DIETARY PHYTOCHEMICALS IN CANCER PREVENTION AND TREATMENT

ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY

Editorial Board:

NA THAN BACK, State University of New York at Buffalo

IRUN R. COHEN, The Weizmann Institute of Science

DA VID KRITCHEVSKY, Wistar Institute

ABEL LAJTHA, N. S. Kline Institute for Psychiatric Research

RODOLFO PAOLETTI, University of Milan

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DIETARY PHYTOCHEMICALS IN CANCER PREVENTION AND TREATMENT

Edited under the auspices of the

American Institute for Cancer Research Washington, D.C.

PLENUM PRESS· NEW YORK AND LONDON

Library of Congress Cataloging-In-Publication Data

Dietary phytochemlcals In cancer preventIon and treatment I edited under the auspices of the American Institute for Cancer Research.

p. cm. -- (Advances in experimental medicine and biology; v. 401)

·Proceedings of the American Institute for Cancer Research's sixth annual research Conference on Dietary Phytochemicals in Cancer Prevention and Treatment, held August 31-Sept.ember 1,1995. in Washington, D.C."--T.p. verso.

Includes bibliographical references and index. 1. Cancer--Chemoprevention--Congresses. 2. Cancer--Nutritional

aspects--Congresses. 3. Botanical chemistry--Congresses. 4. Cancer--Diet therapy--Congresses. I. American Institute for Cancer Research. II. Conference on Dietary Phytochemicals in Cancer Prevention and Treatment (1995 : .Washington, D.C.) III. Series.

[DNLM: 1. Neoplasms--prevention & control--congresses. 2. Plants, Edible--chemistry--congresses. 3. Anticarcinogenic Agents­-congresses. WI AD559 v.401 1996 I OZ 200 D5655 1996J RC268.15.D53 1996 616.99·4--dc20 DNLM/DLC for Library of Congress

l American Institute for Cancer Research

96-24704 CIP

Proceedings of the American Institute for Cancer Research's Sixth Annual Research Conference on Dietary Phytochemicals in Cancer Prevention and Treatment, held August 31 - September I, 1995, in Washington, D.C.

ISBN-13: 978-1-4613-8034-4 e-ISBN-13: 978-1-4613-0399-2 DOl: 10.1007/978-1-4613-0399-2

© 1996 Plenum Press, New York

Softcover reprint of the hardcover 1 st edition 1996

A Division of Plenum Publishing Corporation 233 Spring Street, New York, N. Y. 10013

1098765432

All rights reserved

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

PREFACE

The sixth annual research conference of the American Institute for Cancer Research was held August 31 and September 1, 1995, at the Loews L'Enfant Plaza Hotel in Washing­ton, DC. In view of the promising leads in the diet/nutrition and cancer research field, the conference was devoted to "Dietary Phytochemicals in Cancer Prevention and Treatment." The number of sessions was increased over that in previous conferences in order to accommodate the topics of interest. The conference overview, entitled "Plants and Cancer: Food, Fiber, and Phytochemicals," provided a framework for the following sessions. In addition, the attendees were reminded that for several decades epidemiologists have noted a lower risk of lung, esophageal, stomach, and colon cancer in populations consuming diets high in fruits and vegetables. However, isolation and ingestion of individual protective factors are not the preferred action since the complexity of the food and the matrix in which nutritional factors are embedded are important. The individual sessions then provided more insight as to why eating fruits and vegetables is associated with a lower risk of cancer.

