Flavonoid Fractions from Cranberry Extract Inhibit Growth of Human

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Chapter 12

Flavonoid Fractions from Cranberry Extract Inhibit Growth of Human Tumor Cell Lines Peter J. Ferguson,1,6,* David J. Freeman,2 and James Koropatnick1,3,4,5,6 1Department

of Oncology, 2Department of Medicine, 3Department of Physiology & Pharmacology, 4Department of Microbiology & Immunology, 5Department of Pathology, The University of Western Ontario, Cancer Research Laboratories - VRL, London Regional Cancer Program - LHSC, 790 Commissioners Road East, London, Ontario, Canada N6A 4L6 6Cancer Research Laboratory Program (Lawson Health Research Institute and London Regional Cancer Program), Cancer Research Laboratories - VRL, London Regional Cancer Program - LHSC, 790 Commissioners Road East, London, Ontario, Canada N6A 4L6 *[email protected]

In spite of improvements in cancer chemotherapy, approximately 50% of cancers are not cured and improvement is needed. Given the association of diets high in fruits and vegetables with lower cancer risk, these foodstuffs have been investigated as sources of compounds to both prevent cancer and treat established tumors. Flavonoids, found in high amounts in cranberries, contribute to improved human health. A flavonoid-rich extract of cranberries (Fr6) and isolated polymers of the flavan-3-ol class of cranberry flavonoids (proanthocyanidins, PACs) were investigated for anticancer activity. Both Fr6 and PACs inhibited proliferation of various human cancer cell lines when grown in culture (in vitro). When Fr6 or PACs were administered by injection to mice bearing explants of human tumor cell lines, growth of the tumors was delayed. When treating the prostate tumor cell line DU145 explant, 50% of tumors completely regressed. Both Fr6 and PACs were able to block cell cycle progression and induce cell death. Subfractionation of Fr6 by high performance liquid chromatography yielded fractions containing 3 detectable © 2010 American Chemical Society In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


peaks that exhibited most of the in vitro anticancer activity. Work continues on identifying these compounds, which appear to differ chemically from those identified in other laboratories. It is proposed that several components of cranberry will be identified for use in humans as a cancer preventive dietary supplement and, potentially, as a treatment for established tumors.

Introduction Major progress has been made in understanding the biology of human cancers, with concomitant design and discovery of novel chemotherapy agents. This has yielded a twofold increase in the number of approved anticancer drugs over the past 10 years. In spite of these gains, there remains a need for agents that can significantly improve life expectancy, either by eradicating tumors completely or by maintaining tumors in a dormant, harmless state. Currently, improvement in patient survival achieved using second and third line treatments is measured in only months or even weeks, at a cost of toxicity to non-cancerous tissues.With a very few important exceptions, cancer cures remain as elusive as they were 10 years ago. Therefore, the quest continues for agents that can inhibit tumor growth and allow patients a normal life expectancy. Treatment with agents that demonstrate selective toxicity to cancer cells while sparing normal cells, or when used concomitantly could enhance the selectivity of commonly used anticancer agents, would be ideal. To this end, many investigators have attempted to isolate components of edible plants as possible agents to selectively control or eradicate cancer without the toxic sequelae (1–7). Cranberries (Vaccinia macrocarpa) are a rich source of phytochemicals with known health benefits (8–10) . Extracts of this berry display antiproliferative activity against human tumor cell lines both in vitro and in vivo (8, 11–20). In a preliminary study conducted as collaboration between Ocean Spray, Inc. (Lakeville-Middleboro, MA), and KGK Synergize, Inc. (London, Ontario, Canada), it was observed that ingestion of cranberry juice or freeze-dried presscake slowed the growth and metastatic spread of human tumor cell line explants in immunodeficient (nude) mice (21). Since that time, our group has further characterized this in vivo inhibition of tumor growth and investigated mechanisms involved in the inhibition of proliferation and induction of cell death by cranberry extracts in human tumor cell lines. Collectively, our results and those of other research groups suggest that cranberries contain constituents that can inhibit tumor growth in vivo, to the point of inducing complete eradication of some tumors.

