The Immune-modulatory and Anti-carcinogenic Mechanisms of the Flavonoid Apigenin Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Daniel A. Arango Tamayo, B.S. Graduate Program of Molecular Cellular and Developmental Biology The Ohio State University 2015 Dissertation Committee: Dr. Andrea I. Doseff, Advisor Dr. Erich Grotewold, Co-Advisor Dr. Denis Guttridge Dr. Tsonwin Hai Dr. Dawn Chandler Copyright by Daniel Arango 2015 Abstract Dietary phytochemicals provide health benefits against several cancers and inflammatory diseases. Flavonoids are amongst the most abundant dietary phytochemicals emerging as key anti-carcinogenic and anti-inflammatory molecules. Yet, the mechanisms underlying their anti-cancer and anti-inflammatory activities are poorly defined. The goal of this project was to study the immune-modulatory and anti- carcinogenic mechanisms of the flavonoid apigenin. I investigated the modes of action of apigenin using different model systems including a monocytic leukemia cell line, breast cancer cell lines, macrophages and mouse models of inflammation and breast cancer development. In monocytic leukemia, I found that apigenin induces DNA strand breaks leading to the activation of a DNA damage response pathway that results in cell cycle arrest and induction of apoptosis. Using mouse models of inflammation, I showed that apigenin reduces lipopolysaccharide (LPS)-induced lethality by inhibiting the activity of the transcription factor NF-κB and the expression of the pro-inflammatory molecules miR-155 and TNFα. I established, using a pre-clinical mouse model of breast cancer development, that the immune-modulatory and anti-carcinogenic activities of apigenin work in concert to delay breast tumor progression and metastasis by dually acting on malignant and immune cells. My results show that apigenin induces apoptosis ii and inhibits proliferation in breast tumors as well as halts macrophages infiltration in the tumor microenvironment by reducing the expression of NF-κB-dependent chemokines and promoting apoptosis in blood monocytes, the macrophage progenitors. Moreover, I implemented the use of a newly formulated celery-based apigenin-rich diet in mouse models of inflammation and breast cancer demonstrating that this diet, as well as apigenin, have anti-inflammatory and anti-carcinogenic activities by immune-modulating monocytes and macrophages and inducing apoptosis in cancer cells. To investigate the molecular mechanisms underlying the biological effects of apigenin, we developed of a new genome-wide approach to identify direct targets of this flavonoid. From these studies, I identified 160 candidate targets of apigenin that revealed unexpected mechanisms on how this dietary phytochemical modulates cellular functions such as apoptosis, immune and DNA damage response signaling pathways. In addition, I observed that apigenin interacts with RNA binding proteins including the heterogeneous nuclear RiboNucleoProtein A2 (hnRNPA2) and affects splicing genome-wide, providing a novel mechanism on how this flavone regulates apoptotic cell fate through modulation of mRNA processing. Altogether, this investigation offers a fresh view on how flavonoids influence human health, by impacting multiple cellular targets with moderate affinity. Thus, in contrast to pharmaceutical drugs selected to have high affinity and specificity for main hubs of biological pathways, the effect of flavonoids would be distributed across the entire cellular network with consequent benefits on human health. In addition, these results support the use of functional foods rich in flavonoids as an alternative for the treatment and prevention of inflammatory diseases and cancer. iii Dedication To my parents For planting the seed of curiosity in me A mis padres Por sembrar la semilla de la curiosidad en mi iv Acknowledgements I am deeply thankful with my advisor Dr. Andrea Doseff for her guidance, scientific education and advices throughout these years and to my co-advisor Dr. Erich Grotewold for his collaboration and scientific contribution to my education. My sincere appreciation to Dr. Dennis Guttridge, Dr. Dawn Chandler and Dr. Tsonwin Hai for accepting being in my committee and their scientific contributions to my education. I am especially grateful with Dr. Timothy Eubank for his help and collaboration with animal models of cancer. Especial gratitude to Dr. Kengo Morohashi for teaching me his expertise on PD-seq and for his incredible contribution to the identification of apigenin targets. I would like to thank Dr. Alper Yilmaz, Dr. Xiaokui Mo, Mrs. Katherine Mejia-Guerra, Mr. Erich Mukundi and Mr. Francisco Padilla-Obregon for their help with bioinformatical analyses. My