The chemotherapeutic consequences of tumor pH in breast carcinoma model systems Item Type text; Dissertation-Reproduction (electronic) Authors Mahoney, Brent P. Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 06/10/2021 04:25:37 Link to Item http://hdl.handle.net/10150/280195 INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. 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ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 THE CHEMOTHERAPEUTIC CONSEQUENCES OF TUMOR pH IN BREAST CARCINOMA MODEL SYSTEMS By Brent Paul Mahoney CopjTight © Brent Paul Mahoney 2002 A Dissertation Submitted to the Facuhy of the GRADUATE INTERDISCIPLINARY PROGRAM IN CANCER BIOLOGY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2002 UMI Number: 3073244 Copyright 2002 by Mahoney, Brent Paul All rights reserved. 0 UMI UMI Microform 3073244 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Infonmation and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor. Ml 48106-1346 2 THE UNIVERSITY OF ARIZONA ® GRADUATE COLLEGE As members of the Final Examination Committee, we certify that we have read the dissertation prepared by Brent P. Mahoney entitled The r.hemotherappnM'p rnngpgiipnppg of Tnmny pH in r.arc-innma Model SvsI-pitir and recommend that it be accepted as fulfilling the dissertation Moment for the Degree of Philosophy Robe Date ;tc William Bella Harris Bernstein Date Date Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my dix^cc^ens^and recommend that it be accepted as fulfilling the dissertation f^iremet JjItiLi. Disse: ion D pr Robert Gillies Date^ 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the copyright holder. 4 ACKNOWLEDGMENTS I would first and foremost like to acknowledge my graduate advisor, Dr. Robert J. Gillies, for his support and supervision of this research. I thank Dr. Gillies for his mentoring, providing both support and guidance during our many scientific discussions about this project. I would also like to thank my committee members for all of their advice during committee meetings; Dr. William Bellamy, Dr. Charles Taylor, Dr. Harris Bernstien, and Dr. Janet Decker. I would particularly like to Dr. Bellamy and Dr. Taylor for providing helpful discussions and technical assistance for this work. I would like to thank the entire Gillies laboratory for the lively intellectual discussions and useful technical advice during weekly lab meetings. I especially thank Brenda Baggett for her technical assistance over the years; Dr. Paul Schornack and Charles Fahr for their input on my research during our group meetings; Dr. Natarajan Raghunand for his assistance with the NMR and animal studies; and Xi He, Dr. Natarajan Raghunand, Dr. Paul Schornack, Charles Fahr, Dave Morse and Dr. Nancy Aiken for their contributions during discussions about this work. I would like to thank Anne Cione and Merry Warner for providing secretarial assistance. I would also like to acknowledge Ron Lynch and Jesse Martinez for their helpful advice during scientific discussions. I thank all of my colleagues in the Arizona Cancer Center for making the basement an enjoyable place to work. 5 DEDICATION To my parents Brian and Linda Mahoney and my wife Dr. Paula Inserra-Mahoney. Your love, encouragement, guidance and support will never be forgotten, I would also like to dedicate this dissertation to my grandmother Angela Mahoney who passed away form pancreatic cancer February 7, 1999. 6 TABLE OF CONTENTS LIST OF FIGURES 9 ABSTRACT 15 I - INTRODUCTION - A REVIEW OF THE LITERATURE 17 A) An Overview of Breast Cancer 17 B) Chemotherapy and Breast Cancer 19 C) Characterization of Chemotherapeutic Drugs and their pH Dependent Behavior.— 21 i) Doxorubicin 22 ii) Daunorubicin 30 iii) Mitoxantrone 35 iv) Paclitaxel 41 V) Vincristine „..4 6 vi) Vinblastine 50 vii) Cyclophosphamide 5 5 viii) Chlorambucil 62 ix) 5-Fluorouracil 67 D) Tumor pH - Observations and Measurements.