University of the Pacific Scholarly Commons University of the Pacific Theses and Dissertations Graduate School 1982 Effects of high and low dose warfarin sodium on implanted spontaneous CΓéâH Joan-Marie Deweese-Mays University of the Pacific Follow this and additional works at: https://scholarlycommons.pacific.edu/uop_etds Part of the Medicine and Health Sciences Commons Recommended Citation Deweese-Mays, Joan-Marie. (1982). Effects of high and low dose warfarin sodium on implanted spontaneous CΓéâH. University of the Pacific, Thesis. https://scholarlycommons.pacific.edu/uop_etds/ 2082 This Thesis is brought to you for free and open access by the Graduate School at Scholarly Commons. It has been accepted for inclusion in University of the Pacific Theses and Dissertations by an authorized administrator of Scholarly Commons. For more information, please contact [email protected]. EFFECTS OF HIGH AND LOW DOSE WARFARIN SODIUM ON IMPLANTED SPONTANEOUS C3H/HeJ MOUSE MAMMARY TUMOR GROWTH AND RELATED FACTORS A Thesis Presented to the Faculty of the Graduate School University of the Pacific In Partial Fulf~llment of the Requirements for the Degree Master of Science by Joan-Marie Deweese-Mays December-1982 This thesis, written and submitted by Joan-Marie Deweese-Mays is approved for recommendation to the Committee on Graduate Studies, University of the Pacific. Department Thesis Committee: Chairman Dated-------------------------- December 3, 1982 This thesis, written and submitted by Joan-Marie Deweese-Mays is approved for recommendation to the Committee on Graduate Studles, University of the Pacific. Qor~ Thesis Committee: Dated---------------------------------------- December 3, 1982 ACKNOWLEDGMENTS Sincere gratitude is extended to Dr. Katherine Knapp for her special interest and guidance throughout my studies at the University of the Pacific, during our research, and in preparation of this thesis. Appreciation is also extended to Drs. James Blankenship, Alice Hunter, Warren Schneide:!:", and Kathryn Taubert for their professional guidance and providing advice and careful analysis leading to the comple­ tion of this thesis. Special thanks goes to my parents for their love, support, and for instilling the understanding of the impor­ tance of education; to my sister for always being a good friend and listening to countless lectures about mice, tumors, and warfarin; and to my husband for his tremendous love and belief in me. An additional thank you is extended to Sharon Young, Pharm. D, and Elson Kim who have provided vital technical and professional assistance. i TABLE OF CONTENTS Page LIST OF TABLES . iii LIST OF FIGURES iv INTRODUCTION 1 RATIONALE 3 LITERATURE SURVEY 5 MATERIALS AND METHODS 40 RESULTS 51 DISCUSSION 66 CONCLUSION 73 REFERENCES 75 ii LIST OF TABLES Table I Experimental Schedule . 45 II Criteria for Vascularity Rating in Tumor 48 Locale . III Criteria for Attachment, Edema Fluid, and 49 Local Vasodilation Evaluations IV Summary of Mean Tumor Implant Weights, 52 Whole Blood Clotting Times, and Percent Change in Tumor Weight . v Attachment and Vascularity Evaluations 58 VI Local Vasodilation Evaluation~ 64 VII Edema Fluid Evaluations 65 iii LIS'l' OF FIGURES .Figure I Dvorak Model Postulates of 3 of the Tumor Capabilities . 9 II Common Synonyms of Blood Clotting Factors 16 III Structural Formula of Warfarin Sodium 18 IV Structure and Origin of Vitamin Kl 19 v Structure and Origin of Vitamin K2 20 VI Structure and.Origin of Vitamin K3 21 VII Pathway of Thrombus Formation 23 VIII Action of Warfarin upon Thrombus Formation 24 IX Activity of Vitamin K in Clot Formation 25 X Effect of Altered Blood Coagulability on the Development of Tumor Colonies in Various Animal Systems (Modified from Hilgard, 1976) 30 XI Whole Blood Clotting Times 54 XII Mean Percent of Original Tumor Mass 57 XIII Mean Attachment of Tumor 60 XIV Mean Vascular Rating . 62 iv INTRODUCTION For over two hundred years there has been documentation of the extreme concern and quest for a method to restrain cancer, a condition which has plagued both men and animals (Karnofsky, 1948). The primary obstruction to the control and eradication of this class of disease has been the lack of a thorough understanding of the mechanisms involved in neo­ plastic establishment, survival, and growth, although the presence of tumor-specific transplantation antigens and an immunocompetent host has been established (Smith, 1975). A number of workers have observed the existence of a fibrin investment early in the development of implanted and spontaneously arising tumors (Hiramoto, 1960; Dvorak, 1979). As early as 1878, Billroth detected tumor cells encased inside a fibrin deposit (Zacharski, 1979). Tumors have been observed to possess the ability to stimulate the