198 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Materials and Methods


Preparation of Cranberry Extracts Fr6 and PACs Cranberry extract and fractions were generously provided by Ocean Spray, Inc. Fraction 6 (Fr6) was isolated by standard chromatographic techniques from cranberry presscake as described previously (13). (Presscake is the remaining wet hulls after the juice is squeezed out.) Briefly, a 25EC-water extract of blended cranberry presscake was eluted through a reverse-phase C18 Flash 40M cartridge column (Biotage, Inc., Charlottesville, VA). After elution of inactive material using methanol and water, the Fr6 fraction was eluted using 1% acetic acid/methanol and freeze-dried. The proanthocyanidins (PAC) fraction was isolated from a whole cranberry extract by a similar process, except that the flavonoid-rich fraction (equivalent of Fr6) was subsequently eluted through an LH20 column using 50% ethanol followed by 80% acetone. The concentrations used hereafter refer to those of the freeze-dried powder on a wt:v basis, after solubilizing in the appropriate solvent (DMSO for cell culture, 10% DMSO in water for mouse injections).

Reverse Phase Chromatography of Fr6 As a first step in attempting to isolate individual antiproliferative components from Fr6, a sample of Fr6 (100 µg in 20 µl of methanol) was subjected to reversephase chromatography on a C18 column eluted over 20 min with a gradient of 10% to 90% methanol in 0.04% triethylamine/formic acid-buffered water (pH 3.0). Fractions of 4 ml (2 min) were collected, lyophilized, reconstituted in 10% DMSO, and tested for antiproliferative activity as described below. The final concentration of individual fractions tested on the cells was equivalent to a 60 µg/ml treatment of total Fr6.

In Vitro Assays of Antiproliferative Activity Cell culture medium and fetal bovine serum were purchased from Invitrogen, Inc. (Burlington, Ontario, Canada). Cell culture plasticware was obtained from Invitrogen, Fisher Scientific (Unionville, Ontario), and VWR International (Mississauga, Ontario). The MDA-MB-435 cell line was generously provided by Dr. Janet Price, University of Texas M. D. Anderson Cancer Center (Houston, TX). All other cell lines were purchased from American Type Culture Collection (Rockville, MD). Cultured cell lines were maintained and cytotoxicity assays conducted as described previously (13). Briefly, proliferation was assayed in 96-well plates by staining of cells using the vital stain alamarBlue™ (BioSource International, Inc., Camarillo, CA), and quantitated by fluorescence on a Wallac Victor plate reader (Perkin Elmer, Boston, MA). Concentrations of extract that inhibited proliferation of cultured cells by 50% (IC50 values) were determined by interpolation of plotted data. 199 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


In Vivo Experiments Female immunocompromised NCR-nu/nu mice, 4-6 weeks old (Taconic, Germantown, NY), were housed in a temperature- and humidity-controlled environment, with a 12 h light:dark cycle, in a pathogen-free isolation facility regulated and controlled by the University of Western Ontario. The mice were maintained according to the "Guide for the Care and Use of Laboratory Animals," set by the Canadian Council on Animal Care and adopted by The University of Western Ontario. Mice were fed a standard laboratory diet ad libitum. Tumors were established by harvesting rapidly proliferating, cultured cells, and injecting them subcutaneously into the right flank. Numbers of cells injected, in 75 :l saline, for each cell line were: U87, 1 x 106; HT-29, 5 x 106; DU145, 4 x 106. When primary tumors become palpable, their maximum length and width were measured every 2 days with calipers. Volume was calculated assuming an ellipsoidal tumor shape (length x width2 x B/6). When tumors became larger than 1 cm3, mice were euthanized by CO2 asphyxiation. Control groups (8 mice) and groups that received cranberry extracts (6 mice) were administered vehicle (80 :l of 10% DMSO in water), PAC (100 mg/kg), or Fr6 (250 mg/kg), respectively, delivered intraperitoneally (i.p.) in a volume of not more than 80 :l of 10% DMSO in water. Animals in control groups did not exhibit any detrimental effects. Injections were on the following days after tumor implant: 3, 5, 7, 10, 12, 14, 17, 19, 21, 24 (total of 10 injections).

Flow Cytometric Assay of DNA (Cell Cycle) Distribution Fr6- or PAC-treatment of U87 cells in 75-cm2 flasks was initiated by introduction of 0.2 volume of a sixfold concentration of Fr6 or PAC in growth medium. Following the indicated incubation period, cells were harvested by trypsinization, and nuclei were prepared, treated with RNase A, and stained using propidium iodide (PI) (22). The nuclei were analyzed by flow cytometry (XL-MCL Flow Cytometer, Beckman Coulter, Hialeah, FL). DNA (cell cycle) distributions were analyzed using Multicycle for Windows Advanced DNA Cell Cycle Analysis software (Phoenix Flow Systems, San Diego, CA).

