![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
,
,3,,
* Departments of Pharmacology and Toxicology and
Surgery,
** Comprehensive Cancer Center Mass Spectrometry Shared Facility,
Center for Nutrient-Gene Interaction in Cancer Prevention, University of Alabama at Birmingham and
Purdue University-University of Alabama at Birmingham Botanicals Center for Age-related Disease, Birmingham, AL 35294
3To whom correspondence should be addressed. E-mail: helenkim{at}uab.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT Materials and methodsResultsDiscussionLITERATURE CITED |
|---|
KEY WORDS: • cancer • DMBA • MNU • grapes • proanthocyanidins
Despite tremendous advances in medicine, cancer remains a major health concern in the United States and the rest of the world (1). In many cases, cancer is a long drawn-out disease that emotionally drains both patient and family. Although enormous energies have been invested in treating existing cancer by chemotherapy (with some success in certain cancers), prevention of cancer is the preferred option. Because the incidence of cancer varies dramatically from country to country (2), and epidemiological studies show that ethnicity is at most a small part of the overall risk equation (3), much emphasis has been placed on understanding the environmental factors that influence cancer risk. Of these, diet is considered to have a major role. Epidemiological studies have suggested that certain dietary components are associated with lower cancer risk (4,5). These include vitamins as well as other phytochemicals, particularly polyphenols. Because phytochemicals are safe at levels found in the diet, they are of interest to those investigating the role of chemoprevention in lowering cancer risk.
Animal models of cancer have been used to investigate the chemopreventive potential of phytochemicals and related synthetic molecules, such as 13-cis-retinoic acid and N-(4-hydroxyphenyl)retinamide (6,7). However, the role of the diet in which a potential chemopreventive agent is administered has not been systematically addressed in studies using animal models of cancer. Indeed, in most animal studies the administered chemicals are mixed into diets with little consideration of whether the composition of the diet was a determining factor in the experimental outcome. Paradoxically, the American public places great emphasis on the quality and composition of the diet, particularly with regard to the prevention of chronic disease. This diet consciousness is reflected in the advocacy by both the National Institutes of Health and the National Cancer Institute for diets containing at least 5 daily servings of fruits and vegetables, both rich sources of phytochemicals (8). In the mid 1980s concerns about variation in the laboratory diet negatively affecting the efficacy of potential chemopreventive agents in cancer research led to the use of AIN-76A diet, a purified diet based on casein as the sole source of protein, in place of laboratory dry food diets. The chemoprevention experiments that followed using AIN-76A indicated a notably higher incidence and numbers of tumors in N-methyl-N-nitrosourea (MNU)4 -induced mammary cancer in rats (9). This led to the appreciation that the soy component of the rodent diet may have a chemopreventive effect (10). However, during studies to determine synergies between chemoprevention agents administered in the AIN-76A diet, certain well-described chemoprevention agents did not exhibit their predicted effects (C. J. Grubbs, University of Alabama at Birmingham, 1987, unpublished data). Inspection of previous data revealed that the experiments detecting the chemopreventive activities of these agents had been carried out using a laboratory dry food diet. These data provided a rationale for directly examining the role that the diet plays in chemoprevention experiments.
The diet contains many types of phytochemicals, including vitamins and other compounds that cannot be synthesized by humans or animals. Currently, the health benefits of vitamins are widely appreciated, but the benefits of other phytochemicals such as the polyphenols have yet to be clearly understood or accepted, in part because of the complexity of their actions. The polyphenols include a wide range of closely related compounds synthesized by plants; these include the flavonoids found in tea leaves (catechins), isoflavonoids in soybeans (genistein and daidzein), and stilbenes in red grapes (resveratrol). Each of these has been shown to have anticancer properties in cell culture models of cancer (11–13).
