Protease Inhibitors as Cancer Chemopreventive Agents

Protease Inhibitors as Cancer Chemopreventive Agents

Edited by

Walter Troll New York University Medical Center New York, New York

and

Ann R. Kennedy University of Pennsylvania School of Medicine Philadelphia, Pennsylvania

Springer Science+Business Media, LLC

Llbrary of Congress Cataloglng-ln-Publlcatlon Data

Protease lnhlbitors as cancer chemopreventive agents I edited by Walter Troll, Ann R, Kennedy.

p. cm. Inc I udes b 1 b li ograph 1 ca I references and 1 ndex. ISBN 978-1-4613-6249-4 ISBN 978-1-4615-2882-1 (eBook)

1. Sancer--Chemoprevention. 2. Proteolytlc enzyme lnhlbltors­-Therapeutic use. 1. Troll, Walter. II. Kennedy, Ann R.

[DNLM, 1. Protease Inhibltors--therapeutic use. 2. Protease Inhlbitors--metabolism. 3. Neoplasms--preventlon & control. OU 138 P9885 1993J RC288.15.P77 1993 818.99·405--dc20 DNLM/DLC for Library of Congress

ISBN 978-1-4613-6249-4

© 1993 Springer Science+Business Media New York Originally published by Plenum Press New York in 1993 Softcover reprint of the hardcover 1 st edition 1993

AII rights reserved

93-24959 CIP

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

DOI 10.1007/978-1-4615-2882-1

Contributors

Takaaki Aoyagi Japan

Institute of Microbial Chemistry, Shinagawa-ku, Tokyo 141,

Sila Banerjee Department of Obstetrics and Gynecology, New York Univer-sity Medical Center, New York, New York 10016

Anne H. Bates Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710

Paul C. Billings Department of Radiation Oncology, University of Pennsyl-vania School of Medicine, Philadelphia, Pennsylvania 19104

Yehudith Birk Department of Biochemistry and Human Nutrition, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Donald E. Bowman Departments of Biochemistry and Molecular Biology, School of Medicine, Indiana University, Indianapolis, Indiana 46202-5122

David L. Brandon Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710

Ann F. Chambers The London Regional Cancer Centre, University of West-ern Ontario, London, Ontario, Canada N6A 4L6

Janice D. Chang Department of Biology, Massachusetts Institute of Tech-nology, Cambridge, Massachusetts 02139

Rita Colella Department of Biological Sciences, Rutgers University, Pis-cataway, New Jersey 08855. Present address: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, Kentucky 40292

v

vi CONTRIBUTORS

Pelayo Correa Department of Pathology, Louisiana State University Medical Center, New Orleans, Louisiana 70112

Lydia Cox Department of Environmental Medicine, New York University Medical Center, New York, New York 10016

Diane C. Currie Department of Environmental Medicine, New York Univer-sity Medical Center, New York, New York 10016

David T. Denhardt Department of Biological Sciences, Rutgers University, Piscataway, New Jersey 08855

Thomas H. Finlay Department of Obstetrics and Gynecology, New York University Medical Center, New York, New York 10016

Peter Flecker Institute for Physiological Chemistry, University of Mainz, W-6500 Mainz, Germany

Elizabeth Fontham Department of Pathology, Louisiana State University Medical Center, New Orleans, Louisiana 70112

Krystyna Frenkel Departments of Environmental Medicine and Pathology, New York University Medical Center, New York, New York 10016

Mendel Friedman Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710

Seymour J. Garte Department of Environmental Medicine, New York Uni-versity Medical Center, New York, New York 10016

Susan S. Kadner Department of Obstetrics and Gynecology, New York Uni-versity Medical Center, New York, New York 10016

Joseph Katz Department of Obstetrics and Gynecology, New York Univer-sity Medical Center, New York, New York 10016

Ann R. Kennedy Department of Radiation Oncology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Mortimer Levitz Department of Obstetrics and Gynecology, New York Uni-versity Medical Center, New York, New York 10016

