Anticancer effect of leaves of kigelia africana on breast cancer




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ANTICANCER EFFECT OF LEAVES OF KIGELIA AFRICANA ON BREAST CANCER”

Synopsis for registration of M.pharm Dissertation



Submitted to

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE

KARNATAKA

logo

In partial fulfilment

of the requirement for the Degree of

Master of Pharmacy In Pharmacology
Under the Guidance of

Dr. Nagarathna P.K.M.

Asst.Professor In Pharmacology
BY

N. SRI SAINADH

kcp logo new imege

Department of Pharmacology

Karnataka College of Pharmacy, Bangalore-64

2012-13
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE.

ANNEXURE-II




PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION






1.


Name of the candidate and address


N.Sri Sainadh

S/O: N. Srinivasa Rao

# Qt No: 102/E, Steel Plant

Visakhapatnam-530032

Andhra Pradesh




2.


Name of the Institution

Karnataka College of Pharmacy,

Thirumenahalli,

Bengaluru-560064,

Karnataka.



3 .

Course of study and subject


M.Pharm-Pharmacology



4.

Date of the admission



18-07-2011



5.

Title of the topic


ANTICANCER EFFECT OF LEAVES OF KIGELIA AFRICANA ON BREAST CANCER”







6

BRIEF RESUME OF THE INTENDED WORK




6.1 – NEED FOR THE STUDY:

Cancer is a major cause of death worldwide and causes serious problems in human life, including mental and physical agony and economic strain. Therefore, many kinds of cancer therapies, including various anticancer agents, have been developed. In all types of cancer, genetic alterations give rise to changes in expression, activation or localisation of regulatory proteins in the cells. They then affect the signalling pathways that alter their response to regulatory stimuli and allow the unrestricted cell growth. However, they also have several problems such as serious side effects and drug resistance1. To resolve these difficulties, development of anticancer agents and improvement of cancer treatment are very important. Accordingly, screening of natural products as potential anticancer agents, in the form of functional foods or nutraceuticals has become an important undertaking.

The NCI in vitro primary screen consists of a panel of 60 different human tumor cell lines against which compounds are tested over a defined range of concentrations to determine the relative degree of growth inhibition or cytotoxicity against each cell line. The design and operation of the screen is such that, for each compound tested, both the absolute and relative sensitivities of individual cell lines comprising the screen are sufficiently reproducible that a characteristic profile or "fingerprint” of cellular response is generated. Depending upon the extent of differential cellular response, the profile may contain much information which is useful for further research3 .

6.2 REVIEW OF LITERATURE:

Cancer is developed due to some molecular changes within the cell, and is becoming the second major cause of death in the human after cardiovascular disease4.. About 7.6 million people died from cancer in the world during 2007 (American cancer society, 2007). Hence there is an urgent need for developing new approaches and drugs to prevent as well as cure this devastating disease. Scientists are now developing drugs that target the unique makeup mechanism of cancer cells. A number of natural products have been studied for anti cancer activity on various experimental models5..

Cancer cell lines have been extensively used in various cancer researches. The following breast cancer cell lines are used HBL-100, MCF-76, MCF-7ADR7, HS578T8, MDA-MB-2319-10, MDA-MD-43511-12, MDA-N,BT-549, T47-D13.
A thorough and complete literature search on Kigelia Africana Linn. was performed from the chemical abstracts, national and international journals, E-library, internet and other research materials. Kigelia Africana is a medicinal plant of the Bignoniaceae family. It is widely distributed in Eastern Ghats and Deccan plateau in India. The leaves of Kigelia Africana are used in traditional medicine for relieving headache, anti-inflammatory activity, preservative, anti-bacterial activity. General pharmacological studies revealed analgesic, inflammatory, anti- microbial activity, anti-malarial activity, and anti-fungal activity, anti-ulcer activity of aqueous and ethanolic extracts of the leaves Kigelia Africana15. The anti-inflammatory and wound healing properties of the crude alcoholic extract of the leaves of Kigelia Africana in acute inflammation model has been reported16. The roots and bark are astringent and the roots are reported to be used as febrifuge. The phytoconstituents present in this plant are kigelinole, isokigelinole, pinnatal, isopinnatal, B-Sitosterol, ferulic acid and lapachol17. The anticancer activity of this plant is not reported and hence in this study we have undertaken the anticancer activity of leaves extract of Kigelia Africana.

