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Evaluation of Anticancer Activities in Indian Seaweeds

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Evaluation of Anticancer Activities in Indian Seaweeds EVALUATION OF ANTICANCER ACTIVITIES IN INDIAN SEAWEEDS Thesis Submitted to the University of Goa For the award of DOCTOR OF PHILOSOPHY IN MARINE SCIENCES By RASHMI C. VINAYAK., M.Sc Under the guidance of Dr. ANIL CHATTERJI NATIONAL INSTITUTE OF OCEANOGRAPHY, BIOLOGICAL OCEANOGRAPHY DIVISION, DONA PAULA- 403 004, GOA, INDIA February, 2011 1 2 CERTIFICATE I here by certify that the thesis entitled “EVALUATION OF ANTICANCER ACTIVITIES IN INDIAN SEAWEEDS”, submitted by Ms. RASHMI C. VINAYAK for the degree of DOCTOR OF PHILOSOPHY IN MARINE SCIENCES to the University of Goa is the result of the research work carried out by me under the guidance of Dr. ANIL CHATTERJI, National institute of Oceanography, Goa. I further declare that the results of this investigation have not been previously submitted for any degree or fellowship in any university or institution. (Dr. ANIL CHATTERJI) Place: NIO, Goa Date: 7/1/2013 3 DECLARATION I here by declare that the thesis entitled “EVALUATION OF ANTICANCER ACTIVITIES IN INDIAN SEAWEEDS”, submitted to the University of Goa for the degree of DOCTOR OF PHILOSOPHY IN MARINE SCIENCES is the result of the research work carried out by me under the guidance of Dr. ANIL CHATTERJI, National institute of Oceanography, Goa. I further declare that the results of this investigation have not been previously submitted for any degree or fellowship in any university or institution. (RASHMI C. VINAYAK) Place: NIO, Goa Date: 7/1/2013 4 ACKNOWLEDGEMENTS I wish to express my deep sense of gratefulness and sincere thanks to my research supervisor and guide Dr. Anil Chatterji, Scientist and Deputy Director, Biological Oceanography Division, National Institute of Oceanography, Dona Paula, Goa, for his meticulous guidance, constant encouragements and motivating advice throughout my research tenure. I wish to place on the record my deep sense of appreciation for intellectual freedom, comprehensive positive criticism and timely support given to me. I express my sincere thanks to Dr. Satish R. Shetye, Director, National Institute of Oceanography, Dona Paula, Goa, for giving me an opportunity to be associated with this institute and his silent support for the research work carried for my thesis. I am greatly indebted to Dr. Bharathi P. Salimath, Professor of Biotechnology and Chairperson, Department of Applied Botany and Biotechnology, University of Mysore, to pursue a part of my research work in her department at the University of Mysore. I am also thankful for her guidance, suggestions, expert appraisal and constant encouragement with extreme patience throughout this investigation. I am extremely indebted to Dr S. Wahidulla, Dr. P. S. Parameswaran, and Dr. Lisette of National Institute of Oceanography, Dona Paula, and Goa, for their guidance and help during my research studies. I am extremely thankful to Dr. Z. A. Ansari, Dr. Virupax Banakar of National Institute of Oceanography, Dona Paula, and Goa, for their timely help and advice during the course of my research work. I am grateful to Prof. H. B. Menon, Head, Department of Marine Sciences, Goa University, for all the moral support and encouragement during entire period of research work. My sincere thanks to Dr. C. U. Rivonkar, Reader, Department of Marine Sciences, Goa University, for all the help rendered to me during entire programme. I wish to express my sincere thanks to the catalyst to my work, namely, all my dear colleagues in the Aquaculture laboratory, especially Dr. A. S. Sabu, Dr. Sreekumar P. K, Dr. Savita Kotnala, Dr. Sumita Sharma, Dr. Shantanu Kulkarni, Ms Reena Rodrigues, Ms Madhurima Wakankar, Mr. Loknath Dora, Mr. 5 Cassius Andrade, Mr.Uddhav Suryavanshi, and Mr. Hrishikesh Pawar for their constant support and encouragements given to me without whom this thesis would have never been possible. I am very much thankful to Mr. Thippeswamy G, Mrs. Sheela M.L, Ms. Sunitha R., Mr. Shivakumar S, Mr. Prasanna Kumar S, Mr. Ramakrishna M. K, Mr. Akhilesh Kumar, Ms. Saritha D’Souza, Mrs. Kaveri K and Ms Shilpa P. They have helped me in many ways and will always be thankful for their kind help and moral support provided to me for carrying out my experimental work. I wish to acknowledge my thanks to Mr. Keshav Tari, Mr. Dinesh Shirwaikar, Mr. Suresh Gawas and Mr. P. R. Kurle for their help, companionship and encouragement during my research period in Aquaculture lab. I gratefully acknowledge CSIR for awarding me the Research Fellowship that helped me to carry out the thesis work. I am grateful