The first of these sessions was on "Isothiocyanates" that induce both the Phase I and Phase II enzymes that increase detoxification and conjugation reactions, thus causing more rapid removal of any xenobiotic or carcinogen. Thus, less carcinogen is available for interaction with DNA or other critical cellular macromolecules. Natural isothiocyanates include phenethylisothiocyanate (PEITC) and benzylisothiocyanate (BITC) that occur as glucosinolates, bound in a complex fashion to the sugar glucose, in turnips, watercress, or cabbage. Chewing or mincing the vegetable causes the endogenous vegetable enzyme myrosinase to hydrolyze the glucosinolate; a rearrangement affords the free isothiocyanate that is the chemopreventive agent. In both mice and rats PEITC inhibited lung tumors from 4-(methylnitrosamino )-I-(3-pyridyl)-I-butanone (NNK), one of the major lung carcinogens in tobacco smoke, but it had no effect against lung tumors from benzo( a )pyrene (BP), another carcinogen in tobacco smoke. However, BITC inhibited the lung tumorigenicity of BP in mice. In addicted smokers who ate watercress at each meal for several days, there was higher urinary excretion of detoxified conjugates of NNK, indicating that the PEITC from the watercress blocked the metabolic activation pathway ofNNK and increased the detoxifica­tion mode. The effect of isothiocyanates was explored further with a different animal model, namely esophageal cancer induced in rats by nitrosomethylbenzylamine (NMBA). Although PEITC was a fairly good inhibitor ofNMBA, if given before and during NMBA administra­tion, increasing the carbon chain from phenethyl to phenylpropyl (PPITC) afforded an even more potent compound. However further increases in chain length had little effect or even increased esophageal tumor response. Correspondingly, rats given PEITC or PPITC had lower levels of DNA-methylated products (06-methylguanine) in the esophagi than did control rats or those receiving other isothiocyanates.

v

vi Preface

Overall, some isothiocyanates inhibited esophageal carcinogenesis while others were more effective against lung carcinogens. Further investigation may delineate the separate mechanisms responsible and establish the role of isothiocyanates as chemopreventive substances.

Polyphenols, which are ubiquitous in plant products, were the subject of another session. Various precursors, including caffeic, ferulic, hydroxycinnamic acids, and others, are present in plants; when the plant material is ingested, the bacteria in the gastrointestinal tract convert these precursors to diols and lactones that have antioxidant properties. In model experiments plant phenolics showed antioxidant effects, inhibited nitrosation of secondary amines from endogenous nitrite, trapped electrophiles, inhibited the cyclooxgenase pathway of arachidonic acid metabolism, inhibited protein kinases, and showed antiestrogenic activ­ity. More specifically, the polyphenols in green tea, epicatechin, epigallocatechin, and their gallate esters were noted as inhibiting the initiation, promotion, and progression stages of mouse skin carcinogenesis. Mechanistically, they depressed the activity ofP450-dependent enzymes and decreased the levels of carcinogen-DNA adducts in the skin. In addition, extracts of green tea protected against tumor formation in lung, esophagus, gastrointestinal tract, liver, and other internal organs in model systems for both mice and rats. However, epidemiological studies of populations drinking tea versus those using other beverages are not conclusive as to whether green tea affords protection in humans.

Flavonoids, a subset of plant polyphenols, were the basis for presentations on somewhat diverse and complex investigations. An overview provided some insight into the interaction between estrogens and phytoestrogens, such as genistein and lignans, the rela­tionship to breast cancer, and the factors involved in breast cancer risk, including age at menarche, pregnancy, and age at menopause. Estrogens act through estrogen receptors that may have different conformations, depending on whether the interacting substances are agonists, partial agonists, or antagonists. Some flavonoids can modulate the effects of estrogen by binding to the receptor or by directing the metabolism of estrogen toward the catechol estrogen path, yielding antiestrogens. Some natural products such as zearalenone act like estrogens and may represent a deleterious factor.

A study on genistein showed that although it reduced mammary tumors in female rats given a potent carcinogen, it did not appear to act via inhibition of a protein kinase. Labeled genistein was extensively recirculated in rats, and only a fifth of the label was excreted in a week. In humans consumption of a soy beverage led to plasma levels of genistein considered sufficient to regulate proliferation of epithelial cells in the breast and thus provide a chemopreventive effect.