Flow Cytometric Assay of Apoptosis Cultures were prepared and exposed to Fr6 or PAC as described above. Cells were harvested and, without fixing, stained with fluorescein-conjugated annexin-V (BD Biosciences, Mississauga, ON, Canada) and PI. Cells positive for annexin V and/or PI were considered to be dead or dying.


Table I. Antiproliferative activity of Fr6 and PAC against a panel of human tumor cell lines (Adapted with permission from references (13) and (14). Copyrights 2004 American Society for Nutrition and 2006 Lawrence Erlbaum Associates, Inc., respectively.)


IC50 (:g/ml) Cell Line

Tissue

Fr6

PAC

U87

glioma

128 ± 25 (7)a

47.8 ± 12.2 (7)

HT-29

colon

168 ± 69 (4)

79 ± 5 (2)

DU145

prostate (AR+)b

234 ± 75 (4)

95.8 ± 20 (5)

LNCaP

prostate (AR-)

9.9 ± 4.2 (3)

MCF-7

breast (ER+)

147 ± 22 (3)

MDA-MB-435

breast (ER-)

212 ± 50. (4)

melanoma

147 ± 47 (4)

SCLC

21.1 ± 1.9 (4)

SK-MEL-5 DMS114 a

mean ± SD (n). lung carcinoma.

b

AR - androgen receptor; ER - estrogen receptor; SCLC - small cell

Statistical Analysis Differences in end-points, between treated and control groups, were compared using an unpaired, two-tailed, Student’s t-test for groups with unequal variances (Microsoft Excel). The end-points were: mean tumor sizes; length of time to reach specified tumor volumes; cell cycle distribution; size of populations of dead and dying cells; relative proliferation of extract-treated cells. In all analyses, differences were considered to be statistically significant if the P value was less than 0.05.

Results A series of human tumor cell lines was screened for sensitivity to cranberry preparations Fr6 and PAC. The cell lines reported in Table I are representative of tumors from 6 different tissues. Sensitivity to cranberry extracts was similar among cell lines, with the exception of the high sensitivity of LNCaP and DMS114 lines to Fr6. Before testing Fr6 and PAC against tumors in nude mice, it was necessary to determine safe doses at which these preparations could be used in animals. Doses of 100 mg/kg PAC and 250 mg/kg Fr6, injected i.p. every 2 days for 3 weeks, caused minor toxicities that were manageable and from which the mice recovered: skin petechiae that resolved spontaneously after 3 or 4 days, and weight loss (less than 20%) from which the animals recovered during or immediately after the treatment period. 201 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 1. Effect of PAC treatment on time required for HT-29 tumors to reach milestone sizes in mice. HT-29 colon carcinoma cells (5 x 106) were implanted subcutaneously into the flank of female nude mice, and mice received 10 treatments with PAC (control n = 8, treated n = 6; means ± SEM). The length of time for tumors to reach the volumes indicated were determined by interpolation of plotted sizes of tumors versus time, for each individual animal. *: P < 0.05. (Reproduced with permission from reference (14). Copyright 2006 Lawrence Erlbaum Associates, Inc.)

The ability of PAC to inhibit tumor growth in vivo was then investigated using tumor cell lines derived from three different tissues: colon, prostate and brain. For these experiments, mice were given 10 injections over 3 weeks, beginning 3 days following inoculation of tumor cells. PAC significantly inhibited the growth of both HT-29 colon tumor and DU145 prostate tumor in nude mice (Figures 1 and 2). PAC significantly delayed HT-29 tumor growth by approximately 10 to 14 d, equivalent to a delay of 75% (the time to reach a volume of 200 mm3) to 34% (time to reach 1000 mm3) (Fig. 1). The average size of DU145 prostate tumors at 73 d was decreased by treatment (Figure 2). All control tumors reached a size of 1000 mm3 (at which point the animals must be euthanized) by 52 to 108 d. None of the control tumors regressed in size prior to reaching 1000 mm3. However, of the group of 4 PAC-treated tumors, only one reached a size of 600 mm3. Two of the PAC-treated tumors reached sizes of 400 and 500 mm3, respectively, but completely regressed by 108 d. A third PAC-treated tumor (585 mm3 at maximum size) had shrunk to 285 mm3 when the experiment was ended at 120 d, 96 days after treatment had ended. PAC treatment of U87 glioma explant-bearing mice increased the time required for the U87 tumors to reach milestone sizes of 400, 500 and 600 mm3 by approximately 48-59% (5 to 7.5 days; P < 0.05; data not shown). This cell line was selected for further characterization of effect of Fr6 and PACs on tumor cell proliferation.



