CHAPTER I1

MATERIALS AND METHODS

CHAPTER - 2

MATERIALS AND METHODS

A. MATERIALS

I. Test Compounds

1. Iscador - M (3%, Batch No. ch-B50611).

Iscador an extract of the semiparasitic plant Viscum alburl~ was

purchased from AG. Arlesheim, Switzerland.

2. Curcumin

Cur umin the colouring material of Turmeric was gifted by Kancor

Ltd. Angamaly, Kerala.

Curcumin l i~osome re war at ion (137)

Phosphatidyl Choline - 315mg

Cholesterol - 40.6mg

Curcumin - 35mg

Dissolve in Chloroform: methanol - 17.5m1 (3 : l )

Evaporated using a vaccum rotor evaporator and unilamellar

liposome of curcumin suspended in 17.5ml PBS.

3. Piper longltr71

Dried powder of the above plants were purchased from Amala

Ayurvedic Pharmacy, Thrissur.

11. Chemicals

Dulbeco's modified Eagle medium 1 Eagles minimum essential medium

(MEMI Rosewell Park Memorial Institute -Himedia

Medium (RPMI- 1640) laboratories

Hanks Balanced salt solution Mumbai, Fluid thioglycollate medium India. Trypsin S o d i m Caseinate

Foetal calf serum - Biological Industries Kibbutz, Bethhaemek, Israel.

Concanavalin A - Sigma Chemicals, USA

Collagen Type I - Sigma, gifted by Dr. Muralee Nair

Phy tohaemaglutinin Lipopoly sacharide Difco laboratories, USA Freunds complete adjuvant

Hepes Buffer Acrylamide Bis-acrylamide 1 - Sisco Research PPO Laboratories, Murnbai. POPOP

'H - thymidine Board of radiation and Sodium chromate - and Isotope technology (Nn, CrSZO,) BARC, Mumbai

Anti-asialo GM I antibody - Accurate chemical and Scientific corporation, New York.

Crystal violet, Trypan blue - Romali, Mumbai

Coomasie brilliant blue paraffin wax Eosin Leishman's stain ].- Hydroxyproline -

Sialic acid

Alpha Naphthyl Acetate Pararosaniline

Cyclophosphamide -

Nitrocellulose filters -

Polycarbonate filters

Diagnostic, Research and Reagent kits

a) Drabkins reagent -

b) Folin Ciocalta~l -

C) Gamma glutamy 1 transferase -

E. Merck, India

Gifted by Dr.K.Balasubramaniam. Welcome Research Lab, CMC, Vellore, Tamilnadu.

Gifted by Dr. A.S. Balasubrarnaniam Department of Neurological Sciences, CMC, Vellore.

Loba chemie, Mumbai.

Khandelwal Industries Mumbai.

0.22nm, 13nm diameter pore size.

Millipore pvt. Ltd

~ F M poresize, 13nm diameter

Costar Co, Canihridge MA, USA.

Ethnor diagnostics, Mumbai.

Sisco research laboratories Mumbai.

AVT diagnostics, Madras.

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.

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d) IFN - y 1 I

IL - 2 1

I GM -CSF ELISA kits

TNF - a Endogen, USA

ICAM- I

I11 Reagents

a) Phospate Buffered Saline (PBS)

NaCl - 8.00g

KC1 - 0.20g

Na,H P0,.2H,O - 1.44g

KH, PO, - 0.20g

Distilled Water - lOOOml

pH was adjusted to 7.2 with 1 N HC1 or NaOH.

b) PBS - EDTA Soluuon

EDTA - 20mg

PBS - 1OOml

C) Trypsin solution

trypsin - 200mg

glucose - 20n1g

PBS-EDTA - IOOml

d) Alsever's solution

Dextrose - 2.05g

Sodium citrate - 0.80g

NaCl - 0.42g

Distilled water - 100ml

pH adjusted to 6.1 with 10% Citric acid.

&

.

e) Neutral Buffered formalin solution

Na,H PO,. 2H,O - 16.378

NaH PO,. H,O - lOOmg

Formalin lOOml

Distilled water - 900ml

Stains

f) Trypan blue

Trypan blue - lOOmg

Saline (0.9%) - 1 OOml

g) Eosin

Eosin Y - 500mg

Ethanol - 1OOml (Final volume)

Eosin was dissolved in 5ml of distilled water and made upto lOOml with ethanol.

h) Harris haema!oxylin

Haematoxylin - 5g

Ethyl alcohol - 50ml

Potassium alum - 50mg

Potassium iodide - 50mg

Distilled water - 950ml.

Haematoxylin was dissolved in alcohol using gentle heat. The alum.

was dissolved in distilled water by heating with frequent stirring and

kept overnight at 4OC. Alcoholic haematoxylin was added to the alum

solution. The mixture was cooled and potassium was added and filtered.

i ) Crystal violet

Crystal violet - 50mg

Methanol - 20ml

Distilled water - 80ml

3 3

J i Scintillation Fluid

PPO - 2.5gm

Naphthalene - lOOgm

Dioxan - 1 OOOml

li IV Animals

Balblc mice and C57BLl6 mice (4-6 weeks old) were purchased I ~ from National Institute of Nutrition, Hyderabad.