6.3 OBJECTIVE OF THE STUDY:

1. Extraction- Methanolic extraction of leaves of Kigelia Africana.

2. To carry out in-vitro and in-vivo anticancer effect of extract of Kigelia Africana.

6.4 SOURCE OF DATA:

Whole work is planned to generate data from laboratory studies i.e. experiments are performed as described in references. Experimental studies in journals and in text books available with college, IISc and other libraries. Literature is searched from various web sites in the internet.



6.5 MATERIALS AND METHODS:

Preparation of extract

Powdered leaves was subjected to successive extraction in a Soxhlet extractor with methanol. The extract obtained was concentrated in a rotary shaker evaporator to dryness to get a constant weight



Cell Lines & Culture
HBL-100(ICLC No.HTL 00004)-Breast Myoepithelial Tumour the cell lines will be purchased from the National Center for Cell Science, Pune university campus, Pune.. All the cell lines were cultured at 370C, 5% CO2 in RPMI 1640 media with L-glutamine, supplemented with 10% of fetal calf serum, 100U/ml pencillin and 100µg/ml streptomycin18.
MTT ASSAY

Cell proliferation activity of extract of Kigelia Africana will be carried out by MTT assay which estimate the effect of the various extracts on the growth of cell in vitro. Measurement of cell in viability and proliferation forms should be used as basis for this invitro assay. The reduction of terazolium salt now widely accepted to examine cell proliferation the yellow coloured terazolium, MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5,diphenylterazolium bromide], will reduce metabolically active cell in part by the action of dehydrogenase enzymes to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple colour zone will solubilise and quantify by spectrophotometric method. When metabolic event leads to necrosis or apoptosis in cell, the MTT Should be dissolved in PBS at a concentration of 5mg/ml. Then, 50µg of the MTT solution will be added to each well of the 96-well culture plate, containing the 100-µl medium and incubated at 37oC for 4hr. This medium should be removed carefully without disturbing the purple coloured formazan crystals; 50ml of dimethyl sulphoxide (DMSO) will be added to each well and mixed thoroughly to dissolve the crystals of the formazan. Then these plates should be seen on a microplate reader at a wavelength of 670nm. The readings should be presented as optical density (OD).The growth inhibition of the cells by the extracts will be identified. The cell proliferation activity is to be qualified on MCF-7 cell line, by using positive and negative controls (positive control-17-b-estradiol; negative control-culture medium only).



SULFORHODAMINE B ASSAY

Sulforhodamine B (SRB) (Sigma–Aldrich Chemie GmbH, Munich, Germany) can be used to test the effects of active compounds on cell growth and viability. based on the method described by vichai and kirtikara , compounds 1–8 should be dissolved in dimethylsulfoxide (DMSO) before diluting with the growth medium to a final DMSO concentration of <0.05%. The cancer cells will be seeded into 96 well plates in the growth medium at 3000 cells/well. After 24 h of incubation, the medium to be replaced with a fresh growth medium containing the test compounds 1–8 (0, 25, 50, 100 and 200 lm). The cells will be incubated for another 48 h. The cells should be fixed with TCA by gently adding 50 µl TCA (50%) to each well to a final TCA concentration of 10% with subsequent incubation for 1 h at 4˚c. The plates should be washed 5 times with deionised water and air dried. The dried plates should be stained with 100 µl of 0.4%N(w/v) SRB prepared in 1% (v/v) acetic acid for 10 min at room temperature. The plates will be rinsed quickly four times with 1% acetic acid to remove unbound dye, followed by air-drying until no moisture should be visible. The bound dye will be solubilised in 2 mM Tris base (100 µl/well) for 5 min on a shaker. Optical densities should read on a microplate reader at 562 nm.