Daniel S. Longnecker Department of Pathology, Dartmouth Medical School, Hanover, New Hampshire 03755

Joan Motz Department of Environmental Medicine, New York University Medical Center, New York, New York 10016

Joseph Nickels, Jr. Department of Microbiology and Molecular Genetics, Rutgers University, New Brunswick, New Jersey 08903

CONTRIBUTORS vii

Uma Raju Department of Obstetrics and Gynecology, New York University Medical Center, New York, New York 10016

George Ritchie Department of Physics, Rider College, Lawrenceville, New Jersey 08648

B. D. Roebuck Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755

Lorraine T. Schepis Department of Molecular Biology, Princeton University, Princeton, New Jersey 08530

Tomio Takeuchi Japan

Institute of Microbial Chemistry, Shinagawa-ku, Tokyo 141,

Snait Tamir Department of Obstetrics and Gynecology, New York University Medical Center, New York, New York 10016

Wataru Tanaka Japan

Institute of Microbial Chemistry, Shinagawa-ku, Tokyo 141,

Walter Troll Department of Environmental Medicine, New York University Medical Center, New York, New York 10016

Kazuo Umezawa Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Kohoku-ku, Yokohama 223, Japan

Jonathan Yavelow Department of Biology, Rider College, Lawrenceville, New Jersey 08648

Preface

Protease inhibitors (PIs) are widely distributed in plants and animals, and have a variety of functions, which include preventing digestion of seeds by insects and modifying blood clotting in animals. After it was noted that synthetic and natural inhibitors suppress two-stage carcinogenesis and breast cancer, extensive work investigating PIs as chemopreventive agents was started.

PIs are unique in that they interfere with cancer development in a variety of ways, including suppression of oxygen radicals, oncogenes, and metastases. Epidemiologic evidence supports their prevention of major human cancers in populations that consume foods containing them. Their supervised use in humans is on the threshold of development.

The epidemiologic discovery of the importance of lentils and other seeds rich in PIs in preventing many human cancers allowed us to look at the action of PIs as chemopreventive agents, as reviewed in Chapter I (Fontham and Correa). Chapter 2 (Kennedy) discusses the role of natural PIs (e.g., the Bowman-Birk inhibitor) as anticarcinogens and the possible limitations of their use. In Chapter 3 (Kennedy), the transformation of C3HI lOTlh cells caused by carcinogens and promoters is shown to be suppressed by PIs. Bowman (Chapter 4) relates the discovery of inhibitors in soybeans that are distinct from the Kunitz inhibitor, and the occurrence of a similar inhibitor in peanuts and other legumes. Chapter 5 (Birk) is an overview of PIs of plant origin and their role in human nutrition. In Chapter 6, Brandon and colleagues report on the measurement of soybean trypsin inhibitors by monoclonal antibodies, their possible use for cellular delivery, and the effect of food processing on the survival of the inhibitors. Chapter 7 (Um­ezawa et at.) concerns low-molecular-weight inhibitors, isolated from strep­tomycetes, that have been shown to suppress cancer in animal and some human experiments and are also useful for in vitro and oncogene expression studies. PI synthesis by MCF-7 breast cancer cell lines is discussed in Chapter 8 (Finlay et al.). ai-Trypsin and chymotrypsin inhibitors synthesized by breast cancers may inhibit metastasis. In Chapter 9, Flecker analyzes the structure-activity relation-