Il V Cell lines

L929 (Mouse lung fibroblast) cells, K562 ( leukemic cells) and

B16F-10 (metastatic melanoma) were obtained from National Centre

for cell science, Pune India.

Daltons lymphoma ascites (DLA) cells (arose as a spontaneous carcinoma

of thymus) were obtained from Cancer Institute, Adayar. Ehrlich ascites

tumour (EAT) cells were obtained from Cancer Institute, Mumbai. Both

the cells were maintained as ascites tumour in Swiss albino mice.

VI Instruments

1 . Blind well chamber

(Modified Boyden chamber) - Nuclepore, Cambridge, USA.

2. Cooling Centrifuge - Remi, Chennai

3. CO, Incubator - Napco, Canada

4. Disc electroplioresis tank - Balaji Scientific Services, Madras.

5. ELISA reader - Stat fax

6. Gamma ray spectrometer

7. Inverted microscope

8. Laminar flow hood

9. Liquid scintillation counter

10. Research microscope

11. Rotary microtome (radical)

12. Sonicator

13. Spectro Photometer

14. Tissue hon~ogenizer

- ECI, India.

- Wilovert, Germany

- Klenzaids Contamination control Pvt. Ltd. Gujarat.

- Rack Beta, LKB, Walac

- Meiji, Japan

- Lab agencies, Kerala.

- Labline instruments, Illinois, USA.

- SL 150, ELICO Pvt. Ltd. India.

- York Scientific Industries, Delhi.

B. METHODS

I. Tissue Culture

1.a. sterilization of glass wares

All glass wares and filtration apparatus used for tissue culture

purposes, were soaked in a solution of Extran (1%) overnight, cleaned

using brush and washed thoroughly under running water. All the glass

wares were rinsed in distilled water and oven dried. These were then

autoclaved at a pressure of 15 poundslsquare inch for 15 minutes, dried

and used for experiments.

1.b. Preparation of culture media:

DMEM (9.98 gm/L), MEM (10.3glL) and RPMI (lO.3gmlL) were

prepared in double distilled water, pH adjusted to 7.2, and filtered under

positive pressure using a 0.22pm cellulose filter. Sterility of the medium

was tested using fluid thioglycollati. medium. For this lOml sterile

thioglycollale (929.8g/L) medium was inoculated with lml of medium

prepared and incubated at 37OC for 6 days, and checked for visible

contanlination. Antibiotics - Penicillin (1001U/ml) and streptomycin

(100mglml) were added to the medium prior to use.

1.c. Maintenance of cell lines in tissue culture:

1. L-929 cells ( I 13)

The spent medium was removed from the confluent bottles and the

cells were washed thrice with 2ml of PBS-EDTA. Iml of trypsin solution

containing 0.02%. EDTA was added and incubated for 3-4 minutes, at

37OC and the bottles were tapped to dislodge the cells. MEM (5ml)

containing 10% goat serum and antibiotics (complete medium) was

added. Cells were dispersed to single cell suspension by repeated pipetting

and an aliquot of cell suspension was added to fresh tissue culture bottles

containing l0ml of complete medium and incubated at 37OC. Cells were

subcultured every week.

2. K-562 cells (1 13)

The cell suspension was mixed well, dispersed the clumps by

repeated pipetting. The cells were counted and IXIOh cells were seeded

to fresh bottles containing lOml of RPMI-1640 with 10% FCS and

incubated at 37OC. The cells were subcultured every third day.

3. B 16F-10 melanoma cells ( 1 13)

The spent medium was removed from confluent bottles and the

cells were washed three times with PBS. One ml trypsin solution free of

EDTA was added and incubated for 3-4 minutes at 37 '~ . Bottles were

thcrl tapped to dislodge the cells.DMEM (5ml) containing 10% FCS and

f l 3

antibiotics (complete medium) was added and cells were dispersed to

i single cell suspension by repeated pipetting An aliquote of the cell 1 I

1 I suspension was added to fresh culture bottles containing lOml of complete i I I I medium and incubated at 37°C. The cells were subcultured \,cry week. ~

1

i 11. Maintenance of Experimental Animals. i

I

C57BL16 mice, and Balblc mice were used for the experiments.

They were housed in ventillated cages and fed with pelletted mouse chow

(Lipron, India) and water ad libitum.

I 111. Maintenance of tumours in animals. I

DLA and EAT cells were propagated in the peritoneal cavity of !

swiss albino mice and maintained by transplanting the cells every two I I

I weeks. Tumour cells were aspirated from the peritoneal cavity, washed

I with PBS and 1 x lob cells were injected, intraperitoneally to induce ascites I tumour.

B16F-10 melanoma cells were propagated in C57BLl6 mice as

transplantable solid tumours. 1x106 cells were injected subcutaneously

I to the hind limb of the mice. After 10-15 days, the animal was sacrificed, ~ tumour mass mashed and processed in PBS. lx106 viable cells were

I injected to another set of animals.