sincere acknowledgment to the former and current members of Doseff and Grotewold laboratories, especially Dr. Arti Parihar, Dr. Horacio Cardenas, Dr. Greg Hostetler, Dr. Oliver Voss, Dr. Yadira Malavez, Dr. Antje Feller, Dr. M. Elba Gonzalez-Mejia, Dr. Wei Li, Dr. Marcelo Pereira, Dr. Isabel Casas, Ms. Silvia Duarte, Ms. Catalina Pineda, Mr. Luis D. Prada and Mr. Roberto Alers. It was a pleasure to work with each of them. Especial thanks to Mr. Bledi Brahimaj for his help on cloning all FRET constructs and Ms. Joanna Li for their help with protein purifications and pull downs. I would like to thank the visiting scholars Ms. Mayra Diosa-Toro and Ms. Laura v Rojas for their contributions on the miRNA manuscript and Mrs. Giovanna Merchand for her help with macrophage phenotyping in the PyMT model. I would like also to thank Drs. Tom Schmittgen and Jinmai Jiang for their help with microRNAs. Thanks to Drs. Jessica Cooperstone and Ken Riedl for their help with the preparations of celery-based apigenin diets. Especial gratitude to Dr. Kouji Kuramochi for providing the apigenin beads. I am very grateful with Drs. Wolf Frommer, Lexie Friend, Adrian R. Krainer, Ann C. Williams and Philip B. Wedegaertner for constructs. Finally and I would like to acknowledge my funding sources, Pelotonia, the Food Innovation Center and the Public Health Preparedness for Infectious Diseases Program. vi Vita July, 28th 1984………………………………..…Medellin, Colombia. 2001 – 2006…………………………………….B.S., Biology. University of Antioquia. Medellin, Colombia. 2008 – 2015……………………………………Graduate Research Associate. Working towards Ph.D., Molecular Cellular and Developmental Biology. The Ohio State University, Columbus, OH. Publications Arango D, Diosa-Toro M, Rojas-Hernandez LS, Cooperstone JL, Schwartz SJ, Mo X, Jiang J, Schmittgen TD, Doseff AI. Dietary apigenin reduces LPS-induced expression of mir-155 restoring immune balance during inflammation. 2015. Mol Nutr Food Res 59: 763-772. Arango D*, Morohashi K*, Yilmaz A, Kuramochi K, Parihar A, Brahimaj B, Grotewold E, Doseff AI. Molecular bases for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets. 2013. Proc Natl Acad Sci 110: E2153-E2162. Duarte S*, Arango D*, Parihar A, Hamel P, Yasmeen R, Doseff AI. Apigenin protects endothelial cells from lipopolysaccharide (LPS)-induced inflammation by decreasing caspase-3 activation and modulating mitochondrial function. 2013. Int J Mol Sci 14: 17664-17679. Arango D, Parihar A, Villamena FA, Wang L, Freitas MA, Grotewold E, Doseff AI. Apigenin induces DNA damage through the PKCδ-dependent activation of ATM and H2AX causing down-regulation of genes involved in cell cycle control and DNA repair. 2012. Biochem Pharmacol 84: 1571-1580. Hostetler G, Riedl K, Cardenas H, Diosa-Toro M, Arango D, Schwartz SJ, Doseff AI. Flavone deglycosylation increases their anti-inflammatory activity and absorption. 2012. Mol Nutr Food Res 56: 558-569. *Shared first authorship. vii Fields of Study Major Field: Molecular, Cellular and Developmental Biology viii Table of Contents Abstract ……………………………………………………………………..…………… ii Dedication………………………………………………………………………………...iv Acknowledgements ……………………………………...………………………………. v Vita………………………………………………………………………….……..…….vii Table of contents…………...……………………………………………….……..……...ix List of Figures…………………………………………………………………………..xvii List of Tables…………………………………………………………………………...xxii Chapter 1. Introduction…………………..…………..…………..…………..…………... 1 1.1 Origin of monocytes and macrophages……………………………………….. 2 1.2 Role of monocytes and macrophages in inflammation……………………….. 3 1.3 Malignant transformation of monocytic cells……...…………………............. 5 1.4 Regulation of apoptosis…………………...……...…………………................ 6 1.5 Mechanisms of breast carcinogenesis…..……...……………………........…... 8 1.5.1 Role of monocytes and macrophages in breast cancer………………….. 10 1.5.2 Role of NF-κB in breast cancer…………………….………………….... 12 1.5.3 Transcriptome alterations in breast cancer……………………………… 13 ix 1.5.3.1 Alternative splicing of RNA…………………..……………….......... 14 1.6 Flavonoids ……………..……………..……………..………………………...... 15 1.6.1 Anti-carcinogenic activity of apigenin……………..……..………..….... 16 1.6.2 Apigenin and inflammation ………………………..……..…………...... 18 1.6.3 Molecular targets of apigenin………………………..……..…………… 18 Chapter 2. Material and Methods……………..…….…………………….…………….. 26 2.1 Chemicals and Reagents……………..…………………………..………....... 26 2.2 Cell Lines and Culture……………..…………………………..…………….. 28 2.3 Analysis of cell cycle and proliferation ……………..……..………………... 29 2.4 Caspase-3 activity and apoptosis ……………..……..………………………. 30 2.5 Intracellular measurement of ROS……………..……..……………………… 31 2.6 Alkaline comet assay……………..……..……………………………….......