STUDY OF ANTIPROLIFERATION AND INDUCTION OF APOPTOSIS BY JATROPHONE AND CURCUSONE B ON HUMAN CANCER CELLS
A dissertation submitted in partial fulfilment of the requirements for the degree of Master of
Kulliyyah of Medicine
International Islamic University Malaysia
Jatropha species have long been used as traditional medicines but only few scientific studies in the field of cancer therapy are available. This study was designed to evaluate the antiproliferative and apoptotic properties of jatrophone from the stem bark of Jatropha gossipyfolia and curcusone B from the stem bark of Jatropha curcas on human cancer cells. The pure compounds were screened on oral (HSC3), lung (H1299) and leukaemia (K562) cell lines. Cells were cultured in the presence of jatrophone and curcusone B at various concentrations and the rate of antiproliferation was determined with MTT assay for monolayer cells and Trypan Blue Exclusion assay for suspension cells. Agarose gel electrophoresis was used to detect DNA fragmentation as a hallmark of apoptotic cell death. The study revealed that curcusone B had significant antiproliferative activity against K562 and H1299 cells at an estimated IC50 of 6 ug/ml and 15 ug/ml respectively. Jatrophone did not significantly inhibit proliferation of K562 and H1299 cells despite an impressive decrease in percentage cell viability. None of the compounds showed activity against HSC3 cells.
DNA purified from K562 and H1299 cells treated or untreated with 100 ug/ml of curcasone B for 48h and 72h was loaded onto agarose gels for electrophoresis. No DNA ladder pattern was detected regardless of the duration of treatment. Curcusone B from the stem bark of Jatropha curcas has potential as an anticancer agent but the mode of cell death needs to be studied using a multi-parameter approach.
ﻦﻜﻟ ،ﺔﻳﺪﻴﻠﻘﺘﻟا ﺔﻳودﻷا ﺔﺑﺎﺜﻤﺑ ﺎﻓوﺮﺘﺠﻟا عاﻮﻧأ ﺖﻣﺪﺨﺘﺳا، ﺔﻠﻳﻮﻃ ةﺮﺘﻓ ﺬﻨﻣ نﺎﻃﺮﺴﻟا جﻼﻋ لﺎﺠﻣ ﻲﻓ ﺔﺣﺎﺘﻤﻟا ﺔﻴﻤﻠﻌﻟا تﺎﺳارﺪﻟا ﻦﻣ ﻞﻴﻠﻘﻟا .
ﺺﺋﺎﺼﺧ ﻢﻴﻴﻘﺘﻟ ةﺪﻌﻣ ﺔﺳارﺪﻟا ﺮﻴﺛﺄﺗ
عوﺬﺟ ةﺮﺸﻗ ﻦﻣ جﺮﺨﺘﺴﻤﻟا نﻮﻓوﺮﺘﺟ
ﻟا ﻲﻟﻮﻔﻴﺒﻴﺳﻮﺟ ﺎﻓوﺮﺘﺠ ا
عوﺬﺟ ةﺮﺸﻗ ﻦﻣ جﺮﺨﺘﺴﻤﻟا ءﺎﺑ نﻮﺳﺎﻗﺮﻗ و ﺎﻓوﺮﺘﺟ
ﺔﻳﺮﺸﺒﻟا نﺎﻃﺮﺴﻟا ﺎﻳﻼﺧ ﻲﻠﻋ سﺎﻗﺮﻗ ﻊﻨﻣ ﻲﻠﻋ ﺎﻤﻬﺗرﺪﻗ و
ﻟا نﺎﻃﺮﺴﻟا ةﺪﻳﺪﺠ
ﺺﺋﺎﺼﺨﺑ فوﺮﻌﻤﻟا ﺎﻬﻟ ﺞﻣﺮﺒﻤﻟا تﻮﻤﻟا ﺎﻤﻬﺒﺒﺴﺗ و
ءﺎﺑ نﻮﺳﺎﻗﺮﻗو نﻮﻓﺮﺘﺠﻟ ﻚﻴﺗﻮﺘﺑأ و ﻒﻴﺗاﺮﻴﻔﻴﻟوﺮﺒﺘﻧأ .