Figure 2. Effect of PAC treatment on growth of DU145 tumors in mice. DU145 prostate carcinoma cells (4 x 106) were implanted subcutaneously into the flank of female, nude mice, and mice received 10 treatments with PAC. (control n = 8, treated n = 6; means ± SEM) *: P < 0.05. (Reproduced with permission from reference (14). Copyright 2006 Lawrence Erlbaum Associates, Inc.)

Fr6 and PAC were tested for their ability to alter cell cycle kinetics of U87 cells by analyzing DNA distribution by flow cytometry after 24 h of exposure (Figure 3). The tested concentrations of Fr6 and PAC inhibited proliferation (i.e., decreased cell numbers at 4 d compared to untreated control cells). Both Fr6 and PAC induced a significant decrease in the proportion of cells in S-phase by 24 h, an increase in G1 fraction, and no change in the G2/M fraction. The ability of Fr6 and PAC to induce cell death was tested using a flow cytometric, double-staining method for cells actively undergoing apoptosis and dead cells. Cells that were positive for annexin V and PI were considered dying or dead, including those killed by non-apoptotic mechanisms, and are collectively referred to as “dead and dying cells” (Figure 4). At concentrations that caused inhibition of proliferation by 4 d, both cranberry extract preparations induced significant cell death. Paclitaxel was included as a positive control. Even though Fr6 comprises only 5% of the ingredients in the cranberry presscake extract, it appears that most of Fr6 consists of components that do not have cytotoxic activity. Figure 5 is a chromatogram developed during the fractionation of Fr6. Each fraction was tested for cytotoxic activity against the sensitive LNCaP prostate cancer cell line. Two fractions that contained less than 10% of the material in Fr6 were found to contain the majority of inhibitory activity. Furthermore, a preliminary chromatographic subfractionation of the strongest fraction from this elution indicates that a minor component of this fraction may contain most of the inhibitory activity (Freeman and Ferguson, data not shown).



Figure 3. Cell cycle distribution analysis of Fr6-treated and PAC-treated U87 cells. Data points represent summarized data from 3 to 5 separate determinations (means ± SD). The percentages of cells in G0/G1 phase, S phase and G2/M phases were determined at 24 h. Relative cell density (96-well plate, alamar Blue assay) was determined at 4 d for each exposure for each experiment. IC50 values for Fr6 and PAC for this set of experiments were, respectively, 128.4 ± 25.1 mg/L and 47.8 ± 12.2 mg/L (means ± SD, n = 5). *: P < 0.05 compared to DMSO control. (Adapted with permission from reference (14). Copyright 2006 Lawrence Erlbaum Associates, Inc.)

