 2 wk following cell injection (4 x 104 cells/injection). Seven days later, i.p. injections of either QRN (300 or 1000 µg) or PBS vehicle containing 20% polyethylene glycol (PEG 400) and 2% Tween 80 were performed daily (d 1–14, between 0900 and 1200). In addition, on d 9, ice-cold liquid Matrigel (356231, BD Biosciences,) impregnated with 100 µg/L recombinant murine VEGF (450-32, PeproTech) and 50,000 U/L heparin were administered to the left flank region of mice via s.c. injection (0.5 mL/injection). On d 15, all mice were killed by CO2 asphyxiation; the Matrigel plugs were recovered and localized tumors excised. Matrigel plugs were then solubilized in PBS containing 0.1% Triton X-100 and 50,000 U/L heparin for analysis of hemoglobin using Pointe Scientific hemoglobin standard and reagent (H7504) and control (H7506) sets according to the manufacturer's instructions. Tumor tissue mass was recorded prior to fixation (with 10% neutral buffered formalin for detection of von Willebrand factor; alternatively with Zinc fixative, 550523, BD Biosciences Pharmingen, for CD31 detection) for subsequent analysis by immunohistochemistry.
Immunohistochemistry.
Following routine paraffin-embedding and sectioning of fixed tumors, 5-µm slices (taken 1 mm deep from the tumor periphery) were allowed to adhere to positively charged microscope slides prior to deparaffinization, hydration, and antigen retrieval with BD Retrievagen A (550524, BD Biosciences Pharmingen). Following nonspecific blocking with 10% serum from the secondary antibody host species, the slides were incubated with primary antibody (CD31: 550274, BD Biosciences Pharmingen; alternatively, von Willebrand factor: AB7356, Chemicon International) for 1 h in a humidified chamber, prior to incubation with biotinylated secondary antibody (30 min at room temperature), application of streptavidin-horseradish peroxidase (30 min), and incubation with 3, 3‘-diaminobenzidine substrate (5 min) according to the manufacturer’s instructions (BD Biosciences Pharmingen). Following ethanol dehydration and xylene clearing, coverslips were affixed and the slides analyzed by light microscopy. Staining of endothelial cells was quantified by densitometric analysis (47) of light microscopy images after background elimination using Adobe Photoshop on an Apple Macintosh G4 computer.
Statistical analysis.
Data derived from experiments comparing only 2 means were analyzed by an independent t test and the accompanying test for homogeneity of variance. In the case of experiments requiring the comparison of >2 means, ANOVA was performed followed by Tukey's Studentized Range (Honestly Significant Difference) for the pair-wise comparisons. Data found to have unequal variances were reanalyzed with the Welch ANOVA F-test. All statistical calculations were completed using JMP IN (SAS Institute). Replicated experiments (each carried out at different times and with n 
3 per group) were analyzed independently, and the data presented are those obtained in a single experiment. 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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20 µmol/L. The inhibitory effect of QRN on DNA synthesis was significant (P < 0.05) at concentrations 30 µmol/L, yet a 100 µmol/L QRN dose was required to diminish [3H]thymidine incorporation to background levels. In both control and QRN treated samples, BAE cells remained adherent to the tissue culture flask, with essentially no cells floating in the culture medium. Because the 100 µmol/L QRN dose completely abolished DNA synthesis, this dose was chosen as the maximum concentration for use in all subsequent cell culture dose-response experiments, as well as for characterization of the time-dependent actions of QRN.