ix

x PREFACE

ships of the Bowman-Birk inhibitor of serine proteinases, with a view toward the design of new cancer chemopreventive agents. Troll (Chapter 10) discusses the prevention of cancer by vitamin B3 compounds, PIs that are available for human use. Billings (Chapter 11) details approaches to studying the target en­zymes of anticarcinogenic PIs. In Chapter 12, Colella and colleagues document the anticarcinogenic activities of naturally occurring cysteine proteinase inhibi­tors. These PIs prevent metastasis of tumors and are distinct from serine PIs. Yavelow and colleagues (Chapter 13) identify a chymotrypsin cell membrane enzyme that acts as a possible receptor for chymotrypsin inhibitors. Frenkel (Chapter 14) describes the role of reactive oxygen species in biological damage and the effect of some chemopreventive agents. Chapters 15 (Garte et al.) and 16 (Chang and Kennedy) discuss PI suppression of the actions induced by the ras and c-myc oncogene, respectively. Chapter 17 (Levitz et al. ) reviews the role of esterases in steroid hormone turnover. Esterases in breast cyst fluid hydrolyze steroid esters, modifying their action. The action of PIs in inducing pancreatic cancer in animal models is reviewed by Roebuck and Longnecker (Chapter 18).

Contents

Chapter 1

The Epidemiologic Approach to the Study of Protease Inhibitors

Elizabeth Fontham and Pelayo Correa

1. Introduction ............................................. . 2. Descriptive Epidemiology .................................. 1 3. Analytical Epidemiology ................................... 2 4. Tools Needed for Epidemiologic Studies ...................... 4 5. Intervention Studies ....................................... 5 6. Cancer Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 7. Epilogue ................................................ 6 8. References............................................... 7

Chapter 2

Anticarcinogenic Activity of Protease Inhibitors: Overview 9

Ann R. Kennedy

1. Review of Data Showing Anticarcinogenic Activity of Protease Inhibitors ................................................ 9

2. Potential Adverse Effects of Anticarcinogenic Protease Inhibitors ........................................ 15 2.1. Effects of Protease Inhibitors on Growth .................. 15 2.2. Effects of Protease Inhibitors on the Pancreas· . . . . . . . . . . . . . . 16 2.3. Potential Effects of Protease Inhibitors on the

Immune System ...................................... 22 3. The Soybean-Derived BBI as an Anticarcinogenic Agent. . . . . . . . . 23

3.1. Review of Data on the Anticarcinogenic Activity of BBI . . . . . 23

xi

xii CONTENTS

3.2. Limitations of BBI as a Human Cancer Chemopreventive Agent............................................... 44

4. Mechanism of Action of the Anticarcinogenic Protease Inhibitors ........................................ 50

5. Summary ................................................ 54 6. References ............................................... 55

Chapter 3

In Vitro Studies of Anticarcinogenic Protease Inhibitors. . . . . . . . . . . . . 65

Ann R. Kennedy

1. Introduction .............................................. 65 2. Mechanism of Action of the Anticarcinogenic Protease Inhibitors in

the Suppression of Transformation in Vitro .................... 80 3. References ............................................... 86

Chapter 4

Discovery and Background of the Bowman-Birk Protease Inhibitors ........................................... 93

Donald E. Bowman

1. Origin of Interest in Legumes ............................... 93 2. Unexpected Protease Inhibition by Amylase

Inhibition Preparations ..................................... 93 3. References............................................... 95

Chapter 5

Protease Inhibitors of Plant Origin and Role of Protease Inhibitors in Human Nutrition: Overview ................................... 97

Yehudith Birk

1. Introduction 2. Protease Inhibitors from Legume Seeds ...................... .

2.1. STI ................................................ . 2.2. BBI ............................................... .

3. Protease Inhibitors from Other Plant Sources .................. . 3.1. Potato Protease Inhibitors ............................. . 3.2. Squash Protease Inhibitors ............................. . 3.3. Protease Inhibitors in Cereals .......................... .

4. Role in Nutrition ......................................... . 4.1. Effect on Growth .................................... . 4.2. Effects on the Pancreas ............................... .

