Identification, screening and exploring potentials of PKC directed molecules in anti-cancer drug development A thesis submitted in partial fulfilment of the requirement for the degree of Doctor of Philosophy by Suman Jyoti Deka Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati Guwahati-781039, Assam, India TH-1913_11610611 Indian Institute of Technology Guwahati. Department of Biosciences and Bioengineering. Statement I hereby declare that the matter embodied in this thesis entitled “Identification, screening and exploring potentials of PKC directed molecules in anti-cancer drug development” is a cumulative account of the results of investigations carried out in the Department of Biosciences & Bioengineering, Indian Institute of Technology, Guwahati, India, under the joint supervision of Dr. Vishal Trivedi and Professor Rakhi Chaturvedi. In keeping with the general practice of reporting scientific observations, due acknowledgements have been made wherever the work of other investigators are referred. Suman Jyoti Deka February 2017 Roll no: 11610611 TH-1913_11610611 Indian Institute of Technology Guwahati. Department of Biosciences and Bioengineering. Certificate It is certified that the work described in this thesis entitled, “Identification, screening and exploring potentials of PKC directed molecules in anti- cancer drug development”, by Mr. Suman Jyoti Deka (Roll no: 11610611), submitted to Indian Institute of Technology, Guwahati, India, for the award of the degree of Doctor of Philosophy, is an authentic record of results obtained from the research work carried out under our joint supervision at the Department of Biosciences & Bioengineering, Indian Institute of Technology, Guwahati, Assam, India. This work has not been submitted elsewhere for a degree. Dr. Vishal Trivedi Prof. Rakhi Chaturvedi (Supervisor 1) (Supervisor 2) TH-1913_11610611 Acknowledgements It is a great pleasure to acknowledge whoever is involved and supported me to complete my research work in time. I would like to thank Dr. Vishal Trivedi and Professor Rakhi Chaturvedi for their support and trust in my abilities, and the chance to do this project in Malaria Research Group. Nothing of this would have been possible without their support and I am thankful to their nearly endless patience and guidance. I would also like to thank my doctoral committee members Dr. Nitin Chaudhary, Dr. Sanjukta Patra and Dr. Shrikrishna N Joshi for their cyclic evaluation and valuable suggestions for this work. I am also thankful to Dr. Vibin Ramakrishnan, who provided me important guidance to carry out part of my research work. I would like to thank all my Malaria Research Group colleagues Dr. Rohitas Deshmukh, Dr. Kimjolly Lhouvum, Mr S.N.Balaji, Mr. Ankur Mishra, Mr. Kankgan Kalita, Mr. Sooram Banesh, Mr. Anil Kumar, Ms. Vimee Raturi, Mr. Sushant Kumar, Ms. Ankita Hazarika, Mr. Anish Jain, Mr. Sourav Layek, Mr. KMN Prasad, Mrs. Swagata Nag, Mrs. Ananya Bhowmick and Ms. Pallavi for their support. I would like to thank all research scholars from Department of Biosciences & Bioengineering and specifically members belonging to Dr. Vibin Ramakrishnan’s Lab, Dr. Rakhi Chaturvedi’s Lab, Dr. V.K Dubey’s lab, Dr. A.M. Limaye’s Lab and Dr. B. Anand’s lab who have helped me in my research. I would like to thank the Department of Biosciences & Bioengineering, IIT Guwahati for giving me an opportunity for the PhD Program as well as providing descent infrastructure and good research environment for my work. I would like to thank NTRF and DAE-BRNS for providing financial support to carry out my research work. I would also like to thank Indian Institute of Technology, Guwahati, for providing financial assistantship. Finally, I would like to thank my lovable family members and my dear friends, who gave immense care and support for my career growth. -Suman Jyoti Deka TH-1913_11610611 Table of Contents Table of contents i-ii List of Figures and Tables iii-vi Abbreviations vii Units viii Chapter 1. Potentials of PKC in cancer development and therapeutic outcomes 1-61 1.1 Introduction 2 1.2 Cancer facts and statistics 3-4 1.3 Cancer development and progression 4-8 1.4 Mechanism of tumor formation 8-9 1.5 Different factors contributing to cancer development 9-11 1.6 How cancer spreads to different parts of the body? 11-13 1.7 Different therapeutic approaches to treat cancer 14-22 1.8 Emergence of PKC as a master regulator in carcinogenesis 22-26 1.9 Structural and Biochemical Details of PKC 26-34 1.10 Targeting of PKC for anti-cancer drug development 34-43 1.11 Aim and scope of the proposed work 43-45 1.12 References 45-61 Chapter 2. Experimental procedures 62-76 2.1 Introduction 63 2.2 Cell culture and treatments 63 2.3 Cell viability