The original goal of this study was to evaluate the chemopreventive potential of grape seed extract (GSE), a preparation extracted from grape seeds that is highly enriched in the proanthocyanidins (Fig. 1). These complex polyphenols have in vitro antioxidant activity (14); inhibit aromatase enzyme activity (15); inhibit the growth of cancer cells in cell culture (16,17); and prevent or attenuate disease in various animal models of disease, including atherosclerosis (18), cataract formation (19), and skin cancer (20). Because GSE inhibits the growth of human breast cancer cells in culture (16,17), we hypothesized that GSE would have chemopreventive activity in an animal model of breast cancer. In an initial experiment, however, GSE, at up to 5% by weight administered in AIN-76A diet, did not prevent the incidence or number of mammary tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA), an established carcinogen for the initiation of mammary tumors (21). Similar results were observed in an experiment with genistein, where up to 1 g/kg AIN-76A diet had no effect on MNU-induced mammary cancer. This led to the decision to reexamine the effect of these 2 polyphenol preparations when administered in a laboratory diet (4% rodent diet, Teklad Industries).
|
|
Materials and methods |
|---|
|
TOPABSTRACTMaterials and methodsResultsDiscussionLITERATURE CITED |
|---|
DMBA and Helix pomatia ß-glucuronidase/sulfatase were obtained from Sigma-Aldrich Chemical. MNU was purchased from Ash Stevens. Daidzein was purchased from LC-Labs. Equol, dihydrodaidzein, and O-desmethylangolensin were provided by Dr. Kristiina Wahaala, University of Helsinki, Finland.
Grape seed extract preparation. A powdered GSE preparation was provided by Kikkoman and stored at 4°C in light-tight containers until used. The composition of this preparation was previously described (14) to consist of >95% flavanols, of which 86% were proanthocyanidins.
Genistein. A concentrate enriched (40% by weight) in genistin, the ß-glucoside of genistein, derived from soy molasses was prepared and generously donated by Protein Technologies International (now Solae). Genistin was further purified as follows (22). The concentrate was decolorized by boiling with activated charcoal. After volume reduction by evaporation, it was subjected to fractional crystallization from hot 60% aqueous ethanol. The purity of the final product was determined to be >98% genistin from its absorbance at 262 nm. It was then hydrolyzed by refluxing in methanol:1 mol/L HCl. Genistein was extracted into diethyl ether and after the solvent was removed, it was recrystallized from 60% aqueous ethanol. The final product was analyzed by reverse-phase HPLC and was found to contain >98% genistein.
Animals. Sprague-Dawley rats aged 21 d were purchased from Charles River and fed an AIN-76A diet (Teklad Industries). For the second round of experiments, they were fed a 4% rodent diet (Teklad Industries). They were housed in animal quarters at 22°C with a 12-h light/dark cycle. Animals were given free access to water and the powdered diets. These animal studies were approved by the UAB Institutional Animal Care and Use Committee.
GSE dose selection study.
At age 35 d the rats were segregated into 5 groups receiving diet supplemented with 0%, 0.1%, 1%, 5%, or 10% GSE. The animals were maintained on these diets for 6 wk, after which they were killed by carbon dioxide asphyxiation and necropsied. Body weight gain was monitored weekly and organs were weighed at necropsy, snap frozen in liquid nitrogen, and archived at –80°C. No differences in body weights or organ weights were detected after 6 wk; this was confirmed during the subsequent chemoprevention study reported here. The maximum tolerated dose for GSE was determined to be 10% GSE in the diet. Although in several animal studies the GSE was administered at 
1%, we decided to test as high a dose as the animals could tolerate, to maximize the chances of obtaining chemopreventive activity. Because there appeared to be a very slight reduction (although not statistically significant) in brain weight normalized to 100 g body weight at the 10% GSE dose, doses up to 5% but not 10% were chosen for the chemoprevention study.
A similar study was performed where genistein was added to the AIN-76A diet from age 25 d. No toxic effects were observed in a 6-wk study with doses up to 2.0 g/kg diet. This was the highest dose used in subsequent studies.
Chemoprevention studies. Female rats (20 per group) were given AIN-76A diet supplemented with either 0%, 1.25%, or 5% GSE, beginning at age 35 d. At age 50 d, DMBA (80 mg/kg body weight) in sesame oil was administered by gavage to all animals. Two additional groups of 5 animals each received AIN-76A alone or AIN-76A plus 5% GSE but did not receive DMBA. All animals were maintained in their dietary groups for 120 d after DMBA administration, during which time body weights were monitored and tumors were palpated and counted weekly. Two days before sacrifice, 5 animals selected randomly from each group were placed in metabolic cages, and urine was collected over 48 h into 50-mL plastic tubes sitting in crushed dry ice. Urine samples were collected at 24 and 48 h and stored at –80°C until analyzed. At 120 d post-DMBA, the animals were killed by carbon dioxide asphyxiation and final body and organ weights as well as mammary tumor numbers and sizes were recorded. For the second round of analysis, the same doses of GSE were administered in powdered 4% Teklad rodent diet; all other variables were the same as for the initial study where GSE was given in the AIN-76A diet.