I IV. Preparation of B16F-10 cells for in vivo studies. 1 ! For in v i v o studies turnour mass from animal was resected and

I

I

I processed to get a single cell suspension. Tumour mass was mashed and

the pieces were forced through a fine steel mesh using PBS. The cells I were separated from RBC's and then suspended in PBS to required cell I I I

number and used for in vivo experiments after determining cell viability ! (114). I I I I \ .- i /

V. Preparation of cells for in vitro studies.

a. B I6F-I0 cells

The tumour cells were harvested from subconfluent cultures (50-

80% confluency). The spent medium was removed, the monolayers were

washed three times with PBS and the cells were harvested by mechanical

dissociation using a rubber police man. The cell concentration was

adjusted to required number. Cell suspensions with 90% viability were

used for in virro experiments.

b. L929 cells.

The spent medium was removed, the monolayers were washed,

three times with PBS and the cells were layered by lml of trypsin

conkning EDTA, and incubated at 37OC for 3-4 minutes. Cells were

dislodged by tapping the bottle and a single cell suspension was prepared

by adding lOml MEM (complete medium) and repeated pipetting. Cell

viability checked by trypan blue dye exclusion method(ll5) and used

for experiment.

VI. Determination of cell viability

Cell viability was determined by trypan blue dye exclusion

method(ll5). lml of cell suspension was mixed with O.lml of 1% trypan

blue, kept for 2-3 iiiinutes and loaded on a haemocytometer. Viable cells

exclude trypan blue dye, while non-viabile cells take up the dye and thus

appeared blue in colour. The number of stained and unstained cells were

counted separately.

'30 Dead cells = No. of dead cells x 100 No. of viable cells + No. of dead cells

VII. I n vitro cytotoxicity studies

Tumour cells (1x106) were added to test tubes containing various

conc. of test compounds and the final volume was made upto lml with

PBS. Control tubes contained tumour cells only. The assay tubes were

then incubated at 37OC for 3h, and the percentage of dead cells were

determined by trypan blue dye exclusion method.

VIII.Long term ill vitro cytotoxicity studies by tissue culture.

B16F-10 melanoma cells growing in log phase was used for this

experiment. Cells were collected by trypsinization and 50,000 cells were

seeded into tissue culture bottles containing lOml complete medium and

incubated at 3 7 T . After 24 hours various concentrations of the test

compounds were added to the bottles and incubated for six more days.

After incubation period viable cells were collected by trypsinization and

counted using a haemocytometer.

% viable cells = TIC x 100

Where 'T' is the number of cells in the treated bottles and 'C' the

number of cells in control bottles.

IX. Hematological Parameters

1 . Determination of Hemoglobin (1 16)

Princiole

Ferricyanide forms methaemoglobin with hemoglobin which is

co~:..erted to cyanmethaemoglobin by cyanide which has an absorption

at 540nm(l16).

I

.......... - .... I

I Procedure 1

!

0.02ml of blood was mixed with 5ml of Drabkin's reagent and I I I 1 allowed to stand lor 5 minutes Optical density (0.D) was measured

against a reagent blank. ! Calculations

i GmYc of Hb = 0.D of the test x 251 x Con, of Std I

0 . D of the Std. 1000 i ~ 2. Determination of total count of leukocyte.

I Princiole 1 I

i The cells were diluted in Turk's fluid which contains a weak acid

to lyse RBC and a stain for staining the cells. The number of cells in the I I I

large four corner squares were counted ( I 16). i !

Procedure

I Blood (0.02ml) diluted in 0.38ml of Turk's fluid and loaded onto I I the counting chamber. The cells were allowed to settle at the bottom of

the chamber for 2 minutes and counted under a microscope using lox

objective.

Calculations

I To~al leukocyte counts/mm3 = cells counted x 20 x I Q ! ~ i 4

I 3. Differential count of leukocytes (1 16)

1 Procedure

A thin film of blood was made by spreading a drop of blood evenly I

I ! across a clean glass slide using a glass spreader. Few drops of Leishman's

i \-

I

I

htain \vas poul-cd OYCI- thc slide and kept for 3mts. The stain was dilutecl

with distilled water and kept for IOmts, washed in tap water and allowed

to air dry. Various types of cells were scored using the morphology under

oil immersion using IOOx objective and a total of 100 cells were counted.

X. Immunological Parameters

I . General Techniques

a. Collection and preparation of SRBC ( 1 17)

Sheep blood was freshly collected from the slaughters house in

equal volumes of Alsever's solution and stored at 4OC for not more than

one week. Cells were washed three times with PBS (pH 7.2), supernatant

discarded and the pellet was suspended in Hanks Balanced Salt

SolutioniHBSS).

b. Trysinization of SRBC.

10 parts of 4% SRBC and one part of 1% trypsin solution were

incubated at 37% for 30mts. After incubation the cells were washed twice

in PBS (pH 7.2) and resuspended at a concentration of 2%.

c. Preparation of anti SRBC antibody.

A young healthy rabbit was injected intradermally with 2% SRBC

i n saline which was niixed with Freunds complete adjuvant in a ratio of

I %. A booster dose was given four weeks after the initial dose. Next day

after the booster dose animal was bled and serum separated, heat

inactivated and checked the antibody titre. According to the antibody

titre value, serum was diluted and used for the experiments.

d. PrepcLration of spleen cells

All the procedures were done under sterile conditions. The animals

(mice) were sacrificed, the skin was cleaned with rectified spirit. An

incision was made on the left side just below the last rib and spleen was

removed without any adherent tissues.