assay 63 2.4 Cell-cycle analysis 64 2.5 Acridine Orange and Propidium Iodide (PI) staining of apoptotic and dead 64 cells 2.6 DNA-fragmentation assay 64-65 2.7 Intracellular ROS measurement 65 2.8 Immuno-localization of PKCα in MDAMB-231 cells 65-66 2.9 Preparation of membrane and cytosolic fractions 66 2.10 Immuno-blotting to detect PKC-α translocation 66-67 2.11 Lactate Dehydrogenase Assay 67-68 2.12 Immuno-blotting to detect phospho-threonine proteins 68 2.13 Measurement of change in mitochondrial membrane potential 68 2.14 Immuno-localization to study cyt-c release 68-69 2.15 Caspase-3 assay 69 2.16 Caspase-9 assay 69-70 2.17 Estimation of lipid peroxidation level 70 2.18 Estimation of protein carbonyl level 70-71 2.19 Detection of 5'-nucleotidase in membrane fraction 71 2.20 References 71-72 2.21 Appendix I 72-73 2.22 Appendix II 73-76 Chapter 3: Identification of PKC-directed ligands from different sources 77-99 3.1 Introduction 78-79 3.2 Experimental procedures 79-81 3.3 Results 82-91 i TH-1913_11610611 3.4 Discussion 91-94 3.5 References 94-96 3.6 Appendix I 96-99 Chapter 4: PKC-directed molecules affects breast cancer cells in multiple ways 100-120 4.1 Introduction 101-102 4.2 Experimental procedures 102-103 4.3 Results 103-115 4.4 Discussion 116-118 4.5 References 119-120 Chapter 5: Mechanistic details of PKC directed ligand molecules 121-155 5.1 Introduction 122-123 5.2 Experimental procedures 123-125 5.3 Results 125-149 5.4 Discussion 150-154 5.5 References 154-155 List of publications and conferences attended Reprints of published articles ii TH-1913_11610611 List of Figures Figure No. Figure Captions Page No. Figure 1.1 Schematic illustration of tumor formation 2 Figure 1.2 Cancer incidence rates in India 4 Figure 1.3 Frequent cancer induction sites in the human body 5 Figure 1.4 Schematic diagram to explain the process of cancer spread from their 12 site of origin to the other parts of the body Figure 1.5 Frequent metastatic destination of common cancer types in humans. 13 Figure 1.6 Different available cancer therapies to treat cancer patients 14 Figure 1.7 Some of the classes of popular chemotherapeutic drugs 15 Figure 1.8 Schematic diagram to explain the mechanism of action of alkylating 16 agents as anti-cancer molecules. Figure 1.9 Schematic diagram to explain the mechanism of action of paclitexal as 17 anti-cancer agent. Figure 1.10 Mechanism of action of Herceptin 19 Figure 1.11 Schematic illustration of photodynamic therapy 21 Figure 1.12 PKC isozymes regulating important signalling pathways 23 Figure 1.13 Structure of PKC-βII (PDB ID:3PFQ) 27 Figure 1.14 General Structure of PKC 28 Figure 1.15 Different PKC isozymes with diverse building blocks. 30 Figure 1.16 A schematic model to depict PKC maturation, signalling and 33 degradation Figure 1.17 Chemical Structures of few selected PKC agonists 36 Figure 1.18 Structures of selected PKC antagonists. 39 Figure 1.19 Structures of selected PKC antagonists (Continued) 43 Figure 3.1 Illustration of experimental approach pursued in Chapter 3 79 Figure 3.2 Chemical structure of selected drugs. 85 Figure 3.3 Structures of a few selected top-hit phytochemicals 87 Figure 3.4 Selected molecules fit well into the C1b domain 88 Figure 3.5 Molecular interaction of PKC-α with Danazol, Flunarizine and 89 Cinnarizine Figure 3.6 Molecular interaction of PKC-α with phytochemicals 90 Figure 3.7 Chemical structure of different alkyl cinnamates with their respective 93 compound codes Figure 4.1 Schematic representation of hypothesis that leads to the experimental 101 strategy in chapter4 Figure 4.2 Morphological deformities in MDAMB-231 or MCF-7 breast cancer 104 cells treated individually with different drugs. Figure 4.3 Morphological deformities in MDAMB-231 or MCF-7 breast cancer 106 cells treated individually with different phytochemicals. Figure 4.4 Morphological deformities in MDAMB-231 or MCF-7 breast cancer 107 cells treated individually with different alkyl cinnamates. Figure 4.5 Drug and phytochemical PKC-directed molecules disturb the cell-cycle 109 of breast cancer cells iii TH-1913_11610611 Figure 4.6 Alkyl-cinnamates disturb cell-cycle in MDAMB-231 cells 111 Figure 4.7 PKC-directed molecules reduce the viability of triple negative and 113 ER+ve breast cancer cells in a dose dependent manner. Figure 4.8 Alkyl cinnamates reduce the viability of triple negative and ER+ve 115 breast cancer cells in a dose dependent manner Figure 4.9 Probable pathway of PKC-directed ligands mediated loss of cellular- 118 viability in breast cancer cells Figure 5.1 Schematic diagram about different questions explored in Chapter 5. 122 Figure 5.2 Danazol, Flunarizine and Cinnarizine induce translocation of PKC-α to 126 the plasma membrane.