In the genistein chemoprevention studies, for the first experiment female Sprague-Dawley rats were started on isoflavone-free AIN-76A diet at age 22 d. Diet supplementation with genistein (0, 0.8, and 1.6 g/kg diet) was started 3 d later in groups of 30 animals. At age 50 d, the direct-acting carcinogen MNU dissolved in acidified saline (pH 5) at 50 mg/kg body weight was administered intravenously into the jugular vein (23). In the second experiment, the animals were fed a 4% Teklad diet throughout the study. Supplementation with genistein (0, 0.2, and 2.0 g/kg diet) began on day 25. All animals were monitored weekly for overall health and their body weights; tumors were palpated and counted weekly. At the end of the experiment (108 d post-MNU), the animals were killed by carbon dioxide asphyxiation and final body and organ weights as well as mammary tumor incidence, number, and sizes were recorded. Tumors were histologically classified as adenocarcinomas or benign. Five random animals from each group were anesthetized with ether and blood (1–2 mL) was removed by cardiac puncture. After being allowed to clot for 1 h, the blood samples were centrifuged at 3000 x g for 10 min and the serum was carefully removed and stored at –80°C until further analysis.
Analysis of isoflavones
Diet samples (0.5 g) were extracted with 10 volumes of ice-cold 80% aqueous methanol for 2 h. The ice-cold temperature prevented degradation of the isoflavone malonyl-ß-glucosides into their corresponding ß-glucosides, which occurs at room temperature or during extraction with boiling solvents (24). Dichlorofluorescein was added to the extraction solvent as an internal standard. The diet:solvent mixtures were centrifuged at 3000 x g for 10 min and samples (20 µL) of the supernatant were analyzed by HPLC on a Hewlett Packard 1100 instrument (Agilent Technologies). Analyses were carried out on a 25 cm x 4.6 mm i.d. Rainin C8 reverse-phase column equilibrated with 10% aqueous acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA). Bound compounds were eluted by a linear 10–50% gradient of acetonitrile in 0.1% TFA over 30 min. The column was regenerated by washing with 90% aqueous acetonitrile-0.1% TFA and then reequilibrated with 10% aqueous acetonitrile-0.1% TFA. The mobile phase flow rate was 1.5 mL/min. Eluted compounds were detected by absorbance using a diode array detector. Quantitation was determined from their absorbances at 262 nm. All areas were corrected for that of the internal standard and were compared with those in a mixture of known isoflavone standards. Data were recorded in aglucone units as nmol/g of diet.