Mechanistic studies on quercetin, a polyhydroxyflavone with wide distribution in fruits and plants, showed that it has multiple biochemical effects in mammalian cells, increasing cyclic adenosine monophosphate (cAMP), decreasing protein kinases, decreasing cAMP and cyclic guanosine monophosphate phosphodiesterases, and interacting with some estrogen binding sites. In H-ras-transformed mouse 3T3 fibroblastic cells, quercetin sup­pressed growth in a dose-dependent manner, perhaps by inactivating the p21 protein in the ras-transformed cells. In human mammary carcinoma cells carrying mutant p53, quercetin also decreased cell growth, probably by inhibiting translation of the p53 protein.

Other widely distributed plant products are the monoterpenes, d-limonene and perillyl alcohol, both of which have shown inhibitory action on carcinogenesis in animal models. These compounds affect isoprenylation of specific proteins that are ras gene products. d-limonene and mevinolin, which also affects protein isoprenylation, inhibited the growth of the CT-26 mouse colon tumor line in culture. Given in the diet, these agents decreased the growth of colonies ofCT-26 cells that had been implanted in the spleen. Perillyl alcohol, a metabolite of d-limonene, was effective in chemoprevention of chemically induced rat mammary cancer, as well as in reducing significantly the growth of a transplant-

Preface vii

able pancreatic ductal adenocarcinoma in hamsters or even causing complete regression. Further mechanistic studies on the monoterpenes revealed that d-limonene impaired DNA synthesis in myeloid and lymphoid leukemia cell lines and did not cause accumulation of an unmodified RAS protein. Perillyl alcohol also decreased RAS protein levels, possibly by impairment of RAS transcription. However, the monoterpenes had no effect on hy­droxymethyglutaryl coenzyme A reductase activity, an indication that they apparently do not inhibit farnesyl protein transferase.

Another diverse class of chemopreventive agents includes the organosulfides, present in Allium and Brassica vegetables. Many organosulfides are considered safe and are allowed as food additives. An overview of various epidemiological and laboratory studies showed stronger evidence for the preventive effects of garlic in animal carcinogenesis models than did the epidemiological reports. Although organosulfides from garlic or garlic extracts inhibited the growth of various tumor cell lines in culture or transplantable tumors in animals, extrapolation to reasonable levels of human consumption from the amounts found effective in the animal experiments is dubious. In Allium plants, the organosulfur compounds occur as S-allylcysteine S-oxides; when the plant is crushed or cut, C-S lyase enzymes (alliinases) convert the oxides to sulfenic acids that condense to form thiosulfinate esters. One of these had antitumor activity in animals. Selenium and selenoamino acids are also found in garlic, onions, broccoli, and cabbage. Separating the various sulfur and selenium constituents of these vegetables has been a challenge from the chemical aspect. Delineation of the dynamics of the metabolic disposition of these substances has also been demanding. Although animal experiments have shown inhibitory effects of the garlic constituents, in other situations they have enhanced or promoted tumor activity. Thus, extrapolation to humans must be ap­proached cautiously. Nevertheless, S-allylcysteine, a water-soluble compound from garlic, decreased the incidence of dimethylhydrazine-induced colon tumors in mice and the nucleo­toxicity from the carcinogen. The garlic agents are strong inducers of gluthathione-S-trans­ferase, an enzyme involved in detoxification of many electrophiles. Furthermore, the garlic organosulfur compounds appear to suppress CYP2E I, the specific isoform ofP450 that often governs the metabolic activation of several carcinogens.

A final paper in this session described combining the known chemopreventive effects of selenium, as selenite or selenomethionine, with the active constituents from garlic. Growing garlic bulbs in a greenhouse medium highly enriched with sodium selenite or selenate yielded garlic with selenium concentrations ranging up to 1300 ppm versus the usual 0.03 ppm in ordinary garlic. This high-selenium garlic was more effective than natural garlic in preventing mammary cancer in rats treated with 7, 12-dimethylbenz (a )anthracene and was not toxic. Further trials led to the conclusion that the effect was due to the selenium rather than the garlic. The high-selenium garlic led to approximately a 50% decrease in carcino­gen-DNA adducts, correlating reasonably well with the decrease in tumor incidence. This special garlic did not cause any large increase in selenium levels in tissues, affect selenoen­zymes, or have an apparent toxic action.

