3.1 chemicals and reagents - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/10450/8/08_chapter...

Anticancer Potential of Spiro-Isoxazolidine Derivatives of Parthenin

Materials and Methods

31

3.1 CHEMICALS AND REAGENTS

3.1.1. Chemicals procured from Sigma chemical co. St. Louis, MO, USA

RPMI-1460, L-glutamine, penicillin, streptomycin, HEPES, pyruvic acid, 2-

Mercapto-ethanol, diaminofluoresceine-2-diacetate (DAF-2-DA), 2,7-

dichlorofluoresceine diacetate (DCFH-DA), 3-(4, 5-diamethyl-2-thiazolyl) 2, 5 -

diphenyl-2H-tetrazolium) (MTT), fetal calf serum (FCS), Rhodamine- 123, DMSO,

fixer, quickdraw blotting paper, kodak professional HC-110 developer, bovine serum

albumin, BSA, protease inhibitors cocktail, hoechst-33258, D, L-buthionine-S, R-

sulfoximine (BSO), propidium iodide, DNase-free RNase, proteinase K, phenol

:chloroform: isoamylalcohol, camptothecin, ethidium bromide, trolox, cyclosporin,

EDTA, bromophenol blue, agarose, etoposide, staurosporine, doxorubicin, phosphate

buffer saline (PBS), glycerol, tween-20, triton-X, nonidet P-40,

phenylmethanesulfonyl fluoride (PMSF), sulphanilamide, orthophosphoric acid,

sodium orthovandate, dithiothreitol (DTT), protein marker chemichromeTM , 5-

Fluorouracil, gentamycin, mitomycin C, paclitaxel, penicillin, sulphorhodamine blue

(SRB) and trypsin.

3.1.2. Reagents and antibodies procured from BD Biosciences, Sandigo, USA

AnnexinV-FITC apoptosis detection kit, caspase-3,-8, -9 inhibitors and apoAlert

caspases-3,-8,-9 assay kits were from B.D. clontech, USA.

3.1.3. Anti-human antibodies procured from Santa Cruz, California, USA

Mouse anti-human antibodies to NFκB p65, IκB-α, Cytochrome-C, Bax, PARP-1,

Bcl-2, Bcl-xL, β-Actin, goat anti-human antibodies to ICAD, goat anti-mouse IgG-

HRP and mouse anti-goat IgG-HRP.

3.1.4. Chemicals and reagents procured from Bio-Rad Laboratories, California,

USA



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Acrylamide/ bisacryl amide, Tris, SDS, ammonium per sulphate, TEMED, BioRad

protein Assay kit, Immunoblot TM PVDF membrane (0.2µM), glycine and other

electrophoresis reagents.

3.1.5. Reagents and antibodies procured from Amersham Bioscience, USA

ECL plus western blot detection system, high performance chemiluminescence’s

film, and hyper cassette.

3.1.6. Chemicals and reagents procured from other companies

Tris buffer and Sodium bicarbonate (NaHCO3) from HiMedia, India and

Trichloroacetic acid, Glacial acetic acid and Isopropyl alcohol from South India

Surgical Company Ltd., India. All other chemicals used were of analytical grade and

available locally.

3.2. EQUIPMENTS

Table 3.1: List of Equipments

Instrument Manufacturer

Biosafety cabinet, level-II,AC2-4A1 Esco Micro Pvt Ltd, Singapore

CO2 incubator, Thermo Forma-II-3141 Thermo Forma, USA

Centrifuge Bench top, 2-16K SIGMA, Germany

Inverted microscope, 1X-70 Olympus, Japan

Digital camera, DP12-2 Olympus, Japan

UV-visible spectrophotometer, Specord-250 Analytik Jena, Germany

Flow cytometer, BD-FACS Caliber BD Biosciences, USA

Deep freezer, DFV-1200 Widson Scientific Works, India

Elisa plate reader, Multi-scan spectrum Thermo Forma, Finland

Electron microscope, 100CXII JEOL, Japan

Ultracentrifuge- L8-80 Beckman, USA



33

Sample evaporator, SpeedVac Thermo, USA

Microwave oven, Microsynth Milestones, USA

Gel imaging system, Gel Doc-XR-170-8170 Bio-Rad, USA

SDS-PAGE- assembly, Mini-Protean-Tetra

cell

Bio-Rad, USA

Tissue homogenizer, T18 Ultra-Turrax IKA,Germany

Vortex mixer, Vibrofix VF1 IKA,Germany

Weighing balance, CP324-S Sartorius, Germany

Autoclave, Reico Reliance Instruments Corporation,

India

Gel Rocker, Ultra-rocker-166-0719 Bio-Rad, USA

Water Bath, TW-12 Julabo, Germany

Vacuum pump, X10422050 Millipore, India

Filtration assembly, #27154-032 Millipore, India

Cryo-container, BA-35 IBP, India

Magnetic stirrer cum hot plate, RET-3188800 IKA, Germany

pH meter, Thermo orion-420A+ Thermo Orian, USA

Fluorimeter, LS-50 Perkin Elmer, USA

HPLC, LC-10ATVP Shimadzu, Japan

Water bath shaker, KS 501 IKA, Germany

3.3. APPARATUS

96-well flat bottom tissue culture plates (Grenier)

Cryo 1C Freezing Container (Mr. Frosty, Sigma)

Cryovials (Grenier)

Glass Pipettes (10ml, 5ml) (Borosil)

Glass Strips 25mm x 40cm, 6mm thick ( LKB)



34

Measuring cylinder (25ml, 50 ml, 100 ml) ( Borosil)

Media Glass bottles (2 L, 1L, 500ml, 250ml, 100ml) (Borosil)

Microcentrifuge Tubes (1.5 ml) (Eppendorf)

Micropipettes (2-20l, 20-200l, 200-1000l) (BRAND)

Pipettes Dispensor (BRAND)

Sterile Centrifuge Tubes (15ml, 50ml) (Grenier)

Sterile disposable Syringes (10ml, 5ml, 2ml) (Dispovan)

Syringe Driven Filter Unit (0.22) (Millipore)

Tips for Micropipettes (2-20l, 20-200l, 200-1000l) (Tarson)

Tissue culture flasks – 25 Cm2 (T-25) (Grenier)

Tissue culture flasks – 75 Cm2 (T-75) (Nunc)

Tissue culture flasks - 150 Cm2 (T-150) (Iwaki)



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3.4 SYNTHESIS OF SPIRO-ISOXAZOLIDINE DERIVATIVES OF

PARTHENIN

Spiro-isoxazolidine derivatives of parthenin were synthesized in the Synthetic

Organic Chemistry Division of Indian Institute of Integrative Medicine, Jammu.

Seven compounds viz. SLPAR 3, 7, 13, 14, 16, 17 and 18 were subjected to in-vitro

cytotoxicity against human cancer cell lines. Following is the general scheme for

synthesis of spiro-isoxazolidine derivatives.

Fig. 3.1 Synthesis of spiro-isoxazolidine derivative of parthenin wherein, the value of R/R' is selected from the group consisting of hydrogen, alkyl substituents viz., methyl, ethyl, propyl and the higher homologues either linear or branched, including alicyclic such as cyclopentane, cyclohexane or higher membered rings, fused rings, aryl/ heteroaryl substituted alkyl groups including benzylic or its higher homologues including unsaturated groups such as prenyl, cinnamyl, crotyl group; and R/R' may be aryl groups at the first position of isoxazolidine ring in structure viz., Phenyl or substituted phenyl, napthyl, anthracenyl, phenathrenyl, or other heteroaromatic ring systems such as pyridinyl, indolyl, benzofuryl, furyl, theophenyl, oxazolyl, isoxazolyl, or any other single or fused ring heteroaromatic systems.



36

3.4.1. Structures of four selected compounds

OO

N

O O

Cl

SL-PAR-3

Fig. 3.2 [A] N-(Phenyl)-C-(4-chlorophenyl)-spiro-isoxazolidinyl parthenin

OO

N

O O

Br

SL-PAR-7

Fig. 3.2 [B] N-(Phenyl)-C-(4-bromophenyl)-spiro-isoxazolidinyl parthein



37

OO

N

O O

MeOBr

SL-PAR-13

Fig. 3.2 [C] N-(Phenyl)-C-(5-bromo-2-methoxyphenyl)-spiro-isoxazolidinyl parthein

OO

N

O O

CN

SL-PAR-14

Fig. 3.2 [D] N-(Phenyl)-C-(4-cyanophenyl)-spiro-isoxazolidinyl parthein



38

3.5 IN-VITRO ANTI CANCER STUDIES

3.5.1. SOLUTIONS AND BUFFERS

Phosphate buffered saline (PBS)

7.20 g of NaCl, 1.48 g of Na2HPO4 and 0.43 g of KH2PO4 was dissolved in 1 L of

distilled water. pH was adjusted to 7.2.