Serum and urine samples (2 x 0.5 mL) obtained from genistein and GSE-treated animals were analyzed by LC-electrospray ionization mass spectrometry (LC-ESI-MS) (25,26). In brief, phenolphthalein glucuronide, 4-methylumbelliferyl sulfate, and apigenin were added as internal controls to each serum sample before hydrolysis with 400 U of ß-glucuronidase and aryl sulfatase in 150 mmol/L ammonium acetate buffer, pH 5, for 16 h at 37°C. After acidification with glacial acetic acid, the samples were extracted with hexane to remove neutral lipids and then with diethyl ether to recover the isoflavonoid aglucones. For the samples from the GSE-treated animals, the metabolites were initially extracted with ethyl acetate instead of diethyl ether, then evaporated to dryness, and finally reconstituted in 80% aqueous methanol. Aliquots were analyzed by LC-ESI-MS using multiple-reaction ion monitoring (MRM) for specific detection of daidzein, equol, O-desmethylangolensin, dihydrodaidzein, genistein, catechin, and methylcatechins. Analyses were performed using a Shimadzu SIL-HT gradient HPLC and a Sciex API III triple quadrupole mass spectrometer. Chromatography was carried out on a 10 cm x 2.1 mm i.d. C8 reverse-phase column under isocratic conditions using a 35% aqueous acetonitrile in 10 mmol/L ammonium acetate at a flow rate of 0.4 mL/min. The eluate was split so that 100 µL/min was diverted to the IonsprayTM interface operating at –2700 V. MRM experiments for catechin and its metabolites were performed in the positive ion mode. Selected molecular ions were passed to a collision cell containing Ar/N2 (90:10) and fragment ions detected in the third quadrupole. Isoflavonoids were detected using MRM with the following precursor ion/product ion transitions: daidzein (m/z 253/223), dihydrodaidzein (m/z 255/149), O-desmethylangolensin (m/z 257/108), equol (m/z 241/119), genistein (m/z 269/133), apigenin (m/z 269/149), phenolphthalein (m/z 317/93), and 4-methylumbelliferone (m/z 175/125). Similarly, MRM analysis was conducted by monitoring transitions from precursor ion to product ion—291/139 (catechin), 291/123 (catechin), 305/139 (methylcatechin), and 305/137 (methylcatechin). Areas under the peaks were determined using the MacQuan program provided by Sciex. They were normalized to the area of the internal standard apigenin and also to areas for known standards prepared fresh for each analysis. Mean concentrations and standard deviations were calculated for duplicate aliquots of each sample. The limits of detection for each isoflavonoid were 4–15 nmol/L starting with 500 µL serum.
Statistical analysis
The significance of the effects of GSE and genistein in this study was evaluated using either Student’s t test (2-way comparisons with P < 0.05 considered a significant difference) or Tukey’s adjustment for multiple comparisons (27).
|
Results |
|---|
|
TOPABSTRACTMaterials and methodsResultsDiscussionLITERATURE CITED |
|---|
|
|
|
|
|
|
|
|
|
|
Discussion |
|---|
|
TOPABSTRACTMaterials and methodsResultsDiscussionLITERATURE CITED |
|---|
Topical application of a DMSO-soluble extract of proanthocyanidin-enriched GSE significantly protected against phorbol ester-induced tumor promotion in a mouse model of skin cancer (19), suggesting that anticancer cell growth activity resided in this oligomeric polyphenol fraction from grape seeds. In addition, the GSE oligomers were suggested to be the active agents inhibiting growth of MDA-MB468 cells (16). However, the uptake of the oligomeric proanthocyanidins was not demonstrated in either study. Moreover, catechin and epicatechin were 10% of the GSE extract used in these experiments, raising the possibility that the inhibition of growth of the cells may have been due to the monomeric polyphenols. ESI-MS analysis in the present study revealed that whereas GSE contained oligomers (up to heptamers) of polyphenols, as well as monomeric catechins, only monomeric catechin-based polyphenols and their metabolites were detected in the urine (under the same extraction conditions) (Fig. 3). Follow-up experiments need to be carried out using proanthocyanidin preparations from grape seed depleted of monomeric catechins to confirm that the catechin in the urine from animals that had ingested GSE was generated by metabolism of the higher oligomers. Our initial ESI-MS results suggest that the proanthocyanidins in GSE are metabolized to catechins and catechin derivatives, and that these metabolites may be the chemopreventive agents. These results are consistent with 2 previous studies in rats in which ingested procyanidin dimers were not detected in the urine (34,35). Our findings are also consistent with those of Rice-Evans and co-workers (36), who found monomers but not higher oligomers of epicatechin in the brains of rats after GSE ingestion. However, uptake and metabolism of procyanidins may be different in humans because procyanidin B1 dimers were detected in serum after GSE intake (37); similarly, procyanidin B2 dimer was detected in plasma after consumption of flavanol-rich cocoa (38).
Recently, Eng et al. (15) found that pretreatment of athymic ovariectomized mice with a polyphenol preparation enriched in procyanidin-dimers purified from red wine inhibited proliferation of MCF-7 cells overexpressing aromatase. The suggestion that the procyanidin dimers inhibited the cells via inhibition of aromatase was supported by the dimers directly inhibiting the enzyme in vitro (15). The failure to detect procyanidin dimers in the animals in the present study, however, suggests that monomeric catechin metabolites of the proanthocyanidins may also inhibit aromatase or that other targets in addition to aromatase may be important. This is consistent with the observation that other monomeric flavonoids inhibit aromatase in vitro (39).