Spleen was cut into small pieces and teased over a stainless steel

mesh in cold PBS or HBSS. Clumps were allowed to settle in a centrifuge

tube. kept in ice for 2 minutes. Supernatant was collected, washed three

times in HBSS and resuspended in RPMI-1640 at required concentrations.

I e. Preparation of bone-marrow cells

All the procedures were done under sterile conditions. Mice were

sacrificed by cervical dislocation and fixed on a board with fore and

hind limbs fully stretched. The skin and flesh overlying the limbs were

removed and the femur was exposed. The shaft of the femur was separated

from both ends and the bone-marrow was flushed out of the cavity by

passing a jet of medium or HBSS with 2% FCS through one end of the

shaft using a 26G needle and syringe. Flushed bone-marrow was made

into a single cell suspension by repeated pipetting. It was then centrifuged

and suspended at required cell concentrations in RPMI-1640.

f . Preparat~on of thymus cells

All the procedures were done under sterile conditions. Animals

were sacrificed by cervical dislocation. The slun was cleaned and body

was incised at the upper part above the heart. Thymus is a bilobed structure

and was detached, suspended in HBSS, and processed the same way for

spleen and the thyniocytes were suspended in RPMI-1640 containing

10% foetal c;di seru~n. I ~1

g. Preparatioll of peritoneal macrophages.

Peritoneal macrophages were elicited by injecting 0.2ml of 5%

sodium caseinate solution. After five days animals were sacrificed by I

cervial dislocation. : i l l the processes \\,ere done under sterile conditions.

Mice were fixed on a board, skin was removed and the peritoneum was

exposed. The peritoneal cavity was distended by injecting 5ml of RPMI-

1640. i'eritoneal cavity was gently prodded and the peritoneal fluid which

contained macrophages was aspirated. The cells were washed and

suspended in RPMI-1640 to the desired cell concentration.

2. Determination of Circulating Antibody Titre

Principle

The non-agglutinated SRBC will settle to the bottom of the well as ;,

a clear "button" while agglutinated cells settle as a diffuse "mat". The

maximum dilution of anti-sera at which clear agglutination observed 1

gives the titre of the antibody ( 1 18).

Procedure

0.05ml of antisera was serially diluted in round bottom 96-well

plates in JIBS (pH 7.2). 0. lml of trypsinized SRBC was addcd and final

volume was adjusted to 0.2ml using PBS and incubated at room

termperature for 3h. The dilution at which clear agglutination seen was

noted.

3. Detei-mination of Antibody Forming Cclls

Princiole

Antibodies produced by the lymphoid cells from animals

immunized with SRBC cause lysis of red cells in its vicinity (plaques) in

a semi-solid support in the presence of complement (119).

d . ~ Procedure

0.5ml of agarose (0.5%) was distributed into tubes kept at 45°C

and 0.0Sml SRBC (756) and 0.05ml spleen cells (8x10' cellslml) were

added and mixed well. The contents were poured over a glass slide, I

spread in an area of 10cm' and the gel was allowed to solidify. The

I slides were kept in an incubation rack filled with fresh rabbit serum i I I ( 1 : 10 diluted in PBS, pH 7.2) as a source fo complement and incubated

for lh at 37OC. The number of plaques were counted using a colony ! !

counter and represented as plaque forming cells per lob spleen cells. I

4. Assay for lymphocyte, thymus cell and bonemorrow cell I ! I

i proliferation.

1 Principle i I Mitogens can stimulate restiny lymphocytes to undergo a series of I

biochenlical and physiological changes and are converted to blast - like ' I I

i ! cells. This process leads to cell division and can be quantitated by 'H i

I !

th) midine uptake method (120). I

Procedure ~ I ~

All the techniques were sterile during the experiment. Spleen cells

/ thymus cells/ bonemorrow cell (106/ml) were incubated with and without I

i ! mitovens in a final volume, 3 ml of RPMI - 1640 medium supplemented 1

with 10%. FCS and antibiotics in a humidified atmosphere containing

5% CO, at 37°C for 48h. The concentration of mitogens added were

PHA 6 pglml, 3pglml and Con A 10 pglml, 5 pglml. 2 pci of 3H thymidine

~ 1

was added to each vial and incubated further for 18h under the same

conditions. The cultures were centrifiged at 1500 rpm for 10 minutes , I !

supernatant discarded and the pellets were precipitated twice with 2 ~rl I 1 i

1

f l

cold 1 0 7 ~ TCA. Pellets were dissolved in 0.5 ml of 0.5 N Na OH and

0.3 ml was transfered to loml scintill~tion fluid, kept overnight in dark.

Counts per minute (CPM) was measured in a liquid scitillation Counter

(Rack Beta, LKB).

Calculations

Transformation index = Mean CPM of transformed Mean CPM of control

5. "Cr-release assays

Cr- release assay was used to determine the cytotoxicity mediated

by immune effector cellls such as natural killer cells and cells expressing

Fc receptors (ADCC) and was performed in round bottom titre plates

(121).

Princiule

51Cr binds to cytoplasmic proteins after diffusing through the cell

membrane and is released only when the cell membrane is sufficiently

damaged.