Trichloro acetic acid (TCA)

50% TCA solution was prepared by dissolving 500 g of TCA in 1 L double distilled

water.

Acetic acid (1%)

1 ml of acetic acid was dissolved in 100 ml of double distilled water.

SRB Dye

4 g (0.4%) SRB dye was dissolved in 1 L of 1% acetic acid.

Tris-buffer

1.21 g of Tris (10 mM) was dissolved in 950 ml distilled water, pH was adjusted to

10.5.

DNA extraction buffer

10 mM Tris HCl of pH 8.0, 100 mM NaCl, 5 mM EDTA and 5% Triton x 100 were

used to make STET buffer in double distilled water. 400 µg/ml RNase and 200

µg/ml Proteinase K were used after treatment of STET to degrade RNA and Protein

in the cells.

DNA buffer (TE)

10 mM Tris HCl and 1 mM EDTA of pH 7.4 in double distilled water.

DNA running buffer (TAE)

0.4 M Tris HCl, 0.01 mM EDTA and 0.2 M acetic acid in double distilled water.



39

Sample loading buffer (6x)

0.25% Bromophenol Blue, 30% Glycerol, 1.0 M Tris HCl buffer of pH 8.0, 12.1 g

/100 ml Tris in double distilled water. The pH was adjusted with HCl and solution

was diluted to the required molarity.

PAGE running buffer (10x)

30.3 g Tris base, 144.1 g Glycine and 10.0 g SDS were dissolved in the 1 L of

double distilled water. The pH of the stock solution diluted to 1x was 8.3.

TBST 1X (Tris buffer saline-Tween)

10 mM Tris HCl pH 8.0, 150 mM NaCl and 0.1% Tween-20 in double distilled

water.

Transfer buffer (1x)

25 mM Tris Base and 192 mM Glycine in double distilled water. 20% Methanol

(added freshly)

Blocking buffer (1x)

10 mM Tris HCl pH 8.0, 150 mM NaCl, 0.1% Tween-20 and 5% Fat less dry milk in

double distilled water.

SDS sample buffer (5x)

1 ml Tris HCl (pH 6.8), 0.8 ml Glycerol, 1.6 ml SDS, 0.4 ml 2-mercaptoethanol, 0.2

ml Bromophenol blue and 4 ml HPLC grade water. Total volume: 8ml.

Acrylamide/bisacrylamide solution (30%/0.8%)

30.0 g Acrylamide, 0.8g N, N’-methylene-bisacrylamide was dissolved in 60 ml

double distilled water and the final volume was made up to 100 ml.

Separating gel (10%)

4.0 ml HPLC grade water, 2.5 ml Tris HCl pH 8.8, 0.05 ml SDS, 3.4 ml

Acrylamide/Bis Acryl amide, 0.05 ml Ammonium per sulphate, 0.005 ml TEMED



40

(Ammonium per sulphate and TEMED was added after degassing of solution. Total

monomer volume was 10 ml for two gels)

Stacking gel (5%)

3.4 ml double distilled water, 0.63 ml Tris HCl pH 6.8, 0.025 ml SDS, 0.83 ml

Acrylamide/Bis-Acrylamide, 0.05 ml Ammonium per sulphate and 0.005 ml

TEMED (Add Ammonium per sulphate and TEMED after degassing of solution.

Total monomer volume is 10 ml enough for two gels)

Lysis buffer for Cytosolic and Mitochondrial lysates

For Cytosolic lysate-

5 mM NaCl, 8 mM Na2HPO4, 1 mM NaH2PO4, 1 mM EDTA, 350 µg/ml Digitonin

and 10% Eukaryotic protease inhibitor cocktail in double distilled water.

For Mitochondrial lysate-

20 mM Tris HCl (pH 8.0), 137 mM NaCl, 2 mM EDTA, 10 % Glycerol, 1 %

Nonidet P-40, 1 mM PMSF, 1 mM Sodium orthovanadate, 1 mM Dithiothreitol and

10 % Eukaryotic protease inhibitor cocktail in double distilled water.

RIPA buffer

50 mM Tris HCl (pH 7.4), 150 mM NaCl, 1 % Triton X-100, 0.1 % SDS, 5 mM

EDTA, 30 mM Na2HPO4, 50 mM NaF, 0.5 mM NaPO4, 2 mM PMSF and 10 %

Eukaryotic protease inhibitor cocktail in double distilled water.

Lysis buffer for Nuclear lysate:

Hypotonic buffer:

10 mM HEPES/KOH pH 7.9, 2 mM MgCl2, 0.1 mM EDTA, 10 mM KCl, 1 mM

Dithiotheritol, 0.5 mM PMSF and 1 % Eukaryotic protease inhibitor cocktail (v/v) in

double distilled water.

Saline buffer:

50 mM HEPES/KOH pH 7.9, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10 %

Glycerol, 1 mM DTT, 0.5 mM PMSF in double distilled water.



41

3.5.2 SOURCE FOR HUMAN CANCER CELL LINES

Human cancer cell lines (Table 3.2) were obtained from National Cancer Institute

(NCI), Fredrick (USA) and National Center for Cell Science (NCCS), Pune (India).

3.5.2.1 CELL CULTURING

3.5.2.1.1 Handling of cell lines

Original stock of human cancer cell lines was received in frozen state and

immediately transferred to liquid nitrogen. The stock obtained in tissue culture flasks

was collected in centrifuge tubes as many adherent cells tend to detach during travel.

This suspension was centrifuged (1500 rpm, 5 min, 4ºC) to collect cells. The pellet

was suspended in fresh 20 ml media and the mixture was introduced in a T75 flask

which was incubated at 37ºC and time to time observed for apparent contamination

and proper growth under microscope. At sub-confluent stage the cells were harvested

and cryo-preserved.

3.5.2.1.2 Revival of cell lines

Cryovials were removed from the liquid nitrogen container and thawed in water bath

at 370C. Cells were suspended aseptically in 8 ml of fresh complete medium in TCF-

25 and cells were incubated in CO2 incubator at 370C temperature, 5% CO2

atmosphere and 90% RH.

3.5.2.1.3 Routine maintenance

The culture was observed daily for proper growth, apparent contamination and

periodic medium change. Cells were periodically fed with pre-warmed (370C)

medium. Medium was replaced depending upon the drop of pH etc. For suspension

cultures after every 1-2 days, cells were diluted with fresh media. The dilutions were

based on the density of the cells. Typically 1:4 to 1:20 dilutions were found to be

appropriate for most cell lines. For adherent cultures feeding for adherent cultures

was done by removing the old medium and replacing it with fresh medium.



42

3.5.2.2 SUBCULTURING

3.5.2.2.1. Adherent cell lines

The sub-culturing involved the detachment of cells from the growth surface

(substratum) of the culture flask and re-inoculation of the cells into in new culture

flasks. The medium of the flask having sub-confluent growth was changed one day

in advance. Medium was aspirated from the flask and cells were rinsed with PBS. 3

ml of trypsin-EDTA [0.05% in PBS (pH 7.4) containing 0.02% EDTA] was added

and cells were incubated at 37°C for 2-5min. The flask was observed for the

detachment of the cells and complete growth medium (1.0 ml, pre-warmed at 37°C)

was added to the flask immediately after detachment of the cells. Further, cells were

collected in centrifuge tube and centrifuged at 200g for 5 min and the cell pellet was

re-suspended in complete growth media. Experiments were performed with cells

cultivated not longer than twenty passages. Further passaging of immortalized cell

lines is possible but the risk of genotypic changes increases. Cells were either used

for in-vitro experiments of stored for further use.

3.5.2.2.2 Suspension cell lines

The medium was withdrawn from the flask aseptically and centrifuged at 1100 rpm

for 10 min, the pellet was re-suspended in fresh medium and divided into new TCF

and further incubated in CO2 incubator.

3.5.2.3 PRESERVATION AND STORAGE (CRYOPRESERVATION)

Microbial contamination or genotypic changes may appear in long-term cell cultures,

leading to the loss of well-characterized cell lines. To prevent cell loss, cells can be

frozen and stored (cryo-preservation) almost indefinitely at a very low temperature in

liquid nitrogen (-196°C). Healthy cells with 98% viability were chosen and cell

density was adjusted to 1 x 107. Cells were pelleted by centrifugation (200g, 10 min,

4ºC) and were suspended in freezing medium. Cell suspension was transferred to

cryovials (1.5 ml).



43

Freezing medium- (suspension cells) 9.5 ml of FCS was mixed with 0.5 ml of

DMSO.