Earlier experiments showed that prepubertal exposure to genistein afforded significant protection against DMBA-induced mammary tumorigenesis (29). However, whereas administering genistein in AIN-76A to adult rats that had been bred and grown in a genistein-free environment did not protect against carcinogen-induced mammary tumors, exposure to genistein during neonatal and prepubertal development combined with exposure in adult life afforded greater protection against DMBA than did the neonatal and prepubertal exposure alone (30). The results presented here suggest that the genistein administered only to the adult rats might have had efficacy against the DMBA-induced breast cancer had it been administered under different dietary conditions. Most investigators obtain the animals for experimentation from commercial breeders and have no control of the diet during the critical early days of development; this could be an important source of variation in their experiments because prepubertal exposure of rats to genistein leads to increased mammary differentiation (29,40).
The mechanism by which dietary composition influences chemopreventive efficacy of GSE or genistein remains to be determined. The AIN-76A and 4% Teklad diets, although providing roughly the same amount of protein and same overall nutritional content, differ substantially in the complexity and source of their protein (milk casein for AIN-76A vs. a mixture of soy, wheat, and barley proteins for the Teklad lab diet) as well as in a number of other components. Therefore, it is not unreasonable that different bioactivities might be detected for the same chemical administered in the 2 diets.
The 4% Teklad diet contained 
0.4 mg/g diet isoflavones (Table 1). The animals on this diet, unsupplemented, were thus exposed to a basal level of isoflavones, as shown by a mean genistein serum concentration of 372 nmol/L. Switching from this diet to the isoflavone-free AIN-76A diet may have been the basis for the increased number of MNU-induced mammary tumors observed in previous animal studies (9). In the present study, the supplementation of the 4% Teklad diet with genistein increased the serum genistein concentration to 1182 nmol/L for the 0.2 g/kg dietary dose and to 5327 nmol/L for the 2.0 g/kg dose. These increases were directly proportional to the dose of genistein (Fig. 9).
|
In summary, we demonstrate that GSE is chemopreventive against DMBA-induced breast cancer in adult rats, that genistein exhibits similar activity in the MNU rat model of breast cancer, and that the effects of both grape seed and genistein depend on the diet in which they are administered. The diet dependency of the chemopreventive actions of grape seed and genistein suggests that previous studies where no chemopreventive activity was noted for a compound of interest may warrant retesting in a different diet to ensure that the sought bioactivity was not missed. The modulatory role of the diet in these rat studies suggests that clinical chemopreventive or chemotherapeutic interventions against breast or other cancers might need to consider the overall diet of the patient while such interventions are administered.
|
FOOTNOTES |
|---|
2 This research was supported by a grant (DAMD17-01-0469) to HK from the US Army Medical Research and Materiel Command and by a subcontract from Purdue University to HK and SB as part of the Purdue/UAB Botanicals Center for Age-Related Disease, that was supported by grant (P50 AT00477) to Connie Weaver (P.I.) from the National Center for Complementary and Alternative Medicine. Support for the purchase and installation of the mass spectrometer used at UAB was provided by a Shared Instrumentation grant (S10 RR06487) from the National Center for Research Resources and by funds from the UAB Office of the Provost. Ongoing support for the operation of the mass spectrometry facility has been provided by a grant (P30 CA13148) from the National Cancer Institute to the UAB Comprehensive Cancer Center (Al Lobuglio, PI). This work was presented in part at the 2000 annual meeting of the American Association for Cancer Research.