تﺎﺒآﺮﻤﻟا ﺺﺤﻓ ﺖﻤﺗ
ﺧ ﻲﻠﻋ ﻢﻔﻟا نﺎﻃﺮﺴﻟا ﺔﻴﻠﺧ ةﻮﻄ
ﺔﻴﻠﺧ ةﻮﻄﺧ ﻲﻠﻋو
ﻲﻠﻋ و ﺔﺋﺮﻟﻻا نﺎﻃﺮﺳ
و نﻮﻓﺮﺘﺟ دﻮﺟو ﻲﻓ ةﺮﻀﺤﺘﺴﻣ ﺎﻳﻼﺨﻟا ﺖﻧﺎآو مﺪﻟا نﺎﻃﺮﺴﻟا ةﻮﻄﺧ ءﺎﺑ نﻮﺳﺎﻗﺮﻗ ﺔﻔﻠﺘﺨﻣ تاﺰﻴآﺮﺘﺑ
. ﺎﻳﻼﺧ ﺮﺛﺎﻜﺗ ﻊﻨﻣ ﻲﻠﻋ ﺎﻤﻬﺗرﺪﻗ ﺮﻳﺪﻘﺘﻟ و
ﺔﻘﺒﻄﻟا يدﺎﺣﻸﻟا ﺎﻳﻼﺤﻠﻟ ﻲﺳأ ﻲﺘﻴﺘﻣإ راﺪﻘﻣ مﺪﺨﺘﺳا ةﺪﻳﺪﺠﻟا نﺎﻃﺮﺴﻟا و
ﻖﻴﻠﻌﺘﻟا ﺎﻳﻼﺨﻠﻟ ﻲﺳأ ﻦﺷﻮﻠﻜﻴﺴآإ .
ﻞﻴﺟ زورﺎﻏأ ﺖﻣﺪﺨﺘﺳا
ﺲﻴﺴﻴﻟﻮﻓوﺮﺘﻜﻟإ ﻦﻋ ﻒﺸﻜﻠﻟ
ﺔﻤﺴﻟا ﻩرﺎﺒﺘﻋﺈﺑ يوﻮﻨﻟا ﺾﻤﺤﻟا ﺖﻴﺘﻔﺗ
ﻲﺴﻳأ راﺪﻘﻤﺑ ءﺎﺑ نﻮﺳﺎﻗﺮﻗ نﺄﺑ ﺔﺳارﺪﻟا ﺖﻔﺸآو ﺔﺘﻴﻤﻟا ﺎﻳﻼﺨﻠﻟ ﺔﺠﻣﺮﺒﻤﻟا ﻲﺘﻔﻴﻓ ماﺮﻏوﺮﻜﻴﻣ ﺖﺳ ﻦﻣ
/ ماﺮﻏوﺮﻜﻴﻣ ةﺮﺸﻋ ﺔﺴﻤﺧ و ﻞﻣ /
ةرﺪﻗ ﺎﻬﻳﺪﻟ ﻞﻣ
ﺧ جﺎﺘﻧإ ﻊﻨﻣ ﻲﻓ ةﺮﻴﺒآ نﺎﻃﺮﺳ ﺎﻳﻼ
ﺎﻳﻼﺧو مﺪﻟا ﻲﻟاﻮﺘﻟا ﻲﻠﻋ ﺔﺋﺮﻟا نﺎﻃﺮﺳ
ةرﺪﻗ ﻻو مﺪﻟا نﺎﻃﺮﺳ ﺎﻳﻼﺧ ةرﺪﻗ ﺊﻄﺒﻳ ﻢﻟ ﻪﻧﺈﻓ نﻮﻓوﺮﺘﺠﻟا ظﻮﺤﻠﻣ ﻞﻜﺸﺑو ﻲﻓ ﺪﺟو يﺬﻟا ﻞﺋﺎﻬﻟا ضﺎﻔﺤﻧﻹا ﻦﻣ ﻢﻏﺮﻟا ﻲﻠﻋ ﺮﺛﺎﻜﺘﻟا ﻲﻠﻋ ﺔﺋﺮﻟا نﺎﻃﺮﺳ نﺎﻃﺮﺳ ﺎﻳﻼﺧ ﺪﺿ تﺎﺒآﺮﻤﻟا ﻦﻣ طﺎﺸﻧ يأ ﺮﻬﻈﻳ ﻢﻟ و ﺎﻳﻼﺨﻟا ﻦﻣ ﺔﺋﺎﻣ ﻞآ ﻢﻔﻟا . ﻘﻨﺗ ﻢﺗ ﺔﺋﺮﻟا نﺎﻃﺮﺳ ﺎﻳﻼﺧو مﺪﻟا نﺎﻃﺮﺳ ﺎﻳﻼﺧ ﻦﻣ يوﻮﻨﻟا ﺾﻤﺤﻟا ﺔﻴ
ماﺮﻏوﺮﻜﻴﻣ ﺔﺋﺎﻣ ﻊﻣ ﺔﺠﻟﺎﻌﻣ ﺮﻴﻏ و ﺔﺠﻟﺎﻌﻣ /
ءﺎﺑ نﻮﺳﺎﻗﺮﻗ ﻦﻣ ﻞﻣ ةﺪﻤﻟ
و ﺔﻴﻠﻤﻌﺑ مﺎﻴﻘﻠﻟ ﻞﻴﺟ زورﺎﻏأ ﻲﻠﻋ ﺔﻋﺎﺳ72
إ ﺲﻴﺴﻳرﻮﻓوﺮﺘﻜﻟ ﻢﺘﻳ ﻢﻟو
جﻼﻌﻟا ةﺪﻣ ﻦﻋ ﺮﻈﻨﻟا ﺾﻐﺑ يوﻮﻨﻟا ﺾﻤﺣ ﻂﻤﻧ ﻢﻠﺳ ﻲﻠﻋ فﺎﺸﺘآﻹا .
نﻮﺳﺎﻗﺮﻗ نأ ﺎﻧﺪﺟو ءﺎﺑ
سﺎﻗﺮﻗ ﺎﻓوﺮﺘﺠﻟا عوﺬﺟ ةﺮﺸﻗ ﻦﻣ جﺮﺨﺘﺴﻤﻟا ﺎﻬﻳﺪﻟ
نأ يﻮﺳ نﺎﻃﺮﺴﻠﻟ دﺎﻀﻤآ ﻪﻣاﺪﺨﺘﺳا ﻦﻜﻤﻳ و نﺎﻃﺮﺴﻟا ﺪﺿ ةﺮﻴﺒآ ةرﺪﻗ ﺖﻗﻮﻟا ﺲﻔﻧ ﻲﻓ ﺔﻔﻠﺘﺨﻣ ﺐﻴﻟﺎﺳﺄﺑ ﺎﻳﻼﺨﻟا تﻮﻣ ﺔﻘﻳﺮﻃ سرﺪﺗ .
I certify that I have supervised and read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Master of Medical Sciences.
Muhammad Taher Supervisor
Anil Kumar Saxena Supervisor
I certify that I have read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Master of Medical Sciences.
This dissertation was submitted to the Department of Pharmacology and is accepted as a partial fulfilment of the requirements for the degree of Master of Medical Sciences.
Pakeer Oothuman Sayed Ahmed Head, Department of Basic Medical Sciences.
This dissertation was submitted to the Kulliyah of Medicine and is accepted as a partial fulfilment of the requirements for the degree of Master of Medical Sciences.