Freezing medium- (Adherent cells) 2 ml of FCS and 0.5 ml of DMSO was added to

7.5 ml Growth medium.

The cells were slowly cooled at the optimal rate of 1°C per minute with the help of

low tech device called Mr. Frosty. The cryovials containing the cells were placed in

the Mr. Frosty and placed in the -80°C freezer. Once the container has achieved a

temperature of -80°C (overnight) the vials were removed from the Mr. Frosty and

were immediately placed in the liquid nitrogen storage container.

Table 3.2: Human cancer cell lines used for the study

Tissue Cell line Medium Positive control

Leukamia HL-60 RPMI Camptothecin

Leukamia THP-1 RPMI Camptothecin

Leukamia Molt-4 RPMI Camptothecin

Colon HCT-15 RPMI 5-Flurouracil

Colon Colo-205 RPMI 5-Flurouracil

Colorectal CaCo-2 MEM Mitomycin-C

Protrate PC-3 RPMI Mitomycin-C

Skin A375 RPMI DDP, Taxol

Lung A549 RPMI DDP, Taxol

Breast MCF-7 MEM Adriamycin (Doxorubicin}

Breast T-47D MEM Adriamycin (Doxorubicin}

Cervix SiHa RPMI DDP, 5-Flourouracil



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Cervix HeLa RPMI DDP, 5-Flourouracil

3.5.3 PREPARATION OF TEST MATERIAL FOR IN-VITRO ASSAYS

3.5.3.1 Stock solution

Stock solutions of test compounds were prepared in DMSO. A stock solution of

10mM of each compound was prepared and stored at 40C for further use.

3.5.3.2 Working solution

Working solution was prepared on the day of experiment. The stock solution was

serially diluted with complete growth medium containing 50µg/ml Gentamycin to

obtain working test solutions of required concentration. Gentamycin was added to

control the microbial contamination.

3.5.4 SULPHORHODAMINE BLUE (SRB) ASSAY

The SRB assay is a colorimetric assay for cytotoxicity screening. This assay provides

a rapid and sensitive method for measuring the drug-induced cytotoxicity in both

attached and suspension cultures in 96 well microtiter plates (Skehan et al., 1990).

Cytotoxicity testing was based on mammalian cell lines under active growth and

mitotic division. Cells were cultured in a 96 well tissue culture plates and the cell

growth which depends upon the rate of multiplication was measured indirectly by the

intensity of the color of the dye which was directly proportional to the number of

cells present. In the cytotoxicity assay the rate of cell growth of a cancer cell line in

presence and absence of the test substance was compared after a specified time.

Ideally several different cancer cell lines can be used so that selectivity can be

assessed. This gives an indication of potential usefulness in a clinical setting.

3.5.4.1 Principle

Sulphorhodamine Blue (SRB) is a water-soluble, pink aminoxanthine dye that binds

to the basic amino acid residues of cellular proteins in the plasma membrane in



45

acidic medium. The adsorbed dye is dissolved in alkaline medium and color intensity

is measured spectrophotometrically to determine relative cell growth or viability in

treated and untreated cells. Thus colorimetric measurement of the bound dye

provides an estimate of the total protein mass that is directly related to the cell

number. The greater the number of cells, the greater amount of dye is adsorbed

which finally gives more intense color and greater absorbance. The method is being

currently used by National Cancer Institute, Frederick, USA in their screening

program to assess cell survival.

3.5.4.2 Methodology

The cells at sub-confluent stage were harvested from the flask by treatment with

trypsin [0.05% in PBS (pH 7.4) containing 0.02% EDTA]. Cells with viability of

more than 98% as determined by trypan blue exclusion, were used for determination

of cytotoxicity The cell suspension of 1x105cells/ml was prepared in complete

growth medium. Stock solutions (10 mM) of compounds were prepared in DMSO.

The stock solutions were serially diluted with complete growth medium containing

50µg/ml of gentamycin to obtain working test solutions of required concentrations.

In-vitro cytotoxicity against human cancer cell lines of different tissues was

determined (Monks et al., 1991) using 96-well tissue culture plates. The 100µl of

cell suspension was added to each well (single sample in 5 wells). The cells were

allowed to grow in carbon dioxide incubator (37°C, 5% CO2, 90% RH) for 24 h. Test

materials in complete growth medium (100µl) were added after 24 h of incubation to

the wells containing cell suspension. The plates were further incubated for 48 h in a

carbon dioxide incubator. The cell growth was stopped by gently layering

trichloroacetic acid (50%, 50µl) on top of the medium in all the wells. The plates

were incubated at 4oC for one hour to fix the cells attached to the bottom of the

wells. The liquid of all the wells was gently pipetted out and discarded. The plates

were washed five times with distilled water to remove trichloroacetic acid, growth

medium, low molecular weight metabolites and serum proteins etc and air-dried. The

plates were stained with Sulforhodamine B dye (0.4% in 1% acetic acid, 100µl) for

30min. The plates were washed five times with 1% acetic acid and then air-dried.



46

(Skehan et al., 1990). The adsorbed dye was dissolved in Tris HCl Buffer (100l,

0.01M, pH 10.4) and plates were gently stirred for 10min on a mechanical stirrer.

The optical density (OD) was recorded on ELISA reader at 540 nm.

3.5.4.3 Calculations

The cell growth was determined by subtracting average Absorbance (OD) value of

respective blank from the average Absorbance (OD) value of experimental set.

Percent growth in presence of test material was calculated considering the growth in

absence of any test material as 100% and in turn percent growth inhibition in

presence of test material was calculated.

Percent growth in the presence of test material =

Δ OD in the presence of test material X 100 Δ OD in the absence of test material

Percent growth inhibition in the presence of test material = 100 – percent growth in

the presence of test material

3.5.5 CELL PROLIFERATION ASSAY USING MTT

3.5.5.1 Trypan blue exclusion

The viability of cells was determined by standard trypan blue exclusion assay. A

visual count was made of the number of live and dead cells using haemo-cytometer

following staining with trypan blue (0.4% in PBS) and percentage of live vs. dead

cells was determined. Cell with >95% viability were selected for the studies.

3.5.5.2 Principle

This assay is a quantitative colorimetric method for determination of cell survival

and proliferation. The assessed parameter is the metabolic activity of viable cells.

Metabolically active cells reduce pale yellow tetrazolium salt (MTT) to a dark blue

water-insoluble formazan which can be, after solubilisation with DMSO, directly



47

quantified. The absorbance of the formazan directly correlates with the number of

viable cells (Mosmann, 1983).

3.5.5.3 Methodology

The cells were plated in 96-well plates at a density of 2.0 x 104 in 200µl of medium

per well. Cultures were incubated with different concentrations of test material and

incubated for 24 or 48h. The medium was replaced with fresh medium containing

100µg/ml of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)

for 4h. The supernatant was aspirated and MTT-formazon crystals dissolved in 100μl

DMSO; OD measured at λ 570 nm on ELISA reader (Thermo Labs, USA). Cell

growth was calculated by comparing the absorbance of treated versus untreated cells.

3.5.6 MICROSCOPY

3.5.6.1. Morphology of apoptotic cells

Cell shrinkage, membrane blebbing and the formation of apoptotic bodies are

characteristic events during apoptosis which can easily be detected by light

microscopy. Cells (1x106 cells/ml/well; 24-well tissue culture plate) were either

untreated or treated with test material as per treatment regimen. Cells were viewed at

a 60X- magnification with an Inverted microscope (Olympus-N-71, Japan).

3.5.6.2 Hoechst 33258 staining of cells for nuclear morphology

Cells undergoing apoptosis depicts an increase in chromatin condensation.

Morphologically, the nuclei of apoptotic cells become smaller than those of normal

cells and become hyper-fluorescent when labeled with some nuclear stains. The

bisbenzimide dye Hoechst 33258 is a cell-permeant, adenosine/thymidine (AT)

selective, minor groove-binding DNA dye that brightly stains the condensed

chromatin of apoptotic cells while staining the looser chromatin of viable cells only

moderately. Treated and untreated control cells (2x106 cells) were collected,

centrifuged at 300xg for 5 min and washed twice with PBS. Cells were gently

suspended in 100 µl of PBS and fixed in 400µl cold acetic acid: methanol (1:3, v/v)

overnight at 4°C. Cells were washed again in 1ml of fixing solution, suspended in



48

the residual volume of about 50µl, spread on a clean slide and dried over night at

room temperature. One ml of staining solution (Hoechst 33258, 10 µg/ml 0.01M

citric acid and 0.45M disodium phosphate containing 0.05% Tween-20) was poured

on each slide and stained for 30 min under subdued light at room temperature. Slides

were washed under gentle flow of tap water, rinsed with distilled water followed by

PBS. While wet, 50 µl of mounting fluid (PBS: glycerol, 1:1) was poured over the

center of slide and covered with glass cover slip. The slides were sealed and

observed for any nuclear morphological alterations and apoptotic bodies under

inverted fluorescence microscope (Olympus 1X70, magnification 60x) using UV

excitation.