Labelling of target cells

The traget cells K562 (lo6) and SRBC (lo7) were washed twice in

RPMl - 1640 and were resuspended in few drops of FCS. 100 pci of

Na,"Cr 0, was added and incubated at 37OC for 1 h on a shaker. The

cells were washed in medium twice and allowed to incubate in larger

volumes (5ml) of rilediurn for 30mts at 4°C. Cells were washed twice in

medium and resuspended in complete medium at a concentration of 1x10'

cellsiml.

5. a. Determination of Natural killer cell - mediated cytotoxicity. ~ Labelled target cells (K562,O. 1 ml) and equal volumes of various

i dilutions of spleen cells (to yield effector : target ratios of 100:1, 50.1

and 25: 1 ) were added to 96 well round bottome titre plates. Final volume

was adjusted to 0.2 ml with RPMI-1640 supplemented with 10% FCS

and incubated at 37°C for 4h. Titre plates were centrifugedfor 15 minutes,

supernatant (100bl) collected and radioactivity measured in a gamma

ray spectro meter.

The following control tubes were kept along with each experiment.

Spontaneous release (SR) - wells contained only target cells and i !

medium ~ Total release ( I 'R) - wells contained target cells, medium and 0.1

ml of IN HCI. i

Calculations

%Lysis = Ex~erimental release-spontaneous release X 100 Total release - Spontaneous release

b. Determination of antibody - dependent cellular cytotoxicity.

0 . l m l of labelled SRBC(target cells) and 0 . l m l of spleen

cells(effector cells) were added to get an effector target ratios of 100: 1 ,

50:1, and 25: l .

0.05ml of anti-sera against SRBC was added and incubated at 37°C

for 4h. The final volume was made upto 0.2ml with complc~e medium.

0.lml of the supernatant was counted and percenl lysis was

calculated as above controls.

Spontaneous release (SR) - the tubes contained labelled cells,

antibody and medium.

Total release (TR) - the tubes contained labelled cells and O.lml of I

46

P I 1

6 . Determination of antibody - dependent complement - mediated 1 1 I

cytotoxicity. i

I

Princiole

When tumour cells are incubated with specific antibodies in

presence of complement, the classical pathway will be activated leading

to the lysis of target cells (119).

Procedure

Antiserum was diluted in RPMI-1640 to get 1:1, 1:2 and 1:4

dilutions of the antibody and O.lml of the serum was mixed with loh

A cells.

0.05ml of 1: 10 diluted fresh rabbit serum as a source of complement

was added and the final volume was made upto 2ml and incubated at

37OC for 3h. The cells were centrifuged and lml of the supernatant

discm-.led and cytotoxicity was assessed by trypan blue exclusion method

( 1 15).

7. Determination of Macrophage-mediated cytotoxicity

Princiule

Activated macrophages can mediate cytotoxicity towards tumour

cell lines such as L-929 cells which can be assessed by checking the

morphology of tumour cells after staining with crystal violet (122).

Procedure ll 0. lml of L-929 cell suspension (5x103 cellslwell) was cultured in

microtitre plates. Cells suspended in MEM containing 10% goat serum

and antibiotics were incubated at 37OC in 5% CO, atmosphere.

47 n* P

I

Peritoneal r~lacrophages were elicited by injecting 0.2ml, 5% of I !

sodium caseinate intraperitoneally and were harvested on fifth day as I

described earlier. 1 I

Lung macrophages were collected by mashing the lungs and forcing

the tissue through a steel mesh using medium. Cells were collected and

incubated at 37OC in petridishes for lh in CO, atmosphere. Unattached

cells were removed by washing with PBS and attached cells which are / macrophages were collected using a rubber policeman. Cells were

counted and suspended in required concentrations. Macrophage

suspension (2 .5~10 ' cells/ml) was added to each well and incubated for

i' 48h at 37OC in CO, atmosphere.

In order to check the effect on theTNF production by macrophages, 1 I

macrophages (2 .5~10" cells/well) were incubated at 37OC for 24h in i presence of test compounds alone. Supernatant collected after 24h was

added to wells containing L 929 cells and incubated for 48h under the

same conditions. After incubation medium was removed, cells were

washed with PBS (37OC), fixed by 5% formalin and stained using 0.05%

crystal violet in 20% methanol (20minutes each). Excess stain was washed

using PBS and morphology checked under an inverted microscope.

8. Determination of phagocytic activity ( 117)

Principle

Macrophages when activated can engulf the target cells more

effectively than normal macrophages and other phagocytic cells.

Procedure

Peritoneal macrophages were elicited with sodium caseinate (0.21nl,

5%) in Balblc mice. Macrophages were harvested on fifth day, washed

and incubated with opsonized SRBC (SRBC pre-treated with anti SRBC

antibody) on glass slides for l h at 37OC in CO, atmosphere. The slides

were then washed with PBS, stained with Giemsa stain and examined

under microscope using oil emulsion.

9. Determination of Delayed type hypersensitivity (123)

Princinle

An individual when immunized with an antigen and again injected

with the same antigen later will show a hypersensitivity reaction that

developes slowly.