Mohammed Fauzi Abdul Rani Dean, Kulliyyah of Medicine
I hereby declare that this dissertation is the result of my own investigations, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.
Signature ……… Date ………..
INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA
DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH
Copyright © 2010 by Sauba Nakazibwe. All rights reserved.
STUDY OF ANTIPROLIFERATION AND INDUCTION OF APOPTOSIS OF JATROPHONE AND CURCASONE B ON HUMAN CANCER CELLS
No part of this unpublished research may be reproduced, stored in retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior permission of the copyright holder except s provided below.
1. Any material contained in or derived from this unpublished research may only be used by others in their writing with due acknowledgement.
2. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.
3. The IIUM library will have the right to make, store in a retrieval system and supply copies of this unpublished research if requested by other universities and research libraries.
Affirmed by Sauba Nakazibwe.
a light that guides my foot step My mother,
no reward is good enough for you The memory of my father, may Allah have mercy on your soul
In the Name of Allah, The Beneficent, The Most Merciful.
All perfect praise is for Allah, the Most Gracious who has made this work possible.
I extend my sincere appreciation to the Islamic Development Bank Group for sponsoring my study at IIUM and to the Islamic University in Uganda for the support extended towards the same. Special thanks to my supervisors Dr. Muhammad Taher and Assoc.Dr. Anil Kumar Saxena for the knowledge and guidance you have shared with me throughout the course of this project.