4 Abbreviations used: DMBA, 7,12-dimethylbenz[a]anthracene; GSE, grape seed extract; LC-ESI-MS, LC-electrospray ionization mass spectrometry; MNU, N-methyl-N-nitrosourea; MRM, multiple-reaction ion monitoring; TFA, trifluoroacetic acid.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTMaterials and methodsResultsDiscussionLITERATURE CITED |
|---|
1. Arias, E., Anderson, R. N., Kung, H. C., Murphy, S. L. & Kochanek, K. D. (2003) Deaths: final data for 2001. Natl. Vital Stat. Rep. 52:1-115.[Medline]
2. Shibuya, K., Mathers, C. D., Boschi-Pinto, C., Lopez, A. D. & Murray, C. J. (2002) Global and regional estimates of cancer mortality and incidence by site: II. Results for the global burden of disease 2000. BMC Cancer 2:37.[Medline]
3. Maskarinec, G. (2000) Breast cancer-interaction between ethnicity and environment. In Vivo 14:115-123.[Medline]
4. Doll, R. (1980) The epidemiology of cancer. Cancer 45:2475-2485.[Medline]
5. Gerber, M. (2001) The comprehensive approach to diet: a critical review. Nutrition 131:3051S-3055S.
6. Tallman, M. S. & Wiernik, P. H. (1992) Retinoids in cancer treatment. J. Clin. Pharmacol. 32:868-888.[Abstract]
7. Moon, R. C., Thompson, H. J., Becci, P. J., Grubbs, C. J., Gander, R. J., Newton, D. L., Smith, J. M., Phillips, S. L., Henderson, W. R., Mullen, L. T., Brown, C. C. & Sporn, M. B. (1979) N-(4-Hydroxyphenyl)retinamide, a new retinoid for prevention of breast cancer in the rat. Cancer Res. 39:1339-1346.
8. Eat 5 to 9 A Day for Better Health [Online] National Cancer Institute http://www.5aday.gov/ [accessed Aug. 17, 2004].
9. Grubbs, C. J., Juliana, M. M., Hill, D. L. & Whitaker, L. M. (1987) Effect of laboratory diets on methylnitrosourea (MNU)-induced mammary cancers and on the efficacy of a chemopreventive agent (retinyl acetate). Proc. Am. Assoc. Cancer Res. 28:160.
10. Barnes, S., Grubbs, C., Setchell, K.D.R. & Carlson, J. (1990) Soybeans inhibit mammary tumors in models of breast cancer. Prog. Clin. Biol. Res. 347:239-253.[Medline]
11. Park, O. J. & Surh, Y. J. (2004) Chemopreventive potential of epigallocatechin gallate and genistein: evidence from epidemiological and laboratory studies. Toxicol. Lett. 150:43-56.[Medline]
12. Barnes, S. & Peterson, T. G. (1995) Biochemical targets of the isoflavone genistein in tumor cell lines. Proc. Soc. Exp. Biol. Med. 208:103-108.[Medline]
13. Mgbonyebi, O. P., Russo, J. & Russo, I. H. (1998) Antiproliferative effect of synthetic resveratrol on human breast epithelial cells. Int. J. Oncol. 12:865-869.[Medline]
14. Yamaguchi, F., Yoshimura, Y., Nakazawa, H. & Ariga, T. (1999) Free radical scavenging activity of grape seed extract and antioxidants by electron spin resonance spectrometry in an H2O2/NaOH/DMSO system. J. Agric. Food Chem. 47:2544-2548.[Medline]
15. Eng, E. T., Ye, J., Williams, D., Phung, S., Moore, R. E., Young, M. K., Gruntmanis, U., Braunstein, G. & Chen, S. (2003) Suppression of estrogen biosynthesis by procyanidin dimers in red wine and grape seeds. Cancer Res. 63:8516-8522.
16. Agarwal, C., Sharma, Y., Zhao, J. & Agarwal, R. (2000) A polyphenolic fraction from grape seeds causes irreversible growth inhibition of breast carcinoma MDA-MB468 cells by inhibiting mitogen-activated protein kinases activation and inducing G1 arrest and differentiation. Clin. Cancer Res. 6:2921-2930.
17. Sharma, G., Tyagi, A. K., Singh, R. P., Chan, D. C. & Agarwal, R. (2004) Synergistic anti-cancer effects of grape seed extract and conventional cytotoxic agent doxorubicin against human breast carcinoma cells. Breast Cancer Res. Treat. 85:1-12.[Medline]
18. Zhao, J., Wang, J., Chen, Y. & Agarwal, R. (1999) Anti-tumor-promoting activity of a polyphenolic fraction isolated from grape seeds in the mouse skin two-stage initiation-promotion protocol and identification of procyanidin B5–3'-gallate as the most effective antioxidant constituent. Carcinogenesis 20:1737-1745.