The last session was devoted to the practical aspects of including phytochemicals in the U.S. diet, their role in cancer prevention, and whether dietary changes are needed. The following cautions were presented: Definitive data on the toxicity in animals and humans are not available, nor are data on bioavailability and levels ofphytochemicals in diets; food composition tables are not refined enough to permit estimates of consumption of phyto­chemicals; phytochemical content of foods differs depending on many factors; many Americans fail to meet current recommendations for including fruits, vegetables, and grains in their diets; and there still are questions on any recommendations for including more or specific phytochemicals in diets. Appropriately, the National Cancer Institute had launched the National 5 A Day for Better Health Program, which encourages people to eat a combination of five servings of fruits and vegetables each day. Although it is not known

viii Preface

which combinations of phytochemicals provide a chemopreventive effect, epidemiological studies indicate lower risks of various types of cancer in those eating more fruits and vegetables. The NCI is cooperating with the media, retail stores, the food industry, the research and evaluation segment, and the community through state health agencies in order to deliver the message about diet and cancer prevention to Americans.

The viewpoint of part of the food industry on phytochemicals was expressed in the remaining presentations. Official guidelines on dietary intake have urged Americans to consume less high-fat animal products and increase intake of fruits, vegetables, beans, and grains. The current USDA Food Guide Pyramid emphasizes grains as the foundation of the diet, followed by vegetables and fruits, then dairy and meat, with fats and sweets to be used sparingly. However, it still allows fairly generous portions of animal foods and includes beans, seeds, and nuts with meat and dairy foods. Since beans, seeds, and nuts have different nutrient, fiber, and phytochemical contents from those in meats, there should be revision of the guidelines. A transition from animal-based to plant-based diets should be emphasized. However, the food industry should not overpromise with phytochemicals, and since about half of the population does not eat sufficient fruits and vegetables or meet dietary guidelines, the recommended dietary levels for vitamins should be revised or reconsidered. In addition, there may be a need to develop hypernutritious foods to improve diets. As a sequel, improved diets would lead to disease prevention and lower healthcare costs, as well as new markets and new types of business for the food industry. However, some beneficial foods, such as high-fiber cereals and brans, especially wheat, are already available in grocery stores. In model animal experiments, dietary fiber has inhibited colon cancer, and it aids in removal of circulating estrogens, thus decreasing a risk factor for breast cancer.

At the poster session there were 72 presentations on various aspects of research in the phytochemical/cancer area. An additional evening session was devoted to a workshop, "Diet, Nutrition and Cancer Prevention: Research Opportunities, Approaches and Pitfalls." This session covered such topics as strengthening research on diet and cancer prevention, approaches to increase funding, improving grant applications, and opening lines of commu­nication between funding agencies and research workers.

To summarize, many varied types of foods containing desirable phytochemicals with cancer preventive activities are readily available. Because of the concerns voiced during the last session of the conference, it is not prudent to isolate these substances and use them alone. Thus, educational motivation is needed to encourage everyone to eat a variety of fruits, vegetables, and grain products each day to ensure an approach to better health.

The Editors

CONTENTS

Chapter 1 Chemoprevention of Lung Cancer by Isothiocyanates ....................... . Stephen S. Hecht

Abstract ............................................................. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Inhibition ofNNK-Induced Lung Tumorigenesis by PEITC . . . . . . . . . . . . . . .. 2 Inhibition of BaP-Induced Lung Tumorigenesis by BITC . . . . . . . . . . . . . . . . . . 6 Effects of Watercress Consumption on NNK Metabolism in Smokers. . . . . . . . 7

Summary............................................................ 8 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Chapter 2 Isothiocyanates as Inhibitors of Esophageal Cancer ......................... 13 Gary D. Stoner and Mark A. Morse

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 13 Methods ............................................................. 15

Chemicals ....................................................... 15 Animals ......................................................... 15 Experimental Diets ................................................ 17 Tumorigenesis Bioassays ........................................... 17 Analysis of DNA Adducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 17 Statistical Analysis ................................................ 18