Many thanks to colleagues Dr. Farah Ibrahim Khalil, Dr. Ahmad A. Elbadri, Dr. Muhammad Hassan Yankuzo and Br. Osama Althunibat for the timely help and
A heartfelt gratitude to my family and to all my dear friends for being there, every time. Jazakumullahu khairan.
TABLE OF CONTENTS
Abstract ... ii
Abstract in Arabic ... iii
Approval Page ... iv
Declaration ... v
Copyright page ... vi
Dedication ... vii
Acknowledgement ... viii
Table of Contents ... ix
CHAPTER ONE: INTRODUCTION ... 1
1.1 Background ... 1
1.2 Hypothesis ... 3
1.2.1 General Objective ... 4
2.3.1 Specific Objective ... 4
CHAPTER TWO: LITERATURE REVIEW ... 5
2.1 Cancer World Burden ... 5
2.2 Conventional Cancer Treatments ... 6
2.3 Plants in Drug Development ... 6
2.3.1 Plants Metabolites as Anticancer Agents ... 7
2.4 The Genus Jatropha ... 8
2.4.1 Jatropha curcas ... 10
220.127.116.11 Biological Activities of J. curcas ... 11
2.4.2 Jatropha gossypifolia ... 12
18.104.22.168 Biological Activities of J. gossypifolia ... 13
2.5 Jatrophone and Curcusone B ... 13
2.6 Cell Culture in Cancer Research ... 14
2.6.1 Cell Death ... 15
CHAPTER THREE: MATERIALS AND METHODS ... 17
3.1Materials ... 17
3.1.1 Apparatus and Equipment ... 17
3.1.2 Chemicals ... 17
3.1.3 Cell Lines ... 18
3.2 Methodology ... 18
3.2.1 Plant Materials ... 18
3.2.2 Sample Preparation for Viability Assay ... 19
3.2.3 Cell Culture and Maintenance ... 19
22.214.171.124 Preparation of Culture Media ... 19
126.96.36.199 Monolayer Cells ... 19
188.8.131.52 Suspension Cells ... 20
184.108.40.206 Cell Counting ... 21
220.127.116.11.1 Cell Counting Procedure ... 21
3.2.4 Cell Proliferation Assay ... 22
18.104.22.168 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide Assay ... 22
22.214.171.124.1 MTT Assay Procedure ... 23
126.96.36.199.2 Calculation ... 24
188.8.131.52 Trypan Blue Exclusion Assay ... 24
184.108.40.206.1 Trypan Blue Exclusion Assay Procedure ... 25
3.2.5 DNA Fragmentation Analysis ... 25
220.127.116.11 Harvesting the cells ... 26
18.104.22.168 DNA extraction ... 26
22.214.171.124.1 DNA Extraction Procedure ... 26
3.2.6 Gel electrophoresis ... 27
126.96.36.199 Gel Electrophoresis Procedure ... 28
3.3 Data Analysis ... 28
CHAPTER FOUR: RESULTS ... 29
4.1 Antiproliferation Activity ... 29
4.1.1 Effect of Jatrophone and Curcusone B on the Proliferation of Human Leukemia Cell Line (K562) ... 27
4.1.2 Effect of Jatrophone and Curcusone B on the Proliferation of Human Non-Small Cell Lung (H1299) and Human Tongue Squamous (HSC3) Cell Lines ... 31
4.2 DNA Fragmentation Analysis ... 36
4.2.1 Agarose Gel Electrophoresis ... 36
CHAPTER FIVE: DISCUSSION ... 38
5.1 Antiproliferation Activity ... 39
5.1.1 Curcusone B ... 40
5.1.2 Jatrophone ... 41
5.2 DNA Fragmentation ... 42
5.2.1 Possible Explanations for the Absence of Laddering ... 44
5.2.2 Multi-Parameter Approach in Detection of Apoptosis ... 45
CHAPTER SIX: CONCLUSION ... 47
BIBLIOGRAPHY ... 49
APPENDIX A: PUBLICATION ... 55
LIST OF FIGURES
Table No. Page No.