Discussion The presence of compounds with anticancer activity in safely consumed foods conservatively suggests that agents might be isolated that selectively kill cancer cells, sparing normal tissues and achieving local tumor control or cures. Crude extracts and individual components obtained from fruits and berries are able to not only slow cell growth, but can kill cancer cells outright. However, these activities are observed at concentrations that are not likely achievable through normal dietary consumption. The observed capacity of components to induce cell death suggests that anticancer activity may entail, at least at higher doses, some adverse effect 204 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 4. Induction of cell death in Fr6- and PAC-treated U87 cells. Panel A: after 48 h, cells were tested by staining with annexin V and propidium iodide (Panel A). Bars indicate the percent of the entire population of cells that was dead and dying, summarized from 3 to 4 experiments (means ± SD). Panel B: relative proliferation of treated cells, as a function of live cell density after 4 d (n = 3, means ± SD). Pac’l, paclitaxel. *: P < 0.05 compared to DMSO control. (Adapted with permission from reference (14). Copyright 2006 Lawrence Erlbaum Associates, Inc.) on normal cells. Nevertheless, the optimism about anticancer phytochemicals has been fortified by demonstration that crude extracts and purified components inhibit tumor growth in tumor-bearing mice without untoward toxicity to the animal. Therefore, efforts are increasing to purify, identify and characterize individual components of many different fruits and berries with the goal of obtaining agents that prevent cancer formation or progression, or kill tumor cells outright. Several groups of investigators have made significant contributions to our understanding of the anticancer activity of cranberries. This literature has been reviewed elegantly and thoroughly by Dr. Neto (9, 23, 24), and so will not be discussed comprehensively in this report. However, it is worthwhile to place these findings in an historical perspective. Following the initial observation by Guthrie that cranberry products could inhibit growth of a human tumor cell line explant in nude mice (3, 21), various groups demonstrated growth inhibition and induction of cell death in tumor cell lines in vitro (11–14, 16–20, 25–27). Cranberry extracts also inhibit cellular activities associated with malignant progression and tumor aggressiveness (15). Most of these studies utilized extracts that were enriched in flavonoids and/or PACs, but it was not known which components were responsible for the cytotoxic activity. The individual components have now been well characterized, and some are strong candidates as mediators of anticancer activity. Cranberries contain compounds from each of 205 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 5. Inhibition of proliferation of LNCaP prostate tumor cell line by subfractions of Fr6, separated by HPLC. Fractions of 4 ml were lyophilized, reconstituted in 10% DMSO, and assayed for the ability to inhibit proliferation of LNCaP cells (closed boxes). The graph of the HPLC elution represents ultraviolet absorbance of the various peaks that were eluted. four major groups of flavonoids: proanthocyanidins, anthocyanins, flavan-3-ols (catechins) and flavonols (28–32). Glycosides of quercetin and myricetin qualify as potential candidate anticancer compounds from cranberry (8, 24, 33–37), but have not been unequivocally demonstrated as such in purified cranberry extracts. Some purified components have, in fact, been demonstrated to inhibit tumor cell growth, including hydroxycinnamoyl ursolic acid (11) and a family of PACs (related by their subunit structure and ranging in size from 2 to 12 units in length) (12, 27). The definitive experiment will be to demonstrate antitumor activity of these compounds in vivo. The extracts used in the studies reported herein were obtained from cranberry presscake (the material remaining following the extraction of juice) (Fr6) or from homogenized whole berry (PAC). The initial extract from whole cranberry or from presscake contains sugars, proteins, acids, and waxy materials that have no antiproliferative activity. These are separated from Fr6 and PACs by the initial chromatographic process. Both sources are believed to yield the majority of flavonoids in cranberry, but PACs are expected to be exclusive of flavonols and their glycosides. Both Fr6 and PACs significantly inhibited proliferation of tumor cell lines in vitro (13, 14). PACs inhibited the growth of 3 cell lines in vivo, and Fr6 inhibited growth of U87 in vivo. The effect of the PAC fraction in inducing regression of explant tumors of the androgen receptor-negative cell line DU145 raises the possiblity of a clinical role for this family of polymeric epicatechins. Prostate tumors are generally refractory to chemotherapy after they have converted from being anti-androgen responsive to non-responsive (38), indicating a major need for alternate therapy. Plant and fruit flavonoids inhibit proliferation of the DU145 cell line (30, 39). Therefore some prostate tumors may be sensitive to these components. The mechanisms by which flavonoids inhibit tumor growth are only partially understood. Flavonoids can induce cell death or cell cycle arrest depending on the 206 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


flavonoid and on the concentration (5, 40–44). Also, arrest may occur in G1 or G2/M, depending on the cell type (5, 13, 14, 40). This variation between G1 and G2/M arrest was observed between our cell lines using the same preparation of Fr6 (13, 14). Optimal use of flavonoids to control cancer will require significant progress in mechanistic studies. Besides our demonstration of antitumor activity in vivo, there has been only one other report of in vivo inhibition of tumor explant growth (19). The observed in vivo anticancer activity of cranberry extract, with minimal toxicity to the host animal, suggests that certain components of cranberries may have selective inhibitory activity against tumor cells and therefore may have clinical applicability as a treatment against established tumors. Cranberry also demonstrates cancer preventive activity (23, 45), a property that could be an important basis of a preventative dietary supplement. Therefore, cranberry or components thereof are expected to act as cancer preventive agents, not only through their antioxidant properties [which are not discussed above but have been reviewed elsewhere (8, 24, 25)], but possibly through cytotoxic action against microscopic tumors or premalignant foci. Such small clusters of tumor cells, particularly those in a proliferative state, may be more sensitive to low concentrations of circulating cranberry constituents achieved by dietary consumption. More needs to be understood, however, about the pharmacokinetics of active components, what concentrations are achievable when consumed safely and comfortably in the diet, and whether the isolated active components will function as efficiently as a mixture. Clearly, there remain several important questions to be answered by future studies in order to optimize benefit from this valuable berry.

