Anti- Tumor assay / Screening of Anticancer Drugs

Anti-Tumor Assay
PRESENTED BY :
PRATIK UMESH PARIKH
MASTER OF PHARMAC Y
(PHARMAC EU TIC AL BIOTECHNOLO G Y)
PRESENTEDBY : PRATIKUMESH PARIKH
GUIDED BY :
DR. S. B. DAHIKAR SIR
CELLULAR & MOLECULAR BIOLOGY

Contents :
Introduction of cancer
Need for novel anti cancer drugs
In-vitro assay
In- Vivo assay
Reference

Introduction of Cancer:
•Cancer is a disease which is characterized by uncontrolled proliferation of cells
that have transformed from the normal cells of the body.
• Factors:
External Factors – chemicals, radiation, bacteria, viruses, and lifestyle
Internal Factors – hormones, immune conditions, and inherited mutations

• Although 92 approved anticancer drugs are available today for the treatment of
more than 200 different tumor entities, effective therapies for most of these
tumors are lacking.
• They are mostly cytotoxic in nature and act by a very limited number of
molecular mechanisms.
• Thus, the need for novel drugs to treat malignant disease requiring systemic
therapy is still pressing.
• A pre selection, called the screening process, is therefore required.
• The goal of screening assay is to test the ability of a compound to kill cells, it
should be able to discriminate b/t replicating & non replicating cell

Need for Novel Anti-Cancer Drugs:
•Development of multidrug resistance in patients.
• Long-term treatment with cancer drugs is also associated with severe side
effects.
• Cytotoxic drugs have the potential to be very harmful to the body unless they
are very specific to cancer cells.
• New drugs that will be more selective for cancer cells

Anti-tumor Assay models:
In-vitro Assay In-vivo Assay

In-Vitro Methods:
1. Tetrazolium salt assay
2. Sulphorhodamine B assay
3. 3H-Thymidine uptake assay
4. Fluorescence
5. Dye exclusion test
6. Clonogenic test
7. Cell counting assay
8. Morphological assay

1. Tetrazolium salt assay
•This assay is a sensitive, quantitative and reliable colorimetric assay that
measures viability, proliferation and activation of cells.
• The assay is based on the capacity of mitochondrial dehydrogenase enzymes in
living cells to convert the yellow water-soluble substrate 3-(4,5-dimethylthiazol-
2yl)-2,5-diphenyl tetrazolium bromide (MTT) into a dark blue formazan product
which is insoluble in water.

• The amount of formazan produced is directly proportional to the cell number in
range of cell lines

2. Sulphorhodamine B assay
PRINCIPLE
• Ability of SRB to bind to protein components of cells fixed to tissue culture
plates by TCA
•SRB : bright pink aminoxanthine dye with 2 sulfonic groups
•Binding of SRB is stoichiometric
•Amount of dye extracted from stained cells proportional to the cell mass

Method:
• The Sulphorhodamine B assay measures whole-culture protein content, which
should be proportional to the cell number.
• Cell culture are stained with a protein staining dye, Sulphorhodamine B.
• SRB is a bright pink anionic dye that binds to basic amino acid of cell.
• Unbound dye is then removed by washing with acetic acid.
• During the dead cell either lyse or are lost during procedure, the amount of SRB
binding is proportional to the number of live cells left in a culture after drug
exposure.

3. 3H-Thymidine uptake assay
PRINCIPLE
• 3H- thymidine incorporated into new strands of DNA
•Scintillation beta- counter used to measure radioactivity in DNA recovered from
the cells in order to determine extent of cell division occurred in response to
test agent
• Direct measures of proliferation

Method:
• Tumor cell suspensions exposed to the drug for 5 days
•3H- thymidine added during final 48 hrs
•Replicating cells incorporate 3H- thymidine into their DNA
•Determined either by autoradiography / liquid scintillation counter
•Generate DNA histograms ploidy status of cells
•Non replicating or dead cells will not be counted

4. Dye exclusion test
• To determine cell viability in which dilute solution of dyes ( trypan blue, eosin Y,
nigrosin, Alcian blue) is mixed with suspensions of cells
• Cells exclude dye :- alive
• Cells that stain:- dead
• Indicate structural integrity of cell membrane
• Weisenthal and colleagues used novel combination of fast green dye & eosin –
hematoxylin with more promising results, in patients with hematological
malignancies (CLL)

5. Cologenic Assay
• In-vitro cell survival assay based on the ability of single cell to grow into a
colony
•Assay tests every cells in population for its ability to undergo unlimited division
•Imp. for drugs act by arresting check points in cell cycle

In-Vivo Methods:
1. Carcinogen induced models
2. Viral infection models
3. Transplantation Models
4. Genetically Engineered Mouse Models
5. In vivo hollow fibre assay