2.1 Jatropha curcas 10
2.2 Jatropha gossipyfolia 10
2.3 Molecule of Jatrophone 14
2.4 Molecule of Curcusone B 14
4.1 Effect of Jatrophone on the proliferation of K562 cell line 30 4.2 Effect of Curcusone B on the proliferation of K562 cell line 31 4.3 K562 cells before treatment 31
4.4 K562 cells after treatment with 100 µg/ml 31
4.5 H1299 cells confluent 33
4.6 H1299 cells after treatment with Curcusone B 100 µg/ml 33 4.7 Effect of Curcusone B on the proliferation of H1299 cell line 33
4.8 Effect of Jatrophone on the proliferation of H1299 cell line 34 4.9 Effect of Jatrophone on the proliferation of HSC3 cell line 35 4.10 Effect of Curcusone B on the proliferation of HSC3 cell line 35 4.11 DNA electrophoresis of untreated and Curcusone B treated
K562 and H1299 cells
LIST OF TABLES
Table No. Page No.
2.1 Examples of chemical groups of plant derived
natural products with documented anticancer /antitumor
3.1 Cells used in the experiment 18
4.1 Viability of K62 cell line after treatment with
Jatrophone, Curcusone B and DMSO 29
4.2 Mean absorbance obtained after treatment of H1299
cell line with different compound concentrations 32 4.3 Mean absorbance obtained after treatment of HSC3
cell line with different compound concentrations 34
LIST OF ABBREVIATIONS AND SYMBOLS
µg/ml microgram per milliliter
IC50 inhibitory concentration 50
EDTA ethylenediamine tetra acetic acid
NaHCO3 sodium hydrogen carbonate
HIV human immune-deficiency virus
CO2 carbon dioxide
DMSO dimethyl sulfoxide
FBS fetal bovine serum
DMEM Dulbecco’s Modified Eagle Medium
RPMI Rosewell Park Memorial Institute
MTT 3-(4, 5-dimethylthiazol-2-1)-2, 5-diphenyltetrazolium bromide
PBS phosphate buffer saline
ATCC American Type Culture Collection
CHAPTER ONE INTRODUCTION
Cancer is a generic term for a large group of diseases characterized by a shift in the control mechanisms that govern cell survival, proliferation, and differentiation. It can affect any part of the body (Katzung, 2007; World Health Organization [WHO], 2009). Cancer arises from a change (mutation) in one single cell within a tissue. The change may be started by external agents and inherited genetic factors (WHO, 2009) i.e. cancer arises due to specific effects of environmental factors (such as smoking or diet) on a certain genetic background. In the hormonally related cancers like breast and prostate cancer, genetics seem to be much a more powerful factor than lifestyle (Kintzios and Baberaki, 2004). Among the most prominent features common to cells that have undergone neoplasmic transformation is the ability to proliferate excessively and form local tumors that can compress or invade adjacent normal structures.
Cytotoxic chemotherapy has been one of the mainstays of cancer treatment for many years and continues to be very important (Cancer Research, UK, 2009). Indeed chemotherapy is the most rapidly developing field of cancer treatment, with new drugs being constantly tested and screened including plant metabolites (Kintzios and Barberaki, 2004). In this regard, medicinal plants offer enormous prospects for discovering new compounds with therapeutic properties in the search for new anticancer drugs
Many tropical plants have interesting biological activities with potential therapeutic applications (Moongkardi, Kosem, Kaslunga, Luanratana, Pongpan and
Neungton, 2004). Jatropha gossypifolia and Jatropha curcas belong to the family
“Euphorbiacea”. The species occur in Africa, Latin America, India and parts of South East Asia (Maurice, 1993; Igbinosa O., Igbinosa E., and Aiyegoro, 2009; Emil Akbar Zahira Yaakob, Siti Kartom Kamarudin, Manal Ismail and Jumat Salmon, 2009) where they have been used in traditional medicine to cure various ailments (Maurice, 1993; Burkill, 1994). Studies have revealed that J. curcas plant not only contains phytochemicals such as saponins, steroids, tannins, glycosides alkaloids and flavonoids but also possesses antimicrobial and anticancer activity (Igbinosa et al., 2009). The leaves of J. gossypifolia have been found to contain histamine, apigenin, vitexin, isovitexin and tannins. The bark contains the alkaloids and lignins were found in its stems and aerial parts. Octapeptides occur in the latex. The plant also contains lectins, phorpolesters and curcin (Siddiqui, Verma, Rather, Jabeen and Meghvansi, 2009; Panda, Gaur, Kori, Tyangi, Nema, Sharma and Jain, 2009).