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6 times that of the vehicle-administered controls. In order to further characterize this effect of QRN on BAE cell G2/M progression, we evaluated the endothelial cell mitotic index in response to QRN treatment and found QRN doses as low as 10 µmol/L to trigger cell cycle arrest within mitosis (maximal response at 30 µmol/L QRN, with total mitotic cells 8-fold of the control, data not shown). Moreover, at QRN doses 10 µmol/L, 90% or more of these mitotic cells were blocked prior to metaphase (Fig. 3C,E,G). The cells, however, appear to only pause in this premetaphase state, and eventually transition into an aberrant anaphase (Fig. 3D,F) and undergo micronucleation (Fig. 3H). Also, QRN exposure was found to severely disrupt the polymerization of both cytoplasmic and mitotic endothelial cell microtubules (Fig. 4). Within 24 h, compared with controls, treatment of BAE cells with 100 µmol/L QRN led to only diffuse staining of the cytoplasmic microtubules of interphase cells (Fig. 4D), as well as mitotic microtubules of cells displaying condensed and distinguishable chromosomes. Moreover, aberrant mitotic figures (Fig. 4E,F) including condensed chromosomes malaligned at the equatorial metaphase plate were observed concomitant with mitotic spindles lacking complete microtubule polymerization (Fig. 4F).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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50% inactivation of eNOS within 30 min (Fig. 2A), an effect that was overcome within 24 h. Moreover, we further demonstrated that QRN-induced suppression of eNOS activity involves inhibition of eNOS phosphorylation at Ser 617 (Fig. 2B), a key eNOS regulatory site known to be phosphorylated immediately downstream of Akt (50–53). At doses as low as 10 µmol/L, QRN concomitantly induced significant inhibition of BAE cell Akt (Ser 473) phosphorylation, which strongly suggests that QRN acts to inhibit the enzymatic activity of eNOS via its purported role as an inhibitor of phosphoinositide 3-kinase (54–57). It should be noted that although these data strongly indicate quercetin's ability to blunt the basal activity of eNOS, they do not preclude the possibility that quercetin might also scavenge superoxide and thereby protect nitric oxide from inactivation, as was previously suggested (58). A recent report indicates that blood plasma concentrations may reach in excess of 20 µmol/L QRN and QRN metabolites in response to QRN-rich diets, and some tissues appear to possess elevated ß-glucuronidase activities capable of deconjugating QRN glucuronides, thereby yielding localized tissue QRN aglycone concentrations higher than those observed in plasma (59). Therefore, given adequate QRN intakes coupled with a half-life of 4 h (60) and enzymatic deconjugation in some tissues (59), plasma and tissue QRN levels of at least 10 µmol/L appear likely achievable via the diet (61). Due to the recoverable nature (i.e., within 24 h) of eNOS activity inhibition by QRN, we hypothesized that other mechanisms might also contribute to QRN's antiproliferative action in endothelial cells. Similar to previous studies with other cell types, we found BAE cells to accumulate within the G2/M phase of the cell cycle in response to QRN treatment. This G2/M accumulation, although not evident within the relatively brief 30-min exposure time found to strongly suppress eNOS activity, occurred much later following 24 h of treatment with 100 µmol/L QRN, an effect that appeared to persist after 48 h (Table 1). In order to discern whether QRN-induced G2/M accumulation was actually cell cycle arrest within the G2 of interphase or within mitosis, we next quantified the mitotic indices of BAE cell cultures exposed to increasing dosages of QRN. At doses as low as 10 µmol/L, we found QRN-treated cultures to display an abundance of mitotic figures, the vast majority of which were indicative of early M-phase cell cycle arrest prior to metaphase (Fig. 3). Also, the relatively few postmetaphase mitotic figures were aberrant, and micronuclei suggestive of cell death by mitotic catastrophe (62,63) were evident at higher QRN doses (Fig. 3H). Chronic exposure of BAE cells to QRN, therefore, appears to result in a dose-dependent early M-phase, rather than the G2 phase, cell cycle block, or pause, that is ultimately followed by an aberrant continuation into later stages of mitosis leading to disproportionate DNA division and micronucleation. Furthermore, although we acknowledge the possibility that QRN may be metabolized within 24 h into a form that allows recovery of eNOS activity yet contributes or even facilitates the M-phase arrest, we conclude that the mechanism by which prolonged or chronic QRN exposures suppress BAE cell proliferation (Fig. 1) ultimately involves early mitotic arrest of the endothelial cell cycle.
