Wayne State University Wayne State University Dissertations 1-1-2014 Molecular Studies On The Anti-Tumor Effects Of Metal-Based Complexes: Involvement Of The Ubiquitin-Proteasome And Apoptotic Pathways Sara M. Schmitt Wayne State University, Follow this and additional works at: http://digitalcommons.wayne.edu/oa_dissertations Part of the Molecular Biology Commons, and the Oncology Commons Recommended Citation Schmitt, Sara M., "Molecular Studies On The Anti-Tumor Effects Of Metal-Based Complexes: Involvement Of The Ubiquitin- Proteasome And Apoptotic Pathways" (2014). Wayne State University Dissertations. Paper 915. This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. MOLECULAR STUDIES ON THE ANTI-TUMOR EFFECTS OF METAL- BASED COMPLEXES: INVOLVEMENT OF THE UBIQUITIN- PROTEASOME AND APOPTOTIC PATHWAYS by SARA M. SCHMITT DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2014 MAJOR: CANCER BIOLOGY Approved by: Advisor Date ©COPYRIGHT BY SARA M. SCHMITT 2014 All Rights Reserved DEDICATION For my family. For my parents: Mom and Dad, thank you for your never-ending love and support. You have always inspired me to succeed and I would not be who or where I am today without you. I am lucky to have had parents that always let me try anything (within reason) at least once, and for letting me decide when I had taken on too much. Thank you for always being there to cheer me on or pick me up when I was down, even if it meant dropping everything else to be there. Thank you for always encouraging me and pushing me to succeed, especially in those times when I was ready to give up. Thank you for always believing in me and knowing that I could, and would, succeed if I put my mind to it. Words cannot express how grateful I am for all of the sacrifices you have made for me throughout my life. To my sisters: Christa and Libbie, thank you for remaining the unique women that you are and for reminding me to have a little fun sometimes. Thank you for always cheering me on and for the little notes of encouragement when you couldn’t be there in person. Finally, thank you for forcing me to be a better version of myself so that you had someone to look up to and be proud of. For my husband: Curtis, you have been with me throughout this whole process and I never would have gotten through this without your love and support. I am so grateful for your patience and encouragement through all of my ups and downs. Thank you for always letting me follow my path and for pushing me to try new things. Your generally stress-free attitude towards life has grounded me and for that I am extremely grateful. Thank you for being you and for allowing me to be me. ii ACKNOWLEDGEMENTS This dissertation would not have been possible without the mentorship of Dr. Q. Ping Dou, who accepted me into his lab and gave me the guidance necessary to complete such a work. Thank you for giving me the many opportunities to participate in such a variety of projects as well as for trusting me to independently write research articles, reviews, editorials and book chapters. I know that these opportunities, as well as the collaborations that I have participated in, have expanded my skills and will be useful in the future. Thank you to my committee members, Dr. Julie Boerner, Dr. Sreenivasa Chinni, Dr. Arun Rishi and Dr. Jeremy Kodanko, for all of your guidance in all matters of my graduate studies. Your questions and advice have been invaluable throughout this process. Thank you also to the Graduate Program in Cancer Biology, especially individually Dr. Larry Matherly, Dr. George Brush and Dr. Malathy Shekhar, for everything you’ve done for me. I know that my time in this program will afford me success in the future. Thank you to Dajena Tomco and Dr. Claudio Verani, as well as Dr. Jai Prakash and Dr. Jeremy Kodanko, from the Department of Chemistry at Wayne State University and Dr. Chiara Nardon and Dr. Delores Fregona from the University of Padova for synthesizing the many compounds for me to work with. Our collaborations have been extremely productive and I am glad that I was given the opportunity to work with all of you. Thank you to Dr. Christine Neslund- Dudas and Dr. Bharati Mitra for formulating the ALAD project and for allowing me to contribute to the early discussions and final product. I would be remiss if I did not thank all of the members of the Dou lab, both past and present. Special thanks to Dr. Min Shen and Dr. Daniela (Buac) Ventro for always discussing data and iii offering suggestions, as well as to Dr. Di Chen and Cindy Cui for training and helping me with experiments when needed. Thank you also to Jian Zuo for your collaboration on the XIAP