Jatrophone, a naturally occurring diterpene isolated from jatropha species, exhibits a broad spectrum of biological actions and has received considerable attention due to its significant antitumor activity (Moraes, Rumjanek, and. Calixto, 1996;
D’Alagni, Petris, Marinni-Bettolo, and Temussi, 1983). Curcasone B is one of the four curcasones isolated from the roots of J. curcas and belongs to the rotophorbolane class of compounds (Naengchomnong et al., 1986). Ardiyansyah & Taher (2009) isolated pure compounds jatrophone and curcasone B from the stem barks of J.gossypifolia and J.curcas respectively. Findings from the study suggested both pure compounds could possess potential antimicrobial and anticancer activity.
There is no publication available on the possible antiproliferation activity of jatophone from the stembark of J.gossypifolia or curcusones from J.curcason human cancer cells. The possibility of obtainng such findings along with the compounds’
possible mechanism of cell death are high and as such may provide interesting information on the plants’ activities as anticancer agents.
Killing malignant cells is the number one goal of almost all forms of cancer therapy in use today. Previous studies performed over the past 15 years have demonstrated that all the agents currently utilized to treat cancer can induce apoptosis in susceptible cell types in vitro and in vivo. In the era of modern cancer therapy, researchers have developed innumerable ways to kill cancerous cells by triggering apoptosis, a genetically programmed form of cell suicide (IIgar and Arican 2009).
Understanding of whether the cell death program is engaged after exposure to pure compounds from J.gossypifolia and J.curcas may offer a novel approach to further evaluation of the plants’ potential as antitumor agents.
In this study, anticancer activity was studied using cell culture technique. The parameter of proliferation speed which showed the compounds’ antiproliferative effects was determined with Trypan Blue Exclusion assay for suspension cells and by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for monolayer cells. Where the compounds exhibited significant antiproliferation activity, the cells were evaluated for biochemical evidence of apoptosis by detection of DNA fragmentation with agarose gel electrophoresis.
The pure compounds jatrophone, from the stem bark of J.gossypifolia, and curcasone B, from the stem bark of J.curcas, could inhibit cancer cell growth by inducing apoptotic cell death.
4 1.2.1 General Objective
To evaluate the antiproliferative and apoptotic effects of pure compounds jatrophone and curcusone B from the stem barks of J.gossypifolia and J.curcas, respectively;
using human tongue squamous cell carcinoma line (HSC3), human non-small cell lung carcinoma, (H1299) and chronic myelogeneous leukaemia (K562) cell lines.
1.2.2 Specific Objective
To determine if the antiproliferation activity of jatropone and curcusone B is due to induction of apoptosis.
2.1 WORLD CANCER BURDEN
Cancer, a group of neoplasmic diseases broadly characterized by abnormalities of cellular differentiation, maturation and control of growth (Chandrasoma & Taylor, 2000), is a leading cause of death worldwide. In 2007, cancer accounted for 7.9 million deaths, around 13% of all deaths that year (WH0, 2009). Kintzios & Barberaki (2004) estimate that six million people lose their lives to cancer annually and that one of four citizens of a developed country will be stricken by the disease sometime in their lifetime.
However cancer is no longer considered a Western disease. The world cancer report, which is the most comprehensive global examination of the disease to date, reveals that cancer has emerged as a major public health problem in developing countries, matching its effect in industrialized nations. More than 50 per cent of the world’s cancer burden, in terms of both number of cases and deaths, already occurs in developing countries (WHO, 2003). Globally, 400 new incidents are said to emerge per 100,000 people each year while the number of deaths are projected to continue rising to approximately 12 million by the year 2030 (Kintzios & Barberaki, 2004;
WHO, 2009). An annual investment of $217b is neededto meet the shortfall in global spending on cancer care and treatment (McColl, 2009). In Malaysia cancer is the fourth leading cause of death among medically certifieddeaths. It is estimated at an annual incidence of 30,000 (Lim, 2002).