Results .............................................................. 18 Effects of PElTC on NMBA-Induced Esophageal Tumorigenesis ........... 18 Effects of Various Arylalkyl Isothiocyanates on NMBA-Induced Esophageal

Tumorigenesis ................................................. 19 Effects of Dietary Isothiocyanates on NMBA-Induced Methylation ......... 20

Discussion ........................................................... 20 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 21 References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 21

ix

x Contents

Chapter 3 Plant Phenolics as Potential Cancer Prevention Agents . . . . . . . . . . . . . . . . . . . . . .. 25 Harold 1. Newmark

Abstract ............................................................. 25 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 25 Occurrence of Plant Phenolic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 26 Chemical Properties of Plant Phenolic Compounds. . . . . . . . . . . . . . . . . .. . . . . . . .. 28 Anti-Carcinogenic Properties ............................................ 28

The Antioxidant Hypothesis ......................................... 28 Inhibition of Nitro sat ion ............................................ 29 Electrophile Radical Trap Hypothesis ................................. 29 Arachidonic Metabolism Modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 29 Cell Protein Kinase Inhibition ....................................... 30

Inhibition of Carcinogenesis: Animal Experiments. . . . . . . . . . . . . . . . . . . . . . . . . .. 30 Problems and Adverse Effects ........................................... 31 Endogenous Production ................................................ 31 Multifunctional Biochemical Activities of Plant Phenolics. . . . . . . . . . . . . . . . . . . .. 32 Needs and Objective for Future Research .................................. 32 References ........................................................... 33

Chapter 4 Cancer Chemoprevention by Polyphenols in Green Tea and Artichoke ......... 35 Rajesh Agarwal and Hasan Mukhtar

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 35 Cancer Chemoprevention by Green Tea. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 36

General Background ............................................... 36 Anti-Carcinogenic Effect of Green Tea in Mouse Skin Model of

Carcinogenesis ................................................. 37 Anti-Carcinogenic Effect of Green Tea in Internal Body Organs ............ 40 Potential Anti-Carcinogenic Effect of Green Tea in Prostate Cancer ......... 42 Epidemiological Studies on Tea Consumption and Human Cancer .......... 43

Cancer Chemoprevention by Silymarin .................................... 44 General Background ............................................... 44 Anti-Carcinogenic Effect ofSilymarin in Mouse Skin Model of Carcinogenesis .. 44

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 46 References ........................................................... 46

Chapter 5 Effects of Tea on Carcinogenesis in Animal Models and Humans. . . . . . . . . . . . . .. 51 Chung S. Yang, Laishun Chen, Mao-Jung Lee, and Janelle M. Landau

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 51 Inhibition of Carcinogenesis in Animal Models by Tea . . . . . . . . . . . . . . . . . . . . . . .. 51

Lung Tumorigenesis ............................................... 52 Esophageal Carcinogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 53 Gastric Carcinogenesis ............................................. 53 Formation and Growth of Skin Tumors ................................ 54 Other Laboratory Studies ........................................... 54

Contents xi

Epidemiological Studies on Tea and Cancer ................................ 54 Tea Consumption and Esophageal Cancer in Humans . . . . . . . . . . . . . . . . . . . .. 54 Tea Consumption and Stomach Cancer in Humans ....................... 55

Active Components and Possible Mechanisms for the Inhibition of Tumorigenesis by Tea ........................................................... 56

Concluding Remarks ................................................... 57 Acknowledgments ..................................................... 58 References ........................................................... 58

Chapter 6 Estrogens, Phytoestrogens, and Breast Cancer ................ . . . . . . . . . . . . .. 63 Robert Clarke, Leena Hilakivi-Clarke, Elizabeth Cho, Mattie R. James, and Fabio Leonessa

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 63 Estrogens and Phytoestrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 64

What Is an Estrogen? ............................................... 64 What Is a Phytoestrogen? ........................................... 65 Where Are Compounds with Phytoestrogenic Activity? ................... 65