19. Yamakoshi, J., Saito, M., Kataoka, S. & Tokutake, S. (2002) Procyanidin-rich extract from grape seeds prevents cataract formation in hereditary cataractous (ICR/f) rats. J. Agric. Food Chemistry 50:4983-4988.[Medline]
20. Bomser, J. A., Singletary, K. W., Wallig, M. A. & Smith, M.A.L. (1999) Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds. Clin. Cancer Res. 135:151-157.
21. Huggins, C., Moon, R. C. & Morii, S. (1962) Extinction of experimental mammary cancer. I. Estradiol-17 beta and progesterone. Proc. Natl. Acad. Sci. U.S.A. 48:379-386.
22. Peterson, T. G. & Barnes, S. (1991) Genistein inhibition of the growth of human breast cancer cells: independence from estrogen receptors and the multi-drug resistance gene. Biochem. Biophys. Res. Commun. 179:661-667.[Medline]
23. Moon, R. C., Grubbs, C. J., Sporn, M. B. & Goodman, D. G. (1977) Retinyl acetate inhibits mammary carcinogenesis induced by N-methyl-N-nitrosourea. Nature 267:620-621.[Medline]
24. Coward, L., Smith, M., Kirk, M. & Barnes, S. (1998) Chemical modification of isoflavones in soy foods during cooking and processing. Am. J. Clin. Nutr. 68:1486S-1491S.[Abstract]
25. Coward, L., Kirk, M., Albin, N. & Barnes, S. (1996) Analysis of plasma isoflavones by reversed-phase HPLC-multiple reaction ion monitoring-mass spectrometry. Clin. Chim. Acta 247:121-142.[Medline]
26. Urban, D., Irwin, W., Kirk, M., Markiewicz, M. A., Myers, R., Smith, M., Weiss, H., Grizzle, W. E. & Barnes, S. (2001) The effect of isolated soy protein on plasma biomarkers in elderly men with elevated serum prostate specific antigen. J. Urol. 165:294-300.[Medline]
27. Tukey, J. (1991) The philosophy of multiple comparisons. Stat. Sci. 6:100-116.
28. Cren-Olive, C., Deprez, S., Lebrun, S., Coddeville, B. & Rolando, C. (2000) Characterization of methylation site of monomethylflavan-3-ols by liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 14:2312-2319.[Medline]
29. Fritz, W. A., Coward, L., Wang, J. & Lamartiniere, C. A. (1998) Dietary genistein: perinatal mammary cancer prevention, bioavailability and toxicity testing in the rat. Carcinogenesis 19:2151-2158.
30. Lamartiniere, C. A., Cotroneo, M. S., Fritz, W. A., Wang, J., Mentor-Marcel, R. & Elgavish, A. (2002) Genistein chemoprevention: timing and mechanisms of action in murine mammary and prostate. J. Nutr. 132:552S-558S.
31. Masquelier, J., Michaud, J., Laparra, J. & Dumon, M. C. (1979) Flavonoids et pycnogenols. Intl. J. Vitamin Nutritional Research 49:307-311.
32. Murray, M. & Pizzorno, J. (1999) Procyanidolic oligomers. Murray, M. Pizzorno, J. eds. The Textbook of Natural Medicine 2nd ed. 1999:899-904 Churchill Livingston London, UK. .