97 98 98 98

101 101 101 101 102 102 102

CONTENTS xiii

5. Therapeutic Potential of Protease Inhibitors .................... 103 6. References ....................... . . . . . . . . . . . . . . . . . . . . . . . . 104

Chapter 6

Antigenicity of Soybean Protease Inhibitors. . . . . . . . . . . . . . . . . . . . . . . 107

David L. Brandon. Anne H. Bates. and Mendel Friedman

1. Introduction .................................... . . . . . . . . . . 107 2. Kunitz Trypsin Inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

2.1. Immunochemistry of KTI .............................. 108 2.2. Specificities of Monoclonal Antibodies to KTI ............. 109

3. Immunochemistry of the Bowman-Birk Inhibitor. . . . . . . . . . . . . . . 113 3.1. Sources and Treatment of Protease Inhibitors .............. 114 3.2. Polyclonal Antibodies ................................. 114 3.3. Monoclonal Antibodies to BBI .......................... 114 3.4. Relationship of Inhibitory Activities and Antigenicity. . . . . . . . 116

4. Immunoassays of KTI and BBl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 4.1. Antibody Titrations. Inhibition ELISA, and Competition

ELISA. . . . . . . . . . . . . . . . . . . . . .. . . . .. . . . . . . . . . . . . . . . . . . 117 4.2. Sandwich ELISA ..................................... 117 4.3. Availability of Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

5. Examples of Use of Monoclonal Antibodies to Analyze Soy Foods and Germ Plasm .......................................... 119 5.1. Autoclaved Soy Meal .................................. 119 5.2. Infant Formula ....................................... 120 5.3. Immunoaffinity Methods ............................... 121 5.4. Screening the Genus Glycine. . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

6. Other Effects of Food Processing on Antigenicity of KTI . . . . . . . . . 122 6.1. Chemical Changes during Food Processing ................ 122 6.2. Effects of Alkali ...................................... 124 6.3. Effects of Carbohydrates ............................... 124

7. Conclusions .............................................. 125 8. References............................................... 126

Chapter 7

Low-Molecular-Weight Protease Inhibitors of Microbial Origin 131

Kazuo Umezawa. Takaaki Aoyagi. Wataru Tanaka. and Tomio Takeuchi

1. Introduction.............................................. 131 2. Endopeptidase Inhibitors ................................... 132 3. Exopeptidase Inhibitors .................................... 135 4. References............................................... 138

xiv CONTENTS

Chapter 8

Protease Inhibitor Synthesis by MCF-7 Breast Cancer Cells 141

Thomas H. Finlay, Susan S. Kadner, and Snait Tamir

1. Introduction and Scope of the Chapter ........................ 141 2. <XI-AT and <XI-ACHY ...................................... 144

2.1. Function and Properties ................................ 144 2.2. Site of Synthesis ...................................... 145 2.3. Regulation of Synthesis ................................ 146

3. <XI-AT and <XI-ACHY in MCF-7 Cells ........................ 147 3.1. MCF-7 Breast Cancer Cell Line ......................... 147 3.2. Synthesis of <XeAT and <x~-ACHY by MCF-7 Cells ......... 148 3.3. Kinetics of <XI-AT and <XI-ACHY Synthesis and Secretion .... 149 3.4. Stimulation of <XI-AT and <XeACHY Synthesis by Steroid

Hormones, Inflammatory Mediators, and Growth Factors .... 149 3 .. 5. Production of <XI-AT and <XI-ACHY by Various MCF-7

Sublines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 4. Perspectives and Conclusions ............................... 153 5. References ............................................... 154

Chapter 9

Analysis of Structure-Activity Relationships of the Bowman-Birk Inhibitor of Serine Proteinases: Toward a Rational Design of New Cancer Chemopreventive Agents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Peter Flecker

1. Introduction .............................................. 161 2. Biological Aspects ........................................ 162 3. Prospects of Protein Engineering for the Design of Anticarcinogenic

Agents........ . .. ..... .. ....... ................ ..... .. .. 164 4. A New Approach to BBI-TYpe Proteinase Inhibitors by Synthetic

Gene Technology ......................................... 165 5. Trypsin-Sepharose as a Matrix with Complementary Structure Is