The Interactions of Estrogens and Phytoestrogens with Estrogen Receptors. . . . . .. 67 Structure-Activity Relationships of Estrogen Receptor Ligands. . . . . . . . . . . .. 67 Relative Potencies ............................................... " 68 Bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 69 Agonists, Partial Agonists, and Antagonists. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 70

The Nature and Diversity of Estrogenic Responses. . . . . . . . . . . . . . . . . . . . . . . . . .. 70 What Is an Estrogenic Response? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 70 The Diversity of Estrogenic Responses ................................ 71 What Is a Phytoestrogenic Response? ................................. 71

Estrogenic Stimuli in the Normal Breast ................................... 72 Estrogens and the Normal Breast ..................................... 72 Estrogen and Mammary Gland Development ........................... 72 Estrogenic Exposures throughout Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 73 Sources of Estrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 73

Estrogenic Stimuli in the Malignant Breast ................................. 75 Early Life and Breast Cancer ........................................ 75 Early Phytoestrogen Exposure and Mammary Tumors .................... 76

Mechanisms of Action of Phytoestrogens in Breast Cancer .................... 77 Conclusions and Future Prospects ........................................ 78 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 79 References ..................................................... . . . . .. 79

Chapter 7 Soy Isoflavonoids and Cancer Prevention: Underlying Biochemical and Pharmacological Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 87 Stephen Barnes, JeffSfakianos, Lori Coward, and Marion Kirk

Abstract ............................................................. 87 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 88 Materials and Methods ................................................. 89

Materials ........................................................ 89

xii Contents

Animals ......................................................... 89 Soy Feeding Studies ............................................... 89 HPLC Analysis of Genistein Metabolites in Bile . . . . . . . . . . . . . . . . . . . . . . . .. 90 Analysis of Isoflavones in Plasma .................................... 90 HPLC-Mass Spectrometry .......................................... 90 Results .......................................................... 91 Intestinal Uptake and Biliary Recovery of Genistein. . . . . . . . . . . . . . . . . . . . .. 91 Plasma Isoflavones following a Soy Beverage .......................... 92

Discussion ........................................................... 93 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 99 References ........................................................... 99

Chapter 8 Quercetin as a Modulator of the Cellular Neoplastic Phenotype: Effects on the Expression of Mutated H-ras andp53 in Rodent and Human Cells ............. 101 Matias A. Avila, Jose Cansado, K. William Harter, Juan A. Velasco, and Vicente Notario

Introduction .......................................................... 101 Materials and Methods ................................................. 102

Cell Lines, Culture Conditions, and Quercetin Treatment .................. 102 Growth Rate, Clonogenic Assays, and FACS Analysis .................... 102 Northern Blot Hybridization Analysis ................................. 102 Protein Analyses .................................................. 102 Other Experimental Procedures ...................................... 103

Results .............................................................. 103 Effect of Quercetin on ras-Transformed NIH/3T3 Cells ................... 103 Effect of Quercetin on Human Mammary Carcinoma Cells Carrying Mutant

p53 .......................................................... 105 Discussion ........................................................... 107 Acknowledgments ..................................................... 108 References ........................................................... 1 09

Chapter 9 Effects of Monoterpenes and Mevinolin on Murine Colon Tumor CT-26 in Vitro and Its Hepatic "Metastases" in Vivo ...................................... 111 Selwyn A. Broitman, John Wilkinson IV, Sonia Cerda, and Steven K. Branch

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Materials and Methods ................................................. 118

Cell Culture ...................................................... 118 Preparation of Lipoprotein-Deficient Serum (LPDS) ..................... 118 Preparation of MEV ............................................... 118 Protein Determinations ............................................. 118 Inhibition of Cell Growth with MEV and Perillyl Alcohol Combinations ..... 118 Cholesterol Synthesis Assay ......................................... 119 Protein Isoprenylation Assay ........................................ 119 Tissue Culture for Mouse Implantation ................................ 120 Diets Used for Animal Studies ....................................... 120 Direct Hepatic Implantation ......................................... 120

Contents xlii

Hepatic Colonization from Intrasplenic Inoculation ...................... 120 Measurement of HMG CoA Reductase Activity in Liver and CT-26