33. Singletary, K. W., Stansbury, M. J., Giusti, M., Van Breemen, R. B., Wallig, M. & Rimando, A. (2003) Inhibition of rat mammary tumorigenesis by Concord grape juice constituents. J. Agric. Food Chem. 51:7280-7286.[Medline]
34. Nakamura, Y. & Tonogai, Y. (2003) Metabolism of grape seed polyphenol in the rat. J. Agric. Food Chem. 51:7215-7225.[Medline]
35. Donovan, J. L., Manach, C., Rios, L., Morand, C., Scalbert, A. & Remesy, C. (2002) Procyanidins are not bioavailable in rats fed a single meal containing a grapeseed extract or the procyanidin dimer B3. Br. J. Nutr. 87:299-306.[Medline]
36. Abd El Mohsen, M. M., Kuhnle, G., Rechner, A. R., Schroeter, H., Rose, S., Jenner, P. & Rice-Evans, C. A. (2002) Uptake and metabolism of epicatechin and its access to the brain after oral ingestion. Free Rad. Biol. Med. 33:1693-1702.[Medline]
37. Sano, A., Yamakoshi, J., Tokutake, S., Tobe, K., Kubota, Y. & Kikuchi, M. (2003) Procyanidin B1 is detected in human serum after intake of proanthocyanidin-rich grape seed extract. Biosci. Biotechnol. Biochem. 67:1140-1143.[Medline]
38. Holt, R. R., Lazarus, S. A., Sullards, M. C., Zhu, Q. Y., Schramm, D. D., Hammerstone, J. F., Fraga, C. G., Schmitz, H. H. & Keen, C. L. (2002) Procyanidin dimer B2 [epicatechin-(4beta-8)-epicatechin] in human plasma after the consumption of a flavanol-rich cocoa. Am. J. Clin. Nutr. 76:798-804.
39. Pelissero, C., Lenczowski, M. J., Chinzi, D., Davail-Cuisset, B., Sumpter, J. P. & Fostier, A. (1996) Effects of flavonoids on aromatase activity, an in vitro study. J. Steroid Biochem. Mol. Biol. 57:215-223.[Medline]
40. Cotroneo, M. S., Wang, J., Fritz, W. A., Eltoum, I. E. & Lamartiniere, C. A. (2002) Genistein action in the prepubertal mammary gland in a chemoprevention model. Carcinogenesis 23:1467-1474.
This article has been cited by other articles:
|
|
 |
|
  Y. Su, K. Shankar, and R. C. M. Simmen Early Soy Exposure via Maternal Diet Regulates Rat Mammary Epithelial Differentiation by Paracrine Signaling from Stromal Adipocytes J. Nutr., May 1, 2009; 139(5): 945 - 951. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Messina, S. Watanabe, and K. D. R. Setchell Report on the 8th International Symposium on the Role of Soy in Health Promotion and Chronic Disease Prevention and Treatment J. Nutr., April 1, 2009; 139(4): 796S - 802S. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Gao, A. Budhraja, S. Cheng, H. Yao, Z. Zhang, and X. Shi Induction of Apoptosis in Human Leukemia Cells by Grape Seed Extract Occurs via Activation of c-Jun NH2-Terminal Kinase Clin. Cancer Res., January 1, 2009; 15(1): 140 - 149. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Wen, J. Lu, K. Zhang, and S. Chen Grape Seed Extract Inhibits Angiogenesis via Suppression of the Vascular Endothelial Growth Factor Receptor Signaling Pathway Cancer Prevention Research, December 1, 2008; 1(7): 554 - 561. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Chatelain, S. Phippen, J. McCabe, C. A. Teeters, S. O'Malley, and K. Kingsley Cranberry and Grape Seed Extracts Inhibit the Proliferative Phenotype of Oral Squamous Cell Carcinomas Evid. Based Complement. Altern. Med., July 23, 2008; (2008) nen047v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Su, R. R. Eason, Y. Geng, S. Till, T. M. Badger, and R. C.M. Simmen In utero exposure to maternal diets containing soy protein isolate, but not genistein alone, protects young adult rat offspring from NMU-induced mammary tumorigenesis Carcinogenesis, May 1, 2007; 28(5): 1046 - 1051. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. C. Neto Cranberry and Its Phytochemicals: A Review of In Vitro Anticancer Studies J. Nutr., January 1, 2007; 137(1): 186S - 193S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kaur, R. P. Singh, M. Gu, R. Agarwal, and C. Agarwal Grape Seed Extract Inhibits In vitro and In vivo Growth of Human Colorectal Carcinoma Cells. Clin. Cancer Res., October 15, 2006; 12(20): 6194 - 6202. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Mantena, M. S. Baliga, and S. K. Katiyar Grape seed proanthocyanidins induce apoptosis and inhibit metastasis of highly metastatic breast carcinoma cells Carcinogenesis, August 1, 2006; 27(8): 1682 - 1691. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kim New Nutrition, Proteomics, and How Both Can Enhance Studies in Cancer Prevention and Therapy J. Nutr., November 1, 2005; 135(11): 2715 - 2718. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