project. Finally, thank you to the friends I’ve made here, without whom this experience would have lacked any sanity. Elizabeth Tovar, thank you for always being there for me no matter what, I am so lucky and grateful to have a wonderful friend like you. Thank you again to Daniela, for being not only a great colleague, but also a friend; your advice was always appreciated. iv TABLE OF CONTENTS Dedication ii Acknowledgements iii List of Tables vii List of Figures viii Chapter 1. Introduction 1 Ubiquitin-Proteasome Pathway 1 Apoptosis 8 Proteasome Inhibitors 10 δ-Aminolevulinic Acid Dehydratase 24 Metals in Cancer Development 28 Metal-based Complexes as Proteasome Inhibitors 29 Proteasome Inhibition via Metal Chelation 35 Chapter 2. Materials and Methods 39 Chapter 3. The Heme Synthesis Enzyme δ-Aminolevulinic Acid Dehydratase (ALAD) as an Endogenous Inhibitor of the Proteasome: Involvement of ALAD-20S Proteasome Complexes in Ubiquitination and Acetylation of Proteasomal α2 Subunits 48 Results 49 Discussion 61 Chapter 4. Metal Complexes Targeting the Proteasomal Core 65 4.1 Effects of Tethered Ligands and of Metal Oxidation State on the Interactions of Cobalt Complexes with the 26S Proteasome Results 76 Discussion 84 v 4.2 Gold(III)-Dithiocarbamato Peptidomimetics in the Forefront of the Targeted Anticancer Therapy: Preclinical Studies against Human Breast Neoplasia Results 85 Discussion 101 4.3 Inhibition of the 26S Proteasome as a Possible Mechanism for Toxicity of Heavy Metal Species Results 103 Discussion 111 4.4 Differential Effects of Ga(III)- and Zn(II)-Tethered Ligands on Proteasome Activity and Apoptosis in Cultured Prostate Cancer Cells Results 113 Discussion 121 4.5 Nitroxoline Inhibits the Tumor Proteasome and Suppresses Tumor Growth Results 122 Discussion 132 Chapter 5. Metal Chelators Destabilize the E3 Ligase X-linked Inhibitor of Apoptosis (XIAP): Novel Polypyridyl Chelators Deplete Cellular Zinc and Destabilize the X-linked Inhibitor of Apoptosis Protein (XIAP) Prior to Induction of Apoptosis in Human Prostate and Breast Cancer Cells 133 Results 134 Discussion 147 References 150 Abstract 189 Publications 191 Autobiographical Statement 194 vi LIST OF TABLES Table 1. Structures of Compounds Targeting Upstream Factors. 23 Table 2. AuD6 and 8 inhibit proteasome activity under cell-free conditions. 90 vii LIST OF FIGURES Figure 1. Structure of the 26S Proteasome. 4 Figure 2. Ubiquitin-Proteasome Pathway. 7 Figure 3. The UPP and Apoptosis. 9 Figure 4. Structures of Peptide Aldehyde Proteasome Inhibitors. 11 Figure 5. Structures of Naturally Occurring Proteasome Inhibitors. 13 Figure 6. Structure of Bortezomib. 15 Figure 7. Structures of Next-Generation Proteasome Inhibitors. 20 Figure 8. Heme Biosythesis and ALAD Structure. 26 Figure 9. Structures of Gallium Complexes. 32 Figure 10. Structures of Gold Complexes. 34 Figure 11. Structures of Metal Chelators Used as Proteasome Inhibitors. 38 Figure 12. ALAD binds the 20S proteasome in place of the 19S regulatory cap and is associated with ubiquitinated forms of α2 in cultured prostate cancer cells. 51 Figure 13. ALAD binds the 20S proteasome and is associated with ubiquitinated forms of α2 in human erythrocytes. 52 Figure 14. ALAD is both an inhibitor and a target of the proteasome. 54 Figure 15. SAHA treatment enhances the ALAD-proteasome interaction, associated with acetylation of ubiquitinated α2 subunits. 56 Figure 16. SAHA-treatment promotes nuclear localization of ALAD and modified α2. 58 Figure 17. Purified ALAD promotes ubiquitination of proteasomal α2 under cell free conditions. 60 Figure 18. Proposed Mechanism of ALAD-Proteasome Binding. 64 Figure 19. Structures of Cobalt Compounds. 67 Figure 20. Structures of New Gold(III) Complexes. 69 viii Figure 21. Structures of Heavy Metal Complexes. 71 Figure 22. Structures of Tethered Ga(III) and Zn(II) Complexes. 73 Figure 23. Structures of Nitroxoline and Clioquinol. 75 Figure 24. Complex 3, the Co(III) species, inhibits PC-3 cell proliferation. 77 Figure 25. Complex 3 inhibits purified 20S proteasome activity. 79 Figure 26. Complex 3 inhibits cellular proteasome activity. 81 Figure 27. Complex 3 induces apoptosis in PC-3 cells. 83 Figure 28. Aud6 and AuD8 are potent inhibitors of cellular proliferation. 86 Figure 29. Trolox enhances the anti-proliferative ability of AuD6. 88 Figure 30. AuD6 and 8 inhibit intact cellular proteasome in MDA-MB-231 cells. 92 Figure 31. AuD6 and 8 inhibit intact cellular proteasome and induce apoptosis in MDA-MB-231 cells. 94 Figure 32. AuD6 and 8 induce apoptosis in MDA-MB-231 cells. 96 Figure 33. AuD8 inhibits the proteasome and tumor growth in vivo. 98 Figure 34.