Implanted in the Liver of Ba1b/C Host .............................. 121 Results and Discussion ................................................. 121 Summary ............................................................ 127 Acknowledgments ..................................................... 128 References ........................................................... 128

Chapter 10 Antitumorigenic Effects of Limonene and Perillyl Alcohol against Pancreatic and Breast Cancer ...................................................... 131 Pamela 1. Crowell, A. Siar Ayoubi, and Yvette D. Burke

Abstract ............................................................. 131 Dietary Sources of Monoterpenes ......................................... 131 Antitumor Activity of Limonene ......................................... 132 Limonene Metabolism .................................................. 132 Structure-Activity Relationships among Monoterpenes ....................... 133 Chemotherapeutic Effects ofPerillyl Alcohol ............................... 133 Mechanisms of Action of Perillyl Alcohol and Limonene ...................... 134 Summary ............................................................ 135 References ........................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

Chapter 11 Monoterpenes as Regulators of Malignant Cell Proliferation .................. 137 Raymond J. Hohl

Abstract ............................................................. 137 Monoterpenes ........................................................ 137 ras and Its Relationship to the Cholesterol Biosynthetic Pathway ............... 138 Anti-ras Activity by the Monoterpenes .................................... 140 Applications of the Monoterpenes to Clinical Trials .......................... 143 Acknowledgment ...................................................... 144 References ........................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

Chapter 12 Organosulfur Compounds and Cancer ..................................... 147 Michael A. Lea

Introduction .......................................................... 147 Epidemiological Studies of Garlic and Cancer .............................. 147 Inhibition of Carcinogenesis by Garlic and Garlic Constituents ................. 148 Effects of Garlic and Garlic Constituents on Metabolism ...................... 148

Carcinogen Metabolism ............................................ 148 Polyamine Metabolism ............................................. 149 Sufbydryl Groups and Glutathione S-Transferase ........................ 149 Other Actions that May Be Related to Cancer ........................... 150

Effects on Tumor Growth and Differentiation ............................... 150 Summary ............................................................ 151 References ............................................. . . . . . . . . . . . . . . 151

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Chapter 13 Recent Results in the Organosulfur and Organoselenium Chemistry of Genus Allium and Brassica Plants: Relevance for Cancer Prevention ................. 155 Eric Block

Introduction .......................................................... 155 Organosulfur and Organoselenium Compounds in Allium and Brassica spp. . ..... 155 Selenium Biochemistry ................................................. 156 Analysis of Selenium in Allium spp ........................................ 158 Headspace Analysis .................................................... 159 Selenoamino Acid Analysis .............................................. 160 Human Garlic Breath Analysis ........................................... 163 Cancer Preventative Activity ............................................ 163

Epidemiological Evidence .......................................... 163 Antibacterial Activity Relevant to Cancer Prevention ..................... 164 Cytotoxicity and Anti-Tumor Activity ................................. 165 Antioxidant Activity ............................................... 166 Tumor Promotion Activity .......................................... 166

Acknowledgment ...................................................... 166 References ........................................................... 166

Chapter 14 Allium Vegetables and the Potential for Chemoprevention of Cancer ........... 171 Michael 1. Wargovich and Naoto Uda

Introduction .......................................................... 171 Early Studies Indicating Possible Cancer Prevention Effects for Organosulfur

Compounds ...................................................... 172 Further Evidence for Chemoprevention: Effects of Allium Volatiles in Other

Animal Models and Carcinogen Detoxification Systems .................. 173 Recent Studies on the Effects of Allium-Derived Organosulfur Compounds on

Carcinogen Metabolism ............................................ 174 Epidemiological and Clinical Studies Related to the Cancer Preventives Effects of

Allium Vegetables ................................................. 175 Acknowledgments ..................................................... 176 References ........................................................... 176

Chapter 15 The Attributes of Selenium-Enriched Garlic in Cancer Prevention ............. 179 Clement Ip and Donald J. Lisk