Acknowledgments Supported by a grant from the American Institute for Cancer Research. The authors would like to acknowledge the technical and intellectual contributions of the following people, some of whom were co-authors on previous manuscripts from which portions of this article were reproduced: Najla Guthrie, Dr. Ela Kurowska, Ling He, Jennifer MacDonald, and Dr. Rinee Mukherjee, KGK Synergize, Inc.; Wendy Brown and Michael Keeney, London Health Sciences Centre; Drs. Ann Chambers, David Bailey, and Karen Morley, The University of Western Ontario; Dr. Marge Leahy, David Cunningham, Richard Turk, Donna Ratacik, and Robin Roderick, Ocean Spray, Inc.

Abbreviations AR, androgen receptor; ER, estrogen receptor; Fr6, Fraction 6 of cranberry presscake extract; IC50, concentration that inhibits proliferation by 50%; i.p., intraperitoneally; PAC, proanthocyanidin fraction from whole cranberry; PI, propidium iodide.


References 1. 2.


3.

4.

5.

6.

7.

8.

9. 10. 11.

12.

13.

14.

15.

Waladkhani, A. R.; Clemens, M. R. Effect of dietary phytochemicals on cancer development. Int. J. Mol. Med. 1998, 1, 747–753. Hollman, P. C.; Katan, M. B. Health effects and bioavailability of dietary flavonoids. Free Radical. Res. 1999, 31 (Suppl.), S75–S80. Guthrie, N.; Carroll, K. K. Inhibition of Human Breast Cancer Cell Growth and Metastasis in Nude Mice by Citrus Juices and Their Constituent Flavonoids. In Biological Oxidants and Antioxidants. Molecular Mechanisms and Health Effects; Packer, L., Ong, A.S.H., Eds.; AOCS Press: Champaign, IL, 1998; pp 310−316. Brownson, D. M.; Azios, N. G.; Fuqua, B. K.; Dharmawardhane, S. F.; Mabry, T. J. Flavonoid effects relevant to cancer. J. Nutr. 2002, 132, 3482S–3489S. Choi, J. A.; Kim, J. Y.; Lee, J. Y.; Kang, C. M.; Kwon, H. J.; Yoo, Y. D.; Kim, T. W.; Lee, Y. S.; Lee, S. J. Induction of cell cycle arrest and apoptosis in human breast cancer cells by quercetin. Int. J. Oncol. 2001, 19, 837–844. Liao, S.; Umekita, Y.; Guo, J.; Kokontis, J. M.; Hiipakka, R. A. Growth inhibition and regression of human prostate and breast tumors in athymic mice by tea epigallocatechin gallate. Cancer Lett. 1995, 96, 239–243. Chen, Z. P.; Schell, J. B.; Ho, C. T.; Chen, K. Y. Green tea epigallocatechin gallate shows a pronounced growth inhibitory effect on cancerous cells but not on their normal counterparts. Cancer Lett. 1998, 129, 173–179. He, X.; Liu, R. H. Cranberry phytochemicals: isolation, structure elucidation, and their antiproliferative and antioxidant activities. J. Agric. Food Chem. 2006, 54, 7069–7074. Neto, C. C. Cranberry and its phytochemicals: a review of in vitro anticancer studies. J. Nutr. 2007, 137, 186S–193S. Henig, S. Y.; Leahy, M. H. Cranberry juice and urinary-tract health: science supports folklore. Nutrition 2000, 16, 684–687. Murphy, B. T.; MacKinnon, S. L.; Yan, X.; Hammond, G. B.; Vaisberg, A. J.; Neto, C. C. Identification of triterpene hydroxycinnamates with in vitro antitumor activity from whole cranberry fruit (Vaccinium marcocarpon). J. Agric. Food Chem. 2003, 51, 3541–3545. Seeram, N. P.; Adams, L. S.; Hardy, M. L.; Heber, D. Total cranberry extract versus its phytochemical constituents: Antiproliferative and synergistic effects against human tumor cell lines. J. Agric. Food Chem. 2004, 52, 2512–2517. Ferguson, P. J.; Kurowska, E.; Freeman, D. J.; Chambers, A. F.; Koropatnick, D. J. A flavonoid fraction from cranberry extract inhibits proliferation of human tumor cell lines. J. Nutr. 2004, 134, 1529–1535. Ferguson, P. J.; Kurowska, E.; Freeman, D. J.; Chambers, A. F.; Koropatnick, D. J. In vivo inhibition of growth of human tumor lines by flavonoid fractions from cranberry extract. Nutr. Cancer 2006, 56, 86–94. Kondo, M.; Lamoureaux, T. L.; Neto, C. C.; Hurta, R. A. R.; Curtis, S.; Matchett, M. D.; Yeung, H.; Sweeney-Nixon, M. I.; Vaisberg, A. J. Proanthocyanidins, anthocyanins, and triterpenoids from cranberry fruits: 208 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

16.