3.5.7 DNA AGAROSE GEL ELECROPHORESIS

Apoptosis was also assessed by electrophoresis of extracted genomic DNA from

cells. Briefly, 2x106 cells after various treatments were centrifuged at 100xg for

10min, and washed in PBS containing 20 mM EDTA. The pellet was lysed in 250µl

of lysis buffer (100 mM NaCl, 5mM EDTA, 10mM Tris HCl, pH 8.0, 5% Triton X-

100) containing 400µg/ml DNase-free RNase and incubated at 37oC for 90min

followed by 1h incubation with proteinase-K (200 µg/ml) at 50oC for 1h. The DNA

was extracted with 150µl of phenol for 1min and centrifuged 13000xg for 2min. The

aqueous phase was further extracted with phenol: chloroform: isoamylalcohol

(25:24:1) and centrifuged. DNA was precipitated from aqueous phase with 3

volumes of chilled alcohol and 0.3M sodium acetate at 20oC overnight. The

precipitate was centrifuged at 13000xg for 10 min. The DNA pellet was washed in

80% alcohol, dried, dissolved in 50µl TE buffer, mixed in loading buffer and

electrophoresed in 1.8% agarose gel at 50V for 1.5h in TAE buffer (Kumar et al.,

2008).

3.5.8 FRAP ASSAY

The total antioxidant potential of samples was determined by measuring the ferric

reducing antioxidant power (FRAP assay). FRAP assay uses antioxidants as



49

reductants in a redox-linked colorimetric method, employing an easily reduced

oxidant system present in stoichiometric excess. Reaction mixture containing acetate

buffer (0.3mM), TPTZ (10mM) and ferric chloride (20mM) were mixed in the ratio

10:1:1, respectively. 900µl of this reagent was mixed with 100µl of test compound at

concentrations 0.5, 2, 5, 10 and 20 μM in separate test tubes. This solution is

incubated at 37ºC for 10 min and absorbance is taken at 595 nm (Benzie and Strain,

1996).

3.5.9 FLOW CYTOMETRY

3.5.9.1. Principle

In a flow cytometric system, large numbers of cells or particles flow within a laminar

fluid stream in a single file passing a laser beam where they are individually

evaluated. As the focused laser beam interacts with a cell, scattered light and, in the

case of using fluorescent antibodies or dyes, fluorescence signals are created at the

same time. The electronic signals are converted into digital values and are illustrated

in dot plots or histogram plots. All measurements were performed on a FACS-

CALIBER (Becton Dickinson USA), equipped with a 488 nm argon-ion laser, using

Cell Quest software.

3.5.9.2. DNA content and cell cycle phase distribution

DNA fragmentation constitutes one biochemical hallmark of apoptosis. Thus,

measurement of DNA content makes it possible to identify apoptotic cells, to

recognize the cell cycle phase specificity and to quantitate apoptosis. For flow

cytometry analysis of the relative nuclear DNA content, the fluorescent dye

propidium iodide (PI), which becomes highly fluorescent after binding to DNA, is

most commonly used. After permeabilisation PI binds to DNA in cells at all stages of

the cell cycle, and the intensity with which a cell nucleus emits fluorescent light is

directly proportional to its DNA content. The results of the measurement are

illustrated in a histogram, where the number of cells (counts) is plotted against the

relative fluorescence intensity of PI (FL-2; λem: 585 nm; red fluorescence) (Bhushan



50

et al., 2006). The histogram reflects the cell cycle distribution of the cell population.

Staining normal untreated cells with PI, most of the cells are in G0/G1 phase (DNA

content: 2n) and emit light at a uniform frequency, depicted in the prominent G0/G1

peak of the histogram. Cells in G2/M phase (DNA content: 2 x 2n) emit light with

double intensity of the G0/G1 cells and therefore appear at higher values. Cells

passing through the S phase (DNA content between 2n and 2 x 2n) emit light of an

intensity range that falls between the G0/G1 peak and G2/M peak. Cells treated with

the test material were collected, washed in PBS, fixed in 70% cold ethanol and

placed at −20 °C overnight. Cells were washed with PBS, subjected to proteinase-K

and RNase digestion followed by staining of clean nuclear materials (nuclei) with

propidium iodide using procedures and reagents as described in the instruction

manual of the Cycle Test plus DNA reagent kit (Becton Dickinson, USA). The

preparations were analyzed for DNA content using BD-LSR flow cytometer. Data

were collected in list mode for 10,000 events for FL2-A vs. FL2-W. Apoptotic nuclei

appear as a broad hypodiploid DNA peak at lower fluorescence intensity compared

to nuclei in G0/G1 phase (Krishan, 1975).

3.5.9.3. Flow cytometric analysis of apoptosis and necrosis using annexin V/PI

dual staining

During apoptosis a number of changes in cell surface markers occur. An early event

is the loss of asymmetry in cell membrane phospholipids, altering both the

hydrophobicity and charge of the membrane surface. In living cells, the distribution

of phospholipids is asymmetric, with the inner membrane containing anionic

phospholipids (e.g. phosphatidylserine, PS) and the outer membrane having mostly

neutral phospholipids. Upon induction of apoptosis, however, the amount of PS on

the outer surface of the membrane increases. Annexin V, a calcium-dependent

phospholipid-binding protein, has a high affinity for PS (Vermes et al., 1995).

Hence, FITC-labeled Annexin V can be used to identify apoptotic cells by flow

cytometry. Additional incubation with propidium iodide (PI) is used to distinguish

between viable, early apoptotic, necrotic or late apoptotic cells. Necrotic or late

apoptotic cells will bind Annexin V-FITC and stain with PI because of membrane



51

rupture while PI will be excluded from viable (FITC negative) and early apoptotic

(FITC positive) cells. Detection of apoptosis by Annexin V-FITC was performed

using a Annexin V-FITC Apoptosis Detection Kit from BD Pharmingin (Sandigo

USA) according to the manufacturer’s instruction. Cells treated with test material

were washed twice with PBS and then resuspended in 100µl of the binding buffer

provided with the apoptosis detection kit. Cells were stained with annexinV-FITC

antibody and PI and scanned for fluorescence intensity in (FL-1; λ em: 530 nm) for

FITC and (FL-2; λ em: 585 nm) for PI channels. The fraction of cell population in

different quadrants was analyzed using quadrant statistics. Cells in the lower right

quadrant represented apoptosis and in the upper right quadrant represented post

apoptotic necrosis.

3.5.9.4. Flow cytometeric analysis of reactive oxygen species (ROS)

Apoptosis can be initiated by oxidative stress mediated by the generation of reactive

oxygen species (ROS). For the investigation of ROS levels carboxylated 2’7’-

dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) is commonly used.

Carboxy-H2DCFDA is a cell-permeant indicator for reactive oxygen species,

particularly peroxide, that is non-fluorescent until the acetate groups are removed by

intracellular esterases and oxidation occurs within the cell yielding a green

fluorescent product (Rothe and Valet, 1996). The dye has two negative charges at

physiological pH, allowing for better retention by cells. Cells treated with test

material and untreated controls were incubated for 30 min in a CO2 incubator with

10 µM of H2DCFDA. The dye was removed by centrifugation and washing with

PBS (2X). The pellets were resuspended in adequate volume of PBS (0.5 ml/sample)

and transferred to polypropylene tubes and placed into a water-bath (37°C). To

estimate intracellular peroxide production, fluorescence intensity (FL-1; λem: 530

nm) was recorded (Malik et al., 2007). Cells incubated with carboxy-H2DCFDA

only were employed to monitor basal peroxide synthesis. Fluorescence intensity was

expressed in Dot plot quadrants.

3.5.9.5. Measurement of mitochondrial membrane potential



52

Mitochondrial dysfunction within the apoptotic process is often associated with loss

of the mitochondrial inner transmembrane potential. One possibility to visualise

alterations in the mitochondrial membrane potential is staining with potentiometric

fluorescent dyes. Changes in mitochondrial transmembrane potential (Ψmt) as a

result of mitochondrial perturbation were measured after staining with Rhodamine-

123 (Royall and Ischiropoulos, 1993). Cells after various treatments in 12-well plate

were incubated with medium containing Rhodamine-123 (5 μg /ml; stock, 1mg/ml

PBS) for 1 h. Cells were washed in PBS and centrifuged at 100xg for 5 min and

suspended in sheath fluid. Immediately before analysis, propidium iodide (5 μg /ml;

stock 1mg/ml PBS) was added to the samples. The intensity of fluoresecence from

10,000 events was analyzed in FL-1 channel on flow cytometer.