2.2 CONVENTIONAL CANCER TREATMENTS
Surgery, radiotherapy and cytotoxic chemotherapy have been the mainstays of cancer treatment for many years and continue to be very important (Cancer Research UK, 2009). Surgery, the earliest and most widely used therapy, is used for the excision of the tumor. Radiation (X-rays, gamma rays) of a cancerous tumor, thus causing cancer cell death or apoptosis preserves the anatomical structures surrounding the tumor and also destroys non-visible cancer cells. Chemotherapy, which is based on the systemic administration of anticancer drugs that travel throughout the body via blood circulatory system, aims to wipe out all cancerous colonies within the patient’s body, including metastasized cancer cells. Chemotherapy is the most rapidly developing field of cancer treatment with new drugs being constantly tested and screened. These include, but are not limited to, plant metabolites (Kintzios and Barberaki, 2004). The majority of cancers are curable and thus the search for cures and/or more effective treatments is of utmost significance.
2.3 PLANTS IN DRUG DEVELOPMENT
Plants have formed the basis of sophisticated traditional medicine systems that have been in existence for thousands of years in countries such as China and India. These plant-based systems continue to play an essential role in health care. It has been estimated by the World Health Organisation that approximately 80% of the world’s inhabitants rely on traditional medicines for their primary healthcare (Romeo, 1999).
Plants have a long history of use in the treatment of cancer. Of the plants-derived anticancer drugs in clinical use, the best known include the Vinca alkaloids (vinblastine and vincristine), the taxanes (Taxol), and the camptothecins (topotecan), derivedfrom the Madagascan periwinkle plant Catharantus roseus, thePacific yew
Taxus brevifolia, and the Chinese tree Camptothecaacuminata, respectively (Dennis, Adriana and Gilberto, 2000; Cragg and Newman, 2001).
Higher plants continue to retain their historical significance as important sources of novel compounds. Their role is twofold in the development of new drugs:
they may become the base for the development of a medicine, a natural blue print for the development of new drugs or; a phytomedicine to be used for the treatment of diseases (Igbinosa et al., 2009).
The search for new anti-cancer drugs is one of the most prominent research areas of natural products (Mesquita, 2009). The goals of using plants as sources of therapeutic agents include isolation of bioactive compounds for direct use as drugs (Fabricant and Farnsworth, 2001). Research is focusing on the search for new molecular prototypes, new types of chemotherapeutic agents and plant medicines are proving to be excellent sources of these new compounds. Medicinal plant drug discovery therefore continues to provide new and important leads against various pharmacological targets including cancer (Balunas, 2005). Currently, more than 30 compounds of natural origin are in different phases of clinical trials for the treatment of different types of cancer and more than 60% of the available cancer drugs are derived from natural products (Gordaliza, 2007).
2.3.1 Plant Metabolites as Anticancer Agents
The chemical constituents of the plant cell that exert biological activities on human and animal cells fall into two distinct groups, depending on their relative concentration in the plant body, as well as their major function: primary metabolites, the accumulation of which satisfies nutritional and structural needs, and secondary
metabolites, which act as hormones, pharmaceuticals and toxins (Kintzios and Baberaki, 2004).
Secondary metabolites are compounds belonging to extremely varied chemical groups, such as organic acids, aromatic compounds, trepenoids, steroids, flavonoids, alkaloids, carbonyles etc. Some secondary metabolites are considered as metabolic waste products, for example, alkaloids may function as nitrogen waste products (Kintzios and Baberaki, 2004).
However, a significant portion of the products derived from secondary pathways serve either as protective agents against various pathogens (eg insects, fungi or bacteria) or growth regulatory molecules (eg hormone like substances that stimulate or inhibit cell division and morphogenesis). Due to these physiological functions, secondary metabolites are potential anticancer drugs, since their direct toxicity is produced on cancer cells or the course of tumor development is modulated and eventually inhibited (Kintzios and Baberaki, 2004). Table 2.1 shows examples of plant-derived natural products with documented anticancer/antitumor properties that exist today.
2.4 THE GENUS JATROPHA
J. gossypifolia and J. curcas belong to the family “Euphorbiacea”. Other species are J. glandulifera, J. tanjorensis, J. multifida, J. podagrica and J. intergerrima (Oduola T., Adeosun, Oduola T.A., and Oyeniyi, 2005). The species occur in Africa, Latin America, India and parts of South East Asia (Maurice, 1993; Burkill, 1994;
Igbinosa et al, 2009; Emil Akbar et al, 2009). J.curcas is by far the most widely used species in traditional medicine, although others are more available to investigators because of their use as ornamental garden plants and as hedges. The species appear to
have similar uses in folk medicine, same chemical constituents, and similar pharmacological activity (Maurice, 1993).