prolifera­ tion of host vessels to meet their metabolic needs in a number of clinical and experimental systems including man, mice, rats, and rabbits (Gimbrone, 1974; Folkman, 1975, 1976; Auerbach, 1976; Gullino, 1978; Chodak, 1980). Studies exam­ ining the vascularization of tumors have confirmed tumor growth dependence upon a vascular supply (Folkman, 1976; Gullino, 1981). Experiments in which the tumor is physically separated from other tissues demonstrate the tumor is still l 2 capable of stimulating vascular proliferation through the barrier--supporting the theory that tumor cells release a diffusible tumor angiogenesis factor (Folkman, 1976). The isolated tumor angiogenesis factor has mitogenic effects upon capillary endothelial cells and can cause the formation of tubular networks of blood vessel endothelial cells in vitro (Folkman, 1980). Although a vast amount of research has been conducted in this area, numerous questions still exist in the understanding of the relationship of tumor establishment and growth. RATIONALE Previous experiments in the laboratory under the direc­ tion of Dr. Katherine K. Knapp looked at the effects of sodium heparin upon the growth and morphology of implanted spontaneous C3HjHeJ mouse mammary tumors. These experiments demonstrated that heparin anticoagulation was accompanied by reduced tumor growth and vascularization (Knapp et al., 1981). The concept of tumor dependence upon angiogenesis for growth has led to the proposal that a heparin-induced alteration in the host's normal blood coagulation pathway could modulate fibrin encasement of the tumor and circumvent its establishment. This was thought to be a~complished through a reduction in tumor vascularization as a result of the decreased fibrin encasement of the tumor (Knapp et al., 1981; Banaja, 1982). Continuing the study of the relationships between fibrin investment of the tumor, vascularity, and tumor growth, we decided to investigate the relationship of war­ farin sodium anticoagulation with tumor growth and vascu­ larization. It was reasoned that if the previously observed altered tumor growth was due to heparin's anticoagulant effect rather than a direct effect upon the tumor, another anticoagulant with a different mechanism of action would have the same tumor growth reducing capabilities. Warfarin sodium produces reduced fibrin pqlymer formation by a 3 4 mechanism entirely different from that of heparin. Hepar~n's immediate anticoagulant activity results from a blockade of thrombin's activity on fibrinogen, prevention of prothrombin conversion to thrombin, and a reduction in platelet adhe­ siveness (Vigran, 1965; Gilman, 1980). Warfarin's delayed activity, however, is through an inhibition of vitamin K activity leading to reduced synthesis of several clotting factors (Gilman, 1980). With the decision to use warfarin sodium, experiments were designed to test the hypothesis that a reduction or prevention of fibrin formation and thus tumor encasement with this polymer would alter tumor growth. It was also hypothesized that, accompanying the altered tumor growth, several macroscopic factors including tumor vascularization, extent of tumor attachment, vasodilation of host blood vessels in the locale of the implanted tumor, and local edema fluid would be altered. Experiments were conducted to determine the relationship between the dose of warfarin sodium administered and the degree of alteration of tumor growth and the related factors. An inverse dose­ response relationship between dose of warfarin and tumor growth and the related parameters was hypothesized. LITERATURE SURVEY Studies of Dvorak Part of the foundation for our studies developed from investigations conducted by Harold Dvorak and his associates at Harvard University Medical School. Using line 10 guinea pig hepatocarcinoma and relying upon his observations from these studies, Dvorak postulated that a tumor has the capacity to promote its growth through the manipulation of its host's clotting system (1979, 1979a). Dvorak demon­ strated that the tumor cells released four factors which have the capacity to effectively alter the host's immune and infla~matory mechanisms. These alterations entail an increase in vascular permea~ility in the tumor locale, stim­ ulation of blood coagulation, a stimulatory effect upon plasminogen and inhibition of macrophage migration (Dvorak, 1979, 1979a). The first factor distinguished was the vascular permea­ bility factor (Dvorak, 1979, 1979a). This factor was postu­ lated to contribute to the tumor's establishment of a fibrin encasement by permitting the escape of plasma and thus clotting factors into the area of the tumor (Dvorak, 1979, 1979a). Its activity has been thought to be upon the capil­ lary endothelial cells (Dvorak, 1979,