17.

18.

19.

20.

21. 22.

23. 24. 25.

26.

27.

28.

29.

Antitumor activity and effects on matrix metalloproteinase expression. J. Nutr. 2004, 134 (12S), 3538S. Seeram, N. P.; Adams, L. S.; Zhang, Y.; Lee, R.; Sand, D.; Scheuller, H. S.; Heber, D. Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro. J. Agric. Food Chem. 2006, 54, 9329–9333. Boivin, D.; Blanchette, M.; Barrette, S.; Moghrabi, A.; Béliveau, R. Inhibition of cancer cell proliferation and suppression of TNF-induced activation of NF6B by edible berry juice. Anticancer Res. 2007, 27, 937–948. Kresty, L. A.; Howell, A. B.; Baird, M. Cranberry proanthocyanidins induce apoptosis and inhibit acid-induced proliferation of human esophageal adenocarcinoma cells. J. Agric. Food Chem. 2008, 56, 676–680. Hochman, N.; Houri-Haddad, Y.; Koblinski, J.; Wahl, L.; Roniger, M.; Bar-Sinai, A.; Weiss, E. I.; Hochman, J. Cranberry juice constituents impair lymphoma growth and augment the generation of antilymphoma antibodies in syngeneic mice. Nutr. Cancer 2008, 60, 511–517. Liu, M.; Lin, L. Q.; Song, B. B.; Wang, L. F.; Zhang, C. P.; Zhao, J. L.; Liu, J. R. Cranberry phytochemical extract inhibits SGC-7901 cell growth and human tumor xenografts in Balb/c nu/nu mice. J. Agric. Food Chem. 2009, 57, 762–768. Guthrie, N. Effect of cranberry juice and products on human breast cancer cell growth. FASEB J. 2000, 14, 531.13. Vindeløv, L. L.; Christensen, I. J.; Nissen, N. I. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 1983, 3, 323–327. Neto, C. C. Cranberry and blueberry: evidence for protective effects against cancer and vascular diseases. Mol. Nutr. Food Res. 2007, 51, 652–664. Neto, C. C.; Amoroso, J. W.; Liberty, A. M. Anticancer activities of cranberry phytochemicals: an update. Mol. Nutr. Food Res. 2008, 52, S18–S27. Yan, X.; Murphy, B. T.; Hammond, G. B.; Vinson, J. A.; Neto, C. C. Antioxidant activities and antitumor screening of extracts from cranberry fruit (Vaccinium macrocarpon). J. Agric. Food Chem. 2002, 50, 5844–5849. Sun, J.; Liu, R. H. Cranberry phytochemical extracts induce cell cycle arrest and apoptosis in human MCF-7 breast cancer cells. Cancer Lett. 2006, 241, 124–134. Singh, A. P.; Singh, R. K.; Kim, K. K.; Satyan, K. S.; Nussbaum, R.; Torres, M.; Brard, L.; Vorsa, N. Cranberry proanthocyanidins are cytotoxic to human cancer cells and sensitize platinum-resistant ovarian cancer cells to paraplatin. Phytother. Res. 2009, 23, 1066–1074. Foo, L. Y.; Lu, Y.; Howell, A. B.; Vorsa, N. A-type proanthocyanidin trimers from cranberry that inhibit adherence of uropathogenic P-fimbriated Escherichia coli. J. Nat. Prod. 2000, 63, 1225–1228. Hammerstone, J. F.; Lazarus, S. A.; Schmitz, H. H. Procyanidin content and variation in some commonly consumed foods. J. Nutr. 2000, 130 (8S Suppl), 2086S–2092S. 209 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