Introduction .......................................................... 179 Cultivation of High Selenium Garlic ...................................... 180 Studies of Mammary Cancer Prevention by High Selenium Garlic .............. 180 The Anticarcinogenic Efficacy of High Selenium Garlic Is Primarily Attributable

to the Effect of Selenium ............................................ 181 Nutritional Bioavailability of Selenium from High Selenium Garlic ............. 182 Tissue Selenium Accumulation and Tolerance Studies in Rats Fed High Selenium

Garlic ........................................................... 184 Discussion ........................................................... 185

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Acknowledgment ...................................................... 186 References ........................................................... 186

Chapter 16 Is There a Need to Change the American Diet? .............................. 189 Johanna Dwyer

Introduction .......................................................... 189 Science Base for Evaluating Evidence ..................................... 189 Expert Recommendations ............................................... 190

What Experts Recommend Today ..................................... 190 Are We Meeting Expert Recommendations? ............................ 190 Are New Recommendations Needed? ................................. 191

Next Steps ........................................................... 195 Four Paths ....................................................... 195 Wait for More Evidence to Accumulate and Let Dietary Intakes "Float" ...... 195 Increase Intakes of Foods Naturally Containing Phytochemicals ............ 195 Make Phytochemical Fortified Foods More Available and Eat Them ......... 196 Promote Phytochemical Supplements ................................. 196

Conclusions .......................................................... 196 Acknowledgment ...................................................... 197 References ........................................................... 197

Chapter 17 The National 5 A Day for Better Health Program ............................ 199

Jerianne Heimendinger and Daria Chapel sky

Introduction and Rationale .............................................. 199 Program Design ....................................................... 200 Program Components .................................................. 201

Media ........................................................... 202 Retail ........................................................... 202 Community ...................................................... 203 Research and Evaluation ............................................ 203

Program Materials ..................................................... 204 Conclusion ........................................................... 204 References ........................................................... 205

Chapter 18 Nutritional Implications of Dietary Phytochemicals .......................... 207 Mark Messina and Virginia Messina

The Golden Ages of Nutrition ............................................ 207 Phytochemicals and Their Impact on Dietary Guidelines ...................... 208 Changing Views of Diet and Foods ....................................... 209 Phytochemical Supplements and Phytochemical Food Fortification ............. 210 Summary ............................................................ 211 References ........................................................... 211

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Chapter 19 Designer Foods: Is There a Role for Supplementation/Fortification? ........... 213 John W. Finley

Designer Foods ....................................................... 213 Dietary Guidelines ..................................................... 215 Materials with Perceived Efficacy ........................................ 219 Conclusion ........................................................... 220 References ........................................................... 220

Chapter 20 Wheat Bran, Colon Cancer, and Breast Cancer: What Do We Have? What Do We Need? ............................................................. 221 Richard M. Black

Introduction .......................................................... 221 Colon Cancer ......................................................... 222 Breast Cancer ......................................................... 224 Phytochemicals ....................................................... 227 Conclusion ........................................................... 227 References ........................................................... 228

Chapter 21 Diet, Nutrition, and Cancer Prevention: Research Opportunities, Approaches, and Pitfalls ............................................................ 231 Chung S. Yang, Barbara C. Pence, Michael J. Wargovich, and Janelle M. Landau

Introduction .......................................................... 231 Important, but Not Well Funded, Research Field ............................. 231 Common Weaknesses and Problems in Grant Applications ..................... 232 Ways to Strengthen Research on Diet and Cancer Prevention .................. 233 New Opportunities and Approaches ....................................... 234 Approaches to Increase Funding .......................................... 235

Justifying the Importance of the Research .............................. 235 Increasing Support and Fund Raising .................................. 235 Strengthening the Study Sections and Review Panels ..................... 235 Writing Better Grant Applications .................................... 236 Keeping the Lines of Communication Open ............................ 236

Acknowledgment ...................................................... 236 References ........................................................... 236 Appendix 1 .......................................................... 237 Appendix 2 .......................................................... 237 Appendix 3 .......................................................... 238 Appendix 4 .......................................................... 238

Abstracts . ............................................................. 239

Index . ................................................................ 325