Examples of chemical groups of plant derived natural products with documented anticancer /antitumor properties
Chemical group Species Target disease/cell line Mode of action Aldehydes Cinnamomum Human cancer lines, SW-620
P-388, esophageal Tubulin inhibitor Annonaceous
Annona muricata Prostate adenocarcinoma Cytotoxic Goniothalamus sp. Breast cancer, in vitro various
Flavanoids Acrougehia porter KB Cytotoxic
P-338, KB Cytotoxic
Glycosides Plumeria rubra (iridoids)
P-388, KB Cytotoxic
Various cancer cell lines Cytotoxic
Lignans Brucea sp KB, P-388 Cytotoxic
Wikstroemia foetida P-388 Cytotoxic
Lipids Nigella sativa Elrilch ascites carcinoma Cytotoxic in vitro Quinones Kigelia pinnata In vitro melanoma, renal cell
Tumor inhibitor Koelreuteria henyi Src-Her-2/neu, ras oncognes Tumor inhibitor Phenols Gossypium indicum Murine B 16 melanoma, L
Cytotoxic Polysaccharides Brucea javanica Leukemia, lung, colon, CNS,
Cytotoxic Terpenoids and
Crocus savitus KB, P-388, human prostate, pancreatic, in vitro
Melia sp. Carcinoma,
sarcoma, leukemia, AS49, VA13
Apoptotic/inhibits DNA synthesis
Human leukemia, stem, lung, P-388, L 1210
Protein kinase C activator
Source: Kintzios and Barberaki (2004)
A distinct differentiation between the two plants can be made by macroscopic examination of the leaves and the variations in the dominant color of the flowers.
J.curcas has yellowish-green flowers while J.gossipyfolia Linn. has deep red purple in-lax cymes (Maurice, 1993) as seen in figures 2.1 and 2.2.
2.4.1 Jatropha curcas
Jatropha curcas, also known as physic nut, purging nut or pig nut, is a multipurpose bush/small tree with many attributes, multiple uses and considerable potential.
Different parts of this plant have been traditionally used for various purposes including therapeutic uses. Leaves and roots are used as a remedy for cancer, as an abortifacient, antiseptic, diuretic, purgative, external application for the treatment of skin diseases, sores, rheumatism, piles and as an antidote for snake-bites. The nut of the plant has also been used traditionally for the treatment of many ailments including burns, convulsions, fever and inflammation. Other uses from its oil include soap making, illumination, cosmetics, organic manure and most recently as a potential
Fig 2.2: J.gossipyfolia
Fig 2.1: J.curcas Source:
alternative to diesel. In spite of the myriad of ethnomedical uses to which various parts of the J. curcas plant have been put, it is important to note that toxic properties have also been adduced to parts of the plant, especially the seed (Kumar and Sharma, 2005; Osoniyi and Onajobi, 2003).
Regarding the chemical constituents of J.curcas there is a massive body of evidences which clearly demonstrate that saponins, steroids, tannins, glycosides, alkaloids, and flavonoids occur in this plant and some of them have relevant biological activities (Igbinosa et al,. 2009).
188.8.131.52 Biological Activities of J. curcas
Some of the ethnomedical uses of J. curcas have received support from the results of scientific investigations in recent times. For example the crushed leaf is applied onto cuts and bleeding wounds to achieve haemostasis. This was investigated by Osoniyi and Onajobi (2003) and results showed that whole latex significantly (P<0.01) reduced the clotting time of human blood while diluted latex prolonged the clotting time; a finding that was confirmed with prothrombin time (PT) and activated partial thromboplastin time (APTT) tests on plasma. Solvent partitioning of the latex with ethyl acetate and butanol led to a partial separation of the two opposing activities: at low concentrations, the ethyl acetate fraction exhibited a procoagulant activity, while the butanol fraction had the highest anticoagulant activity.
Other studies have shown that the sap and crushed leaves of J.curcas possessed anti-parasitic activity, while water extract of the branches strongly inhibited HIV - induced cytopathic effects with low cytotoxicity (Igbinosa et al, 2009). In a study by Lin, Yan, Tang and Chen., (2003), curcin a compound with antitumor effects isolated from seeds of J.curcas was shown to be a novel cystein-containing ribosome-