Procedure

Animals were immunized with SRBC (lxlOR cells in O.lml) by

injecting intraperitoneally. On 5th day animals were given a challenging

dose of SRBC (1xI0%cells in 2Oml) in the left hind foot pad. Thickness

of the paw was measured before challenge and 24h. after challenging

dose. The difference will indicate (increase in the thickness of foot pad)

delayed type hypersensitivity reaction.

10. Determination of a-naphthyl acetate esterase activity(l24).

Principle

'The enzyme hydrolyses the substrate to form an invisible primary

reaction product. The complex is coupled with the diazonium salt to

produce coloured final reaction product.

Procedure

Animals were sacrificed by cervical dislocation. Skin and flesh

removed from the thigh of the animal. Bone marrow cells were flushed

in PBS with 10% goat serum using a syringe. Cells were counted and

thin smears prepared on glass slides. The smear was air dried and fixed

using 37% formaldehyde. Slides were incubated in a reaction buffer

containing pararosaniline, sodium nitrate and a-naphthyl esterase at room

temperature. Smear slides were counter stained with hematoxylin for 2

minutes. a-esterase positive cells take up a yellowish brown colour and

cells were counted under microscope using oil emulsion.

XI ANTIMETASTATIC STUDIES

1. Determination of the invasion of B16F-10 melanoma cells in

animal model.

Studies on the invasive property of tumour cells (in vivo) were

done in C57BLl6 mice. Pulmonary colony forming ability of B 16F- 10 1

cells were carried out as described by Fidler (125). C57BLI6 mice were

injected with B 16F- 10 cells (lx106) through the lateral tail vein. Animals

were sacrificed on 21st day. Metatasis of lungs were determined by a)

counting the metatatic focii on the surface of the lungs. b) by measuring I

biochemical parameters such as lung collagen hydroxyproline, serum

sialic acid, and serum y-glutamyl transferase c) histo pathological analysis

of lurizs f) by determining the rate of survival.

1.a. Biochemical parameters

Estimation of Protein

Princiole

This assay relies on the formation of protein copper complex and

reduction of phosphomolybdate-phosphotungstate reagent (Folin

Ciocaltau reagent) by tyrosine and tyrosine and tryptophan residues of

protein ( 126).

Reagents

solution A

1. Sodium potassium tartarate - Iml(2%)

CuSO, - l m l ( l % )

Na,CO, - 98m1(2% in 0.1N NaOH)

Solution B

Folins phenol reagent - Diluted 1: l with distilled water

Procedure

'5 20pl sample and different concentrations of standard BSA

(150pg, 100pg, 50pg, 25pg) were made upto 1.2ml with distilled water

6ml solution A was added. Incubated at room temperature for 10 minutes. Iacqbaturl c& Ywm fCmpcl~'4fu*r & 20 ~ b .

Vortex mixed the reaction mixture and 300p1 solution B added"0ptical

density read at 660nrn.

Estimation of Hvdroxvuroline (1271

Principle

Hydroxyproline present in samples were oxidised by chlorarnin T.

The coloured product is more stable in the presence of high concentration

of isopronanol.

1. Oxidant solution

Sodium acetate - 5.7g

Trisodium citrate - 3.758

Citric acid -

Isoproy jnol - 38.5m1

Distilled water - 61.5ml

I 2. Erllch Reagent A

P-dimethyl amino - 4.4g

benzaldehy de

Perchloric acid - 10.2g (60%)

Isopropanol - 25ml (final volume)

3. Chloramin T - 1.75g125ml oxidant solution

prepared on the day of use).

Procedure

20p1 of the sample was diluted to 3ml using Isopropanol and to

this lml of oxidant solution was added. After 4 minutes, 2ml Erlich A

reagent was added, and the reaction mixture was heated for 21 minutes

at 60°C. After one hour optical density was recorded at 562nm using a

spectro photometer.

I Protein bound serum sialic acid estimation (128)

Principle

Acid hydrolysis of serum for liberation of sialic acid forms a

coloured compound with thiobarbituric acid.

Reagents

1. HZSO, - 0.2N

2. Periodic acid - 25pM in 62.5mM H,S04

3. Sodium arsenite - 0.2% in 0.5M HC1

I

: I I

- -

f r 3

I I ! I

4. Thiobarhituric acid - 6% (pH 9) ! ! I

5. Dimethyl sulphoxide - (DMSO) !

I Procedure 1 ! I

1

1 20011 of sample was mixed with equal volume of 0.2N H,SO, and I ! hydrolyzed for lh at 80°C. To this hydrolysate 50ml periodic a c i d ( 2 5 ~ M )

i was added and incubated for 30 minutes at 37OC. To this reaction mixture I

i I 5 0 ~ 1 of sodium arsenite was added, followed by 1 0 0 ~ 1 of thiobarbituric i

i acid and was heated in a boiling water bath for 7.5 minutes. After heating 1

~ !

40011 of DMSO was added to intensify the colour and read at 549nm 1

I

! and 532nm. ! I

Estimation of Gamma glutamvl transferase ( 1 29)

1 Princiule

p-nitro aniline ---t, y-glutamyl-p-nitroanilide + acceptor - y-glutamyl acceptor + .

: p-nitro aniline.