3.5.9.6. Flow cytometric analysis of intracellular nitric oxide using DAF-2-DA

Intracellular nitric oxide was measured by employing a low molecular weight

fluorescent probe diaminofluoresceine 2-diacetate (DAF-2-DA), which is membrane

permeable and usually serves as a reporter of nitric oxide synthase activity.

Immediately after NO is generated inside the cells, it binds with the chromophore to

yield strong fluorescent signal that can be measured in the green (FL-1) channel of

the flow cytometer. Cells (2.0 x 106/3ml/well of 6-well plate) were incubated for 30

min with DAF-2-DA (10µM) before the treatment of test material. Cells were

collected, washed in PBS and analyzed on flow cytometer in FL-1 channel for

evaluation of NO positive cell population.

3.5.10 CASPASE ACTIVITY ASSAYS

Most of the proteolytic cleavages during apoptosis results from the activation of

caspases, a family of cysteine-dependent proteases. These enzymes recognize

specific tetra- or pentapeptide motifs in their substrates and cleave exclusively on the

carboxyl side of aspartate residues. Caspase activation can be measured by applying

a synthetic peptide substrate which is coupled to a fluorophor. Cleavage of the

fluorogenic substrate by the activated enzyme leads to increased fluorescence. The



53

generated fluorophore is proportional to the concentration of activated caspase. 7-

amino-4-trifluoromethyl coumarin (AFC) is a commonly used fluorophore. The

liberation of AFC shows a blue to green shift in fluorescence at an excitation

wavelength of 400nm and an emission wavelength of 505 nm while the liberation of

AMC shows blue to green shift at an excitation wavelength of 380nm and an

emission wavelength of 440nm respectively. Cells (2x106) were incubated with test

material for the indicated concentrations and time periods. At the end of treatment

cells were washed in PBS and pellet lysed in cell lysis buffer. Activities of caspase-

3, -8 and -9 in the cell lysates were determined fluorometrically using BD Apoalert

caspase fluorescent assay kits. Caspase-3 and -8 employed fluorochome conjugated

peptides DEVD-AFC and IETD-AFC as substrates, respectively while caspase-9

employed LEHD-AMC. Release of AFC (7-amino-4-trifluoromethyl coumarin) and

AMC (7-aminomethylcoumarin) were assayed according to the instructions provided

in the Manual by the supplier. Specific inhibitors were used as negative control to

determine whether fluorescence intensity changes were specific for the activity of

caspases. The peptide based inhibitors used were DEVD-CHO for caspase-3, IETD-

fmk for caspase-8 and LEHD-CHO for caspase-9.

3.5.11 IMMUNOBLOTTING

3.5.11.1. Preparation of cytosolic and mitochondrial lysates of HL-60 cells

In apoptosis, factors from the intermembrane space of mitochondria are released into

the cytosol where they form part of activation complexes for caspases (e.g.

cytochrome c, Smac) or they translocate into the nucleus where they participate in

DNA fragmentation (AIF). To detect the occurrence of certain proteins in the

cytoplasm the separation of cytosolic and mitochondrial fraction is necessary. HL-60

cells were collected after treatment and washed twice with PBS. Cytosolic fractions

were obtained by selective plasma membrane permeabilization with digitonin (Wang

et al., 2002). Briefly, 3 x106 cells were lysed for 2 min in 200µl of lysis buffer (75

mM NaCl, 8 mM Na2HPO4, 1 mM NaH2PO4, 1 mM EDTA, and 350 µg/ml

digitonin).The digitonin treated cells were centrifuged at 12,000g for 1 min. The



54

supernatant from each sample was mixed with an equal volume of 2x gel-loading

sample buffer for Western blot analysis. For mitochondrial proteins expression, the

above remaining pellets were dissolved in ice-cold 200 µl of lysis buffer containing

1% Nonidet P-40, 20 mM Tris-HCl (pH 8.0), 10% glycerol, 137 mM NaCl, 2 mM

EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 mM sodium

orthovanadate and Protease inhibitor Cocktail: 1µl/106 cells, and incubated on ice for

30 min. After centrifugation at 12,000x g for 10 min at 4°C, the cell lysates were

transferred to fresh tubes and stored at -80°C for immunoblotting of proteins

(Bhushan et al., 2009). The protein contents were determined using Bradford reagent

(Bio-Rad protein assay kit) and aliquots normalized to equal quantities before

loading.

3.5.11.2. Preparation of total cell lysate

Test material treated cells (3x106) were harvested and resuspended in 0.2 ml of RIPA

buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 5 mM

EDTA, 30 mM Na2HPO4, 50 mM NaF, 0.5 mM NaPO4, 2 mM phenylmethylsulfonyl

fluoride, and 10% protease cocktail inhibitor). Cells were incubated on ice for 30

min, vortexed and centrifuged at 12000xg for 15 min. Supernatants were collected

and stored at -800C (Han et al., 2004). The protein contents were determined using

Bradford reagent (Bio-Rad protein assay kit) and aliquots normalized to equal

quantities before loading.

3.5.11.3. Preparation of nuclear extracts

Cells (1x107/10ml/100mm plate) were incubated with test material for the predefined

time period. At the end of treatment, cells were collected and washed twice with PBS

(100xg, 5 min, 40C) and suspend in 400 µl ice cold hypotonic buffer for 10 min on

ice. Suspension was vortexed and centrifuged at 15000xg for 30 sec at 4°C .The

supernatant was discarded and the nuclear pellet was gently resuspended in to 100µl

of ice cold saline buffer on ice for 20 min. Cells suspension was vortexed and

centrifuged at 15000xg for 5 min at 4°C. The supernatant (nuclear lysate) was stored

at -700C and their protein contents assayed. The protein contents were determined



55

using Bradford reagent (Bio-Rad protein assay kit) and aliquots normalized to equal

quantities before loading.

3.5.11.4. SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Separation of proteins was performed by denaturating SDS-polyacrylamide gel

electrophoresis. This technique allows the electrophoretic separation of denaturated

proteins according to their size. SDS, a highly negative charged detergent, solubilises

proteins and leads to a constant net charge per mass unit. Hence, SDS-polypeptide

complexes migrate toward the anode through the polyacrylamide gel according to

their molecular weight. In addition, the differences in molecular shape are

compensated by the loss of the tertiary and secondary structures because of the

disruption of the hydrogen bonds and unfolding of the molecules. By adding of a

reducing agent like dithiothreitol (DTT) disulfide bonds are cleaved and proteins are

totally unfolded. The molecular weight of the investigated proteins is estimated by

applying molecular weight standards (ChemichromeTM western control, Sigma).

Preparation of separating gel (10%): Plate of desired thickness (1.0 or 1.5 cm) was

fixed into the plate fixing assembly (BioRad). Separating gel solution was added in

between the plate and the upper layer of gel solution was overloaded with water

saturated n-butanol. Whole assembly was kept aside for 45 min. The stacking gel

solution (5%) was prepared. Percentage of acrylamide/bisacrylamide gel was chosen

according to the expected molecular weight of the protein of interest. Upper layer of

n-butanol was washed out properly, rinsed with water and stacking gel solution was

added. Immediately gel comb (1or 1.5 cm) was fixed, and gel was allowed to get

polymerized for 30 min. Comb was gently removed, plate was fixed into SDS-PAGE

electrophoresis assembly and gel running buffer was added. Electrophoresis was

carried out in a vertical apparatus Mini Protean II (BioRad). Two gel runs were

performed in parallel.

3.5.11.5. Western Blot Analysis

The protein lysates along with standard protein marker were subjected to

discontinuous SDS-PAGE analysis. Proteins aliquots (50µg) were resolved on SDS-



56

PAGE, run at 60V (PowerPacTM HC High current power supply, BioRad), for 3h.

The resolved proteins were electro transferred to polyvinylidene difluoride (PVDF)

membranes (Bio-RAD) in to Western bloting transfer frames in the following

manner: Sponge-Blotting paper-gel-PVDF membrane-western blotting paper-sponge

and transfered overnight at 4°C at 30V in transfer buffer. Non-specific bindings of

the membrane were blocked by incubation with 5% non-fat milk in Tris-buffered

saline ((10mM Tris-HCl, 150mM NaCl) containing 0.1% Tween-20 (TBST) for 1 h

at room temperature. The blots were probed with respective primary anti-human

antibodies for 2 h (1:1000 dilutions) and washed three times with TBST. The blots

were then incubated with horseradish peroxidase conjugated respective secondary

antibodies for 1 h (1:1000 dilution), washed again three times with TBST. PVDF

membrane was incubated in to ECL Pus western blot detection reagent (ECL kit,

Amersham Biosciences) for 5 min on a transparency sheet, in dark. PVDF membrane

was placed in to the Hyper Cassette and superimposed with high performance

chemiluminescence’s film in the dark room for the 2 min and the protein signals

were developed on to the high performance chemiluminescence’s X-ray film by

using developer and fixed by processing chemical fixer. The film was washed out

gently with water and dried. The density of the bands was arbitrarily quantified using

Quantity One software of Bio-RAD gel documentation system.