1. Carcinogen Induced Model
DMBA induced mouse skin papillomas
•Two stage experimental carcinogenesis
› Initiator – DMBA(dimethylbenz[a]anthracene),
› Promotor – TPA (12-O-tetradecanoyl-phorbol-13acetate)
•Mice : Single dose – 2.5 µg of DMBA , 5 to 10 μg of TPA in 0.2 ml of acetone
twice weekly.
•Papilloma begins to appear after 8 to 10 weaks
•Tumor incidence & multiplicity of treatment group is compared with DMBA
control group

Control Model DMBA Model

Method
 Mice are topically applied a single dose of 2.5 µg DMBA in acetone, followed by 5-10 µg
of TPA in 0.2 ml acetone twice weekly on the same site starting one week after DMBA
application.
 Percent tumor incidence and multiplicity of treatment groups is compared with DMBA
control group.
 Drug under test can be administered either topically or oral route.
 The tumor incidence in this model is usually about 100% in DMBA controls.
 In repeated topical application of DMBA Alone has also been shown to induced
carcinogenesis.
 Drug efficacy is measured as percent reduction in carcinoma incidence, compared with
that of carcinogen control.

2) MNU induced rat mammary gland CA
• MNU Induces hormone dependent tumors.
• Single i.v of 50 mg/kg of methylnitrosourea(MNU)
given to 50 days old Sprague-Dawley rats.
• Adenocarcinoma will be produced within 180
days of post carcinogen in 75 to 95% cases
• Drug efficacy is measured
• Drawback – cannot detect inhibition of
carcinogen activation
• Better model for human breast cancer

3) Method Involving cell line :
• Specified number of particular cell line inoculated into sensitive mouse strain
• Tumors develop rapidly thus time saving
• Effective drug retard tumor growth and increase life span of animal
• L-1210, P-388, B-16 cell lines- host mouse strain BDF1
• Sarcoma-180 – Swiss albino mouse

4) Xenografts :
• Human tumors(lung, breast, colon, ovary, brain, HCC) are optimized in mouse cell lines
• Directly injected below the skin of the mouse
• Drugs showing activity in hollow fiber model are administered at various dosages
• Compounds that kill or slowdown growth of specific tumor with minimal toxicity-
proceed to next stage of testing
• 1. Spheroid culture of LuCap 147-induced prostate cancer model
• 2.Integration free-induced pluripotent stem cell model high throughput screening drug
induced cell cycle arrest Apoptosis can be demonstrated in spheroid cultures

5) EAC Cells by Liquid Tumour Model (Ehrlich Ascites Carcinoma)
on (Swiss albino mice) EXPERIMENTAL PROTOCOL
• Induction of ascitic carcinoma - The ascitic tumor bearing mice (donor) were
used . The ascitic fluid was drawn using an 18 gauge needle into a sterile syringe.
A small amount of tumor fluid was tested for microbial contamination. Tumor
viability was determined by tryphan blue exclusion test and cells were counted
using haemocytometer. The ascitic fluid was suitably diluted with saline to get a
concentration of 10 million cells/ml of tumor cell suspension. 250 µl of this fluid
was injected in each mouse by i.p. route to obtain ascitic tumor

• The mice were weighed on the day of tumor inoculation and then for each
three days.
• Cisplatin was injected on two alternative days 1st and 3rd day after tumor
inoculation (intraperitoneally).
• The drugs were administered after 24 hours of tumor inoculation and were
admistered till 9th day intraperitoneally.
• On 15th day blood was collected from the animal through the retroorbital
plexus to determine the heamatological parameters and lipid profil

Parameters monitored :
1) % Decrease in weight variation compared to control.
2) Median survival time (MST) and percentage increase in lifespan (%ILS).
3) Mean survival time (MEST) and percentage increase in lifespan (%ILS).
4) Cell viability test. (% Survivors of malignant cells in ascitic fluid).
5) Haematological parameters a. Total W.B.C. and differential leukocyte counts.
b. Total R.B.C. and Hemoglobin content.

Cell viability test :
• Tryphan blue exclusion test is based on the principle that the living cell membrane has the ability to prevent the entry
of the dye. Hence they remain unstained and can be easily distinguished from the dead cells, which take the dye.
• On day15 after tumor inoculation 0.1 ml of ascitic fluid was taped and subjected to Tryphan Blue Assay to assess the
number of viable tumor cells
• Equal volumes of EAC cell suspension and 0.1% Tryphan blue solution were mixed thoroughly. The diluted suspension
was charged into Haemocytometer
• The viable (unstained) were counted in WBC chamber under microscope and mean number of cells in 4 chambers
were counted.
•Total number of cells =Mean number of cells x dilution factor x104

Reference :
1. Drug screening methods by S K Gupta, 3rd edition, page no: 170-187
2. Drug discovery and evaluation: pharmacological assays by Hans Gerhard
Vogel , 3rd edition
3. Stephen Hus, Fu Xin Yu et al , A mechanism based in in-vitro anticancer drug
screening approach by , 46

Anti- Tumor assay / Screening of Anticancer Drugs

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