Reagents

I L-y-glutamy I-p-nitroanilide - 2.5mM

I glycyl glycine - 20mM

: Tris-HCl (pH.8) - 0.05M

NaCl - 75mM

A,

i

Gamma glutamyl transpeptidase catalyzes the transfer of y-glutamyl

moiety of a y-glutamyl donor to a variety of acceptors.

2y (Glutamyl-p-nitroanilide)

1 y-glutamyl-y-glutamyl-p-nitroanilide+

Procedure

The standard assay mixture contained (lml) 0.05M Tris HC1.75mM

NaC1, 2.3mM-L-y-glutamyl-p-nitroanilide and 20mM glycyl glycine

along with 25p1 sample. The rate of release of p-nitroaniline wah

measured at an optical density of 410nm using a spectrophotometer.

I .b. tIistopathologica1 Analysis

The tissueb as soon as they are removed were fixed in 10% neutral

formalin for at least 4h. The tissues were dehydrated in alcohol series.

cleared in xylene and embedded in paraffin. About 5-6pm thick sections

were taken on a glass slide and stained with hematoxylin and eosin and

visualized under the microscope for histological changes ( ' 30.

1 .c. Determination of the rate of survival.

Animals were injected with B 16F-10 melanoma cells (IxlOh)

intravenously. The mortality of animals was noted and the percentage

increase in lifespan (%ILS) was calculated from the formula % ILS = T-

CICx100 where 'T' is the number of days drug treated animals survived

and 'C' the number of days control animals survived.

2. Adoptive Imrnunothera~v In TUrnour Bearing Animals

Princi~le

Adoptive immunotherapy is based on the observations that

lymphocytes activated by interleukin-2 proliferate vigourously and at

the same time develop into very potent killer cells capable of lysing a

large variety of malignant cells while sparing normal cells (131).

f B i I

I 1

Procedure

1 a) In vivo activation of splenocytes: Balblc mice were treated

with non toxic concentration of test compound for five d a y Aher the I !

i

fifth day animals were sacrificed and the spleen was processed to single

cell suspension.

b) In virro activation of splenocytes: Spleen cells were cultured

in presence of non toxic concentration of the test compound for 48h in

RPMI-1640 medium supplemented with 10% FCS and antibiotics. After I

the incubation cells were washed with PBS and suspended as 1x10' cells/

ml. The activated splenocytes(l0%ells in 0.lml) were injected into

turnour bearing animals through the lateral tail vein.

~ The animals were sacrificed after 21 days of tumour cell inoculation

and the effect of the adoptive immunotherapy was assessed from tumour

nodule counts, biochemical parameters and survival of the tumour bearing

animals as mentioned above.

3. In vitro antimetastatic studies

3.~1. Collagen matrix invasion assay.

Invasion of collagen matrix by tumour cells was carried out using

modified, Boy den chambers (Blind well chambers) as described by Albini

ri ul (144). The lower compartment of the chamber was filled with

DMEM without FC:; and the nitrocellulose filters were placed above

this. Poly carbonate filters coated with 25pg collagen type I were placed

above nitrbiellulose filters in Blind well chamber. B 16F-10 cells (1x10'

1 /.lSm! were prepared and suspended in DMEM without F C S The cell

1 suspension was added to the upper chamber and incubated at 3 7 T for ~ 1

A J

i I

i i

12h in a 5% CO, atmosphere. After the incubation period medium from

the upper chamber was removed and the cells on the upper side of the

filters were removed by a cotton swab.The filters were then fixed in

methanol for 1 minute and stained ( I minute) with crystal violet. Cells

that penetrated the polycarbonate filters were counted in 10 fields under

i

a microscope. Results were calculated as percentage inhibition of invasion

using the formula

% Inhibition of = Mean number of x 100 Invasions 100- migrating cells in test

Mean number of migrating cells in control

3.b. Tumour cell motility assay

B16F-I0 cells (1x1OS10.15ml) were seeded on the upper

compartment of Blind well chamber, containing polycarbonate filters

without collagen i. lating. Chambers were incubated at 37OC for 24h.

Migrated cells were collected from the lower chamber and counted using

a haemocj tometer. Results were calculated as

' i ~ Motility = Mean number of x 100 migrating cells in test

Mean number of migrating cells in control.

3.d. Tumour Cell adhesion assay (132)

Transformed cells have higher adhesive attachment rates to a variety

I of homotypic or heterotypic cell substrates. Metastatic cells were always

found to have higher rates of homotypic attachment (133) 1

T i - \\

i Procedure I ' I B16F-10 melanoma cells (lxlOS/well) were added to 24 well flat i

I bottom titre plates, pre-coated with collagen type I (25 pglwell) and I I

! incubated for 5h at 37OC, in 5% CO, atmosphere. After incubation 1

medium was removed and the swells were washed with PBS. Adhering - -- I 1 cells were fixed with 5% folarnaldehyde and stained using crystal violet i

fol. 20 min. each. The cells were counted under an inverted microscope. 1 3.C. Gelatin Zyn~~~graphy (134)

!