3.5.11.6 Protein Estimation

Bio-Rad protein assay kit solvent (5x) was diluted to 1x with HPLC grade water and

filtered through Whatman filter No. 1. 190 µl of diluted reagent and 50µl of protein

sample were added into each well of 96 well plates and mixed properly. After 5min

of addition, absorbance was recorded at 595 nm using Elisa plate reader. A

calibration curve of BSA (1-100 µg) was generated using the procedure explained as

above and straight line equation was developed. Amount of sample protein was

calculated using the standard calibration curve equation.



57

3.5.12 DNA TOPOISOMERASE ASSAY

Supercoiled PryG DNA was incubated with 50 μM and 100 μM of SLPAR7 and four

units of human topoisomerase I & II separately (TOPOGEN), in relaxation buffer

containing 10 mM Tris–HCl, pH 7.9, 10 mM EDTA, 1.5 mM NaCl, 0.1 mg/ml BSA,

1 mM spermidine and 50% glycerol. Camptothecin (10 μM) and etoposide (100 μM)

were used as positive controls for topoisomerase I & II, respectively. Each reaction

volume was made up to 20μl with water, incubated at 37ºC for 30 min and reaction

was stopped by the addition of SDS to a final concentration of 1% which was treated

further with proteinase K (20 mg/ml) for 15 min. Products were resolved by 1%

agarose gel electrophoresis in TAE buffer (40 mM Tris-acetate, pH 8.0, 1 mM

EDTA) and stained with 0.5 μg/ml ethidium bromide (EtBr) for 15 min and

destained with distilled water for 15 min at room temperature (Muller and Hoepner,

1985).



58

3.6 IN- VIVO ANTI CANCER STUDIES

The studies for in-vivo anti cancer activity in mouse models were conducted as per

the protocols of National cancer Institute (NCI), USA (Geran et al., 1972). The in-

vivo experiments were based upon growth inhibition of tumor cells and the size of

solid tumor in treated animals in comparison to tumor cell growth/ the size of solid

tumor in normal control (normal saline treated).

3.6.1 TUMOR MODELS USED IN THE STUDY

Following tumor models were used for in-vivo anticancer activity screening study

1. Ehrlich Ascitic Carcinoma

2. Ehrlich Tumor (Solid)

3. Sarcoma-180 (Ascites)

4. Sarcoma-180 (Solid)

5. P388 Lymphocytic leukemia

3.6.2 MAINTENANCE AND SELECTION OF ANIMALS

Inbred BALB/c (Fig 3.22 A), non-inbred Swiss albino (Fig 3.22 B), DBA/2 and

CDF1 mice from in house colonies were maintained at anticancer vivarium of Indian

Institute of Integrative Medicine. The animals were housed in standard

polycarbonate cages (Fig 3.22 C). The room temperature and humidity of the room

in which animals were kept were maintained at 23±20C and 50±5% respectively.

Animals were provided commercially available pelleted feed and water ad lib. All

the protocols were approved by Institutional Animal Ethics Committee prior to the

experimentation.



59

Fig 3.3: A- Inbred BALB/c mice, B- Swiss albino, C- Polycarbonate cage

3.6.2.1 Animals

The weight and age of selected animals was 18-23 g and 2 months respectively. The

animals were healthy, free from disease. Animals of the same sex were used for all

treatment and control groups of the particular experiment.

3.6.2.2 Positive control

5-fluorouracil at 20 and 22 mg/kg i.p. was used a positive control for ascetic and

solid tumor models respectively (positive control group).

3.6.2.3 Normal control

Normal saline (0.85%) at 0.2 ml/animal i.p. was used as normal control for tumor

bearing animals (normal control group).

3.6.2.4 Preparation of test material

The required quantity of test material was weighed accurately by using analytical

balance and was transferred to glass mortar and pestle. Gum acacia (1 % of total

volume of formulation) was added as suspending agent. The weighed material was

triturated and aqueous fine suspension corresponding to 5, 10, 20 and 25 mg/kg was

prepared by using HPLC grade water. Test material was administered via i.p. route

to the ascetic and solid tumor bearing animals.

3.6.3 ASCITIC TUMOR MODELS

3.6.3.1 Propagation of Ehrlich Ascitic Carcinoma and Sarcoma 180 ascites

A B C



60

Peritoneal fluid was collected from animals bearing 8-10 days old Ehrlich Ascites

Carcinoma (Geran et al, 1972) (Fig 3.23). The fluid was diluted with normal saline

to make 1x107 cells/0.2ml of ascitic fluid. These cells were transplanted in the

peritoneal cavity of non-inbred Swiss mice for propagation. When the ascitic tumor

grew 8-10 days old, again peritoneal fluid was collected and 1×107 cells were

transplanted in the peritoneal cavity of fresh non-inbred Swiss mice selected for

experiments. For Sarcoma-180 cells, similar procedure was adopted except that the

cells were transplanted in the peritoneal cavity of BALB/C mice.

Fig 3.4: Mice bearing Ehrlich ascitic carcinoma

3.6.3.2 Test protocol

Test samples were evaluated for their in-vivo anticancer activity against Ehrlich

ascitic carcinoma as per protocol described by Geran et al, 1972.

Day 0

Peritoneal fluid was collected from animals bearing 8-10 days old Ehrlich ascites

carcinoma and the number of tumor cells per ml of ascites fluid were determined by

counting the cells with the help of hemocytometer. The ascetic fluid was diluted with

normal saline to obtain 1x 107 cells in 0.2 ml of fluid. Each animal selected for the

experiment was injected with 1x107 EAC cells contained in maximum 0.2 ml of

ascetic fluid, intraperitoneally.

Day 1



61

On day 1, tumor induced animals randomized and divided in treatment and control

group. Normal control group consisted of 10-15 animals whereas treatment and

positive control group consisted of seven animals each. Animals in each group were

weighed individually. Based on the average body weight, test materials were

formulated as described in section 3.6.2.4. Test drugs (0.2 ml) were administered

from day 1 to day 9 as described below.

Day 1-4

Animals in the treatment and control group were treated with respective test drugs at

predefined time.

Day 5

Animals in each group were weighed and based on the average body weight of each

group, suspension of test drugs were prepared in 1% gum acacia for the next 5 days.

The animals were administered with the dose at fixed time as earlier.

Day 6-9

Animals in the treated and control groups were administered with respective test

drugs at a fixed time.

Day 12 Evaluation

For evaluation of the experiment, animals in each group were sacrificed by cervical

dislocation. The ascitic fluid from each animal was collected in a pre-weighed

graduated centrifuge tube with the help of funnel, the volume and weight of ascitic

fluid from each animal was recorded. The number of tumor cells in ascitic fluid was

counted with the help of hemocytometer and the total number of tumor cells per

animal was also calculated. Total number of tumor cells present in the peritoneal

fluid of each animal was calculated and percent tumor growth inhibition was

calculated as follows.



62

Calculations

Percent tumor

growth inhibition =

Average no. of cells in control group –

Average no. of cells in treated group X 100

Average no. of cells in control group

3.6.4 SOLID TUMOR MODELS

Two solid tumor models viz., Ehrlich Tumor (solid) and Sarcoma 180 (solid) were

used to investigate the in-vivo tumor potential of test samples.

Fig 3.5: Sarcoma 180 (solid) tumor bearing mice

3.6.4.1. Test Protocol

Day 0

Ehrlich ascites carcinoma and Sarcoma-180 (ascites) cells were propagated in mice

as described earlier. For induction of solid tumors peritoneal fluid of the required

tumor was collected from animals bearing 8-10 days old Ehrlich ascites carcinoma or

Sarcoma-180 (ascites). The number of tumor cells per ml of ascetic fluid was

determined with the help of hemocytometer. The ascetic fluid was diluted with

normal saline in such a way that 0.2 ml of fluid contained 1 x 107 EAC/ Sarcoma-

180 cells. The animals selected for the experiment were injected intramuscularly in



63

the right thigh with 0.2 ml of ascetic fluid containing 1 x 107 EAC or Sarcoma- 180

cells.