to my parents, sisters for the constant support and encouragement given to me at every step of the way, without whom this thesis would never have been possible. Last but not the least; I would like to thank the almighty God for providing me with the inner strength to make this thesis a success. (RASHMI C. VINAYAK.) Place: NIO, Goa Date: 7/1/2013 6 TABLE OF CONTENTS Chapter Page No. No. DECLARATION CERTIFICATE ACKNOWLEDGEMENTS 1. GENERAL INTRODUCTION 1.1. BACKGROUND AND SIGNIFICANCE 1 1.1.1. Cancer Statistics 2 1.1.2. Causes of cancer 3 1.1.3. Cancer prevention 3 1.1.4. Cancer treatment 4 1.1.5. Anticancer agents 4 1.1.6. The need for new anticancer drugs 5 1.1.7. Natural products in cancer chemotherapy 6 1.1.8. Marine natural products as anticancer agents: their 7 source, collection and screening 1.2. APOPTOSIS: A HISTORICAL OVERVIEW 11 1.2.1. Morphological features of apoptosis 11 1.2.2. Physiological significance of apoptosis 15 1.2.3. Molecular mechanisms of apoptotic signaling pathways 15 1.2.4. Final pathway- Caspases 16 1.2.5. Targeting apoptosis pathways in cancer therapy 17 1.3. FREE RADICALS AND DISEASES 20 1.3.1. Antioxidants 22 1.3.2. Antioxidant mechanism 22 1.3.2.1 Primary (chain-breaking) antioxidant 22 1.3.2.2. Secondary antioxidant 23 1.3.3. Types of antioxidants 24 1.3.3.1. Synthetic antioxidant 24 1.3.3.2. Natural antioxidant 24 1.3.3.3. Dietary antioxidants 25 7 1.3.3.4. Endogenous antioxidants 26 1.3.4. Phytochemicals 26 1.3.4.1 Phenolic compounds 27 1.3.5. Measuring antioxidant activity 28 1.4. TUMOR ANGIOGENESIS 30 1.4.2. The angiogenic 'switch' 32 1.4.3. Mechanisms of the angiogenic switch 33 1.4.4. Angiogenesis: Activators and inhibitors 35 1.4.5. Angiogenesis inhibitors as a strategy in the treatment of 39 cancer 1.5. OBJECTIVES AND SCOPE OF THE CURRENT 44 INVESTIGATION 2. SCREENING FOR CYTOTOXIC AND PRO-APOPTOTIC ACTIVITY IN SEAWEEDS COLLECTED FROM THE COASTS OFF GOA AND MALWAN (MAHARASHTRA) 2.1 INTRODUCTION 45 2.2. MATERIALS AND METHODS 48 2.2. 1. Animals, Cell lines and Chemicals 48 2.2. 2. Collection and identification of Seaweeds 48 2.2.3. Processing and preparation of methanol extract from 49 seaweed samples 2.2.4. In-vitro culture of cancer cell lines 49 2.2.5. Hatching of Brine shrimp 50 2.2.6. Brine shrimp lethality test 50 2.2.7. MTT assay 51 2.2.8. Wright's - Giemsa staining 52 2.2. 9. Annexin V apoptosis detection assay 52 2.2.10. Propidium Iodide (PI) staining for apoptosis detection 53 2.2.11. DNA fragmentation assay 53 2.2.12 Statistical analysis 54 2.3. RESULTS 55 2.3.1. Collection and identification of seaweeds collected from 55 8 the coasts of Goa and Malwan (Maharashtra) 2.3.2. S. marginatum extract (SME) inhibits proliferation of 61 HeLa and Hep-2 cells, and is lethal to brine shrimps in-vitro 2.3.3. S. marginatum extract (SME) induces apoptosis in 62 Giemsa, Annexin V and Propidium iodide staining assay of EAT cells 2.3.4. S. marginatum extract (SME) induces DNA 62 fragmentation in EAT cells by apoptosis 2.4. DISCUSSION 71 3. SCREENING FOR ANTI-OXIDANT ACTIVITY IN SEAWEEDS COLLECTED FROM THE COASTS OF GOA AND MALWAN (MAHARASHTRA) 3.1. INTRODUCTION 74 3.2. MATERIALS AND METHODS 78 3.2.1. Chemicals 78 3.2.2. Collection and identification of Seaweeds 78 3.2.3. Processing and preparation of methanol extract from 79 seaweed samples 3.2.4. DPPH radical scavenging assay 79 3.2.5. Total Polyphenolic content 79 3.2.6. Reducing power assay 80 3.2.7. Ferrous ion chelation assay 80 3.2.8. Statistical analysis 81 3.3. RESULTS 81 3.3.1. DPPH radical scavenging activity 81 3.3.2. Total Polyphenolic Content (TPC) of seaweed extracts 82 3.3.3. Reducing power activity 82 3.3.4. Ferrous ion chelating activity 83 3.4. DISCUSSION 91 9 4. SCREENING FOR ANTI-ANGIOGENIC ACTIVITY IN SEAWEEDS COLLECTED FROM THE COASTS OF GOA AND MALWAN (MAHARASHTRA) 4.1. INTRODUCTION 99 4.2. MATERIALS AND METHODS 101 4.2.1. Collection and identification of Seaweeds 101 4.2.2. Processing and preparation of methanol extract from 101 seaweed samples 4.2.3. Animals, in-vivo tumor model, cell lines and chemicals 101 4.2.4 In-vitro culture of EAT, BeWo, HUVEC and HEK-293 102 cells. 4.2.5. Thymidine incorporation assay 102 4.2.6. Matrigel tube formation assay 103 4.2.7. Migration of HUVEC’s in oil drop assay 103 4.2.8. Migration of HUVEC’s in wound-healing assay 105 4.2.9. Chorio-allantoic membrane assay (CAM) 105 4.2.10. Corneal Neovascularization (CNV) assay 106 4.2.11. Record of body weight, ascites volume, cell number and peritoneal angiogenesis of EAT hearing mice treated 107 with SME 4.2.12. Immunohistolgical analysis (H and E staining) 107 4.2.13. Immunohistolgical analysis (CD 31 staining) 108 4.2.14.
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