30. Kampa, M.; Hatzoglou, A.; Notas, G.; Damianaki, A.; Bakogeorgou, E.; Gemetzi, C.; Kouroumalis, E.; Martin, P. M.; Castanas, E. Wine antioxidant polyphenols inhibit the proliferation of human prostate cancer cell lines. Nutr. Cancer 2000, 37, 223–233. 31. Kandil, F. E.; Smith, M. A.; Rogers, R. B.; Pepin, M. F.; Song, L. L.; Pezzuto, J. M.; Seigler, D. S. Composition of a chemopreventive proanthocyanidin-rich fraction from cranberry fruits responsible for the inhibition of 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity. J. Agric. Food Chem. 2002, 50, 1063–1069. 32. Gu, L.; Kelm, M.; Hammerstone, J. F.; Beecher, G.; Cunningham, D.; Vannozzi, S.; Prior, R. L. Fractionation of polymeric procyanidins from lowbush blueberry and quantification of procyanidins in selected foods with an optimized normal-phase HPLC-MS fluorescent detection method. J. Agric. Food Chem. 2002, 50, 4852–4860. 33. Yang, J. H.; Hsia, T. C.; Kuo, H. M.; Chao, P. D.; Chou, C. C.; Wei, Y. H.; Chung, J. G. Inhibition of lung cancer cell growth by quercetin glucuronides via G2/M arrest and induction of apoptosis. Drug Metab. Dispos. 2006, 34, 296–304. 34. Lu, J.; Papp, L. V.; Fang, J.; Rodriguez-Nieto, S.; Zhivotovsky, B.; Holmgren, A. Inhibition of mammalian thioredoxin reductase by some flavonoids: Implications for myricetin and quercetin anticancer activity. Cancer Res. 2006, 66, 4410–4418. 35. De Leo, M.; Braca, A.; Sanogo, R.; Cardile, V.; DeTommasi, N.; Russo, A. Antiproliferative activity of Pteleopsis suberosa leaf extract and its flavonoid components in human prostate carcinoma cells. Planta Med. 2006, 72, 604–610. 36. Kuntz, S.; Wenzel, U.; Daniel, H. Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity, and apoptosis in human colon cancer cell lines. Eur. J. Nutr. 1999, 38, 133–142. 37. Ichimatsu, D.; Nomura, M.; Nakamura, S.; Moritani, S.; Yokogawa, K.; Kobayashi, S.; Nishioka, T.; Miyamoto, K. Structure-activity relationship of flavonoids for inhibition of epidermal growth factor-induced transformation of JB6 Cl 41 cells. Mol. Carcinog. 2007, 46, 436–445. 38. Lee, E. C.; Tenniswood, M. P. Emergence of metastatic hormone-refractory disease in prostate cancer after anti-androgen therapy. J. Cell. Biochem. 2004, 91, 662–670. 39. Tyagi, A.; Agarwal, C.; Agarwal, R. The cancer preventive flavonoid silibinin causes hypophosphorylation of Rb/p107 and Rb2/p130 via modulation of cell cycle regulators in human prostate carcinoma DU145 cells. Cell Cycle 2002, 1, 137–142. 40. Casagrande, F.; Darbon, J. M. Effects of structurally related flavonoids on cell cycle progression of human melanoma cells: Regulation of cyclin-dependent kinases CDK2 and CDK1. Biochem. Pharmacol. 2001, 61, 1205–1215. 41. Bhatia, N.; Agarwal, R. Detrimental effect of cancer preventive phytochemicals silymarin, genistein and epigallocatechin 3-gallate on 210 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

42.


43.

44.

45.

epigenetic events in human prostate carcinoma DU145 cells. Prostate 2001, 46, 98–107. Gupta, S.; Ahmad, N.; Nieminen, A. L.; Mukhtar, H. Growth inhibition, cell-cycle dysregulation, and induction of apoptosis by green tea constituent (-)-epigallocatechin-3-gallate in androgen-sensitive and androgen-insensitive human prostate carcinoma cells. Toxicol. Appl. Pharmacol. 2000, 164, 82–90. Park, J. H.; Oh, E. J.; Choi, Y. H.; Kang, C. D.; Kang, H. S.; Kim, D. K.; Kang, K. I.; Yoo, M. A. Synergistic effects of dexamethasone and genistein on the expression of Cdk inhibitor p21WAF1/CIP1 in human hepatocellular and colorectal carcinoma cells. Int. J. Oncol. 2001, 18, 997–1002. Murai, T.; Nakagawa, Y.; Maeda, H.; Terada, K. Altered regulation of cell cycle machinery involved in interleukin-1-induced G(1) and G(2) phase growth arrest of A375S2 human melanoma cells. J. Biol. Chem. 2001, 276, 6797–6806. Prasain, J. K.; Jones, K.; Moore, R.; Barnes, S.; Leahy, M.; Roderick, R.; Juliana, M. M.; Grubbs, C. J. Effect of cranberry juice concentrate on chemically-induced urinary bladder cancers. Oncol. Rep. 2008, 19, 1565–1570.


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