Internalization of Extracellular ATP in Cancer Cells and Development of New Generations of Anticancer Glucose Transport Inhibitors

3 Acknowledgments . 5 List of Tables . 10 List of Figures . 11 Abbreviations . 14 Chapter 1 Introduction . 19 1.1 Cancer metabolism and the Warburg effect . 19 1.2 Original hypothesis and current findings of the Warburg effect . 20 1.3 Brief history and current interpretations of the Warburg effect . 21 1.4 The Warburg effect in normal cells and cancer cells . 23 1.5 ROS balancing and the Warburg effect . 24 1.6 Gene regulation and the Warburg effect . 26 1.7 Oxygen supply, ATP synthesis and the Warburg effect . 30 1.8 Warburg effect at the tumor microenvironment scale . 33 1.9 The Warburg effect as a potential target for cancer treatment . 36 1.10 Conclusion . 43 Chapter 2: Internalization Of Extracellular ATP In Cancer Cells . 45 2.1 Introduction . 45 2.2. Materials and methods . 47 2.2.1 Compounds and cell lines . 47 2.2.2 ATP rescue study and inATP measurement . 48 2.2.3 Protein analysis, clonogenic and flow cytometry assays of cancer cells . 48 2.2.4 Doseand time-dependence studies of ATP . 49 2.2. 5 Fluorescence microscopy and ATP localization studies . 49 2.2. 6 Oligomycin and glucose deprivation assays – time course study . 50 2.2. 7 Studies with inhibitors of AMPK, transcription, translation . 50 2.2. 8 Macropinocytosis inhibitor study . 50 2.2. 9 Drug resistance study . 51 2.2.10 Experimental designs and statistical analysis . 51 2.3 Results . 51 2.3.1 exATP rescued cancer cells under different metabolic stresses and reduced anticancer efficacy of tyrosine kinase inhibitors . 51 2.3.2 exATP induced doseand timedependent inATP increases in multiple cancer cell lines . 55 2.3.3 Nontumorigenic cells displayed profiles of exATP induced inATP increase different from those of cancer cells . 56 8 2.3.4 Colocalization of ATP analog and high-molecular-weight (HMW) dextran in A549 cells . 58 2.3.5 inATP was increased by exATP even when OXPHOS and/or glycolysis were blocked . 60 2.3.6 exATP induced changes in levels and phosphorylation of AMPK . 63 2.3. 7 inATP increase was not dependent on AMPK, transcription, or translation . 65 2.3. 8 Inhibiting macropinocytosis reduced inATP levels and anticancer activity of TKIs . 66 2.4 Discussion . 68 Chapter 3 Inhibitors Of Glucose Transport And Glycolysis As Novel Anticancer Therapeutics . 76 3.1 Cancer and cancer metabolism . 76 3.2 Glucose transport and glucose metabolism in cancer cells – the Warburg effect . 79 3.3 Anticancer therapeutics targeting glycolysis and its connected pathways . 80 3.4 Glucose transporters (GLUTs) and upregulation of GLUTs in cancer . 95 3.5 Glucose transporter 1 (GLUT1) inhibitors . 101 3.6 Future directions and challenges . 105 Chapter 4: Development Of New Generations Of Anticancer Glucose Transport Inhibitors . 109 4.1 Introduction . 109 4.2 Methods and materials . 110 4.2.1 Cell culture . 110 4.2.2 Compound preparation . 111 4.2.3 Glucose uptake assay . 111 4.2.4 Cell viability assay (MTT assay) . 112 4.2. 5 Compounds stability in serum test . 112 4.2. 6 Western blot analysis . 112 4.2. 7 Cell cycle analysis . 113 4.2. 8 RNA isolation and realtime PCR . 114 4.2. 9 Statistical analysis . 115 4.3 Results . 115 4.3.1 WZB-173, a lead compound of a new generation of glucose transport inhibitors, is much more stable than WZB-117 . 115 4.3.2 WZB-173 induced cell cycles arrested at G1 phase in A549 human lung cancer cells through the regulation of phosphorylation and total Rb protein . 120 4.3.3 DRB series compounds as a new generation of glucose transport inhibitors 124 4.3.4 DRB-18, a lead compound of new generation of glucose transport inhibitors, was much more potent than WZB-117 in NCI-60 cancer cell panels . 130 4.4 Disscussion . 141 Chapter 5: Future Work . 145 5.1 Introduction . 145 5. 2 Study of exATP in cancer cells and tumors . 146 5.2.1 Mechanism study of ATP internalization . 146 9 5.2.2 Identification of sources of ATP increase in intratumoral spaces . 154 5.2.3 Mechanism study of exATP-induced drug resistance . 157 5.2.4 Studies on exATP-induced metastasis . 159 5.3 Development of new generations of glucose transporter inhibitors . 160 5.3.1 Optimization study of GLUT1 inhibitors . 160 5.3.2 Identification of targets . 161 5.3.3 Mechanisms study of GLUT1 inhibitors . 161 5.3.4 Animal studies for safety and efficacy . 162 5.3.5 Optimization of screening strategy . 163 5.3.6 Optimization of the selectivity of GLUT1 inhibitors . 164 5.4 Links between ATP internalization and GLUT 1 inhibitors studies . 165 References . 168 Appendix 1. Structures And Parameters Of DRB Series Compounds . 202 Appendix 2. NCI-60 Cancer Cells Panel Test of WZB-117 (10 μM) .