On the basis of these findings, we then hypothesized that the underlying mechanism by which QRN triggers BAE cell M-phase arrest involves disruption of normal mitotic microtubule polymerization, as was extensively reported in other cell types in response to mitosis-arresting agents known to preclude normal metaphase, lead to aberrant later mitotic stages, and ultimately trigger micronucleation and catastrophic cell death (39,43,44). Immunofluorescence tubulin staining clearly indicated QRN's ability to concurrently disrupt normal mitotic (as well as cytoplasmic) microtubule polymerization and induce early M-phase cell cycle arrest.
Given our findings of QRN-induced eNOS suppression and mitotic cell cycle arrest in endothelial cell culture, we hypothesized that QRN would suppress endothelial cell proliferation in vivo and hence limit blood vessel growth associated with tumor progression. In female Balb/C mice, the organization of tumor-associated endothelial cell markers was suppressed by QRN (Fig. 5A-D), and we observed a relative lack of organized blood vessel growth compared with vehicle-administered controls. In order to control for the possibility that QRN-induced inhibition of angiogenesis in vivo is merely a secondary effect resulting from the arrest of cancer cell cycling and tumor growth (15–19,21), we concurrently examined hemoglobin content of Matrigel implants impregnated with VEGF yet devoid of cancerous cells. Although it is possible that the growth factor concentration within the vicinity of EMT6 cell tumors may be greater than the level of VEGF incorporated in the Matrigel implants, our observation of strong and significant (P < 0.05) inhibition of hemoglobin infiltration into Matrigel plugs (Fig. 5E), together with suppression of tumor-associated angiogenic markers, indicates that the endothelial cell cycle (and perhaps eNOS activity per se) is a likely target of QRN not only in cell culture but also in the whole animal.
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FOOTNOTES |
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2 Supported by grant number 05A079 (to S.J.T.J) from the American Institute for Cancer Research, and by NHLBI00059 (to R.C.V.) from the NIH.
4 Abbreviations used: BAE, bovine aortic endothelial; BSA, bovine serum albumin; CYP1A2, cytochrome P450 isozyme 1A2; DMSO, dimethyl sulfoxide; eNOS, endothelial nitric oxide synthase; QRN, quercetin; VEGF, vascular endothelial growth factor.
Manuscript received 10 August 2005. Initial review completed 30 September 2005. Revision accepted 23 January 2006.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Lamson D, Brignall M. Antioxidants and cancer III: quercetin. Altern Med Rev. 2000;5:196–208.[Medline]
2. Sellappan S, Akoh C. Flavonoids and antioxidant capacity of Georgia-grown Vidalia onions. J Agric Food Chem. 2002;50:5338–42.[Medline]
3. Lee K, Kim Y, Kim D, Lee H, Lee C. Major phenolics in apple and their contribution to the total antioxidant capacity. J Agric Food Chem. 2003;51:6516–20.[Medline]
4. Alonso-Salces R, Ndjoko K, Queiroz E, Ioset J, Hostettmann K, Berrueta L, Gallo B, Vicente F. On-line characterisation of apple polyphenols by liquid chromatography coupled with mass spectrometry and ultraviolet absorbance detection. J Chromatogr A. 2004;1046:89–100.[Medline]
5. Jin D, Hakamata H, Takahashi K, Kotani A, Kusu F. Determination of quercetin in human plasma after ingestion of commercial canned green tea by semi-micro HPLC with electrochemical detection. Biomed Chromatogr. 2004;18:662–6.[Medline]
6. Miean K, Mohamed S. Flavonoid (myricetin, quercetin, kaempferol, luteolin, and apigenin) content of edible tropical plants. J Agric Food Chem. 2001;49:3106–12.[Medline]
7. Ross JA, Kasum CM. Dietary flavonoids: bioavailability, metabolic effects, and safety. Annu Rev Nutr. 2002;22:19–34.[Medline]
8. Arabbi P, Genovese M, Lajolo F. Flavonoids in vegetable foods commonly consumed in Brazil and estimated ingestion by the Brazilian population. J Agric Food Chem. 2004;52:1124–31.[Medline]
9. Freudenheim JL, Marshall JL, Vena JE, Laughlin R, Brasure J, Swanson MK Nemoto T, Graham S. Premenopausal breast cancer risk and intake of vegetables, fruits, and related nutrients. J Natl Cancer Inst. 1996;88:340–8.