METHODOLOGY FOR BREAST CANCER

7,12 DMBA induced Mammary tumors : Female Wistar rats weighing 100-110gm will be used for the study. All the animals will be purchased from IISC, Bangalore. After procuring, the animals would be acclimatized for 10 days at normal laboratory condition. All animals will be allowed free access to tap water and pellet diet and maintained at room temperature in polyethylene cages

Cancer will be induced in female Wistar rats (120-140gm) at the age of 50-57 days by intravenous injection of 2 mg of DMBA dissolved in 0.05 ml of DMSO on the 1st, 4th, and 7th day.

DMBA will be dissolved by gentle heating (in a dark incubator at approximately 70°cfor 10 min) in DMSO (40 mg/ml). The solution will then immediately poured into bottles made of dark brown glass and each with a capacity of 10 ml, and for additional protection against light the containers were covered with aluminum foil. In addition to these precautions, the bottles will be kept, except for the duration of the injections, in complete darkness at room ternperature16. All the animals would allowed free access to tap water and pellet diet and maintained at room temperature in polyethylene cages .

The rats would be divided into four groups consisting of six rats each



Group 1: Administered vehicle - Normal control.

Group 2: Administered DMBA (6mg/animal) - DMBA control

Group 3: Administered Steroidal extract of Kigelia Africana

Group 4: Reference standard , Tamoxifen (0.2mg/kg;ip;day/30days).

Tamoxifen will be dissolved in peanut oil and (0.2mg Tamoxifen will be dissolved in peanut oil and (0.2mg/kg;ip;day/30days)administered to rat .The rats would develop tumors at the age of 140-160 days, i.e after 3 months of DMBA injection with tumor size of 9 to 11mm in diameter. The rats would be treated with test and standard drug for 30 days Beginning on the 1st day of drug treatment and every day the mean tumour diameter will be recorded for each rat. After the 30 days of treatment .the animals will be sacrificed and breast tissue was collected. The breast tissue homogenate will be analysed for oxidative stress parameters, SOD, CAT, LPO, GSH and histopathological analysis of breast21.


ENZYME MARKERS

Anticancer potency in breast myoepithelial cells will be seen after 48 h incubation by decreasing the cell viability and reduction in the activities of the marker enzymes γ-GT, and 5’-nucleotidase.



STATISTICAL ANALYSIS:

The statistical significance of the results will be analyzed by ANOVA. p<0.05 will indicate the significance of the result.21



7.1 - Does The Study Require Any Investigation To Be Conducted On Patients Or Animals?

If So, Please Describe Briefly.

No,the entire experimental models do not require usage of laboratory animals.



7.2 - Has Ethical Clearance Been Obtained From Your Institution In Case Of 7.2?

Applied to IAEC.




REFERENCES

  1. Sethi T, West R, McNeill A, Raw M. Smoking cessation guidelines for health professionals. An update. 2000;55:987–99.

  2. Kaefer CM, Milner JM. The role of herbs and spices in cancer prevention. J. Nutr. Biochem. 19. 2008; 347–361.

  3. Michael R, Boyd, Kenneth D, Paull. Some Practical Considerations and Applications of the National Cancer Institute In Vitro Anticancer Drug Discovery Screen DRUG DEVELOPMENT RESEARCH. 1995;34: 91-109.

  4. Jackson BG. Mechanism based target identification and drug discovery in Cancer research. Science 2000; 287-1969.

  5. Ramakrishna Y, Manohar AL, Mamata P, Shreekanth KG. Plant and novel antitumor agents. A Review. Indian Drugs 1984;21:178-85.

  6. Soule HD, Vazquez J, Long A, Albert S, Brennan M. A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst 1973;51: 1409-1416.

  7. Cohen JS, Lyon RC, Chen C, Faustino PJ, Batist G, Shoemaker M, Rubalcaba E, Cowan KH. Differences in phosphate metabolite levels in drug-sensitive and resistant human breast cancer cell lines determined by 3'P magnetic resonance spectroscopy. Cancer Res 1986; 46: 4087-4090.

