Investigating the Anticancer Effects of Ficus Religiosa Plant Leaves
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Republic of Iraq Ministry of Higher Education and Scientific Research Al-Nahrain University, College of Science Investigating the Anticancer Effects of Ficus religiosa Plant Leaves A dissertation Submitted to the Council of College of Science, Al-Nahrain University, in partial fulfillment of the Requirements for the Degree of Doctorate of Philosophy in Science, Biotechnology By Niran Alaa Ibrahim B.Sc. Biotechnology/ University of AL-Nahrain /2003 M.Sc. Biotechnology/ University of AL-Nahrain/ 2007 Supervised by Dr. Khulood W.Abbood Dr. Nahi Y. Yaseen Professor Professor October Rabiaa-Thani 2013 1435 Supervisors Certification We, certify that this dissertation entitled ''Investigating the Anticancer Effects of Ficus religiosa Plant Leaves '' under our supervision at the College of Science / Al-Nahrain University as a partial fulfillment of the requirements for the Degree of Doctorate of Philosophy in Biotechnology. Supervisors Signature: Signature: Dr. Khulood W. Abbood Dr. Nahi Y. Yaseen Scientific Degree: Professor Scientific Degree: Professor Date: Date: In view of the available recommendations, I forward this dissertation for debate by the examining committee. Signature: Name: Dr. Hameed M. Jasim Scientific Degree: Professor Title: Head of Biotechnology Department Date: Committee Certification We, the examining committee certify that we have read this dissertation entitled ''Investigating the Anticancer Effects of Ficus religiosa Plant Leaves'' and examined the student ''Niran Alaa Ibrahim'' in its contents and that in our opinion; it is accepted for the Degree of Doctorate of Philosophy in Biotechnology. (Chairman) Signature: Name: Dr. Saad M. Shuker Scientific Degree: Professor Date: (Member) (Member) Signature: Signature: Name: Dr. Ali H. Ad'hiah Name: Dr. Hameed M. Jasim Scientific Degree: Professor Scientific Degree: Professor Date: Date: (Member) (Member) Signature: Signature: Name: Dr. Mohsen H. Riesan Name: Dr. Intesar T. Numan Scientific Degree: Assist. Prof. Scientific Degree: Assist. Prof. Date: Date: (Member / Supervisor) (Member / Supervisor) Signature: Signature: Name: Dr. Khulood W. Abbood Name: Dr. Nahi Y. Yaseen Scientific Degree: Professor Scientific Degree: Professor Date: Date: I, hereby certify upon the decision of the examining committee. Signature: Name: Dr. Hadi M. A. Abood Scientific Degree: Assist. Professor Title: Dean of College of Science Date: Acknowledgments At the beginning thanks to Almighty Allah who showered upon me all His blessings for the completion of this work First and foremost, I pay my heartfelt tribute to my supervisors Prof. Dr. Khulood W. Abbood and Prof. Dr. Nahi Y. yaseen, for giving me the best possible introduction to the world of science; their support, patience and motivation made this work not only possible, but also very enjoyable. I wish here to thank Dr. Hameed Al Dulaimi (Head of Biotechnology Department), and biotechnology department staffs. With respect to my center of work “the Iraqi Center for Cancer and Medical Genetic Research” there are no words which can attempt to describe my gratitude towards my colleagues and superiors. I am very grateful to Ministry of higher education and scientific research for their facilities to finish this work. The work presented here was performed at the Laboratory of Pharmacology, Department of Pharmacy, Western University of Health science, California, USA. Sincere gratitude is also extended to Prof. Dr. Moses S.S. Chow (Director, Center for Advancement of Drug research and Evaluation) and Assist Prof. Dr. Zhijun Wang for their innate sense of enthusiasm and encouragement, particularly in times of difficulty, proved a source of motivation and helped in remaining focused on the task at hand. I am particularly appreciative for their personal support and their interest in my career, support that have been instrumental in helping me to realize my future undertaking. For this I am truly grateful. Indeed, I express my sincere gratitude to all the other colleagues, who were working at the institute, I express my thanks to Dr. Mohammed Al-Takriti (Associate Professor of Anatomy College of Osteopathic Medicine of the Pacific and College of Optometry) and Dr. wael A. Khamas (Professor of Anatomy and Histology College ofVeterinary Medicine),Western University of Health Science, California, USA, for their patience, time and guidance through certain laboratory procedures, all their help, technical support and advice with the immunohistochmical analysis , also my sincere thanks go to them for invaluable assistance, suggestions and evaluations of histopathology and immunohistochemistry