Required for Refolding of Proteinase Inhibitor Variants .......... 168 6. BBI Has Structural Similarity to the COOH-Terminal Part of the B

Chain of Laminin ......................................... 172 7. Conclusions .............................................. 173 8. References............................................... 174

CONTENTS xv

Chapter 10

Prevention of Cancer by Vitamin B 3 (Nicotinamide and Nicotinic Acid): A Protease Inhibitor Available in Pure Form ................ 177

Walter Troll

1. Introduction .............................................. 177 2. Dietary PIs Suppress Cancer ................................ 178 3. Interference with Oxyradical Formation ....................... 178 4. Suppression of Transformation-Caused Oncogene Transfection .... 180 5. Suppression of Carcinogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

5.1. Suppression of Two-Stage Carcinogenesis by Applying PIs to Mouse Skin .......................................... 181

5.2. Suppression of Two-Stage Carcinogenesis and Breast Cancer in Rodents by Feeding Soybean Diets .................... 181

5.3. Suppression of Tumor Promotion in Mice by Feeding NA-Supplemented Diets ................................... 185

6. Mechanism of Cancer Prevention by Feeding PIs ............... 185 7. Concluding Remarks ...................................... 186 8. References ............................................... 187

Chapter II

Approaches to Studying the Target Enzymes of Anticarcinogenic Protease Inhibitors ........................................... 191

Paul C. Billings

1. Introduction ............................................. . 2. Results and Discussion .................................... .

2.1. Defined Substrates as Tools to Identify Proteolytic Activities Which Are Inhibited by the Anticarcinogenic Protease Inhibitors ........................................... .

2.2. The Use of Affinity Chromatography to Identify Proteins Which Specifically Interact with Anticarcinogenic Protease Inhibitors ........................................... .

3. References

Chapter I2

Anticarcinogenic Activities of Naturally Occurring Cysteine

191 192

192

194 197

Proteinase Inhibitors ......................................... 199

Rita Colella, Ann F. Chambers, and David T. Denhardt

1. Introduction .............................................. 199 2. Cysteine Proteinases ....................................... 200

xvi CONTENTS

3. The Cystatin Superfamily. . . . . . . . . . . . . . . . . .. . . . .. . . . . . . . . . . . 202 4. Mode ofInhibition ........................................ 205 5. Cystatins and Metastasis ................................... 206 6. References............................................... 211

Chapter 13

Cell Membrane Enzymes Containing Chymotrypsin-like Activity. . . . . . 217

Jonathan Yavelow, Lorraine T. Schepis, Joseph Nickels, Jr., and George Ritchie

1. Introduction .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 2. Effect of Chymotrypsin Inhibitors on in Vitro

Cell Transformation ....................................... 218 3. Properties of Cell Membrane Enzymes Containing Chymotrypsin-

like Activity ............................................. 220 4. Potential Cellular Substrates for Membrane Chymotrypsin-like

Enzymes ................................................ 221 5. Fluorescence Anisotropy as a Tool for Studying the

Anticarcinogenic Action of Chymostatin ...................... 222 6. Conclusions .............................................. 223 7. References ............................................... 223

Chapter 14

The Role of Reactive Oxygen Species in Biological Damage and the Effect of Some Chemopreventive Agents . . . . . . . . . . . . . . . . . . . . . . . . . . 227

Krystyna Frenkel

1. Formation of Reactive Oxygen Species ....................... 227 2. Formation of H20 2 and Oxidized DNA Bases .................. 229

2.1. By Thmor Promoter-Stimulated PMNs .................... 229 2.2. By Hepatic Microsomes .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

3. Inhibition of H20 2 Formation by Stimulated PMNs ............. 235 3.1. Inhibition by Protease Inhibitors ......................... 237 3.2. Inhibition by Sarcophytol A ............................ 242

4. References ............................................... 243

Chapter 15

Protease Inhibitor Suppression of ras Oncogene-Induced Transformation .............................................. 251

Seymour J. Garte, Lydia Cox, Diane C. Currie, Joan Motz, and Walter Troll

1. Introduction .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 2. Methods ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

2.1. Cell Culture and DNA Isolation ......................... 253

CONTENTS

2.2. DNA-Mediated Transfection ........................... . 2.3. Characterization of Transformed Phenotype ............... . 2.4. Southern Blot Hybridization ........................... . 2.5. Northern Blot Hybridization ........................... .