  8. Hackett AJ, Smith HS, Springer EL, Owens RB, Nelson-Rees WA, Riggs JL, Gardner MB. Two syngeneic cell lines from human breast tissue: The aneuploid mammary epithelial (Hs578T) and the diploid myoepithelial (Hs578Bst) cell lines. J Natl Cancer Inst 1977; 58: 1795-1806.

  9. Cailleau R, Young R, Olive M, Reeves WJ Jr. Breast tumor cell lines from pleural effusions. J Natl Cancer Inst 1974; 53: 661-674.

  10. Siciliano MJ, Barker PE, Cailleau R. Mutually exclusive genetic signatures of human breast tumor cell lines with a common chromosomal marker, Cancer Res 1979 39: 919-922.

  11. Cailleau R, Olive M, Cruciger QVJ. Long-term human breast carcinoma cell lines of metastatic origin. Preliminary characterization In Vitro 1978;14: 911-915.

  12. Brinkley BR, Beall PT, Wible LJ, Mace ML, Turner DS, Cailleau RM. Variations in cell form and cytoskeleton in human breast carcinoma cells in vitro. Cancer Res 1980;40: 3118-3129.

  13. Keydar I, Chen I, Karby S, Weiss FR, Delarea J, Radu M, Chaitcik S, Brenner HJ. Establishment and characterization of a cell line of human breast carcinoma origin. Eur J Cancer 1979;15: 659-670.

  14. Chanda YR. The wealth of India: A dictionary of Indian Raw materials and Industrial products; Publication and Information Dirctoratorate, CSIR, NewDelhi. 1982; 520-521.

  15. Binutu OA, Adesogan KE, Okogun JI (1996), Antibacterial and antifungal from Kigelia pinnata plant Med 62: 352-3.

  16. Dr John Wilkinson, Director, Herbal Sciences International Ltd, UK. Antiinflammatory and wound healing activities of the crude alcoholic extract and flavonoids of Kigelia Africanax.

  17. Sangitha saini, Harmeet Kaur, Bharat Verma, Ripudaman, S.K.Singh. Natural product Radiance, Vol 8 (2), 2009, pp. 190-197.

  18. Mitrofan LM, Pelkonen J, Monkkonen J. The level of ATP analog and isopentenyl pyrophosphate correlates with zoledronic acid-induced apoptosis in cancer cells in vitro. Bone 45 2009;1153–1160.

  19. Mosamann,Tim. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods .1983;65(1-2):55-63.

  20. Decker T, Lohmann-Matthes ML. A quick and simple method for the quantitation of lactase dehydrogenase release in measurements of cellular cytotoxicity and tumour necrosis factor (TNF) activity. Journal of Immunological Methods. 1988;61-9.

  21. Kulkarni SK. Practical Pharmacology and clinical pharmacy, Vallabh Publication SU-221 Pitampur Delhi 2007.

  22. Roghayeh Abbasalipourkabir, Arash Deghan, Aref Salehzadeh. Induction of mammary tumors using 7,12 DMBA, African Journal of Biotechnology Vol.9(28), pp.4491-4498, 12 july, 2010.




9


Signature of the candidate



N. SRI SAINADH


10


Remarks of the Guide : The topic selected for dissertation is satisfactory. Adequate equipment and chemicals are available to carry out the project work.


11


Name and Designation:





11.1

Guide

Dr.Nagarathna P.K.M,

Assist.Professor ,

Dept Of Pharmacology,

Karnataka College Of Pharmacy,

Bengaluru-64.





11.2



Signature of Guide




Dr.Nagarathna P.K.M.




11.3

Co-Guide








11.4

Signature of Co-Guide






11.5

Head of the Department


Dr. Raju Koneri,

Professor & Dean,

Dept Of Pharmacology,

Karnataka College Of Pharmacy,

Bengaluru-64.






11.6



Signature of HOD



Dr. RAJU KONERI


12


12.1


Remarks of the Principal : All the required facilities will be provided to carry out dissertation work under the supervision of guide.




12.2



Principal

Dr. K. RAMESH,

Director,

Karnataka College Of Pharmacy,

Thirumenhalli,



Bangalore.





12.3



Signature of the Principal


K.RAMESH













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