part. I have been lucky to work with an amazing group of people and I am very grateful to the members of Pharmacy; especially Mr. Steven Wang for their help and support over the duration of the research. I am indebted to Dr. Ahmed Khamees and Dr. Yaseen Ismael for their support and friendship. Mrs. Ruqaia Al-Aezzy, for offering their support and encouragement. My special thanks to Dr. Faris S. Yuseef in Baghdad University for his offering, support, advice and encouragement. Of course, my sincere appreciation and respect goes to Dr. Gassan (Head of Biotechnology Department, AL- Technologia University) for personal help, time and advice. In particular I am also eternally grateful to my father, my mother, my sister and my brother. Their love, unlimited support, guidance and prayers throughout my life kept the internal desire for knowledge at all times. Last but not least, an enormous thanks to my Mother for her love and support over all these years, and the hard work and her prayers; I could achieve nothing without her. Summary Ficus religiosa plant is an important medical plant and traditionally used to treat various diseases including mastitis, otitis media, pharyngolaryngitis, urethritis, dysmenorrhea and diabetis. The present study was conducted to evaluate anti-tumour properties of Ficus religiosa leaves collected from Baghdad, Iraq. 1- Test different extracts of Ficus religiosa plant (chloroform, butyl alcohol, absolute methanol and a crud aqueous extracts) to determine the cytotoxic effect on breast cancer (MCF7), prostate cancer sensitive to docetaxel (PC3) and resistance to docetaxel (PC3-TxR), leukemic cancer cells sensitive to daunorubicin (K562) and resistance to daunurobicin (K562Dox) cancer cell lines using sulforhodamine B assay. Then choose the best extract caused cancer cell inhibition to study chemosensitizing effect on cancer cell lines using sulforhodamine B assay. HPLC-DAD was used to determinate the active compounds in plant extract and study its ability for inhibition P-gp, EGFR, NrF2, Id1, Id2, Id3, Patch1 and Gli2 genes expression on PC3-TxR cells using RT-PCR and Western blot. 2- Investigate the toxic effect of extract in vivo by determination of LD50 and evaluate anti-tumour (histopathologically and immunohistochemically for P-gp gene expression) in SCID mice. The in vitro results of inhibition concentration (IC50) against cell lines demonstrated that treatment of cell cultures with four Ficus religiosa extracts decreased inhibitor concentrate (IC50) of the cells significantly (P≤0.05) and the effect was dose. The chloroform extract with low IC50 caused high toxic against PC3-TxR while a crud aqueous extract had less toxicity against cancer cell lines. The chloroform extract revealed that the extract exhibited strong chemosensitizing effect (CE) on PC3-TxR cell line than other cancer cell lines. Chemical analysis of chloroform extract was carried out using high performance liquid chromatography (HPLC-DAD) analysis. Accordingly, five different compounds (quercitin, myrecitin, tannic acid, gallic acid and serotonin) were characterized. The results displayed tannic acid and serotonin in the extract, while the other pure compounds have not been reported and there were many other compounds in the extract were considered unknown. The highest concentration of tannic acid was observed in the extract while serotonin was less concentration. Moreover, the chloroform extract had potent inhibitory cytotoxic effected against the proliferation of PC3-TxR cancer cells, through down-regulation of P-gp, Id1 and Id2 genes expression and the effect was markedly detect after 24 hours of treatment. The Western blot result confirmed chloroform extract inhibit P-gp protein production in PC3-TxR cancer cell lines. Lethal dose (LD50) of chloroform extract of Ficus religiosa leaves was 11.3 g.Kg-1 and the results showed the chloroform extract was significantly effective in reducing tumour size in a dose-dependent manner, when compared with mice intravenous (i.v) with docetaxel (20 mg.Kg-1) once weekly which associated with adverse side effects as well as observed reduction in mice weights more than 30% compared with negative and positive groups. Histopathological examinations of tumour sections from treated mice revealed decreased of mitosis and increased vascularization of cells. The P-gp gene showed a positive expression in tumour cells treated with docetaxel alone (positive control), while it showed a decreased expression in tumour sections of animals treated orally with the dose 3.8 g.kg-1 and at the dose 7.5 g.kg-1. The results revealed that Ficus religiosa extract has anticancer activity against cancer cell in vivo and in vitro. 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