Day 1

On day 1, the animals injected with EAC or Sarcoma-180 cells were randomized and

divided in different treatment and control groups. Normal control group contained

10-15 animals and all other groups (treatment and positive control) contained seven

animals each. Animals in each group were weighed and an average body weight of

animals in each group was worked out. Based on the average body weight, test drugs

were prepared in 1 % gum acacia for four days. Test drugs were administered from

day 1 to day 9 as described below.

Day 1-4

Animals in the treatment and control group were treated with respective test drugs at

predetermined time.

Day 5

Animals in each group were weighed and based on the average body weight for each

group, suspensions of test drugs were prepared in 1 % gum acacia for the next 5

days. The animals were administered with the test drugs at the fixed time as earlier.

Day 6-9

Animals in the treated and control groups were administered with respective test

drugs at a fixed time.

Day 13

The evaluation was done on 13th day. Before evaluation, hair were removed from

tumor bearing thigh of each animal.

Shortest and largest diameters of the tumor were measured with the help of Vernier

Caliper and tumor weight was calculated for individual animal.



64

Tumor weight (mg) = Length (mm) x [width] (mm)]2

2

Percent tumor

growth inhibition =

Average tumor weight of control group –

Average tumor weight of treated group X 100

Average tumor weight of control group

3.6.5. P388 LYMPHOCYTIC LEUKAMIA MODEL

P388 lymphocytic leukemia cells grown in the peritoneal cavity of DBA/2 female

mice were collected from the animal harbouring 6-7 days old ascites. For testing,

CDF1 males were used. 1 x 106 cells were injected intraperitoneally in 24 CDF1

males weighing 18-23 g on day 0. The next day, animals were randomized and

divided into four groups, containing 6 animals each. Group I & II were treated with

SLAPR7 at the dose of 5 mg/kg (i.p.) and 10 mg/kg (i.p.), respectively for 9

consecutive days. Group III was treated with 5-Fluorouracil (20 mg/kg) and it served

as positive control. Group IV was tumor bearing control and it received 0.2 ml

normal saline (i.p.) for 9 consecutive days. The median survival time of animals in

each group was calculated using the prescribed formula and % T/C values were

arrived at.

Acceptable median survival time of control group was taken as 8-11 days.

Minimum criteria of activity were as follows:

T/C < 86% Toxicity

T/C ≥ 125 % Moderate Activity

T/C ≥ 150 % Significant Activity



65

3.6.6 SAMPLES EVALUATED

SLPAR7 was evaluated against Ehrlich Ascitic Carcinoma, Sarcoma 180 (ascites),

Ehrlich Tumor (solid), P388 lymphocytic leukamia and Sarcoma-180 (solid) at

various dose levels.

Table 3.3: Different doses of SLPAR7 in various murine models

Model Type SLPAR7 Dose (mg/kg, i.p.)

Ehrlich Ascites Carcinoma

(EAC)

Ascitic 10, 20 and 30

P388 Lymphocytic leukemia Leukamia 5 and 10

Ehrlich tumor Solid 10 and 25

Sarcoma- 180 Ascites 10 and 20

Sarcoma-180 Solid 20 and 25



66

3.7 TOXICITY STUDIES

Toxicity study is the most important aspect of the new drug development program as

far as the safety evaluation is concerned. It is important to study the safety evaluation

of a drug candidate for prolonged use particularly when the nature of disease under

consideration is chronic. Furthermore, acute studies are the primary requirements for

the safety of a new drug molecule. The present study includes acute toxicity

(observation for 14 days) in swiss mice (Female, 22-25g body weight, 8-10 weeks

age) maintained in regulated environmental conditions (well-ventilated with > 10 air

changes/h; 12-h light/dark photoperiod; temperature 28 ± 2ºC; relative humidity, 60

± 10%), according to CPCSEA guidelines. Animal experiments were approved by

Institutional Animal Ethics Committee and were performed as per the Guidelines for

Animal Care as recommended by the Indian National Academy, New Delhi (1992).

Animals were fed with standard pelleted diet (Ashirwad Industries, Chandigarh,

India) and sterilized water was provided ad libitum. Seven days after acclimatization,

animals were used. In addition to daily observation on the general behavior of the

animals, food and water intake, and weekly body weight changes; macroscopic

observations and organ to body weight ratios were studied in detail at the termination

of the study.

3.7.1 Objectives

The purpose of this study is to assess the toxicological profile of SLPAR7 after

single dose administration via oral route to Swiss Albino mice. The animals were

observed for 14 days depending on the occurrence of toxic symptoms. The results of

acute toxicity study were useful for selection of doses for repeated dose toxicity

study and may also provide preliminary information on the target organ of toxicity. It

is also a useful parameter for establishing the Therapeutic Index (ie LD50/ED50) of

drugs and xenobiotics.



67

3.7.2 Guidelines

The study was performed in compliance with the OECD (Organization for Economic

Cooperation and Development) Guidelines for the testing of chemicals (No. 420,

Section 4: Health Effects) Acute Oral Toxicity- Fixed Dose Method” Adopted on

17th December 2001.

3.7.3. Dose of SLPAR7 used for toxicity study

Sighting study:

Dose: Treatment – Female - 5 mg/kg body weight

- 50mg/kg body weight



Main study:

Dose : Treatment- Female – 2000mg/kg body weight

Dose volume : 10ml/kg

Vehicle : Normal saline

Procedure : The test substance was suspended in distilled water to obtain 0.5, 5,

30.0 and 200.0 mg/ml strength of suspensions. The test substance was administered

in the dose volume of 10ml/kg body weight. The formulation was prepared fresh on

the day of dosing.

3.7.4. Test System

No. of animals per study : Sighting study- One for each study and

Main study- Five.

Acclimatization : Seven days prior to dosing

Veterinary examination : Before allocation of animals to different doses after

the completion of acclimatization period



68

Housing & Environment

Sighting study: One animal per polypropylene cage.

Main study: A total of 5 disease free Swiss female mice, were maintained in

regulated environmental conditions per polypropylene cage.

Randomization and numbering of animals

10 healthy female mice, acclimatized to laboratory conditions for 7 days prior to

dosing, were used in this study. Animals were randomly assigned to the cages and

the individual animal was marked with picric acid. The females were nulliparous and

nonpregnent.

Preparation of animals

The mice were deprived of feed for 15-18 hours before and 3 hours after the

administration of the test substance. Water was provided ad libitum.

3.7.5. Rationale for Selection of Albino Swiss mice as test system

1. One of the recommended rodent species by the regulatory authorities for

conducting preclinical toxicity studies among rodents, as it is a sensitive species for

expression of toxic responses.

2. This is because literature surveys of conventional LD50 tests show that usually

there is little difference in sensitivity between the sexes, but in those cases where

differences are observed, females are generally slightly more sensitive.

3.7.6. Route of administration and reason for choice

Oral route of administration is the proposed therapeutic route of administration in

human being.



69

3.7.7. Justification for selection of doses

Sighting study:

Dose

(mg/kg body weight)

No. of

animals

Mortality

0 1 0/1

5 1 0/1

50 1 0/1

300 1 0/1

2000 1 0/1

Results: Based on the sighting study following dose was selected for the main study.

Main study:

Dose

(mg/kg body weight)

No. of

animals

0 5

2000 5



70

3.7.8. Experimental procedure

The test substance, suspended in normal saline was administered to mice as per SOP.

Allocation of animals:

Sighting study:

Species

/strain

Group No. No. of

Animal

Dose

(mg/kg)

Concentration

(mg/ml)

Route

Mice/

Swiss Albino

Control 1 0 -

Oral I 1 5 0.5

II 1 50 5

III 1 300 30

IV 1 2000 200

Main study:

Species

/strain

Group No. No. of

Animal

Dose

(mg/kg)

Concentration

(mg/ml)

Route

Mice/

Swiss Albino

Control 5 0/N.S. 0

Oral I 5 2000 200



71

3.7.9. Observations

Clinical signs of intoxication: Observations of clinical signs were made at 0.5, 1, 2,

4 and 24 h after dosing on day 1 and once daily thereafter for 13 days at

approximately same time.

Cage side observations included mortality, changes in the skin, fur, eyes and mucous

membrane. It also included respiratory, circulatory, autonomic and central nervous

system and behavior pattern. Particular attention was directed to the observation of

tremors, convulsion, salivation, diarrhea, lethargy, sleep and coma.

Mortality- The mortality was checked twice daily.

Body weight- Individual animal body weight was recorded following the period of

fasting on day 0, weekly thereafter and at termination on day 14. Changes in the

body weights were calculated and recorded.

Gross necropsy- At the day of termination, all animals were sacrificed by CO2

inhalation. Following sacrifice a thorough gross necropsy of all organs were

performed on all animals.