— 73 E) Causes of Acidic Tumor pH 75 F) Modulators of Tumor pH in vivo. 77 G) Consequences of Tumor Acidity 78 H) Ionizing Radiation Therapy and Tumor pH 81 I) Hypothermic Therapy and Tumor pH 83 J) Tumor pH as a Novel Therapeutic Target 85 K) Cancer Genes and Tumor pH 87 L) V-ATPase Can Inhibit Apoptosis 88 M) Apoptosis and Cell Cycle Blockade 89 II - MATERIALS AND METHODS 92 a) Cell and Tiomor Growth 92 b) Magnetic Resonance Spectroscopy 92 c) Measurement of Octanol Partition Coeffecient94 d) Measurement of Intracellular pH In Vitro. 95 e) D r\xg Upta ice..H.».......»..*.».......•...... *96 f) Drug Cytotoxicity 97 7 g) Calculation of Theoretical Drug Distributions 97 h) Tumor Growth Statistics 98 i) Doxorubicin Biodistribution in MCF-7 Mammary Tumor Bearing SCID Mice 99 j) Doxorubicin Biodistribution in C3H Mammary Tumor Bearing C3H/hen Mice 102 k) Mitoxantrone Biodistribution in C3H Mammary Tumor Bearing C3H/hen Mice 104 1) C3H/hen Mouse-C3H Mammary Carcinoma Model. 106 m) Gavage Administration of NaHC03/NH4Cl 107 n) Intraperitoneal Administration of NaHCOa/Glucose 107 o) Localized in vivo MR Spectroscopy 108 p) Chemotherapy 109 III - ION TRAPPING AND THE CHEMOTHERAPEUTIC CONSEQUENCES OF EXTRACELLULAR pH MODULATION IN VITRO... Ill Introduction Ill Results 118 Theoretical Drug Distribution 118 Characterization of the MCF-7 In Vitro Model System 126 In Vitro Drug Accumulation 130 In Vitro Drug Accumulation in Resistant Model Systems 134 In Vitro Toxicity 141 Discussion 161 IV - THE PHARMACOKINETIC CONSEQUENCES OF METABOLIC ALKALOSIS ENHANCED CHEMOTHERAPY WITHIN IN VIVO BREAST CARCINOMA MODEL SYSTEMS 161 Irrtro^iuctjLon.•...• 161 Results 161 MCF-7 Tumor pH Modulation by Chronic Sodium Bicarbonate Ad Librixim In SCID Mice 163 Acute Modulation of C3H Mammary Carcinoma Tumor pH in C3H/hen Mice with B^carbona t • • • • 166 The Effect of Chronic 200 mM Ad Librium Sodium Bicarbonate Administration on MCF-7 Tumor Growth in SCID Mice 181 The Effect of Acute Alkalization, via Intraperitoneal Injection of 0.7 ml 8- of a IM NAHCO3, on the Growth Rate of C3H Mammary Carcinoma in C3H/hen Mice 184 The Effect Of Chronic Metabolic Alkalosis On Doxorubicin's Anti-Tumor Activity in the MCF-7 Human Mammary Carcinoma In Vivo Model System 187 Acute Alkalization with Bicarbonate Enhances Mitoxantrone's Anti-Tumor Activity in C3H Mammary Carcinoma In Vivo. 192 The Effect Of Our Acute Tumor Alkalization Protocol, i.e. the Intraperitoneal Injection of 0.7 ml of a IM NaHC03, on Dose Limiting Toxicity to the Host, C3H/Hen Mice 200 The Effect Of Acute Tumor Alkalinization Via Intraperitoneal Injection of 0.7 ml Of IM NaHCOa, On Paclitaxels C3H Mammary Carcinoma Anti-Tumor Activity In C3H/Hen Mice 209 Discussion 217 V - THE EFFECT OF METABOLIC ALKALOSIS ON DRUG BIODISTRIBUTION IN VIVO 223 Introduction ..223 Results 227 Mitoxantrone Biodistribution in C3H Mammary Tumor Bearing C3H/hen Mice 227 Doxorubicin Biodistribution in MCF-7 Mammary Tumor Bearing SCID Mice 243 Doxorubicin Biodistribution in C3H Mammary Tumor Bearing C3H/hen Mice 259 Discussion.™ 275 VI - CONCLUSIONS 280 VII - REFERENCES 284 9 LIST OF FIGURES Figure 1.1 Doxorubicin 29 Figure 1.2 Daunorubicin 34 Figure 1.3 Mitoxantrone 40 Figure 1.4 Paclitaxel 45 Figure 1.5 Vincristine 49 Figure 1.6 Vinblastine 54 Figure 1.7 Cyclophosphamide 61 Figure 1.8 Chlorambucil 66 Figure 1.9 5-Flourouracil 72 Figure 3.1 Predicted Partitioning of Mitoxantrone (pKa =8.2) 121 Figure 3.2 Predicted Partitioning of Chlorambucil (pKa = 5.8) 122 Figure 3.3 ^'*C Doxorxabicin Partition Coeffecient 123 Figure 3.4 Flourescent Measurement of Doxoriibicin's Partition Coeffecient 124 Figure 3.5 ^"'c Mitoxantrone Partition Coeffecient ..125 Figure 3.6 MCF-7 Cell Density Following Either Acid or Base Exposure..~.M....M.n............................M................u.......M.......~....... 128 Figure 3.7 Intracellular pH of MCF-7 Cells as a Fxinction of Extracellular pH 129 Figure 3 .8 In vitro "c Doxorxibicin Accumulation in P^^«»F 7 11 S .....M....M.».M....H....n.......»....m.M....»..........M.............M.M...........• 13 2 Figure 3 .9 In Vitro ^"^C Mitoxantrone Accumulation in MCF-7 Cells 133 10 Figure 3,10 In.