3. Results ................................................. . 3.1. Dose Response of Protease Inhibitors on ras

Transformation ...................................... . 3.2. Effects on aph Gene Transfection ....................... . 3.3. Time Course Studies ................................. . 3.4. Influence of Cell Proliferation .......................... . 3.5. Subculture and Serum Conditions ....................... . 3.6. Reversibility ........................................ . 3.7. ras Gene Expression ................................. . 3.8. Retinoic Acid Effects ................................. .

4. Discussion 5. References

Chapter 16

xvii

253 253 254 254 255

255 256 256 257 257 258 258 258 259 261

Suppression of c-myc by Anticarcinogenic Protease Inhibitors . . . . . . . . 265

Janice D. Chang and Ann R. Kennedy

1. Introduction .............................................. 265 2. The Oncogene c-myc and Malignant Transformation. . . . . . . . . . . . . 265

2.1. Activation of c-myc ................................... 266 2.2. Function of the Normal c-myc Gene. . . . . . . . . . . . . . . . . . . . . . 267 2.3. Mechanism of c-myc Regulation. . . . . . . . . . . . . . . . . . . . . . . . . 270

3. c-myc Expression Is Reduced in Protease Inhibitor-Treated Proliferating Mouse Fibroblasts .............................. 270

4. Cells Progress through the Cell Cycle in the Absence of an Increase in c-myc RNA Levels ...................................... 272

5. Antipain Increases the Half-Life of the c-myc Message .......... 274 6. Possible Mechanisms by Which Protease Inhibitors Regulate c-myc

Expression ............................................... 275 7. Concluding Remarks ...................................... 276 8. References ............................................... 277

Chapter 17

A Role for Esterases in Steroid Hormone Turnover. . . . . . . . . . . . . . . . . 281

Mortimer Levitz, Sila Banerjee, Joseph Katz, Uma Raju, and Thomas H. Finlay

1. Introduction and Scope of the Chapter ........................ 281 2. Endogenous Steroid Esters .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282

xviii CONTENTS

2.l. Steroid Esters in Body Fluids ........................... 282 2.2. Steroid Esters in Tissues ............................... 282 2.3. Steroid Fatty Acid Ester Production in Vitro ............... 283

3. Steroid Ester Metabolism: Physiological Implications ............ 283 3.1. In Man..... ..... .. .. .. .. ....... ...... ........ ..... .. 283 3.2. In MCF-7 Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

4. Esterification and Deesterification Enzymes. . . . . . . . . . . . . . . . . . . . 284 5. Transesterification ......................................... 285 6. Breast Cyst Fluid Esterase .................................. 287 7. Perspectives and Conclusions ............................... 289 8. References............................................... 292

Chapter 18

Protease Inhibitors and Pancreatic Carcinogenesis .. . . . . . . . . . . . . . . . 295

B. D. Roebuck and Daniel S. Longnecker

1. Pancreatic Cancer ......................................... 295 1.1. Human Disease ....................................... 295 1.2. Animal Models of Pancreatic Cancer ..................... 296 1.3. Cholecystokinin Hypothesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297

2. Response of the Pancreas to Trypsin Inhibitors ................. 298 2.l. Soybean Products ..................................... 298 2.2. Camostate (FOY-305) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

3. Carcinogenesis and Cholecystokinin .......................... 299 3.1. Rat Pancreas ......................................... 299 3.2. Other Species Including Humans ........................ 300

4. Conclusion............................................... 300 5. References ............................................... 301

Index...................................................... 305