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3.8 PHARMACOKINETIC PROFILING

Pharmacokinetics, sometimes abbreviated as PK, (from Ancient Greek pharmakon

"drug" and kinetikos "to do with motion") is a branch of Pharmacology dedicated to

the determination of the fate of substances administered externally to a living

organism. In practice this discipline is applied mainly to drug substances.

Pharmacokinetics includes the study of the mechanisms of absorption and

distribution of an administered drug, the rate at which a drug action begins and the

duration of the effect, the chemical changes of the substance in the body and the

effects and routes of excretion of the metabolites of the drug. In general it is the

study of what the body does to the drug and the measurement of drug in biological

fluids.

3.8.1 Development and validation of HPLC protocol for the determination of

SLPAR7 in mice plasma.

3.8.1.1 Instrumentation: Chromatographic analysis was performed on Shimadzu

HPLC system equipped with a diode array detector (SPD-M10AVP), solvent

delivery module (LC-10ATVP), online degasser (DGU-14A), an auto-injector (SIL-

10ADVP), flow channel system (FCV-14AH) and system controller (SCL-10AVP)

using a reversed-phase HPLC column (RP-18, 250×4.6 mm, 5µm particle size,

Sigma, USA). (Software for data analysis: VP V6.12 SP2).

3.8.1.2 Selection of columns (stationary phases) and mobile phases: On the basis

of physico-chemical properties of SLPAR7, various mobile phases were tested on

the lipophilic stationary phases for best possible resolution. The columns (stationary

phases) of various packing materials and particle sizes from different manufacturers

were used. The organic solvents like MeOH, acetonitrile (ACN) and isopropyl

alcohol (IPA) along with organic modifier such as acetate, citrate, and phosphate

buffers were investigated as mobile phase composition for their ability to resolve the



73

SLPAR7. The best resolution was using RP-18, 5μM Column and ACN: H2O; 60:40

as mobile phase. The same was used in the present study.

3.8.1.3 General: The solutions were prepared by weighing suitable quantity of

SLPAR7 using pre-calibrated analytical balance. The weighed quantity was

transferred to volumetric flask and solubilized using HPLC grade ACN. The HPLC

column was equilibrated with mobile phase composition for at least 2 h before start

of analysis. The external standard method was utilized for quantification.

3.8.1.4 Preparation of reference solutions: 50 mg of SLPAR7 was exactly weighed

and dissolved in 50 ml of ACN using sonication to achieve 1 mg/ml strength. 15 min

of sonication was done to facilitate homogenous mixing.

3.8.1.5 Preparation of calibration standards (CAL STD): The reference solution

of 1 mg/ml was diluted to make working solutions for CAL STD of 2, 10, 20, 50,

100, 200 and 400 μg/ml by diluting with ACN. Now, 50 μl of working solutions for

CAL STD (2, 10, 20, 50, 100, 200 and 400 μg/ml) were spiked in 950 μl of blank

plasma to achieve CAL STD of 0.1, 0.5, 1.0, 2.5, 5.0, 10.0 and 20.0 μg/ml

respectively.

3.8.1.6 Preparation of quality control standards (QC STD): The reference

solution of 1 mg/ml was diluted to make working solutions for QC STD of 4, 180

and 360 μg/ml by diluting with ACN. Now, 50 μl volume of these working solutions

(4, 180 and 360 μg/ml) were spiked in 950 μl of blank plasma to achieve QC STD of

0.2, 9.0 and 18.0 μg/ml respectively.

3.8.1.7 Preparation of system suitability standards (SS STD): The reference

solution of 1 mg/ml was diluted to prepare working solution for SS STD of 20 μg/ml

by dilution with ACN. Now, 50 μl of this working solution was mixed with 950 μl of

mobile phase (ACN: H2O; 60:40) to achieve SS STD of 1.0 μg/ml.



74

3.8.1.8 Recovery procedure: The Precipitaion technique was optimized for

recovery of SLPAR7 from plasma. The reference solution of 1 mg/ml was diluted to

make 100 and 200 µg/ml working stocks in ACN. 25 µl of the working stocks

mentioned above were spiked in 475 µl blank plasma samples to achieve 2.5 and 5

μg/ml strength respectively (Recovery Samples). 3 ml each of HPLC grade

Acetonitrile (ACN), Di-Chloro Methane (DCM) and Ethyl Acetate (EA) were added

using micro-pipettes and the samples were vortexed for 2 min., centrifuged (5000

rpm, 10 min) and supernatant collected. The separated organic layer was allowed to

dry using solvent evaporator (Thermo Electron Corporation, USA). The dry samples

thus obtained were reconstituted in 300 µl mobile phase, filtered through 0.45 µm

syringe filters (Millipore, USA) and injected into HPLC system for analyzing

SLPAR7. Finally ACN was selected for further studies because of better recovery of

SLPAR7 in ACN as compared to DCM and EA.

3.8.1.9 Validation: The method was validated in accordance with guidelines of

International Conference on Harmonization (ICH). The parameters assessed were

linearity, range, accuracy, precision, specificity, limit of quantitation and robustness.

A. System suitability: The SS STD (1 µg/ml) were utilized for the test. The system

suitability test was performed using nine replicate injections before analysis of

samples. The acceptance parameters were less than 0.5 % and 1.5 % relative

standard deviation (R.S.D.) for retention time and peak area respectively along

with more than 3500 theoretical plates.

B. Recovery: The spiked plasma samples (Recovery Samples) of SLPAR7 were

analyzed and the peak area obtained for each sample was fitted mathematically

into amount vs. peak area co-relation.

C. Linearity and range: Seven point calibration curves were constructed using

CAL STD over a pre-defined conc. range of SLPAR7. The peak areas vs. conc.



75

plots were subjected to linear least square regression analysis. The intra and

inter-day linearity was established.

D. Accuracy and precision: The accuracy and precision of method was estimated

by analyzing QC STD. The intra and inter-day accuracy was established by

evaluating nominal and mean measured conc. of QC STD which were compared

and expressed as difference% (diff.%). The diff.% was calculated by using

following formula. Diff.% = [(Mean measured conc. - Nominal conc.)/Nominal

conc.] x 100. The intra and inter-day precision was established by analyzing nine

replicates each of 3 QC STD at 3 different time intervals in a day and on three

consecutive days respectively. It was expressed in terms of % RSD.

E. Limit of quantitation (LOQ): The lowest conc. of calibration curves with

acceptable accuracy and precision were reported as LOQ for the analyte.

Further, it was confirmed by signal to noise (S/N) ratio values. The signal 3

times noise value was treated as limit of detection (LOD) and 9 times noise

value as LOQ.

F. Robustness: The robustness of both the methods was evaluated by analyzing

QC STD after varying the mobile phase composition. The ACN volume in total

mobile phase was modified in between 55-65 %. The acceptance criteria were

less than 2 % variation in the final results after modification in mobile phase

composition.

G. Specificity: The specificity was assessed using spiked plasma samples. The

SLPAR7 working solutions were spiked into plasma samples so as to check

plasma artifacts interference in SLPAR7 estimation.

3.8.2 Pharmacokinetic study of SLPAR7 in mice plasma



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3.8.2.1 Preparation of drug solution for i.p. administration: 20 mg of SLPAR7

was weighed accurately by using analytical balance and was transferred to glass

mortar and pestle. Normal saline with gum acacia (not more than 2%) was added as

suspending agent. The weighed material was triturated and aqueous fine suspension

was prepared.

3.8.2.2 Storage: Solution was prepared fresh on each day of experiment, and stored

in amber colored glass vial (20 ml capacity) away from direct light exposure.

3.8.2.3 Procedure for blood samples collection: The animals were marked with

marking solution (5 % picric acid) for individual identification, and weights were

recorded using an animal weighing balance. The required volumes of drug solution

was administered intraperitonealy using syringe and needle. The exact time of test

compound administration was noted. Blood samples (500 µl) were collected in

heparinized glass tubes via jugular vein at pre-defined time intervals, from individual

animal. The blood samples were centrifuged at 5000 rpm for 10 min and the plasma

was collected. The plasma samples (250 µl each) were processed for the recovery of

drug(s) according to pre-optimized procedure.

3.8.2.4 HPLC analysis: The recovered and dry samples were reconstituted in mobile

phase (300 µl), filtered through 0.45 µm syringe filter and analyzed for SLPAR7 by

HPLC.

3.9 STATISTICAL ANALYSIS

Data are presented as mean ± S.D. of the number of experiments indicated. For both

in-vitro and in-vivo experiments comparisons were made between control and treated

groups unless otherwise indicated using unpaired Student’s t-test and p values < 0.01

was considered significant.

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