10. Franceschi S, Parpinel M, La Vecchia C, Favero A, Talamini R, Negri E. Role of different types of vegetables and fruit in the prevention of cancer of the colon, rectum, and breast. Epidemiology. 1998;9:338–41.[Medline]
11. Franceschi S, Favero A, La Vecchia C, Negri E, Dal Maso L, Salvini S, Decarli A, Giacosa A. Influence of food groups and food diversity on breast cancer risk in Italy. Int J Cancer. 1995;63:785–9.[Medline]
12. Pierce J, Faerber S, Wright F, Newman V, Flatt S, Kealey S, Rock C, Hryniuk W, Greenberg E. Feasibility of a randomized trial of a high-vegetable diet to prevent breast cancer recurrence. Nutr Cancer. 1997;28:282–8.[Medline]
13. Rajendran M, Manisankar P, Gandhidasan R, Murugesan R. Free radicals scavenging efficiency of a few naturally occurring flavonoids: a comparative study. J Agric Food Chem. 2004;52:7389–94.[Medline]
14. Bacon JR, Williamson G, Garner RC, Lappin G, Langouet S, Bao Y. Sulforaphane and quercetin modulate PhIP-DNA adduct formation in human HepG2 cells and hepatocytes. Carcinogenesis. 2003;24:1903–11.
15. Scambia G, Ranelletti F, Panici P, Piantelli M, Bonanno G, De Vincenzo R, Ferrandina G, Rumi C, Larocca L, Mancuso S. Inhibitory effect of quercetin on OVCA 433 cells and presence of type II oestrogen binding sites in primary ovarian tumours and cultured cells. Br J Cancer. 1990;62:942–6.[Medline]
16. Yoshida M, Sakai T, Hosokawa N, Marui N, Matsumoto K, Fujioka A, Nishino H, Aoike A. The effect of quercetin on cell cycle progression and growth of human gastric cancer cells. FEBS Lett. 1990;260:10–3.[Medline]
17. Ranelletti F, Ricci R, Larocca L, Maggiano N, Capelli A, Scambia G, Benedetti-Panici P, Mancuso S, Rumi C, Piantelli M. Growth-inhibitory effect of quercetin and presence of type-II estrogen-binding sites in human colon-cancer cell lines and primary colorectal tumors. Int J Cancer. 1992;50:486–92.[Medline]
18. Ferrandina G, Almadori G, Maggiano N, Lanza P, Ferlini C, Cattani P, Piantelli M, Scambia G, Ranelletti F. Growth-inhibitory effect of tamoxifen and quercetin and presence of type II estrogen binding sites in human laryngeal cancer cell lines and primary laryngeal tumors. Int J Cancer. 1998;77:747–54.[Medline]
19. Rong Y, Yang E, Zhang K, Mack P. Quercetin-induced apoptosis in the monoblastoid cell line U937 in vitro and the regulation of heat shock proteins expression. Anticancer Res. 2000;20:4339–45.[Medline]
20. Gupta K, Panda D. Perturbation of microtubule polymerization by quercetin through tubulin binding: a novel mechanism of its antiproliferative activity. Biochemistry. 2002;41:13029–38.[Medline]
21. Richter M, Ebermann R, Marian B. Quercetin-induced apoptosis in colorectal tumor cells: possible role of EGF receptor signaling. Nutr Cancer. 1999;34:88–99.[Medline]
22. Igura K, Ohta T, Kuroda Y, Kaji K. Resveratrol and quercetin inhibit angiogenesis in vitro. Cancer Lett. 2001;171:11–6.[Medline]
23. Tan W-f, Lin L-p, Li M-h, Zhang Y-X, Tong Y-g, Xiao D, Ding J. Quercetin, a dietary-derived flavonoid, possesses antiangiogenic potential. Eur J Pharmacol. 2003;459:255–62.[Medline]
24. Noiri E, Peresleni T, Srivastava N, Weber P, Bahou WF, Peunova N, Goligorsky MS. Nitric oxide is necessary for a switch from stationary to locomoting phenotype in epithelial cells. Am J Physiol Cell Physiol. 1996;270:C794–802.