j 1

Princi~le i

Proteases of tumour cell lysate were initially resolved on SDS

i i I

I polyacrylamide gels which were incorporated with gelatin. Following

incubation of the gel in the activation buffer, proteases separated on the

I I i

I gel will break down the gelatin and appear as transparent zones or i

I i

clearings against n dark back ground (upon staining) I I Reagents

I I

1. 0.25 M sucrose - 0.01M Tris-HCI buffer, pH 7.4

Sucrose - 85.57g

I Tris-HCI - 1.21g

Distilled watcr - 1 OOOml (Final volume)

I

I 2. O.IM Tris-HCI. pH-8.0, lOmM CaCl,. 1

i C ~ C I I , ~ H , O - - 1.47g

1 Tris - 12.lg

I i

pH adjusted with conc.HCI i I I I Distilled water - 1OOOml (Final volume) i I 1

I

\L-- 4

~ -

3. Trypsin solution

Trypsin-75pgIml in 0.1M Tris-HCI, with lOmM CaCI2, pH8.

4. Activation Buffer (0.1M Tris-HCI, lOmM CaCI,, pH7.8)

Tris-HCI - 12.lg

CaCl ? ' 2H,O - - 1.47g

Distilled water - 1 OOOml (Final volume)

5. Preparation of gels

11% polyacrylamide gels with 0.1% SDS and 0.6% gelatin

29.2% acrylarnide + 0.5% bisacrylamide - 1 lrnl

0.1M Tris-HC1, pH8.8 - 1.2ml

70% SDS - 0.15ml

20% Ammonium per sulphate - 0.10ml

Gelatin (180mg/2ml

distilled water, heated to dissolve) 2ml

Distilled water - 6.505m1

TEMED - 0.045ml

Mix and pour at room temperature

5% stacking gel

29.2% acrylamide+

0.5% bisacrylamide - 1.67ml

0.lM Tris-HC1, pH 8.8 - 1.75ml

20% SDS - 0.10ml

20'70 Ammonium persulphate - 0.10ml

~ ~

Distilled water - 6.36ml

TEMED - 0.02ml

Mix and pour above the 1 1 % get at room temperature.

6. Sample Buffer (2x)

Glycerol - Iml

1M Tris-HCI, pH6.8 - 0.25ml

20% SDS - Iml

Bromophenol blue - 1.65mg

(tracking dye)

Made upto 5ml with distilled water.

7. Running Buffer

Tris Base - 3g

SDS - 2g

Glycine - 14.2g

Made upto 1L with distilled water.

8. 2% Triton X- 100

Triton X- 100 - 2ml

0.1M Tris-HC1, pH 7.8 - I OOml (Final volume)

9. IOmM EDTA Solution

EDTA. Na, - 372.24mg

0.1 M Tris-HCI. pH 7.8 - 1 OOOml (Final volume)

10. Destaining solution

Methanol: Acetic acid : water - 50:10:40

1 1. 0.2% Coomassie Blue

Coomassie Blue - 0.2g

Destaining solution - l00ml

Pro~~edure:

Gelatin zymo"raphy was followed according to the procedure of

Habres and Billings ( 1 35) with some modifications. The medium from

sub- contluent (70%) bottles of B16F-10 tumour cells were removed,

cells were then washed with serum free medium and incubated in serum

free medium (DMEM) at 37OC for 24h.

After the incubation cells were detached by mechanical dissociation,

with a rubber police man and the cells were suspended in 0.5ml sucrose

buffer. Cells were lysed using sonicator with 6 burst for 10 seconds each

on i i 2 . The tumour cell lysate was centrifuged at 4OC, 5000g for lominutes

and after determining the protein concentration, supernatant (equivalent

to 100yg protein) containing the proteases were activated with trypsin

(75 pglml. 5y1 trypsin solution for 100 yg protein) for 30 minutes at

room temperature. Trypsin treated and untreated samples (equivalent to

10Oyg protein) were mixed with an equal-volume of sample buffer (2x1,

without reducing agent, loaded on 0.1% SDS-11% polycirylamide gels

containing 0.6% gelatin and electrophoresed at 10°C for 3h with a constant

current of 2mA per tube until the tracking dye (Bromophenol Blue)

reached the periphery of the gels.

The gels were then washed with 2% Triton X-100 on a shaker at

20-25OC for three changes of 30 minutes each, to remove the SDS which

could interfere with proteolytic activity. This was followed by Zh washing

with activation buffer and the gels were finally incubated ir the same

buffer at 37OC for 18h, in the presence and absence of test compound.

Gels were stained with 0.2% coornassie blue for 2h, destained to visualize

the clear bands against a dark background.

XII. Statistical Analysis

Statistical significance of the data was calculated using students 't '

test (136). To determine the significance between group (X) and group

B 9

I (Y) the value 't' was found from the equation: I

i 1 t - X-V !

i s l + L I i nx ny !

I

where I ~ I x - Mean of Sample (X) i i i

y - Mean of Sample (Y) i

nx - Sample size (X)

ny - Sample size (Y)

S was found out from the equation

i S = [nx- I ) sx2+(nv- l )sv2 (nx+ny)-2 i

1 where

sx - Standard deviation of x

: sy - Standard deviation of y

By knowing the degree of freedom (nx-ny-2) statistical significance (P value) was deduced from the "t" distribution table.

I I I i I

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