25. Zollner S, Aberle S, Harvey SE, Polokoff MA, Rubanyi G. Changes of endothelial nitric oxide synthase level and activity during endothelial cell proliferation. Endothelium. 2000;7:169–84.[Medline]
26. Fukumura D, Gohongi T, Kadambi A, Izumi Y, Ang J, Yun C, Buerk D, Huang P, Jain R. Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability. Proc Natl Acad Sci U S A. 2001;98:2604–9.
27. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature. 2000;407:249–57.[Medline]
28. Ferrara N. Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney Int. 1999;56:794–814.[Medline]
29. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med. 2000;6:389–98.[Medline]
30. Ignarro L, Cirino G, Casini A, Napoli C. Nitric oxide as a signaling molecule in the vascular system: an overview. J Cardiovasc Pharmacol. 1999;34:879–86.[Medline]
31. Fukumura D, Jain R. Role of nitric oxide in angiogenesis and microcirculation in tumors. Cancer Metastasis Rev. 1998;17:77–89.[Medline]
32. Hood JD, Meininger CJ, Ziche M, Granger HJ. VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells. Am J Physiol Heart Circ Physiol. 1998;274:H1054–8.
33. Kroll J, Waltenberger J. VEGF-A induces expression of eNOS and iNOS in endothelial cells via VEGF receptor-2 (KDR). Biochem Biophys Res Commun. 1998;252:743–6.[Medline]
34. Papapetropoulos A, Garcia-Cardena G, Madri JA, Sessa WC. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest. 1997;100:3131–9.[Medline]
35. Morbidelli L, Donnini S, Ziche M. Role of nitric oxide in the modulation of angiogenesis. Curr Pharm Des. 2003;9:521–30.[Medline]
36. Buckley BJ, Mirza Z, Whorton AR. Regulation of Ca(2+)-dependent nitric oxide synthase in bovine aortic endothelial cells. Am J Physiol Cell Physiol. 1995;269:C757–65.
37. Ziche M, Morbidelli L. Nitric oxide and angiogenesis. J Neurooncol. 2000;50:139–48.[Medline]
38. Lee PC, Salyapongse AN, Bragdon GA, Shears, II LL, Watkins SC, Edington HDJ, Billiar TR. Impaired wound healing and angiogenesis in eNOS-deficient mice. Am J Physiol Heart Circ Physiol. 1999;277:H1600–8.
39. Jordan MA. Mechanism of action of antitumor drugs that interact with microtubules and tubulin. Curr Med Chem Anticancer Agents. 2002;2:1–17.
40. Jordan MA, Kamath K, Manna T, Okouneva T, Miller HP, Davis C, Littlefield BA, Wilson L. The primary antimitotic mechanism of action of the synthetic halichondrin E7389 is suppression of microtubule growth. Mol Cancer Ther. 2005;4:1086–95.
41. Jordan M, Wendell K, Gardiner S, Derry W, Copp H, Wilson L. Mitotic block induced in HeLa cells by low concentrations of paclitaxel (Taxol) results in abnormal mitotic exit and apoptotic cell death. Cancer Res. 1996;56:816–25.
42. Zhang HY, Gu YY, Li ZG, Jia YH, Yuan L, Li SY, An GS, Ni JH, Jia HT. Exposure of human lung cancer cells to 8-chloro-adenosine induces G2/M arrest and mitotic catastrophe. Neoplasia. 2004;6:802–12.[Medline]
43. Jackson SJ, Singletary KW. Sulforaphane: a naturally occurring mammary carcinoma mitotic inhibitor, which disrupts tubulin polymerization. Carcinogenesis. 2004;25:219–27.
44. Jackson SJ, Singletary KW. Sulforaphane inhibits human MCF-7 mammary cancer cell mitotic progression and tubulin polymerization. J Nutr. 2004;134:2229–36.
45. Belleri M, Ribatti D, Nicoli S, Cotelli F, Forti L, Vannini V, Stivala LA, Presta M. Antiangiogenic and vascular-targeting activity of the microtubule-destabilizing trans-resveratrol derivative 3,5,4'-trimethoxystilbene. Mol Pharmacol. 2005;67:1451–9.
46. Venema RC, Sayegh HS, Arnal JF, Harrison DG. Role of the Enzyme calmodulin-binding domain in membrane association and phospholipid inhibition of endothelial nitric oxide synthase. J Biol Chem. 1995;270:14705–11.
47. NIH Image website[cited August 1, 2005]; Available from: http://rsb.info.nih.gov/nih-image/
48. Reeves PG, Nielsen FH, Fahey GC Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr. 1993;123:1939–51.
49. Chiesi M, Schwaller R. Inhibition of constitutive endothelial NO-synthase activity by tannin and quercetin. Biochem Pharmacol. 1995;49:495–501.[Medline]
50. Michell B, Griffiths J, Mitchelhill K, Rodriguez-Crespo I, Tiganis T, Bozinovski S, de Montellano P, Kemp B, Pearson R. The Akt kinase signals directly to endothelial nitric oxide synthase. Curr Biol. 1999;9:845–8.[Medline]
51. Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 1999;399:597–601.[Medline]
52. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher A. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999;399:601–5.[Medline]
53. Michell BJ, Harris MB, Chen ZP, Ju H, Venema VJ, Blackstone MA, Huang W, Venema RC, Kemp BE. Identification of regulatory sites of phosphorylation of the bovine endothelial nitric-oxide synthase at serine 617 and serine 635. J Biol Chem. 2002;277:42344–51.
54. Matter W, Brown R, Vlahos C. The inhibition of phosphatidylinositol 3-kinase by quercetin and analogs. Biochem Biophys Res Commun. 1992;186:624–31.[Medline]
55. Vlahos C, Matter W, Hui K, Brown R. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4- morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem. 1994;269:5241–8.
56. Agullo G, Gamet-Payrastre L, Manenti S, Viala C, Rémésy C, Chap H, Payrastre B. Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: a comparison with tyrosine kinase and protein kinase C inhibition. Biochem Pharmacol. 1997;53:1649–57.[Medline]
57. Walker E, Pacold M, Perisic O, Stephens L, Hawkins P, Wymann M, Williams R. Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol Cell. 2000;6:909–19.[Medline]
58. Lopez-Lopez G, Moreno L, Cogolludo A, Galisteo M, Ibarra M, Duarte J, Lodi F, Tamargo J, Perez-Vizcaino F. Nitric oxide (NO) scavenging and NO protecting effects of quercetin and their biological significance in vascular smooth muscle cells. Mol Pharmacol. 2004;65:851–9.
59. de Boer V, Dihal A, van der Woude H, Arts I, Wolffram S, Alink G, Rietjens I, Keijer J, Hollman P. Tissue distribution of quercetin in rats and pigs. J Nutr. 2005;135:1718–25.
60. Ader P, Wessmann A, Wolffram S. Bioavailability and metabolism of the flavonol quercetin in the pig. Free Radic Biol Med. 2000;28:1056–67.[Medline]
61. Manach C, Morand C, Demigné C, Texier O, Regerat F, Rémésy C. Bioavailability of rutin and quercetin in rats. FEBS Lett. 1997;409:12–6.[Medline]
62. Park SS, Eom YW, Choi KS. Cdc2 and Cdk2 play critical roles in low dose doxorubicin-induced cell death through mitotic catastrophe but not in high dose doxorubicin-induced apoptosis. Biochem Biophys Res Commun. 2005;334:1014–21.[Medline]
63. Roninson IB, Broude EV, Chang BD. If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells. Drug Resist Updat. 2001;4:303–13.[Medline]
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